Encyclopedia of Agrochemicals, 3 Volume Set

ENCYCLOPEDIA OF AGROCHEMICALS VOLUME 1 ENCYCLOPEDIA OF AGROCHEMICALS Editor-in-Chief Jack R. Plimmer Editorial Staf...

Author: Jack R. Plimmer

300 downloads 2338 Views 15MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

ENCYCLOPEDIA OF

AGROCHEMICALS VOLUME 1

ENCYCLOPEDIA OF AGROCHEMICALS Editor-in-Chief Jack R. Plimmer

Editorial Staff Vice-President, STM Books: Janet Bailey Executive Editor: Jacqueline I. Kroschwitz

Associate Editor Derek W. Gammon California EPA Associate Editor Nancy R. Ragsdale Agricultural Research Service, USDA

Director, Book Production and Manufacturing: Camille P. Carter Managing Editor: Shirley Thomas Publishing Technology Associate Manager, Books: David Blount Illustration Manager: Dean Gonzalez Editorial Assistant: Audrey Roker

ENCYCLOPEDIA OF

AGROCHEMICALS VOLUME 1 Jack R. Plimmer Derek W. Gammon Nancy N. Ragsdale

The Encyclopedia of Agrochemicals is available Online at www.mrw.interscience.wiley.com/eoa

A John Wiley & Sons, Inc., Publication

Copyright  2003 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, e-mail: [email protected]. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services please contact our Customer Care Department within the U.S. at 877-762-2974, outside the U.S. at 317-572-3993 or fax 317-572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print, however, may not be available in electronic format. Library of Congress Cataloging in Publication Data: ISBN 0-471-19363-1 Encyclopedia of agrochemicals / Jack R. Plimmer, editor-in-chief. p. cm. ISBN 0-471-19363-1 (cloth) 1. Agricultural chemicals—Encyclopedias. I. Plimmer, Jack R., 1927S584.4 .E53 2002 631.8 03—dc21 2002027418 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

PREFACE The Encyclopedia of Agrochemicals covers chemical technology relating to pesticides, fertilizers, and other chemicals germane to this topic. Because the scope of this field has broadened considerably with deeper understanding of the environmental implications of chemical use and its ecological consequences, these aspects have been included. Such knowledge has stimulated greater regulatory activity and increased the need for toxicological information, which we have included. However, manufacturing technology has been excluded from the Encyclopedia. The Encyclopedia is a compilation of many specially commissioned original articles. It also includes definitions: The work is intended to serve principally as a source of chemical information but toxicology, metabolism, biotechnology, regulatory and environmental aspects have been included. The widespread use of synthetic pesticides began approximately 50 years ago and we have included articles on classes of compounds which, although no longer in use, have provided experience of the environmental implications of the use of xenobiotics. Moreover in many cases, especially for some organochlorine insecticides, their persistence combined with widespread use has led to sites of contaminated land and water, continuing to the present. However, it should also be borne in mind that many of these insecticides have played a major role in restricting diseases such as malaria, yellow fever, dengue fever, plague, Chargas’ disease, African river blindness, typhus, sleeping sickness etc. Such success could not have been achieved had these agents not been initially developed as agrochemicals. The years immediately following the close of World War II witnessed almost explosive growth in the development and application of synthetic organic chemicals to the control of insect pests, plant-disease causing organisms, and weeds. The subsequent challenges to pest management systems that relied predominantly on chemical control arose both from environmental concerns and from the development of resistance among pest species. Alternative strategies were needed and these developments were accompanied by the establishment of regulatory agencies whose major goal was to mitigate the harmful effects of pesticide use, whether real or perceived. Industry responded to this evolutionary process by seeking to develop molecules that met criteria of improved human and environmental safety. In recent years biotechnology has provided solutions by modifying crops so that they would express insecticidal toxins or resist the action of herbicides that had met the approval of regulatory authorities. These changes have been reflected in the market place. The acreage treated with pesticides remains high but amounts applied have decreased as chemicals become more efficacious and application becomes more efficient. The pesticide market continues to flourish and in the United States where over 1.2 billion pounds of active ingredient were used in 1997, pesticides account for about 4.5% of total farm production costs (1). The situation in the US was the subject of a report by a National Research

Council Committee on the Future Role of Pesticides in US Agriculture which concluded that ‘‘chemical pesticides will continue to play a role in pest management in the foreseeable future, in part because the environmental compatibility of products is increasing—particularly with genes that protect crops, chemicals with new modes of action, and non pesticide management techniques’’ (2). Although the pesticide market has leveled off in N. America and Europe, growth in the remainder of the world reflects the continued demand. China, in particular, has increased its production of pesticide chemicals and production of the active ingredients of pesticides grew from 395 thousand metric tons in 1997 to 696 thousand metric tons in 2001 (3). The degree of sophistication associated with pesticide use in Europe and N. America must be matched in the lesser developed countries by increased education and training if the full benefits of this technology are to be realized. The preparation of this work relied on the cooperation of experts world wide. We would like to thank our many contributors who have devoted time to the preparation of articles. This has been a particularly difficult time for those who work in industrial research. During its preparation there were many changes in industry that resulted in consolidation of programs and redirection of research effort. Thanks are due to colleagues in industry who have prepared manuscripts as well as coping with organizational changes and shifts in research emphasis. Our contributors in government and academia have also felt the general pressures of reduced support for science. We recognized that gaps in coverage were inevitable because the exigencies of publication did not always harmonize with the schedules of the many experts who were consulted during the preparation of the Encyclopedia. We acknowledge the help and advice of many colleagues. Among them Dr. Terry Roberts of JSCN International was involved in the conceptual and planning stages, Dr. Steve Duke provided guidance and help with herbicide entries, Dr. Istvan Ujvary assisted in writing or commissioning articles. Many definitions have been included. Where indicated in the text these have been published by IUPAC in the IUPAC Report on Pesticides (36), Glossary of Terms relating to Pesticides, Pure and Appl. Chem. 68:N06, pp. 1167–1193 (1996). (1) A. L. Aspelin and A. H. Grube, Pesticides Industry Sales and Usage: 1996 and 1997 Market Estimates, Biological and Economic Analysis Division, Office of Pesticide Programs, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC 20460, November 1999, 733-R-99-001. (2) Committee on the Future Role of Pesticides in US Agriculture, ‘‘The Future Role of Pesticides in US Agriculture’’, National Research Council, Board on Agriculture and Natural Resources, National Academy Press, Washington, D.C. p.2 (2000). (3) Chemical and Engineering News, 80, 82 (2002). Jack R. Plimmer v

CONTRIBUTORS Heinrich Dittmar, BASF Aktiengesellschaft, Ludwigshafen, Germany, Plant Nutrition

Basil Acock, USDA-ARS, Beltsville, Maryland, USDA-ARS Pesticide Properties Database Thomas Addiscott, Rothamsted Experimental Station, Harpenden, Herts, United Kingdom, Nitrate in Groundwater David B. Alexander, University of Portland, Portland, Oregon, Soil Bacteria Reza Asiaie, Roche Molecular Systems, Somerville, New Jersey, Capillary Electrophoresis Michael L. Avery, National Wildlife Research Center, Gainesville, Florida, Avian Repellents Joseph E. Bailey, Arlington, Virginia, Food Quality Protection Act of 1996 David L. Balkwill, Florida State University, Tallahassee, Florida, Subsurface Microbial Communities: Diversity of Culturable Microorganisms Jerry J. Baron, Rutgers, The State University of New Jersey, North Brunswick, New Jersey, Minor Use Pesticides, Registration Anton Baudoin, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, Chemotherapy of Plant Diseases Donald H. Beermann, University of Nebraska, Lincoln, Nebraska, Growth Regulators, Animals G. Knauf-Beiter, Syngenta Crop Protection AG, Basel, Switzerland, Fungicides, Phenylpyrroles Ravi G. Bhat, University of California, Davis, California, Tillage Eula Bingham, Regulations and Guidelines in the Workplace Robert S. Boethling, Office of Pollution Prevention and Toxics, U.S. Environmental Protection Agency, Washington, District of Columbia, Chemical Properties Estimation Douglas Boyette, USDA, ARS, Southern Weed Science Res. Unit, Stoneville, Mississippi, Herbicides, Biotechnology for Control of Weeds ¨ Walter Brandlein, BASF Aktiengesellschaft, Ludwigshafen, Germany, Plant Nutrition Keith J. Brent, Bristol, England, Fungicides, An Overview Richard Bromilow, Rothamsted, Harpenden, Herts, United Kingdom, Soil Persistence Matthew W. Brooks, AG-CHEM Consulting, LLC, Clifton, Virginia, Standard Evaluation Procedures Thomas M. Brown, GenéCTAr.com, LLC, Clemson, South Carolina, Insect Resistance to Insecticides Charles T. Bryson, USDA-ARS, Stoneville, Mississippi, Weed Species Alan Buckle, (formerly Novartis Crop Protection, Basel), Hergiswill, Switzerland, Rodenticides Carolee T. Bull, USDA–ARS, Salinas, California, Biological Control of Plant Diseases H. Buschhaus, Aventis CropScience GmbH, Regulatory Affairs Europe, Frankfurt am Main, Germany, Fungicides, Carbamates Antonio Caballero, Estación Experimental del Zaid´ın, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes Thomas M. Cahill, Canadian Environmental Modelling Centre, Trent University, Peterborough, Ontario, Canada, Fugacity Modeling C. Camara, University Complutense of Madrid, Madrid, Spain, Ethylenebisthiocarbamates (Analysis) D. A. Carlson, University of Florida, Gainesville, Florida, Repellents David J. Chitwood, USDA-ARS, Beltsville, Maryland, Nematicides J. Marshall Clark, University of Massachusetts, Amherst, Massachusetts, Turfgrass Pesticides: Management and Environmental Issues Ralf Conrad, Max-Planck-Institut fur ¨ Terrestrische Mikrobiologie, Marburg, Germany, Flooded Soils ´ Tibor Cserhati, Hungarian Academy of Sciences, Budapest, Hungary, Chromatography, HPLC; Chromatography, TLC Vilmos Czikkely, BASF Aktiengesellschaft, Ludwigshafen, Germany, Plant Nutrition Franck E. Dayan, USDA-ARS Natural Products Utilization Research Unit, University, Mississippi, Herbicides, Carotenoid Biosynthesis Inhibitors; Herbicides, Cinmethylin; Herbicides, Protoporphyrinogen Oxidase Inhibitors A. J. Dewar, Arthur Dewar Associates, United Kingdom, EU Registration Directive

Kelley J. Donham, , Agricultural Hygiene Manfred Drach, BASF Aktiengesellschaft, Limburgerhof, Germany, Plant Nutrition Stephen O. Duke, USDA, ARS, Natural Products Utilization Res. Unit, University, Mississippi, Herbicides, Biotechnology for Control of Weeds; Herbicides, Carotenoid Biosynthesis Inhibitors; Herbicides, Cinmethylin; Herbicides, Protoporphyrinogen Oxidase Inhibitors Estrella Duque, Estación Experimental del Zaidıń, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes Clive Edwards, The Ohio State University, Columbus, Ohio, Microcosms Janice W. Edwards, Monsanto Company, St. Louis, Missouri, Genetic Engineering, Plants Thomas Egli, Swiss Federal Institute for Environmental Science and Technology, Dubendorf, ¨ Switzerland, Metabolism of Mixtures of Organic Pollutants Monica Elliott, Montana State University, Bozeman, Montana, PCR Manuel Espinosa-Urbel, Estación Experimental del Zaidıń, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes ´ nez, ˜ Abraham Esteve-Nu Estación Experimental del Zaidıń, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes Morifusa Eto, Fukuoka, Japan, Organophosphorus Insecticides Allan Felsot, Washington State University Tri-Cities, Richland, Washington, LD50 ´ Esther Forgacs, Hungarian Academy of Sciences, Budapest, Hungary, Chromatography, HPLC; Chromatography, TLC Peter Frenzel, Max-Planck-Institut fur ¨ Terrestrische Mikrobiologie, Marburg, Germany, Flooded Soils L. J. Shane Friesen, University of Guelph, Guelph, Ontario, Canada, Herbicides, Plant Hormone Mimics—Auxins Derek W. Gammon, California EPA, Sacramento, California, Herbicides: Inhibitors of Glutamine Synthetase; Insecticides, Organochlorines B. Clifford Gerwick, Dow Agrosciences, Indianapolis, Indiana, Herbicides, Identification of Biologically Active Materials Ulrich Gisi, Syngenta Crop Protection, Basel, Switzerland, Fungicides, Phenylamides/Acycalanines David J. Glass, D. Glass Associates, Inc., Needham, Massachusetts, Regulation of the Commercial Use of Microorganisms Dayan B. Goodenowe, Yol Bolsum Inc., Rycroft, Alberta, Canada, LC/MS, Pesticide Residue Analysis L. Faye Grimsley, Regulations and Guidelines in the Workplace Reinhold Gutser, Lehrstuhl fur ¨ Pflanzenernahrung, ¨ Technische Universitat ¨ Munchen-Weihenstephan, ¨ Freising, Germany, Plant Nutrition ¨ Reinhardt Hahndel, BASF Aktiengesellschaft, Limburgerhof, Germany, Plant Nutrition Christopher Hall, University of Guelph, Guelph, Ontario, Canada, Herbicides, Plant Hormone Mimics—Auxins Raymond Hammerschmidt, Michigan State University, East Lansing, Michigan, Juglone; Preexisting Defense Chemicals Kriton Hatzios, Virginia Polytechnic Institute, Blacksburg, Virginia, Herbicides, Plant Resistance to Herbicides Matthew J. Henry, Dow AgroSciences, Indianapolis, Indiana, Fungicides, Quinoxyfen Joan M. Henson, Montana State University, Bozeman, Montana, PCR Albert E. Herner, USDA-ARS, Beltsville, Maryland, USDA-ARS Pesticide Properties Database Abe Hiroshi, Tokyo University of Agriculture, Tokyo, Japan, Brassinosteroids Robert E. Hoagland, USDA, ARS, SWSRU, Stoneville, Mississippi, Metabolism of Herbicides Derek W. Hollomon, University of Bristol, Bristol, United Kingdom, Fungicides, 2-Aminopyrimidines; Fungicides, Fungal Resistance to Chemical Controls

vii

viii

CONTRIBUTORS

Robert E. Holm, Rutgers, The State University of New Jersey, North Brunswick, New Jersey, Minor Use Pesticides, Registration William R. Horwath, University of California, Davis, California, Biomass: Soil Microbial Biomass Philip H. Howard, Syracuse Research Corporation, Environmental Science Center, North Syracuse, New York, Chemical Properties Estimation Hideo Ishii, National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, Japan, Fungicides, Tubulin-Binding Compounds Nicholas Jarvis, Swedish University of Agricultural Sciences, Uppsala, Sweden, Macropore Flow Models; Macropore and Preferential Flow Paul Jepson, Oregon State University, Corvallis, Oregon, Natural Enemies Philip J. Jewess, IACR–Rothamsted, Harpenden, Hertfordshire, United Kingdom, Acetylcholine Receptors; Acetylcholinesterase; Chitin Biosynthesis Inhibitors Madan M. Joshi, DuPont Agricultural Products, Wilmington, Delaware, Fungicides, Cymoxanil Shinzo Kagabu, Gifu University, Gifu, Japan, Insecticides, Imidacloprid Philip C. Kearney, USDA-ARS, Beltsville, Maryland, Soil Chemistry of Pesticides Ivan R. Kennedy, University of Sydney, Sydney, Australia, Immunoassays Allen Kerr, University of Adelaide, Adelaide, Australia, Agrocin 84 Sushil K. Khetan, New Delhi, India, Storage Stability Ganesh M. Kishore, Monsanto Company, St. Louis, Missouri, Genetic Engineering, Plants Kimiko Klein, California Environmental Protection Agency, Sacramento, California, Remediation William A. Kleschick, Dow AgroSciences LLC, Indianapolis, Indiana, Combinatorial Chemistry as Applied to The Discovery of Agrochemicals ¨ Gunter Kluge, Bundesministerium fur ¨ Ernahrung, ¨ Landwirtschaft und Forsten, Bonn, Germany, Plant Nutrition ´ ´ Institut Rudjer Boˇsković, Zagreb, Croatia, Auxins, Biserka Kojic-Prodi c, Indole Auxins ¨ Wolfram Koller, Cornell University, Ithaca, New York, Fungicides, Sterol Biosynthesis Inhibitors William C. Koskinen, USDA-Agricultural Research Service, Soil Chemistry of Pesticides Sagar V. Krupa, University of Minnesota, St. Paul, Minnesota, Ethylene Alexander J. Krynitsky, U. S. Environmental Protection Agency, Fort Meade, Maryland, LC/MS, Pesticide Residue Analysis Friedrich Kuhlmann, Institut fur ¨ Landwirtschaftliche Betriebslehre, Giessen, Germany, Plant Nutrition Paul J. Kuhn, Syngenta Crop Protection, Vero Beach, Florida, Fungicides, Inhibitors of Mitochondrial Energy Production Karl-Friedrich Kummer, BASF Aktiengesellschaft, Limburgerhof, Germany, Plant Nutrition Walter Kunz, Syngenta Crop Protection AG, Basel, Switzerland, Chemical Activators of Disease Resistance Norio Kurihara, Kyoto, Japan, Chirality and Chiral Pesticides Leslie C. Lane, University of Nebraska, Lincoln, Nebraska, Electrophoresis Bernd Lennartz, University Rostock, Rostock, Germany, Soil, Movement of Pesticides In Pierre Leroux, Institut National de la Recherche Agronomique, Versailles, France, Fungicides, Anilopyrimidines; Fungicides, Dicarboximides Gavin B. Lewis, JSC International Ltd., Harrogate, North Yorkshire, United Kingdom, Natural Enemies Leslie C. Lewis, Agricultural Research Service, USDA, Ankeny, Iowa, and Iowa State University, Ames, Iowa, Biological Control, Survey Morton Lippmann, Pathways and Measuring Exposure to Toxic Substances David A. Lipson, San Diego State University, San Diego, California, Kinetics of Microbial Processes and Population Growth J. P. Little, Aventis CropScience, Ongar, Essex, United Kingdom, Herbicides, Inhibiting 4-Hydroxyphenylpyruvate Dioxygenase (HPPD) John Lydon, USDA, ARS, Sustainable Agricultural Systems Res. Unit, Beltsville, Maryland, Herbicides, Biotechnology for Control of Weeds H. Lyr, Eberswalde, Germany, Fungicides, Aromatic Hydrocarbons Donald Mackay, Canadian Environmental Modelling Centre, Trent University, Peterborough, Ontario, Canada, Fugacity Modeling

Neil Mackay, Cambridge Environmental Assessments, Boxworth, United Kingdom, Groundwater Modeling Tools Eugene L. Madsen, Cornell University, Ithaca, New York, Biodegradability: Assessment Volker Magnus, Institut Rudjer Boˇsković, Zagreb, Croatia, Auxins, Indole Auxins Ashok Kumar Malik, G.N.D. University, Amritsar, Punjab, India, Ethylenebisthiocarbamates (Analysis) Otis C. Maloy, Washington State University, Pullman, Washington, Chemotherapy of Plant Diseases; Disinfection; Disinfestation George M. Markle, Rutgers, The State University of New Jersey, North Brunswick, New Jersey, Minor Use Pesticides, Registration Albertus Martijn, CIPAC, Ruurlo, The Netherlands, CIPAC Konrad Mengel, Justus-Liebig-Universitat ¨ Giessen, Institute for Plant Nutrition, Giessen, Germany, Plant Nutrition Julius J. Menn, Highland, Maryland, Biopesticides Robert L. Metcalf, University of Illinois, Urbana, Illinois, Genetic Control of Insects William M. Meylan, Syracuse Research Corporation, Environmental Science Center, North Syracuse, New York, Chemical Properties Estimation ´ Carmen Michan, Estación Experimental del Zaid´ın, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes Carol J. Miller, Wayne State University, Detroit, Michigan, Groundwater Monitoring Pierre Mineau, National Wildlife Research Centre, Canadian Wildlife Service, Ottawa, Ontario, Canada, Avian Species Amitava Mitra, University of Nebraska, Lincoln, Nebraska, Genetic Engineering William T. Molin, USDA-ARS, Stoneville, Mississippi, Herbicides, Pyridine Carboxylate Thomas B. Moorman, USDA Agricultural Research Service, National Soil Tilth Laboratory, Ames, Iowa, Soil Distribution of Microorganisms Takayuki Motoyama, RIKEN (The Institute of Physical and Chemical Research), Wako, Japan, Fungicides, Melanin Biosynthesis Inhibitors ¨ Hermann Muhlfeld, Formerly Chemische Fabrik Kalk GmbH, Köln, Germany, Plant Nutrition ¨ Franz Muller, (formerly Novartis Crop Protection, Basel), Hergiswill, Switzerland, Genetic Control of Insects; Rodenticides David D. Myrold, Oregon State University, Corvallis, Oregon, Nitrogen Cycle in Soil William E. Newton, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, Nitrogen Fixation Ralph L. Nicholson, Purdue University, West Lafayette, Indiana, Phytoalexin Titus Niedermaier, Formerly BASF Aktiengesellschaft, Ludwigshafen, Germany, Plant Nutrition Naoko Nishikawa-Koseki, Tokyo University of Agriculture, Tokyo, Japan, Brassinosteroids Patrick Noland, ABC Laboratories Europe, Coleraine, Ireland, Solid Phase Extraction Walter Oettmeier, Ruhr—Universitaet Bochum, Bochum, Germany, Herbicides, Inhibitors of Photosynthesis at Photosystem II Offiah O. Offiah, Maryland Department of Agriculture, Annapolis, Maryland, Analysis—Instrumentation and Techniques Anna Oliva, Second University of Naples, Caserta, Italy, Herbicides, Biotechnology for Control of Weeds Michael O’ Malley, University of California, Davis, California, Epidemiology of Pesticide Exposure Michael Oostendorp, Syngenta Crop Protection AG, Basel, Switzerland, Chemical Activators of Disease Resistance Ann B. Orth, FMC Corporation, Princeton, New Jersey, Fungicides, Inhibitors of Mitochondrial Energy Production Craig Osteen, Economic Research Service, USDA, Washington, District of Columbia, Economic Issues of US Agricultural Pesticide Use Merritt Padgitt, Economic Research Service, USDA, Washington, District of Columbia, Economic Issues of US Agricultural Pesticide Use Ken E. Pallett, Aventis CropScience, Frankfurt am Main, Germany, Herbicides, Asulam; Herbicides, Inhibiting 4-Hydroxyphenylpyruvate Dioxygenase (HPPD) ´ Laszl o´ Pap, Agro-Chemie Ltd., Budapest, Hungary, Pyrethroids

CONTRIBUTORS Marshall H. Parker, Dow AgroSciences LLC, Indianapolis, Indiana, Combinatorial Chemistry as Applied to The Discovery of Agrochemicals Juergen Pauluhn, Bayer AG, Wuppertal, Germany, Inhalation Toxicity Mikael Pell, Swedish University of Agricultural Sciences, Uppsala, Sweden, Toxicity Testing in Soils Wilfried Pestemer, Federal Biological Research Centre of Agriculture and Forestry, Berlin, Germany, Bioassays: Phytotoxicity to Succeeding Crops Ch. Pillonel, Syngenta Crop Protection AG, Basel, Switzerland, Fungicides, Phenylpyrroles Jack R. Plimmer, Tampa, Florida, Insecticides, Organochlorines; Herbicides: Inhibitors of Glutamine Synthetase; Herbicides, An Overview; Insecticidal Carbamates Ernest L. Plummer, FMC Corporation, Princeton, New Jersey, QSAR William Popendorf, Agricultural Hygiene ¨ Formerly BASF Aktiengesellschaft, Limburgerhof, Germany, Hans Prun, Plant Nutrition Jos M. Raaijmakers, Wageningen University, Wageningen, The Netherlands, Rhizosphere ´ ´ Marıa-Isabel Ramos-Gonzalez, Estación Experimental del Zaid´ın, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes Juan L. Ramos, Estación Experimental del Zaid´ın, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes P. T. Reeves, National Registration Authority for Agricultural and Veterinary Chemicals, Kingston, Australia, Animal Health Products Jon B. Reid, Dibenzo-p-Dioxins: 2,3,7,8-Tetrachlorodibenzo-p-Dioxin William A. Rickelton, Cytec Canada Inc., Phosphine and Its Derivatives Joanne G. Romagni, USDA-ARS Natural Products Utilization Research Unit, University, Mississippi, Herbicides, Cinmethylin Erin N. Rosskopf, USDA–ARS, USHRL, Fort Pierce, Florida, Biological Control of Weeds Robert P. Sabba, USDA-ARS, Fargo, North Dakota, Herbicides, Cellulose Biosynthesis Inhibitors Hudan Safarpour, American Cyanamid Company, Princeton, New Jersey, Stephen H. Safe, Texas A&M University, College Station, Texas, Chlorocarbons and Chlorohydrocarbons—Toxic Aromatics Mitsuru Sasaki, Kobe University, Kobe-shi Hyogo-ken, Japan, Fungicides, Organophosphorus Compounds Brian E. Scheffler, USDA, ARS, Natural Products Utilization Res. Unit, University, Mississippi, Herbicides, Biotechnology for Control of Weeds Heinrich W. Scherer, Universitat ¨ Bonn, Agrikulturchemisches Institut, Bonn, Germany, Plant Nutrition Steven K. Schmidt, University of Colorado, Boulder, Colorado, Kinetics of Microbial Processes and Population Growth Mark R. Schmitt, Trenton, New Jersey, Fungicides, Inhibitors of Mitochondrial Energy Production Ana Segura, Estación Experimental del Zaid´ın, Granada, Spain, Biodegradation of Xenobiotics by Engineered Microbes Dale Shaner, USDA-ARS, Colorado State University, Fort Collins, Colorado, Herbicides, Imidazolinone Daniel R. Shelton, USDA-Agricultural Research Service, Soil Chemistry of Pesticides Malcolm C. Shurtleff, University of Illinois, Urbana–Champaign, Illinois, Texas A&M University, College Station, Texas, Aflatoxin; Fumonisins; Mycotoxin and Mycotoxicoses; Vomitoxin James J. Sims, University of California, Riverside, California, Fungicides, Soil Fumigants John H. Skerritt, Australian Centre for International Agricultural Research, Canberra, Australia, Immunoassays Jeffrey L. Smith, USDA–ARS, Washington State University, Pullman, Washington, Soil Quality, The Role of Microorganisms David M. Stark, Monsanto Company, St. Louis, Missouri, Genetic Engineering, Plants J. R. Startin, Central Science Laboratory, Sand Hutton, York, United Kingdom, MALDI Paul Staswick, University of Nebraska, Lincoln, Nebraska, Jasmonic Acid Theodor Staub, Syngenta Crop Protection AG, Basel, Switzerland, Chemical Activators of Disease Resistance ¨ Gunter Steffens, Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Oldenburg, Germany, Plant Nutrition A. Steinemann, Syngenta Crop Protection AG, Basel, Switzerland, Fungicides, Phenylpyrroles

ix

Hugo Steinhauser, Formerly Lehrstuhl fur ¨ Wirtschaftslehre des, Landbaues, Technische Universitat ¨ Munchen, ¨ Freising, Germany, Plant Nutrition Steven J. Stout, BASF Corporation, Princeton, New Jersey, LC/MS, Pesticide Residue Analysis James Stry, Dupont Crop Protection, Newark, Delaware, LC/MS, Pesticide Residue Analysis Krishna V. Subbarao, University of California, Davis, California, Tillage George W. Sundin, Michigan State University, East Lansing, Michigan, Antibiotic Resistance; Antibiotics; Streptomycin; Tetracycline Susan D. Sutton, Miami University, Oxford, Ohio, Quantification of Microbial Biomass M. Ali Tabatabai, Iowa State University, Ames, Iowa, Soil Enzymes Michito Tagawa, Nissan Chemical Ind. Ltd., Shiraoka, Japan, Fungicides, Antibiotics Max E. Tate, University of Adelaide, Adelaide, Australia, Agrocin 84 Alan W. Taylor, USDA-ARS, College Park, Maryland, Fertilizers; Volatility of Pesticides J. Richard M. Thacker, Biological Sciences, University of Paisley, Paisley, Scotland, Pesticide Adjuvants Lennart Torstensson, Swedish University of Agricultural Sciences, Uppsala, Sweden, Toxicity Testing in Soils Martin E. Trenkel, Eusserthal, Germany, Plant Nutrition Ron Turco, Purdue University, West Lafayette, Indiana, Biodegradation in Soil James A. Turner, Dow AgroSciences LLC, Indianapolis, Indiana, Combinatorial Chemistry as Applied to The Discovery of Agrochemicals B. G. Tweedy, Jamestown, North Carolina, Fungicides, Copper Compounds and Sulfur Yasuhiko Uesugi, Tokyo, Japan , Fungicides, Multisite Inhibitors—Broad Spectrum Surface Protectants ´ Ujvary, ´ Istvan Hungarian Academy of Sciences, Budapest, Hungary, Natural Product Pesticides; Propesticides Karl-Heinz Ullrich, BASF Aktiengesellschaft, Limburgerhof, Germany, Plant Nutrition Marilyn Underwood, California Department of Health Services, Oakland, California, Remediation Don Valentine, Ridgefield, Connecticut, Soil Conditioners: Agricultural Applications Jan Roelof van der Meer, Swiss Federal Institute for Environmental Science and Technology (EAWAG), Dubendorf, ¨ Switzerland, Metabolic Pathways Evolution Laura L. Van Eerd, University of Guelph, Guelph, Ontario, Canada, Herbicides, Plant Hormone Mimics—Auxins Dick Van Elsas, Plant Research International B.V., Wageningen, The Netherlands, Genetically Modified Microorganisms (GMM) in Soil Environments Leo Van Overbeek, Plant Research International B.V., Wageningen, The Netherlands, Genetically Modified Microorganisms (GMM) in Soil Environments Kevin C. Vaughn, USDA-ARS, Stoneville, Mississippi, Herbicides, Pyridine Carboxylate; Photosystem I Energy Diverters Anne K. Vidaver, University of Nebraska, Lincoln, Nebraska, Bacteriocins Ralf Vosskamp, BASF Aktiengesellschaft, Limburgerhof, Germany, Plant Nutrition Shuo Wang, University of Sydney, Sydney, Australia, Immunoassays Monte R. Weimer, Dow Agrosciences, Indianapolis, Indiana, Herbicides, Identification of Biologically Active Materials Wilfried Werner, Agrikulturchemisches Institut der Universitat ¨ Bonn, Bonn, Germany, Plant Nutrition Richard Wilkins, University of Newcastle, Newcastle upon Tyne, United Kingdom, Controlled Release Formulations of Pesticides M. F. Wilson, Central Science Laboratory, Sand Hutton, York, United Kingdom, MALDI Thomas S. Woods, DuPont Agricultural Enterprise, Wilmington, Delaware, Pesticide Formulations Isamu Yamaguchi, RIKEN (The Institute of Physical and Chemical Research), Wako, Japan, Fungicides, Melanin Biosynthesis Inhibitors; Fungicides, Antibiotics Hendrik Ypema, BASF Corporation, Research Triangle Park, North Carolina, Fungicides, Dimethomorph; Fungicides, Ferimzone; Fungicides, Hymexazol

x

CONTRIBUTORS

Brian Gerald Young, Southern Illinois University, Carbondale, Illinois, Herbicide Adjuvants Robert M. Zablotowicz, USDA, ARS, SWSRU, Stoneville, Mississippi, Metabolism of Herbicides

Hugo Ziegler, Pentapharm, Basel, Switzerland, Fungicides, Phenylamides/Acycalanines Stephen H. Zinder, Cornell University, Ithaca, New York, Reductive Dehalogenation

ENCYCLOPEDIA OF


A ABIOTIC DEGRADATION

THE INSECT CHOLINERGIC SYSTEM

Degradation of a pesticide via purely physical or chemical mechanisms (IUPAC).

The nervous system of higher animals consists of the central and peripheral systems. Neurons (nerve cells) convey signals along cell processes (axons) by electrical (ionic) means and between cells by chemical transmission using neurotransmitter substances. Signaling between cells takes place at specific locations (synapses) where the two cell membranes are close together (Fig. 1). When an electrical signal reaches the presynaptic membrane of the synapse, it stimulates the release of a neurotransmitter. The electrical current is thus converted into a chemical signal. The neurotransmitter then diffuses across the synaptic cleft and binds to specific receptors on the postsynaptic membrane of the receiving nerve cell. On binding, it induces a conformational change in the receptor protein that stimulates the opening of specific ion channels in the membrane, either directly via integral ion channels or indirectly through a receptor coupled to a second protein known as a G-protein that opens ion channels via the production of a second intracellular transmitter substance. The signal is thus converted back into electrical energy. Some synapses are excitatory, where the opening of the ion channel leads to a rapid influx of cations (sodium and calcium) resulting in the depolarization of the membrane. If the depolarization reaches a certain level, it triggers the opening of further ion channels that are sensitive to the membrane potential. This signal constitutes a so-called action potential, and the depolarization moves rapidly along the axonal membrane in a self-perpetuating cascade (Fig. 1). Other synapses are inhibitory. These modulate excitatory signals using channels specific for an anion (usually chloride) and hyperpolarize the postsynaptic membrane. Animals use many different substances as neurotransmitters, and one neuron may have synapses specific for several different compounds. They are all low molecular weight and usually nitrogenous compounds, and their biosynthesis and removal from the synapse is finely regulated. In all animals, acetylcholine (1) is one of the most important, and synapses using it are known as cholinergic synapses. These are located in the central nervous system, peripheral ganglia, and some nerve gland junctions. In vertebrates, although not in insects, the neuromuscular junction is cholinergic. In vertebrates, nAChRs are expressed in both central and peripheral nervous systems but only in the central nervous system of insects.

ABSORBANCE (OF LIGHT). See BEER-LAMBERT LAW

ACARICIDES. See MITICIDES

ACCEPTABLE DAILY INTAKE (ADI) Estimate of the amount of a pesticide in food and drinking water that can be ingested daily over a lifetime by humans without appreciable health risk. It is usually expressed in milligrams per kilogram of body weight. This term has been replaced by Reference Dose (IUPAC).

ACCURACY (OF MEASUREMENT) Closeness of agreement between the result of a measurement and the (conventional) true value of the measurand (IUPAC).

ACETYLCHOLINE RECEPTORS PHILIP J. JEWESS IACR-Rothamsted, Harpenden Hertfordshire United Kingdom

The insect cholinergic system has proven a fruitful area for insecticide discovery. It contains two proteins that are targets for insecticides: the enzyme acetylcholinesterase and the nicotinic acetylcholine receptor (nAChR). The former is the site of action of two major classes of insecticides: organophosphates (OPs) and carbamates. At present, these comprise a major proportion of world insecticide sales; however, their toxicology is being increasingly called into question and few agrochemical companies would now attempt to develop a new insecticide of this type. In contrast, recently discovered compounds that interact with the nicotinic acetylcholine receptor (the neonicotinoids and the microbial natural products, spinosins) have very favorable vertebrate toxicology and are an expanding field of research and development.

CH3

O

N+ CH3

CH3 O

CH3 (1)

Cholinergic synapses have two enzymes, which synthesize and destroy acetylcholine, and two types of acetylcholine receptor proteins. The synthesis enzyme is called 1

2

ACETYLCHOLINE RECEPTORS

insect nAChR. Additionally, only the nAChR has proven a fruitful target for insecticides; consequently, the mAChR will not be discussed in this article. Axon

N+(CH3)3

Na+

H Sodium channel (pyrethroids, DDT)

CH3

CH3

N (2)

ACh mAChR Presynaptic membrane Synaptic cleft

Synaptic vesicles ACh ACh Choline

CAT

ACh

HO (3)

ACTION OF THE nAChR Choline

Na+

O

N

ACh Acetic acid

Postsynaptic membrane AChE K+ nAChR (OPs, carbamates) (neonicotinoids, nicotine, nereistoxin)

Figure 1. Schematic diagram of a cholinergic synapse shows the molecular targets of some insecticides: nAChR (nicotinic acetylcholine receptor), AChE (acetylcholinesterase), the sodium channel, and other components of the cholinergic system: ACh (acetylcholine), CAT (choline acetyl transferase), and mAChR (muscarinic acetylcholine receptor).

choline acetyltransferase (CAT) and effects the acetylation of choline by acetylcoenzyme A (acetylCoA). Acetylcholine is stored in vesicles in the presynaptic neuron. These fuse with presynaptic membrane upon stimulation by a nerve signal, thus, generating a pulse of neurotransmitter, which diffuses across the membrane. Acetylcholine may either bind reversibly to one of two different types of acetylcholine receptors on the postsynaptic membrane or be destroyed by the acetylcholine-hydrolyzing enzyme, acetylcholinesterase. Choline, generated by this reaction, is recycled and used to make more acetylcholine via CAT in the presynaptic neuron. The two receptors are the nAChR, which is responsible for fast transient transmission of impulses and the muscarinic acetylcholine receptor (mAChR), which is a G protein–coupled receptor and used where slower but maintained stimulation is required. Although these two receptors have different structures and actions, the terms ‘‘nicotinic’’ and ‘‘muscarinic’’ refer to mammalian pharmacology and to the two different drugs, nicotine (2) and muscarine (3) originally used to characterize them. Although it is clear that receptors structurally and functionally related to the mAChRs of vertebrates also exist in insects, the pharmacology of insects is different to vertebrates, and muscarinic drugs as defined by vertebrate pharmacology also interact with the

Nicotinic acetylcholine receptors are protein complexes in the outer membrane of nerve cells that mediate fast neural signaling between cells by acting as selective gateways for particular ions. Resting nerve cells have an unequal distribution of sodium and potassium ions across their membranes, maintained by an active pumping mechanism. The membrane of the resting nerve cell is also much more permeable to potassium than to sodium. The asymmetric distribution of these ions, together with the membrane’s differential permeability, generates an electrical potential. Under normal circumstances, there is a higher concentration of potassium ions inside the nerve cell. When a neurotransmitter (acetylcholine in the case of the nAChR) binds to the agonist binding site on the receptor protein, it triggers a conformational change that creates an aqueous pathway through the membrane and allows the flow of sodium and potassium ions down their respective electrochemical gradients. This process normally lasts less than a millisecond, and the open channel is only a few atom diameters wide. The size restriction and the charged groups at either end of the pore screen out ions of the wrong charge and the amino acid side chains lining the pore facilitate passage of the correct ions. If a compound other than the normal physiological agonist binds, the period of opening may be altered or the opening prevented altogether (antagonist action). INSECTICIDES AND OTHER COMPOUNDS THAT INTERACT WITH THE nAChR Most insecticides, whether of natural or purely synthetic origin, interact with the nervous system. Although many other specific targets such as chitin biosynthesis and the insect endocrine system have been investigated and have produced useful commercial products, it is likely that neurotoxins will still provide the bulk of insecticides in the near future. Apart from acetylcholinesterase, the other important neuronal targets are the gamma aminobutyric acid (GABA) receptor, the target for cyclodienes and fipronil; the chloride-gating glutamate receptor, the target of avermectins; and the voltage-gated sodium channel, the target for DDT, pyrethrins, and pyrethroids. Many compounds, particularly natural products, have been identified as interacting with the nAChR. Of these, the most important pharmacologically and historically is (S)-nicotine, an alkaloid found in the tobacco plant


(Nicotiana tabacum, Solanaceae) that has been used to control insects for at least 200 years and can justifiably be called the first organic insecticide. Although other, similar alkaloids are found in N. tabacum and related plants, nicotine is the most insecticidal; however, many of its properties are less than ideal, with lack of persistence due to high volatility and with high acute mammalian toxicity limiting its safety. Nicotine acts as an agonist of the nAChR, binding and effecting a similar conformational change to acetylcholine and, hence, causing the ion channel to open. The consequence is repeated firing of the nerve cells causing muscle twitching and convulsions. Additionally, with prolonged exposure and at higher concentrations, it may antagonize the action of acetylcholine by desensitization of the nAChR and, hence, causing a neural block. Consequently, nicotine poisoning initially gives symptoms of hyperactivity later followed by paralysis. However, despite much knowledge about its mode of action, chemistry, and molecular modeling, no useful insecticide has been developed from its lead (1,2). Neither has one been developed from the many other active compounds isolated from plants, animals, and microorganisms such as methyllycaconitine (4) isolated

from Delphinium spp. (Scrophulariaceae). This is a highly active antagonist of the insect nAChR, but it has moderate insecticidal activity and its complex structure is not an ideal lead for analog synthesis. Another interesting natural product is epibatidine (5), isolated from the skin of the frog Epipedobates tricolor. It has the same chloropyridyl substituent as the insecticide imidacloprid (see below) and is one of the most potent vertebrate cholinergic ligands known. It also binds strongly to nAChR preparations from some insect species but not others. However, two groups of insecticides acting on the nAChR have been developed from natural-product leads. The first was the dithiolane nereistoxin (6), a compound isolated from the marine annelid worm Lumbriconereis heteropoda, which was found to contain an insect-paralyzing factor. This led to the development of insecticides such as cartap (7), which is a nereistoxin generator and acts as a proinsecticide. Nereistoxin is an antagonist of the nAChR. As insecticides, nereistoxin analogs are relatively minor products. More recently, the macrolide antibiotic spinosad (8) (a mixture of spinosins A and D) produced by the actinomycete bacterium Saccharopolyspora spinosa has been developed as an insecticide by Dow Agrosciences.

H OCH3 CH3O C2H5

H OCH3 H

N

NH

OH H COO

OH OCH3

O

CH3

S

H

N

N

Cl

O

S

CH3

CH3 (4)

(5)

(6)

(CH3)2N O H2N

S

H2N

S

CH3

CH3

O

O

CH3

N

O O

O H H

O

CH3

O

C2H5 (7)

N

N

NH NNO2

(9)

O

H

H (8)

Cl

3

CH3(H)

OCH3 CH3

OCH3 OCH3

4


Its complex structure precludes economical chemical synthesis, and so it is made by fermentation. It also binds to the nAChR but not to the acetylcholine binding site. However, in current commercial terms, the most important group of insecticides by far that target the nAChR is the neonicotinoid class exemplified by imidacloprid (9). DEVELOPMENT OF THE NEONICOTINOIDS The development history of these compounds has been extensively reviewed (3,4). From a low-level insecticide lead 2-(dibromonitromethyl)-3-methyl pyridine (10) was detected by random screening, Shell developed the highly active but photochemically unstable nitromethylene compound nithiazine (11) (5), which was mainly active on caterpillars but not commercialized. Further development, principally by Nihon (now Nihon Bayer), eventually led to the commercialization of imidacloprid, which is principally active against Homopteran sucking pests (aphids, hoppers, and whiteflies). To date, three other neonicotinoids have reached the marketplace (acetamiprid, nitenpyram, and thiamethoxam), and imidacloprid has become the topselling insecticide in the world. They are increasingly displacing OP and carbamate compounds to control pests on major crops, and their success has led to renewed interest in the nAChR by the agrochemical industry.

CH3 CBr2 N (10)

NO2

α-bungarotoxin, has been used extensively to characterize vertebrate nAChRs because it selectively binds to certain populations of vertebrate receptors. It is one of a family of small neurotoxic proteins (MW ∼ 8,000 Da) isolated from the venom of elapid snakes (cobras and their relatives). α-Bungarotoxin also binds to certain insect nAChRs (8,9), although a separate α-bungarotoxin–insensitive class of nAChR has been found in some species (9). It binds very tightly to the receptor and competitively blocks the binding of acetylcholine; consequently, radiolabeled α-bungarotoxin has been used to design competitive binding assays to measure the in vitro potencies of competitive ligands such as the neonicotinoids. However, no information concerning the agonist or antagonist nature of test compounds is obtained using these methods. Binding studies using both α-bungarotoxin and imidacloprid have indicated that some insect receptors that do not bind αbungarotoxin with high affinity do bind imidacloprid (10). Such bungarotoxin-insensitive nicotinic receptors have also been identified using electrophysiology (11). Structure–activity relationships for neonicotinoids have been shown to be more satisfactory using imidacloprid as the ligand (10) because the insecticidal activity correlates better with the in vitro data. The selective binding of the nAChR to such α-snake toxins (and imidacloprid analogs) has also been used to purify the receptor by affinity chromatography. INTERACTIONS OF NEONICOTINOIDS WITH THE nAChR

HN

S CHNO2 (11)

TESTING COMPOUNDS THAT ACT ON THE nAChR Two main methods have been used for the nAChR:electrophysiological tests and competitive binding assays. These methods give different types of information about the interactions between the ligand and the test system. Several types of electrophysiological tests of varying degrees of sophistication have been used to test nitromethylenes and neonicotinoids. These range from whole nerve assays such as the cockroach ventral nerve cord (6), identified single neurons (7) to single receptor molecules isolated on glass electrodes analyzed by the patch-clamp technique. Electrophysiological testing is used for detailed mode of action studies, but each preparation can usually only be used to test a single compound, so that it is impracticable to test large numbers of materials. However, these methods can give detailed informations about modes of action such as whether the test ligand in an agonist or an antagonist (or both) and, in the case of patch-clamp, detailed information about channel conduction states. Conversely, competitive-binding studies are capable of automation and can be used to test large numbers of compounds quickly; however, they give very limited information about mode of action. The snake toxin,

Nithiazine and other members of the nitromethylene family were shown at an early stage in their development to be cholinergic agonists (and antagonists) by electrophysiology (6). However, unlike nicotine, they mostly act as partial agonists and are open channel blockers, a property resulting from their partial positive charge. These compounds also bind to insect nAChRs much more effectively than do those from vertebrates resulting in intrinsic selectivity. Early biochemical studies using a competitivebinding assay with α-bungarotoxin also showed that they bound to a receptor having the properties of a nAChR isolated from housefly heads. This has been corroborated by further electrophysiological data and experiments on cloned and expressed acetylcholine receptors as follows. The binding of [3 H]imidacloprid to housefly head membranes is inhibited by both nicotinic and muscarinic ligands (12). Specific binding to preparations from a number of other insects species has also been reported, including Myzus persicae (13), Nephotettix cincticeps (13), Bemisia tabaci (14,15) (Homoptera), Manduca sexta (13), Heliothis virescens (13) (Lepidoptera); Lucilia sericata (13), Drosophila melanogaster (13,16) (Diptera), Periplaneta americana (13) (Orthoptera), and Ctenocephalides felis (13) (Siphonaptera). Two classes of receptors have been detected in Homopteran insects, a low and high affinity site (13,14), but only one in nonHomopterans. Electrophysiological studies on neurons of Periplaneta americana (cockroach) and Schistocerca gregaria (locust) indicated that imidacloprid and other neonicotinoids have both agonist (17–19) and antagonist activities (17,20), but were without activity on glutamate


or voltage-gated sodium channels. There is electrophysiological evidence for several types of nAChR in insects, and it has been reported that imidacloprid acts on three pharmacologically distinct nAChR populations in the same ganglion of Periplaneta americana nerve cord (21). Additionally, neonicotinoids were active against a cloned expressed subunit of an insect nAChR (19,22). Their insecticidal efficacies, electrophysiological activity, and binding to the receptor are broadly correlated (7,23). This indicates that the primary mode of action of neonicotinoids and other nitromethylene compounds such as nithiazine is by interaction with the insect nAChR; however, the interactions are complex due to the existence of several types of receptor in insects and the dual agonist/antagonist action of these compounds. THE nAChR AS PART OF A LIGAND-GATED ION CHANNEL SUPERFAMILY OF RECEPTORS Due to its importance in human pharmacology, most of the information on the nAChR has come from work on vertebrates. Much initial work examined the receptors isolated from the electric organ of the electric ray Torpedo californica, which is a particularly rich source. Although only a few insect nAChR genes have been cloned and sequenced, it is inferred from homology studies that the insect neuronal receptor is similar to the vertebrate neuromuscular nAChR exemplified by the Torpedo spp. protein. Molecular genetic studies using DNA homology have shown that the nAChR is one of a superfamily of ligand-gated ion channels with differing selectivities for both transmitter molecules and ions. These all share a pseudosymmetrical arrangement of three to nine homologous protein subunits around a central ion pore. In the vertebrate neuromuscular receptor, the subunits are typically of four different types; the subunit responsible for binding the transmitter substance is designated the α-subunit. Other important members of this superfamily are the GABA receptor, which gates chloride and bicarbonate ions, the 5hydroxytryptamine receptor (sodium and potassium ions) and the glycine receptor (chloride and bicarbonate ions). These receptors can also be subdivided on the basis of homology and pharmacology. It thus seems probable that evolution has produced a large number of neuroreceptors of differing pharmacology from an ancestral form having only one type of subunit encoded by a single gene (24,25). STRUCTURE OF A VERTEBRATE nAChR The macromolecular structure of the nAChR from Torpedo californica was first reported in 1993 (26). The spatial arrangement of the subunits was determined to ˚ resolution using electron microscopy (Fig. 2). The 9A Torpedo spp. nAChR is a transmembrane glycoprotein with a MW of about 300,000 Da. It is composed of two identical α and three distinct non-α (β, γ , and δ) subunits around an axis of pseudosymmetry in a pentamer of α2 βγ δ. Either the δ or γ subunit is located between the

5

two α subunits. The four types of subunits have been cloned and sequenced. They are homologous, glycosylated, phosphorylated, and share a very similar secondary structure. The receptor can be divided into three main regions. 1. The N-terminal region is hydrophilic and protrudes into the synaptic cleft. This comprises a large disulphide-linked loop between cysteines 128 and 142, some N-glycosylation sites, and the ligandbinding site. Subunits designated α have two adjacent cysteines 192 and 193 that form a vicinal disulphide bond. This is critical for ligand binding, and all subunits in other receptors of the superfamily that possess these cysteines are probably ligandbinding subunits. 2. The transmembrane region forms the ion pore. It comprises four highly conserved membranespanning domains designated M1–M4. M1 and M2 have α-helical structures, and there is evidence that M2 lines the pore. 3. A large hydrophilic segment between M3 and M4 protrudes into the cytoplasm of the cell. This is the least conserved portion of the protein, and there are many phosphorylation sites that are important for regulation and desensitization of the receptor.

Top view d

b

ACh a

a g ACh

Sectional view

M+ −

Synaptic side

−

M2

M2 −

Membrane

− Cytoplasmic side

Rapsyn

Connection to cytoskeleton

Figure 2. Schematic diagram of the nAChR complex shows the pentameric stoichiometry of the complex, two acetylcholine binding sites (ACh) between the α and δ- and the α and γ -subunits and the transmembrane α-helix forming the cation-selective channel (M2). Redrawn and simplified from Hucho et al. (1996).

6


There is also an additional protein, of MW 42,000 Da called rapsyn, that appears to span the subunits at the cytoplasmic end. It is probably important in linking the receptor to the cytoskeleton (27). THE LIGAND-BINDING SITES There is much interest in identifying ligand-binding sites, particularly the acetylcholine binding site located on the hydrophilic extracellular domain of the nAChR, because it may be possible to design new effector molecules de novo should the site be known in sufficient detail. Apart from the main agonist-binding site, a number of other peripheral sites have been inferred. These include domains to which noncompetitive inhibitors such as local anaesthetics bind. Peripheral sites have been located on both faces of the ion channel and on the protein-membrane interface and act either through direct blocking of the channel or through induction of a conformation change in the protein structure. Compounds that bind to the former sites are the so-called luminal noncompetitive inhibitors. These bind to rings of amino acids forming the ion channel (see below), when the channel is in the closed state. Examples are chlorpromazine (12) and phencyclidine (13), which interact with the serine, threonine, and valine rings, and meproadifen (14), which binds to the nonpolar valine and extracellular rings. Examples of compounds that bind to sites outside the channel lumen are quinacridine (15) and ethidium (16), which bind to separate distinct sites on the receptor. In addition to the above exogenous compounds, the receptor may also be modulated by endogenous substances, such as the neurotransmitter 5hydroxytryptamine (serotonin) (17), which binds to the lumen of the ion channel. Of particular interest is the phenomenon whereby agonists (including acetylcholine) at high (millimolar) concentrations also bind to the peripheral sites and effectively self-inhibit their own action. These sites have been identified as the ion channel and the nonluminal site to which quinacridine binds. The

pharmacology of peripheral binding sites on the nAChR has been reviewed by Arias (28). However, most effort has concentrated on the acetylcholine-binding site and its structure has been inferred from photo-affinity labeling, site-directed mutagenesis, and the X-ray structure of a soluble homologous acetylcholine-binding protein purified from the freshwater snail Lymnaea stagnalis (29) (see below). Two acetylcholine-binding sites are located in deep clefts between the α- and δ-subunits and the α- and γ -subunits on each nAChR molecule. Consequently, the two sites are nonidentical. This is corroborated by the sites having different affinities for the competitive antagonist d-tubocurarine (18). A six-loop model has been proposed as contributing to the binding site (30). Amino acids important in acetylcholine binding include three tyrosine residues (93, 190, 198), one tryptophan (149), and the cysteine pair (192, 193) located on the α-subunit and single tryptophan, tyrosine, aspartic acid, and glutamic acid residues on the δ- or γ -subunits (Fig. 3). With the possible contribution of an additional tryptophan and tyrosine residue from the α-subunit, it can be seen that the acetylcholine-binding site is a highly hydrophobic domain (see the entry on acetylcholinesterase). As with many other integral membrane proteins, it has not been possible to obtain crystals of the Torpedo spp. nAChR of sufficient quality for high resolution X-ray crystallography. However, the structure of a pentameric-soluble acetylcholinebinding protein isolated from Lymnaea stagnalis has been ˚ (29). This has many of solved to a resolution of 2.7 A the properties of the nAChR α-subunit, including binding nicotinic agonists and antagonists such as acetylcholine, nicotine; d-tubocurarine, and bungarotoxin; quite high homology, and the presence of the vicinal cysteine pair characteristic of ligand-binding receptor subunits. It does not, however, form ion channels. Of particular interest was the finding that the buffer component, HEPES (N2-hydroxyethylpiperazine-N -2-ethanesulphonic acid) was

S Cl

N

N

(C2H5)2

O

N+

O

CH3

N(CH3)2 (12)

(13)

(14)

HO HN

N(C2H5)2

H2N

N H

OCH3 Cl

NH2

N (15)

NH2

NH2

(16)

(17)


7

Synaptic side d- or g NH2 subunit

a-subunit

glu −

− glu

H2N val

W

Y

s s

Y C s s

C Y

E

s s

leu

Ion channel n n

leu

ser

OHHO

ser

thr

glu

bound to the crystal with its quaternary ammonium ion π -stacked onto tryptophan 143 (homologous to tryptophan 149 in the Torpedo spp. α-subunit). It has thus been inferred that binding of the positively charged nitrogen atom of cholinergic ligands to the nAChR involves an interaction with the face side of the π -system of a tryptophan residue analogously to acetylcholinesterase.

O

CH3

N+

OCH3

OH H

H

CH3

OH

asp

OH

−

thr

HO

−

−

−

glu asp

Cytoplasmic side Figure 4. Schematic diagram of the ion channel formed by the M2 transmembrane α-helices. Only those amino acids lining the channel are shown: aspartic acid (asp), glutamic acid (glu), serine (ser), threonine (thr), valine (val), and leucine (leu). Amino acid side chains have been designated − (negative charge, asp and glu), OH (hydroxyl group, ser and thr) and n (neutral, leu and val). The zigzag lines represent the α-helices.

MOLECULAR GENETIC STUDIES ON INSECT nAChRS

N+

O CH3O

val

D

Figure 3. Proposed loop model for the acetylcholine binding site between the α- and δ- or γ -subunits (30). The amino acids are designated by their single letter codes: C, cysteine; D, aspartic acid; E, glutamic acid; W, tryptophan; Y, tyrosine.

CH3

n

Y

Acetylcholine

W

n

H

(18) THE ION CHANNEL It is postulated that the ion channel is formed from five M2 helices, one from each subunit. A model in which the ion channel is formed from several rings of amino acid residues has been proposed (30). The helices that form the membrane-spanning domains are bent toward each other near the middle, forming a narrow constriction in the pore (26). At each turn of the M2 α-helix, a particular amino acid side chain is aligned toward the pore. From the synaptic side to the cytoplasmic face, the amino acids lining the channel are glutamic acid, valine, leucine, serine, threonine, glutamic acid, and aspartic acid (Fig. 4). Consequently, negatively charged residues line both ends of the channel, whereas neutral (valine or leucine) or polar (threonine or serine) residues line the center. Mutagenesis studies show that substitution of these charged and polar residues can change ion selectivity and that the nature of the residues lining the pore can confer either cation or anion selectivity.

Insect nAChRs are diverse in structure, as are those from vertebrates. In general, the subunits of insect nAChRs resemble those of vertebrate neuronal, rather than neuromuscular nAChRs. Cloned subunits from insect nAChRs have been assigned as α or non-α on the basis of the presence or the absence of the vicinal cysteine pair. Genes encoding nAChR subunits have been cloned from Drosophila melanogaster (3α and 2 non-α), the locusts Schistocerca gregaria (22) (1α, 1 non-α) and Locusta migratoria (31) (4α and 1 non-α), the moths Manduca sexta (32) (1α) and Heliothis virescens (31) (1α), and the aphid Myzus persicae (33,34) (5α, 1 non-α). The situation is consequently complex, because it appears that insects have several types of nAChR subunits that could associate to form channels of disparate pharmacology, and this could explain some of the complex binding and electrophysiology seen with the insect cholinergic system. REGULATION OF THE RECEPTOR Electrophysiological and biochemical studies indicate that the nAChR may exist in a number of discrete and interconvertible states: resting, active, and two desensitized states. In the active state, the channel spontaneously opens and the receptor has low affinity

8

ACETYLCHOLINESTERASE

for acetylcholine. In the desensitized states, the channel is closed and has high sensitivity for acetylcholine. Competitive antagonists bind to the receptor in its resting state. These states exist in a dynamic equilibrium in the absence of any ligand binding. When acetylcholine or another agonist binds, the channel opens and the receptor is stabilized in its active (low affinity) state. However, competitive antagonists cause the channel to open and the protein is stabilized in its desensitized state. As stated above, some ligands, in particular nicotinoids, may act as both agonists, causing channel opening, and antagonists, causing a desensitized block of the receptor. Minor structural modifications to nitromethylene compounds and other neonicotinoids have been shown to produce compounds with either or both of these properties.

BIBLIOGRAPHY 1. I. Yamamoto, Adv. Pest. Control Res. 6: 231–260 (1965). 2. I. Yamamoto et al., Agric. Biol. Chem. 26: 709–716 (1962). 3. W. D. Kollmeyer et al., in I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, New York, 1999, pp. 71–90. 4. S. Kagabu, in I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, New York, 1999, pp. 91–106.

19. C. A. Leech, P. Jewess, J. Marshall, and D. B. Sattelle, FEBS Lett. 290: 90–94 (1991). 20. D. Bai et al., Pestic. Sci. 33: 197–204 (1991). 21. S. D. Buckingham et al., J. Exp. Biol. 200: 2685–2692 (1997). 22. J. Marshall et al., EMBO J. 9: 4391–4398 (1990). 23. M. Tomizawa, M. Lati, and J. E. Casida, in I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, New York, 1999, pp. 271–292. 24. M. O. Ortells and G. G. Lunt, Trends Neurosci. 18: 121–127 (1995). 25. N. Lenovere and J. P. Changeux, J. Molec. Evolution. 40: 155–172 (1995). 26. N. Unwin, J. Mol. Biol. 229: 1101–1124 (1993). 27. W. D. Phillips, Clin. Exp. Pharmacol. Physiol. 22: 961–965 (1995). 28. H. R. Arias, Biochim. Biophys. Acta—Reviews on Biomembranes 1376: 173–220 (1998). 29. K. Brejc et al., Nature 411: 269–276 (2001). 30. H. R. Arias, Brain Res. Rev. 25: 133–191 (1997). 31. B. Hermsen et al., J. Biol. Chem. 273: 18394–19404 (1998). 32. H. M. Eastham et al., Eur. J. Neurosci. 10: 879–889 (1998). 33. F. Sgard et al., J. Neurochem. 71: 903–912 (1998). 34. Y. Huang et al., J. Neurochem. 73: 380–389 (1999).

5. S. B. Soloway et al., in H. Geissbuehler, ed., Advances in Pesticide Science, Vol. 2, 4th edn., International Congress of Pesticide Chemistry, Pergamon, Oxford, 1979, pp. 206–217.

FURTHER READING

6. M. E. Schroeder and R. F. Flattum Pestic. Biochem. Physiol. 22: 148–160 (1984).

Yamamoto, I. and Casida, J. E., eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, New York, 1999, p. 300.

7. S. D. Buckingham et al., Neuropharmacology 34: 591–597 (1995). 8. D. B. Sattelle, Adv. Insect Physiol. 15: 215–315 (1980). 9. L. L. Restifo and K. White, Adv. Insect Physiol. 22: 115–219 (1990). 10. D. Wollweber and K.Tietjen, in I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, New York, 1999, pp. 109–125. 11. D. B. Sattelle, J. A. David, I. D. Harrow, and B. Hue, in D. B. Sattelle, L. M. Hall, and J. G. Hildebrand, eds., Receptors for Neurotransmitters, Hormones and Pheromones in Insects, Elsevier, Amsterdam, 1980, pp. 125–139. 12. M.-Y. Liu and J. E. Casida, Pestic. Biochem. Biophys. 40: 40–46 (1993). 13. R. J. Lind, M. S. Clough, S. E. Reynolds, and F. G. P. Earley, Pestic. Biochem. Physiol. 62: 3–14 (1998). 14. R. J. Lind et al., in Neurotox ‘98: Progress in Neuropharmacology and Neurotoxicology of Pesticides and Drugs, Oxford, UK, 1998. 15. S. L. Chao, T. J. Dennehy, and J. E. Casida, J. Econ. Entomol. 90: 879–882 (1997). 16. M. Tomizawa, B. Latli, and J. E. Casida, J. Neurochem. 67: 1669–1676 (1996). 17. J. A. Benson, J. Exp. Biol. 170: 203–233 (1992). 18. D. B. Sattelle et al., Proc. Royal Soc. Lond. ser. B 237: 501–5141 (1989).

This publication is the proceedings of a symposium held in Las Vegas, Nevada, USA, in September 1997. It mainly covers the development of the nicotinoid, nitromethylene, and neonicotinoid insecticides, although several chapters touch on their interactions with the receptor. Arias, H. R., Brain Res, Rev. 25: 133–191 (1997). Hucho, F., Tsetlin, V. I., and Machold, J., Eur. J. Biochem. 238: 539–557 (1996). Benson, J., in McFarlane, N. R, ed., Progress and Prospects in Insect Control, British Crop Protection Council, BPC Monograph No. 43, London, 1989, pp. 59–69. Sattelle, D. B., in Kerkut, A. and Gilbert, L. I., eds., Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 11, Pergamon Press, New York, 1985, pp. 395–435.

ACETYLCHOLINESTERASE PHILIP J. JEWESS IACR-Rothamsted, Harpenden, Hertfordshire United Kingdom

The enzyme acetylcholinesterase (EC 3.1.1.7) occurs widely in both vertebrate and invertebrate nervous systems and is the target site for two major insecticide classes:


organophosphates (OPs) and carbamates. Although a family of esterases, which catalyze the hydrolysis of various choline esters, is present in all animals, the membranebound enzyme that specifically hydrolyzes the neurotransmitter acetylcholine (1) is toxicologically important. Failure to degrade acetylcholine to choline and acetic acid in cholinergic synapses results in a build up of acetylcholine. This binds to the nicotinic acetylcholine receptor (nAChR) and saturates the binding site, causing the ion channel of the receptor to remain open (Fig. 1) (see the entry on the acetylcholine receptor). Consequently, ions continue to pass through the synaptic membrane, which results in membrane depolarization, continuous neuronal firing, convulsions, and ultimately death of the organism. Additionally, the nAChR may become desensitized when exposed to continuously high concentrations of acetylcholine, so preventing transmission of nerve impulses and resulting in paralysis. Vertebrates possess a second acetylcholine-hydrolyzing enzyme (butyrylcholinesterase; EC 3.1.1.8), which differs in its substrate specificity and its susceptibility to different inhibitors. It occurs in muscle endplates, certain other synapses, and as a soluble protein in blood serum. In invertebrates, the situation is less clear, because insect acetylcholinesterase catalyzes the hydrolysis of butyrylcholine at an appreciable rate. However, insect acetylcholinesterase is inhibited by the plantderived toxin physostigmine (eserine) (2), such inhibition being an accepted diagnostic of true acetylcholinesterase. Butyrylcholinesterase is apparently absent from most insects, although it has been reported from aphids (1). The physiological significance of butyrylcholinesterase is not definitely known, although it may act as a scavenger for cholinergic compounds.

CH3 CH3

CH3

O

N+

O

CH3 (1)

O CH3

O

C

NHCH3

N

N H CH3 CH3

9

insecticide resistance (see below). As well as its importance in crop protection, the enzyme is the target site for the highly toxic nerve agents such as sarin (3), from which the insecticidal OPs were developed in the 1940s. Reversible inhibitors of acetylcholinesterase have also been used to treat a number of medical conditions, including myasthenia gravis, glaucoma, and Alzheimer’s disease. These applications, together with the fact that assays for the enzyme are easy to perform, have resulted in AChE being the subject of intense scientific scrutiny from the 1950s to the present day. Wilson’s pioneering work in the 1950s established the mechanism of action and the importance of an acetylated enzyme intermediate in the hydrolysis of acetylcholine and suggested a physical model of the active site (2–9). This scientific effort eventually culminated in the determination of the 3-D structure of the enzyme isolated from the electric ray Torpedo californica. There is now much research on cloning, expression, and resolution of the 3-D structures of the enzyme from different species, as well as work investigating how inhibitors and substrates interact with amino acid residues in the active site.

O iC3H7O

P

F

CH3 (3) STRUCTURE OF THE ENZYME Many of the definitive structural studies on the structure of acetylcholinesterase have been performed on the purified protein isolated from the electroplax (electric organ) of the electric eel (Electrophorus electricus) or electric rays (Torpedo spp.). These tissues are extraordinarily rich in both acetylcholinesterase and the nicotinic acetylcholine receptor. This naturally abundant source of two membrane-associated proteins has meant that structural studies have become possible, whereas with many other membrane proteins, it has had to await cloning and high level eukaryotic expression in order to obtain sufficient material. Attachment to the Synaptic Membrane

(2) Insects have a predominantly cholinergic nervous system and are particularly sensitive to anti-cholinesterases. However, OPs and carbamates that inhibit acetylcholinesterase also have appreciable but variable toxicity toward vertebrates. The insecticidal selectivity of commercial insecticides is largely due to differential metabolic detoxification. The sensitivity of the enzyme from target (insect pest) and nontarget (vertebrate) organisms is generally of lesser importance, although the selection of acetylcholinesterase that is insensitive to OPs and carbamates is becoming important as a mechanism for

Torpedo acetylcholinesterase consists of two identical protein molecules joined by a disulphide bond, each of which is anchored to the postsynaptic membrane through a phosphatidyl inositol moiety. The phosphatidyl inositol group is attached to each monomer through an intervening oligosaccharide linked to the C-terminal cysteine of the enzyme (10) (Fig. 1). This form also occurs in the insect cholinergic synapse. The molecular weight of each monomer is 68,000, including the sugar function, and comprises 537 amino acids. Detailed studies on acetylcholinesterase from other sources and tissues have shown that many molecular forms exist that differ in quaternary structure, oligomeric state, and the way in which they are anchored to the membrane.

10


ACh ACh Acetylcholine ACh ACh Presynaptic

ACh Figure 1. Schematic diagram of a cholinergic synapse. An acetylcholinesterase (AChE) dimer is anchored to the post synaptic membrane. Acetylcholine (ACh) is secreted in vesicles in the presynaptic cytoplasm. These fuse with the membrane and release acetylcholine, which diffuses across the synapse. Acetylcholine is either hydrolyzed to choline and acetic acid by AChE or binds to the nicotinic acetylcholine receptor (AChR), causing the integral cation-selective channel to open in the postsynaptic membrane.

ACh

Na+

ACh s s

ACh Choline + acetic acid

Membrane anchor

ACh

Postsynaptic

A common alternative form that occurs in vertebrates but not insects has the catalytic subunits arranged as tetramers that are linked via disulphide bonds to a collagen molecule. This is bound to a heparin-like proteoglycan in the matrix. The consequences of these different modes of membrane-anchoring are that enzymes from different sources may either dissociate from the membrane easily or require detergents to solublize them. Once solublized, the enzyme tends to reassociate into several different oligomeric states if the detergent is removed. Nonionic detergents such as Tween-20 or Triton X-100 are usually used to make solublized preparations, although the enzyme phospholipase c may be used to cleave the phosphatidyl inositol group to yield a detergent-free soluble preparation. There is little evidence that membrane-anchoring significantly influences the conformation of the protein; consequently, in vitro kinetic studies on solublized enzyme can be considered relevant to the in vivo situation. The Active Site Early studies indicated the active site contained two subsites, the ‘‘esteratic’’ and ‘‘anionic’’ site corresponding, respectively, to the catalytic functional site and a quaternary ammonium binding pocket. The active-site serine, with which OPs react, was unequivocally identified using radiolabeled tagging with [32 P]diisopropylfluorophosphate as serine 200 in the Torpedo enzyme. Kinetic and chemical studies had also implicated the specific histidine residue required for catalytic activity. The anionic subsite, which binds the charged quaternary ammonium group of acetylcholine, was widely assumed to be an acidic residue (aspartic or glutamic acid) that would bind electrostatically to the substrate and to charged inhibitors. However, some affinity-labeling studies indicated that the anionic

site was in fact uncharged and corresponded with aromatic residues in the protein. In addition to these two subsites, acetylcholinesterase possesses an additional site (or sites) that binds quaternary ammonium ligands. This so-called peripheral anionic site has been implicated in inhibition by bis-quaternary ligands such as decamethonium (4) and in substrate inhibition, whereby the enzyme is inhibited by excess acetylcholine.

CH3 CH3

CH3 CH3 N+ CH3

N+ CH3 (4)

The Structure Derived from X-Ray Crystallography The X-ray–derived structure of the Torpedo enzyme published in 1991 (11) contained many surprises, particularly the confirmation that aromatic rather than acidic residues are involved in binding quaternary ammonium groups. These finding are summarized as follows. ˚ The enzyme has an ellipsoidal shape (45 × 65 × 65 A) consisting of 12 central β-sheets surrounded by 14αhelices. The protein is pierced by a deep and narrow cleft (designated the active site gorge) that penetrates ˚ into the enzyme. The active site, comprising the 20 A catalytic triad, serine 200, histidine 440, and glutamic acid 327, is toward the bottom of the gorge. The presence of glutamic rather than aspartic acid as a component of the catalytic triad is highly unusual among serine esterases. The carbonyl group of acetylcholine is bound by the peptide chain nitrogens of alanine 201, with contributions from glycines 118 and 119 forming the


only hinted at by kinetic or affinity studies and indicate a complex active site that can potentially bind many types of effector molecule. This results in complex kinetics and pharmacology that are the subject of intense research and may also form the basis of understanding how other acetylcholine-binding proteins such as the acetylcholine receptors function.

Serine 200

CH3 CH3

O

O

N+

CH3

O−

CH3

H H “oxyanion hole”

H

N H tryptophan 84

CATALYTIC MECHANISM Acetylcholinesterase, like other serine esterases, catalyzes the hydrolysis of its substrate via an unstable acylenzyme intermediate, whereby the hydroxyl group of a serine residue in the active site is acylated. Serine acts as a nucleophile in the hydrolysis mechanism and is activated via an acid (glutamic acid 327) residue and a histidine (his 440) acting as both an acid and a base (Fig. 3). These three components (serine 200, glutamic acid 327, and histidine 440) make up the catalytic triad of acetylcholinesterase and are totally conserved in all forms of the enzyme, being essential for enzymatic activity. An initial reversible ‘‘Michaelis’’ complex is formed during the course of hydrolysis (Fig. 3, step Km ). This rapidly reacts to release choline and form the acetylated enzyme (Fig. 3, step k2 ), which is in turn rapidly hydrolyzed to regenerate the enzyme (Fig. 3, k3 ). The mechanism deduced from the structure of the Torpedo enzyme confirms that the negatively charged glutamic acid 327 activates serine 200, forming a powerful nucleophile. This attacks the carbonyl group of acetylcholine, resulting in the formation of an acetylated enzyme. Deacetylation requires a residue in the active site to activate a water molecule that is bound by two glutamic acid residues (443 and 199). Attack on the acetylated serine by

Figure 2. Schematic representation of the active site of acetylcholinesterase. The acetylcholine hydrolysis transition state is shown covalently bonded to serine 200. Tryptophan 84 is now known to be principally responsible for binding the quaternary ammonium group and represents the ‘‘anionic site’’ of earlier reports. The ester carbonyl group is hydrogen-bonded to three peptide chain nitrogen atoms and is represented by the ‘‘oxyanion hole.’’ A 3-D representation of the active site of the Torpedo enzyme is depicted in Sussman et al. (11).

so-called oxyanion hole (Fig. 2). The active-site gorge is lined with 14 aromatic residues thought to be involved in substrate ‘‘guidance.’’ The main residue responsible for binding the quaternary ammonium moiety of acetylcholine is a tryptophan (trp 84) residue in the base of the gorge. There is one acid residue in the active site (a glutamic acid) that could possibly bind the quaternary ammonium moiety of acetylcholine, but there is good evidence that it does not do so. Hydrophobic residues near the rim of the gorge have been identified as the peripheral anionic site by affinity labeling methods. The structure of the active site is shown schematically in Figure 2. These studies reveal a structure

Choline

O Acetylcholine N+

O

CH3 O Acetylcholine Km + Acetylcholinesterase

11

:N

H

N

O

−O

H

O

O

k2

ser 200

his 440

ser 200

glu 327

Michaelis complex

CH3 O

H

+

N

N

N:

HO

his 440

O

glu 327

CH3

H+

:N

H

Acetyl enzyme intermediate

Formation of acetyl enzyme

O Acetic acid O−

N+

HO

CH3

H

O H

−

O

O

O

O

H :N

N

H

HO

k3

ser 200

his 440 Regenerated enzyme

glu 327

ser 200

his 440

Hydrolysis of acetyl enzyme

Figure 3. Mechanism of the hydrolysis of acetylcholine by acetylcholinesterase.

glu 327

O

12


inhibition of the enzyme) is often quoted when comparing the activities of inhibitors but is comparatively meaningless for such progressive inhibitors and depends on the length of time the compound is incubated with the enzyme. Dephosphorylation (Fig. 4, step k3 ) rates are dependent on the nature of the phosphorylating group and the source and nature of the enzyme. Half-lives for the reactivation of dimethylphosphorylated acetylcholinesterases are typically 5 to 10 hours, although much longer (50 to 100 days) for the diethylphosphorylated enzyme. Acetylcholinesterase is not inhibited by P=S containing phosphorothioates [e.g., parathion (5)], which, when purified and free of the P=O (oxon) analogs, have negligible anti-acetylcholinesterase activity (12). Activation by conversion of the P=S group to P=O is therefore necessary for the compounds to be toxic. Carbamates (e.g., carbofuran; 6) and carbamoyloximes (e.g., methomyl; 7) also inhibit acetylcholinesterase by acylating (carbamoylating) the active-site serine but are reactivated through decarbamoylation at a much faster rate than OPs. Typical reactivation half-lives are 1–2 hours for N-methylcarbamates and rather longer for N,N-dimethylcarbamates (pirimicarb (8) and the carbamoyltriazole, triazamate; 9). An additional reaction of particular toxicological significance is ‘‘aging’’ of OP-inhibited enzymes, whereby the dialkyphosphoryl group is subject to dealkylation via C−O bond cleavage leading to the formation of a monoalkylphosphorylated enzyme, which is not hydrolyzed and causes completely irreversible inhibition of the enzyme (Fig. 4). Whether the OP-inhibited acetylcholinesterase ages significantly is dependent on the nature of the alkyl groups attached to the phosphorus atom. In general, branched (secondary and tertiary) alkyl groups are lost more easily than are n-alkyl ones, leading to more rapid aging. This irreversibly inhibited enzyme is very important in

this water molecule leads to a high-energy tetrahedral transition state that then dissociates, releasing acetate and regenerating the enzyme. The speed of these reactions makes acetylcholinesterase one of the most efficient esterases known; a necessary attribute for modulating rapid nerve impulses by removing acetylcholine from the synapse once its mission is accomplished. MECHANISM OF INHIBITION BY OPs AND CARBAMATES The reaction of OPs with acetylcholinesterase involves a mechanism analogous to that of substrate hydrolysis. This involves nucleophilic attack of the active site serine hydroxyl on the phosphorus atom, cleavage of the P−O or P−S bond, followed by phosphorylation of the serine, and concomitant release of the ‘‘leaving group.’’ The same active-site serine residue (200) is acetylated by acetylcholine during acetylcholine hydrolysis; however, although the acetylated enzyme is very rapidly hydrolyzed, the phosphorylated enzyme is hydrolyzed at an extremely slow (although measurable) rate and results in inhibition of the enzyme. OPs therefore show a time-dependent inhibition of the target protein that mimics the mechanism of normal esterase function. After the reversible interaction with the active site (Fig. 4, step KI ), the reversible Michaelis complex reacts with loss of the leaving group X to form a phosphorylated enzyme (Fig. 4, step k2 ). This phosphorylation step is fast, with a typical first-order rate constant of 1 to 2 sec−1 , so that the overall second-order rate constant for inhibition (ki = k2 /KI ) is usually measured. It is also probable that OPs act as such efficient inhibitors of acyl hydrolases because they mimic the tetrahedral structure of the transition state formed during hydrolysis and thus bind efficiently to the active site prior to phosphorylating it. The IC50 (the concentration of inhibitor producing 50%

O

O

R1O

R1O P

X

P

X

Kl

R2 O

O ser

k2

R2O

+

O

XH + O

R1O k3

R1O

R2O

P H

O

H

R2O

ser Michaelis complex

OH

+ O

O

ser phosphorylated enzyme

−

P

H

ser reactivated enzyme

O

O P

R2O

+

R1OH

O ser aged enzyme

Figure 4. Reaction of OPs with acetylcholinesterase. The other two components of the catalytic triad, histidine 440 and glutamic acid 327, are omitted for the sake of clarity.


the chronic toxicity of OPs, because the aged enzyme is not reactivated by antidotes such as 2-PAM (10) (see below). Hence, recovery from ‘‘aged’’ OP poisoning requires de novo synthesis of more acetylcholinesterase enzyme. An X-ray crystallographic study on an aged OP complex has attributed its stability to its formal negative charge, extra hydrogen bonds to the oxygen attached to phosphorus, and histidine 440 being wrongly aligned in order to act as a general base catalyst (13). It also mimics the tetrahedral structure of the deacetylation transition state, which is stabilized by the enzyme. Carbamoylated acetylcholinesterase is not dealkylated, and hence, inhibition by carbamates can be described as pseudoirreversible because, although covalently modified, the inhibited enzyme is slowly regenerated.

S P

NO2

O

C2H5O (5) O O

C

NHCH3 CH3 O

CH3

(6) O O

S

C

NHCH3

N CH3 (7) O O CH3 CH3

C

N(CH3)2

N N

N(CH3)2

(8) O C

N(CH3)2 O

N N n-C4H9

An example is 2-PAM (pyridinium-2-aldoxime methiodide; 10), which is used as an antidote in cases of OP poisoning. 2-PAM binds strongly to the enzyme and takes the place of water as the nucleophile in step k3 (Fig. 4), being phosphorylated on the OH group in the course of the reaction.

N+

CH

N OH

CH3 (10)

INSECTICIDE RESISTANCE CAUSED BY MODIFIED ACETYLCHOLINESTERASE (MACE)

C2H5O

CH3

13

N

SCH2C

OC2H5

(9) REACTIVATION OF INHIBITED ENZYME Certain nucleophilic cationic compounds accelerate the reactivation of phosphorylated acetylcholinesterase.

Resistance to OP and carbamate insecticides is becoming increasingly important in both crop protection and control of public health pests. This often takes the form of increased rates of detoxification by hydrolases (esterases), mixed function oxidases, or glutathione S-transferases but may also be due to target-site resistance caused by a mutation of acetylcholinesterase. This makes the enzyme less susceptible to inhibition by the insecticide and has been termed MACE (modified acetylcholinesterase). When it occurs with an additional metabolic mechanism, it often results in insects becoming immune to the insecticide at any dose. MACE has been identified in a number of arthropod species, including red spider mite (Tetranychus urticae) (14), cattle tick (Boophilus microplus) (15), green rice hopper (Nephotettix cincticeps) (16), aphids (Myzus persicae (17), and Aphis gossypii (18)), tobacco whitefly (Bemisia tabaci) (19), the common fruit fly (Drosophila melanogaster) (20), several species of mosquito (21), Colorado potato beetle (Leptinotarsa decemlineata) (22), and the moths (Plutella xylostella (23) and Helicoverpa armigera (24)). MACE potentially causes resistance to all OP and carbamate insecticides, although the degree of insensitivity conferred by a given mutation may vary considerably between different compounds. Indeed, a particular insect strain may be susceptible to some insecticides, whereas other related compounds are highly resisted. Insensitive forms of the enzyme have been found in the same species and differ in their resistance for the same OP or carbamate insecticide. Some mutant forms of acetylcholinesterase are actually more efficient esterases than the wild type, although most mutations occurring near the active site have a tradeoff, causing the enzyme to be a less efficient catalyst with a higher Km and lower kcat . This means that the substrate will not compete with the inhibitor so efficiently. Consequently, resistance ratios in vivo may not be as high as predicted from the differences in the bimolecular rate constants (ki ) for inhibition. Most of the detailed molecular studies have been performed on Drosophila (25) and housefly strains (26), from which the enzymes have been cloned and compared with the Torpedo enzyme. Four specific mutations have been identified in the housefly that involve the mutation of

14


an amino acid to a more bulky one (valine to leucine, glycine to alanine, and phenylalanine to tyrosine). This particular mechanism may involve a steric restriction of the active site that denies access to the more bulky inhibitors while maintaining access for the smaller substrate molecule. Mutations often occur in combinations conferring added resistance. Heterozygotes are sometimes nearly as resistant as homozygotes because inhibition of the enzyme by >80% has been found to be necessary to cause death (27). With a problem as important as targetsite resistance, it is important that its presence is detected at an early stage in field pest populations before they build up large numbers of resistant insects. To this end, simple biochemical monitoring methods are available to test individual insects against specific insecticides (28). Because individual insects can be tested, these will detect resistance at a much earlier stage than conventional bioassays. Biochemical tests will also detect heterozygous as well as homozygous individuals and allow resistance genes in the population to be monitored and managed. TOXICOLOGICAL CONSEQUENCES OF ACETYLCHOLINESTERASE INHIBITION Since the patenting of parathion in 1944, many dozens of acetylcholinesterase-inhibiting OP and carbamate insecticides have been launched onto the agrochemical market. Many have also been withdrawn for various reasons. The 11th edition of The Pesticide Manual lists 96 OPs and carbamate insecticides and nematicides, these having acute mammalian toxicities (AOR) ranging from less than 1 mg kg−1 (the carbamoyloxime aldicarb) to 1,375–2,800 mg kg−1 (the OP malathion). In all cases, their acute mode of action is acetylcholinesterase inhibition, although chronic toxicity may be due to other causes (see below). The toxicology of all anti-acetylcholinesterase agrochemicals, especially OPs, is currently under intense scrutiny, with regulatory authorities requesting resubmission of data for their continued usage in Europe. OPs have often been especially stigmatized as being ‘‘nerve gases,’’ which is unfortunate as many are very useful and safe insecticides. They are also used in almost every crop outlet and in many public health and veterinary applications. Typical symptoms of acetylcholinesterase inhibition following administration of large doses of OPs or carbamates to mammals are defecation, urination, lacrymation, and muscular twitching followed by clonic spasms. The cause of death is usually asphyxiation. Human exposure is usually monitored by measuring the level of cholinesterases in the blood serum or erythrocytes by the method of Ellman (29) using acetylthiocholine as the substrate and a chromogenic disulphide, which reacts with the thiocholine produced during the course of the reaction. It may take several days or weeks to recover baseline levels after OP poisoning. Erythrocyte cholinesterase is a particularly sensitive test and may often remain depressed long after neurological symptoms and evidence of urine metabolites have gone. Progressive neuronal degeneration, usually referred to as organophosphate-induced delayed neuropathy (OIDN), which is characterized by ataxia caused by

degeneration of long nerve fibers, is not due to inhibition of acetylcholinesterase. It is caused by inhibition of neuropathy target esterase (NTE), which is a protein of unknown function but possibly a nerve cell receptor. Such effects have only been found to occur after ingestion of high doses of OPs and subsequent recovery from the symptoms of acute poisoning and not after chronic low-level exposure. Only some OPs produce delayed neuropathy because only ‘‘aged’’ enzyme (Fig. 4) results in progressive and irreversible neural degeneration. Anecdotal accounts of neural degeneration after chronic exposure to moderate doses of OPs (e.g., via sheep dipping) have an unknown cause and in only a few cases have blood cholinesterase levels been measured to assess likely degrees of exposure. BIBLIOGRAPHY 1. A. P. Breskin et al., Insect Biochem. 15: 309–314 (1985). 2. I. B. Wilson and F. Bergman, J. Biol. Chem. 185: 479–489 (1950). 3. I. B. Wilson and F. Bergman, J. Biol. Chem. 186: 683–692 (1950). 4. F. Bergman, I. B. Wilson, and D. Nachmansohn, J. Biol. Chem. 186: 693–703 (1950). 5. I. B. Wilson, F. Bergman, and D. Nachmansohn, J. Biol. Chem. 186: 781–790 (1950). 6. I. B. Wilson, J. Biol. Chem. 190: 111–117 (1950). 7. F. Bergman, I. B. Wilson, and D. Nachmansohn, Biochim. Biophys. Acta 6: 217–224 (1950). 8. I. B. Wilson, Biochim. Biophys. Acta 7: 466–470 (1950). 9. I. B. Wilson, Biochim. Biophys. Acta 7: 520–525 (1951). 10. I. Silman and A. H. Futerman, Eur. J. Biochem. 170: 11–22 (1987). 11. J. L. Sussman et al., Science 253: 872–879 (1991). 12. J. R. Coats, Chemtech 23: 25–29 (1993). 13. C. B. Millard et al., Biochemistry 38: 7032–7039 (1999). 14. H. R. Smissaert, Science 143: 129–131 (1964). 15. W. J. Roulston, H. J. Schnitzerling, and C. A. Schuntner, Aust. J. Biol. Sci. 21: 759–767 (1968). 16. H. Hama and S. Iwata, Appl. Entomol. Zool. 6: 183–191 (1971). 17. G. D. Moores, G. J. Devine, and A. L. Devonshire, Pestic. Biochem. Physiol. 49: 114–120 (1994). 18. G. D. Moores, X. W. Gao, I. Denholm, and A. L. Devonshire, Pestic. Biochem. Physiol. 56: 102–110 (1996). 19. F. J. Byrne and A. L. Devonshire, Pestic. Biochem. Physiol. 45: 34–42 (1993). 20. D. Fournier et al., J. Mol. Biol. 210: 15–22 (1989). 21. R. H. ffrench-Constant and B. C. Bonning, Med. Vet. Ent. 3: 9–16 (1989). 22. J. M. Wierenga and R. M. Hollingworth, Econ. Entomol. 86: 673–679 (1993). 23. V. Noppun, T. Miyataand, and T. Saito, Appl. Entomol. Zool. 22: 116–18 (1987). 24. R. V. Gunning, G. D. Moores, and A. L. Devonshire, Pestic. Biochem. Physiol. 55: 21–28 (1996).

ACUTE TOXICITY 25. A. Mutero, M. Pralavorio, J. M. Bride, and D. Fournier, Proc. Natl. Acad. Sci. USA 91: 5922–5926 (1994).

ACS

26. A. L. Devonshire, F. J. Byrne, G. D. Moores, and M. S. Williamson, in B. P. Doctor et al., eds., Structure and Function of Cholinesterases, Plenum Press, New York, 1998, pp. 491–496.

American Chemical Society

27. H. R. Smissaert, F. M. A. El Hamid, and W. P. J. Evermeer, Biochem. Parmacol. 24: 1043–1047 (1975). 28. G. D. Moores, A. L. Devonshire, and I. Denholm, Bull. Ent. Res. 78: 537–544 (1988). 29. G. L. Ellman, K. D. Courtney, V. Andres, and R. M. Featherstone, Biochem. Pharmacol. 7: 88–95 (1961).

FURTHER READING The literature on acetylcholinesterase is vast and includes papers on protein structure, physiology, toxicology, pharmacology, molecular genetics, and enzyme kinetics. There are many reports concerned with its inhibition by carbamate and OP insecticides, and these may also be of interest to agrochemical scientists and toxicologists. The following books and reviews cover both fundamental and applied aspects of the chemistry and biology of this very important enzyme.

15

ACTION LEVEL (REGULATORY) (IUPAC) For food commodities, an administrative Maximum Residue Limit (MRL) used by regulatory authorities to initiate action where no legally defined MRL has been established or where a MRL has been exceeded. For the environment, concentration of a pesticide in air, soil, or water at which emergency measures or preventative actions are to be taken (after Duffus, 1993).

BIBLIOGRAPHY J. H. Duffus, Glossary for Chemists of Terms used in Toxicology, Pure Appl. Chem. 65: 2003–2122 (1993).

Doctor, B. P. et al., eds., Structure and Function of Cholinesterases, Plenum Press, New York, 1998, p. 630.

ACTION LIMITS (ANALYTICAL QUALITY CONTROL)

This publication is the proceedings of the 6th International meeting on the topic held in La Jolla, California, in March 1998. It contains excellent review articles by the foremost researchers in the field and are well referenced.

Limits for measurement on reference material of spiked samples, which indicate when an analytical procedure is not performing adequately and requires immediate action before data can be reported (IUPAC).

Quinn, D. M., Chem. Rev. 87: 955–979 (1987). Rosenberry, T. L., Adv. Enzymol. 43: 103–218 (1975).

ACTIVATION

A general review. Aldridge, W. N. and Reiner, E., Enzyme Inhibitors as Substrates, North-Holland, Amsterdam, 1972, p. 328. This book describes fundamental studies on the mechanisms and kinetics of esterase inhibition. O’Brien, R. D., Toxic Phosphorus Esters, Academic Press, New York, 1960, p. 434. Although dated and mainly concerned with OP insecticides, Chapter 3 contains a useful description of the reaction of OPs with acetylcholinesterase.

ACID EQUIVALENT Refers to that part of a formulation that theoretically can be converted to the acid (Klingman et al., 1975) The total organic acid content expressed in terms of the active acid (CIPAC).

Metabolic or chemical reaction of a molecule that transforms it simply into a product of higher biological activity.

ACTIVE INGREDIENT (ai) Pesticide present in a formulation as defined by the common name (IUPAC). The part of a pesticide formulation from which the biological effect is obtained (FAO). Active Ingredient as defined in the data requirements for USEPA registration (Part 158.153) means any substance (or group of structurally similar substances, if specified by the Agency) that will prevent, destroy, repel, or mitigate any pest, or that functions as a plant regulator, desiccant, or defoliant within the meaning of FIFRA sec. 2a. Repellents, attractants, etc., are covered by pesticide legislation and are included in discussions of pesticides.

BIBLIOGRAPHY

ACUTE TOXICITY

G. C. Klingman, F. M. Ashton, and L. J. Noordhoff, Weed Sciences, Wiley, New York, 1975, p. 115.

Ability of a substance to cause adverse effects within a short period following dosing or exposure (IUPAC).

16

ADVERSE EFFECT

ADVERSE EFFECT Change in morphology, physiology, growth development, or lifespan of an organism that results in impairment of functional capacity or that increases susceptibility to the harmful effects of other environmental influences (Gold, 1987). BIBLIOGRAPHY V. Gold et al. (eds), International Union of Pure and Applied Chemistry, Compendium of Chemical Terminology. IUPAC Recommendations, Blackwell Scientific Publications, Oxford, UK, 1987.

AEROSOL A suspension of fine particles, solid or liquid, in a gas, fog, or smoke (IUPAC). System of fine solid or liquid particles (5000 EU/m

Routine activities that might lead to chronic symptoms such as nonallergic asthma-like syndrome, bronchitis (cough with phlegm), and chronic inflammation of the mucus membranes include indoor swine or poultry production, work in dairy barns, mushroom production, or handling unspoiled grain. Expected aerosol levels in chronic settings are shown as follows: Total Aerosols 1–10 mg/m

3

Endotoxin 100–500 EU/m

Microbes 3

4

10 –107 CFU/m3

Organic dust from agricultural operations is a complex mixture of biologically active materials (115,117). It is usually quite difficult to identify which of the agents in the dust are responsible for the given condition(s). Some research has shown that grain mites or animal dander are important relative to atopic asthma (140–142). Endotoxin is the primary agent related to bronchitis, asthma-like syndrome, MMI, and ODTS (125,144,145). Glucans are emerging as agents of chronic inflammation like endotoxin and also like endotoxin are components of the cell wall of microbial organisms (74,117,145). Control of agricultural dusts should follow the classic IH paradigm: rely first on reduction of the dust source, second on ventilation or other pathway control, and third on personal protection. Reduction of acute exposure to organic dusts may involve applying moisture to the top of the material to reduce its aerosolization when disturbed; to apply this principle to some farm operations (e.g., silo unloading) may require special techniques (146,147). Good

housekeeping to reduce dust accumulation can help. High ventilation of animal confinement or other farm buildings is often resisted by operators who prefer to conserve heat in cold winter climates (148,149). And the application of negative exhaust pressures can pull hazardous agents into a building from covered manure pits or naturally exhausted heaters (148). The principles of respirator use in agriculture is similar to that in any other industry, except that there are few trained persons available either on an individual farm or even in the rural community to direct the selection, assure proper fit, and supervise the respiratory program (126,150). Pesticides The term ‘‘agricultural chemicals’’ refers to fertilizers and pesticides. While fertilizers comprise the largest category of agricultural chemicals, pesticides are the traditional ‘‘whipping boy’’ of environmental and occupational health concerns for farmers, especially for farm workers. Although chemicals do indeed present hazards when misused, it should be apparent herein that pesticides and fertilizers represent only a narrow spectrum of the occupational risks within agriculture. The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) refers to pesticides as ‘‘economic poisons’’ intended to prevent, destroy, repel, or mitigate ‘‘any insects, rodents, nematodes, fungi, or weeds or any other form of life declared to be pests, . . . and any substance or mixture of substances intended for use as a plant regulator, defoliant, or desiccant.’’ Toxicologically, the major field-use agricultural pesticides can be broken down into six major chemical groups of organophosphate, carbamate and thiocarbamate, and chlorinated insecticides and phenoxy-aliphatics, triazines, and bipyridyl herbicides (70,102,105). Although reviews of their toxicities are readily available (e.g., 102–105), the industrial hygiene aspects of their use practices, levels of exposure, and the efficacy of exposure controls are less accessible. Pesticides can present a hazard to applicators, to harvesters reentering a sprayed field, and to offsite rural residents via air, water, and even food contamination. Methods to assess exposure include direct methods via dermal patches (151–154), skin washes (151,152,154–156), dietary surveillance (157), and fluorescent tracers (154,158). Indirect exposure assessment methods include biochemical responses such as change in cholinesterase activity (152,159,160), urinary excretion (152,161–164), and DNA changes (160). And epidemiologic methods to assess response include morbidity (165–169) and mortality (165,170–173) studies. Archetypical of the difficulty of investigating health effects among diversely exposed and dispersed populations was the discovery of testicular atrophy and sperm count depression among applicators of the nematocide dibromochloropropane (174,175) following its initial discovery by and among pesticide formulators (176). Differences in the above methods of assessment complicate comparisons among the multiple routes of exposure contributing to farmers’ total doses. Dermal, inhalation, and ingestion are all possible routes of exposure;

AGRICULTURAL HYGIENE

however when outdoors, the dermal route predominates over inhalation (>99% of the total dose) during application (177–181), harvest (152,182,183), and even in cases of local environmental contamination via spray drift (184–185). Indoor agricultural uses of pesticides such as grain fumigation (183,186) and especially greenhouses (183,187,188) represents a specialized environment where airborne exposure can dominate. Ingestion of pesticides can occur through the contamination of food at work (189). Dermal exposure assessment has been a developing area of research. Some studies have found favorable comparisons between direct and indirect methods of assessment, while others have found differences or a lack of correlation. It is often forgotten that correlations should not be expected between measured chemicals that were prevented from actually reaching the skin by the collection media and any measured metabolic excretion of what is absorbed by the same subjects. Not surprisingly, levels of exposure to pesticides during application vary by task. Broadly speaking, variations in exposures within the tasks shown in Table 7 are unrelated to the particular chemical being used but are a function of the pesticide formulation and concentration, the application process and equipment, clothing, personal techniques amenable to education, and uncontrolled conditions like weather and foliage (180). Taking advantage of this principle, the Pesticide Handlers Exposure Database (PHED) is a relatively new tool to predict dermal and inhalation exposure to mixers, loaders, applicators, and flaggers based on a database of previously measured values (181). Studies of health effects from pesticides are predominantly mortality studies (see Section 2.10 on Cancer). Morbidity studies include acute poisoning reporting (105,190, 191), a smaller number of investigations into subtle, chronic neurotoxic effects of pesticides (166,168,169), and even fewer on seasonal cholinesterase inhibitions (152,192). For instance, acute pesticide poisoning accounted for 10% of all hospital admittances of farmers and agricultural workers in Colorado, Iowa, and South Carolina during 1971–1973; this rate extrapolated to 9.1 per 100,000, for the 3-year study period. Organophosphates were responsible for 64% of these observed cases (189). Studies in both the United States and abroad have shown that only about 25% of the acute pesticide poisoning fatalities are of occupational origin. Of the remaining 75% in California, nearly 60% were children, frequently due to improperly stored insecticides (193); in the Third World, 75% were suicides (194). Elevated frequencies of suicides (even among U.S. farmers) (16,195), indicates that rural life in general and

Table 7. Ranges of Pesticide Dermal Task Flaggers Mixer–loaders Applicators Harvesters

Range, mg/hr 0.03–300 10–100 2–10 0.5–30

27

farming in particular is stressful; in less developed countries, pesticides are merely an available, convenient, and perhaps economic vehicle for suicide. Pesticide exposure controls for field applications include engineering/mechanical controls (110,177,196,197) but in practice seem to stress personal protection from clothing, gloves, and respirators (156,158,180). In contrast to performance based programs implied by the TLV and regulated by OSHA via the PEL, pesticide usage as regulated by EPA is specified in the label instructions to users. All users of pesticides should be trained to read and follow the label instructions. Unfortunately, the historical focus of pesticide label instructions has been the respiratory route of exposure, despite the repeated finding by direct measurements that the dermal route tends to be at least 100 times larger than the respiratory route (152,178–180). High rates of dermal depositions coupled with most insecticide’s high rate of absorption through intact skin (by design), indicates that reducing dermal exposures is usually the most important component of control. Herbicides are not as dermally absorbable, but even for compounds like paraquat, the skin can be an important route of dosing if improper use practices are allowed (69). The impact of many but not all of these controls can be estimated from PHED (181). One of the unanswered questions is the impact of incomplete removal of pesticides by home laundering (198–201); at least one study (201) seems to be on the track of quantifying the fraction of the residual pesticide bound to clothing after machine washing that is transferable to the wearer, but the ability to predict a dose and an acceptable threshold remains elusive. Because of the acute danger when organophosphate (OP) pesticides are in use (and to a lesser degree carbamates), the existence of a cholinesterase monitoring program is important. Where surveillance is in place and protection breaks down, preplanned medical management is essential. Fortunately, guidelines for such monitoring and diagnosis of OP poisoning are well established (152,158,160,202,203). The ‘‘reentry hazard’’ (going into a field after a pesticide application) represents a long unrecognized hazard to harvesters from field residues. It was initially implicitly assumed and only later shown that the rate of dermal exposures to harvesters is proportionate to the field residue, usually the foliar residue (152,182,183,204). Thus in principle, exposure control for harvesters could be based on measured residues. However, applications must be planned to allow certain required field activities to be scheduled, and widespread field residue sampling has not been feasible. Therefore, the use of ‘‘reentry intervals’’ between application and harvest came be the protective tool. A poorly reported 25-year history of sporadic groups of harvesters having symptoms of organophosphate pesticide poisoning was often attributed to poor sanitation, water, or food poisoning (6,189), thus delaying the recognition of a significant flaw in the reentry interval strategy (152). The classic study by Milby et al. (205) typifies the evolutionary impact of new analytic technologies upon the investigation and understanding of pesticide hazards. In this case using the then-new process of gas chromatography found for

28


the first time that a more toxic ‘‘oxon’’ analogue of the applied thio-phosphate insecticide sometimes forms in field residues. Since that time, the high variability of oxon production in leaf residues (206) and its importance to harvester acute poisoning has been clarified and much better (although still perhaps incompletely) integrated into the field worker protection strategy (152,183,207). A wide variety of disinfectants (also classified by EPA as a pesticide) are used in livestock operations, especially dairy farms and large hog buildings (106). They include chlorine, quaternary ammonia compounds, organic iodines, cresol-based compounds, and formaldehyde emitters, and often one of a variety of detergents. Certain individuals may develop contact dermatitis or an allergic contact dermatitis from these chemicals (208). Prevention of dermatoses can be based on selection and use of chemicals that are not known as irritants or sensitizers. Chemical resistant gloves should be worn as a rule during operations that require repeated contact with the chemicals, such as cleaning milking equipment. Protective hand creams are a better supplement to the use of gloves than they are an alternative (209). Hog farmers who use quaternary ammonium disinfectants have shown increased bronchial hyper-responsiveness (210). Veterinary Biologicals, Antibiotics, and Pharmaceuticals Biologicals are made from living products to enhance the immunity of an animal to a specific infectious disease or diseases. They may be live attenuated microbes, killed viruses (vaccines), killed bacteria (bacterins), or inactivated bacterial toxins (toxoids). All of the above products are intended to enhance the active immunity of the host. These products may also contain adjuvants which enhance the immunogenicity of the products. Another group of biologicals enhances the passive immunity of the host by injecting antibodies produced in another animal. These products may be crude blood sera from a hyperimmunized animal (antiserum), more refined globulin fractions of the sera, or genetically engineered products. The main risk groups are those involved in livestock production and related veterinary care who administer these products to animals. Besides veterinarians and their assistants, farmers, ranchers, their family members, and employees all may be at risk (211). Operations involving swine, poultry, beef, dairy cattle, and sheep all may have an inherent risk for exposure. A government-regulated disease control program in effect for certain diseases (e.g., brucellosis, pseudorabies) requires that a veterinarian administer the biological. Otherwise the producer, as well as the veterinarian, may administer any of these biologicals. The hazard is associated with either accidental inoculation, splashing the product into the eyes or mucous membrane, or contamination of the broken skin (211,212). In one survey, almost 10% of veterinarians reported a needle stick injury per year (212). The result may be an infection (certain live products), inflammation, or an allergic reaction. Inflammation or allergic reactions may occur from inoculating either live or killed products, the adjuvant, or the foreign protein in the product (211).

Inoculation may also introduce surface organisms beneath the skin where they can induce infection. And inoculation with a dirty needle has the extra risk of causing infections of environmental origin. The primary products that have been associated with occupational illnesses include brucellosis strain 19, Escherichia coli bacterins, Jhone’s disease bacterin, erysipelas vaccines, contagious ecthyma vaccine, and Newcastle disease vaccine. The most frequent reports of occupational illnesses associated with biologicals involve veterinarians using brucellosis strain 19, which is a live product containing an adjuvant (213). Veterinarians have become ill either by splashing the material in their eyes or by accidental needle sticks. The results may be infection, inflammation, and allergic reaction. The infection mimics the acute infection seen from acquisition of brucellosis directly from either cattle or swine (214). If the person had a previous exposure to brucellosis (many veterinarians practicing before the mid-60s had previous exposures), they may develop severe inflammatory and allergic reactions in addition to an infection (213). The reaction is characterized by severe localized swelling and pain extending from the site of the inoculation. The swelling and allergic reaction must be treated in addition to the infection in these cases. Disability may last for days to weeks in the worst cases. Newcastle disease and contagious ecthyma (orf) vaccines are live products used in chickens and sheep, respectively. Newcastle vaccine is applied inside poultry buildings via a nebulizer. Workers who contaminate their eyes with this vaccine may acquire a moderate conjunctivitis with influenza-like systemic symptoms. Orf vaccine can cause the same pox-like lesions at the site of inoculation as a naturally acquired infection. Both of these diseases are self-limited and disability will only last for a few days, unless the orf lesions are numerous (215,216). Jhone’s, E. coli, and most erysipelas biologicals are bacterins, and therefore injuries induced by these products are limited to the inflammatory response induced by the adjuvants. Control of injuries associated with biologicals revolves around good animal handling techniques and facilities, because most of the accidental needle punctures are secondary to uncontrolled and untimely movements of stressed and improperly restrained animals. The proper construction of animal handling facilities has been reviewed by Grandin (217). The use of pneumatic syringes, lock-on needle hubs, and multiple-dose syringes will also help reduce injuries. Eye protection is indicated in many instances, and a full face respirator is necessary for aerosolized vaccines such as Newcastle. Antibiotics are products derived (or synthesized) from living organisms, mainly mold species of the genus Streptomyces. Antibacterials are chemical compounds not from living organisms, but used in the same manner to treat infectious diseases therapeutically. They are also used widely at lower levels in livestock production to improve the rate of weight gain and feed efficiency in cattle, swine, and poultry. Livestock producers, veterinarians, and feed manufacturers and formulators are commonly exposed to these agents by direct contact


with antibiotic-containing feeds, or via aerosol exposure within livestock buildings, within feed preparation areas on the farm, or in feed manufacturing plants. There are two main occupational hazards: allergic reactions and the development of antibiotic-resistant infections. There are many different products used as feed additives, but the main ones include penicillin, tetracycline, sulfamethazine, erythromycin, and virginiamycin. These same products plus many more are used therapeutically. Penicillin is the primary agent that may induce an allergic reaction manifest in the form of a skin reaction from direct contact, or possibly a systemic reaction from inhalation or inoculation. A variety of these agents may induce development of resistant organisms in the gut flora of exposed individuals. The resulting health impact of this is not clear. However, there have been some cases of severe resistant salmonellosis traced to direct animal contact (218) and in people who were treated with antibiotics for a condition unrelated to salmonella. The latter case is a result of an overgrowth of the resistant organisms secondary to the antibiotic treatment. Although the full importance of antibiotics as an agricultural health hazard is unknown, it is prudent to take some control measures. Feed formulation, grinding, mixing, and storing operations should be in closed systems. General dust control procedures should be utilized in both feed preparation areas and in animal feeding operations. Until dust control procedures are proven effective, dust masks should be worn in conjunction with other engineering and work practice procedures. In addition, an emphasis should be placed on removal of those antibiotics used in human health from feed additives and a rotation of the particular type of antibiotic used should be considered. There are numerous pharmaceuticals used in livestock production and veterinary practice. These products are largely available without prescription and thus, workers at all levels may be using these, and like biologicals, accidental needle sticks or other exposures may occur. Two products in particular are of concern for pregnant women—Oxytocin and Protaglandins. Accidental inoculation with either of these products could cause abortion (213). These products are commonly used in swine, beef, and dairy production. Zoonoses Zoonoses are infectious diseases common to animals and man. At least 24 of the over 150 such diseases known worldwide are occupational hazards for agricultural workers in North America (114,219–221). Some of these diseases may be contracted directly from animals, whereas many are contracted from the natural environment that is part of the farmer’s workplace. A list of recognized agricultural zoonoses was prepared by Donham and Horvath elsewhere (12). The agricultural worker’s risk of acquiring a zoonotic infection varies with the type and species of animal and the geographic location (12,220,221). For example, dairy farmers in North America are at risk to acquire

29

ringworm, milker’s nodules, or leptospirosis. Beef cattle producers are more prone to acquire rabies, anthrax, or salmonella. Swine producers are at risk for contracting swine influenza, streptococcus suis, or erysipelotrix rheusiopathiae (erysipeloid). Besides livestock producers, those doing related service work (e.g., veterinarians) or animal processing are also at risk for certain zoonotic infections (220). Turkey processing workers are known to be at risk particularly for ornithosis, red-meat processing workers for brucellosis and leptospirosis, and hair and hide processors for anthrax (10,114,222). Control of these infections in the production phase depends largely on an awareness of the specific hazards, good preventive veterinary care, hazard communication, and medical backup, especially in cases where serological monitoring of animals or people may be indicated. For livestock producers, close animal health monitoring and veterinary preventive practices are best. In processing, early identification of infected animals as they come into the plant and appropriate handling of them is important. In some cases sanitation and personal protection are important. The key is developing both an understanding of certain generic features of this group of diseases and an awareness of conditions and agricultural activities that increase infection risks within specific locations (as reviewed elsewhere, Refs. 114,219,223). Such an awareness is essential to enable the hygienist to anticipate, recognize, evaluate, and design a control program for zoonotic infections. Skin Diseases Diseases of the skin are very common in agriculture (208,209, 224–228). Compared to other occupational groups, farmers have a proportionately higher prevalence of skin diseases (227), and in some regions skin diseases are the most common condition reported by agricultural workers (228). Common agricultural skin diseases, causative agents, and suggested methods of control are listed in Table 8. Irritant contact dermatitis is the most common type of agricultural dermatoses (209,224–231). There is no particular subgroup of agricultural workers that is free from contacting a substance that may cause an inflammatory response to the skin. Irritant substances are ubiquitous and include ammonia fertilizers, several insecticides and fungicides, a few herbicides, soaps, petroleum products, and solvents (209,226). Avoidance schemes must include work practices to eliminate or reduce exposure to the most irritative substances and/or the use of personal protection equipment. Delayed allergic contact dermatitis is typified by poison ivy or poison oak reactions. These are exquisite sensitizers, and nearly 60% of the general population is capable of reacting to these allergens. Only a few herbicides and pesticides are sensitizers (208,209). Several of these substances may produce a more immediate allergic response, but it is difficult to control exposure to sensitizers because just a small amount of the allergen may produce a reaction. Sun-induced dermatoses include sunburn and skin cancers (173,209,232). Acute sunburn may be prevented

Table 8. Skin Conditions of Agricultural Workers: The Principal Source, Symptoms, and Prevention Classification Irritant contact dermatitis

Allergic contact dermatitis

Photocontact dermatitis (including both photo irritant and photoallergic contact dermatitis)

Source or Agent (Examples) Ammonia fertilizers

Animal feed additives (ethoxquin, cobalt) Insecticides (inorganic sulfur, petroleum, coal tar derivatives) Plants (bulbs of tulips, hyacinths, onion, garlic, carrots, asparagus, celery, parsnips, lettuce) Herbicides (trichloroacetic acid, paraquat) Fumigants (ethylene oxide and methyl bromide) Herbicides (propachlor, thiram, maleic hydrazide, randox, barban, nitrofen, dazomet, lasso) Insecticides (pyrethrum, rotenone, malathion, phenothiazine, naled, ditalimfos, omite, dazomet, dinobuton) Antibiotics (penicillin, spiromycin, phenothiazine) Plants (poison ivy, poison oak, poison sumac, ragweed) Creosote

Description of Condition

Control

Dermatitis mainly on hands, arms, and other points of contact

Assure proper dilution of chemicals; wear protective clothing; wash hands, arms, and other contact areas frequently

Acute inflammatory response with swelling, possibly reddish elevated eruptions, blisters, pruritus; usually on hands and arms

Same as above, plus: wash clothes that contact offending substances; any work practice change that will limit contact with offending substance

From skin exposure to agent followed by exposure to sunlight; dose dependent, furocoumarin causes blisters followed by hyperpigmentation in bizarre, streaked pattern

Wash hands and contact areas of skin; protective clothing (e.g., gloves and long-sleeved shirt)

Feed additives (phenothiazine) Plants containing furocoumarins (carrots, celery, parsley, parsnips, limes, lemons); ragweed, oleoresins

30

Table 8. (Contiuned) Classification

Source or Agent (Examples)

Sun-induced dermatoses

Ultraviolet radiation

Infectious dermatoses

Cattle, swine, rodent animal ringworm (Trychophyton verrucosum, Microsporum nanum, T. metagophytes, respectively).

Heat-induced dermatoses

Arthropod-induced dermatoses

Sheep pox virus (orf or contagious ecthyma). Cattle pseudocowpox virus (milker’s nodules). Moist, hot environments

Chiggers, animal mites, grain mites, Hymenoptera (bees, wasps, hornets, yellow jackets, fire ants).

Description of Condition

Control

Includes sunburn; wrinkling of skin; actinic keratoses; squamous cell carcinoma; basal cell carcinoma Ringworm: highly inflamed, scaly lesions on hands, arms, face, and head; Orf: lesions on hands and arms, develop as red papules, progress to an ulcerative lesion; Milker’s nodules: multiple solitary, wartlike lesions on hands and arms.

Protective clothing (wide-brimmed hat, long-sleeved shirt); sunscreen (e.g., paraaminobenzoic acid) Appropriate veterinary treatment and prevention, e.g., good sanitation of animal environment; wear protective clothing when handling infected animals.

Miliaria rubra (prickly heat): an exanthematous eruption of the skin caused by inflammation of eccrine sweat glands mainly under the arms and around the belt line Red maculas, papules, pruritic lesions, possibly vesicles; sensitivity may vary with repeated exposure; anaphylactic reaction possible

Wear loose-fitting, well-ventilated clothing; ventilate the work environment; daily bathing with a good soap

31

Wear light-colored, nonflowery clothing; avoid perfumes; use insect repellent (e.g., diethyltoluamide)

32


by the use of sunscreens and protective clothing. More important is the cumulative effect of sun exposure, which may produce a variety of lesions about the face and arms. Skin thickening, wrinkling, and actinic keratoses are common in older farm workers; the latter is a precancerous lesion. Twenty-five percent of the preneoplastic lesions may develop into squamous cell carcinomas, the second most common skin cancer after basal cell carcinoma. Squamous cell carcinomas do not tend to be malignant unless they occur on the lip but usually require surgical removal. Basal cell carcinomas are more common but have a low tendency to become malignant. Melanomas have a high tendency to metastasize, but fortunately are the least common of these skin tumors. The risk for melanomas are related to the frequency of sunburns (high exposures), while the other sun induced skin lesions are due to the cumulative chronic (without burning) sun exposure. Heat-induced dermatoses are not generally very serious, but they can be quite uncomfortable and recovery may take several days. The primary problem with heat is an inflammation of the eccrine sweat ducts, resulting in a pruritic eruption called prickly heat or miliaria rubra (233). Infections of the skin are primarily a result of viruses and fungal agents of animal origin. Ringworm of cattle (Trychophyton verrucosum) is a common agricultural fungal skin infection (234). The pox viruses are the next major source of infection. The virus in sheep that produces contagious ecthyma (sore mouth) produces orf in humans, and the virus in cattle that produces pseudocowpox produces milker’s nodules in humans (235). Chiggers, grain mites, animal mites, bees, and wasps all can cause significant injury to the skin of agricultural workers (233,236). The lesions vary from a mild selflimited skin rash from chiggers and mites to an anaphylactic reaction from stings of bees and wasps. Physical Agents It should come as no surprise that mechanization has had a major impact upon noise-induced hearing loss among farmers. Today, it is a novel experience (perhaps at a farm show) to watch a draft team pulling a plow and to hear the sod being broken and the soil turning over. Numerous surveys show that farmers today suffer a higher incidence of hearing loss compared to other occupational groups (e.g., Refs. 58,237,238). Even some nonmechanized farming practices can result in high noise exposure levels, as can be seen in Table 9. Sullivan et al. (241) conducted a year-long study of the noise environment of agricultural workers on six Nebraska farms and 67 farm workers. Thirty-eight percent of their machines produced sound levels in excess of 90 dB. To cope with farming’s temporal variability, they time-weight averaged over monthly intervals and found 39% of farm workers exceeded 8-hour 90 dB OSHA limits for 15% of the months (241). Only slightly lower levels of exposure were reported more recently for New York dairy farmers (242). Noise exposure is reduced in tractors with cabs; a study in Wisconsin found noise in 75% of tractors without cabs exceeded 90 dBA versus 18% with cabs (243). This study also found that partially opening the back cab windows

Table 9. Typical Noise Levels During Selected Farming Operationa Activity Chain saws Vane-axial grain drying fan Combine at full throttle Corn grinder Squealing sows Bed chopper Hay choppers and balers Grain storage bin construction Tractor at full throttle Next to tractor On seat, no cab (75% > 90 dBA) In cab Harvestore unloader/conveyer Milking parlor a

dBA 105–112 100–110 102–107 94–103 95–102 94–102 95–100 60–98 102 93 82–85 85 76–84

Adapted from Refs. 239,240.

increased the average noise level by 1.7 dB; operating the radio with the windows closed increased cab noise by an average of 3.1 dB; and completely opening the windows increased the average noise level by 4.5 dB. Other traditional methods used to prevent noise-induced hearing loss among general industrial workers (a hous described in CHAPTER 20) are broadly applicable to farmers. Obstacles to such interventions include the long-term capital investments characteristic of large mechanized pieces of equipment and the limited resources available to reach such a large, voluntary audience (244). Heat, vibration, and ergonomic hazards are all prevalent in agriculture. Heat (and cold in many regions) is a seasonal stressor for outdoor workers generally (see also CHAPTER 23). Heat-induced illness is rarely reported for farmers (12). Among the few examples are elevated heatstroke reported by West (32), more than 2% of workers compensation claims for production agriculture were heat related reported by Jensen (245), and elevated PMRs from exposure to heat or cold reported by Une et al. (246). Given the exposure of farmers to extremes of both heat and cold, these few reports probably reflect the poorto-no epidemiologic surveillance of this population, a large measure of self-selection within the work force, and perhaps a limited measure of self-control in their work hours. Whole body vibration (WBV) is very common on tractors. A review of WBV on tractors found pathological radiological changes in the spine and that complaints of low-back pain were found to be associated with both total years and hours per year of tractor driving (61). A more recent investigation involving 577 of 732 male tractor drivers employed by two Dutch companies found the prevalence of self-reported back pain (most often in the lower back) was approximately 10% higher in subjects who drove tractors exposed to WBV versus those not exposed, and the prevalence increased significantly with vibration dose (63). Thus, whole body vibration appears to interact with twisting of the spine and a prolonged sitting posture to increase the prevalence of lower back pain (60,63,64). Vibration can be greatly reduced by properly designing the tractor seat (66,247). Segmental vibration among


farmers is most common from chain saws, although many hand tools also contribute. Exposure of farmers to such hand tools is usually limited to short periods (248), but the actual incidence of vibratory white fingers among farmers has not been reported. As discussed in Section 2.3, ergonomics is only beginning to have a major impact on agriculture (66–68), notably on the agricultural tractor cab, mechanized milking equipment, and the banning of the short hoe. Systematic study of additional hazards for disabled farmers returning to work is also in its infancy (249,250). Cancer Compared to the general population, farmers have lower overall cancer rates (37,173,251–253). They also have lower rates for the most common cancers related to smoking, viz., lung, esophageal, and mouth (251), consistent with the observation that only approximately 17% of farmers smoke compared to 34% of the general population (35,173). In spite of this lower overall rate, positive associations often appear between farming or even rural life and several less common cancers including leukemia, non-Hodgkin’s lymphoma, multiple myeloma, Hodgkin’s disease, and cancers of the lip, skin, prostate, stomach, and brain (37,173,253–257). According to the extensive 1991 review by Blair and Zahm, the statistical evidence for these associations is variable across studies and usually lacking a clear etiology (173). Easily the strongest evidence is for lip cancer which, along with skin cancer, is quite clearly related to sun exposure (10,50,209). A small but innovative project tested the farmer’s acceptability and sun protection characteristics of eleven different hats (258). Each hat had both positive and negative characteristics, and none was ideal. A baseball cap modified with a removable back flap was rated highest overall by the farmers. Many of these skin cancers are preventable through education adequate to break some strong cultural norms. Determining the risk factors for other cancers has been a very difficult problem because of cancer’s long latency periods, difficulties in obtaining accurate exposureclassification data, and probably the intrinsic variability in farming. Risk factors most extensively studied are for the reticuloendothelial cancers. A recent meta-analysis of leukemia found a pooled risk ratio of 1.09 for farming (256). Individual studies have found leukemia to be linked to exposure to dairy cattle, poultry, corn production, fertilizers, and animal pesticides (173). An early suspicion of a link to bovine leukemia virus via cattle has not been confirmed (259). Although farmers showed excess multiple myelomas in twelve of sixteen studies, they have been linked to pesticides in only two (173). The annual incidence of non-Hodgkin’s lymphoma (NHL) in the United States rose from 5.9 per 100,000 people in 1950 to 9.3 in 1975, and 13.7 in 1989 (255). About half of the 21 cited studies showed excess NHL among farmers, and about half of these were significant but at a relative risk of less than two-fold (173). Non-Hodgkin’s lymphoma was associated with exposure to the phenoxy acetic acid herbicide 2,4-D in North America but not in Europe or New Zealand (173). Hodgkin’s disease seems

33

typically to be slightly elevated but the least frequent of the reticuloendothelial-lymphatic neoplasms; risk factors have been linked to phenoxy acetic herbicides and to grain dust (173). No environmental factor was linked to prostrate cancer, but it is the most common cancer of those for which farmers are sometimes found to be at an elevated risk. Clarifying the risk factors for agricultural cancers will require a great deal more research. Until further information is available, about the only thing the hygienist can tell farmers with certainty is that they can reduce their risk of skin cancer by wearing protective clothing, sun screen, and installing shade devices on their tractors and other pertinent equipment. Mental Stress Farmers die of suicide at a greater frequency (195) and suffer more frequent mental disability relative to other occupations (52). A 1985 study regarding social concerns within farm families suggests dysfunctional families, divorce, alcohol abuse, and children having problems are all more common within the farm community (260). The Iowa Farm Family Survey of 1988 indicated that farmers rated stress as one of their major concerns (85). Compounding inherent, endemic stressors in farming are episodic events such as the farm economic crisis of 1982–1987 and the drought of 1988–1989 (261), and increasing globalization, industrialization, and change in commodity support prices of the mid and late 1990s (18,262). Mental stress not only should be considered an important occupational health issue for farm families but may also contribute to more frequent injuries (263). Agriculture has always been plagued by economic uncertainty and the constant eroding of profit margins, requiring more to be produced with less labor to assure a livelihood. Additionally, most types of farming include a series of work-cycle peaks (e.g., Table 2) that can be complicated by adverse weather conditions and machinery breakdowns (264). The stoic and independent nature of many farmers makes them reluctant to talk to anybody about these problems, let alone seek professional help (265). The organized support systems typically available in urban centers is not present in most rural communities, and the extended family and social makeup of the rural community is not the support structure that it once was (18,27). To make ends meet economically in today’s farm families, it is very common for one or both spouses to work full or part time off the farm (see Section 1.1). This increases family stress and creates a child-care problem. All too often children are found in the workplace, which is difficult to supervise and where too often they become accident victims (8,38,39). Control of mental stresses is certainly difficult (27). A few innovative, largely pro-active programs are being piloted in communities scattered around the country but there needs to be greater activity in this area (265,266). One such program in Iowa is the ‘‘Sharing Help Awareness United Network (SHAUN),’’ which seeks out farm families in trouble such as having experienced an injured family member, and gets them together for discussion and mutual support. This promises to be a successful way to get help to

34


the stoic independent farmer. The agricultural hygienist needs to be aware of ‘‘farm psychology’’ and mental health resources, and find ways to deal with this aspect of farm health (27). Emerging Hazards The ability to deliver effective prevention programs to the farm community should include the ability to anticipate developing occupational hazards. New genetically engineered crops, livestock, pesticides, and hormones will substantially increase productivity, forcing less efficient farmers out of business and concentrating agriculture even further into larger and fewer operations. Although consolidation will increase the need for hired labor, farm mechanization will eliminate many of the menial labor-intensive operations with which hired farm labor is primarily involved today. The trend for farm employees to become more technically skilled, and the need for temporary migrant labor to diminish, will probably continue (33). For example, the operation of indoor methods of livestock production requires rather specialized year-round labor, but the longer daily exposure to organic dust has created health hazards with which farmers have never before had to deal. The farm manager must become aware of these hazards and of the opportunities and responsibilities to control this working environment, the provision of preventive health and safety services will not only enhance productivity in these environments but may well be necessary for the farm’s sustained profitability. Whatever changes with technology, many risks in agriculture will continue to be of biological origin. For instance, Lyme disease, a tick-transmitted disease first recognized in the northeastern states, is now recognized in much of the upper Midwest (22,219,267,268). This generalized illness from the organism Borrelia burgdorferi can result in prolonged arthritic disability. Although no studies of U.S. farmers have been reported, two British blood serology studies have shown farmers (particularly those with cattle) to be at high risk of exposure (269,270). Its latency and dynamic environmental prevalence precludes an accurate assessment of its potentially large impact at this time. Hantavirus is a recurring problem in the arid regions of the southwestern states. Outbreaks of the resulting ‘‘hemorrhagic fever with renal syndrome’’ or ‘‘hantavirus pulmonary syndrome’’ have been reported among farmers elsewhere in the world (271), but none of the blood serology samples from fifty-seven randomly sampled farmers in New Mexico and Arizona tested positive for hantavirus antibodies (272). The risk of farmers to Creutzfeldt-Jakob Disease (CJD) from exposure to cattle sick with bovine spongiform encephalopathy (BSE) is best described as controversial. BSE has been a great concern to cattle production in the U.K. The authors of a third case report of a farmer who acquired CJD estimated the probability of three or more farmers in the U.K. acquiring CJD at between 0.002 and 0.09, leading them to conclude that farmers are at increased risk (273). However, another group of authors calculated the incidence of CJD in five European countries to be virtually the same (circa 0.75 cases per million person years), leading them to conclude that contact with BSE afflicted cattle is not a risk factor

for farmers (274). The uncertainty with regard to exposure and the small numbers make it much too early to rely on either conclusion. Aflatoxin is known to be an extremely toxic carcinogen in at least eight species of test animals but has long thought to represent only an oral risk to man. A recent follow-up of a small (60–70 persons) cohort exposed to roughly 5 pg/m3 aflatoxin (that is 10−12 gram) on airborne organic dust in a peanut and linseed oil processing plant showed a 2.5 to 4.4 increased risk of cancer of all types for different exposed time periods (275). This diverse pattern of human toxic responses to aflatoxin is not inconsistent with the cancer findings previously described herein (69). The presence of aflatoxin in corn dust (276–278), its measured routine exposures of about 5 ng/m3 within enclosed swine buildings and peak exposures of well over 1000 ng/m3 while cleaning out grain bins (278), and the increased risk of Aspergillus flavus infestation during drought conditions (277) suggest that airborne agricultural exposures to aflatoxin should be a long-term concern. Another anticipated manifestation of climatic changes (including drought) is that continued ozone depletion will increase the ultraviolet light exposure to the farm population which will result in their greater risk for skin cancer. Pesticides and nitrate fertilizers are known to contaminate rural water supplies (82–85), raising a hypothetical concern for increased toxic effects at some point in the future. This concern and others are likely to lead to decreasing use of pesticides and high-volume fertilizers, to be replaced by integrated pest management and genetically engineered tools, leading to decreased soil tillage and exposure to machinery. Whatever the potential health hazards of these new products, agricultural workers are likely to receive the highest exposures and exhibit the first adverse effects. Suffice it to say that not only should agricultural hygienists keep informed generally about new technologies and specific products that become available, but they are among the best qualified to anticipate their hazards and feasible controls. INTERVENTION Lessons from General Industry The industrial hygiene paradigm of anticipation, recognition, evaluation, and control can, in principle, be applied to agriculture with the following translations: • Anticipating health and safety hazards is the preventive application of a dose–response knowledge database. Response data for hazards unique to agriculture must be generated by either mandatory or funded research surveillance systems. Transferring experience from other industries requires either a knowledge of dose (exposures generated by a given act), the ability to assess dose in real time, or an assumption of worst case. It is unfortunate that in widely diverse settings (characteristic of agriculture), the worst case is much worse than the average case. This ‘‘belt and suspenders’’ approach is characteristic


of the specification standards implied by EPA’s pesticide users labels. Overly restrictive controls for everyone necessary to protect against the worst case can be perceived as contrary to the intrinsically risk-taking philosophy of farming. Thus, a great deal of probability salesmanship would have to go into preventive programs based on anticipating the worst case, unless a solid understanding of exposure mechanisms is established. • Recognizing the incidence of injury, disease, and fatalities requires the systematic application of existing methods. Decades ago Knapp complained about the lack of good scientific epidemiologic studies in agriculture (22). Much of what was reported above is new, but data generated in agriculture (especially for health hazards) is fragmented in time and geography and reflects current and in some cases old technologies. Technologies evolve, and even if current risks were known, the dispersed and locally innovative nature of agriculture would bolster a natural bias toward the use of new unevaluated technologies (279). • Methods exist to evaluate essentially all the agricultural hazards noted above (e.g., Refs, 70,139,131,158, 240,277). Although usually rewarding in the long run, agriculture is an inherently risky venture with a slow economic rate of return. The value of evaluation is dimmed by the psychological perception that risks are intrinsic to farming and the cost of voluntary prevention is not competitive in the short term. Even in general industry, a person with less wealth will be more willing to accept job risks; e.g., the average blue collar worker would accept perceived risky jobs for $900/yr more (in 1980 dollars) (279). The concept of evaluation within risk management can best be sold not only on the economic costs of retraining and even relocating, but also on psychological grounds imparted by the break with tradition and the loss of prestige and self-image of not being physically able to farm. • Implementing controls requires resources. Among the resources readily accessible to the producer are time, land, water (usually), a wide variety of equipment, and the innovative skills to use them. Money is not on this list. The farmers’ view of time and money have been inexorably bound up in the dichotomy of farming as a way of life versus a business. As one of Wilder’s characters said about new technology in the 1800s, ‘‘All it saves is time, son. And what good is time with nothing to do’’ (1). Agricultural income is limited to those commodities producible by a given land and climate and marketable via the existing infrastructures. By and large, an individual farmer can increase income only by producing more (compatible with a strong work ethic typical of highly agricultural communities), producing better (a weakly marketable option such as ‘‘natural’’ produce), or producing cheaper (an option conducive to operating without ‘‘optional’’ protection features). The widespread use of exposure controls will require external policies and strategies.

35

Control Policies and Strategies The provision of industrial hygiene (and expanded safety) services to agriculture could be initiated via some combination of governmental requirements, private economic incentives, and/or organized producer (‘‘grassroots’’) demands. The lack of U.S. governmental interest is dramatized by the 1986 comparison of federal expenditures in Table 10 and the long-standing fragmentation of responsibility for agricultural health and safety among multiple agencies (4,280). National governmental interest subsequent to a major policy conference and report (85) may remain, but should disinterest return, one must look to private forces to initiate interest in or actually provide preventive occupational health and safety services to the agricultural industry. Generic options and approaches for any agency to implement such services are outlined below. The model most likely to succeed will depend on the local culture and broader political issues. Research Research on any or all of the traditional elements of anticipation, recognition, evaluation, or control can contribute to society’s knowledge, but because of the technical and often interdisciplinary and specialized nature of health research and the equally segmented and organizationally flat nature of agriculture, such knowledge is either not available or not used by policy administrators and is often unusable by individual farmers who constitute agriculture. A large communication gap exists between agricultural, general industrial, environmental, and medical researchers who often publish in diverse literature. Thus collectively a great deal more is known by some than by any one or all. Education Policies Education is the least restrictive but most passive preventive measure. In the best of circumstances, education is the cornerstone of creating an interest to recognize, a desire to evaluate, and a commitment of resources to control health and safety hazards (282). Agricultural health and safety education has been federally supported, albeit weakly for over 50 years. Education’s inability by itself to reduce injury and accidental death in pace with other industries as shown in Figure 1, may be attributed to a lack of resources for good, relevant research, the often dichotomous nature of dissemination between the technical literature and the farming literature, ineffective marketing of risk

Table 10. Distribution of $210 M Total Federal Expenditures for Protective Labor Services in Fiscal Year 1986a

Mining General industry Agriculture a

Ref. 281.

$/Worker

$/Death

$/Disabling Injury

182.0 4.34 0.30

363,400 39,770 606

4540.00 231.00 5.71

36


management to farmers, and the sheer magnitude of the target population (26,283). The problem is not solely the fault of agriculture. Certification Certification of chemicals, equipment, implements, or structures via a voluntary standards process offers the next least restrictive option to prevent unsafe practices or environments. FIEI (the Farm Implement and Equipment Institute) is one organization that has acted to adopt consensus safety and health production standards for agriculture. In certain markets, labor-management agreements have become de facto certification requirements. Many insurers, but not all banks or lenders, actively enforce rudimentary on-farm certification requirements in their dealings with farmers. A variation on voluntary compliance is the delegation of Pesticide Certification Training (required by the U.S. EPA for the purchase of certain commercial pesticides) to many land grant universities. Although the threat of liability litigation can hinder expansion of voluntary certification standards, farm consolidation and incorporation can encourage them. Cost Reduction. Deductions in the cost of doing business are a potential incentive to encourage safer production practices. One model for such incentives is a government tax subsidy (e.g., the 1970s energy conservation tax deduction in the United States and a 1980s workers compensation insurance safety equipment rebate program in Ontario). Another model is insurance discounts for farms meeting certain safety/health criteria (284). Related approaches could include reduced costs by financial lenders who might raise a pro-active farmer’s credit rating or health care providers lowering costs for farm families who participate in preventive health and safety services. The potential benefits of this latter approach was tested in rural Iowa (285). The current policy of the major farm equipment manufacturers to offer retrofit safety options to their products at manufacturer’s cost (without profit) is yet another example. Taxation Taxation can take various forms to create for producers either a financial disincentive to continue to use relatively unsafe practices or a financial incentive to choose to use relatively safe practices. The first form of this option is currently being used via governmental taxes to fund the Occupational Safety and Health Administration (OSHA) and preventive health and safety research (note that farmers too are paying for OSHA but are not receiving its benefits). Voluntary tax (sometimes referred to as a ‘‘mill tax’’) is an alternative program with control and benefits vested in the taxing organization such as a commodity group, the Farm Bureau, a rural coop business, or the integrated system of on-farm and clinical health ¨ services funded in Sweden via Lantbrukshalsan (286). An intermediate example can be found in the funding of the ‘‘Farmsafe’’ educational services through workers compensation fees in Ontario (287).

Regulation Regulation in the United States implies either specification or performance standards, a system of inspectors, and (usually) financial penalties. Governmental control is characteristically political, bureaucratic, and restrictive. Although the passage of occupational health and safety legislation for general industry required broad political support, agriculture is the only sector to have purposefully precluded itself from most OSHA requirements (perhaps to its detriment). The U.S. EPA (as authorized by FIFRA legislation) has completely eliminated exposure to a small number of hazardous agents by cancellation of pesticide registrations, and has implemented specification standards via applicator certification and label use requirements (with only weak enforcement). The temporal and geographic diversity of agriculture (e.g., Table 2) creates a bureaucratic dilemma for any agency attempting to impose controls via a specification standard: the attempt to protect employees in one crop or region requires overly restrictive protection in other settings. The impact of OSHA’s 1988 Field Sanitation standard (29 CFR 1928.110) varies from dramatic to largely redundant, depending upon the setting. Although the OSHA Act states a preference toward performance standards, the level of exposure and compliance during an agricultural operation is difficult to assess or inspect because of its temporal transience. To the degree that agricultural regulations expand, perhaps a combination of specification standards assuring only a minimum level of protection with solid awareness education can be more palatable and therefore effective. Model Programs The development of preventive occupational health and safety services to U.S. agriculture suffers from a lack of both a clear governmental policy at the top and local leadership to express an interest at the bottom. Available services are fragmented, such as the various programs for migrant workers (which primarily stress acute medical services and occupational illness and injury education, but not hygiene); the Cooperative Extension Service (which is largely limited to one person per state to disseminate agricultural health and safety educational materials); and the Farm Bureau (which in part provides its members with services similar to the Extension Service). These activities are limited in scope to awareness-level information dissemination. They help but are clearly inadequate, considering the breadth and size of the industry’s hazards. In the late 1970s, Finland (62,288) and Sweden (286) initiated model programs to deliver comprehensive occupational health services to their farm families. Sweden’s ¨ Lantbrukshalsan clinics provide medical surveillance, medical treatment, preventive physiotherapy, education, and on-the-farm industrial hygiene and safety services. Via these voluntary but subsidized programs, the majority of farmers in these countries now have access to occupational services similar to those in general industry. These countries have been the example for Norway, Denmark, The Netherlands, and other countries who are establishing similar programs (289). France and Germany also have farm programs but they are not nearly so comprehensive;


their programs are primarily through their insurance systems and concentrate on medical issues in France and equipment safety features in Germany. Australia is initiating a new program modeled after the Scandinavian approach. Ontario and Saskatchewan in Canada have well-developed programs based primarily on education but include some on-the-farm hygiene and safety services. Suffice it to say that the small independent programs in the United States are quite behind all of these countries in providing services to farmers. The University of Iowa has had an active research and teaching activity at the Institute of Agricultural Medicine since 1955 (5). In 1987, two state-funded model projects were initiated to deliver comprehensive services through community hospitals with consultation, training of their medical staff, farm educational program development, and referral services provided by university based core staff. One of these programs (the Iowa Agricultural Health and Safety Network) has now been expanded to a total of twentytwo community sites (285). Evaluations are in progress to determine if this community mechanism is a feasible option to provide needed services. A further expansion of this program called the ‘‘Certified Safe Farm’’ is being initiated, whereby an operator will be eligible for insurance incentives if their farm safety inspection rating is sufficiently high to become ‘‘certified’’ (284). The New York Center for Agricultural Medicine and Health at Cooperstown, New York was initiated in 1990, and is attempting to provide services out of their hospital, and network with several other regional hospitals (290). The Marshfield Medical Clinic at Marshfield, Wisconsin, has been active in treating farmers with occupational illnesses and doing research in agricultural lung illnesses (291). They have more recently expanded their activities in farmer education. These institutions (Iowa, New York, and Wisconsin) receive funding from NIOSH as part of their Agricultural Health Center programs, along with Kentucky, Florida, Texas, California, and Washington state). South Carolina has initiated a program that unites the land grant university extension safety specialists with the medical school to deliver health and safety education programs (292). This Agro-medicine Program expands the traditional extension approach, and programs have now expanded to several Atlantic and Midwestern states. A greater emphasis must be placed on model programs offering comprehensive, interdisciplinary services rather than the piecemeal programs of the past. Policy strategies to implement services from research to education via consultation, certification, taxation, or regulation need coordination and strong leadership. Such calls have been made for decades (280). The fact that most new agribusiness and food employees are not agricultural school graduates (293) suggests that such leadership is likely to come from other backgrounds, perhaps even industrial hygiene. CONCLUSION The future of agricultural hygiene will continue to be affected by the economic and technologic forces that

37

have promoted the progressive consolidation of farms into larger, more capital intensive operations (3,18,28,29). New technologies will require more training, and from consolidation will evolve the stratification of agricultural producers into managers and hired employees. These economic and social forces should stimulate a growing interest in product safety, in occupational safety and health, and in more complete management services to both the traditional and the consolidated farm. Evidence for such interest is already seen internationally (289) and in the United States by increased funding at the national level, at the state levels, (e.g., in California, Iowa, Minnesota, and New York), and at local levels centered around rural community hospitals (285). A host of new and diverse professionals are becoming interested in the field, as noted by the recently formed National Coalition for Occupational Safety and Health (85). These forces and disciplines are already blending advantageously with the traditional extension services characteristic of U.S. land grant colleges and universities. It is hoped that this transition to more specialized and comprehensive services will include agricultural hygiene as a growing opportunity. BIBLIOGRAPHY 1. L. I. Wilder, Farmer Boy, Harper and Row, New York, 1933/1971. 2. L. W. Knapp, Arch. Environ. Hlth. 13 (4): 501–506 (1966). 3. S. S. Batie and R. G. Healy, Sci. Am. 248(2): 45–53 (1983). 4. T. A. Knudson, A Harvest of Harm, an award-winning series of six feature articles in the Des Moines Register, Des Moines, IA, Sept. 16–30, 1984. 5. L. Lawhorne, J. Iowa Med. Soc. 66(10): 409–418 (1976). 6. D. J. Murphy, Prof. Safety 26(12): 11–15 (1981). 7. National Safety Council, Accident Facts, NSC, Chicago, IL, (1950–1998). 8. M. A. Purschwitz and W. E. Field, Am. J. Ind. Med. 18(2): 179–192 (1990). 9. T. W. Kelsey, Am. J. Publ. Hlth. 84(7): 1171–1177 (1994). 10. C. F. Mutel and K. J. Donham, Medical Practices in Rural Communities, Springer, New York, 1983, pp. 77–78. 11. K. J. Donham, Am. J. Ind. Med. 18(2): 107–119 (1990). 12. K. J. Donham and E. Horvath, Agricultural Occupational Medicine, in Occupational Medicine, 2 ed., C. Zenz, 1988, pp. 933–957. 13. C. J. Chisholm, D. J. Bottoms, M. J. Dwyer, J. A. Lines, and R. T. Whyte, Safety Sci., 15(4–6): 225–248 (1992). 14. J. B. Sullivan, Jr., M. Gonzales, G. R. Krieger, and C. F. Runge, Health-Related Hazards of Agriculture in J. B. Sullivan, Jr., and G. R. Krieger, eds., Hazardous Materials Toxicology, Clinical Principles of Environmental Health, Williams and Wilkins, Baltimore, Maryland, 1992, pp. 642–666. 15. J. E. Zejda, H. H. McDuffie, and J. A. Dosman, Western J. Med. 158(1): 56–63 (1993). 16. D. Pratt and J. May, Agricultural Occupational Medicine in C. Zenz, O. B. Dickerson, and E. P. Horvath, Jr., eds., Occupational Medicine, 3 ed., Mosby-Year Book, Inc., St. Louis, MO, 1994, pp. 883–902.

38


17. J. Merchant, S. Reynolds, and C. Zwerling, Work in Agriculture in C. McDonald, ed., Epidemiology of Work Related Diseases, BMJ Publishing Group, London, 1995, pp. 267–292. 18. D. E. Albrecht and S. H. Murdock, The Sociology of U.S. Agriculture, Iowa State University Press, Ames, IA, 1990. 19. H. M. Deer, C. E. McJilton, and P. K. Harein, Am. Ind. Hyg. Assoc. J. 48(6): 586–593 (1987). 20. B. Leistikow, W. Pettit, K. Donham, J. Merchant, and W. Popendorf, Respiratory Risks in Poultry Farmers in J. A. Dosman and D. W. Cockcroft, eds., Principles of Health and Safety in Agriculture, CRC Press Inc., Boca Raton, FL, 1989, pp. 62–65. 21. C. S. Rhodes, Health Concerns in Large Animal Veterinarians in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Lewis Publishers, Boca Raton, FL, 1995, pp. 339–342.

40. S. P. Baker, B. O’Neill, and R. S. Karpf, The Injury Fact Book, Heath and Co., Lexington, MA, 1984. 41. S. Richardson and S. May-Lambert, J.Agromed. 4(3/4): 257–267 (1997). 42. D. Richardson, D. Loomis, S. H. Wolf, and E. Gregory, Am. J. Ind. Med. 31(4): 452–458 (1997). 43. S. G. Pratt, S. M. Kisner, and J. C. Helmkamp, J. Occup. Envir. Med. 38(1): 70–76 (1996). 44. C. J. Lehtola, K. J. Donham, and S. J. Marley, Tractor Risk Abatement and Control: A Community-Based Intervention for Reducing Agricultural Tractor-Related Fatalities and Injuries. in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Lewis Publishers, Boca Raton, FL, 1995, pp. 385–389. 45. J. R. Myers and K. A. Snyder, J. Agric. Safety Hlth. 1(3): 185–197 (1995).

22. L. W. Knapp, J. Occup. Med. 7(11): 545–553 (1965).

46. A. Thelin, Am. J. Ind. Med. 18(4): 523–526 (1990).

23. K. J. Donham and C. F. Mutel, J. Family Pract. 14: 511–520 (1982).

47. S. R. Browning, H. Truszczynska, D. Reed, and R. H. McKnight, Am. J. Ind. Med. 33(4): 341–353 (1998).

24. M. B. Schenker, J. Publ. Hlth. Policy 17(3): 275–305 (1996).

48. S. G. Gerberich, R. W. Gibson, P. D. Gunderson, L. R. French, F. Martin, J. A. True, J. Shutske, C. M. Renier, and W. P. Carr, Regional Rural Injury Study (RRIS): A Population Based Effort. in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Supplement, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 1994, pp. 195–200.

25. L. J. Fuortes, J. A. Merchant, S. Van Lier, L. F. Burmeister, and J. Muldoon, Am. J. Ind. Med. 18(2): 211–222 (1990). 26. K. Thu, K. J. Donham, D. Yoder, and L. Ogilvie, Am. J. Ind. Med. 18(4): 427–431 (1990). 27. J. L. Ellis and P. R. Gordon, Occup. Med.: State Art Rev. 6(3): 493–502 (1991). 28. L. Tweeten, Science 219: 1037–1041 (1983). 29. P. L. Martin and A. L. Olmstead, Science 227: 601–606 (1985). 30. B. Kneen, Farmageddon: Food and the Culture of Biotechnology, New Society Publ., Stony, CT, 1999. 31. P. H. Poma, Iowa Med. J. 126: 451–458 (1979). 32. I. West, Arch. Environ. Hlth. 9: 92–98 (1964). 33. D. Villarejo, Health Issues for Farm Employees in the United States, in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Lewis Publishers, Boca Raton, FL, 1995, pp. 479–483.

49. P. Demers and L. Rosenstock, Am. J. Pub. Hlth. 81(12): 1656–1658 (1991). 50. A. F. Hoskin and T. A. Miller, J. Safety Res. 11(1): 2–13 (1979). 51. J. A. Burkart, C. F. Egleston, and R. J. Voss, The Rural Health Study: A Comparison of Hospital Experience Between Farmers and Non Farmers in a Rural Area of Minnesota, DHEW Publ. No. (NIOSH) 78-184, 1978. 52. L. D. Haber, J. Chronic Dis. 24: 482–483 (1971). 53. J. Kennedy and T. J. Fishback, Occupational Characteristics of Disabled Workers: Social Security Disability Benefits Awards to Workers during 1969–1972, U.S. DHHS Publ. No. (NIOSH) 80-145, U.S. Government Printing Office, Washington, DC, 1980.

34. L. K. Isaacs and T. L. Bean, An Overview of the Ohio Migrant Farmworker Safety Needs Assessment, J. Agric. Safety Hlth. 1(4): 261–272 (1995).

54. R. M. Brackbill, L. L. Cameron, and V. Behrens, Am. J. Epid. 139(11): 1055–1066 (1994).

35. P. R. Pomrehn, R. B. Wallace, and L. F. Burmeister, J. Am. Med. Assoc. 248: 1073–1076 (1982).

55. J. Ekholm, R. Nisell, U. P. Arborelius, O. Svensson, and G. Nemeth, Ergonomics, 28(4): 665–682 (1985).

36. A. D. Stark, H.-G. Chang, E. F. Fitzgerald, K. Riccardi, and R. R. Stone, Arch. Environ. Hlth. 42(4): 204–212 (1987).

56. C. L. Anderson, P. S. Treuhaft, W. E. Pierce, and E. P. Horvath, Degenerative Knee Disease among Dairy Farmers, in J. A. Dosman and D. W. Cockcroft, eds., Principles of Health and Safety in Agriculture, CRC Press, Boca Raton, FL, 1989, pp. 367–379.

37. A. Blair, S. H. Zahm, N. E. Pearce, E. F. Heineman, and J. F. Fraumeni, Jr., Scand. J. Work, Envir. Hlth. 18(4): 209–215 (1992). 38. L. Stallones, Fatal Injuries among Adult Males and Children on Colorado Farms, 1980–1988, in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Supplement, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 1994, pp. 207–212. 39. F. P. Rivara, Pediatrics, 76(4): 567–573 (1985).

57. P. Louyot and R. Savin, Rev. Rhum. Mal. Osteoartic. 33: 625–632 (1966). 58. A. Thelin, Scand. J. Soc. Med. 8(Suppl. 22): 5–25 (1980). 59. P. Croft, D. Coggon, M. Cruddas, and C. Cooper, Br. Med. J. 304(6837): 1269–1272 (1992). 60. G. Barbieri, S. Mattioli, S. Grillo, A. M. Geminiani, G. Mancini, and G. B. Raffi, Spinal Diseases in an Italian Tractor Drivers Group in H. H. McDuffie, J. A. Dosman,

AGRICULTURAL HYGIENE K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Lewis Publishers, Boca Raton, FL, 1995, pp. 319–323. 61. C. Hulshof and B. V. vanZanten, Int. Arch. Occup. Environ. Hlth. 59: 205–220 (1987). 62. K. Husman, V. Notkola, R. Virolainen, K. Tupi, J. Nuutinen, J. Penttinen, and J. Heikkonen, Scand. J. Work. Env. Hlth. 14(Supp. 1): 118–120 (1988). 63. H. C. Boshuizen, P. M. Bongers, and C. T. J. Hulshof, Intr. Arch. Occup. Envir. Hlth. 62(2): 109–115 (1990). 64. M. Bovenzi and A. Betta, Appl. Ergonomics 25(4): 231–241 (1994). 65. G. B. Raffi, V. Lodi, G. Malenchini, M. Missere, M. Naldi, S. Tabanelli, F. Violante, G. Minak, Jr., V. D’Elia, and M. Montesi, Arch. Ind. Hyg. Toxicol. 47(1): 19–23 (1996).

39

86. W. E. Burnett, Environ. Sci. Technol. 3(8): 744–749 (1969). 87. J. A. Merkel, T. E. Hazen, and J. R. Miner, Trans. ASAE 12: 310–315 (1969). 88. W. C. Banwart and J. M. Brenner, J. Environ. Qual. 4(3): 363–366 (1975). 89. K. J. Donham, M. Rubino, T. D. Thedell, and J. Kammermeyer, J. Occup. Med. 19(6): 383–387 (1977). 90. K. J. Donham and W. Popendorf, Am. Ind. Hyg. Assoc. J. 46: 658–661 (1985). 91. D. L. Morese and M. A. Woodbury, J. Am. Med. Assoc. 245(1): 63–64 (1981). 92. K. J. Donham, L. W. Knapp, R. Monson, and K. Gustafson, J. Occup. Med. 24(2): 142–145 (1982). 93. S. R. Hagley and D. L. South, Med.J. Australia 2: 459–460 (1983).

66. J. Matthews, J. Soc. Occup. Med. 33(3): 126–136 (1983).

94. Anonymous, MMWR 38(33): 583–586 (1989).

67. N. NevalaPuranen, J. Occup. Rehab. 6(3): 191–200 (1996).

95. J. Zhu, D. S. Bundy, X. W. Li, and N. Rashid, J. Environ. Sci. Hlth. Part A: A32(3): 605–619 (1997).

68. N. NevalaPuranen, (1995).

Appl.

Ergonomics

26(6):

411–415

69. W. Popendorf, K. J. Donham, D. N. Easton, and J. Silk, Am. Ind. Hyg. Assoc. J. 46(3): 154–161 (1985).

96. E. R. Hayhurst and E. Scott, J. Am. Med. Assoc. 63: 1570–1572 (1914). 97. R. R. Grayson, Ann. Int. Med. 45: 393–408 (1956).

70. W. Popendorf and S. J. Reynolds, Industrial Hygiene Evaluations in Agriculture, in R. Langley et al., eds., Health and Safety in Agriculture, Forestry, and Fisheries, Government Institutes Inc., Rockville MD, 1997, pp. 439–468.

98. T. Lowry and L. M. Schuman, J. Am. Med. Assoc. 162: 153–160 (1956).

71. D. K. Olson and S. M. Bark, AAOHN J. 44(4): 198–204 (1996).

100. E. D. Horvath, G. A. do Pico, and R. A. Barbee et al., J. Occup. Med. 20: 103–110 (1978).

72. X. W. Li, J. Environ. Sci. Hlth. Part A A32: (9–10), 2449–2469 (1997).

101. F. L. Zwemer, Jr., D. S. Pratt, and J. J. May, Am. Rev. Resp. Dis. 146(3): 650–653 (1992).

73. S. G. Von Essen and K. J. Donham, Respiratory Diseases Related to Work in Agriculture. in R. Langley, W. Meggs, R. McLymore, and G. Roberson, eds., Health and Safety in Agriculture, Forestry, and Fisheries. Government Institution, Inc., Rockville, MD, 1997, pp. 353–384.

102. M. Moses, Pesticides, in W. H. Rom, ed., Environmental and Occupational Medicine, Little, Brown, Boston, MA, 1983, pp. 547–571.

99. W. H. Peterson, R. H. Burris, S. Rameshchandra, and H. N. Little, Agric. Food Chem. 6: 121–126 (1958).

103. M. Moses, AAOHN J. 37(3): 115–130 (1989).

75. A. B. Spencer and M. G. Gressel, Am. Ind. Hyg. Assoc. J. 54(11): 671–677 (1993).

104. J. A. Legaspi and C. Zenz, ‘‘Occupational Health Aspects of Pesticides. Clinical and Hygienic Principles’’ in C. Zenz, O. B. Dickerson, and E. P. Horvath, Jr., eds., Occupational Medicine, 3 ed., Mosby-Year Book, Inc., St. Louis, MO, 1994, pp. 617–653.

76. S. Helmers, F. H. Top, and L. W. Knapp, J. Iowa Med. Soc. 61(5): 271–280 (1971).

105. C. S. Shaver and T. Tong, Occup. Med. State Art Rev. 6(3): 391–413 (1991).

77. I. Kass, N. Zamel, C. A. Dobry, and M. Holzer, Chest, 62: 282–285 (1972).

106. W. Popendorf and M. Selim, Am. Ind. Hyg. Assoc. J. 56(11): 1111–1120 (1995).

78. J. A. Davies, Ed., Ground Water Protection SW 8-86, U.S. Environmental Protection Agency, 1980, pp. 1–19.

107. W. Popendorf, A. Pryor, and H. R. Wenk, Ann. Am. Conf. Gov. Ind. Hyg. 2: 101–115 (1982).

79. W. E. Donahoe, Pediatrics 3: 308–311 (1949).

108. W. Popendorf and H. R. Wenk, Chrysotile Asbestos in a Vehicular Recreation Area: A Case Study, in H. G. Wilshire and R. H. Webb, eds., Environmental Effects of Off-Road Vehicles—Impacts and Management in Arid Regions, Springer, New York, 1983, pp. 375–396.

74. M. B. Schenker, D. Christiani, Y. Cormier, et al. Am. J. Resp. Critical Care Med. 158(5): S1–S76 (1998).

80. D. H. K. Lee, Environ. Res. 3(5–6): 484–511 1970. 81. J. White, Jr., J. Agric. Food Chem. 23: 886–891 (1975). 82. B. Gabel, R. Kozicki, U. Lahl, A. Podbielski, B. Stachel, and S. Struss, Chemosphere 11(11): 1147–1154 (1982). 83. R. Zaldivar, Experientia 33: 264–265 (1977).

109. R. H. Casterton, Ann. Am. Conf. Gov. Ind. Hyg. 2: 121–127 (1982).

84. R. C. Shank, Toxicol. Appl. Pharmacology 31: 361–368 (1975).

110. M. J. Nieuwenhuijsen and M. B. Schenker, Am. Ind. Hyg. Assoc. J. 59(1): 9–13 (1998).

85. J. A. Merchant, B. C. Kross, K. J. Donham, and D. S. Pratt, Agriculture at Risk: A Report to the Nation, National Coalition for Agricultural Safety and Health, Iowa City, IA, 1989.

111. C. M. Stopford and W. Stopford, Appl. Occup. Envir. Hyg. 10(3): 196–199 (1995). 112. R. P. Sherwin, M. L. Barman, and J. L. Abrahams, Lab. Invest. 40(5): 576–582 (1979).

40


113. W. M. Johnson, J. Occup. Med. 23(5): 367–374 (1981). 114. K. J. Donham, Int.J. Zoonoses 12: 163–191 (1985). 115. R. Rylander, Y. Peterson, K. J. Donham, eds., Am.J. Ind. Med. 10: 193–340 (1986). 116. R. Rylander and Y. Peterson, eds., Am.J. Ind. Med. 17(1): 1–148 (1990). 117. Lacey and J. Dutkiewicz, J. Aerosol Sci. 25(8): 1371–1404 (1994). 118. K. J. Donham, Health Hazards of Pork Producers in Livestock Confinement Buildings: From Recognition to Control in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Lewis Publishers, Boca Raton, FL, 1995, pp. 43–48. 119. M. F. Carvalheiro, Y. Peterson, E. Rubenowitz, and R. Rylander, Am.J. Ind. Med. 27(1): 65–74 (1995). 120. E. N. Schachter, ‘‘Byssinosis and Other Textile DustRelated Lung Diseases,’’ in L. Rosenstock and M. R. Cullen, eds., Textbook of Clinical Occupational and Environmental Medicine, Saunders Company, Philadelphia, 1994, pp. 209–224. 121. R. M. Niven and C. A. C. Pickering, Thorax 51(6): 632–637 (1996). 122. L. A. Lindesmith, J. N. Fink, and E. P. Horvath, Jr., ‘‘Hypersensitivity Pneumonitis’’ in C. Zenz, ed., Occupational Medicine: Principles and Practical Applications, 2 ed., Year Book Medical Publishers, Inc., Chicago, IL, 1988, pp. 226–234. 123. V. P. Kurup, Immunology and Allergy Clinics of North America 9(2): 285–306 (1989). 124. K. J. Donham, L. J. Scallon, W. Popendorf, M. W. Treuhaft, and R. C. Roberts, Am. Ind. Hyg. Assoc. J. 47(7): 404–410 (1986).

138. M. Arden-Jones, Ann. Am. Conf. Gov. Ind. Hyg. 2: 172–182 (1982). 139. J. J. Marx, J. Guernsey, D. A. Emanuel, J. A. Merchant, D. P. Morgan, and M. Kryda, Am. J. Ind. Med. 18(3): 263–268 (1990). 140. I. I. Lutsky, G. L. Baum, H. Teichtahl et al. Eur. J. Respir. Dis. 69: 29–35 (1986). 141. A. D. Blainey, M. D. Topping, S. Ollier, and R. J. Davies, Thorax 43: 697–702 (1988). 142. M. Van Hage-Hamsten, E. Ihre, O. Zetterstrom, and S. G. Johansson, Allergy 43: 545–551 (1988). 143. K. J. Donham, Am. J. Ind. Med. 10: 205–220 (1986). 144. S. A. Olenchock, J. J. May, D. S. Pratt, L. A. Piacitelli, and J. E. Parker, Am. J. Ind. Med. 18(3): 279–284 (1990). 145. J. Milanowski, Inhalation Toxicol. 9(4): 369–388 (1997). 146. R. D. Watson, Am. J. Ind. Med. 10(3): 229–243 (1986). 147. D. S. Pratt, L. Stallones, D. Darrow, and J. J. May, Am. J. Ind. Med. 10: 328–329 (1986). 148. J. J. R. Feddes and E. M. Barber, Agricultural Engineering Solutions to Problems of Air Contaminants in Farm Silos and Animal Buildings, in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety. Workplace, Environment, Sustainability, CRC Press Inc., Boca Raton, FL, 1995, pp. 527–533. 149. X. W. Li, J. Environ. Sci. Hlth. Part A A32(9–10): 2449–2469 (1997). 150. W. Popendorf, Nat. Hog Farmer 35(5): 38–48 (1990). 151. W. F. Durham and H. R. Wolfe, Bull. World Health Org. 26: 75–91 (1962). 152. W. Popendorf and J. T.Leffingwell, Residues Rev. 82: 125–201 (1982).

125. J. R. Mulhausen, C. E. McJilton, P. T. Redig, and K. A. Janni, Am. Ind. Hyg. Assoc. J. 48(11): 894–899 (1987).

153. M. Hussain, K. Yoshida, M. Atiemo, and D. Johnston, Arch. Environ. Contam. Toxicol. 19(2): 197–204 (1990).

126. W. Popendorf, J. A. Merchant, S. Leonard, L. F. Burmeister, and S. A. Olenchock, J. Appl. Occup. Envir. Hyg. 10(7): 595–605 (1995).

154. G. Chester, in P. B. Curry, S. Iyengar, P. A. Maloney, and M. Maroni, eds., Methods of Pesticide Exposure Assessment, Plenum Press, New York, 1995, pp. 29–49.

127. K. J. Donham, J. A. Merchant, D. Lassise, W. J. Popendorf, and L. F. Burmeister, Am.J. Ind. Med. 18(3): 241–261 (1990).

155. R. R. Keenan and S. B. Cole, Am. Ind. Hyg. Assoc. J. 43(7): 473–476 (1982).

128. K. J. Donham, Sem. Resp. Med. 14(1): 49–59 (1993). 129. K. J. Donham, S. J. Reynolds, P. Whitten, J. A. Merchant, L. Burmeister, and W. J. Popendorf, Am.J. Ind. Med., 27(3): 405–418 (1995). 130. K. J. Donham, D. Cumro, S. J. Reynolds, P. Whitten, J. A. Merchant, and L. F. Burmeister, J. Occup. Environ. Med., In press, 1999. 131. W. Popendorf, Am.J. Ind. Med. 10(3): 251–259 (1986). 132. S. J. Reynolds and D. K. Milton, Appl. Occup. Envir. Hyg. 8(9): 761–767 (1993). 133. S. Von Essen, R. A. Robbins, A. B. Thompson, and S. I. Rennard, J. Toxicol. Clinical Toxicol. 2(4): 389–420 (1990). 134. P. Malmberg and A. Rask-Andersen, Sem. Resp. Med. 14(1): 38–48 (1993). 135. A. Rask-Anderson, Br. J. Ind. Med. 46: 233–238 (1989). 136. J. M. Campbell, Br. Med. J. 2: 1143–1144 (1932). 137. E. O. Terho, Am. J. Ind. Med. 10: 329 (1986).

156. J. E. Davies, V. H. Freed, H. F. Enos, R. C. Duncan, A. Barquet, C. Morgade, L. J. Peters, and J. X. Danauskas, J. Occup. Med. 24(6): 464–468 (1982). 157. W. J. Hayes, Monitoring Food and People for Pesticide Content, in Scientific Aspects of Pest Control, National Research Council, National Academy of Sciences, Washington, D.C., Publ. No. 1402, 1966, pp. 314–342. 158. R. A. Fenske and S. G. Bimbaum, Am. Ind. Hyg. Assoc. J. 58(9): 636–645 (1997). 159. M. J. Coye, J. A. Lowe, and K. T. Maddy, J. Occup. Med. 28(8): 619–627 (1986). 160. W. A. Anwar, Environ. Hlth Perspectives, 105(Supp. 4): 801–806 (1997). 161. W. F. Durham, H. R. Wolfe, and J. W. Elliott, Arch. Environ. Hlth 24: 381–387 (1972). 162. M. J. Coye, J. A. Lowe, and K. J. Maddy, J. Occup. Med. 28(8): 628–636 (1986). 163. H. N. Nigg and J. H. Stamper, ‘‘Biological Monitoring for Pesticide Dose Determination. Historical Perspectives,

AGRICULTURAL HYGIENE Current Practices, and New Approaches,’’ in R. G. M. Wang, C. A. Franklin, R. C. Honeycutt and J. C. Reinert, eds., Biological Monitoring for Pesticide Exposure: Measurement, Estimation, and Risk Reduction, ACS Symposium Series 382, American Chemical Society, Washington, DC, 1989, pp. 6–27. 164. W. J. Murray and C. A. Franklin, Monitoring for Exposure to Anticholinesterase-Inhibiting Organophosphorus and Carbamate Compounds by Urine Analysis, in B. Ballantyne and T. C. Marrs, eds., Clinical and Experimental Toxicology of Organophosphates and Carbamates, ButterworthHeinemann, Ltd., Oxford, England, 1992, pp. 430–445. 165. D. P. Morgan, L. I. Lin, and H. H. Saikaly, Arch. Environ. Contam. Toxicol. 9(3): 349–382 (1980). 166. J. L. De Bleecker, J. L. De Reuck, and J. L. Willems, Clinical Neurology and Neurosurgery, 94(2): 93–103 (1992). 167. V. F. Garry, J. T. Kelly, J. M. Sprafka, S. Edwards, and J. Griffith, Arch. Environ. Hlth. 49(5): 337–343 (1994). 168. D. J. Ecobichon, ‘‘Pesticide-Induced Chronic Toxicity: Fact or Myth?’’ in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Lewis Publishers, Boca Raton, FL, 1995, pp. 119–126. 169. M. C. Keifer and R. K. Mahurin, Occup. Med. State Art Rev. 12(2): 291–304 (1997). 170. H. H. Wang and B. MacMahon, J. Occup. Med. 21(11: 741–744 (1979).

41

M. Maroni, eds., Methods of Pesticide Exposure Assessment, Plenum Press, New York, 1995, pp. 9–19. 184. A. W. Taylor, J. Air Pollut. Control Assoc. 28: 922–927 (1978). 185. W. M. Draper, R. D. Gibson, and J. C. Street, Bull. Environ. Contam. Toxicol. 26: 537–543 (1981). 186. D. Zaebst, P. Morelli-Schroth, and L. Blade, ‘‘Summary of Recent Environmental Assessments of Exposure to Grain Fumigants at Export, Inland, and Country Elevators,’’ in J. A. Dosman and D. W. Cockcroft, eds. Principles of Health and Safety in Agriculture, CRC Press, Inc., Boca Raton, FL, 1989, pp. 240–243. 187. J. H. Stamper, H. N. Nigg, W. D. Mahon, A. P. Nielsen, and M. D. Royer, Chemosphere 17(5): 1007–1023 (1988). 188. J. Liesivuori, S. Liukkonen, and P. Pirhonen, Scand. J. Work, Environ. Hlth 14(Suppl. 1): 35–36 (1988). 189. J. F. Armstrong, H. R. Wolfe, S. W. Comer, and D. C. Staiff, Bull. Environ. Contam. Toxicol. 10(6): 321–327 (1973). 190. E. P. Savage, ‘‘Acute Pesticide Poisonings,’’ in Pesticide Residue Hazards to Farm Workers, Proceedings of a Workshop Held February 1976, HEW Publ. No. (NIOSH) 76-191, 1976, pp. 63–65. 191. A. H. Hall and B. H. Rumack, ‘‘Incidence, Presentation and Therapeutic Attitudes to Anticholinesterase Poisoning in the USA,’’ in B. Ballantyne and T. C. Marrs, eds., Clinical and Experimental Toxicology of Organophosphates and Carbamates, Butterworth-Heinemann, Ltd., Oxford, England, 1992, pp. 471–481.

171. A. Blair, D. J. Grauman, J. H. Lubin, and J. F. Fraumeni, J. Nat. Cancer Inst. 71(1): 31–37 (1983).

192. W. Popendorf, Am. J. Ind. Med. 18(3): 313–319 (1990).

172. B. MacMahon, R. R. Monson, H. H. Wang, and T. Zheng, J. Occup. Med. 30(5): 429–432 (1988).

193. K. T. Maddy and S. Edmiston, Vet. Human Toxicol. 30(3): 246–254 (1988).

173. A. Blair and S. H. Zahm, Occup. Med. State Art Rev. 6(3): 335–354 (1991).

194. L. B. L. De-Alwis and M. S. L. Salgado, Foren. Sci. Int. 36(1/2): 81–89 (1988).

174. S. H. Sandifer, R. T. Wilkins, C. B. Loadholt, L. G. Lane, and J. C. Eldridge, Bull. Envir. Contam. Toxicol. 23: 703–710 (1979).

195. S. Milham, Occupational Mortality in Washington State, 1950–1971, Vol. I–III: U.S. DHEW Publ, Nos. (NIOSH) 76-175 A,B,C, U.S. Government Printing Office, 1976.

175. R. I. Glass, R. N. Lyness, D. C. Mengle, K. E. Powell, and E. Kahn, Am. J.Epidemiol. 109(3): 346–351 (1979).

196. E. F. Taschenberg, J. B. Bourke, D. F. Minnick, Bull. Environ. Contam. Toxicol. 13(3): 263–268 (1975).

176. D. Whorton, R. M. Krauss, S. Marshall, and T. H. Mibly, Lancet 7: 1259–1261 (1977). 177. G. E. Carman, Y. Iwata, J. L. Pappas, J. R. O’Neal, and F. A. Gunther, Arch. Environ. Contam. Toxicol. 11(6): 651–659 (1982).

197. C. Lunchick, A. P. Nielsen, and J. C. Reinert, ‘‘Engineering Controls and Protective Clothing in the Reduction of Pesticide Exposure to Tractor Drivers,’’ in S. Z. Mansdorf, R. Sager, and A. P. Nielsen, eds., Performance of Protective Clothing: Second Symposium, American Society for Testing and Materials, Philadelphia, 1988, pp. 605–610.

178. H. N. Nigg and J. H. Stamper, Arch. Environ. Contam. Toxicol. 12: 477–482 (1983).

198. J. F. Stone and H. M. Stahr, J. Environ. Hlth 51(5): 273–276 (1989).

179. J. M. Devine, G. B. Kinoshita, R. P. Peterson, and G. L. Picard, Arch. Environ. Contam. Toxicol. 15: 113–119 (1986).

199. H. E. Braun, R. Frank, and G. M. Ritcey, Bull. Envir. Contam. Toxic. 44(1): 92–99 (1990).

180. W. Popendorf, ‘‘Mechanisms of Clothing Exposure and Dermal Dosing during Spray Application,’’ in S. Z. Mansdorf, R. Sager, and A. P. Nielsen, eds., Performance of Protective Clothing: Second Symposium, American Society for Testing and Materials, Philadelphia, 1988, pp. 611–624.

200. J. P. McBriarty and N. W. Henry, eds. Performance of Protective Clothing: Fourth Volume, American Society for Testing and Materials, Philadelphia, Pennsylvania, ASTM STP 1133, 1992.

181. T. M. Leighton and A. P. Nielsen, Appl. Occup. Environ. Hyg. 10(4): 270–273 (1995). 182. H. N. Nigg, J. H. Stamper, and R. M. Queen, Am. Ind. Hyg. Assoc. J. 45(3): 182–186 (1984). 183. J. J. van Hemmen, Y. G. C. van Golstein Brouwers, and D. H. Brouwer, ‘‘Pesticide Exposure and Re-Entry in Agriculture,’’ in P. B. Curry, S. Iyengar, P. A. Maloney, and

201. Y. Yang and S. Li, Arch. Envir. Contam. Toxicol. 25(2): 279–284 (1993). 202. J. E. Midtling, P. G. Barnett, M. J. Coye, A. R. Velasco, P. Romero, C. L. Clements, M. A. O’Malley, M. W. Tobin, T. G. Rose, and I. H. Monosson, West. J. Med. 142(4): 514–518 (1985). 203. D. P. Morgan, Recognition and Management of Pesticide Poisonings, 4 ed., Health Effects Division, U.S. Environmental

42

AGRICULTURAL HYGIENE Protection Agency, U.S. Government Printing Office, Washington, DC, 1989.

204. R. C. Spear, W. Popendorf, W. F. Spencer, and T. H. Milby, J. Occup. Med. 19(6): 411–414 (1977). 205. T. H. Milby, F. Ottoboni, H. W. Mitchell, J. Am. Med. Assoc. 189(5): 351–356 (1964). 206. W. J. Popendorf and J. T. Leffingwell, J. Agric. Food. Chem. 26(2): 437–441 (1978). 207. W. Popendorf, Rev. Environ. Contam. Toxicol. 128: 71–117, 1992. 208. P. Lisi, Clinics in Dermatology, 10(2): 175–184 (1992). 209. K. Abrams, D. J. Hogan, and H. I. Maibach, Occup. Med. State of the Art Reviews 6(3): 463–492 (1991). 210. P. F. J. Vogelzang, W. J. van der Gulden, L. Preller, M. J. M. Tielen, C. P. van Schayck, and H. Folgering, Intr. Arch. Occup. Envir. Hlth. 70(5): 327–333 (1997).

229. J. C. TeLintum and J. P. Nater, Dermatologica 148: 42–44 (1974). 230. D. Burrows, Br. J. Dermatol. 92: 167–170 (1975). 231. R. D. Peachey, Br. J. Dermatol. 105(Suppl. 21): 45–50 (1981). 232. J. C. Whitakar, W. R. Lee, and J. E. Downes, Br. J. Ind. Med. 36: 43–51 (1979). 233. D. T. Harvey and D. J. Hogan, ‘‘Common Environmental Dermatoses,’’ in S. M. Brooks, M. Gochfeld, J. Herzstein, R. J. Jackson, and M. B. Schenker, eds., Environmental Medicine, Mosby-Year Book, Inc., St. Louis, MO, 1995, pp. 263–281. 234. U. W. Leavell and J. A. Phillips, Arch. Dermatol. 111: 1307–1311 (1975). 235. L. Chmel, J. Buchvald, and M. Valentova, Int. J. Epidemiol. 5(3): 291–295 (1976).

211. R. J. Geller, Vet. Human Toxicol. 32(5): 479–480 (1990).

236. W. L. Krinsky, Int. J. Dermatol. 22(2): 75–91 (1983).

212. J. R. Wilkins, III, and M. E. Bowman, Occup. Med. 47(8): 451–457 (1997).

214. I. Z. Trujillo, A. N. Zavala, J. G. Caceres, and C. Q. Miranda, Infectious Disease Clinics of North America 8(1): 225–241 (1994).

237. M. R. Reesal, L. Hagel, P. Pahwa, D. Domoney, H. McDuffie, and J. A. Dosman, ‘‘Hearing Loss in a Saskatchewan Farm Community’’ in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Supplement, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 1994, pp. 201–206.

215. A. H. Keeney and M. C. Hunter, Arch. Opthalmol. 44: 573–580 (1950).

238. M. Feldman and C. D. E. Downing, Can. Agric. Eng. 14(1): 2–5 (1972).

216. U. W. Leavell, Jr., M. S. McNamara, R. Muelling et al., J. Am. Med. Assoc. 204: 109–116 (1968).

239. H. H. Jones and J. L. Oser, Am. Ind. Hyg. Assoc. J. 29(2): 146–151 (1968).

217. T. Grandin, Vet. Clin. North Am. Food Anim. Pract. 3: 324–336 (1987).

240. D. Tharr, Appl. Occup. Envir. Hyg. 9(8): 525–528 (1994).

213. W. W. Spink, Brucella Abortus 47: 861–873 (1957).

218. R. W. Lyons, C. L. Samples, H. N. DeSilva et al., J. Am. Med. Assoc. 243: 546–547 (1980). 219. P. N. Acha and B. Szyfres, Zoonoses and Communicable Diseases Common to Man and Animals, Scientific Publication No. 354, Pan American Health Organization, Washington DC, 1980. 220. D. Snashall, Br. Med. J. 313(7056): 551–554 (1996).

241. N. W. Sullivan, R. D. Schneider, and K. Von-Bargen, Prof. Safety 26(12): 16–21 (1981). 242. J. W. Dennis and J. J. May, ‘‘Occupational Noise Exposure in Dairy Farming’’ in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety: Workplace, Environment, Sustainability, Lewis Publishers, Boca Raton, FL, 1995, pp. 363–367.

221. K. J. Donham, ‘‘Infectious Diseases Common to Animals and Man of Occupational Significance to Agricultural Workers,’’ in Proceedings of Conference on Agricultural Health and Safety, New York Society for Occupational and Environmental Health, Environmental Sciences Laboratory, New York, 1975, pp. 160–175.

243. J. J. Holt, S. K. Broste, and D. A. Hansen, Laryngoscope 103(3): 258–262 (1993).

222. K. Hedberg, K. White, J. Forfang et al., Am. J. Epidemiol. 130: 569–577 (1989).

246. H. Une, S. H. Schuman, S. T. Caldwell, and N. H. Whitlock, South. Med. J. 80(9): 1137–1140 (1987).

223. P. R. Schnurrenberger and W. T. Hubbert, An Outline of Zoonoses, Iowa State University Press, Ames, IA, 1981.

247. C. W. Suggs, L. F. Stikeleather, and C. F. Abrams, Am Soc. Agric. Eng. 13(5): 608–611 (1970).

224. D. Hogan and P. Lane, Occup. Med. State Art Rev. 1: 285–300 (1986).

248. M. Takamatsu, T. Sakurai, and C. P. Chang, ‘‘Vibration Disease among Farmers Induced by Vibration Tools,’’ Proceedings of the VII International Congress of Rural Medicine, Salt Lake City, Utah, Sept. 17–21, 1978, Int. Assoc. Agric. Med., 188–190 (1978).

225. A. Cellini and A. Offidani, Dermatology 189(2): 129–132 (1994). 226. M. A. O’Malley, Occup. Med. State of the Art Reviews 12(2): 327–345 (1997). 227. C. L. Wand, The Problem of Skin Diseases in Industry, Office of Occupational Safety and Health Statistics, U.S.Department of Labor, U.S. Government Printing Office, Washington, DC, 1978. 228. W. B. Whiting, J. Occup. Med. 17(3): 177–181 (1975).

244. C. E. McJilton and R. A. Aherin, Am. Ind. Hyg. Assoc. J. 43(6): 469–471 (1982). 245. R. C. Jensen, Prof. Safety 28(9): 19–24 (1983).

249. W. E. Field and R. L. Tormoehlen, Appl. Ergonomics 16(3): 179–182 (1985). 250. P. B. Allen, W. E. Field, and M. J. Frick, J. Agric. Safety Hlth. 1(2): 71–81 (1995). 251. L. F. Burmeister, G. D. Everett, S. Van Lier, and P. Isacson, Am. J. Epidemiol. 118: 72–77 (1983).

AGROCHEMICAL

43

252. A. Blair, H. Malker, K. P. Cantor, L. Burmeister, and K. Wiklund, Scand. J. Work Environ. Hlth. 11: 397–407 (1985).

272. P. S. Zeitz, J. M. Graber, R. A. Vorrhees, G. Kioski, L. A. Shands, T. G. Ksiazek, S. Jenison, and R. F. Khabbaz, J. Occup. Envir. Med. 39(5): 463–467 (1997).

253. M. Schenker and S. McCurdy, ‘‘Pesticides, Viruses, and Sunlight in the Etiology of Cancer Among Agricultural Workers,’’ in C. E. Becker and M. J. Coye, eds., Cancer Prevention: Strategies in the Workplace, Hemisphere Publishing, Washington, DC, 1986, pp. 29–37.

273. P. E. M. Smith, M. Zeidler, J. W. Ironside, P. Estibeiro, and T. H. Moss, Lancet 346(8979): 898 (1995).

254. K. P. Cantor, A. Blair, G. Everett, R. Gibson, L. F. Burmeister, L. M. Brown, L. Schuman, and F. R. Dick, Cancer Res. 52(9): 2447–2455 (1992).

274. N. Delasnerie-Laupretre, S. Poser, M. Pocchiari, D. P. W. M. Wientjens, and R. Will, Lancet 346(8979): 898 (1995). 275. R. B. Hayes, J. P. Van Nieuwenhuize, J. W. Raatgever, and F. J. W. Ten Kate, Food Chem. Toxicol. 22(1): 39–43 (1984).

255. D. D. Weisenburger, Ann. Oncology 5(Supp. 1): S19–S24 (1994).

276. W. G. Sorensen, J. P. Simpson, M. J. Peach, T. D. Thedell, and S. A. Olenchock, J. Toxicol Environ. Hlth 7: 669–672 (1981).

256. J. E. Keller-Byrne, S. A. Khuder, and E. A. Schaub, Envir. Res. 71(1): 1–10, 1995.

277. O. Shotwell and W. Burg, Ann. Am. Conf. Gov. Ind. Hyg. 2: 69–86 (1982).

257. L. W. Figgs, M. Dosemeci, and A. Blair, Am. J. Ind. Med. 27(6): 817–835 (1995).

278. M. I. Selim, A. M. Juchems, and W. Popendorf, Am. Ind. Hyg. Assoc. J. 59(4): 252–256 (1998).

258. B. Lee, B. Marlenga, and D. Miech, ‘‘Farmers’ Caps and Hats Project’’ in H. H. McDuffie, J. A. Dosman, K. M. Semchuk, S. A. Olenchock, and A. Senthilselvan, eds., Agricultural Health and Safety. Workplace, Environment, Sustainability, CRC Press Inc., Boca Raton, FL, 1995, pp. 535–539.

279. W. K. Viscusi, Risk By Choice: Regulating Health and Safety in the Workplace, Harvard University Press, MA, 1983. 280. C. M. Berry, Am. J. Public Hlth, 55(3): 424–428 (1965).

260. E. Elam and R. Rauncy, Rural Health Crisis (A Project Report). Northwest Services, Inc., Mound City, MO, 1986.

281. M. A. Purschwitz and W. E. Field, ‘‘Federal Funding for Farm Safety Relative to Other Safety Programs,’’ in Proc. of the 1987 National Institute for Farm Safety Summer Meeting, National Institute for Farm Safety (NIFS), Columbia, MO, 1987.

261. R. R. Swisher, G. H. Elder, F. O. Lorenz, and R. D. Conger, J. Hlth. Soc. Behavior 39(1): 72–89 (1998).

282. L. J. Chapman, R. T. Schuler, T. L. Wilkinson, and C. A. Skjolaas, Am. J. Ind. Med. 28(4): 565–577 (1995).

262. D. J. Donham, ‘‘The Long Arm of the Farm: How an Occupation Structures Exposure and Vulnerability to Stressors Across Role Domains,’’ Sustainable Development in Intensive Livestock Production: Exposure Assessment and Health Outcomes, abs. 4th International Symposium: Rural Health and Safety in a Changing World, held at The Center for Agricultural Medicine, University of Saskatchewan on October 18–22, 1998 in Saskatoon, Canada.

283. D. J. Murphy, N. E. Kiernan, and L. J. Chapman, Am. J. Ind. Med. 29(4): 392–396 (1996).

259. K. J. Donham, L. Burmeister, S. vanLier, and T. Greiner, Am. J. Vet. Res. 48: 235–238 (1987).

263. W. E. Field, Effects of Stress on the Performance of Agricultural Equipment Operators, SAE Tech. Paper Series, 800932, Warrendale, PA, 1980. 264. L. M. Haverstock, ‘‘Farm Stress: Research Considerations,’’ in J. A. Dosman and D. W. Cockcroft, eds., Principles of Health and Safety in Agriculture, CRC Press, Boca Raton, FL, 1989, pp. 381–384. 265. C. N. Larson, S. Kuperman, and R. E. Smith, ‘‘Rural Psychiatry: A Definition of the Field,’’ in J. A. Dosman and D. W. Cockcroft, eds., Principles of Health and Safety in Agriculture, CRC Press, Boca Raton, FL, 1989, pp. 385–388.

284. S. VonEssen, K. Thu, K. J. Donham, ‘‘Insurance Incentives for Safe Farms’’ in K. Donham, R. Rautiainen, S. Scheuman, and J. Lay, eds. Agricultural Health and Safety: Recent Advances, Hawthorne Press, Binghamton, NY, 1997, pp. 125–127. 285. J. Gay, K. Donham, S. Leonard, Am. J. Ind. Med 18(4): 385–389 (1990). 286. S. Höglund, Am. J. Ind. Med. 18(4): 371–378, 1990. 287. Ontario Farm Safety Association, Am. J. Ind. Med. 18(4): 409–411 (1990). 288. K. Husman, V. Notkola, R. Virolainen, J. Nuutinen, K. Tupi, J. Penttinen, and J. Heikkonen, Am. J. Ind. Med. 18(4): 379–384 (1990). 289. S. Höglund, Am. J. Ind. Med. 18(4): 365–370 (1990). 290. D. S. Pratt, Am. J. Ind. Med. 18(4): 391–393 (1990).

266. W. G. Hollister, ‘‘Innovations in Mental Health Service Delivery in Rural Areas,’’ in J. A. Dosman and D. W. Cockcroft, eds., Principles of Health and Safety in Agriculture, CRC Press, Boca Raton, FL, 1989, pp. 399–401.

291. D. A. Emanuel, D. L. Draves, and G. R. Nycz, Am. J. Ind. Med. 18(2): 149–162 (1990).

267. R. Reotutar, J. Am. Vet. Med. Assoc. 194: 1387–1391 (1989).

293. C. Tevis, Successful Farming 87(12): 17 (1989).

268. B. S. Schwartz and M. D. Goldstein, J. Occup. Med. 31: 735–742 (1989).

294. A. Thelin, Ann. Agric. Envir. Med. 2(1): 21–26 (1995).

269. P. Morgan-Capner, S. J. Cutler, D. J. M. Wright, N. Hamlet, D. Nathwani, D. O. Ho-Yen, and E. Walker, Lancet 1(8641): 789–790 (1989). 270. A. G. Baird, J. C. M. Gillies, F. J. Bone, B. A. S. Dale, and N. T. Miscampbell, Br. Med. J. 299(6703): 836–837 (1989). 271. T. F. Tsai, Lab. Animal Sci. 37(4): 428–430 (1987).

292. S. H. Schuman, Am. J. Ind. Med. 18(4): 405–408 (1990).

AGROCHEMICAL Chemical used in crop and food production including pesticide, feed additive, veterinary drug, and related compounds (IUPAC).

44

AGROCIN 84

AGROCIN 84 ALLEN KERR MAX E. TATE University of Adelaide Adelaide, Australia

STRUCTURE AND FUNCTION Agrocin 84 (Fig. 1, either a or b) is an antibiotic produced by the nonpathogenic strain K84 of Rhizobium rhizogenes (formerly Agrobacterium radiobacter biovar 2), its derivatives, and other closely related bacteria. It inhibits the growth of many pathogenic agrobacteria, and strong evidence indicates that it is the major, but not the only, factor in the commercially successful biological control of crown gall caused by pathogenic strains of Rhizobium radiobacter and R. rhizogenes. It has marked specificity and may be considered as the first nonprotein bacteriocin. Agrocin 84 is an N6 , 5 -disubstituted adenine nucleotide with a fraudulent nucleoside sugar. The lower (or alpha) face of the β-D-3 -deoxyarabino-furanosyl group is structurally equivalent to a dideoxynucleotide, and its toxicity depends on the termination of DNA synthesis in susceptible strains of Agrobacterium (1). In the control of crown gall, it would also prevent DNA synthesis required for transfer of T-DNA from bacterial cell to plant cell, which is the basis of crown gall induction. Specificity of agrocin 84 is an uptake phenomenon, susceptible strains taking it up through a high affinity transport system lacking in resistant strains (2). Uptake depends on the N6 ,D-gluco-furanosyl-oxy-phosphoramidate substituent (3). Susceptibility to agrocin 84 is largely confined to pathogenic agrobacteria carrying a nopalinetype Ti-plasmid. Because it seemed biologically illogical for a bacterium to possess a ‘‘suicide’’ gene for the uptake of a toxin, an alternative substrate was sought (4).

HO HO O

O

R3 =

OR2

OH

P OR1

−O

M+

H N 6

N

N O

O

P HO

O 5′

N H OH

N

N

O− M+

O

This proved to be a new kind of opine, which was named agrocinopine A. The agrocinopine A catabolic (acc) region in the Ti-plasmid of strain C58 (a nopaline pathogenic strain) has been located at kb 130 on the Ti-plasmid map. It is almost 8 kb in size and consists of eight open reading frames (ORF), as defined by DNA sequence analysis (5). Five of these ORFs are required for agrocinopine A and agrocin 84 uptake, indicating that a common transport mechanism is involved. Initially, there seems to be very little structural similarity between the two molecules. However, if the 1-O-linkage (Fig. 1a) is replaced by a 2-O-linkage (Fig. 1b), the first five glucose carbons are then exactly analogous to the L-arabinose phosphodiester of agrocinopine A or the six carbons of the glucose phospho-diester of agrocinopine C. This 2O-glucosyl phosphoryl structure (Fig. 1b) for agrocin 84 has not yet been excluded and consequently is the preferred structure. Agrobacteria with an agropine Tiplasmid are normally resistant to agrocin 84, but they do possess an agrocinopine C-specific permease. However, the genes encoding the permease are normally completely repressed, and only after induction by agrocinopine C do these strains become sensitive to agrocin 84 (4). A similar situation applies to strains harboring some Riplasmids, chrysopine-type Ti-plasmids, and mannopine At-plasmids. Although the adenine N6 -substituent is required for uptake of agrocin 84, it is not required for toxicity; this is the role of the 5 -substituent (3). When this substituent is removed, the molecule completely lacks toxicity, although readily taken up into an agrocin 84–susceptible cell (3). A likely explanation is that toxicity depends on the presence of a 5 -phosphoryl group, which could then be converted to triphosphate, a necessary preliminary for incorporation into a DNA chain. GENETICS OF AGROCIN 84 PRODUCTION AND IMMUNITY Synthesis of agrocin 84 in strain K84 is encoded by a 48-kb plasmid called pAgK84. The agrocin 84 synthetic and immunity regions occupy a continuous 20-kb segment of this plasmid, comprising six closely linked loci (6). The immunity regions are also required for biosynthesis, details of which are not known. However, chemical synthesis of the toxic component lacking the N6 -substituent has been achieved (7). There are two distinct immunity regions with no detectable DNA homology, one at each end of the agrocin 84 synthesis region (6). Both regions, when separately transferred to a susceptible cell, render it immune to agrocin 84. Although the mechanisms of immunity have not been established, it has been suggested that one region could be responsible for the rapid export of agrocin 84 from the cell and the other for the addition of the 5 -pentanamide (8).

HO 3′

Figure 1. Alternative structures for agrocin 84. Either (a) R1 = H, R2 = R3 or (b) R1 = R3, R2 = H. M+ is a singly charged cation, e.g., sodium.

CONSTRUCTION OF A TRANSFER-DEFICIENT MUTANT pAgK84 is a conjugative plasmid and when transferred to other strains of Rhizobium gives the recipient strain

ANALYSIS—INSTRUMENTATION AND TECHNIQUES

the ability to synthesize agrocin 84. If nonpathogenic, the recipient can act as a biocontrol agent for crown gall; if pathogenic, it poses a major problem for the continued success of biological control of crown gall. It retains its pathogenicity but now produces agrocin 84 and is immune to it. Such strains are not subject to biological control. This prompted the construction by recombinant DNA technology of a transfer-deficient (Tra− ) deletion mutant of pAgK84 (9). The new strain K1026 contains the mutant plasmid but is otherwise indistinguishable from strain K84; it is now widely used in Australia and America for crown gall control. It was the first genetically engineered organism in the world to be released for commercial use.

45

ALLOMONES Chemicals emitted by one species that modify the behavior of a different species to the benefit of the emitting species. (EPA No. 540/09-89-056, March 1989; USEPA; Washington DC; 1989.)

ANALYSIS—INSTRUMENTATION AND TECHNIQUES OFFIAH O. OFFIAH Maryland Department of Agriculture Annapolis, Maryland

BIBLIOGRAPHY 1. P. K. Das, M. Basu, and G. C. Chatterjee, J. Antibiot. 31: 490–492 (1978). 2. P. J. Murphy and W. P. Roberts, J. Gen. Microbiol. 114: 207–213 (1979). 3. P. J. Murphy, M. E. Tate, and A. Kerr, Eur. J. Biochem. 115: 539–543 (1981). 4. J. G. Ellis and P. J. Murphy, Mol. Gen. Genet. 181: 36–43 (1981). 5. H. Kim and S. K. Farrand, J. Bacteriol. 179: 7559–7572 (1997). 6. C. L. Wang, S. K. Farrand, and I. Hwang, Mol. Plant—Microbe Interact. 7: 472–481 (1994). 7. D. Filippov et al., Tetrah. Lett. 39: 4891–4894 (1998). 8. M. H. Ryder, J. E. Slota, A. Scarim, and S. K. Farrand, J. Bacteriol. 169: 4184–4189 (1987). 9. D. A. Jones et al., Mol. Gen. Genet. 212: 207–214 (1988).

ALGICIDES Substances that control algae in lakes, canals, swimming pools, water tanks, and other sites (USEPA).

ALIQUOT The known fraction of a homogeneous material (1).

BIBLIOGRAPHY 1. W. Horwitz et al., Nomenclature for Sampling in Analytical Chemistry. Pure Appl. Chem. 62: 1193–1208 (1990).

ALLELOCHEMICALS Chemicals inducing interactions between species (e.g., scents, pheromones, toxins). Semiochemicals that mediate interspecific reactions. See Semiochemicals.

Plant protection is an integral part of environmental protection. Plants are an important part of the biosphere, the environment that nurtures life. All living things through exposure and response mechanisms are constantly adapting to their environment to maintain their survival edge. Naturally, different relationships have evolved from these interactions between organisms and their environments. One mutual relationship is the natural selection process that led to what we know today as essential elements for plant growth (e.g., Na, Mg, K, Ca, B, C, N, O, P, S, Se, V, Cr, Mn, Fe, Co, Cu, Zn, Mo, etc.), and the same is true for other organisms, including humans. It follows, therefore, that there are nonessential elements, and among these nonessential elements, some are innocuous (e.g., Sc, Ge, etc.) and some are toxic (e.g., Be, Li, Al, Ni, Cd, Hg, Pb, etc.). Plants, animals, and, consequently, humans have to be protected from these toxic substances, because the food chain tells us that if the plants get sick, subsequently humans will get sick. Chemical analysis is an established scientific approach that makes it possible to identify and quantify elements that make up a material. Even though analysis simply denotes the separation of material into its constituent elements but from a chemical analyst standpoint, chemical analysis is a function of technique, instrumentation, and application. The analyte and matrix of interest dictate the technique, the instrumentation, and the application. The analytes of interest in this case are metals (those elements that tend to form positive ions and, therefore, are electropositive) and metalloids (semimetals). Identifying and quantifying these analytes is efficiently done by a spectroscopic technique. Spectroscopy is the study of methods of producing and analyzing spectra using spectroscopes, spectrometers, spectrographs, and spectrophotometers. Chemical analysis involves the breakdown of molecular structures and the examination of atomic energy levels. Chemical analysis utilizing a spectrometer is a relative technique. It measures light intensity at various wavelengths and not the direct amount of element present. This makes instrumental calibration a necessity. So the act of calibration converts the instrument from a light measuring device to a chemical analysis instrument (1). The focus of this discussion is on atomic spectroscopy as it is applicable to agrochemicals. Three modes of detection

46


will be discussed, namely, absorption spectrum, emission spectrum, and mass spectrum. Most nutrients enter into a plant in a transpiration stream via mass flow and diffusion, and some of these substances can mimic one another; therefore, one way to try to protect plants is to minimize the availability of the toxic substances to plants. Fertilization is a means of making nutrients available to crops and plants, and therefore, it is very important that fertilizers are not laden with toxic substances. Monitoring and minimization of toxic substances can be done through fertilizer analysis. Also, feed analysis can stop contaminated vegetation and food products from becoming a part of the food chain. States such as Maryland and Washington, through their regulatory process, routinely monitor the levels of these toxic substances in feeds and fertilizers. CONCEPTS AND THEORY The fundamental principles of atomic spectroscopy stem from the works of Bunsen and Kirchoff, who postulated that 1) under sufficient excitation, an element will give its own characteristic spectrum, and 2) the elemental vapor presence can be identified based on the presence of its characteristic lines. In addition to these is a Kirchoff postulate that states that emission of radiation at a particular wavelength relates to absorption of radiation at that wavelength. These postulates combined give rise to the fundamental relationship between emission and absorption spectra (2). The explanation of these phenomena was accomplished through the understanding of the atomic structure. The model of an atom according to Bohr is a nucleus consisting of protons and neutrons and orbited by electrons at specified energy levels or discrete orbitals. Every atom comprises available orbitals with characteristic energy levels that electrons can occupy. The preferred stable state is when an electron is closest to the nucleus. The interaction of matter with electromagnetic radiation can lead to perturbation, which in turn can give rise to fascinating phenomena that come from absorption of electromagnetic radiation or thermal energy by the atoms that make up that matter. As a result, the atoms can become excited, leading to an electron moving from ground state orbital to a higher orbital. The removal of the energy source will lead to the electron returning to its ground state orbital with a loss of energy in the form of photons. This is called emission. The character of the changes in the atom will determine the quality and quantity of light emitted. These emission and absorption phenomena are the basis for spectrophotometric analysis. If the source of energy is maintained, leading to continuous absorption of energy, the electron may eventually leave the atom, and this is ionization, and the energy is termed ionization potential. The difference in energy between the two energy levels is the wavelength of the radiation. The electromagnetic energy is described by Planck’s equation.

between wavelength and energy is given by substituting c/λ for v, where c is the speed of light and λ is the wavelength. hc (2) E= λ As the above equation shows, energy is inversely related to wavelength. The characteristic set of energy levels that defines every element serves as a fingerprint for that particular element, and this is reflected by a unique set of absorption and emission wavelengths. This property therefore lends itself to element-specific analytical techniques. The region of electromagnetic spectrum that is important to this technique is from ultraviolet to visible (160–800 nm). Figure 1 is an energy level diagram depicting energy transitions, in which a) represents excitation, b) is ionization, c) is ionization/excitation, d) is atom emission, and e) is ion emission. The property described above can be measured by a spectroscopic technique, thereby obtaining qualitative and quantitative information about a sample. In order to effect this measurement, the instrument (spectrometer) has to be calibrated. A relationship between light intensity and element concentration is established in the form of an equation or a plot. For an atomic emission spectrometer, it is the relationship between emission intensity and analyte concentration. For mass a spectrometer, it is the relationship between mass to charge ratio (intensity) and analyte concentration. For atomic absorption, it is the relationship between absorbance and analyte concentration. Absorbance is the quotient between the log of intensity of incident light and the intensity of transmitted light. log Io (3) A= I where Io is the initial intensity and I is the intensity value after passing through a path length. The Beer–Lambert law gives an empirical relationship between absorbance and concentration. A = abC, (4)

Ion excited state

hn e Ion ground state

Atom excited state

hn

E = hv

(1)

E is the energy between the two levels, h is Planck’s constant, v is frequency of radiation, and the relationship

a

b

c

d

Atom ground state

Figure 1. Drawing of energy levels and transitions of an atom.


where a is the absorptivity constant that varies with wavelength and from substance to substance, b is path length, and C is concentration, and this equation applies to single species.

INSTRUMENTATION A spectrometer today is more than just a spectroscope; it is an integrated system of coupled devices that makes it possible to take a sample and generate data that reflect the elemental makeup of that sample. The discussion will mirror the flow chart in Figure 2, flowing from top to bottom. These are the a) sample introduction section, b) atomization/ionization section, c) signal focusing and discrimination section, d) detection section, and e) data translation section.

SAMPLE INTRODUCTION A sample transport system consists of a pump, a nebulizer, and a spray chamber. There are different types of pumps, but peristaltic pumps are the pumps of choice. They are equipped with tubes and rollers and operate by mimicking the peristaltic motion. The solution does not come in contact with the pump; only the tubing that carries the sample makes contact with the sample (3). The pump facilitates the aspiration of the solution to the nebulizer, even though nebulizers, by virtue of their mechanism of operation, do aspirate solutions. A pump gives the analyst more control of the flow rate of the sample solution, which can be used to offset some of the effects of surface tension, specific gravity, and viscosity that undermine the nebulization of some samples. The

Plasma emission

Atomic absorption

Sample introduction

Sample introduction

Ionization

Lamp

Mass spec. OES

Atomization

Ion optics

Optics

Mass analyzer

Optics

Optics

Detector

Detector

Detector

Data translation Figure 2. Instrumental flow chart.

47

greater demand in analytical work has led to the pump being interfaced with an automatic sampler for liquid analysis. An auto-sampler gives the analyst the freedom of unattended analysis. A nebulizer is the device that generates aerosol from the liquid or solution by combining it with a fast flowing gas, at a specified flow rate, to make a mist/gas mixture that is sprayed into a spray chamber that houses the nebulizer. There are different types of nebulizers, which include pneumatic, ultrasonic, and cross-flow nebulizers. They have their merits and demerits in terms of cost, efficiency, and durability. A spray chamber serves as an aerosol/liquid separator and discriminator in terms of droplet size. The fine homogenous mist generated is directed up to the flame or plasma, whereas the heavy droplets are drained away by gravity to the waste container. Some spray chambers are equipped with impact beads or flow spoilers that increase the efficiency of the separation. The above devices are employed in both atomic absorption and inductively coupled plasma, except when options are for solid sampling, vapor generation, and electrothermal analysis (furnace). Solid sampling is accomplished through glow discharge, spark discharge, and laser ablation. Aerosol is liberated from the solid sample surface with a laser, and the aerosol is fed to the plasma. Cold vapor sample introduction consists of a peristaltic pump and some specified reagents. The pump serves as a reaction device for mixing the sample with acid and a reductant (NaBH4 ). The chemical solution generated is fed to a liquid/gas separator that liberates the gaseous portion into an analytical cell where the absorption takes place. Electrothermal analysis sample transport involves aspiration of the sample by a pneumatic sampler and injecting it into a graphite tube that is situated in the furnace for atomization. ATOMIZATION/IONIZATION Atomization generates individual atoms through a thermal or electrothermal process that includes evaporation and vaporization. The atomization temperature ranges are air/acetylene flame (2100–2400 ◦ C), nitrous/acetylene flame (2600–2800 ◦ C), and furnace (1800–3000 ◦ C). During the atomization process, a light beam is directed through the flame, graphite tube (furnace), or analytical cell (hot vapor), then through a monochromator, and onto a detector where the amount of light absorbed by atoms is measured. The lamp emits a characteristic radiation (the spectrum of analyte of interest), and the analytical lines are the resonance lines that result from those energy transitions that involve the ground state. The amount of absorbed energy at the characteristic wavelength is proportional to the concentration of the element in the sample over a limited concentration range. The common light source is the hollow cathode lamp. An electrodeless discharge lamp is primarily used for volatile elements (3). Most atomic absorption spectrometers can operate in emission mode as well.

48


In the furnace, the sample is pipetted into a graphite tube that is housed in the furnace, and by the use of a computer program (furnace control program), the sample is dried, charred (ashed), and then atomized. When the cycle gets to atomization, the absorption is measured. However, in cold vapor (Hg) and hot vapor (hydride generation for As, Sb, Bi, Se, and Te) techniques, vapor is generated by chemical reaction and fed to the analytical tube, where the absorption is measured (Hg) or atomized and measured (hydride generation). An atomization technique that utilizes the absorption concept has sensitivity (based on 1% absorption) from low ppm to high ppb (flame to furnace). It is not capable of qualitative analysis and is poor in multielement analysis. It can tolerate a high dissolved solids solution and viscous samples and is capable of analyzing all metals. The ionization process is accomplished in a plasma and goes beyond atomization. It is made possible by the very high temperature range (6000–10,000 ◦ C) achieved by the plasma. The plasma vaporizes the aerosol, atomizes the constituent elements, and ionizes the atoms. In this process, a ground state atom is stripped of an electron, resulting in a formation of a positive ion. The atoms, during the process of ionization, emit light of characteristic wavelength with an intensity directly proportional to the concentration. An inductively coupled plasma source consists of a stream of argon gas ionized by an applied radio frequency field that oscillates at about 27.1 MHz. This field is inductively coupled to the ionized gas by a water-cooled coil surrounding a quartz torch that supports and confines plasma (4). Other plasma types are DC arc and microwave-induced plasma (2). There are two ways that the torch can be oriented; the most common is radial, but axial placement seems to increase intensity of radiation (longer pathlength) and, therefore, increase sensitivity and precision for some elements. It may also lead to higher background noise (2). An ionization technique that reflects the concept of emission involves complicated spectra and high background noise. It has moderate sensitivity (low ppm to high ppb) and can tolerate up to 2% dissolved solids. Analysis of all metals and metalloids is possible. Multielement and qualitative analysis are routine.

SIGNAL FOCUSING AND DISCRIMINATION In an optical emission spectrometer, the optics consists of a camera shutter, mirrors, a prism, and a grating. These devices aligned in a fashion dictated by optics makes it possible to collect, disperse, differentiate, and focus the emission radiation from the plasma onto the detector. There are simultaneous polychromator and sequential monochromator spectrometers for sequential and simultaneous monitoring of wavelengths, and wavelengths less than 190 nm require a vacuum optics because oxygen absorbs radiation below 190 nm (3). The emission from the plasma is a combination of all wavelengths of individual atoms and ions in the plasma gas and in the sample. This emission spectrum is therefore separated into individual

component wavelengths using a diffraction grating (6). A recent improvement in optical emission is the introduction of echelle monochromators. It is the use of two dispersive devices, e.g., a prism and a grating, placed one after the other, so that the light dispersed by the prism in one plane is subsequently dispersed by the grating at right angle to the previous plane. The resultant wavelengths are measured by a grid of photomultipliers (2). The ion extraction, filtration, and focusing are accomplished by the ion optics and the quadrupole in the mass spectrometer. The ion optics consists of cones and metal plates. The geometric configuration and spatial arrangement of these devices make it possible to eliminate photons and stray light. The cone orifices range from 0.8 to 1.2 mm, and the plates have electrical potential placed on them, which makes it possible to regulate their voltages. Together, they extract singly charged positive ions from the plasma and provide a well-collimated beam of ions into the mass analyzer while maintaining their kinetic energies. The above configuration serves as an initial barrier to isolate the mass spectrometer from the surrounding atmosphere (1 torr). The second vacuum partition is the quadrupole that operates at 10−4 torr. The vacuumed environment prevents interaction of charged ions with their neighbors. Like an optical emission monochromator, a mass analyzer separates components into their representative spectra, but instead of wavelength, it is in mass/charge ratio. This mass calibration technique provides excellent sensitivity (ppb to ppt) and is capable of isotope ratio determination, but it needs routine calibration and internal standard for drift correction, and it cannot tolerate high dissolved solids (>2%). Analysis of all elements is possible. Multielement and qualitative analysis are routine.

DETECTORS The detector provides an electrical signal that is proportional to the amount of ions or intensity of the emission at a particular wavelength. The most common detectors in the laboratories today for spectrometers are photomultiplier tubes (AA, ICP), Charged Injection Device, Charged Coupled Device (ICP), and Channeltron Electron Multiplier Tube/Active Film Multiplier (MS). Photomultipliers are made of photosensitive material that ejects an electron when struck by a photon. The signal is multiplied by a series of dynodes. A single electron hits one dynode, several electrons are ejected, and the process is repeated several times, resulting in an increase of signal (3). A charged injection device (CID) gives the analyst the ability to view the entire emission spectrum simultaneously. This is made possible by an echelle optical design grating that focuses a high resolution, two-dimensional image onto its surface. It is a single solid-state chip composed of over 250,000 light-gathering detector elements, each of which is only 23 µm in size. A charged coupled device (CCD) is also a combined echelle monochromator and a single chip that is capable of viewing the entire spectrum and differs from CID by the way signal is collected and analyzed. These solid-state


detectors, like photomultiplier tubes, convert incoming light into an electrical signal, but they operate at a much lower voltage level and provide a continuous picture of the spectrum rather than the discrete, single wavelength signal (1). In the third vacuum partition in the mass spectrometer, the channeltron electron multiplier is further isolated from the environment by reducing the pressure to 10−6 torr during operation. In this vacuumed environment, positive ions from the quadrupole are channeled onto the negatively charged detector where they strike a PbO2 coated tube and release secondary electrons. Change in potential carries electrons inward, and they quickly multiply in number to as much as a 108 increase. POEMS, a Plasma Optical Emission Mass Spectrometer, is a combined OES and MS technique that makes it possible to screen samples for concentration range, perform a multitechnique confirmation on elements of interest, and assure an extended working range (% to ppt). DATA TRANSLATION The electrical signal from the detector is converted by an analog-to-digital (A/D) converter to make it usable by the computer. The computer then translates it into a common language datum. APPLICATION Data quality objectives are scientifically based systematic planning tools that establish the criteria for developing data collection designs. It is a documented criterion for defensible decision making before data are collected and therefore assures data quality. Analytical protocol (12), which details the analytical criteria, is an integral part of data quality objectives. These criteria include analyte of interest, matrix, technique, method development, analytical limitations, statistical decisions, and performance goals. These criteria sum up to applicability that focuses on the analyte and matrix of interest. A protocol includes a) methodology, b) interferences, c) troubleshooting, d) quality control, e) safety, and f) waste reduction. METHODOLOGY This is a composite of sample preparation procedures and instrumental procedures. Sample preparation gets the sample in the form that is compatible with the introduction system of the instrument. In other words, the sample preparation procedure describes the necessary steps, reagents, and apparatuses to achieve the above objective. The instrumental procedure is a set of computer instructions (software) that enables the instrument to generate validated data from the sample. Instrumental Methods The parameters needed for instrumental method development are usually a part of the software provided by

49

the instrument manufacturer. The process includes optimization, calibration, and analysis of the samples, taking into consideration quality control initiatives. The method applicability may be specific (one element) or general (multielement), or qualitative or quantitative. The instrumental software makes it possible to develop and store many methods, which then become available at the click of a mouse. The software also provides a database of elemental properties. In the case of a mass spectrometer, the information for the determination of elemental isotope ratio may be used to differentiate a naturally occurring mineral from a manmade one. Sample Preparation Some techniques pride themselves as being noninvasive and nondestructive, but chemical analytical atomic spectroscopy is both invasive and destructive for the simple fact that it quantitates the building blocks of matter. Samples are collected in the form of gas, liquid, or solid that reflects their natural existence (states of matter). Based on the instrumental technique, especially the sample introduction system, the sample aliquot may have to be pretreated to get it in a form that is suitable for the application. Most instruments in use today require liquid samples. Instrumental techniques for direct analysis of gaseous and solid samples are available, but they are not as popular, even though most samples are collected in those forms. Although pretreatment may be contamination prone, it leads to a more homogenous sample and thus less variability. Three basic pretreatment approaches are 1) acidification, 2) extraction, and 3) decomposition. These approaches are operations on the samples that reflect a conceptual definition of the data quality objectives. Acidification: This approach is used primarily on water samples, and its purpose is to stabilize (Table 1) the sample prior to analysis or storage. When and how it is done depends on whether dissolved metals or total metals are required. For dissolved metals, it is done after filtration. Extraction: Solid sample is weighed and extracted with a prescribed extraction solution. A predetermined quantity of the extracting solution is added to a tarred sample and shaken for a prescribed length of time, filtered, and then analyzed. Decomposition: This procedure includes 1) dry ashing, 2) digestion (wet ashing), and 3) fusion. Dry ashing is done in a regular muffle furnace or microwave muffle furnace. A tarred solid sample is heated at 400–650 ◦ C for 2 to 24 hours. The ash is then dissolved in dilute acid to give an analytical solution. This process involves the volatilization of moisture, evaporation of volatile materials, and oxidation of nonvolatile residues, including organic matter. Therefore, this pretreatment is not recommended for analysis of volatile elements like Sb, As, Se, and Hg (3). Digestion is a mineral acid–assisted decomposition of a solid sample that employs hot plates or digestion blocks or microwave ovens. Temperature regulation is an important part of this process. In the case of microwave digestion, pressure regulation is also utilized. The introduction of the microwave oven has quickened

50

ANALYSIS—INSTRUMENTATION AND TECHNIQUES Table 1. Analytical Properties of Some of the Commonly Analyzed Agrochemical Elements ∗ ICP

Elements Boron Sodium Magnesium Aluminum Phosphorus Sulfur Potassium Calcium Chromium Manganese Iron Cobalt Nickel Copper Zinc Arsenic Selenium Molybdenum Cadmium Mercury Lead ∗

Rec. WL (nm)

∗ AA

∗ Mass#

Rec. WL (nm)

AT.Wt (amu)

249.77, 208.96 588.99, 330.24 279.55, 280.27 308.22, 309.22 214.91, 213.62

249.8 589.0 285.2 396.2 213.6

766.49, 404.72 317.93, 315.89 267.72, 357.87 257.61, 293.31 259.94, 238.20 228.62, 238.89 231.60, 341.48 324.75, 327.40 213.86, 206.20 197.20, 193.70 196.03, 203.98 202.03, 203.84 228.80, 226.50 253.65, 435.84 220.35, 283.31

766.5 422.7 357.9 279.5 248.3 240.7 232.0 324.7 213.9 193.7 196.0 313.3 228.8 253.7 217.0

10.81 22.99 24.31 26.98 30.97 32.07 39.10 40.08 52.0 54.94 55.85 58.93 58.69 63.55 65.39 74.92 78.96 95.94 112.4 200.6 207.2

(amu) & % Abundance

11 & 80.1 23 & 100 24 & 79 27 & 100

39 & 93.3 43/44 & 0.14/2.1 52/53 & 83.8/9.5 55 & 100 56/57 & 91.7/2.2 59 & 100 60 & 26.2 63 & 69.2 66 & 27.9 75 & 100 77/82 & 7.6/8.7 95 & 15.9 111 & 12.8 202 & 29.9 208 & 52.4

Matrix HF/H2 O HNO3 HNO3 HCl HNO3 HNO3 HNO3 HNO3 HCl HNO3 HNO3 HNO3 HNO3 HNO3 HNO3 HNO3 HNO3 HNO3 HNO3 HNO3 HNO3

Courtesy of Thermo Jarrell Ash Corporation.

the process, reduced the incidence of cross-contamination, and made possible closed system digestion that may eliminate loss of volatile elements. Also, it has led to a reduction in the amount of acid used and therefore a reduction in acid waste. Fusion is used for acidresistant compounds, which involves fusing it with a basic compound, e.g., alkali hydroxide or carbonate at very high temperatures (above 300 ◦ C) that make it easier dissolve the compound in dilute acid. It is a very severe process. However there is concern for contamination from the alkali compounds. ANALYTICAL INTERFERENCES This is a change in the analyte signal in a sample compared with the analyte signal for the same concentration of that analyte in a calibration solution. Flame Atomic Absorption Spectrometer The interferences includes a) spectra, b) matrix, c) nonspecific, and d) ionization. Spectra interference is when an absorption line of a matrix component overlaps the resonance line for the analyte within the spectra line width of the emission line from the light source (1,7). The correction for spectra interference is the use of an alternative wavelength, making a matrix match or removing the interfering component or the analyte of interest by extraction. Matrix (Physical and Chemical) Physical interference is caused by changes in surface tension and the viscosity of the sample (2). This affects

the nebulization efficiency of the instrument transport system. The correction includes use of a pump, making a matrix match, and use of the standard addition method. Chemical interference occurs when a matrix component reacts with the analyte to alter the rate and extent of formation of free ground-state analyte atoms. This effect can suppress or enhance absorption. Correction includes matrix matching, use of hotter flame, and use of releasing agent or method of standard addition. Nonspecific interference is a background absorption caused by the presence of molecules or particles in the light path that either absorbs or scatters the energy from a hollow cathode lamp. This leads to a high signal (2,7). Correction includes matrix matching and background correction. Ionization interference is caused by too high a temperature removing outer electrons from the atoms (2,7). These atoms are therefore not available for absorption, thereby reducing the signal measured. Correction includes matrix matching, cooler flame, and use of suppressants. Electrothermal Atomic Absorption The variation in magnitude of the transient signal produced during atomization is reflected in the peak area because the matrix components in the sample affect both the rate and extent of atom formation, which can make a marked difference in relation to the standardization solution. The furnace program is used to control the elimination of matrix and to maintain a balance between matrix removal and analyte loss (6,8). Volatile compound formation occurs when the analyte is lost at a relatively low temperature during the drying or ashing stage before atomization. Stable


compound formation is when one or more relatively stable compounds of the analyte are formed. Some elements form stable carbides that do not readily decompose, even at a high atomization temperature, and thus affect the formation of free atoms. Barium, V, Mo, and Ti can still be analyzed, but W, Ta, and Zr cannot be analyzed. Correction is by the use of matrix modification to alter the vaporization temperature of the analyte. Pyrolytic tubes and platforms are used to minimize vapor phase interference and carbide formation, resulting in better separation of atomic signal from the nonatomic signal (7,8). Background noise in a furnace is caused by 1) scatter effects (varying size particles), 2) molecular absorption, 3) atomic absorption, and 4) Zeeman effect absorption. Scatter is due to light scattering caused by either large particles (Mé) or small particles (Rayleigh) present along the optical path. They are more pronounced at a lower wavelength. Correction is achieved through background correction. Deuterium background correction (continuum) uses the asymmetric mode of subtraction where the time between background and sample measurement is 2 ms. It has very good sensitivity and linear range but produces structured background and spectral interferences. Zeeman background correction has a 4 ms timing and uses the polynomial interpolation method of subtraction. It is accurate at much higher absorption levels but may reduce sensitivity and linear range for some elements. Both will correct background up to two absorbance units (1% error at 2 a.u) (8). Atomic emission interferences are 1) spectra and 2) matrix. Spectra are caused by light emitted from sources other than the element of interest, which contributes to net signal intensity (2). Sources include spectra line overlaps, broadened wings of intense spectra lines, nonatomic recombination continuum emission, molecular band emission, and stray light from emission of elements at high concentrations (9). Correction can be effected by use of an alternative wavelength, choice of background positions, and use of the empirical correction method. Matrix (Physical and Chemical) Physical interference is associated with the transport process(es) due to viscosity and surface tension. High dissolved salt can also build up on the nebulizer and the torch, leading to partial clogging of the orifice. Correction is achieved through sample dilution, matrix match calibration, and the method of standard addition. Chemical interference is caused by molecular compound formation, ionization effects, and thermochemical effects resulting from sample vaporization and atomization in the plasma (3). Correction is effected by use of the method of standard addition and matrix match calibration and use of internal standards and interfering element correction (IEC). When background emission intensity varies among samples and standards due to changing matrices, inspect the emission subarray and choose a background position on a flat region, away from the peak so that it is not affected by the wings.

51

Mass spectrometer interferences are a) isobaric element interference, b) isobaric polyatomic ion interference, c) abundance sensitivity, d) matrix oxide interference, e) physical interference, f) mass dependence, and g) space charge effects. Isobaric element interference is caused by overlapping masses from one element ion to another element ion that cannot be separated by the resolution of the spectrometer (5,9). Correction is by use of isobaric correction. Isobaric correction relies on the natural abundance ratio of the various isotopes and requires subtraction of the interfering element measured with a noninterfered isotope. Isobaric polyatomic ion interference is caused by masses from ions containing more than one atom overlapping with the desired element mass/charge ratio that cannot be separated by the resolution of the spectrometer. Polyatomic atoms form mostly in the plasma and sampling interface from the argon plasma gas and constituents in the sample. Mineral acids (Table 1) that are used in digestion and stabilization of samples and standards lead to polyatomic ion interference (9). Correction for this interference includes using primarily nitric acid, where possible, and keeping the concentration of hydrochloric acid below 0.5%. Also, it includes use of isobaric correction. Abundance sensitivity is the measure of the ability of the spectrometer to measure a small ion signal at a mass immediately adjacent to a mass at a very high signal level, as in the determination of U-234 in the presence of U-235 (9). Correction of this type of broadening is achieved by choosing resolution parameters that minimize mass overlap. Matrix oxide interference is the formation of an oxide of the matrix element that can interfere with some isotopes of the element of interest. This is only a problem when elements like Ti, Zr, and Mo, which are known to form refractory oxides, are present in high concentration (10). Correction is achieved by tuning the plasma, for example, the nebulizer gas, to reduce the oxide formation to a tolerable level. Also, a CeO/Ce ratio of less than 2% is a good indication of low oxide formation. Physical interference is any process that changes the transport of sample to the plasma, formation of ions in the plasma, and transmission of the ions through the spectrometer (9). Sample viscosity is the primary source of this interference. In axially viewed plasma, the physical effects also manifest in the element of interest excitation process. This effect is also known as plasma loading. The ion generation regions of the plasma and the ion trajectory through the quadrupole subsystems are of greater consequence. This is manifested in the effect of a high amount of heavyweight ions on lightweight ions. This effect is referred to as mass discrimination or simply the ‘‘Schmelzel effect.’’ Correction is effected by use of solutions containing less than 0.2% dissolved solids and tuning the spectrometer to favor the transmission of lightweight ions. Also, matrix matching and use of internal standards should compensate for these interferences. It is also very critical to match the behavior of the internal standard with that of the ions that will be

52


measured. Generally, mass-to-charge ratio and ionization characteristics should be similar. Mass dependence is changes in ion transmission is a function of mass/charge ratio. This mass effect is produced by kinetic energy differences caused by ions of different mass-to-charge ratios entering the ion optics. Space charge effects result from interaction between ions when brought into tight focus due to the fact that they carry electrical charges, unlike photons. This also affects transmission of certain masses. Ion optic design helps to minimize the effects of both mass dependence and space charge (11).

QUALITY ASSURANCE Quality assurance is the plan and operations that ensure data quality. Generally, quality assurance comprises quality assessment and quality control, and these are detailed in the data quality objectives. Troubleshooting and clean chemistry have become an integral part of quality assurance in light of instrumental sophistication and picogram metal levels demanded by regulatory policies. Troubleshooting in a present-day analytical laboratory serves as a tool in discovering and eliminating the cause of poor quality data. Minimization of downtime and reduction of repair cost are major reasons for troubleshooting, but the most important reason may be to minimize poor quality data. Poor sensitivity, poor precision, signal noise, and erroneous results are causes for troubleshooting. Clean chemistry has become an important component of trace metal analysis. It mandates a clean laboratory environment, clean conscious laboratory personnel, and is cheaper and more attainable financially and otherwise than a clean room. Although clean chemistry requires type A water, clean glassware, and clean sample preparation procedures, a clean room requires the elimination of all metallic devices and isolation of the room from the general laboratory environment. Clean chemistry, therefore, minimizes contamination and provides a suitable environment for trace analysis. A quality assurance plan should include standard operating procedures for all equipment, standards, methods, as well as maintenance records, reagent labels, chain of custody of samples, sample receipt log books, sample storage, and sample handling and disposal. Quality control is the component of quality assurance that is used to monitor the analytical process in terms of reliability. The following items, depending on technique and application, are employed and monitored during analysis: blank samples, spiked samples, duplicate samples, quality control charts, check samples, calibration standards, standard reference materials, laboratory reference standards, internal standards, optimization or tuning standards, interference standards, instrument detection limits, and method detection limits (12).

SAFETY Safety of the environment and the personnel is very important. This includes functioning hoods, proper apparel and shields, and waste handling minimization. AGROCHEMICAL ANALYSIS Fertilizers and feeds are the two main matrices for agrochemical atomic spectrometry techniques. Some other matrices are water, pesticides, and animal drugs. Some of the studies that involve some of these matrices will be discussed below. A recent study (13) showed that the ICP-AES method enabled simultaneous determinations of phosphorus, potassium, and magnesium in fertilizers. Also, that the precision and accuracy of ICP-AES determinations are comparable with those of standard methods, namely, gravimetry and AAS, even though there was a constant systematic error at low levels of K2 O. A threelaboratory method trial evaluated two sample preparation procedures (dry ash digestion and microwave dissolution) and instrumental analysis parameters (GFAAS and ICP-AES) for calcium and lead in Ca supplements. Both sample preparation methods gave accurate and precise results, but microwaves have the advantage with contamination control and digestion time (14). A study (15) showed that the best wavelength for analysis of boron in fertilizers was 208.959 nm because the most sensitive lines (249.773 nm, 249.678 nm) are interfered by K, P, and Fe. An investigation of analysis of total B, Ca, Cu, Fe, Mg, Mn, P, K, and Zn in fertilizers by AES (16) and that of total Al, Fe, Ca, and Mg (17) demonstrated the feasibility of such analysis becoming routine in agrochemical laboratories. The examination of spectral and interelement effects for iron (18), magnesium (19), and sulfur (20) in fertilizers showed that the following analytical wavelengths (238.204 nm), (383.826 nm), and (180.731 nm), respectively, are the best for these metals. Even though these wavelengths may not be the recommended wavelengths (Table 1), different matrices and other types of interferences may necessitate the use of alternative wavelengths. The form (oxidation state) of some elements (As, Se, Cr, V, etc.) rather than the total amount of the element has become very important in terms of their biological activities. This has made speciation (21–24) a very attractive analytical technique. There is therefore a compelling effort to interface atomic spectrometry with separation techniques (e.g., CE, HPLC, GC, IC, etc.). FUTURE OF ANALYSIS Matrix Calibration Soil matrix controls the movement of ions in the unconsolidated portion of the earth crust. The fate of


boron as well as that of other nutrients in the natural matrix do not conform to adsorption/desorption phenomena (25), but instead reflect retention/release characteristics (26). This may be due to the heterogeneity and lability of the natural matrix. A retention/release characteristic can be represented by a plot or an equation that describes the relationship between the concentration (standard addition or spike) of an element and the response of the matrix (matrix properties includes physical texture, chemical pH, species, ionic strength). The slope of the calibration curve reflects the sensitivity of the matrix (capacity for retention or release of the element) to the element of interest. A spectrum of these curves with their respective slopes can be generated to represent a corresponding spectrum of matrices. The feasibility of data generation and storage is possible due to computer technology. A spectrum of a retention/release characteristic covering all soil types and consequently all matrix types can be used to predict nutrient fate in different matrices and therefore the application rate of fertilizers.

53

7. C. W. Fuller, ed., Electrothermal Atomization for Atomic Absorption Spectrometry, Chem. Soc. Burlington House, London, 1997, pp. 62–64. 8. J. C. Van Loon, ed., Analytical Atomic Absorption Spectroscopy—Selected Methods, Academic Press, London, 1980, pp. 43–49. 9. E. H. Evans and J. J. Giglio, J. Anal. At. Spec. 8: 1–18 (1993). 10. M. A. Vaughn and G. Horlick, Appl. Spectrosc. 40: 434–445. 11. D. J. Douglas and S. D. Tanner, in A. Montaser, ed., Fundamental Considerations in ICP-MS, Wiley-VCH, New York, 1998, pp. 615–679. 12. U. S. Environmental Protection Agency, Methods for the Determination of Metals in Environmental Samples, June 1991, 600/4–91/010. 13. M. Hamalova, J. Hodslavska, and P. Janos, J. AOAC Int. 80: 1151–1155 (1997). 14. P. H. Siitonen and H. C. Thompson, Jr., J. AOAC Int. 81: 1233–1238 (1998). 15. R. Matilainen and J. Tummavuori, J. AOAC Int. 78: 598–785 (1995). 16. J. B. Jones, J. Assoc. Off. Anal. Chem. 65: 781–785 (1982).

Performance-Based Measurement System

17. G. B. Hunter, T. C. Woodis, Jr., and F. J. Johnson, J. AOAC 64: 25–27 (1981).

The myriad matrices, with their unique complexities in terms of interferences and quality control initiatives, make application of a uniform protocol very difficult. The impossibility of developing methodology for every matrix makes the concept of a performance-based method very appealing. As long as a method can demonstrate an acceptable level of method performance that is governed by such criteria as accuracy, precision, sensitivity, linear range, and detection limits, this validity gives credence to the quality of the data generated with the method. A performance-based measurement system (27) makes it possible to use any scientifically valid method or technology to demonstrate compliance.

18. R. Matilainen and J. Tummavuori, J. AOAC Int. 79: 22–28 (1996).

BIBLIOGRAPHY

19. R. Matilainen and J. Tummavuori, J. AOAC Int. 78: 1134– 1140 (1995). 20. R. Matilainen and J. Tummavuori, J. AOAC Int. 79: 102– 1035 (1996). 21. K. Van den Broeck, C. VandeCasteele, and J. M. C. Geuns, Anal. Chimica Acta 361: 101–111 (1998). 22. W. Chia-Ching and S. Jen Jiang, Anal. Chimica Acta 357: 211–218 (1997). ¨ 23. M. Ochsenkuhn-Petropulu and P. Schramel, Anal. Chimica Acta 313: 243–252 (1995). 24. M. Shuster and M. Schwarzer, Anal. Chimica Acta 358: 1–11 (1996). 25. M. A. Elrashidi and G. A. O’Connor, Soil Sci. Soc. Am. J. 46: 27 (1982).

1. V. B. E. Thomsen, ed., Modern Spectrochemical Analysis of Metals, ASM International, Ohio, 1996, p. 55.

26. O. Offiah and J. H. Axley, in U. C. Gupta, ed., Boron and Its Role in Crop Production, CRC Press, Florida, 1993, pp. 105–123.

2. J. W. Robinson, ed., Atomic Spectroscopy, 2nd ed., Marcel Dekker Inc., New York, 1996, p. 8.

27. D. E. Kimbrough and R. Spinner, Am. Environ. Lab. 11: 1–9 (1994).

3. K. M. Anderson, ed., Analytical Techniques for Inorganic Contaminants, AOAC International, AOAC Int., suite 400, 2200 Wilson Blvd, Arlington, Virginia 22201, 1999, p. 47. 4. L. S. Clesceri, A. E. Greenberg, and A. D. Eaton, eds., Standard Methods for the Examination of Water and Waste Water, 20th ed., United Book Press Inc., Baltimore, Md., 1998, pp. 3–38. 5. K. W. Jackson and S. Lu, Anal. Chem. 70: 363R–383R (1998). 6. T. J. Gloudenis, Am. Lab. 30: 24s–27s (1998).

FURTHER READING Anderson, K. M., ed., Analytical Techniques for Inorganic Contaminants, AOAC International, AOAC Int., suite 400, 2200 Wilson Blvd, Arlington, Virginia 22201, 1999. Robinson, J. W., ed., Atomic Spectroscopy, 2nd ed., Marcel Dekker Inc., New York, 1996. Thomsen, V. B. E., ed., Modern Spectrochemical Analysis of Metals, ASM International, Ohio, 1996.

54

ANIMAL HEALTH PRODUCTS

ANIMAL HEALTH PRODUCTS P. T. REEVES National Registration Authority for Agricultural and Veterinary Chemicals Kingston, Australia

A vast range of veterinary chemical products are used in the animal health sector. Those that control external parasites (ectoparasites) on domestic animals are known as ectoparasiticides and comprise two major groups—pesticides and systemic insecticides/acaricides. For the purpose of this article, pesticides are defined as veterinary chemical products that are applied topically and act by surface contact with target pests. Systemic insecticides/acaricides are veterinary chemical products that are administered orally (e.g., as feed additives), topically (e.g., as pour-ons and spray-ons), or by injection and are then absorbed systemically and distributed by the host’s circulatory system before acting on external parasites. Endectocides are veterinary chemical products that control both internal and external parasites and are considered here but only from an ectoparasiticidal perspective. The pharmacology of the numerous groups of veterinary drugs that lack ectoparasiticidal effects is the subject of several excellent texts (1,2) and is generally outside the scope of this article. Ectoparasites of companion animals are a major cause of dermatologic problems in animals and act as vectors for various pathogens. Chemical agents are used to control insects (fleas, lice, and flies) and acarines (mites and ticks) on pets and in their immediate environment. Numerous formulations and delivery systems containing a range of active ingredients with different modes of action have been developed. This situation reflects the considerable challenges that are encountered in the control of ectoparasites. For example, it is desirable for antiflea insecticides to demonstrate a persistent effect and the ability to decrease or eliminate life cycle stages in the environment. However, flea allergic dermatitis, which is by far the most common dermatosis in the cat and dog (3), is unlikely to respond solely to an antiflea insecticide that allows the host to be bitten by fleas, albeit infrequently. Similarly, pathogens transmitted by tick vectors are present in tick salivary glands and are transmitted during engorgement. Therefore, an effective acaricide needs the ability to inhibit the attachment and feeding of ticks in addition to being able to kill ticks. Ectoparasites cause production losses in food-producing animals that incur enormous costs to the livestock industry. The estimated cost of ticks and tick-borne diseases worldwide was $US8 billion per annum in 1984 (4). The cattle tick Boophilus microplus is estimated to have caused productivity losses in excess of $US1 billion in South America in 1987 (5) and more than $A100 million in Australia in 1990 (6). Productivity losses exceeding $US730 million have been attributed to the horn fly in North America (7), while the annual cost of the sheep blowfly Lucilia cuprina in Australia has been estimated at $A100–200 million (8,9).

The manifestations of ectoparasites on food-producing animals include anemia; reduced production of meat, milk, and eggs; reduced quality and quantity of fleece; damage to hides (cattle grubs and cockle of sheep pelts from lice infestations); hypersensitivity reactions (to the itchmite Psorergates ovis of sheep); transmission of animal diseases (pinkeye of cattle, encephalitis of horses, anaplasmosis of cattle); and decreased resistance to other diseases. Presently, chemical treatment applied prophylactically, strategically, or tactically remains the main option for ectoparasite control (10). The objective of prophylactic treatment is to prevent infestations from becoming established. Strategic treatments are applied immediately prior to a predicted seasonal increase in a parasite population, whereas tactical treatments control infestations that have been detected at an early stage or prevent infestations from developing after a period of favorable weather conditions. Quarantine control of stock movement from tick-infested areas to tick-free areas is a major method of avoiding tick infestations. FORMULATIONS AND DELIVERY SYSTEMS Part 1: Cats and Dogs Topical, enteral, and parenteral formulations and delivery devices (collars and medallions) are available for control of external parasites on companion animals. These are supplemented by foggers and house sprays that control developmental stages of fleas in the animal’s immediate environment. The active ingredients in these products include adulticidal agents, insect growth regulators, synergists, and repellents that have been developed for their topical and/or systemic modes of action. All the major ectoparasiticidal chemical groups (botanicals, synthetic pyrethroids, carbamates, organophosphates, and organochlorines), as well as insect growth regulators and several newer compounds (imidacloprid, fipronil, and selamectin) are represented in topical formulations. Insecticidal soaps, foams, and shampoos cleanse the skin and coat and control fleas, lice, and ticks. Insecticidal shampoos should remain on the animal for at least 10 min before rinsing in order to optimize their efficacy. Insecticidal rinses, which may be sponged onto the pet after prior bathing and drying or used as dips, provide longer residual activity than insecticidal shampoos. Insecticidal dusting powders are effective and especially convenient for use on cats that resist attempts at shampooing, rinsing, or dipping, or dislike the noise of sprays. Because dusting powders cake when applied to moist skin, their use on moist excoriated skin should be avoided. Pump-action and aerosol insecticidal sprays are available; however, the latter rely on propellants, and their popularity has declined in recent years. Spot-on formulations are applied to the pet’s dorsal midline from where the active ingredient spreads in the sebum, coating the skin and hair, and forms depots in the pilosebaceous units. The persistence observed with spot-on formulations is attributed to their slow release from the sebaceous glands (11). Ear mite infestations in cats and dogs are controlled by otic drops comprised of insecticidal solutions


or suspensions and by systemically acting agents. Creams and ointments containing insecticides are available for control of localized mange, while emulsifiable concentrates diluted with water or mixed with propylene glycol are commonly used to treat generalized demodectic and sarcoptic mange in dogs. Chemicals that reach the skin via the systemic circulation are also used for treating mange on cats and dogs. Enteral and parenteral formulations exert their effects when fleas ingest blood from treated hosts. Examples of such dosage forms include solutions and tablets of cythioate for oral administration to cats and dogs; nitenpyram tablets for cats and dogs; and tablets, oral suspensions and injectable suspensions of lufenuron to control fleas on cats and dogs. Collars and medallions are delivery devices that consist of a plastic matrix impregnated with insecticide; the latter may be an adulticide or insect growth regulator released in a controlled manner over a period of months to control fleas (some collars and medallions also control ticks) on cats and dogs. Microencapsulated formulations of chlorpyrifos, diazinon, and pyrethrins are available for flea control. The process of microencapsulation involves incorporating the active ingredient in microscopic spheres in order to modify the delivery, stability, and/or safety profiles of the compound. The reduced degradation of microencapsulated pyrethrins in ultraviolet light, for example, results in enhanced residual activity. Because the availability of pyrethrins from microencapsulated formulations is reduced, the immediate knockdown activity on insects and acarines is reduced, and safety for the animal is enhanced. Part 2: Horses Pastes and gels are semisolid dosage forms that are particularly convenient for administrating to horses for control of Gastrophilus spp. larvae (bots). Pastes comprise aqueous, vegetable oil, or mineral oil vehicles in which cosolvents may be incorporated to increase the solubility of the active ingredient (typically an organophosphorus compound or macrocyclic lactone). The properties of cohesiveness, plasticity, and syringeability of pastes are attributed to thickeners in the formulation. It is critical that pastes and gels have a pleasant taste, or are tasteless, and display a tendency to adhere when deposited on the posterior portion of the horse’s tongue using a plastic syringe. This lessens the likelihood of the dose being rejected. Desiccation and syneresis (the separation of liquid from a swollen gel indicates that the formulation is unstable) of pastes and gels are commonly addressed by adding humectants and adsorbing agents, respectively, to the formulations. Part 3: Ruminants Numerous ectoparasiticidal dosage forms are available for ruminants. These include oral endectocidal drenches, pour-ons, plunge and shower dip concentrates, and jetting fluids that are suspension concentrates. Dipping and jetting suspension concentrate formulations are diluted with water in accordance with the label directions prior to

55

use. Suspension concentrates consist of insoluble solid active ingredients (normally at high concentration) in water or oil. They facilitate the stability of some active ingredients that are unstable in solution and overcome the concerns of dust associated with wettable powders and of toxic and/or flammable solvents used in some emulsifiable concentrates. Surfactants play several key roles in suspension concentrate formulations, such as during manufacture when they serve as wet milling aids, for wetting the solid particles of the active ingredient, and for dispersing and stabilizing the solid particles in the continuous phase. Flocculation and changes in particle size with time are controlled by the adsorption of surfactant onto the solid particles, whereas the inclusion of thickeners in suspension concentrate formulations provides good long-term stability by increasing viscosity to avoid sedimentation of the solid particles. Emulsifiable concentrates are commonly used for spraying and dipping of cattle and sheep. When poured into water, emulsifiable concentrates spontaneously emulsify. The resultant emulsion consists of fine oil droplets, ranging from approximately 0.5 mm to a few hundred millimeters, as the dispersed phase, in water as the continuous phase. Emulsification relies on surface-active agents concentrating at the oil/water interface and lowering the interfacial tension between the two phases. A common practice is to use blended emulsifiers, i.e., a mixture of an anionic surfactant and a non-ionic surfactant. A second requirement of emulsifiable concentrates is stability of the emulsion. Flocculation of oil droplets leads to a layer of cream that can readily be redispersed by mild agitation. Whereas flocculation can be tolerated in practice, coalescence of droplets, which causes inversion or breaking of the emulsion, cannot be tolerated (12). Both spontaneity of emulsification and stability are strongly affected by the concentration of cations, such as Ca+2 and Mg+2 , that characterize the degree of hardness of the water and may react chemically with the anionic surfactants present. Dip additives such as zinc sulphate, which is used for minimizing the spread of dermatophilosis in sheep, may also impact on the emulsion (13,14). Acaricides are applied to cattle by spray, dip, or pouron to control ticks. With dairy cattle, the preferred application methods are hand-spray, spray race, and pour-on. Hand-spraying generally results in nonuniform coverage of animals and, for this reason, is considered an inefficient method of application (15). Moreover, the exposure of some ticks to sublethal concentrations of acaricide may select for resistant strains. Recirculating and nonrecirculating spray races facilitate whole body spraying and wet cattle to the skin. Plunge dipping of cattle requires a dipping vat, commonly of 10,000 L capacity, for thoroughly immersing the animals in pesticide to control ticks, flies, grubs, lice, and mites. The vats are usually permanent in-ground structures that are shielded from direct sunlight by roofing. High costs are associated with the large quantities of chemicals required to fill the vats; the environmental impact from disposing of dip wash is another disadvantage. Dip chemicals are usually formulated as aqueous solutions, emulsifiable concentrates, suspension concentrates, or

56


wettable powders. The concentration of the chemicals used in dips may decline because of leaching, volatilization, adsorption, decomposition, or metabolism, in addition to in-use ‘‘stripping’’ of the pesticide. Cattle dips have to be cleaned regularly on account of fouling with manure and mud carried into the vat. Some dip chemicals require that stabilizers be added. For example, amitraz hydrolyzes rapidly in acid media and is stabilized by the addition of hydrated lime (Schering Agrichemicals, 1992). The carbamate tickicide, promacyl, is subject to microbial degradation (16); the latter contributes to the declining concentrations of promacyl observed in cattle dips when the ambient temperature ranges from 15 ◦ C to 26 ◦ C (17). Stabilization of promacyl in dips can be achieved by adding dichlorophen, 2,4-benzisothiazolin-2-one, or sucrose or by adjusting the pH of the dip wash to less than 5.0 (17). Pouron formulations will be discussed later in this chapter. Dipping of sheep and cattle is commonly associated with ‘‘stripping’’ of the active ingredient from the dip wash, and this influences the amount of pesticide retained on the animal. ‘‘Stripping’’ is the process whereby pesticide loss from dip wash occurs at a greater rate than water loss and is categorized as mechanical and/or chemical. In the case of sheep, mechanical ‘‘stripping’’ results from the fleece acting as a sieve toward the active ingredient, with the degree of ‘‘filtration’’ being primarily determined by particle size. The practical impact of mechanical ‘‘stripping’’ is apparent from a comparison of solutions and wettable powders. With solutions, the amount of pesticide retained by wool is determined by the volume of dip wash retained and the pesticide concentration of the dip wash. Wettable powders are mixed with water to form a suspension, and the retention of pesticide on the wool from the suspension is determined by the volume of dip wash retained, the pesticide concentration, and also by the particle size of the active ingredient. Finely micronized powders (10 mm) tend to be filtered out by the wool (18). By comparison, chemical ‘‘stripping’’ is due to the preferential absorption of pesticide by the fleece. A complex dip management regimen, which involves ‘‘reinforcement’’ and ‘‘topping-up,’’ applies to ‘‘stripping’’ dips. ‘‘Reinforcement’’ refers to the addition of undiluted chemical product to the dip without the addition of water and compensates for ‘‘stripping’’ by ensuring that the dip concentration remains above the minimum effective concentration as the dip wash reduces in volume. ‘‘Topping-up’’ refers to the addition of water and undiluted chemical product (at the recommended rate) to the sump or dip vat to return the volume to the starting level. Despite the complex management regimes that apply to ‘‘stripping’’ dips, animals dipped early in the operation are exposed to higher concentrations of pesticide than those dipped later. Sherwood et al. (19) recently described a constant concentration dipping approach that led to more predictable wool and tissue residues, improved efficacy against lice, and reduced exposure of dip operators to unnecessarily high dip wash concentrations. However, not all dip pesticides demonstrate ‘‘stripping’’ behavior. Synthetic pyrethroids ‘‘strip’’ less than organophosphorus compounds, so, in the case of dipping with synthetic

pyrethroids, the amount of chemical applied is closely related to the amount of dip wash applied. Non-stripping dips are managed by ‘‘topping-up’’ when the dip or sump volume falls by approximately 25%. Traditionally, synthetic pyrethroids were applied to sheep by plunge and shower dipping and hand-jetting. Hand-jetting of long-wool sheep (sheep with more than 6-weeks wool growth) involves the use of a hand piece (also referred to as a wand) to ‘‘rake’’ a pesticide solution into the wool along the backline and sometimes into the breech or crutch, as well as the poll. The pesticide solution is applied at pressures of 525 to 700 kPa (75–100 psi) to sheep with 3-months wool growth, 770 kPa (110 psi) to sheep with 4-months wool growth, and 900 kPa (130 psi) to sheep with 8-months wool growth; the solution penetrates to the skin. It is recommended that a minimum of 0.5 L of pesticide solution per month’s wool growth is applied. Eradication of body lice from sheep in long wool is rarely achieved by hand-jetting, and the practice is used primarily to reduce lice burdens to levels that cause minimal economic losses. Chemicals that are approved in some countries for application by hand-jetting include cyhalothrin, cyromazine, diazinon, diflubenzuron, ivermectin, propetamphos, rotenone, and temephos. Although dipping and hand-jetting achieve uniform coverage of the animal, these application methods raise concerns relating to worker exposure, environmental contamination, the disposal of unused chemical, and, to a lesser extent, variable animal exposure due to progressive ‘‘stripping’’ of pesticide from the dip wash. These concerns led to topical backliner products gaining wide acceptance. However, subsequent experience with backliners has led to issues peculiar to these products being identified, and these are briefly discussed in the following text. Topical backliner products are formulated as pouron and spray-on preparations and are applied off-shears (within 24 h of shearing) or to long-wool sheep (sheep with more than 6-weeks wool growth). These products do not rely on percutaneous absorption into the bloodstream. Rather, successful protection of sheep against lice depends on the chemical spreading quickly from the application site to remote sites at concentrations that are lethal to lice. However, several workers have reported inefficient diffusion of synthetic pyrethroids from the application site with topical backliners. Johnson et al. (20) reported that when a water-based formulation of deltamethrin was applied to sheep 24 h after shearing, there was very little movement of the synthetic pyrethroid down the staple or away from the application site. Campbell et al. (21) and Johnson et al. (22) reported that some chemicals, when applied as long-wool pour-on treatments, remain largely concentrated in the application strip along the backline of sheep. With off-shears backline treatments, application within 24 h after shearing is recommended in order to optimize diffusion away from the application site. Darwish and coworkers (23) recently confirmed that the secretion of wool grease off-shears is higher (increases in the order of 24% were reported) than at later times and that these increases were associated with an abundance of fresh minimally oxidized wool grease. Based on these findings, these workers postulated that the conditions that


prevail off-shears are conducive to significant diffusion of deltamethrin away from the application site, whereas oxidation of wool grease over the ensuing days entraps any dissolved synthetic pyrethroid, thereby limiting further diffusion. This is consistent with the recommendation to apply off-shears backliner products within 24 h after shearing. Hennessy et al. (24) subsequently achieved both enhanced spread of deltamethrin from the application site and improved efficacy when they formulated the synthetic pyrethroid in nonoxidized sterol and wax ester fractions of wool grease, as compared with a commercial formulation of deltamethrin. Lice feed at the skin surface and, from an efficacy perspective, poor spreading of the synthetic pyrethroid from the application site and carriage of the chemical away from the skin surface as a consequence of wool growth, result in sublethal doses of synthetic pyrethroid being attained in many parts of the fleece. This has the potential to predispose to the selection for synthetic pyrethroid-resistant lice. Early studies of organophosphate sheep blowfly larvicides reported that, in order for these compounds to be highly efficacious, they needed to demonstrate both good larvicidal activity and the ability to translocate. In this context, translocation is the process whereby blowfly larvicides migrate to the base of the fleece where cutaneous myiasis takes place. Du Toit and Fiedler (25) postulated that the mechanism by which organophosphorus compounds translocate involves a process of diffusion down the wool fiber from the originally treated wool onto the new wool that grows after treatment. However, a later study suggested that the mechanism of translocation depends on the physicochemical properties of the chemical and that some larvicides form follicular depots at the time of application and subsequently translocate as a coating on new wool growing out of these follicles (26). Pour-on and spot-on formulations deliver drugs transdermally for control of internal and external parasites on animals. The mechanisms by which specific pesticides penetrate the skin of sheep and cattle are not well documented and only general principles of the barrier function of the skin and of percutaneous drug absorption will be described here. The epidermis consists of five layers (from the deepest to the most superficial, these layers are the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum) with the principal barrier to drug absorption being the outermost layer, the stratum corneum. The cells comprising the stratum basale are the only cells within the epidermis that undergo division, and once having committed to terminal differentiation, they cease dividing and migrate superficially to become keratinocytes in the nonviable stratum corneum. The complex intercellular lipid matrix in which the keratinocytes are embedded is derived from exocytosed lipid from cells in the stratum granulosum. This matrix has a liquid crystalline structure, and its composition varies throughout the epidermis due to the ionic phospholipid content decreasing and the nonionic sphingolipid and neutral lipid content increasing in the direction of the skin surface. Ceremides formed by the enzymatic hydrolysis of sphingomyelin are one of the main components of the layered intercellular

57

lipids found in the stratum corneum (27). Ceremides function as structural lipids, whereas squalenes, waxes, and triglycerides provide the membrane continuity. The principal route of percutaneous absorption for most drugs is the intercellular pathway making the intercellular lipid matrix the primary barrier to absorption. However, based on the scant data available on the penetration of ions through the skin of cattle and sheep, it seems that the transport of ionized solutes is probably more through the shunt pathways (sweat ducts, follicles) (28). The vehicles of pour-on and spot-on formulations play an important role in achieving percutaneous drug absorption. The solubility of the active constituent determines whether the active is suspended in aqueous or organic solvent. The formulation must facilitate: 1) partitioning of the drug out of the vehicle and into the skin, 2) transport of the drug across the skin, and 3) absorption of the drug into the bloodstream. It appears that an empirical approach is taken towards the development of many topical formulations for animal health. The pharmaceutical literature details the roles of penetration enhancers and supersaturated solutions improving the transport of drugs into and across skin. Both surfactants and certain solvents are used as penetration enhancers. Nonionic surfactants, which are favored in topical formulations because they have less potential for skin irritancy compared with ionic surfactants, and certain solvents alter the emulsions coating hair/wool, thereby allowing more drug to reach the skin surface. It is postulated that the mechanism by which skin penetration enhancers increase drug transport across the skin involves increasing the fluidity and/or the hydration of the polar head groups of the lipid bilayers (29). The vehicle of a topical formulation may affect the hydration state of the stratum corneum and percutaneous absorption. Hydration results in water ‘‘opening’’ the compact substance of the stratum corneum and reducing the density of the intracellular structures, thereby decreasing the cells resistance to passive diffusion and allowing substances to permeate more readily than in the normal dehydrated state (30). An increase in the percent hydration from 10% to 50% of the stratum corneum can result in as much as a 10-fold increase in diffusion constants (31). In addition to transdermal absorption of pour-on products, grooming can lead to variable oral absorption of the active ingredient. When Hasker et al. (32) investigated the use of licking behavior of cattle as a means of dose administration, the application of sugar syrup or molasses to the coat of cattle was shown to stimulate self-licking behavior. Mild irritants were proposed to elicit a similar response. Finally, rainfastness is an important characteristic of pour-on and spot-on formulations for use on farm animals and is a function of the formulation. Two types of insecticide-releasing eartags are used for control of flies on cattle. One type has a monolithic structure involving a polymer that provides structural support and functions as a release rate-controlling matrix. The second type is a membrane-based eartag that consists of an insecticidal reservoir with a relatively impermeable backing on one side and a rate-controlling membrane

58


on the other. A square-wave delivery profile has been described for membrane-based eartags (33). Both types rely on the animal’s ear and head movements and grooming activities to transfer insecticide from the surface of the eartag to the animal’s hide or to other animals. The active ingredients in eartags are generally pyrethroids, synergized pyrethroids, organophosphates, or various combinations of these. Back rubbers allow for self-treatment of cattle with insecticide for control of flies. Back rubbers typically consist of burlap (or the equivalent) supported across laneways, gateways, or areas where cattle congregate. Back rubbers are charged by soaking them thoroughly in oil containing pesticide and are recharged as necessary. The active ingredients in back rubbers are generally synthetic pyrethroids, organophosphates, or combinations thereof. The oil retards evaporation of the insecticide and enhances adherence to the animal’s coat (34). Dust bags facilitate the self-treatment of cattle for control of flies and lice. The active ingredient, which is generally a synthetic pyrethroid or organophosphorus compound, is contained in an inner porous bag; an outer weather-proof skirt is fitted over the inner bag. Dust bags are hung in laneways or gateways so that passing cattle will brush against them and be dosed topically with pesticide. Feed additive larvicides are administered to beef and dairy cattle or poultry for control of coprophagous flies. Tetrachlorvinphos, methoprene, diflubenzuron, and cyromazine are the active ingredients approved for such use in the U.S. The larvicidal activity of these chemicals is attributed to unmetabolized compound excreted in the feces. Sustained-release boluses are approved for control of external parasites on ruminants. A methoprene bolus and a diflubenzuron bolus are marketed for control of manure-breeding flies in cattle. The manufacture of these boluses utilizes tabletting technologies, and the boluses are formulated accordingly. The endectocide ivermectin is available as a controlled-release bolus for cattle and a controlled-release capsule for sheep. The essential components of the Ivomec SR bolus for cattle are an exterior semipermeable membrane, an osmotic tablet, a drug reservoir, and a density element that is responsible for retention of the bolus in the reticulorumen (35). When water from the reticulorumen is imbibed through the exterior semipermeable membrane of the bolus, the osmotic tablet swells and forces drug through an exit port. This mechanism results in a steady-state delivery rate of approximately 12 mg ivermectin per day being maintained for some 135 days. By comparison, the Captec capsule for sheep has a polypropylene barrel with a spring-loaded plunger; ivermectin tablets are loaded into the barrel. The barrel has an orifice for drug delivery at one end and a pair of polypropylene folding wings at the other. The wings are taped to the barrel of the capsule during administration and open out when the tape dissolves in the reticulorumen liquor. The wings are the key to its retention in the reticulorumen. After the tablet adjacent to the exit port of the capsule has dissolved, the plunger pushes the

next tablet up against the exit port where it undergoes dissolution; this process is repeated until all tablets have been dissolved. The delivery rate of the capsule is 1.6 mg ivermectin per day (adult sheep) or 0.8 mg ivermectin per day (weaner sheep) for 100 days. CHEMICAL RESIDUES IN FOOD Currently, ectoparasite control relies heavily on the use of chemicals and, even when these are used in accordance with label directions, chemical residues may remain in the meat, offal, milk, and eggs. The key to eliminating unacceptable residues lies in their prevention during all stages of animal production, rather than by screening for contaminated produce and eliminating it immediately before it enters the food chain. In this respect, quality assurance programs and the promotion of responsible chemical use among the farming community are seminal. Extensive regulatory and monitoring systems have been established to ensure that chemical residues in food do not constitute an unacceptable health risk. The safety of veterinary chemical residues in food is determined after a review of toxicologic, metabolic, residue depletion, and dietary exposure data. The maximum residue limit (MRL), expressed as mg/kg of the commodity, is the highest concentration of a residue of a particular chemical that is legally permitted in a food or animal feed. MRLs are regulatory standards that help to monitor the use of the chemical as directed on the label of the registered product. If an MRL is exceeded, it may indicate a misuse of the chemical but does not normally indicate a public health and safety concern. The Codex Alimentarius Commission plays a key role in establishing international standards for veterinary drugs and pesticides in food, thereby protecting consumers and facilitating trade (see also Codex Alimentarius). MRLs for pesticides and veterinary drugs are recommended to the Codex Alimentarius Commission by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA), respectively. The approaches adopted by JMPR and JECFA for establishing MRLs and determining dietary exposure to chemical residues are fundamentally different. This can lead to the residue definitions (marker residues), commodity definitions, and MRLs (tolerances) that are recommended by JMPR and JECFA being different for the same chemical. Harmonization meetings of JMPR and JECFA are addressing this aspect of the standards setting process. JMPR sets MRLs on animal commodities based on either residues in animal feed leading to residues in milk, eggs, and tissues or on residues from direct animal treatment, or both. MRLs are recommended by JMPR following toxicological assessment of the pesticide and its residue and review of residue data from supervised trials, including those that reflect national good agricultural practice (36). In this context, good agricultural practice means that pest control will be achieved without leaving behind in the food more residues than necessary. Dietary exposure assessments are performed to verify that food complying with Codex MRLs is safe for human


consumption. The assessments are based on the use of FAO/WHO food consumption data (food balance sheets data classified into regional diets) and food residue concentration data (MRLs, trials median residues in edible portions and levels in processed foods, allowing for food processing factors). The best estimate of actual dietary exposure is obtained using all available data in a procedure published by WHO in 1997 (37). By comparison, JECFA derives MRLs in the context of good practice in the use of veterinary drugs and recommends MRLs in accordance with the acceptable daily intake (ADI) of the chemical (38). JECFA has adopted a conservative approach to estimating dietary consumption that is based on available intake data at the upper limit of the range for individual consumption of edible tissues and animal products. The model diet used for this purpose is comprised of 300 g meat (as muscle tissue), 100 g liver, 50 g kidney, 50 g fat, 100 g eggs, and 1.5 L milk. Because the daily intake values in the model diet exceed the average consumption (as reported in food balance sheets data), the ADI will not be exceeded from veterinary drug usage. However, a chemical such as abamectin may be used as both an agricultural pesticide and a veterinary drug and, in these circumstances, the ADI should apply to both uses. Recent attention has focused on the potential of chemical residues in food to cause acute toxicological effects. In response to this, JMPR recently introduced acute dietary exposure assessments for pesticides that may represent an acute hazard; JECFA and the Codex Committee on Residues of Veterinary Drugs in Foods are in the process of developing guidelines for residues at injection sites. International trade in animal commodities has been confounded by a need to comply with a diverse range of standards imposed unilaterally by importing countries. Differences among countries in the approval system, the procedures for establishing MRLs, and the use patterns have led to national MRLs for numerous chemical-commodity combinations varying substantially. Furthermore, national MRLs rarely exist for unapproved chemicals. In the case of pesticides, for example, the pest pressure in a particular country may not justify product registration or the establishment of MRLs. The situation has been confounded by Codex standards being adopted internationally only to a limited extent. However, this is changing with the successful conclusion of the Uruguay Round of Multilateral Trade Negotiations and the establishment of the World Trade Organization (WTO). The WTO Agreement on the Application of Sanitary and Phytosanitary Measures (SPS Agreement) cites standards, guidelines, and recommendations of the Codex Alimentarius Commission as reflecting international consensus regarding the requirements to protect human health from food-borne hazards. A WTO member nation that uses a Codex standard is seen as meeting its obligations under the SPS Agreement. However, WTO members reserve the right to adopt standards that are stricter than Codex standards, provided these can be justified scientifically. Different approaches to establishing MRLs for ectoparasiticides are practiced among countries and often within the same country. In the U.S., the method by which a

59

chemical comes in contact with the target parasite determines whether the USFDA or the USEPA is the reviewing agency (39). Chemicals that are administered orally or topically but exert their effects following systemic distribution are assessed by the USFDA as veterinary drugs, whereas those applied topically and act on the pest by surface contact are evaluated as pesticides by the USEPA. In Australia, the JMPR evaluation procedure applies to both agricultural and veterinary chemicals. The system applied in Australia achieves harmonization of residue definitions, commodity definitions, dietary exposure assessments, and MRLs for those chemicals that are used in both agriculture and animal health. PESTICIDE RESIDUES IN WOOL Following the use of pesticides for the control of lice and flystrike in sheep, some residues remain on the greasy wool at shearing. Although the pesticide residues are removed by scouring and do not detract from clean wool, the presence of pesticide residues does raise occupational safety concerns for shearers, human health concerns regarding the presence of residual pesticide in pharmaceutical grades of lanolin, and environmental concerns pertaining to residual pesticides in scouring effluent. An Australian monitoring survey found that approximately 50% of the total residue loading in the national wool clip was attributed to some 5% of sale lots and that late season treatments (close to shearing) were found to be a major contributor to the total residue in the Australian wool clip. Although wound and blowfly strike dressings are often applied relatively close to shearing, these treatments (typically diazinon, chlorfenvinphos, and propetamphos) involve only a relatively small number of fly-struck sheep in a mob and, consequently, are unlikely to have much impact on mean wool residue levels in a flock. A review of all sheep ectoparasiticides commercially available in Australia has been conducted by the National Registration Authority for Agricultural and Veterinary Chemicals (40). Of the off-shears and long-wool backline/spray-on formulations evaluated, those containing synthetic pyrethroids were found to result in the most persistent wool residues. Synthetic pyrethroids wool residues were most persistent following treatment with short-wool dipping formulations, whereas diflubenzuron wool residues were most persistent following application of long-wool jetting formulations. Recently, a mathematical model was reported that predicts the likely consequences of on-farm treatments at any time throughout the wool-growing cycle and determines how late an insecticide may be applied to sheep without creating excessive residues at shearing (41). The model suggests that, as the length of the wool increases, the amount of pesticide applied by dipping increases, as does the protection of the pesticide from environmental degradative factors. The method of application was shown to impact significantly on the rate of pesticide breakdown. For example, application of pesticides by automatic jetting races, which tend to deposit the chemical on the surface of the fleece, is associated with a rapid rate of

60


breakdown of photolabile compounds. Dipping and jetting are associated with slower rates of breakdown due to the pesticides being deposited deep in the fleece. Other studies have reported that organophosphorus compounds undergo faster breakdown when applied to sheep breeds with coarse open fleeces (42), to the backs compared with the sides of sheep (42), and to the surface of fleeces (41). The slow overall breakdown displayed by synthetic pyrethroids results in high levels of residues in wool at shearing. The insect development inhibitors, triflumuron and diflubenzuron, also demonstrate relatively slow rates of breakdown in wool (41). ENVIRONMENTAL IMPACT OF VETERINARY PESTICIDES Valuable lessons have been learned over the past decades with respect to the environmental impact of some ectoparasiticides. For example, the previous legal use of arsenical products to control external parasites on sheep has led to residual contamination of plunge and shower dips and surrounds. Most contamination resulted from dip wash being emptied onto adjacent sheep yards or paddocks. Resolution of contaminated sites is problematic and is often managed by fencing off the contaminated areas. The organochlorine ectoparasiticides also have some serious shortcomings from an environmental perspective; notably, they are chemically stable and persist in the environment. Considerable effort has been made in recent years in Australia to identify properties contaminated with organochlorines and to encourage the property owners to adopt farming enterprises and management practices that mitigate the chance of unacceptable organochlorine residues accumulating in livestock. Today, regulatory authorities conduct rigorous assessments of environmental chemistry, fate, and toxicology during the registration process for pesticides and veterinary drugs. The potential environmental impact of veterinary chemicals is determined by performing environmental hazard assessments. With intensive poultry production, for example, a feed-through chemical to control coprophagous flies may pose an environmental risk when the chicken manure is used as a fertilizer. It is possible that a simple management practice, such as allowing some period of degradation prior to use as manure, will significantly reduce the level of chemical present. Chemicals used in aquaculture are of concern to the environment when used in large volumes in open waters or are released into open waters. Ectoparasiticides to control lice and flies on sheep have implications for the environment when the fleece is scoured. Most pesticides are removed with the grease and dirt fractions during scouring and then discharged in aqueous effluents (see the earlier section on Pesticide Residues in Wool). The effects of fecal residues of antiparasitic drugs on dung fauna and dung degradation have been the subject of numerous studies (43,44,45) and will be considered here in more detail. In 1987, Wall and Strong (46) first described the absence of dung-degrading insects, particularly beetles, in the feces of calves treated with an ivermectin bolus and the failure of dung pats to degrade at normal rates. Since 1987, little evidence has become available to either

confirm or refute ecotoxic effects associated with the use of organophosphates in animals. By comparison, studies by Wardhaugh et al. (44) indicate that fecal residues of several commonly used pyrethroids can be highly toxic to coprophagous insects. Deltamethrin concentrations in the feces of treated cattle are sufficient to inhibit the survival of the bushfly Musca vetustissima for 1 to 2 weeks after treatment. Most attention has focused on the macrocyclic lactones; however, despite this, there is a lack of consensus in the scientific community over the environmental risks associated with their use. The macrocyclic lactones are lipid-soluble drugs that are distributed widely throughout the body with the highest tissue concentrations occurring in adipose tissue and liver. The liver is also the principal organ of metabolism for macrocyclic lactones. Typically, biliary clearance of macrocyclic lactone conjugates is followed by deconjugation in the gut and fecal elimination of the parent compound. The macrocyclic lactones represent a potential threat to the ecosystem on account of their efficacy against coleopterans and dipterans and the fact that the vast majority of the administered dose is excreted in the feces. Quantitation of the overall impact of the macrocyclic lactones on the population dynamics of dung beetles is confounded by fluctuations in insect populations attributable to factors such as temperature changes, rainfall, cattle management, pasture quality, and climate. In addition to mortalities of coprophagous beetles caused by lethal concentrations of drug in the feces, sublethal concentrations may also impact adversely on the life cycle of dung fauna. This is further complicated by the fact that both lethal and sublethal effects of ivermectin differ substantially even between closely related genera of dung beetles (47). In general, the duration of toxic effects of chemical fecal residues on dung fauna reflects the fecal excretion profile of the drug. Excretion profiles differ among drugs, among animal species, and among formulation types. In the case of macrocyclic lactone use in cattle, oral drenches impact less on dung fauna than do subcutaneous injections or pour-on formulations (45,48). The trend towards the use of delivery systems such as intraruminal controlled-release capsules and boluses that deliver drugs for periods of 100 days or more and formulations that facilitate slow drug absorption from the site of administration (injectable formulations) or application (topical formulations) are a concern. The timing and frequency of parasite control programs also influence the environmental impacts. The periodic and infrequent use of macrocyclic lactones in strategic worm programs lessens the exposure of dung beetles to chemical residues in the feces of treated animals. However, the sustainability of dung fauna will benefit from strategic worm programs only if the fecal residues are associated with short-term toxicity. A recent review (49) of the environmental impact of macrocyclic lactones in Australia concluded that, although different products may be associated with different shortterm effects and varying toxicities to dung beetle larvae, the available evidence did not indicate that any of the macrocyclic lactone products had a long-term detrimental effect on dung beetle populations or dung disappearance


rates in the field under Australian conditions. The review also noted that further research is required to better understand the potential environmental impacts of antiparasitic treatments of animals. PESTICIDE RESISTANCE Resistance is a change in the genetic composition of an insect population that results from exposure to an insecticide over a period of time. This change permits members of the resistant population to survive exposure to the specific insecticide. Mechanisms of resistance differ across the various groups of insecticides and pests and, in some instances, across various strains of the same pest. Four mechanisms for synthetic pyrethroid resistance in insects have been described: 1) behavioral resistance where modification of the insect’s behavior allows contact with the insecticide to be avoided, 2) penetration resistance where modification of the insect’s exoskeleton prevents the insecticide from penetrating, 3) site insensitivity where modification of the active site for the insecticide lessens or abolishes sensitivity, and 4) metabolic resistance where detoxification of the insecticide is enhanced and/or metabolic activation of the insecticide is slowed (50). Resistance to organophosphate acaricides in certain strains of the cattle tick Boophilus microplus is ascribed to decreased sensitivity of the target enzyme acetylcholinesterase (51,52,53) and to increased detoxification of the organophosphate insecticide by other strains (54). Numerous conditions select for the development of resistance including: 1) prolonged sublethal exposure to a single insecticide group with a persistent action, 2) multiple generations of the insect being selected, 3) high selection pressure and no refuge for the exposed population, 4) widespread use of the insecticide, and 5) a low population threshold for the application of the control measures (10). Delivery systems and formulations may contribute to resistance via prolonged exposure to insecticide, particularly when sublethal concentrations are involved. The latter is commonly associated with poor efficacy. The selection of resistant fly populations by insecticide-impregnated eartags is a case in point. In this respect, membrane-based eartags may prove valuable in delaying the emergence of resistance because, unlike eartags with a monolithic structure, they are capable of delivering approximately 95% of their insecticidal load at a constant rate (a square wave delivery profile). Sublethal concentrations of pesticide also result from ‘‘stripping’’ of active ingredient from dip wash, leading to the selection of resistant strains of sheep lice. Sublethal concentrations of synthetic pyrethroids are also commonly encountered with lousicides applied to long-wooled sheep and with off-shears pour-on lousicides. Spreadability of the latter from the application site to remote areas is a function of the formulation and its compatibility with the chemistry of the sheep’s skin. Persistent sublethal concentrations are also common with agents whose blood concentration-time profiles are characterized by ‘‘tailing’’ of the terminal elimination phase. Intraruminal delivery systems with square wave delivery profiles for control of the sheep blowfly Lucilia cuprina avoid both

61

‘‘tailing’’ of the terminal elimination phase of the blood concentration-time profile and persistent wool residues; the latter are observed with many pesticides following external applications. Control of Boophilus microplus in Australia has relied heavily on the use of chemicals. This species of cattle tick has become resistant successively to arsenic, DDT, other organochlorines, a range of organophosphates and carbamates, amidines, and finally synthetic pyrethroids (55,56). This is compounded by many tick strains being multiply resistant to different chemicals. The cat flea Ctenocephalides felis has developed resistance to at least five different categories of insecticides including pyrethrins, synthetic pyrethroids, carbamates, organophosphates, and organochlorines (57). The resistance status of an insect population is usually a function of the level of insecticide use, with the resistant phenotypes losing their advantage in the absence of the insecticide. When established or developing parasite resistance is identified, the most effective use of the available pesticides must be achieved with the prime objective of effective control with as few treatments as possible. Several options are available to reduce selection for resistance and to prolong the life of existing pesticides as follows: 1) chemical groups with different modes of action can be rotated, 2) combinations of agents that act synergistically or where one agent potentiates another can be used in combinations, and 3) different agents to combat specific life cycle stages can be used sequentially. Finally, the nonchemical components of resistance management strategies must not be overlooked. Genetics play a pivotal role in resistance to ectoparasites as demonstrated by Bos indicus cattle being less susceptible to cattle ticks compared with Bos taurus breeds and fleece rotresistant sheep being less susceptible to body strike by the sheep blowfly Lucilia cuprina than fleece rot-susceptible sheep. Animal husbandry practices such as crutching, mulesing, and shearing of sheep reduce the feeding and breeding sites of Lucilia cuprina, thereby reducing the susceptibility of sheep to breech strike. Biological control measures including bacteria, protozoa, viruses, and parasitic nematodes have been investigated for the control of many ectoparasites. A reduced dependence on tickicide applications to control Boophilus microplus on cattle is facilitated by anti-tick vaccines, pasture spelling, and the use of disinfection paddocks. ECTOPARASITICIDES USED IN ANIMAL HEALTH Many of the chemicals that are approved for the control of ectoparasites on domestic animals are described below. ADIs and MRLs have been established for many of these chemicals by the Codex Alimentarius Commission; the available values are presented in Table 1. Because these standards are subject to change, it is recommended that the reader consult the relevant Codex Alimentarius Commission’s publications (36,58) and/or website (http://www.fao.org/WAICENT/FAOINFO/ECONOMIC/ESN/codex/default.htm) for current information. The approved uses of the various ectoparasiticides are shown in Tables 2 to 13; however, these tables are not all-inclusive listings and the approved uses vary among countries.

Table 1. Acceptable Daily Intakes and Maximum Residue Limits Established by the Codex Alimentarius Commission for Ectoparasiticides Applied Directly to Animals Substance (Codex Committee) Amitraz (JMPR)

Bendiocarb (JMPR)

ADI (mg/kg bw) 0.01

0.004

Marker Residue (JECFA) or Residue Definition (JMPR)

Animal Species

Tissue

MRL (mg/kg)

Sum of amitraz and N-(2, 4-dimethylphenyl)-N methylformamidine calculated as N-(2, 4-dimethylphenyl)-N methylformamidine

Cattle Cattle, pigs, sheep Mammalian Pigs Sheep

Meat Edible offal

0.05 0.2

Milks Meat Meat

0.01∗ 0.05 0.1

Sum of conjugated /unconjugated bendiocarb, 2,2-dimethyl-1,3benzodioxol-4-ol/N-hydroxymethyl bendiocarb expressed as bendiocarb

Cattle

Fat Kidney Meat Edible offal (except kidney) Milks

0.05∗ 0.2∗ 0.05 0.05∗ 0.05∗

Chlorpyrifos (JMPR)

0.01

Chlorpyrifos

Cattle Mammalian Sheep Turkeys

Meat (in the fat) Milks Meat (in the fat) Meat (in the fat)

2 0.01∗ 0.2 0.2

Cyfluthrin (JECFA)

0.02

Cyfluthrin

Cattle

Muscle, liver, kidney Fat Milk

0.02 0.2 0.04

Cyfluthrin (JMPR)

0.02

Cyfluthrin

Cattle

Milk

0.01

Cypermethrin (JMPR)

0.05

Cypermethrin (sum of isomers)

Mammalian

Edible offal Meat (in the fat) Milks

0.05∗ 0.2 0.05

Cyromazine (JMPR)

0.02

Cyromazine

Poultry Mammalian Poultry Sheep

Eggs Milks Meat Meat

0.2 0.01∗ 0.05∗ 0.05∗

Deltamethrin (JMPR)

0.01

Deltamethrin

Mammalian Mammalian Mammalian

Edible offal Meat (in the fat) Milks

0.05 0.5 0.02

Diazinon (JMPR)

0.002

Diazinon

Cattle, pigs, sheep Mammalian

Meat (in the fat)

0.7

Milks

0.02

Doramectin (JECFA)

0.0005

Doramectin

Cattle

Muscle Liver Kidney Fat

0.01 0.1 0.03 0.15

Fenthion (JMPR)

0.007

Sum of fenthion, its oxygen analogue and their sulphoxides and sulphones, expressed as fenthion

Mammalian

Meat (in the fat) Milks

2 0.05

Fluazuron (JECFA)

0.04

Fluazuron

Cattle

Muscle Liver, kidney Fat

0.2 0.5 7

Flumethrin (JMPR)

0.004

Flumethrin

Cattle Cattle Honey bees

Meat (in the fat) Milks Honey

0.2 0.05 0.005∗

Ivermectin (JECFA)

0.001

H2 BIa

Cattle

Liver Fat

0.1 0.04

Pigs, sheep

Liver Fat

0.015 0.02 (continued overleaf )

62

Table 1. (Continued) Substance (Codex Committee)

ADI (mg/kg bw)

Marker Residue (JECFA) or Residue Definition (JMPR)

Animal Species

Tissue

MRL (mg/kg)

Methoprene (JMPR)

0.1

Methoprene

Cattle Mammalian

Milk Meat (in the fat)

0.05 0.2

Moxidectin (JECFA)

0.002

Moxidectin

Cattle Cattle, sheep

Muscle Liver

0.02 0.1

Kidney Fat

0.05 0.5

Sheep

Muscle

0.05

Permethrin (JMPR)

0.05

Permethrin (sum of isomers)

Mammalian Mammalian

Edible offal Meat (in the fat)

0.1 1

Phosmet (JMPR)

0.01

Sum of phosmet and its oxygen analogue

Cattle Mammalian

Meat (in the fat) Milks

1 0.02∗

ADI = acceptable daily intake; JECFA = Joint FAO/WHO Expert Committee on Food Additives; JMPR = Joint FAO/WHO Meeting on Pesticides Residues; MRL = maximum residue limit; T = temporary. ∗ At or about the limit of quantitation of the analytical method.

Table 2. Botanical Compounds Used as Ectoparasiticides on Animals Compound

Formulation Type/ Application Method

d-Limonene

Rinse, shampoo, spray

Dogs

Fleas, lice, mites, ticks

Pyrethrinsa

Aerosol spray

Birds Cats, dogs Cattle, horses, pigs

Lice, mites Fleas, lice, ticks Flies, mosquitoes

Rotenone

Animal

Target Pest

Collar, medallion

Cats, dogs

Fleas

Cream

Dogs, horses

Flies, mosquitoes

Dusting powder

Birds Cats, dogs Cattle, goats, pigs, sheep

Lice, mites Fleas, lice, ticks Wound dressing

Foams, shampoo, soap

Cats, dogs

Fleas, ticks

Rinse

Birds Cats, dogs

Lice, mites Fleas, lice, ticks

Dusting powderb

Beef cattle, cats, dogs, goats, horses, poultry

Fleas, lice, mites

Hand-jettingc

Sheep (long-wool)

Itch mites, keds, lice

Plunge/shower dip

Sheep

Itch mites

Plunge/shower dipc,d

Sheep

Itch mites, keds, lice

Plunge/shower dipe

Sheep

Blowflies, itch mites, keds, lice

a The majority of formulations includes synergists (piperonyl butoxide, N-octyl bicycloheptene dicarboxamide) ± repellents (di-N-propyl isocinchomeronate, diethyltoluamide, citronella oil). Some formulations contain additional insecticidal agents including botanicals (e.g., rotenone, pennyroyal), carbamates (e.g., carbaryl), insect growth regulators (e.g., methoprene), or organophosphorus compounds (e.g., diazinon). b +sublimed sulphur. c +a synthetic pyrethroid. d +magnesium fluorosilicate and sulphur. e +piperonyl butoxide + an organophosphorus compound.

63

Table 3. Synthetic Pyrethroids Used as Ectoparasiticides on Animals Formulation Type/ Application Method

Compound

Animal

Target Pest

Allethrin

Shampoo

Cats, dogs

Fleas, ticks

Cyfluthrin

Eartag

Cattle

Flies

Cyhalothrin

Backliner Hand-jetting Hand-jettinga Plunge/shower dip Plunge/shower dipa Spraya

Sheep (off-shears) Sheep (long-wool) Sheep (long-wool) Sheep Sheep Goats

Lice Lice Itch mites, keds, lice Keds, lice Itch mites, keds, lice Lice

Cypermethrin

Backliner

Sheep (off-shears) Sheep (long-wool) Cattle Cats, dogs Cattle, deer, dogs, goats, horses, sheep Sheep (long-wool)

Lice Blowfly strike prevention, lice Flies Fleas, ticks Flies, lice, ticks

Goats Sheep (off-shears) Cattle

Lice Keds, lice Flies, ticks

Eartagb Rinseb , shampoob Plunge/shower dipc , sprayc Shower dipd Deltamethrin

Backliner Plunge/shower dipe , spraye Pour-on

Fenvalerate

Eartag, spray Pour-on, spray

Spray

Blowflies, lice

Cattle (beef and dairy)

Flies, lice

Cattle Goats Pigs Sheep Cats, dogs Horses

Flies Lice Lice, mites Keds, lice Fleas, ticks Biting insects, flies, lice, ticks Fleas, ticks Ticks Flies, ticks Ticks Mites

Flumethrin

Collarf , medallionf Plunge dip, spray Pour-on Spray Strips suspended in beehives

Dogs Cattle Cattle Horses Bees

Fluvalinate

Strips suspended in beehives

Bees

Mites

Permethrin

Aerosol sprayg Aerosol sprayb

Birds Cattle, horses Dogs Cats, dogs Cats, dogs Horses

Lice Biting insects, flies Fleas, lice, ticks Fleas Fleas, lice, ticks Flies, ticks

Cattle Goats, sheep Cats, dogs Horses Pigs Dogs

Flies, ear ticks Flies, keds, lice, and ticks Fleas, ticks Biting insects, flies Flies, lice, mites, ticks Fleas, lice, ticks

Pigs

Flies, lice, mites, ticks

Collar Dusting powder Dusting powder, spray, strips, wipes Eartagh Pour-on Shampoob,i Sprayb,j Spray, paint, dip Spot-on, pump sprayb,k , rinseb,k Phenothrin

Dip, dusting powder, paint, spray

Prallethrin

Dusting powder

Cats, dogs

Fleas, ticks

Resmethrin

Shampoo, spray Wipe

Cats, dogs Horses

Fleas, ticks Biting insects, flies

+rotenone. +piperonyl butoxide. c +chlorfenvinphos. d +diazinon. e +ethion. f +propoxur. g +piperonyl butoxide + methoprene. h +chlorpyrifos + piperonyl butoxide. i +piperonyl butoxide + melaleuca oil. j ±synergists (e.g., piperonyl butoxide, N-octyl bicycloheptene dicarboxamide) + repellents (e.g., dibutyl phthalate, diethyltoluamide). k +pyriproxyfen. a b

64

Table 4. Organochlorine Compounds Used as Ectoparasiticides on Animals Formulation Type/ Application Method

Compound

Animal

Target Pest

Lindane

Collar, medallion Spray

Cats, dogs Horses

Fleas Ear ticks

Methoxychlor

Backrubber Dusting powder

Cattle (beef) Cattle (beef and dairy) Dogs Goats, sheep Pigs Cattle (beef) Goats Pigs Sheep Horses

Flies Flies Fleas, ticks Keds, lice Lice Flies, lice, ticks Keds, lice Lice Keds, lice Flies, lice

Spray

Spray, wipe

Table 5. Carbamates Used as Ectoparasiticides on Animals Compound


Animal

Target Pest

Bendiocarb

Collar Dust bag Pour-on

Dogs Cattle (beef and dairy) Cattle (beef and dairy)

Fleas, ticks Flies Lice

Carbaryl

Aerosol spraya Collar Dusting powder Dusting powdera,b Dusting powderc Ear dropsd Lotione Rinsed Shampoo

Cats Cats, dogs Birds Cats, dogs Cats, dogs Poultry Cats, dogs Horses Cats, dogs Cats, dogs

Fleas, lice Fleas Lice, mites Fleas, lice, mites, ticks Fleas, lice, mites, ticks Lice, mites, ticks Ear mites Biting insects, lice, mites Fleas, lice, mites, ticks Fleas, lice, mites, ticks

Fenoxycarb

Spray

Cats, dogs

Fleas

Promacyl

Hand-spray Plunge/spray dip, handspray

Horses Cattle

Flies, ticks Flies, ticks

Propoxur

Collar Collarf , medallionf Dusting powder Spray

Cats Dogs Cats, dogs Cats, dogs

Fleas, ticks Fleas, ticks Fleas Flea, ticks

Thiram

Powder (added to plunge and shower dips to prevent post-dipping lameness)

Sheep

Erysipelothrix rhusiopathiae

+salicylic acid. +piperonyl butoxide + pyrethrins. c +malathion. d +antibacterial and antifungal agents. e +sulphur + zinc oxide. f +flumethrin. a b

65

Table 6. Organophosphorus Compounds Used as Ectoparasiticides on Animals Compound


Animal

Target Pest

Azamethiphos

Spray

Poultry

Lice, mites

Chlorfenvinphos

Aerosol spraya Backrubber Medicated dressing Over-spray Plunge/shower dipb , sprayb Rinse Sprayb

Sheep Cattle Cattle, horses, sheep Cattle Cattle, goats, sheep

Flystrike dressing Flies Wound dressing Flies Flies, lice, ticks

Dogs Deer, horses

Flies, lice, ticks Flies, lice, ticks

Collar Eartag Rinse Soap Spray

Cats, dogs Cattle Dogs Horses Dogs Dogs

Fleas, ticks Lice Fleas, lice, ticks Biting insects, lice Fleas Fleas, ticks

Aerosol spray

Cattle

Chlorpyrifos

Coumaphos

Backrubber Dip, spray

Cattle Cattle

Dust bag Dusting powder, spray

Cattle Horses

Flystrike, screw-worms, ticks Flystrike, screw-worms Flies, grubs, lice, ticks Flies, lice, mites, screw-worms, ticks Flies, lice Biting insects, lice

Cythioate

Oral drops, tablet Oral drops, tablet

Cats Dogs

Fleas Fleas, mites, ticks

Diazinon

Backliner

Sheep (long-wool)

Backrubber, eartag, rubbing post, spray Collar Collarc , medallionc Dusting powderd

Cattle

Blowfly strike prevention, lice Flies, lice

Hand-jetting, plunge/shower dip Medicated dressing Plunge/shower dipe

Sheep

Plunge/shower dipf

Goats Sheep

Rinse Spray

Dogs Goats Pigs Horses

Flystrike dressing Flies, itch mites, keds, lice Lice Blowflies, itch mites, lice, mites Fleas, lice, mites, ticks Lice Lice, mites Lice, flies

Backrubber, spray Collar Oral pasteg Plunge/shower diph , sprayh Pour-on

Cattle Cats, dogs Horses Cattle

Flies, lice, ticks Fleas, ticks Botfly larvae Flies, ticks

Cattle

Grubs, lice

Pour-on, spot-on Spot-on

Cattle Dogs

Grubs, lice Fleas

Goats, pigs, sheep

Spray, swab Dichlorvos

Ethion Famphur Fenthion

Cats, dogs Cats, dogs Cattle, goats, pigs, sheep

Sheep Sheep

Fleas Fleas, ticks Flystrike/wound dressing Blowflies, keds, lice

(continued overleaf )

66

Table 6. (Continued) Formulation Type/ Application Method

Compound Malathion (maldison)

Phosmet

Propetamphos

Temephos

Tetrachlorvinphos

Trichlorfon (metrifonate)

Animal

Target Pest

Dusting powder, spray

Horses

Biting insects, lice, ticks Lice, mites Fleas, lice, mites, ticks

Rinse, spray Rinse, swab

Birds Cats, dogs

Backrubber Dip, dust Dip, pour-on, spray

Cattle Dogs Cattle

Spray

Pigs

Flies, lice, mites, ticks Fleas, mites, ticks Flies, grubs, lice, mites, ticks Lice, mites

Hand-jetting, plunge/shower dip Dressing fluid

Sheep

Blowflies, keds, lice

Sheep

Flystrike/wound dressing

Dusting powder, rinse Hand-jetting, plunge/shower dip Pour-on

Cats, dogs Sheep

Fleas Lice

Cattle

Lice

Backrubber, dust bag, spray Dust bag Dusting powder, spray Eartag Oral larvicide

Cattle

Flies, lice, ticks

Cattle Pigs Cattle Cattle, pigs

Flies, lice Lice Flies Flies

Fish tank medicationi

Aquarium fish

Oral drench Oral granules/pelletsj Oral pasteg,k

Cattle Horses Horses

Anchor worms, copepods, fish lice, flukes GI roundworms Botfly larvae Botfly larvae

GI = gastrointestinal. a +dibutyl phthalate. b +cypermethrin. c +pyriproxyfen. d +pyrethrins + piperonyl butoxide. e +rotenone. f +amitraz. g +oxfendazole. h +deltamethrin. i +formaldehyde solution + methylene blue. j +mebendazole. k +febantel.

Table 7. Formamidines Used as Ectoparasiticides on Animals Compound Amitraz

Formulation Type/ Application Method Collar Plunge dip, spray Plunge/shower dipa Shampoo Spray

a

Animal Dogs Cattle (beef and dairy) Goats Sheep Dogs Pigs Some circus animals, deer, goats, sheep

+diazinon.

67

Target Pest Ticks Lice, mites, ticks Lice, mites Blowflies, itch mites, lice Mites, ticks Lice, mites Ticks

68

ANIMAL HEALTH PRODUCTS Table 8. Insect Growth Regulators and Insect Development Inhibitors Used as Ectoparasiticides on Animals Compound Cyromazine


Animal

Target Pest

Dressing Hand-jetting, plunge/shower dip Oral larvicide Tableta

Sheep Sheep

Flystrike Flystrike prevention

Cattle, poultry Dogs

Manure-breeding flies Heartworm prophylaxis

Dicyclanil

Spray-on

Sheep (off-shears, short-wool, long-wool)

Flystrike prevention

Diflubenzuron

Oral larvicide, sustained-release bolus Hand-jetting, plunge/shower dip

Cattle

Manure-breeding flies

Sheep (short-wool and long-wool)

Flystrike prevention, lice

Fenoxycarb

Spray

Cats, dogs

Fleas

Fluazuron

Pour-on

Beef cattle

Ticks

Lufenuron

Injection Oral suspension Tablet Tabletb

Cats Cats Dogs Dogs

Fleas Fleas Fleas Fleas, GI roundworm, heartworm prophylaxis

Methoprene

Aerosol sprayc Aerosol sprayb Collar Feed premix sustained-release bolus Rinsee

Birds Cats, dogs Cats, dogs Cattle

Lice Fleas, ticks Fleas Manure-breeding flies

Cats, dogs

Fleas, ticks

Collar Collare , medallione Shampoo

Cats Dogs Cats, dogs

Fleas Flea, ticks Fleas

Pyriproxyfen

Teflubenzuron

Oral premix

Salmon

Sea lice

Triflumuron

Backliner

Sheep (off-shears)

Lice

GI = gastrointestinal. a +diethylcarbamazine. b +milbemycin oxime. c +permethrin + piperonyl butoxide. d +N-octyl bicycloheptene dicarboxamide + piperonyl butoxide + pyrethrins. e +diazinon.

Moreover, the claims shown in the tables may apply only to a specific life cycle stage of a parasite or may not differentiate between efficacy claims for ‘‘controls’’ and ‘‘aids in the control of.’’ The label should be consulted for definitive information. Finally, it is most important that the use directions, precautions, warnings, and withholding period(s) on the label are observed at all times. BOTANICAL INSECTICIDES Azadirachtin Kernel extracts from the Indian neem tree (Azadirachta indica) have insecticidal and insect-repellent properties.

The key active ingredient is azadirachtin, a nortriterpenoid that exhibits insect growth regulator effects but no adulticidal activity. Azadirachtin is rapidly degraded by light, oxidation, and alkalinity (59) and is costly to extract and difficult to synthesise (60). The repellent, antifeedant, oviposition inhibitory, and sterilant effects of neem extracts tend to be of short duration and only apply to specific insects under certain conditions (61). Although azadirachtin is registered as an insect growth regulator for food crop use in many countries, studies with azadirachtin on animal ectoparasites have been limited to the sheep biting louse Bovicola ovis (62), the sheep blowfly Lucilia cuprina (63), and the cat flea Ctenocephalides felis (64).

Table 9. Macrocyclic Lactones Used as Endectocides on Animals Compound Abamectin

Doramectin

Eprinomectin

Ivermectin

Formulation Type/ Application Method Injection

Animal

Target Pest

Cattle

GI roundworms, lice, lungworms, ticks

Pigs

GI roundworms, kidney worms, lice, lungworms

Sheep

GI roundworms, itch mites, lungworms, nasal bots

Oral drencha

Horses

Botfly larvae, Draschia spp., GI nematodes, Habronema spp., lungworms, tapeworms

Oral drench

Sheep


Oral drencha,b

Sheep

GI roundworms, itch mites, lungworms, nasal bots, tapeworms, selenium deficiency

Oral paste

Horses

Botfly larvae, Draschia spp., GI roundworms, Habronema spp., lungworms

Oral pastea

Horses

Botfly larvae, Draschia spp., GI roundworms, Habronema spp., lungworms, tapeworms

Pour-on

Cattle

GI roundworms, lice, lungworms, ticks

Injection

Cattle

GI roundworms, grubs, lungworms, lice, mites, ticks

Pigs

GI roundworms, kidney worms, lice, lungworms, mites

Oral drench

Sheep

GI roundworms, lungworms

Pour-on

Cattle

Eyeworms, flies, GI roundworms, lice, lungworms, mites, ticks

Pour-on

Cattle (beef and dairy)

Flies, GI roundworms, lice, lungworms, mites, ticks

Deer

GI roundworms, lungworms

Cats

Heartworm prophylaxis, hookworm

Chewable tablets

Dogs

Heartworm prophylaxis

Chewable tabletsc

Dogs

GI roundworms, heartworm prophylaxis

Controlled-release capsule

Sheep


Hand-jetting

Sheep (long-wooled)

Blowflies, lice

Injection

Cattle

Eyeworms, GI roundworms, grubs, lice, lungworms, mites, screw-worm flies, ticks

Injectiond

Cattle

Eyeworms, GI roundworms, grubs, lice, liver flukes, lungworms, mites, screw-worm flies, ticks

Injection

Pigs

GI roundworms, kidney worms, lice, lungworms, mites

Oral drench

Sheep


Oral paste, tubing liquid

Horses

Botfly larvae, GI roundworms, Habronema spp., lungworms, Onchocerca spp. (continued overleaf )

69

Table 9. (Continued) Compound

Milbemycin oxime

Moxidectin

Selamectin


Animal

Target Pest

Pour-on

Cattle

Flies, GI roundworms, grubs, lice, lungworms, mites, ticks GI roundworms, lungworms GI roundworms, kidney worms, lice, lungworms, mites GI roundworms, grubs, lice, lungworms, mites

Premix

Deer Pigs

Slow release bolus

Cattle

Tablet

Dogs

GI roundworms, heartworm prophylaxis

Tablete

Dogs

Fleas, GI roundworms, heartworm prophylaxis

Injection

Cattle Sheep

Injectionf

Sheep

Oral drench Oral gel

Sheep Horses

Pour-on Tablet

Cattle Deer Dogs

GI roundworms, lice, lungworms, ticks GI roundworms, lungworms, mites, nasal bots Corynebacterium pseudotuberculosis and clostridial diseases, GI roundworms, lungworms, mites, nasal bots GI roundworms, lungworms, mites Botfly larvae, GI roundworms, Habronema spp., Onchocerca spp. GI roundworms, lice, lungworms, ticks GI roundworms Heartworm prophylaxis

Spot-on

Cats

Fleas, ear mites, heartworm prophylaxis, hookworms, roundworms Fleas, ear mites, heartworm prophylaxis, mites

Dogs Spinosad

Dressing Hand-jetting

Sheep (long-wool) Sheep (long-wool)

Flystrike Flystrike prevention, lice

GI = gastrointestinal. a +praziquantel. b +selenium. c +pyrantel embonate. d +clorsulon. e +lufenuron. f +6-in-1 Corynebacterium pseudotuberculosis and Clostridial spp.vaccine.

Table 10. Nitromethylene (chloronicotinyl) Compounds Used as Ectoparasiticides on Animals Compound Imidacloprid Nitenpyram


Animal

Target Pest

Spot-on Tablet

Cats, dogs Cats, dogs

Fleas Fleas

Table 11. Phenylpyrazole Compounds Used as Ectoparasiticides on Animals Compound Fipronil

Formulation Type/ Application Method Spot-on

Animal Cats Dogs Cats, dogs

Spray

70

Target Pest Fleas Fleas, ticks Fleas, ticks


71

Table 12. Inorganic Compounds Used as Ectoparasiticides on Animals Formulation Type/ Application Method

Compound

Animal

Target Pest

Magnesium fluorosilicate + sulphur

Dusting Plunge/shower dipa

Sheep Sheep

Itch mites, lice Itch mites, keds, lice

Sublimed sulphur

Dusting powdera

Beef cattle, cats, dogs, goats, horses, poultry

Fleas, lice, mites

Sulphur

Lotionb

Dogs, horses

Biting insects, lice, mites

a b

powdera

+rotenone. +carbaryl + zinc oxide.

Table 13. Miscellaneous Compounds Used as Ectoparasiticides on Animals Compound Benzyl benzoate


Animal

Target Pest

Lotion

Dogs

Ear mites, mites

Rinse

Birds

Scaly face, scaly leg (Cnemidocoptes pilae)

Chemical structure:

Chemical structures: CH3

O H C H3C

C C

O

O

COOCH3 OH O CH3

CH3

CH3COO CH3OOC

H CH3

H

OH O H

OH

H O

Mol wt: 720.7 Empirical formula: C35 H44 O16 Form: Yellow-green powder with a strong garlic/sulphur odor Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals Registered uses: Nil Citrus Extracts Several insecticidal substances are extracted from fresh peels of citrus fruits. The most important of these is d-limonene; another is linalool. D-limonene, which is a volatile oil, constitutes approximately 98% of orange peel oil by weight and has moderately good knockdown activity against ectoparasites of companion animals. The insecticidal activity of both d-limonene and linalool is enhanced when synergized by piperonyl butoxide. Apart from toxicoses reported in cats (65), d-limonene generally has a high margin of safety.

CH2

C

CH3

CH2 H

O

OH CH

CH2 C

C CH2 H3C Limonene

C H3C

CH3 Linalool

Mol wt: 136.2 (d-limonene); 154.3 (linalool) Empirical formula: C10 H16 (d-limonene); C10 H18 O (linalool) Form: Clear, colorless, mobile liquid with a pleasant citrus fragrance (d-limonene); a liquid (linalool) Acute oral LD50 for rats: >5,000 mg/kg (d-limonene); >2,790 mg/kg (linalool) CODEX ADIs and MRLs: Not established. No products are registered for use on food-producing animals Registered uses: Citrus derivatives are formulated as shampoos and rinses for control of fleas, lice, mites, and ticks on cats (the active ingredient is linalool) and dogs (the active ingredient is d-limonene or linalool). Nicotine Nicotine is an alkaloid obtained from the dried leaves of Nicotiana tabacum and Nicotiana rustica. Nicotine stimulates acetylcholine receptors of the postsynaptic membrane at nerve synapses resulting in depolarization of the membrane. Toxic doses cause stimulation that is rapidly followed by blockade of nerve transmission.

72


Chemical structure:

CH3 N

N

Mol wt: 162.2 Empirical formula: C10 H14 N2 Form: Colorless liquid (darkens rapidly on exposure to light and air) Acute oral LD50 for rats: 50-60 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals Registered uses: Nil (nicotine is no longer used as an insecticide against ectoparasites of animals) Pyrethrins The pyrethrins are the active ingredients in pyrethrum extract, obtained when the flower heads of the pyrethrin flower Chrysanthemum cinerariaefolum are extracted with solvent. They are natural insecticidal esters of chrysanthemic acid and pyrethric acid. Natural pyrethrins include pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I, and jasmolin II. The chrysanthemates (pyrethrin I, cinerin I, and jasmolin I) are generally more potent for insecticidal kill, whereas the pyrethrates (pyrethin II, cinerin II, and jasmolin II) cause more rapid knockdown. When combined with synergists, the pyrethrins are effective at low doses in causing knockdown and kill of a wide variety of pests. Pyrethrins exert their effects primarily by acting on sodium channels in nerves to disturb nerve conductance (see the section on Synthetic Pyrethroids for additional details). Two distinct effects, referred to as type I and type II, have been defined for pyrethrins. Type I effects are characterized by repetitive discharges, whereas type II effects are characterized by membrane depolarization in the absence of repetitive discharges. Clinical signs for dermal, oral, and inhalational routes of pyrethrin exposure include depression, hypersalivation, muscle tremors, vomiting, ataxia, dyspnea, and anorexia. The pyrethrins have low toxicity to mammals, and death after exposure to pyrethrins is rare. Their lability in light and air leads to a lack of residual activity and the need for repeated applications. This has restricted the use of the natural pyrethrins in the animal health sector. Chemical structure:

H3C C R

Rotenone The principal source of rotenone is the tuber root of Derris elliptica; however, it is also extracted from the roots of Derris mallaccensis, Lonchocarpus utilis, and Lonchocarpus uruca. Rotenone is both a stomach and contact poison for arthropods. Its fast knockdown action is attributed to decreasing the availability of nicotinamide adenine dinucleotide to serve as a cofactor in various biochemical pathways including the Krebs cycle, thereby inhibiting the mitochondrial respiratory enzymes. Chemical structure:

CH3

H

C H O H CH3O

O

O

CH2

O

OCH3 Mol wt: 394.4 Empirical formula: C23 H22 O6 Form: Colorless crystals Acute oral LD50 for rats: 132-1,500 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 2 SYNTHETIC PYRETHROIDS

H

CH3 H

Mol wt: 328.4 (pyrethrin I); 372.4 (pyrethrin II); 316.4 (cinerin I); 360.4 (cinerin II); 330.4 (jasmolin I); 374.5 (jasmolin II) Empirical formula: C21 H28 O3 (pyrethrin I); C22 H28 O5 (pyrethrin II); C20 H28 O3 (cinerin I); C21 H28 O5 (cinerin II); C21 H30 O3 (jasmolin I); C22 H30 O5 (jasmolin II) Form: Pale yellow viscous oils (as the crude extracts; dark brown) or tan dusts (as ground flowers) Acute oral LD50 for rats: 2,370 mg/kg (males); 1,030 mg/kg (females) CODEX ADI and MRLs: ADI = 0.04 mg/kg bw per day (set in 1972 and confirmed at periodic review in 1999 by JMPR). Animal commodity MRLs have not been established. Registered uses: See Table 2

CH3

C H

CO2

CH2

HH CH3

C O

R = CH3 or CO2CH3 R1 = CH=CH2 or CH3 or CH2CH3

Chemistry

C

R1

H

Pyrethroids are structural analogues related to the natural pyrethrins. They are generally esters in which both the alcohol (e.g., 1-cyano-3-phenoxybenzyl alcohol, allethrolone) and the carboxylic acid (e.g., chrysanthemic acid, substituted chrysanthemic acid, 2-(4-chlorophenyl)-3methylbutyric acid) may have asymmetric center(s). Most


pyrethroids contain two or three asymmetric centers and, therefore, contain four or eight stereoisomers. The isomers vary considerably in insecticidal activity. The use of natural pyrethrins in agriculture was prevented by their lability in light and air. The main environmental reactions occurring in pyrethroids are: 1) R/S epimerisation, 2) trans/cis isomerisations, 3) reductive dehalogenation including those encountered with halovinyl substituents, 4) photoelimination of carbon dioxide, 5) hydrolytic cleavage of ester and ether bonds, 6) oxidation of the parent pyrethroids and the cleavage products, and 7) dimerization of free radicals (66). The discovery of permethrin in 1973 provided the first evidence that the replacement of light-unstable centers in both the acid and alcohol portions of the pyrethrins by light-stable groups of otherwise similar steric and chemical properties permitted the use of the pyrethroids in agriculture (67). Subsequent efforts in the design of synthetic analogues focused on achieving high levels of insecticidal activity while minimizing costs of synthesis and retaining desirable levels of selective toxicity. Four generations of pyrethroids are now recognized. First generation pyrethroids include allethrin, which was synthesized in 1949. The second-generation pyrethroids include tetramethrin (1965), resmethrin and bioresmethrin (1967), bioallethrin (1969), and phenothrin (1973). They are sensitive to sunlight. The third-generation pyrethroids include fenvalerate (1972) and permethrin (1973). They are stable in sunlight. The fourth-generation pyrethroids include cypermethrin, alpha-cypermethrin, cyfluthrin, cyhalothrin, lambda cyhalothrin, fluvalinate, tralomethrin, and bifenthrin. All of the fourth-generation pyrethroids are photostable. Mode of Action The cellular effects of pyrethrin and pyrethroid insecticides have been postulated to involve interactions with various membrane-related structures including sodium channels, receptor-ionophore complexes, neurotransmitters, and adenosine triphosphatase (for a review, see reference 68). The disturbance of nerve conductance caused by changes in the permeability of sodium channels on nerve membranes is the major mechanism by which synthetic pyrethroids exert their effects and is the only mechanism that will be discussed here. The categorization of pyrethroid actions into type I and type II reflect the different poisoning symptoms and nerve disruptions that are principally attributed to modifications in the activity of sodium channels (69). During normal membrane depolarization, sodium channels open and permit an influx of sodium ions into the nerve axon. Inactivation of the action potential occurs as sodium conductance decreases. Potassium channels in the membrane open at the peak of the action potential so that potassium can move out of the cell. Finally, energy-dependent sodium and potassium pumps return the membrane to the normal resting state. With type I pyrethroids, such as allethrin and tetramethrin, perturbations in sodium conductance result in repetitive discharges. In the process, sodium influx is prolonged, and both the peak sodium current and the steady-state potassium efflux are

73

decreased. The opening of sodium channels is slower than normal, suggesting that sodium channels are affected at their resting or closed states. By comparison, type II pyrethroids such as cypermethrin and fenvalerate act on nerve axons to enhance sodium conductance in the absence of repetitive discharges. Moreover, the amplitude of action potentials is decreased. Type II pyrethroids also act presynaptically by inhibiting inactivation of voltage-dependent sodium channels and post-synaptically by interacting with the nicotine, acetylcholine, and γ -aminobutyric acid receptors. Pharmacokinetics and Metabolic Fate Pyrethroids are lipophilic molecules that generally undergo rapid absorption and distribution following ingestion by mammals and birds. Pyrethroids that are not retained in fat depots are rapidly metabolized and eliminated from the body. In the case of permethrin, the trans isomer is metabolized faster than the cis isomer, with the latter being more persistent in fat, milk, and eggs. Permethrin used in animal health products is generally a 40:60 or 25:75 (cis:trans) isomeric mixture. The enantiomeric pyrethroids, in general, demonstrate pharmacokinetic behavior similar to that of cis- and transpermethrin. All pyrethroids are metabolized by ester hydrolysis and oxidation to metabolites that are excreted as alcohols, phenols, or carboxylic acids and their glycine, sulphate, glucuronide, or glucoside conjugates (69). Interspecies differences in the metabolic pathways of pyrethroids are relatively small although differences do occur in the sites of oxidation and the types of conjugates formed. For example, 4 -hydroxy permethrin and permethrin hydroxylated at the cis- and/or trans-methyl group are important metabolites in chickens, rats, goats, and cows (70–73), whereas glutamic acid conjugates of the acid moiety of 3-phenoxybenzoic acid are important metabolites in cows and goats (71,73). Interspecies differences also occur with the acid moiety of fluvalinate, the conjugation of which involves cholic acid in cows and taurochenodeoxycholic acid in chickens (74) (taurochenodeoxycholic acid is a bile salt formed in the liver by conjugation of chenodeoxycholate with taurine; it acts as a detergent to solubilize fats in the small intestine). Esterase and oxidase inhibitors are used as synergists (see also the section on Synergists) because they increase the potency of pyrethroids. Toxicity Compared with other classes of insecticides, the pyrethroids demonstrate more favorable selectivity for insects over mammals. The respective rat oral LD50 /insect topical LD50 ratios are: methylcarbamates 16, organophosphates 33, organochlorines 91, and pyrethroids 4,500 (39). Nevertheless, the toxicity of pyrethroids to mammals varies enormously with the isomer mixture, formulation, and solvent in which they are applied. In rats, type I pyrethroids generally produce a tremors syndrome that is manifested by aggressive sparring, hyperesthesia, tremors, and prostration. Type II

74


pyrethroids cause a choreoathetosis (jerky and/or rhythmic involuntary movements) and salivation syndrome that is manifested by pawing and burrowing behavior, hypersalivation, and coarse tremors that progress to choreoathetosis and clonic seizures. The clinical signs of synthetic pyrethroid toxicity in animals include vomiting, hypersalivation, muscle tremors, depression, seizures, anorexia, ataxia, and diarrhea. The period of onset of clinical signs typically takes several hours but varies with the compound and the route of exposure. Death after exposure to pyrethroids is rare. Humans dermally exposed to synthetic pyrethroids have reported stinging or burning of the skin that progresses to numbness. Diazepam and mephenesin ameliorate the toxic effects of pyrethroids possibly by facilitating inhibitory pathways. Allethrin Chemical structure:

CH3

O H H3C C

CH3 C

H3C

CH2 CH

H

CH

Chemical structure:

CF3

H C

C Cl

O CH3 C H

H

CN CH

O

O

CH3 Mol wt: 449.9 Empirical formula: C23 H19 ClF3 NO3 Form: Yellow to brown viscous oil (technical grade) Acute oral LD50 for rats: 166 mg/kg (males); 114 mg/kg (females) CODEX ADI and MRLs: ADI = 0.02 mg/kg bw per day (JMPR 1984). Animal commodity MRLs have not been established (evaluated at the 54th Meeting of JECFA) Registered uses: See Table 3 Cypermethrin

CH2

O

Cyhalothrin

O

Chemical structure:

O

Cl

CH3

H C

C

CH3

C

CH

O

O

Cl Mol wt: 302.4 Empirical formula: C19 H26 O3 Form: An orange-yellow viscous liquid Acute oral LD50 for rats: 709 mg/kg (males); 1,042 mg/kg (females) CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals Registered uses: See Table 3

CN

CH3

Cyfluthrin

Mol wt: 416.3 Empirical formula: C22 H19 Cl2 NO3 Form: Odorless crystals (pure); yellow-brown viscous semisolid at ambient temperatures (technical grade) Acute oral LD50 for rats: 250-4,150 mg/kg (pure); 7,180 mg/kg (technical grade) CODEX ADI and MRLs: See Table 1 Registered uses: See Table 3

Chemical structure:

Deltamethrin

H

Cl C Cl

CH3 C

C H

Chemical structure:

CN

O

CH

O

O

C

H CH3

O

Br

F

H C

Br

CH3 C H

H

CN C O H

CH3 Mol wt: 434.3 Empirical formula: C22 H18 Cl2 FNO3 Form: Viscous, partly crystalline, amber oil Acute oral LD50 for rats: ca. 500 mg/kg (in xylol); ca. 900 mg/kg (PEG 400) CODEX ADI and MRLs: See Table 1 Registered uses: See Table 3

Mol wt: 505.2 Empirical formula: C22 H19 Br2 NO3 Form: Colorless crystals Acute oral LD50 for rats: 135- >5,000 mg/kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 3

O


Fenvalerate

Form: Viscous amber oil with a moderate or weak sweetish odor (technical grade) Acute oral LD50 for rats: >3,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 3

Chemical structure:

CN

O

Cl

CH

C

O Permethrin

O

CH

75

Chemical structure:

CH CH3

CH3

O

Cl

Mol wt: 419.9 Empirical formula: C25 H22 ClNO3 Form: Viscous yellow or brown liquid, sometimes partly crystalline at room temperature (technical grade) Acute oral LD50 for rats: 151 mg/kg CODEX ADI and MRLs: ADI = 0.02 mg/kg bw per day (JMPR 1986). Animal commodity MRLs have been established based on residues in feed, not on direct animal treatment. Registered uses: See Table 3 Flumethrin

H C

C

CH3

C O

Cl

CH2

O

CH3 Mol wt: 391.3 Empirical formula: C21 H20 Cl2 O3 Form: Yellow-brown to brown liquid, which sometimes tends to crystallize at room temperature (technical grade) Acute oral LD50 for rats: 430-4,000 mg/kg (cis:trans ca. 40:60); ca. 6,000 mg/kg (cis:trans ca. 20:80) CODEX ADI and MRLs: See Table 1 Registered uses: See Table 3

Chemical structure: Phenothrin

CN

O CH3 C

H Cl C

CH O

Chemical structure:

O

CH3

O

H

CH

F

CH3

C

CH3

CH

C O

CH3

CH2

O

CH3

Cl Mol wt: 510.4 Empirical formula: C28 H22 Cl2 FNO3 Form: Yellowish, highly viscous liquid Acute oral LD50 for rats: 911 mg/kg (males); 662 mg/kg (females) (in arachis oil) CODEX ADI and MRLs: See Table 1 Registered uses: See Table 3 Fluvalinate

Mol wt: 350.5 Empirical formula: C23 H26 O3 Form: Pale yellow to yellow–brown clear liquid with a faint characteristic odor Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: ADI = 0.07 mg/kg bw per day for d-phenothrin (JMPR 1988). Animal commodity MRLs have not been established. Registered uses: See Table 3 Prallethrin

Chemical structure:

Chemical structure:

CN

O Cl O

CH CF3

CH

C

NH

CH3

CH3

H

C

C

CH3 CO2

CH3

Mol wt: 502.9 Empirical formula: C26 H22 ClF3 N2 O3

CH2C

HH

CH3

CH CH3

O

CH3 Mol wt: 300.4 Empirical formula: C19 H24 O3

O

CH

76


Form: Yellow to yellow-brown liquid Acute oral LD50 for rats: 640 mg/kg (males); 460 mg/kg (females) CODEX ADI and MRLs: Not established Registered uses: See Table 3 Resmethrin Chemical structure:

ORGANOCHLORINE COMPOUNDS

O

CH3 C

CH

CH3

C O

CH3

CH2

CH2 O

CH3

Mol wt: 338.4 Empirical formula: C22 H26 O3 Form: Colorless crystals (pure); yellow-brown waxy solid (technical grade) Acute oral LD50 for rats: >2,500 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 3 Tetramethrin Chemical structure:

CH3

CH

O C O

CH3

CH2 N

CH3 O Mol wt: 331.4 Empirical formula: C19 H25 NO4 Form: Colorless crystals with slight pyrethrum-like odor Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: Nil Tralomethrin Chemical structure:

Br CH Br3C

O CH3 C H

H

The organochlorine compounds fall into three groups based on their chemical structures. These are: 1) the diphenyl aliphatic group that includes DDT and methoxychlor; 2) the cyclodiene group that includes aldrin, chlordane, dieldrin, endosulfan, and heptachlor; and 3) a group of heterogeneous compounds without a common structured feature such as lindane, mirex, and toxaphene. The organochlorine pesticides are used primarily as contact poisons; however, their use has declined in recent years, reflecting concerns about their persistence in the environment and bioaccumulation in the food chain. Many developed countries have legislated to restrict or ban the use of organochlorine compounds. The emergence of pest populations resistant to organochlorine compounds has also contributed to their declined use. Only lindane and methoxychlor will be considered here. Lindane

O

CH3 C

Form: Orange-to-yellow resinous solid (technical grade >39%) Acute oral LD50 for rats: 99-3,000 mg/kg depending on the carrier used CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: Nil

CN C O H

O

Cyclohexanehexachloride (HC) has five isomers named after the Greek letters alpha, beta, gamma, delta, and epsilon. Only the gamma isomer has insecticidal activity. Technical material containing ≥99% gamma isomer is known as lindane. Chemical structure:

Cl

Cl

Cl

Cl Cl

Cl

Mol wt: 290.8 Empirical formula: C6 H6 Cl6 Form: Colorless crystals Acute oral LD50 for rats: 88–270 mg /kg (values vary with the test conditions, especially the carrier) CODEX ADI and MRLs: Temporary (1997 to 2000) ADI = 0.001 mg/kg bw per day (JMPR 1997). Animal commodity MRLs have not been established. Registered uses: See Table 4

CH3 Methoxychlor Mol wt: 665.0 Empirical formula: C22 H19 Br4 NO3

Methoxychlor is a structural analogue of DDT but is not as persistent in the environment as DDT.


Chemical structure:

CCl3 CH CH3O

OCH3

Mol wt: 345.7 Empirical formula: C16 H15 Cl3 O2 Form: Colorless crystals; (technical grade, grey powder) Acute oral LD50 for rats: 6,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 4

CARBAMATES Chemistry The first carbamate esters to exhibit insecticidal activity were derivatives of dithiocarbamic acid. A few of these were contact poisons to soft-bodied insects (aphids), whereas others, including thiram, demonstrated antifeedant properties. In subsequent studies with physostigmine and related carbamate compounds, the charge of amine salts and quaternary structures was shown to prevent their penetration through the waxy cuticle and into the fatty nervous system of insects. Several substituted-phenyl monomethylcarbamates were shown to be effective contact toxins for aphids, flies, and thrips. Still later, the oxime carbamates were discovered and shown to be effective contact and systemic insecticides, nematocides and/or miticides. Mode of Action Acetylcholine is a neurotransmitter in cholinergic nerves. In insects, the neuromuscular junction is not cholinergic (unlike that in mammals), and the only known cholinergic synapses are in the central nervous system. The effect of acetylcholine is normally terminated by acetylcholinesterase-mediated hydrolysis leading to the formation of acetic acid and choline. Carbamates inhibit acetylcholinesterase by carbamylating the esteratic site of the enzyme. This results in continued stimulation of cholinergic synapses in the central nervous systems of insects, leading to death. Fenoxycarb is somewhat unusual in as much as it is a carbamate that does not inhibit acetylcholinesterase and instead inhibits the activity of juvenile hormone despite being structurally unrelated. Fenoxycarb is ovicidal and larvicidal at very low concentrations but is not adulticidal.

decomposes with loss of carbon dioxide to give an amine derivative. Numerous microorganisms in vitro have the ability to hydrolyze promacyl either at the amide bond to form promecarb or at the ester bond to form isothymol (16). McDougall and Makin (17) reported that microbial degradation accounts for seasonal declines observed in promacyl concentrations in cattle plunge dips and that stabilization of promacyl can be achieved by adjusting the dip wash pH to 10,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 5

N

S

C N

CH3

S

C

CH3

S

Mol wt: 240.4 Empirical formula: C6 H12 N2 S4 Form: Colorless crystals Acute oral LD50 for rats: 1,800 mg/kg CODEX ADI and MRLs: ADI = 0.01 mg/kg bw per day (JMPR 1992). Animal commodity MRLs have been established for dithiocarbamates, including thiram, but are based on residues in feeds and not direct treatment of animals. Registered uses: See Table 5

Promacyl Chemical structure:

CH3

CH3 CH

O

H3C

CH3

N C

C

O

O

CH2

CH3 CH2

ORGANOPHOSPHORUS COMPOUNDS Chemistry Organophosphorus insecticides are normally ester, amide, or thiol derivatives of phosphoric acid, phosphonic,


thiophosphoric, or dithiophosphoric acids. Given below are seven subclasses of organophosphorus compounds having varying combinations of oxygen, carbon, sulfur, and nitrogen attached to the phosphorous atom.

79

‘‘irreversible,’’ not because the enzyme inhibition itself is irreversible but because the intact organophosphorus ester molecule is not recovered with the enzymatic activity. Pharmacokinetics and Metabolic Fate

O O

P

S O

O

O

P

O O

O

Phosphate

Phosphorothioate

O

S

O

P

O

C Phosphonate

O

P

O

P

N

O Phosphoramidate O

O

C Phosphonothioate

O

P

S

O Phosphorothiolate

S O

P

S

O Phosphorodithioate These groups differ with respect to rates of hydrolysis and isomerization, and these chemical behaviors are reflected as different biological activities. The organophosphorus compounds that are widely used in animal health tend to be phosphates (chlorfenvinphos, dichlorvos, and tetrachlorvinphos), phosphorothioates (coumaphos, cythioate, diazinon, famphur, and fenthion), and phosphorodithioates (malathion and phosmet). The rate of hydrolysis of organophosphorus esters is a function of the nature of the acid and alcohol moieties, pH, and temperature. Due to their reactivity, the organophosphorus ester insecticides generally do not persist in the environment. Mode of Action The mode of action of the organophosphorus esters is similar to that described earlier for carbamates in as much as the primary effect involves the inhibition of acetylcholinesterase. Acetylcholine is a neurotransmitter in the central nervous system of insects, the action of which is terminated by acetylcholinesterase. The hydrolysis of acetylcholine by acetylcholinesterase normally involves acetylation of the serine hydroxyl group at the catalytic site of the enzyme with release of the choline moiety. The hydrolysis of acetylcholine is again catalyzed by acetylcholinesterase following hydrolysis of the acetylated form of the enzyme. The deacetylation half-life is about 0.15 ms. In the presence of an organophosphorus ester, acetylcholinesterase is phosphorylated at the serine hydroxyl group, and, because of the exceedingly slow rate of hydrolysis of the phosphorylated acetylcholinesterase, cholinergic stimulation at synapses continues unabated. The inhibition of acetylcholinesterase by organophosphorus esters is said to be

The metabolism of organophosphorus esters, which comprises both activation and detoxification reactions, is ascribed to a variety of enzyme systems including cytochrome P-450, glutathione transferases, A-esterases (such as paraoxonase), and B-esterases (such as carboxylesterase). An activation reaction that is particularly important is desulfuration of the phosphorothioates. An example of a P = S to P = O conversion is the oxidative desulfuration of parathion, which has little insecticidal activity, to paraoxon, which is a potent insecticide (77). The conversion of trichlorfon to dichlorvos in vivo is a different example. Carboxylesterases are thought to contribute to the detoxification of organophosphorus esters by acting as alternative phosphorylation sites, and, thereby, ‘‘protecting’’ acetylcholinesterase to some extent. The low mammalian toxicity of malathion is attributed to the rapid hydrolysis of its carboxylester groups by liver carboxylesterases. Toxicity A brief account of the relevant autonomic pharmacology is presented here as a basis for understanding the symptomatology of organophosphate toxicoses. The symptoms of toxicity result primarily from the inhibition of acetylcholinesterase. Acetylcholine is the neurotransmitter released by: 1) all preganglionic autonomic nerves (i.e., both sympathetic and parasympathetic); 2) all postganglionic parasympathetic nerves; 3) some postganglionic sympathetic nerves (i.e., thermoregulatory sweat glands and skeletal muscle vasodilator fibers); 4) the nerve to the adrenal medulla; 5) somatic motor nerves to skeletal muscle end-plates; and 6) some neurons in the central nervous system. The division of acetylcholine receptors (cholinoreceptors) into nicotinic and muscarinic sub-types is important because the antidote, atropine, blocks muscarinic but not nicotinic receptors. Muscarinic effects include accommodation for near vision, constriction of the pupils, profuse watery salivation, bronchiolar constriction, bronchosecretion, hypotension (due to bradycardia and vasodilatation), increases in gastrointestinal motility and secretion, contraction of the urinary bladder, and sweating. Nicotinic receptors occur in autonomic ganglia, in the adrenal medulla, and at the skeletal muscle neuromuscular junctions, and the action of acetylcholinesterase inhibition at these sites is relatively weak compared with its effect on muscarinic receptors. The clinical signs of organophosphate toxicity that are mediated via nicotinic receptors include twitching of facial and tongue muscles, progressing to generalized twitching, followed by paralysis. Clinical signs of toxicity involving the central nervous system include depression and tonic/clonic seizures. Death is generally due to respiratory failure. It is noteworthy that chlorpyrifos has caused toxic and lethal effects when administered to bulls (78). The clinical

80


symptoms appeared several days after chlorpyrifos administration and included dullness, inappetance, dehydration, rumen stasis, and rumen distention. In addition, diazinon has caused lethal reactions in companion animals and food-producing animals as a result of contamination with small quantities of water leading to the production of toxic degradation products. Organophosphorus esters also cause toxic effects unrelated to acetylcholinesterase inhibition. For example, wasting that occurs a week or more after treatment can progress to death and is attributable to byproducts of synthesis that are present in organophosphate formulations. Diazinon and malathion are teratogenic in the chicken embryo test, with the teratogenic signs being almost completely alleviated when these organophosphorus compounds are supplemented with nicotinamide or nicotinic acid (75). The most studied of the toxic effects that are unrelated to acetylcholinesterase inhibition is organophosphate-induced delayed neuropathy (OPIDN). This condition is characterized by a dying back of long myelinated nerve axons particularly in the sciatic nerve and within the spinal cord. The onset of symptoms is delayed 10 days or more following treatment, and the damage to individual axons appears to be irreversible. A nerve protein termed neurotoxic esterase or neuropathy target enzyme is the proposed target. Recovery and aging of phosphorylated acetylcholinesterase are two reactions that are important from a toxicity perspective. Recovery refers to the hydrolytic removal of the phosphoryl moiety responsible for regenerating the active enzyme; its rate is greatly enhanced by chemicals such as pralidoxime (2-PAM) that act as acceptors for a second transphosphorylation. Pralidoxime is used in the clinical management of organophosphate intoxications. Over time, however, phosphorylated acetylcholinesterase becomes refractory to chemical reactivation, a process referred to as aging. The aging mechanism is linked to the synthesis of monoalkyl phosphoryl-acetylcholinesterase, which is refractory to chemical reactivation. Azamethiphos Chemical structure:

O

O N

Chemical structure:

O

O P CH3CH2O CH3CH2O

P CH3CH2O CH3CH2O

Cl

O C

C Cl

H

O C

H

C Cl

Cl

Cl

Cl

(E)-isomer

(Z)-isomer

Mol wt: 359.6 Empirical formula: C12 H14 Cl3 O4 P Form: Colorless liquid; (technical grade, amber liquid) Acute oral LD50 for rats: 24-39 mg/kg CODEX ADI and MRLs: ADI = 0.0005 mg/kg bw per day (JMPR 1994); animal commodity MRLs have been revoked (JMPR 1999) Registered uses: See Table 6 Chlorpyrifos Chemical structure:

S P

O N

Cl

Cl

OCH2CH3 OCH2CH3

Cl Mol wt: 350.6 Empirical formula: C9 H11 Cl3 NO3 PS Form: Colorless crystals with a mild mercaptan odor Acute oral LD50 for rats: 135-163 mg/kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 6 Coumaphos Chemical structure:

CH2 O

Chlorfenvinphos

P S

OCH3 OCH3

S P

N Cl Mol wt: 324.7 Empirical formula: C9 H10 ClN2 O5 PS Form: Colorless crystals Acute oral LD50 for rats: 1,180 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 6

O

O

CH3CH2O CH3CH2O

O Cl

CH3 Mol wt: 362.8 Empirical formula: C14 H16 ClO5 PS Form: Colorless crystals Acute oral LD50 for rats: 41 mg/kg (males); 15.5 mg/kg (females)


81

CODEX ADI and MRLs: ADI (JMPR 1980) subsequently withdrawn. Animal commodity MRLs were not established. Registered uses: See Table 6

CODEX ADI and MRLs: ADI = 0.004 mg/kg per day (JMPR 1977, 1993). Meat and milk MRLs have been established based on residues in feed. Registered uses: See Table 6

Cythioate

Famphur

Chemical structure:

Chemical structure:

S P

SO2

O

CH3O CH3O

S

O

CH3

P OCH3 OCH3

N

NH2

SO2

H3C

Mol wt: 297.3 Empirical formula: C8 H12 NO5 PS2 Form: Crystalline solid Acute oral LD50 for rats: 160 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals Registered uses: See Table 6

Mol wt: 325.3 Empirical formula: C10 H16 NO5 PS2 Form: Colorless crystalline powder Acute oral LD50 for rats: 35 mg technical grade/kg (males); 62 mg technical grade/kg (females) CODEX ADI and MRLs: Not established Registered uses: See Table 6

Diazinon

Fenthion

Chemical structure:

Chemical structure:

CH3

S P CH3CH2O CH3CH2O

CH

N

O

S CH3

N

CH3

O

P

OCH3 OCH3

CH3S

CH3 Mol wt: 304.3 Empirical formula: C12 H21 N2 O3 PS Form: Clear colorless oil; (technical grade, yellow oil) Acute oral LD50 for rats: 300-400 mg /kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 6

Mol wt: 278.3 Empirical formula: C10 H15 O3 PS2 Form: Colorless oily liquid (technical grade, brown oily liquid with a mercaptan-like odor) Acute oral LD50 for rats: ca. 250 mg /kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 6

Dichlorvos

Malathion (Maldison)

Chemical structure:

Chemical structure:

O P CH3O CH3O

O

O O

Cl CH

CH3O

C Cl

Mol wt: 221.0 Empirical formula: C4 H7 Cl2 O4 P Form: Color liquid (technical grade, colorless-to-amber liquid with an aromatic odor) Acute oral LD50 for rats: ca. 50 mg /kg

CH3O H3C

CH2

C

S

CH

P S CH2

CH3 CH2 C

O

O

Mol wt: 330.3 Empirical formula: C10 H19 O6 PS2 Form: Clear amber liquid (technical grade) Acute oral LD50 for rats: 1,375-2,800 mg/kg

82


CODEX ADI and MRLs: ADI = 0.3 mg/kg bw per day (JMPR 1997). Animal commodity MRLs have not been established. Registered uses: See Table 6

CODEX ADI and MRLs: Not established (evaluated at the 54th Meeting of JECFA) Registered uses: See Table 6 Tetrachlorvinphos

Phosmet

Chemical structure:

Chemical structure:

O

O S P

CH2

CH3O CH3O

N

S

Cl

O C

C

Cl

O

CH3O CH3O

P

Cl

H

Cl

Mol wt: 317.3 Empirical formula: C11 H12 NO4 PS2 Form: Off-white, crystalline solid Acute oral LD50 for rats: 113 mg/kg (males); 160 mg/kg (females) CODEX ADI and MRLs: See Table 1 Registered uses: See Table 6

Mol wt: 366.0 Empirical formula: C10 H9 Cl4 O4 P Form: Colorless crystalline solid (technical grade) Acute oral LD50 for rats: 4,000-5,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 6 Trichlorfon (Metrifonate)

Propetamphos

Chemical structure:

Chemical structure:

CH3O CH2

O

S

CH3

O

P

C

C

O

C

OH

CH3

CH3O CH3O

CH

P

CH C

CH3

O

Cl

HN

Cl Cl

H

CH3

Mol wt: 281.3 Empirical formula: C10 H20 NO4 PS Form: Yellowish oily liquid (technical grade) Acute oral LD50 for rats: 119 mg/kg (males); 59.5 mg/kg (females) CODEX ADI and MRLs: Not established Registered uses: See Table 6

Mol wt: 257.4 Empirical formula: C4 H8 Cl3 O4 P Form: Colorless crystals with a weak characteristic odor Acute oral LD50 for rats: ca. 250 mg/kg CODEX ADI and MRLs: ADI (JMPR 1978) subsequently withdrawn. Animal commodity MRLs have not been established (evaluated at the 54th Meeting of JECFA) Registered uses: See Table 6

Temephos

FORMAMIDINES

Chemical structure:

Amitraz is the only formamidine pesticide used in animal health. The use of amitraz on cattle has increased markedly in those regions where severe resistance in cattle tick is prevalent. This is attributed firstly, to the compound’s efficacy against synthetic pyrethroid-resistant ticks and secondly to it being a low residue acaricide. Amitraz has been shown to be effective against Notoedres cati; however, it is not registered for use on cats.

H3CO H3CO

S

S

S

P

P O

O

OCH3 OCH3

Mol wt: 466.5 Empirical formula: C16 H20 O6 P2 S3 Form: Colorless crystals (technical grade, brown, viscous liquid) Acute oral LD50 for rats: 4,204 mg/kg (males); >10,000 mg/kg (females)

Chemistry The hydrolysis of amitraz occurs most rapidly in acid media, on exposure to sunlight, and at elevated temperatures. Hydrated lime is used to stabilize amitraz in dips.


Mode of Action The mechanism of action of amitraz has not been completely elucidated, and, presently, a dual mode of action appears most likely. Firstly, the enzyme monoamine oxidase, which metabolizes neurotransmitter amines in mites and ticks, is inhibited. Secondly, octopamine receptors in the central nervous system of ectoparasites are activated by amitraz, thereby modifying tonic muscle contractions. The effect of amitraz is to induce increased neuronal activity, abnormal behavior, detachment, and death of mites and ticks. Pharmacokinetics and Metabolic Fate Amitraz is poorly absorbed when applied topically to animals. By contrast, orally administered amitraz is rapidly and extensively absorbed. The metabolism and excretion of amitraz are also rapid. It is hydrolyzed to N-(2,4-dimethylphenyl)-N -methyl formamidine and 2,4dimethyl formamidine and the final product, 4-amino-3methylbenzoic acid, is converted to non-toxic conjugates. The latter are excreted in the urine and, to a lesser extent, in bile. Toxicity Amitraz displays serotonin (5-hydroxytryptamine) blocking activity and a2 -adrenoceptor agonist activity in animals. The clinical signs associated with intoxication in dogs include sedation, bradycardia, hypotension, hyperglycaemia, hypothermia, and mydriasis. The specific antidote for animal toxicity is the a2 -adrenoceptor antagonist, yohimbine. The toxicity profile of amitraz in the horse includes transient sedation and intestinal stasis that can progress to impaction colic (79). For this reason, amitraz is not approved for use in this species in any country. Amitraz Chemical structure:

CH3

CH3

N

N

N CH

CH3

CH

CH3

CH3

programs. It is important that users of insect growth regulators and insect development inhibitors understand that several weeks may elapse between application and when reductions in insect populations are expected to occur. Because cats and dogs suffering from flea allergy dermatitis remain fully symptomatic for days following a single flea bite, insect growth regulators and insect development inhibitors alone may provide inadequate protection. Nevertheless, these agents play an important role in killing life cycle stages of fleas in the environment of pets that are allergic to fleas. Insect growth regulators are juvenile hormone analogues that function in arthropods to prevent premature metamorphosis of larvae. Ablation of the corpora allata, a small cluster of cells located behind the brain that synthesizes juvenile hormone, induces early pupation and the emergence of dwarfed adults. Implantation of corpora allata cells from young to mature larvae has the opposite effect, with metamorphosis being postponed or suppressed. In veterinary medicine, insect growth regulators are represented by cyromazine, dicyclanil, fenoxycarb, methoprene, and pyriproxyfen. Insect development inhibitors are typically chitinsynthesis inhibitors. Chitin is an amino-sugar polysaccharide present in the exoskeleton of arthropods and is a major component of the chito-protein complex of the cuticle in insects. Inhibition of chitin synthesis may occur via interference of either the enzyme chitin synthetase or the polymerization step. Insect development inhibitors currently used in animal health include diflubenzuron, fluazuron, lufenuron, and triflumuron, all of which are benzoylphenyl urea compounds. Cyromazine Cyromazine, a triazine, is an insect growth regulator whose mode of action is postulated to prevent exuviation. Cyromazine shows a high specificity for dipteran larvae, whereas benzoylphenyl urea compounds usually act against a wide range of insects. When cyromazine is used as a sheep blowfly control agent, eggs laid by female flies on treated sheep hatch normally. However, the flystrike is arrested when larvae that have ingested cyromazine are unable to moult to the second instar. This explains the need to use cyromazine prophylactically and its negligible efficacy against existing strikes.

Mol wt: 293.4 Empirical formula: C19 H23 N3 Form: White/pale yellow crystalline solid Acute oral LD50 for rats: 600-800 mg/kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 7

Chemical structure:

INSECT GROWTH REGULATORS AND INSECT DEVELOPMENT INHIBITORS

Mol wt: 166.2 Empirical formula: C6 H10 N6 Form: Colorless crystals Acute oral LD50 for rats: 3,400 mg/kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 8

The insect growth regulators and insect development inhibitors interfere with the metamorphosis and reproduction of target arthropod pests. They have no adulticidal activity and are, therefore, used in strategic control

83

H2N

NH

N N

N NH2

84


Dicyclanil

Diofenolan

Dicyclanil is a pyrimidine derivative with insect growth regulator activity. It prevents egg hatching and is lethal against first and second instar stages of diptera larvae. In vivo studies against Lucilia cuprina larvae indicate that the potency of dicyclanil is 10-fold higher than the potency of cyromazine (80). The protection period from flystrike by Lucilia cuprina is 14 weeks for cyromazine and 18-24 weeks for dicyclanil. This difference is significant because it means that farmers in some regions of Australia are able to protect their flocks with a single annual treatment of dicyclanil, whereas this could not be achieved using cyromazine (80).

Chemical structure:

Chemical structure:

H2N

N

NH N

NC

CH3 CH2 O O

O CH2 O

Mol wt: 300.3 Empirical formula: C18 H20 O4 Form: Colorless liquid Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: Nil

NH2 Fenoxycarb Mol wt: 190.2 Empirical formula: C8 H10 N6 Form: White powder Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: Not established (evaluated at the 54th Meeting of JECFA) Registered uses: See Table 8 Diflubenzuron Diflubenzuron, a benzoylphenyl urea compound, is an insect development inhibitor. It is applied directly to the manure or indirectly by administration as a feed-through or sustained-release bolus to cattle to control dungbreeding flies including Musca, Stomoxys, and Haematobia species. The parent compound acts on eggs and larvae to interrupt the life cycle. Diflubenzuron is also formulated as a suspension concentrate for control of lice and blowflies on sheep. Chemical structure:

F

O

O

C

C NH

Cl

NH

F Mol wt: 310.7 Empirical formula: C14 H9 ClF2 N2 O2 Form: Colorless crystals (technical grade, off-white to yellow crystals) Acute oral LD50 for rats: >4,600 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 8

See earlier entry under Carbamates. Fluazuron Fluazuron is a benzoylphenyl urea compound that is formulated as a pour-on tickicide for use on beef cattle. This insect development inhibitor is approved in some countries for control of the one-host tick Boophilus microplus. The mode of action of fluazuron makes it especially suitable for strategic tick control programs. Chemical structure:

F

C

C NH F

Cl

O

O

NH

CF3

N

O Cl

Mol wt: 506.2 Empirical formula: C20 H10 Cl2 F5 N3 O3 Form: White to pink, odorless, fine crystalline powder Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 8 Lufenuron Lufenuron is an insect development inhibitor of the benzoylphenyl urea class. It demonstrates activity against fleas that have fed on treated cats and dogs and become exposed to lufenuron in the host’s blood. Lufenuron also has activity by virtue of its presence in adult flea feces, leading to its ingestion by flea larvae. Both activities result in the production of eggs that are unable to hatch, causing significant reductions in flea larvae populations. The lipophilicity of lufenuron leads to its deposition in adipose


tissues of animals from where it is slowly released into the bloodstream. This permits effective blood concentrations to be maintained throughout the recommended oral dosing interval of 1 month. Chemical structure:

F

F

O

O

C

C NH

CH

O

Cl

CF2

CF3

persistent efficacy. For example, a water-based 5.3% pyriproxyfen spot-on formulation applied to cats was reported to completely prevent the hatching of flea eggs for at least 46 days after treatment and continued to provide greater than 96% control until day 60 (57). Because pyriproxyfen is efficacious at very low concentrations, trace amounts of the chemical, when transferred from treated pets to their environments, are sufficient to inhibit the development of larvae. Pyriproxyfen is not associated with adulticidal activity; however, combination products that contain pyriproxyfen plus an adulticide are available.

Cl

NH

Chemical structure:

F

O

Mol wt: 511.2 Empirical formula: C17 H8 Cl2 F8 N2 O3 Form: Colorless crystals Acute oral LD50 for rats: >2,000 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: See Table 8 Methoprene Methoprene is an insect growth regulator that is used in the control of manure-breeding fly larvae in cattle and in flea control in cats and dogs. Drug delivery to cattle involves the application to feed or by administering sustained-release boluses. For flea control in cats and dogs, methoprene is applied topically in collars and sprays or used as a premise treatment. Topical application of methoprene inhibits flea eggs from hatching, increases larval mortality, and eventually leads to the emergence of abnormal pupae and adults. Methoprene is combined with adulticidal agents in some products. Chemical structure:

OCH3

85

CH3

H

CH3 O

CH2 C C CH C C O C H3C CH2 CH2 C CH2 H3C H H

CH3 CH

CH3

Mol wt: 310.5 Empirical formula: C19 H34 O3 Form: Amber liquid (technical grade) Acute oral LD50 for rats: >34,600 mg/kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 8 Pyriproxyfen Pyriproxyfen is a pyridine compound and, in common with fenoxycarb, is a juvenile hormone mimic whose structure is unrelated to natural juvenile hormone. It is an insect growth regulator. Fleas absorb pyriproxyfen either by direct contact or by ingesting blood from a treated animal. Pyriproxyfen formulations demonstrate

CH2

O

N

O

CH CH3

Mol wt: 321.4 Empirical formula: C20 H19 NO3 Form: Colorless crystals Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: ADI = 0.1 mg/kg bw per day (JMPR 1999). Animal commodity MRLs have not been established. No products are registered for use on foodproducing animals. Registered uses: See Table 8 Teflubenzuron Teflubenzuron, an acyl urea derivative, is an insect development inhibitor that acts by interfering with chitin synthesis and, thereby, disrupting the moulting process of insects. Teflubenzuron is used for control of sea lice in salmon. Dosing typically involves mixing 2 g teflubenzuron per kg of pelleted feed and administering daily for 7 days. Chemical structure:

Cl F

O C

C NH

F

F

O

Cl

NH F

Mol wt: 381.1 Empirical formula: C14 H6 Cl2 F4 N2 O2 Form: White to yellowish crystals Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: ADI = 0.01 mg/kg bw per day (JMPR 1994). Animal commodity MRLs have not been established. Registered uses: See Table 8

86


Triflumuron Triflumuron, a benzoylphenyl urea compound, is an insect development inhibitor that inhibits the synthesis of chitin. It is formulated as an off-shears pour-on lousicide for control of the sheep body louse Bovicola ovis. The development of immature lice present in the fleece at application and those that hatch from eggs in the following weeks is prevented. Triflumuron is also used for fly control in livestock housing and for control of fleas around pets. Chemical structure:

OCF3

O

O

C

C NH

NH

Cl Mol wt: 358.7 Empirical formula: C15 H10 ClF3 N2 O3 Form: Colorless odorless powder Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 8 MACROCYCLIC LACTONES The macrocyclic lactone endectocides are derivatives of fermentation products of soil-dwelling bacteria of the genus Streptomyces. Two major groups of compounds, the avermectins and the milbemycins, comprise the macrocyclic lactones; the nemadectins are a subgroup of the milbemycin family. The avermectin endectocides are represented by abamectin, doramectin, eprinomectin, ivermectin, and selamectin; the milbemycins include milbemycin oxime and the nemadectin, moxidectin. These compounds demonstrate activity at very low concentrations against internal parasites (nematodes) and external parasites (arthopods). Macrocyclic lactones have no activity against cestodes, trematodes, or protozoa. Chemistry The avermectins are 16-membered macrocyclic lactones that are characterized by the presence of a spiroketal unit at C-17 to C-28, a hexahydrobenzofuran moiety at C-2 to C-8a, and a bisoleadrosyloxyl disaccharide at C-13. The fermentation organism Streptomyces avermitilis produces a series of natural compounds referred to as avermectin A1 , A2 , B1 , and B2 . The A and B classifications are based on the substitution at C-5; avermectin A has a methoxy substituent at C-5, whereas avermectin B has a hydroxy substituent. The numerical designation is determined by the nature of the bond linking C-22 and C-23. Thus, avermectin A1 and B1 have a double bond, whereas avermectin A2 and B2 have a single bond plus a 23-hydroxy substituent. Avermectins are lipophilic compounds that dissolve in most organic solvents and are practically insoluble in water. They are acid sensitive, and treatment with dilute hydrochloric acid results in the

cleavage of the first of the C-13 sugars. The avermectins are also photosensitive, and exposure to ultraviolet light leads to isomerization of the 8, 9 and 10, 11 double bonds. The milbemycins are 16-membered macrocyclic lactones but, unlike the avermectins, lack a disaccharide group at the C-13 position. They are fermentation products of Streptomyces hygroscopicus aureolacrimosus. The nemadectins are fermentation products of Streptomyces cyaneogriseus noncyanogenus. Structurally, the nemadectins lack a disaccharide group at C-13 but differ from the milbemycins proper because they contain a trisubstituted double bond at C-26 in their side chains. The milbemycins are highly lipophilic drugs that are soluble in organic solvents and insoluble in water. Mode of Action The principal effector mechanism of macrocyclic lactones in invertebrates involves the opening of chloride channels via a specific binding site that is glutamategated. Additionally, macrocyclic lactones may potentiate γ -aminobutyric acid-gated sites that are located near the glutamate-gated chloride channels. The primary site for these actions is the synapse between interneurons and excitatory motor neurons in nematodes and myoneural junctions in arthropods. The overall effect is an increase in membrane permeability to chloride ions that leads to a slight hyperpolarization of the resting potential of postsynaptic cells. The resultant interference with neurological transmission and muscle stimulation in the parasite causes it to become flaccid. Death and/or expulsion of the parasite from the host follows. Pharmacokinetics and Metabolic Fate The lipophilicity of the avermectins strongly influences their pharmacokinetics and metabolic fate. They are well absorbed following oral, parenteral, and topical routes of administration. The formulation of the various dosage forms is critical in controlling the rate of drug release and for ensuring that effective blood concentrations are maintained. The lipophilicity of the avermectin family of compounds facilitates their wide distribution throughout the body (including the predilection sites for gastrointestinal parasites) and deposition in adipose tissues. The slow release of avermectins from fat stores confers their persistence in the body. The major metabolites from hepatic metabolism of ivermectin are 24hydroxymethyl-H2 B1a and -H2 B1b in cattle, sheep, and rats and 2 -O-demethyl-H2 B1a and -H2 B1b in pigs. Additional metabolism of ivermectin occurs in fat, leading to the formation of metabolites that are less polar than the parent drug (81). The avermectins are mainly excreted in bile and feces, regardless of the animal species or the route of administration (82). At least 98% of the ivermectin dose is excreted in feces in both cattle and sheep (83,84). Fecal excretion of unchanged ivermectin has implications for dung fauna (refer also to the earlier section on Environmental Impact of Pesticides). Toxicity The macrocyclic lactones have a wide therapeutic index in mammals. Nevertheless, overdosage may result in


acute toxic signs including ataxia, depression, tremors, mydriasis, and recumbency, progressing to coma and death (85,86). Adverse reactions to ivermectin have been observed that represent sequelae to parasitic mortality. For example, hemorrhage into the spinal canal leading to paresis can result from treating the first instar stage of Hypoderma bovis in cattle, whereas the death of Hypoderma lineatum larvae migrating in the esophagus of cattle may lead to edematous esophagitis and bloat (87).

Abamectin is the common name of avermectin B1 , a fermentation product of Streptomyces avermitilis. Abamectin has been formulated as an injection and a pour-on for cattle, as an oral suspension for sheep, and as an oral paste and tubing liquid for horses. The target pests are summarized in Table 9. Abamectin toxicity has been reported in Murray Grey cattle from one Australian farm (88) and was attributed to enhanced passage of the drug across atypical blood–brain barriers in the affected cattle. The use of abamectin in calves under 16 weeks of age is contraindicated. Chemical structure: H3CO O

O H3CO HO

formulation for subcutaneous administration and a pouron formulation. An injectable formulation is also approved for use on pigs. Chemical structure: H3CO O

H C O 3

CH3

CH3 O

O

CH3 H

O H3C

CH3

R O

O OH

Component B1a, R = CH2CH3 O Component B1b, R = CH3

CH3 OH

Mol wt: 873.1 (avermectin B1a ); 860.1 (avermectin B1b ) Empirical formula: C48 H72 O14 (avermectin B1a ); C47 H70 O14 (avermectin B1b ) Form: Colorless to pale yellow crystals Acute oral LD50 for rats: 10 mg/kg CODEX ADI and MRLs: ADI = 0.002 mg/kg per day (JMPR 1997). Animal commodity MRLs have not been established. Registered uses: See Table 9

O

O

HO

O

H3C

CH3

O OH

O

O

CH3 OH

Mol wt: 899.1 Empirical formula: C50 H74 O14 Form: White to light tan powder Acute oral LD50 for rats: 500–1,000 mg/kg (in aqueous vehicle) CODEX ADI and MRLs: See Table 1 Registered uses: See Table 9 Eprinomectin Eprinomectin is a semisynthetic avermectin that consists of a mixture of two homologues, eprinomectin B1a (4 epi-acetylamino-4 -deoxy-avermectin B1 ; 90%) and eprinomectin B1b (10%). Eprinomectin was developed as a topical endectocide for all categories of cattle including lactating dairy cattle, and the process by which eprinomectin was selected for further development has been described (90). Briefly, several hundred avermectin/milbemycin analogues were screened in a sheep model for efficacy against a range of endoparasites and in lactating dairy cows for a low milk:plasma partitioning ratio. The milk:plasma ratio for eprinomectin is 80%) and H2 B1b (1,600 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: See Table 9

Chemical structure:

Spinosad

CH3O N CH3

CH3 O

CH3

O

H3C H

CH3 CH3 O

O

OH

Spinosad is a fermentation product derived from an Actinomycetes bacterium, Saccharopolyspora spinosa. It is composed of a mixture of spinosyns A and D. Spinosad is registered as an insect-control agent in a variety of crops and, in Australia, is registered for the treatment and prevention of sheep blowfly (Lucilia cuprina) strike and for the control of body lice (Bovicola ovis) in sheep with long wool. Analytical methodology for quantifying spinosad and its metabolites in animal commodities has been reported (99). Chemical structure:

O

H3C

CH3

O

(H3C)2N

CH3

Mol wt: 639.8 Empirical formula: C37 H53 NO8 Form: White to pale yellow crystalline powder Acute oral LD50 for rats: 50 mg/kg CODEX ADI and MRLs: See Table 1 Registered uses: See Table 9

HH

O O O

H3CH2C

O O

HH

H R

Selamectin Selamectin is formulated as a spot-on for cats and dogs to control fleas (Ctenocephalides spp.) and flea-allergy dermatitis, to prevent heartworm disease, and to control ear mites (Otodectes cynotes) in cats and dogs. It is also approved for the treatment and control of sarcoptic mange (Sarcoptes scabiei) in dogs and hookworms (Ancylostoma tubaeformis) and roundworms (Toxocara cati) in cats.

CH3

OCH3OCH3 OCH3

Mol wt: 732.0 (spinosyn A); 746.0 (spinosyn D) Empirical formula: C41 H65 NO10 (spinosyn A); C42 H67 NO10 (spinosyn D) Form: Light grey to white crystalline solid Acute oral LD50 for rats: 2,000-5,000 mg/kg CODEX ADI and MRLs: Not established (evaluated by JMPR in 2001) Registered uses: See Table 9 NITROMETHYLENE COMPOUNDS

Chemical structure:

Imidacloprid

H3CO HO

O

CH3

CH3 O

O H3C

Spinosyn A, R = H Spinosyn D, R = CH3

O

OH

O

H3C O

O OH O

CH3

Imidacloprid is a nitroguanidine compound and belongs to the nitromethylene family of chemicals. The mode of action of imidacloprid involves interference with neurological transmission in insects by binding to the postsynaptic nicotinic acetylcholine receptors. Imidacloprid is available as a spot-on treatment for cats and dogs for flea control, and, following application, it distributes throughout the skin within 6 h (Bayer, 1996). It is not absorbed systemically by the animal, and its adulticidal activity is by contact with fleas. Chemical structure:

N N

HO Mol wt: 770.0 Empirical formula: C43 H63 NO11

NO2

CH2 N Cl

N

N

H

90


Mol wt: 255.7 Empirical formula: C9 H10 ClN5 O2 Form: Colorless crystals with a weak characteristic odor Acute oral LD50 for rats: ca. 450 mg/kg CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: See Table 10 Nitenpyram Nitenpyram is a nitroenamine compound and belongs to the nitromethylene family of chemicals. It has been shown in insects to have noncompetitive antagonist activity at nicotinic acetylcholine receptors and to affect the actions of the associated ion channels. Nitenpyram is the active ingredient in tablets for control of adult fleas in cats and dogs. Chemical structure:

CH3 CH2 N Cl

N

C HN CH3

Cl CN

N F3C

N

O S

Cl H2N

CF3

Mol wt: 437.2 Empirical formula: C12 H4 Cl2 F6 N4 OS Form: White solid Acute oral LD50 for rats: 100 mg/kg CODEX ADI and MRLs: ADI = 0.0002 mg/kg per day (JMPR 1997). Animal commodity MRLs have not been established. No products are registered for use on foodproducing animals Registered uses: See Table 11 INORGANIC COMPOUNDS

CH2 C

Chemical structure:

Boric Acid

H

NO2

Mol wt: 270.7 Empirical formula: C11 H15 ClN4 O2 Form: Pale yellow to light brown crystalline powder Acute oral LD50 for rats: 1,680 mg/kg (males); 1,575 mg/kg (females) CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: See Table 10 PHENYLPYRAZOLE COMPOUNDS Fipronil Fipronil is a phenylpyrazole, the mode of action of which is to inhibit nerve transmission in arthropods by blocking γ -aminobutyric acid-gated chloride channels. Fipronil is available as spray and spot-on formulations to control fleas and ticks on cats and dogs. The adulticidal activity of fipronil accounts for the majority of its activity, although additional activity against flea eggs and larvae results from the presence of fipronil on hairs and debris shed into the environment from treated pets. Autohistoradiography studies (11) into the cutaneous distribution of 14 C-fipronil in the cat and dog following spot-on administration demonstrated that radioactivity was restricted principally to the stratum corneum, the viable epidermis, and the pilosebaceous units. Following its slow release from sebaceous glands, fipronil migrates in the sebum covering the skin and hairs by passive diffusion and was shown to persist on hair for up to 2 months after treatment.

Various borate formulations are used as flea larval stomach poisons and desiccants; they have no effect on flea eggs, pupae, or adults. These compounds are applied to carpeted areas of the home as fine powders with residual powder being removed by vacuuming. The borates can cause toxicoses in pets and humans, particularly if ingested. Chemical structure:

OH B OH

HO

Mol wt: 61.8 Empirical formula: BH3 O3 Form: Colorless, odorless transparent crystals or white granules/powder Acute oral LD50 for rats: 2,666 mg/kg CODEX ADI and MRLs: Not established. The compound is not applied to food-producing animals. Registered uses: Used on carpeted areas of the home Magnesium Fluorosilicate Magnesium fluorosilicate is a water-soluble compound. When used as a sheep dip, it is not subject to mechanical or chemical ‘‘stripping’’ from dip wash. Chemical structure: 2−

F F

F Si

F

F F

Mg2+


Mol wt: 166.4 Empirical formula: F6 MgSi Form: White, efflorescent, odorless crystals Acute oral LD50 for guinea pigs: 200 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 12 Sulphur Sulphur is probably the oldest known effective parasiticide. Elemental sulphur (sublimed sulphur) and lime sulphur, which is a mixture of CaS2 , CaS5 , and CaS2 O3 , have been used for controlling external parasites on animals. The therapeutic properties of topical applications of sulphur are attributed to the production of sulphides and polythionic acids. The topical application of sulphur to animals leads to little or no absorption into the systemic circulation. Chemical structure:

S S S

S

S S

S

91

CODEX ADI and MRLs: Not established. No products are registered for use on food-producing animals. Registered uses: See Table 13 REPELLENTS In contrast to toxicants that act by killing or immobilizing pests, repellents create an unfavorable environment for pests, thereby causing parasites to leave the host. They are used on animals and animal housing to repel biting insects and flies. Repellents are commonly formulated with pyrethrins, synergists, and other repellents to achieve dual ectoparasiticidal and repellent activities. Citronella Oil Citronella oil is present as a repellent in many sprays that contain other repellents, pyrethrins, and synergists. These formulations have insecticidal and repellent actions against biting insects and flies on cattle, dogs, horses, and pigs. The major constituent of citronella oil is citronellal (to which the information below applies). Chemical structure:

S

CH3

Mol wt: 256.5 Empirical formula: S8 Form: Yellow crystalline powder or solid Acute oral LD50 for rats: >5,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See Table 12

CH CH2

Benzyl benzoate is an inexpensive acaricide that is still used, albeit infrequently, as an adjunct in the treatment of sarcoptic mange, demodectic mange, and ear mite infestations in dogs. Its mechanism of action is unknown. Benzyl benzoate is formulated as a lotion for spot treatment of localized infestations and is applied to animals with generalized infestation after they have been clipped, bathed, and while still wet. Benzyl benzoate has no residual effect. It is toxic to cats.

CHO

CH2 CH C CH3

MISCELLANEOUS COMPOUNDS Benzyl Benzoate

CH2

CH3

Mol wt: 154.3 Empirical formula: C10 H18 O Form: Liquid Acute oral LD50 for rats: 2,420 mg/kg CODEX ADI and MRLs: Not established Registered uses: See above Dibutyl Phthalate Dibutyl phthalate is included as an insect repellent in some aerosol sprays used to treat flystrike in sheep. Chemical structure:

Chemical structure:

O

O CH2

C

C

CH2 O

O

O C Mol wt: 212.2 Empirical formula: C14 H12 O2 Form: Colorless oily liquid with faint pleasant aromatic odor Acute oral LD50 for rats: 1,700 mg/kg

CH2 CH2

CH3

CH2 CH2

CH3 CH2

O Mol wt: 278.3 Empirical formula: C16 H22 O4 Form: Colorless oily liquid with a very weak aromatic odor

92


Acute oral LD50 for rats: >6,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See above N, N-Diethyl-m-toluamide (DEET) DEET is an ingredient in sprays that repel biting insects and flies from cattle, dogs, horses, and pigs and from aviaries, lofts, kennels, poultry houses, and stables. Creams containing DEET for application to allergic skin lesions and wounds are also approved. Chemical structure:

CH2CH3 O

N

insecticides, and these enzyme systems are inhibited by synergists. With fast knockdown chemicals such as the pyrethrins, allethrin, and resmethrin, synergists enhance insecticidal activity by prolonging the period of knockdown. Synergists also improve the safety of insecticidal formulations because their presence permits comparable efficacy to be achieved with lower doses of the active ingredient. N-Octyl Bicycloheptene Dicarboxamide N-Octyl bicycloheptene dicarboximide (MGK 264) is a synergist for pyrethrins and some pyrethroids. It is an ingredient of many ectoparasiticidal formulations, some of which contain repellents and other synergists. Chemical structure:

C

H3CH2C

O

Di-n-propyl Isocinchomeronate (MGK 326) MGK 326 is commonly formulated as creams and sprays that contain other repellents, pyrethrins, and synergists. These formulations have similar uses to those described above for DEET.

CH3 CH2

Mol wt: 275.4 Empirical formula: C17 H25 NO2 Form: Very light yellow colored liquid Acute oral LD50 for rats: 2,800 mg/kg CODEX ADI and MRLs: Not established Registered uses: A synergist with pyrethrins and some synthetic pyrethroids Piperonyl Butoxide

C O

Chemical structure:

O N

CH2

C O

O CH2

CH2

Piperonyl butoxide is a classical mixed-function oxidase inhibitor and the most important pyrethroid synergist. The degree of synergism can be very pronounced. For example, the toxicity of pyrethrin I and deltamethrin to houseflies is increased by 300-fold and 10-fold, respectively, with high doses of piperonyl butoxide. Synergism from piperonyl butoxide is not restricted to the pyrethrins and pyrethroids but is also observed with the carbamates, organochlorines, organophosphates, and rotenone (39).

Chemical structure:

CH3

CH2

CH2

O

Mol wt: 191.3 Empirical formula: C12 H17 NO Form: Colorless to amber liquid Acute oral LD50 for rats: ca. 2,000 mg/kg CODEX ADI and MRLs: Not established Registered uses: See above

CH2

CH

N

CH3

CH3

CH2

CH3 CH2

Mol wt: 251.3 Empirical formula: C13 H17 NO4 Form: Amber liquid with a mild aromatic odor Acute oral LD50 for rats: 4,270-5,850 mg/kg CODEX ADI and MRLs: Not established Registered uses: See above SYNERGISTS Synergists per se are not insecticidal but rather enhance the activity of insecticides by inhibiting their metabolic degradation. Components of the cytochrome P-450 system are responsible for the oxidative metabolism of numerous

CH2 CH3

CH2 CH2

O

CH2 O

CH2

CH2 CH2

CH2 O CH2

CH3

CH2

O O

Mol wt: 338.4 Empirical formula: C19 H30 O5 Form: Colorless liquid (technical grade is a yellow oil) Acute oral LD50 for rats: ca. 7,500 mg/kg CODEX ADI and MRLs: ADI = 0.2 mg/kg bw per day (JMPR 1995). Animal commodity MRLs have not been established. Registered uses: A synergist with pyrethrins and synthetic pyrethroids


CONCLUSIONS Chemicals have been, and continue to be, the mainstay of ectoparasite control strategies in animals. However, the development of parasites that are resistant to chemicals and the potential for chemicals to contaminate the environment and leave residues in food and fiber are major concerns associated with the current control strategies. For many years, major research efforts have been directed at developing alternative technologies, particularly antiparasite vaccines. Despite these endeavors, however, few antiparasite vaccines are currently marketed. A commercial vaccine is available against the cattle tick Boophilus microplus. This anti-tick vaccine reduces the build-up of Boophilus microplus on pastures, and, when used in conjunction with existing tick management practices, it allows for a reduced frequency of acaricide applications to cattle. Until the potential of antiparasite vaccines to protect animals is realized, and possibly beyond, an important role for novel systems that can deliver existing and new chemicals in an effective, safe, and cost-effective manner will exist. GLOSSARY Anticholinesterase compound. A chemical that interacts with the enzyme cholinesterase. The latter catalyzes the breakdown of the neurotransmitter acetylcholine released at nerve endings, thereby terminating its effect. In the presence of an anticholinesterase compound, acetylcholine will continue to cause nerve stimulation and muscle contractions, often leading to death of the organism. Arthropod. A segmented invertebrate animal with a head, jointed appendages, and a thickened chitinous cuticle forming an exoskeleton. The major parasitic arthropods of domestic animals include arachnids, such as mites and ticks, and insects, such as flies, keds, and lice. The head and thorax of arachnids, but not insects, are fused. Ataxia. Inability to coordinate voluntary movements; failure of muscle coordination. Bolus. Ruminal boluses are controlled-release delivery systems retained in the reticulorumen by their density or geometry. The majority of ruminal boluses are used to control internal and/or external parasites and to treat nutritional deficiencies. Cholinoreceptor. A receptor in the nervous system that responds to the neurotransmitter acetylcholine; cholinoreceptors are categorized into two subtypes known as muscarinic and nicotinic. Coprophagous. Applies to certain fungi, bacteria, flies, and beetles that grow on or in dung. Dermatosis. Any disease of the skin. Ectoparasite. A parasite that lives on the external surface of an organism. Enantiomers. Optical isomer pairs that rotate the plane of polarized light in opposite directions; the two molecules are not superimposable. See also Isomer.

93

Endectocide. A chemical agent that is used to control both external and internal parasites in animals. Hyperesthesia. Increased sensitivity of nerves that typically results from inflammation or tissue injury and leads to discomfort or pain. Isomers. Also known as structural isomers, are chemical compounds with the same molecular formula. Stereoisomers have the same functional groups but they differ in the arrangement of their atoms in space. There are at least two principal kinds of stereoisomers—geometric and optical isomers. Geometric (cis and trans) isomers display different spatial arrangement about a double bond. Optical isomers are mirror images of each other due to a center, axis, or plane of asymmetry. Although a tetravalent carbon atom with four different ligands attached to it is the most common basis for asymmetric molecules, phosphorus, sulphur, and nitrogen can also form chiral (optically active) molecules. Keratinocyte. A cell in the epidermis of the skin that produces keratin to construct the horny layer of the skin. Larvicide. A chemical or biologic agent that kills the larval stage of an organism. Lipophilic. A substance that demonstrates an affinity for fat; a lipophilic chemical partitions preferentially into fat tissue of an animal. Parenteral. Administration of a drug by a route other than orally or rectally; commonly involves intramuscular, subcutaneous, or intravenous injection or intravenous infusion. Pesticide. Veterinary chemical products used externally on animals are termed ‘‘pesticides’’ in some jurisdictions. Pharmacokinetic. Relating to pharmacokinetics, which is the study of the time course of drug absorption, distribution, metabolism, and excretion. Product. A formulation containing one or more active constituent(s), and possibly nonactive constituent(s), which is intended for application, with or without dilution prior to use, and is labeled with directions for use. Prophylactic. Relating to prophylaxis; measures taken to prevent a disease. Sebum. Material rich in lipids (oils and fats) excreted by sebaceous glands of the skin. Its function is to protect, lubricate, and waterproof the skin and hair and to help prevent desiccation. Syneresis. The contraction of a pharmaceutical gel resulting in the separation of liquid; it is a form of instability in aqueous and nonaqueous gels. Synergist. Acting together, often to produce an effect greater than the sum of the two agents acting separately. Systemic. Involving the whole body; the systemic effect of a drug involves absorption into the bloodstream and distribution by the systemic circulation throughout the whole body.

94


Veterinary drug. A pharmaceutical, pesticide, feed additive, or biological product that is used to treat, prevent, and control animal diseases. BIBLIOGRAPHY

Ectoparasite Treatment. Report to International Wool Secretariat, Agriculture Victoria, Attwood, Canada, 1995. 22. P. W. Johnson, A. Darwish, R. Dixon, and J. W. Steel, Int. J. Parasitol 26: 1369–1374 (1996). 23. A. Darwish, D. R. Hennessy, and C. A. Maxwell, Aust. Vet. J. 77: 667–670 (1999).

1. H. R. Adams Veterinary Pharmacology and Therapeutics, 7th ed., Iowa State University Press, Ames, IA, 2001.

24. D. R. Hennessy, A. Darwish, and C. A. Maxwell, Vet. Parasitol 89: 117–127 (2000).

2. G. C. Brander, D. M. Pugh, R. J. Bywater, and W. L. Jenkins Veterinary Applied Pharmacology and Therapeutics, 5th ed., Bailliere Tindall, London, 1991.

25. R. Du Toit and O. G. H. Fiedler, Nature 168: 608–609 (1951).

3. J. M. McDonald, in R. W. Kirk ed., Current Veterinary Therapy VIII Small Animal Practice, Saunders WB, Philadelphia, 1983, pp. 488–495. 4. S. J. Brown and P. W. Askenase, in D. A. Griffiths and C. E. Bowman eds., Acarology VI, Vol. 2, Ellis Horwood, Chichester, UK, 1984, pp. 1040–1050. 5. S. Horn, Ectoparasites of animals and their impact on the economy of South America, Proc. 23rd World Veterinary Congress, Montreal, Canada, 1987. 6. G. S. Cobon and P. Willadsen, in G. C. Woodrow and M. M. Levine eds., New Generation Vaccines, Marcel Dekker, New York, 1990, pp. 901–917.

26. P. M. Harrison and J. C. Rundle, Sheep Blowfly and Flystrike in Sheep, 2nd National Symposium, Department of Agriculture, Sydney, New South Wales, 1983, pp. 180–185. 27. D. W. Osborne, D. A. Hatzenbuhler, in D. W. Osborne and A. H. Amann, eds., Topical Drug Delivery Formulations, Marcel Dekker, New York, 1990, pp. 69–86. 28. I. H. Pitman and S. J. Roberts, J. Pharm. Sci. 70: 1181–1193 (1981). 29. H. E. Junginger, H. E. Bodde, and F. H. N. de Hann, in D. S. Hsieh, ed., Drug Permeation Enhancement Theory and Applications, Marcel Dekker, New York, 1994, pp. 59–89. 30. J. E. Riviere and J. W. Spoo, in H. R. Adams, ed., Veterinary Pharmacology and Therapeutics, 8th ed., Iowa State University Press, Ames, IA, 2001, pp. 1084–1104.

7. R. O. Drumond, G. Lambert, H. E. Smalley, and C. E. Terrill, in CRC Handbook of Pest Management in Agriculture, Vol. 1, CRC Press, Boca Raton, FL, 1981, pp. 111–117.

31. B. Idson, Drug Metab. Rev. 14: 207–222 (1983).

8. J. H. Arundel and A. K. Sutherland, in J. H. Arundell and A. K. Sutherland, eds., Ectoparasitic Diseases of Sheep, Cattle, Goats and Horses, Vol. 10, Australian Government Publishing Service, Canberra, 1988, pp. 35–60.

33. S. M. Herbig and K. L. Smith, J. Controlled Release 8: 63–72 (1988).

9. T. Beck, B. Moir, and T. Meppem, Q. Rev. Rural. Economy 7: 336–343 (1985). 10. P. W. Johnson, Pharmacological Basis of Veterinary Therapeutics, Proc. 198 Postgraduate Committee in Veterinary Science, University of Sydney, Sydney, 1992, pp. 39–78. 11. P. Birckel, P. Cochet, P. Bernard, and A. Weil, in K. W. Kwochka, T. Willemse, and C. von Tscharner, eds., Advances in Veterinary Dermatology, Vol. 3, Butterworth Heinemann, Oxford, 1998, pp. 571–572. 12. L. Carino and G. Nagy, Pestic. Sci. 2: 20–23 (1971). 13. G. M. Downing, Aust. Vet. J. 70: 392 (1993). 14. P. W. Morcombe et al., Aust. Vet. J. 72: 411–414 (1995). 15. A. M. Spickett and B. H. Firaz, Onderstepoort J. Vet. Res. 59: 203–210 (1992). 16. N. B. Fraser, Thesis. The Microbial Degradation of the Phenyl Carbamate Pesticide Promacyl, Melbourne: University of Melbourne, Australia, 1982. 17. K. W. McDougall and M. V. Makin, Pestic. Sci. 22: 307–315 (1988). 18. C. A. Hall, Symposium on the Australian Sheep Blowfly, Department of Agriculture New South Wales, Sydney, 1974, pp. 57–60.

32. P. J. S. Hasker, P. Roach, and C. J. Howett, Aust. J. Exper. Agr. 29: 765–769 (1989).

34. M. P. Nolan and E. L. Roberson, in N. H. Booth and L. E. McDonald, eds., Veterinary Pharmacology and Therapeutics, 6th ed., Iowa State University Press, Ames, IA, 1988, pp. 969–999. 35. J. R. Zingerman et al., J. Controlled Release 47: 1–11 (1997). 36. Report of the Joint FAO/WHO Food Standards Programme, Codex Committee on Pesticide Residues, Residues of Pesticides in Foods and Animal Feeds, FAO, The Hague, 1999. 37. Report of a FAO/WHO Consultation, Food consumption and exposure assessment of chemicals, WHO, Geneva, 1997. 38. Report of the FAO/WHO Joint Expert Committee on Food Additives (JECFA), Procedures for Recommending Maximum Residue Limits—Residues of Veterinary Drugs in Food (1987–1999), FAO, Rome, 2000. 39. B. L. Blagburn and D. S. Lindsay, in H. R. Adams, ed., Veterinary Pharmacology and Therapeutics, 7th ed., Iowa State University Press, Ames, IA, 2001, pp. 1017–1039. 40. G. Savage, The residue implications of sheep ectoparasiticides. A report for the Woolmark company, National Registration Authority for Agricultural and Veterinary Chemicals, Canberra, 1998. 41. N. J. Campbell et al., Aust. J. Exper. Agric. 38: 441–449 (1998).

19. N. Sherwood, T. Watts, C. Allen, and D. Chandler, Aust. Vet. J. 77: 384–387 (1999).

42. C. G. Rammell and G. R. Bentley, N.Z. J. Agric. Res. 23: 213–218 (1998).

20. P. W. Johnson, A. Darwish, R. Dixon, and J. W. Steel, Int. J. Parasitol 25: 471–482 (1995).

43. R. Herd, L. Strong, and K. Wardhaugh, Vet. Parasitol 48: 1–343 (1993).

21. N. J. Campbell, I. M. Russell, G. S. Roberts, and P. D. Hanrahan, Pesticide Residues in Wool After Late Season

44. K. G. Wardhaugh, B. C. Longstaff, and M. J. Lacey, Aust. Vet. J. 76: 273–280 (1998).

ANIMAL HEALTH PRODUCTS 45. K. G. Wardhaugh and R. J. Mahon, Aust. Vet. J. 76: 70–272 (1998).

95

74. G. B. Quistad, L. E. Stainger, and D. A. Schooley, Nature 296: 462–464 (1982).

46. R. Wall and L. A. Strong, Nature 327: 418–421 (1987).

75. N. H. Proctor and J. E. Casida, Science 190: 580–582 (1975).

47. C. Sommers, B. Gronvold, P. Holter, and P. Nansen, Vet. Parasitol 48: 171–197 (1993).

76. Special Review Program Report of the Chemicals Review and International Harmonisation Section, Chemicals and Nonprescription Medicines Branch, Therapeutic Goods Administration, Review of the Mammalian Toxicology and Metabolism/Toxicokinetics of Carbaryl, Canberra, 2000.

48. R. P. Herd, R. A. Sams, and S. M. Ashcraft, Int. J. Parasitol 26: 1087–1093 (1996). 49. J. W. Steel, Assessment of the effects of the macrocyclic class of chemicals on dung beetles and dung degradation in Australia, NRA Report on the Special Review of Macrocyclic Lactones, National Registration Authority for Agricultural and Veterinary Chemicals, Canberra, 1998. 50. T. A. Miller, Parasitol Today 4: S8–S12 (1988). 51. J. Nolan, H. J. Schnitzerling, and C. A. Schunter, Pestic. Biochem. Physiol. 2: 85–94 (1972). 52. C. A. Schuntner and B. N. Smallman, Pestic. Biochem. Physiol. 2: 78–84 (1972). 53. P. W. Riddles and J. Nolan, in M. J. Howell, ed., Parasitology Quo Vadit Proc. 6th Int. Congress of Parasitol, Australian Academy of Science, Canberra, Brisbane, 1986, pp. 679–687. 54. B. F. Stone, Aust. Vet. J. 48: 345–350 (1972).

77. A. Parkinson, in C. D. Klaassen, ed., Casarett and Doull’s Toxicology: The Basic Science of Poisons, 5th ed., McGrawHill, New York, 1996, pp. 113–186. 78. D. H. Lein et al., Cornell Vet. 72: 5–58 (1982). 79. M. C. Roberts and A. A. Seawright, Aust. Vet. J. 55: 553–554 (1979). 80. F. L. Bowen et al., Aust. Vet. J. 77: 454–460 (1999). 81. S.-H. L. Chiu et al., Drug Metab. Rev. 18: 289–302 (1987). 82. S.-H. L. Chiu and A. Y. H. Lu, in W. C. Campbell, ed., Ivermectin and Abamectin, Springer-Verlag, New York, 1989, pp. 131–143. 83. B. A. Halley, R. J. Nessel, and A. Y. H. Lu, in W. C. Campbell, ed., Ivermectin and Abamectin, Springer-Verlag, New York, 1989, pp. 162–172.

55. J. Nolan and H. J. Schnitzerling, in W. C. Campbell and R. S. Rew, eds., Chemotherapy of Parasitic Disease, Plenum Press, New York, 1986, pp. 603–620.

84. S.-H. L. Chiu et al., J. Agric. Food Chem. 38: 2072–2078 (1990).

56. J. Nolan, W. J. Roulston, and H. J. Schnitzerling, Aust. Vet. J. 55: 463–466 (1979).

85. W. C. Campbell and G. W. Benz, J. Vet. Pharmacol. Ther. 7: 1–16 (1984).

57. D. H. Ross, D. R. Young, R. Young, and R. G. Pennington, Feline Pract. 26: 18–22 (1998).

86. G. R. Lankas and L. R. Gordon, in W. C. Campbell, ed., Ivermectin and Abamectin, Springer-Verlag; New York, 1989, pp. 89–112.

58. FAO Food and Nutrition Paper, Residues of some veterinary drugs in animals and foods, FAO, Rome, 2000. 59. O. Koul, B. I. Isman, and C. M. Ketkar, Can. J. Botany 68: 11–12 (1990). 60. H. Schmutterer, Ann. Rev. Entomol. 35: 271–297 (1990). 61. J. A. Immaraju, Pestic. Sci. 54: 285–289 (1998). 62. A. C. Heath, L. Lampkin, and J. H. Jowett, Med. Vet. Entomol. 4: 407–412 (1995). 63. M. Rice, J. Entomol. Res. 23: 1231–1234 (1989). 64. V. H. Guerrini and C. M. Kriticos, Vet. Parasitol. 74: 289–297 (1998). 65. S. B. Hooser, Toxicology of selected pesticides and chemicals. Vet. Clin. North Am. Small Anim. Pract. 20: 383–385 (1990). 66. J. Miyamoto, Pure Appl. Chem. 53: 1967–2022 (1981). 67. D. M. Soderlund, Xenobiotica 22: 1185–1194 (1992). 68. D. C. Dorman and V. R. Beasley, Vet. Hum. Toxicol. 33: 238–243 (1991). 69. J. E. Casida, D. W. Gammon, A. H. Glickman, and L. J. Lawrence, Ann. Rev. Pharmacol. Toxicol. 23: 413–438 (1983). 70. L. C. Gaughan, T. Unai, and J. E. Casida, J. Agric. Food Chem. 25: 9–17 (1977). 71. L. C. Gaughan, M. E. Ackerman, T. Unai, and J. E. Casida, J. Agric. Food Chem. 26: 613–618 (1978).

87. Q. A. McKellar and H. A. Benchaoui, J. Vet. Pharmacol. Ther. 19: 331–351 (1996). 88. J. T. Seaman, J. S. Eagleson, M. J. Carrigan, and R. F. Webb, Aust. Vet. J. 64: 284–285 (1987). 89. A. C. Goudie et al., Vet. Parasitol. 49: 5–15 (1993). 90. W. L. Shoop et al., Int. J. Parasitol. 26: 1227–1235 (1996). 91. J. D. Pulliam, R. L. Seward, and R. T. Henry, Vet. Med. 80: 33–40 (1985). 92. W. J. Tranquilli, A. J. Paul, R. L. Seward, Am. J. Vet. Res. 50: 769–770 (1989). 93. R. F. Jackson, W. G. Seymour, and R. S. Beckett, in G. F. Oho, ed., Proc. of the Heartworm Symposium, American Heartworm Society, Washington, DC, 1986, pp. 15–18. 94. J. C. Schlotthauer et al., Proc. of the Heartworm Symposium, American Heartworm Society, Washington, DC, 1986, pp. 29–32. 95. R. P. Herd and J. C. Donham, Am. J. Vet. Res. 44: 1102–1105 (1983). 96. W. H. Miller, D. W. Scott, J. R. Wellington, and R. Panic, J. Am. Vet. Med. Assoc. 203: 1426–1429 (1993). 97. D. G. Stansfield and D. I. Hepler, Canine Pract. 16: 11–16 (1991).

72. L. C. Gaughan, R. A. Robinson, and J. E. Casida, J. Agric. Food Chem. 26: 1374–1380 (1978).

98. W. L. Shoop, H. Mrozik, in A. D. Jernigan, L. M. Sadler, and S. A. Brown, eds., Proc. 9th Biennial Symposium, The American Academy of Veterinary Pharmacology and Therapeutics, Kalamazoo, MI, 1994, pp. 35–51.

73. G. W. Ivie and L. M. Hunt, J. Agric. Food Chem. 28: 1131–1138 (1980).

99. S. D. West and L. G. Turner, J. Agric. Food Chem. 46: 4620–4627 (1998).

96

ANTIBIOTIC RESISTANCE

FURTHER READING Blagburn, B. L., Lindsay, D. S., in H. R. Adams, ed., Veterinary Pharmacology and Therapeutics, 8th ed., Iowa State University Press, Ames, IA, 2001, pp. 1017–1039. Klink, P. R., Ferguson, T. H., Magruder, H. A., in G. E. Hardee and J. D. Baggot, eds., Development and Formulation of Veterinary Dosage Forms, 2nd ed., Marcel Dekker, Inc., New York, 1998, pp. 145–229. McKellar, Q. A., Benchaoui, H. A., Avermectins and milbemycins, J. Vet. Pharmacol. Ther. 19: 331–351 (1996). Riviere, J. E., Spoo, J. W., and Adams, H. R., ed., in Veterinary Pharmacology and Therapeutics. 8th ed., Iowa State University Press, Ames, IA, 2001, pp. 1084–1104.

ANTIBIOTIC RESISTANCE GEORGE W. SUNDIN Michigan State University East Lansing, Michigan

The antibiotic streptomycin is an important and effective chemical for the management of bacterial diseases of fruit trees (especially apple), woody ornamentals, and vegetables. Streptomycin was initially discovered in 1944 and was one of the first antibiotics to be utilized in clinical medicine to control human diseases, and is still important as a feed amendment for growth promotion in agricultural animals. The widespread and diverse usage of streptomycin has contributed to the currently observed global streptomycin resistance (SmR) problem. This problem is especially critical in plant disease management, as there are few alternatives to streptomycin available and, as a consequence of increased usage, SmR has been increasingly observed among bacterial plant pathogens. Streptomycin resistance has been reported in the fire blight pathogen of apple and pear, Erwinia amylovora 1), Pseudomonas syringae pv. papulans (blister spot of apple; 2), P. syringae pv. syringae (canker and tip-dieback on woody ornamentals; 3), and Xanthomonas campestris pv. vesicatoria (bacterial spot of tomato and pepper; 4). Streptomycin resistance is a global problem; SmR plant pathogens have been isolated in Argentina, Brazil, Canada, Japan, New Zealand, Taiwan, Tonga, and the United States (1–4). Streptomycin resistance can be conferred by two distinct mechanisms: alteration of the ribosomal binding site of streptomycin as a result of a spontaneous chromosomal mutation (5), or by enzymatic detoxification of the streptomycin molecule through a phosphorylation or adenylylation process (6). Both of these mechanisms have contributed to the evolution of SmR in plant pathogens. A comprehensive genetic study has shown that SmR strains of E. amylovora isolated from apple orchards in the western United States and New Zealand had single chromosomal mutations which conferred resistance to streptomycin of >4,096 mg/L (7). In contrast, SmR in E. amylovora from Michigan (8), P. syringae pv. papulans from New York (2), P. syringae pv. syringae from Oklahoma (3), and X. campestris pv. vesicatoria from Florida

and Argentina (4) is conferred through the expression of a plasmid-encoded tandem gene pair, strA-strB (9). Levels of SmR conferred by strA-strB vary from 75–100 mg/L in P. syringae and X. campestris to up to 1,000 mg/L in the Michigan E. amylovora strains (7,10). It should be noted that even the relatively low level of SmR observed in P. syringae enables strains to survive on plant surfaces sprayed with streptomycin at recommended field rates (11). The strA-strB gene pair was initially described on plasmids of importance in human medicine, and both genes encode proteins that phosphorylate the streptomycin molecule at unique positions (6). Bacterial isolates from humans and animals which encode strA-strB tend to carry the determinant on small, nonconjugative plasmids (9); however, the strA-strB genes from plant-pathogenic bacteria are typically encoded on large conjugative plasmids and contained within the transposable element Tn5393, a 6.7-kb transposon belonging to the Tn3 family (8). Tn5393 encodes genes involved in the transposition process as well as the strA-strB genes (8). The Tn5393 elements studied among the different plant-pathogenic bacterial genera are essentially identical, except for the additional presence of the insertion sequence elements IS1133 and IS6100 in E. amylovora and X. campestris pv. vesicatoria, respectively (10). These IS elements are involved in the expression of strA-strB, and the strA-strB genes are expressed from a promoter sequence located within the Tn5393 transposon in P. syringae (10). Within populations of E. amylovora and P. syringae pv. syringae from agricultural habitats, Tn5393 was detected on plasmids of different sizes or incompatibility groups, suggesting that interplasmid mobilization, presumably by transposition, is a common occurrence (12,13). Sequences hybridizing to strA-strB, sometimes in association with Tn5393 sequences, have also been detected in nontarget gram-negative bacteria isolated from plant tissue and soil in agricultural habitats where streptomycin was utilized as a bactericide (2,14–16), and from regions where streptomycin was presumably never introduced through human usage (16). The widespread presence of strA-strB within nontarget bacteria suggests that these organisms may serve as a reservoir resulting in an increased accessibility of the genes for plant-pathogenic strains. Clearly the strA-strB genes, and Tn5393, have been disseminated on a global scale and among a collection of diverse bacterial genera in a relatively short time period. Tetracycline is an antibiotic that has been utilized in disease management situations in which SmR strains of E. amylovora or P. syringae already exist. However, tetracycline does not appear to be as effective as streptomycin in reducing blossom populations of E. amylovora (17). Additionally, strains of P. syringae with resistance to tetracycline have been isolated from pear orchards in Oregon and Washington (18), suggesting that resistance to this antibiotic will probably develop in orchards where it is applied. Although SmR is increasing in importance, populations of target plant pathogens recovered in recent

ANTIBIOTICS

surveys are not uniformly resistant to the antibiotic. A combination of experimental and, in some cases, anecdotal evidence, however, does suggest that once the SmR phenotype is selected, SmR is stable without antibiotic selection and SmR target plant pathogens are not reduced in fitness when compared to their streptomycin-sensitive counterparts (1,12). Thus the development of alternative management strategies will be critical in situations where SmR plant pathogens exist. Two recent studies have addressed the possible usage of a combination of a SmR biological control strain in combination with streptomycin sprays (19,20). This method may be an effective alternative in controlling mixed SmR and streptomycin-sensitive pathogen populations. BIBLIOGRAPHY 1. M. N. Schroth, S. V. Thomson, and W. J. Moller, Phytopathology 69: 565–568 (1978). 2. J. L. Norelli et al., Appl. Environ. Microbiol. 57: 486–491 (1991). 3. G. W. Sundin and C. L. Bender, Appl. Environ. Microbiol. 59: 1018–1024 (1993). 4. G. V. Minsavage, B. I. Canteros, and R. E. Stall, Phytopathology 80: 719–723(1990). 5. L. E. Bryan, in L. E. Bryan, ed., Antimicrobial Drug Resistance, Academic Press, Orlando, Fla., 1984, pp. 241–277. 6. K. J. Shaw, P. N. Rather, R. S. Hare, and G. H. Miller, Microbiol. Rev. 57: 138–163 (1993). 7. C.-S. Chiou and A. L. Jones, Phytopathology 85: 324–328 (1995). 8. C.-S. Chiou and A. L. Jones, J. Bacteriol. 175: 732–740 (1993). 9. G. W. Sundin and C. L. Bender, Mol. Ecol. 5: 133–143 (1996). 10. G. W. Sundin and C. L. Bender, Appl. Environ. Microbiol. 61: 2891–2897 (1995). 11. G. W. Sundin and C. L. Bender, Can. J. Microbiol. 40: 289–295 (1994). 12. P. S. McManus and A. L. Jones, Phytopathology 84: 627–633 (1994). 13. G. W. Sundin, D. H. Demezas, and C. L. Bender, Appl. Environ. Microbiol. 60: 4421–4431 (1994). 14. T. J. Burr, J. L. Norelli, and C. L. Reid, Plant Dis. 77: 63–66 (1993). 15. P. Sobiczewski, C.-S. Chiou, and A. L. Jones, Plant Dis. 75: 1110–1113 (1991). 16. G. W. Sundin, D. E. Monks, and C. L. Bender, Can. J. Microbiol. 41: 792–799 (1995). 17. P. S. McManus and A. L. Jones, Phytopathology 84: 627–633 (1994). 18. R. A. Spotts and L. A. Cervantes, Plant Dis. 79: 1132–1135 (1995). 19. S. E. Lindow, G. McGourty, and R. Elkins, Phytopathology 86: 841–848 (1996). 20. V. O. Stockwell, K. B. Johnson, and J. E. Loper, Phytopathology 86: 834–840 (1996).

97

ANTIBIOTICS GEORGE W. SUNDIN Michigan State University East Lansing, Michigan

Antibiotics are products of microbial origin that are inhibitory to another microorganism or group of microorganisms. Compared to pesticides, antibiotics used for plant disease control have several desirable properties including reduced effects on nontarget organisms, limited mammalian toxicity, and reduced environmental persistence. However, other aspects such as the lack of systemic transport of antibiotics within plants, and inactivation by abiotic environmental factors have limited the effectiveness of antibiotics in certain plant pathosystems. Since the early 1950s, a number of antibiotics (either antibacterial or antifungal) have shown in vitro activity against plant pathogens, only to fail during field testing. Thus only a few antibiotics have been utilized for plant disease control; it should be noted, however, that these compounds have been highly successful control agents. When antibiotics such as streptomycin and penicillin were initially used in human medicine in the 1940s, these compounds were hailed as miracle drugs because of their ability to kill some of the worst known human bacterial pathogens. As the successes of antibiotic usage increased, the applications of antibiotics became more diverse, and grew to include agricultural uses such as feed amendments for growth promotion in agricultural animals. Plant pathologists also became interested in the prospects of using antibiotics to control plant diseases. Early reports of antibiotic use in agriculture hailed two important aspects regarding disease control—their selective action and their potency (1). The selective action of antibiotics was deemed important in terms of a reduced chance for phytotoxicity and the potency was important in terms of reduced usage and cost. Large-scale testing and utilization of the two major antibiotics for bacterial disease control, streptomycin and oxytetracycline, began in the early 1950s. The most common commercially available formulations were a 37% streptomycin sulfate solution (Agristrep) or a 15% streptomycin sulfate +1.5% oxytetracycline mixture (Agrimycin). These compounds were shown to be effective control agents against a wide variety of diseases including angular leaf spot of cucumber, bacterial blight of celery, bacterial spot of tomato and sweet pepper, bacterial wilt of chrysanthemum, fire blight of apple and pear, halo blight of beans, soft rot and blackleg of potato, walnut blight, and wildfire of tobacco (2). The most common concentration of Agristrep or Agrimycin utilized in spray mixtures in these studies was 100 or 200 ppm. Currently streptomycin and oxytetracycline are still approved for use in U.S. agricultural production. These antibiotics are also utilized for crop protection in other countries such as New Zealand, but are banned from agricultural usage in Europe because of potential antibiotic-resistance problems and the threat of the transfer of resistance genes to human pathogens. It should be noted that the amount of antibiotics applied

98

ANTIFEEDANTS

to U.S. crops represents only 0.006% of the total chemical application (including fungicides, herbicides, and inseticides) (3). The heaviest current use of antibiotics for bacterial control is on fruit trees and ornamental trees primarily because of phytotoxicity problems with coppercontaining bactericides and because of crop value and the destructive nature of certain bacterial diseases including fire blight. Antifungal antibiotics have been studied mainly in Japan, and although a variety of compounds have been characterized, their field potential is typically limited because of phytotoxicity problems (4). Two compounds worth mentioning are blasticidin S and cycloheximide. Blasticidin S is produced by Streptomyces griseochromogenes, and has a wide range of antimicrobial activity (4). This antibiotic was utilized in the Far East beginning in 1961 against the rice blast pathogen Pyricularia oryzae, with effective control achieved at rates of 10–40 ppm (4). Application of blasticidin S at rates above 40 ppm caused phytotoxicity problems; of additional concern for use in rice fields is the toxicity of the compound to fish (LD50 > 40 ppm). Another antibiotic of historic interest is cycloheximide (trade name Actidione), a compound which is produced by S. griseus, the same organism which produces streptomycin. Cycloheximide is a potent inhibitor of translation in eukaryotic cells, and is not active against bacteria. The toxicity of cycloheximide to plant-pathogenic fungi is high, with inhibition of growth and sporulation occurring in the range of 1–20 ppm (5,6). This compound had a minor usage in disease control and was shown to be effective in the control of powdery mildew of bean, cherry leaf spot, and certain turf diseases (1). However, the effectiveness of cycloheximide was more limited when tested against powdery mildew of apple and onion, snow mold of turf, and azalea blight (1). In Japan, cycloheximide was used for at least 22 years (1959 to 1980) for controlling downy mildew of onions (4). The main problem with the use of cycloheximide was phytotoxicity, since this compound is an inhibitor of eukaryotic cells, and thus the use of cycloheximide in plant disease control was discontinued around 1980. It should be noted that cycloheximide is still widely utilized by plant pathologists as an antifungal agent in isolation media for bacteria from plant surfaces and soil (7). A significant problem in the utilization of antibiotics for bacterial disease control in plant, human, and animal systems, is the development of resistance in the target bacterial population. Antibiotic resistance is a global problem whose importance continues to spiral upward. The large-scale usage of antibiotics by the human population has exerted a tremendous selection pressure on bacterial populations. These populations have responded by developing resistance to the antibiotics either by detoxification, selective or nonselective efflux, or by modification of the target site of the antibiotic (8,9). From a human standpoint, the aspect of antibiotic resistance of utmost concern is the apparent ability of bacteria to rapidly and nondiscriminately transfer antibiotic resistance genes among related and unrelated species (10). Recent genetic evidence has shown that many transfer-proficient genetic

elements (plasmids, transposons, integrons) exist, which facilitate this process (10). Indeed the ability of bacterial populations to rapidly evolve resistance to antibiotics has been a critical limiting factor in the development of new antibiotics. Thus the promise of antibiotic usage in human medicine as first viewed in the 1940s has led to the reality of the antibiotic-resistance problem and the difficulty of new antibiotic development. In agriculture, resistance problems have been combatted by utilizing a different compound, or by utilizing the antibiotic in conjunction with a biological control agent to maximize the possibilities of reduction of pathogen populations. The introduction of new antibiotics for use in agriculture is severely limited by the continued clinical importance of the few choices available. Few chemical alternatives are currently available should streptomycin and oxytetracycline resistance become prevalent.

BIBLIOGRAPHY 1. C. Leben and G. W. Keitt, Agric. Food Chem. 2: 234–239 (1954). 2. W. J. Zaumeyer, in Proceedings of First International Conference on the Use of Antibiotics in Agriculture, National Academy of Sciences—National Research Council, Washington, D.C., 1956, pp. 171–187. 3. A. K. Vidaver, Phytopathol. News 27: 6,7 (1993). 4. I. Yamaguchi, in H. Lyr, ed., Modern Selective Fungicides: Properties, Applications, Mechanisms of Action, 2nd ed., Gustav Fischer Verlag, New York, 1995, pp. 415–429. 5. A. W. Henry, R. L. Miller, and E. A. Peterson, Science 115: 90,91 (1952). 6. T. T. McClure and D. Cation, Plant Dis. Rep. 35: 393–395 (1952). 7. N. W. Schaad, ed., Laboratory Guide for Identification of Plant Pathogenic Bacteria, 2nd ed., APS Press, St. Paul, Minn., 1988. 8. H. Nikaido, Science 264: 382–388 (1994). 9. B. G. Spratt, Science 264: 388–393 (1994). 10. J. Davies, Science 264: 375–382 (1994).

ANTIFEEDANTS. See PHEROMONES

ANTIFOULING AGENTS Substances that kill or repel organisms attached to underwater surfaces, such as boat bottoms (USEPA).

ANTIMICROBIALS Substances that kill microorganisms (such as bacteria and viruses) (USEPA).

AUXINS, INDOLE AUXINS

AOAC Association of Official Analytical Chemists. (AOAC International, website http://www.aoac.org)

ARBORICIDE A chemical for killing trees and shrubs (CIPAC).

ASTM American Society for Testing and Materials

ATTRACTANTS Substances that attract pests, for example, to lure an insect or rodent to a trap. However, food is not considered a pesticide when used as an attractant (USEPA).

ATTRACTICIDES. See PHEROMONES

99

are examples illustrating the ways by which genes communicate with the extranuclear world via a complex network of biophysical and biochemical processes to accomplish classical physiological responses. In unraveling these signaling mechanisms in further detail, auxin chemistry provides the tools and critical insight required to prepare new auxins, to deal with impurities in commercial preparations, and to recognize chemical pitfalls such as oxidation, radiolysis, and photochemical reactions, which can invalidate the interpretation of biological experiments. As physiological action depends on auxin structure and concentration, analytical methods that permit identification and quantification have been significantly refined during the past two decades. This has provided new insights into the dynamics of endogenous auxin levels as affected by biosynthesis, conjugation, and degradation. Research on the mechanisms of auxin perception may now be based on X-ray structures for a representative set of auxins, but the molecular architecture of the corresponding receptor protein(s) is still under investigation. Progress at the far end of the signaling cascade includes the identification of a large set of auxin-regulated genes and some of the auxin-responsive elements in their promoters. NATURAL INDOLE AUXINS AND SOME SYNTHETIC ANALOGUES OF AGRICULTURAL RELEVANCE Of the large number of known indole auxins, the selection presented in Figure 1 deserves more general interest.

AUXINS, INDOLE AUXINS 1H-Indole-3-Acetic Acid VOLKER MAGNUS BISERKA KOJIC´ -PRODIC´ Institut Rudjer Boˇsković Zagreb, Croatia

The auxins were the first plant growth regulators discovered (1) and were originally defined as organic acids stimulating stem elongation in standard bioassays. This definition is still useful if new compounds are to be classified, even though auxins are now known to act throughout the life cycle of any single plant cell. The most active endogenous auxins contain the indole ring system. While the physiological processes were still incompletely understood, agricultural chemists set out to prepare synthetic auxin analogues, which could be produced at lower cost, were effective in smaller concentrations, and remained active for longer periods of time under field conditions. However, as residues of these growth regulators keep building up in arable soils, undesirable side effects on human health and ecological equilibria can no longer be neglected. Less invasive methods are needed to maintain and to increase crop productivity, while keeping the consumers and their environment healthy. To devise such strategies, in-depth knowledge on the endogenous regulation of plant growth and development is required. In accord with these practical needs, research on the genetic base of auxin physiology is in precipitous progress; results are turning over so rapidly that there are as yet no firm grounds for a balanced summary. However, there already

1H-indole-3-acetic acid (1), CAS reg. no. 5448-47-5, is also known as heteroauxin, β-indolylacetic acid, indol-3ylacetic acid and is usually abbreviated as IAA. In its systematic name and in those of the compounds listed below, there is disagreement on the use of the suffix ‘‘yl,’’ the prefix ‘‘1H,’’ and of the number ‘‘3’’ and its position. Beyond this listing we will use the chemical names (or standard abbreviations, if any) as shown in bold print, thus following CA conventions, except for the prefix ‘‘1H’’ because 3H- and other tautomers of the indole ring system will not be discussed. IAA has long been known to mimic the biological effects of endogenous auxin, and unequivocal analytical methods have now confirmed its role as a ubiquitous phytohormone. 1H-Indole-3-Propanoic Acid 1H-indole-3-propanoic acid (2), CAS reg. no. 830-96-6, or 3-(indol-3-yl)propionic acid, here abbreviated as IPA, has been found as a minor auxin in pea (2) and squash (3). 1H-Indole-3-Butanoic Acid 1H-indole-3-butanoic acid (3), CAS reg. no. 132-32-4, or 4-(indol-3-yl)butyric acid, with the standard abbreviation, IBA, was first prepared as a synthetic plant growth regulator (4). Sporadic chromatographic evidence then indicated its natural occurrence, which was later confirmed by mass spectroscopy, for plants such as pea, cypress, maize, carrot, tobacco, and Arabidopsis thaliana (L.) Schur. (5).

100


4-Chloro-1H-Indole-3-Acetic Acid

rapa L. ssp. pekinensis [Lour.] Hanelt cv. Kinshu), lettuce (Lactuca sativa L. cv. Gokuwase-CISCO), and rice (Oryza sativa L. cv. Koshihikari), 10−4 to 10−6 M of compound 8 and its above derivatives stimulated root growth but had little effect on the shoot (21,22). If applied to corn seedlings (Zea mays L. cv. Alize) in the same range of concentrations, 8 inhibited both root and shoot growth and, moreover, specifically interfered with the biosynthesis and perception of IBA (23).

4-Chloro-1H-indole-3-acetic acid (4), CAS reg. no. 251961-1, here abbreviated as 4-Cl-IAA, was discovered in ripening pea seeds (6,7), is now well known from other species of the Vicieae tribe of the Fabaceae (Leguminosae) (8), and may be even more widespread, as suggested by its identification in Pinus sylvestris L. (9), a gymnosperm with very loose evolutionary links to the legume family.

PHYSICAL PROPERTIES OF PRACTICALLY IMPORTANT INDOLE AUXINS

7-Chloro-1H-Indole-3-Acetic Acid 7-Chloro-1H-indole-3-acetic acid (5), CAS reg. no. 191241-0, here abbreviated as 7-Cl-IAA, is excreted by the soil microorganisms Pseudomonas aureofaciens and P. pyrrocinia (10,11) and could affect the roots of higher plants.

Indole-3-acetic acid (1). MW: 175.19. Leaflets or crystalline powder from water, mp 168–170 ◦ C. pKa 4.75. Sparingly soluble in cold water or chloroform; freely soluble in alcohols, soluble in acetone and diethylether. UV absorbance (95% ethanol), λλmax (log ε): 222.7 (4.50), 276.3 (shoulder, 3.73), 282.0 (3.76), 290.0 (3.69) nm. Fluorescence (water, pH 7.0); excitation: λmax : 285 nm; emission: λmax : 345 nm. Indole-3-propanoic acid (2). MW: 189.21. White crystals, mp 134–135 ◦ C. Indole-3-butanoic acid (3). MW: 203.24, white or slightly yellow crystals, mp 123–125 ◦ C. Practically insoluble in water and chloroform; soluble in alcohols, diethylether and acetone. 4-Chloroindole-3-acetic acid (4). MW: 209.63, mp 184–187 ◦ C. UV absorbance (95% ethanol), λλmax (log ε): 225.9 (4.55), 277.3 (shoulder, 3.77), 283.4 (3.80), 291.1 (shoulder, 3.75) nm. 7-Chloroindole-3-acetic acid (5). MW: 209.63, mp 181.5–183 ◦ C. UV absorbance (95% ethanol), λλmax (log ε): 223.9 (4.55), 278.0 (shoulder, 3.75), 285.1 (3.78), 294.2 (3.71) nm. 5-Bromoindole-3-acetic acid (6). MW: 254.09, mp 143–145 ◦ C. UV absorbance (95% ethanol), λλmax (log ε): 229.0 (4.47), 284.3 (shoulder, 3.63), 290.6 (3.66), 298.3 (shoulder, 3.57) nm. 5,6-Dichloroindole-3-acetic acid (7). MW: 244.08, mp 189–191 ◦ C. UV absorbance (95% ethanol), λλmax (log ε): 232.4 (4.54), 288.6 (shoulder, 3.71), 295.1 (3.76), 304.3 (3.72) nm. β-Trifluoromethyl-indole-3-propanoic acid (8). MW: 257.21, mp 117–119 ◦ C (racemic). Optical rotation:

5-Bromo-1H-Indole-3-Acetic Acid 5-Bromo-1H-indole-3-acetic acid (6), CAS reg. no. 4043284-6, a synthetic auxin, was recently patented for its efficiency in inducing callus formation and root and shoot regeneration in vitro (12). However, at the whole-plant level, 6 was toxic to Lemna gibba L. and Zea mays L. (13). 5,6-Dichloro-1H-Indole-3-Acetic Acid 5,6-Dichloro-1H-indole-3-acetic acid (7), CAS reg. no. 98640-00-7, is one of the most active (synthetic) indole auxins (14). Patents have been granted for its application in the rooting of cuttings, as a promoter of seed ripening, as a yield-increasing agent in field crops (15), and for maintaining in vitro cultures producing tropane alkaloids (16,17). β-Trifluoromethyl-1H-Indole-3-Propanoic Acid [4,4,4-Trifluoro-3-(Indole-3-)Butyric Acid] β-Trifluoromethyl-1H-indole-3-propanoic acid [4,4,4trifluoro-3-(indole-3-)butyric acid] (8), CAS reg. no. 153233-36-4, and a number of its 2- and benzene-ringsubstituted derivatives, as well as their esters and amides, were patented as potent auxins with organspecific, species-dependent effects. The S-(+)-enantiomer of 8 is more active than its R-(−)-analogue (18). The compound was used to increase the sugar content of citrus fruits (19,20). In seedlings of Chinese cabbage (Brassica

4 5 Figure 1. Examples for indole auxins including the numbering conventions for the indole ring-positions (top formula). Compounds 1 (indole-3-acetic acid), 2 (indole-3-propanoic acid), 3 (indole-3-butanoic acid), and 4 (4-chloroindole-3-acetic acid) have been found in plants; compound 5 (7-chloroindole-3-acetic acid) is a metabolite of soil bacteria. Compounds 6 (5-bromoindole-3-acetic acid) and, in particular, 7 (5,6-dichloroindole-3-acetic acid) are synthetic auxins with high general activity. Compound 8 (β-trifluoromethyl-indole-3-propionic acid) has interesting selective effects and may be an antagonist of endogenous indole-3-butanoic acid.

3

R 6 7

1 N H

(CH2)nCOOH 2

(1): n = 1; R = H (4): n = 1; R = 4-Cl (2): n = 2; R = H (5): n = 1; R = 7-Cl (3): n = 3; R = H (6): n = 1; R = 5-Br CF3

Cl

Cl

CH2COOH

CHCH2COOH

N H

N H

(7)

(8)

AUXINS, INDOLE AUXINS ◦ S-(+)-isomer, [α]20 (c = 2.0, ethanol); R-(−)D +10.4 ◦ 20 isomer, [α]D −10.4 (c = 2.0, ethanol).

BIOLOGICAL PROPERTIES

Length of coleoptile sections in mm

The first step in the discovery of auxins was the observation that the growing zones of roots (24) and grass coleoptiles (25) respond to stimuli perceived by their tips. This effect is based on two essential properties of IAA: stimulation of cell expansion and polar transport. Most cell division in plants occurs in specific tissues called meristems. The small isodiametric cells originally formed expand to their adult size in a defined developmental phase. Accompanying morphological changes include the extension of the cell wall and the formation of a vacuole. In elongating stems and roots, the cells preferentially stretch in one direction; isodiametric cell expansion is more common in other tissues. Auxins were originally defined and quantified by their stimulatory effect on stem elongation in standard bioassays (26,27). Typical, asymmetrically bell-shaped, dose-response curves obtained with oat coleoptile sections floating on auxin solutions diluted to a range of concentrations (Avena coleoptile straight-growth test) are shown in Figure 2. The preferred parameters to be used for comparison of different auxins are the maximal elongation and the halfoptimal concentration. The steeply declining response and growth inhibition at supraoptimal auxin levels has been rationalized by assuming that the cellular recognition sites intended to interact with a set of complementary topological elements of the same auxin molecule (for details refer to the section on structure-activity correlations) start interacting, at high ligand concentrations, with the respective topological elements of different auxin

14

12

10 −9

−8

−7

−6

−5

−4

−3

−log (auxin concentration in mol/L) Figure 2. Dose-response curves for 5,6-dichloroindole-3-acetic acid (7) (stars, full line), indole-3-acetic acid (1) (open circles, broken line), and 4,7-dichloroindole-3-acetic acid (9) (black circles, full line), as obtained in the Avena (oat) coleoptile section straight-growth test. Error bars are standard errors of the mean (n = 10). The horizontal line represents the length of control sections kept in water instead of auxin solutions. Elongation is here defined as the difference in the lengths of auxin-treated and control sections. The half-optimal concentration is reached when the dose response curve has ascended to one-half its maximal value; the corresponding value in the descending part of the curve is disregarded.

101

molecules (28) and, in that case, lose the ability to trigger stem elongation. In addition, supraoptimal auxin levels induce the formation of ethylene, a growth inhibitor in many test systems, and frequently exhibit general toxicity. Weak auxins, such as 4,7-dichloroindole-3-acetic acid (9, Fig. 3), even though less growth-promoting than IAA, compete for the same cellular recognition sites and, thus, inhibit its effect for certain concentration ratios (29). Strongly competitive inhibitors that do not stimulate growth to any noteworthy extent have been termed antiauxins (30). α,α-Dimethyl-5,7-dichloro-1H-indole-3-acetic acid (10) (31) and 4-chlorophenoxyisobutyric acid (11) (32), which are shown in Figure 3, are particularly effective. During expansion growth, the otherwise rigid proteinpolysaccharide network in the cell wall is ‘loosened,’ and the turgor pressure is allowed to inflate the cell to a tissue-specific size. One aspect of this complex process is covered by the widely accepted Acid Growth Theory (33,34). The latter is, at least in part, based on the fact that auxins activate specific plasmalemma ATPase(s), both by upregulating the corresponding genes (35) and by more direct mechanisms (36). The enzymes are mostly believed to be oriented in such a way that the hydrogen ion liberated per ATP molecule hydrolyzed is released into the apoplast, while the ADP and the phosphate remain inside the cell (37). The resulting drop in cell wall pH activates expansins, proteins which relax the strong physicochemical interactions between the cellulose fibers and the hemicellulose matrix, thus permitting them to shift positions (38). Nicks in hemicellulose molecules may also form, and some of their building blocks may be rearranged. As the expanding cell wall increases in mass, its polymer components must also be extended, or formed de novo. Enzymes likely involved in these processes are, indeed, induced by auxins (38). The curving grass coleoptile has remained a convenient model system showing the combined effects of auxin transport and cell elongation. In Went’s classical ‘Avena test’ (26,39), the tip is removed from the coleoptile of an intact seedling, and a tiny block of agar containing auxin is placed on the stump to cover about half the cut surface. The auxin diffusing out of the agar is transported down the coleoptile without much lateral spreading. The cells straight below the agar block thus receive most auxin and elongate fastest, and the coleoptile curves by an angle proportional to the auxin concentration applied. The fact that synthetic auxins with top activity in stem elongation assays can be very ineffective inducers of curvature demonstrates that cell expansion and auxin transport have different molecular mechanisms. Auxins can be translocated in the vascular system in any direction. In contrast, cell-to-cell transport is ‘‘polar’’ and, in the shoot, proceeds ‘‘basipetally’’ (i.e., downward for shoots with standard morphology), at a speed of 5 to 20 mm/h. This process is essential for concerted plant development and under tight genetic control. For instance, mutants carrying nonfunctional alleles of the families of PIN-related genes lack essential components of the auxin transport system. One of the first alleles characterized in Arabidopsis, atpin1, gives rise to inflorescence axes that bear 0–1 flower and literally look like pins (40). Polar IAA

102


CH3 Cl

CH3 CH2COOH

Cl

N H

Cl

Cl

CH2COOH

N H

CCOOH CH3

CCOOH

(9)

O

O

CH3 Cl

(10)

(11)

CH2COOH

Cl

CH2SO3H N H

Cl (12)

(13)

(14)

Figure 3. Chemical tools for studying auxin physiology. 4,7-Dichloroindole-3-acetic acid (9) is a weak auxin (Fig. 2) that, in the proper range of concentrations, inhibits IAA-induced growth because both compounds compete for the same cellular recognition sites. α,α-Dimethyl-5,7-dichloroindole-3-acetic acid (10) and 4-chlorophenoxyisobutyric acid (11) compete even more strongly, have no noteworthy growth-promoting effect and are, thus, termed antiauxins. 2,4-Dichlorophenoxyacetic acid (12), a synthetic auxin analogue and herbicide, is transported into plant cells by uptake carriers but can leave only by passive diffusion. In contrast, naphthalene-1-acetic acid (13) has to enter the cells by diffusion but is exported by efflux carriers. Due to the combined action of the uptake and efflux carriers, auxin is polarly transported through plant tissues. This effect was previously rationalized by the chemiosmotic theory, a concept based on the assumption that auxin anions are more abundant in the neutral cell interior than in the acidic apoplast. Strongly acidic auxins, such as indole-3-methanesulfonic acid (14), did not fit into the chemiosmotic theory because the compound is equally ionized inside and outside a plant cell but is, nevertheless, polarly transported.

transport in these mutants occurs at about 10% the wildtype capacity (40). As inferred from immunolocalization studies employing antibodies to fragments of the PIN proteins, polar auxin transport in differentiated stems normally takes place in the cambium and in specialized cell strands in the xylem parenchyma (41). In the root, IAA moves ‘‘acropetally’’ (toward the tip) in the central cylinder (vascular tissue) and is then, at the tip, fed into the cortex and epidermis in which it moves up again (42). Polar auxin transport used to be rationalized by the chemiosmotic theory. All its versions agree in assuming that auxins, which are acids by definition, enter the plant cell by diffusion in the undissociated state and are then retained as the anion. Indeed, the pH gradient between the apoplast and the protoplasm causes some auxin molecules to dissociate while entering the cell. However, indole-3-methanesulfonic acid (14), shown in Figure 3 as an example for an auxin that is completely dissociated at any physiological pH, is taken up by plant stems and is polarly transported (43). A system of specific uptake carriers and efflux carriers, which is supported by an increasing amount of experimental evidence, is thus supplanting the chemiosmotic theory. Among the synthetic auxins, 2,4-dichlorophenoxyacetic acid (12) is a substrate of the uptake carrier, but not the efflux carrier, in contrast with naphthalene-1-acetic acid (13), which is exported by the efflux carrier but has to

enter the cell by passive diffusion. The two carrier systems respond to different inhibitors, even though selectivity is not always perfect. A set of aryl- and aryloxy-acetic acids containing 1-4 condensed benzene moieties and various ring substituents (mostly halogen) were recently established as inhibitors of the uptake carrier (44). 1 µM 3,4-dichlorophenoxyacetic acid (15), shown as an example in Figure 4, was sufficient to slow down the import of 12 to one-half its original rate, but about 200 times higher concentrations were required to impede the export of 13 to the same extent. The relation of the uptake carrier(s) to the AUX1 gene (not to be confused with the AUX/IAA genes, which are transcriptional regulators) remains to be clarified in detail. The gene was discovered based on a set of mutations (aux1-1, etc.) that enable Arabidopsis seedlings to form a normal root system on agar media containing IAA, 2,4-dichlorophenoxyacetic acid (12) (45) or other auxins that are substrates of the uptake carrier, at concentrations that severely inhibit root growth in wild-type seedlings. The AUX1 protein shows sequence homologies to plasmalemma-based carrier proteins involved in the uptake of amino acids, but its expression appears to be restricted to apical root tissues (46,47). Inhibitors of the efflux carrier are shown in Figure 4. Included are representatives of the phytotropins, which are characterized by a benzoic acid moiety coupled at


COOH O

OH

CH2COOH

COOH

H N

O Cl

O

Cl O (16)

(15)

(17) OH

COOH I HO

COOH

I

HO

O

(19)

its ortho-position, via a variety of linkage groups, to a second aromatic ring system. In the most active ˚ (48). analogues, the ring-to-ring distance is 7.5 A Representative examples are fluorescein (16) and its derivatives and N-naphthylphthalamic acid (17), which is particularly popular for experiments on the laboratory scale. Many structurally related compounds with acidic functionalities also inhibit auxin efflux (49); examples include the morphactins (50) such as 9-hydroxyfluorene9-carboxylic acid (18), 2,3,5-triiodobenzoic acid (19; also a weak auxin and an antiauxin), and a number of endogenous flavonoids (51,52), such as quercetin (20). Based on its affinity for 17 and IAA, a plasmalemma protein localized at the basal end of pea-stem cells (53) was proposed as a component of the efflux carrier. A 23 kDa protein with similar binding properties (but unverified polar distribution) was isolated from the plasma membranes of corn coleoptiles (54). In Arabidopsis thaliana, a corresponding protein (or proteins regulating its biosynthesis and function) may be encoded by the TIR3 gene: a mutation at this locus reduced both auxintransport velocity and the number of binding sites for 17 (55). Transmembrane proteins of the PIN family are also thought to participate in auxin efflux and are localized accordingly, i.e., in Arabidopsis, AtPIN1 (67 kDa) in the vascular tissues of shoots and roots (41), and AtPIN2 (69 kDa) in the root cortex and epidermis (42). Genetic control of auxin efflux also includes RCN1, which encodes a regulatory A subunit of protein phosphatase 2A (a serine/threonine phosphatase) and affects sensitivity to 17 by mechanisms that require further study (56). Examples for physiological processes in which auxin transport plays a decisive role include: phototropism, gravitropism, and morphogenetic events such as stem branching, the differentiation of vascular elements, and embryo development. Even though phototropism and gravitropism deal with different stimuli, the response is, in both cases, assumed to be based on preferential auxin transport along one side of the stem and accelerated cell

OH OH

I HO

(18)

103

O (20)

Figure 4. Inhibitors of auxin transport. 3,4-Dichlorophenoxyacetic acid (15) is one of the most efficient inhibitors of the uptake carriers; the other compounds shown inhibit the efflux carriers. Fluorescein (16) and N-naphthylphthalamic acid (17) are phytotropins, carboxylic acids characterized by a specific arrangement of aromatic ring systems. 9-Hydroxyfluorene-9-carboxylic acid (18), a representative of the morphactins, is usually applied as its methyl ester which is hydrolyzed in situ. 2,3,5-Triiodobenzoic acid (2,3,5-T, 19) also acts as a weak auxin/antiauxin. Quercetin (20), as an example for the endogenous flavonoid transport-inhibitors, contains structural features reminiscent of the phytotropins (e.g., 16,17) but lacks their carboxyl function.

expansion on that side, in perfect analogy to the induction of coleoptile curvature by an asymmetrically placed source of auxin in Went’s Avena test (57). This classical concept, known as the Cholodny-Went theory, is supported by an increasing amount of evidence. The gravitropic response, for example, is clearly blocked by auxin transport inhibitors. Asymmetric auxin transport during gravistimulation was first demonstrated by Dolk (58) and appears to be accompanied by asymmetric IAA distribution within the responding tissues (59). IAA transport is one of the factors that influence stem branching patterns in a fashion traditionally described as apical dominance or apical control. These terms are based on the observation that the development of lateral meristems (axillary buds) is usually inhibited to an extent depending on their distance from an actively dividing apical meristem (57,60). The lateral buds that eventually develop will result in branches oriented at defined angles with respect to gravity (‘‘plagiotropism’’). Genetic control of the various aspects of apical dominance is demonstrated by the unique shapes and branching patterns that characterize many cultivars of crop plants and ornamentals. The pruning techniques used in horticulture to force trees and shrubs into arbitrary shapes exemplify that removal of the terminal meristem may activate dormant lateral buds. In many ornamentals, new flowering branches are induced when previously formed seeds are removed, thus demonstrating that reproductive structures can have morphogenetic effects similar to apical dominance. The crucial processes controlling stem branching have been searched for in the lateral buds involved but now appear more likely to occur in the preexisting stem. During shoot development, vascular strands start forming while the leaf initials are just taking shape. This appears to be regulated by the IAA transported out of the growing leaves as indicated, for instance, by the observations that: 1) leaf removal stops vascular differentiation; 2) the latter can be maintained by

104


two separate cotyledons if exposed to auxin transport inhibitors. Why do plants, in addition to IAA, need other indole auxins, such as 4-Cl-IAA and IBA? For many years, only quantitative differences in their physiological properties were known, but now there are examples for qualitative differences as well. The elongating pea pod, for instance, contains both IAA and 4-Cl-IAA (67), which appear to be provided by the seeds. When those are removed, application of 4-Cl-IAA will sustain continued pod growth. IAA, in contrast, acts, if at all, then as a slight inhibitor (68). Corn roots contain both IAA and IBA, but only the latter is substantially affected by drought stress (69), treatment with compound 8 (23), and infection with an arbuscular mycorrhizal fungus, which profoundly affects root morphology (23,70).

providing an artificial source of auxin which will, moreover, induce vascular strands if applied in parts of the stem that never bore any leaves; 3) inhibitors of auxin transport applied at the leaf base arrest vascular differentiation in the adjacent lower internode(s); and 4) mutants with nonfunctional PIN1 genes form disorganized clusters of vascular elements at the leaf bases (41). When secondary growth starts, IAA gradients appear to be one of the essential factors regulating vascular differentiation (61–63). IAA levels peak in the cambium (up to 7 µg/g fresh weight or 40 µM), dropping more steeply at the phloem than at the xylem side. The xylem cells then appear to continue expanding as long as the IAA level exceeds a certain threshold, whereupon they start differentiating into functional vascular elements. The auxin gradient is supposedly maintained by auxin diffusion from the cambium and slow auxin metabolism in the vascular tissue, but active transport could also be important. When in the life cycle of a seed plant is polar auxin transport established? Immunolocalization of the PIN1 protein indicates increasing polar distribution during the globular stage of embryo development (64). This establishes not only a vertical axis, but, in dicots, also directs formation of the two cotyledons. Arabidopsis embryos homozygous for pin1, and, hence, defective in auxin transport, have fused cotyledons (65). Also, regardless of whether excised from developing seeds of Brassica juncea (L.) Koss. and cultured in a nutrient medium (65) or formed in vitro in carrot suspension culture (66), globular embryos would never develop

CHEMICAL SYNTHESIS Procedures suitable for the preparation of IAA on an industrial scale include the base-catalyzed condensation of indole with chloroacetic acid (71) or with potassium glycolate at 250◦ (72). These reactions are based on the particular reactivity of the indole 3-position towards electrophilic agents, a property which is also exploited in laboratoryscale syntheses. Two general reaction sequences for the preparation of ring-substituted IAAs (73) are illustrated in Figure 5. Conversion of the corresponding indole (21) to 3-(N,N-dialkylaminomethyl)indole 22 under classical Mannich conditions usually proceeds at room temperature. Gramines (22, R = methyl) have been used in the syntheses of most ring-substituted IAAs, but an ethyl residue is

R CH2N

X

R N H

KCN

(22) HCHO + HNR2 + HOAc

X

CH2CN KOH

X N H

N H

(21) HCONMe2 + POCl3

CHO

(26)

KCN

NaBH4

CH2OH

X

N H (24)

N H

(23)

X

CH2COOH

X

CH2

+ N

R

N H (25)

R

(27)

Figure 5. Methods for the preparation of ring-substituted indole-3-acetic acids from appropriately substituted indoles. Reagents and intermediates are discussed in the text.


105

CN (30) CH2

CH2CH2CN

D

D

D

D NH2

D

HNO2

D + N

D

D

D

CH2COOH N H

D

(34)

C COOC2H5

D

D C(H,D)2COOH

D

N

(31)

D

D

CH2 H N

D

D

D

D

(29)

D

D

COOC2H5

N

D

COCH3

D

(28)

D

HC

N H

COOH

D

C(H,D)2COOC2H5

D D

(33)

N H

COOC2H5

(32)

[4,5,6,7-2 H

Figure 6. Preparation of 4 ]indole-3-acetic acid (34), as an example for the application of the Fischer synthesis, a versatile method for the preparation of IAA and its ring-substituted derivatives by acid-catalyzed cyclization of phenylhydrazones. Reagents and intermediates are discussed in the text.

the best choice for R in the preparation of 4-Cl-IAA (4) (74). In the presence of ring-substituents that interfere with the standard Mannich procedure, the presumed active entity, the N,N-dialkyl-N-methylene-ammonium ion (27), may also be prepared separately (75). Displacement of the dialkylamino moiety in 22 with cyanide affords nitrile 23, which is saponified to acid 26. A less popular, but about equally efficient, way of introducing a side chain at the indole 3-position is based on Vilsmeyer-Hack condensation with N,N-dimethylformamide in the presence of phosphorus oxychloride (73). The resulting substituted indole-3-carboxaldehyde (24) may then be reduced (76) to the corresponding indole-3-methanol (25), which reacts with cyanide in essentially the same way as 22 (77). Many of the starting indoles required in the above syntheses are commercially available. A particularly versatile method for the preparation of additional ring-substituted indoles was described by Batcho and Leimgruber (78). Many ring-substituted IAAs have also been prepared from the appropriate phenylhydrazones as illustrated for the case of [4,5,6,7-2 H4 ]IAA (34; Fig. 6) (79). Hydrazone 31 was obtained from commercial [2,3,4,5,62 H5 ]aniline (28) via diazonium ion 29, which underwent Japp-Klingemann condensation (80) with 2-acetyl-4cyanobutyric acid ethyl ester (30) without significant deuterium-protium exchange. Fischer cyclization of 31 in DCl/C2 H5 OD/D2 O afforded diester 32, which was hydrolyzed to 33 and converted to 34 by base-catalyzed 2-decarboxylation with concomitant side-chain dedeuteration. Further applications of the Fischer cyclization in auxin synthesis are discussed by Brown (81). In most cases, low-priced starting materials are readily available, but the acidic catalysts, which are usually required,

inevitably cause concomitant formation of polymeric material (‘‘tar’’). Purification of the crude product thus tends to require time and considerable amounts of solvents and/or chromatographic sorbents, with final yields rarely exceeding 10%–20%. Even lower yields have been reported with ring substituents sensitive to strong acid, such as (ar)alkoxy, and with substituents that inactivate the benzene ring of the starting hydrazones towards electrophilic substitution. 4,6- and 4,7-dichloro-IAA (9) were thus obtained in yields of 7% and 9% (74). Fischer synthesis of the particularly potent auxin 7 afforded the target compound in a 1 : 1 mixture with 4,5-dichloro-IAA (14) because closure of the pyrrole ring, in the step corresponding to the conversion of 31 to 32, may occur in any of the two positions ortho with respect to the hydrazono group. IBA (3) is prepared by condensation of indole with γ -butyrolactone (82), a method that permits modification for the synthesis of ring-substituted IBAs. Alternatively, they may be prepared by Fischer cyclization in essentially the same way as the corresponding IAAs, with straightforward modification of the aliphatic moiety in the starting hydrazones. CHEMICAL STABILITY IAA, both in crystalline form and in solution, needs to be stored in the dark and as cold as possible. Aqueous solutions should be around neutral pH and should not contain any oxidants, including ions such as Fe3+ . Organic solvents should be rigorously purified from peroxides (important for diethyl- and other aliphatic ethers, dioxane, tetrahydrofuran) and other reactive impurities such as: phosgene in chloroform and related haloalkanes, acetic

106


Figure 7. Products formed by chemical transformation of IAA. 3-Methyleneoxindole (35) is formed by chemical oxidation and by the action of peroxidases. 1-Trimethylsilylindole-3-acetic acid methyl ester (36) is one of the volatile derivatives used in gas-chromatographic analysis. The intensely fluorescent 2-methylindolo-α-pyrone (37), formed by acid-catalyzed condensation of IAA and acetic acid anhydride, has been used in quantitative analysis.

CH2

N H

O

CH2COOCH3

O

N H3C

N H

Si

CH3

O CH3

CH3 (35)

anhydride in ethyl acetate, and aldehydes in primary alcohols. It is also worth remembering that the category of aliphatic ethers, as far as peroxide-forming potential is concerned, includes the Cellosolve family of solvents, polyethylene glycol, and detergents such as Tween and certain kinds of Triton. If the above precautions are ignored, IAA quickly turns from colorless to pink or brown, and dilute solutions lose auxin activity. The classical lore on this subject has been summarized by Galston and Hillman (83). Based on published data and their own experiments, Hinman and Lang (84) gave an overview over the oxidation of IAA by peroxidase and a variety of chemical agents (NO2 − , K2 S2 O8 , Fe3+ + O2 , pH 1 + O2 , pH 1 + H2 O2 ), concluding from UV absorbance patterns that the most prominent product was 3-methyleneoxindole (35) (Fig. 7) in all cases examined. The proposed mechanism includes: 1) electron abstraction from the indole nucleus to form a radical cation with the unpaired electron localized at the ring 3-position, 2) reaction with molecular oxygen, and 3) side chain decarboxylation and a complex pattern of rearrangements influenced by external and internal factors. Step 3 also allows for predominant formation of indole-3-carboxaldehyde (24, R = H) and (even though not explicitly stated) indole-3-methanol (25) (R = H), as found by other authors under a different set of conditions. 3-Methyleneoxindole with its extreme tendency toward polymerization (85) was suggested as the parent compound of the ‘‘tars’’ formed on IAA decomposition. Radiochemical yields obtained in the preparation of [14 C]- and [3 H]-labeled indolic compounds tend to decrease as the specific activity increases, because the compounds’ own β-radiation triggers the formation of destructive free radicals. As these are quenched by most solvents, decomposition of radiolabeled indoles is usually more severe when their solutions are taken to dryness or during recrystallization (86). Thus, when a solution of pure L[5(n)-3 H]tryptophan was evaporated, about one-third of the residue was converted to [5(n)-3 H]IAA (87). This is particularly disturbing, as radiolabeled tryptophan has been used many times to ‘‘prove’’ the role of this amino acid in IAA biogenesis. A combination of an antioxidant (1,2dithioethane) and a free-radical quencher (anthracene) efficiently protected radiolabeled IAA during chemical operations (88), but the large excess in which these protectants must be added makes them unsuitable for use during IAA purification from biological material. In crude plant extracts, the organic solvents present and freeradical quenching phytochemicals, such as carotenoids, flavonoids, and stilbenes, will cut off radiolytic chain

(36)

(37)

reactions. The latter can, however, do considerable damage when the plant sample has been purified for auxin analysis. Stable-isotope-labeled precursors should, thus, be preferred in metabolic studies on indole auxins. Even though IAA is decomposed by visible light, the compound absorbs only UV radiation, which does not pass through the walls of standard laboratory glassware. Also, IAA fluoresces intensely if irradiated at its absorbance maximum at 280 nm, which implies that the molecule is largely stable under these conditions. Photosensitizers must be involved in IAA decomposition by visible light. Their effect is particularly disturbing in plant tissue culture in which the hormone levels supplied should be strictly defined. However, about 90% of the IAA originally added to standard Murashige-Skoog medium vanished after 3 days of exposure to white fluorescent light at an intensity required for the explants to proliferate. The photosensitizer present in this system was identified as EDTA-complexed iron, which acts alone or in concert with nitrate (89–91). Both have absorbance maxima around 300 nm, which are, however, sufficiently broad to extend into the visible (violet and blue) region. Accordingly, IAA was largely stable in Murashige-Skoog medium exposed to light that was passed through a yellow filter that removes wavelengths below 450 nm (91). ISOLATION, IDENTIFICATION, AND QUANTITATIVE ANALYSIS Reliable data on the kind and concentration of endogenous auxins provide a rational base for understanding and manipulating their physiological effects. Auxin analyses may also be necessary to check the purity and composition of commercial preparations. In the latter case, thin-layer chromatography is the method to start with. Suitable solvents and spray reagents for the visualization of indole derivatives have been compiled by Kaldewey (92). Reagents based on p-dimethylaminobenzaldehyde, such as the van Urk (1% solution in 1 : 1 HCl/ethanol) and the Ehmann (1 part of van Urk’s reagent + 3 parts of a solution of 2 g FeCl3 ·6H2 O in 500 mL H2 O + 300 mL conc. H2 SO4 ) (93) reagents afford structure-dependent colors (various shades of yellow, red, and blue) and a detection limit around or below 100 ng per spot. When thin-layer chromatography is used in quantitative analysis, or on a preparative scale, acidic solvents should be avoided. The developed chromatogram should be left in a stream of cool air, for a few minutes, and the zones or spots of interest should be collected while the sorbent is still somewhat moist (not dripping wet). Further precautions are discussed in 94. For radioactive indole auxins,


recoveries on cellulose layers tend to be superior to silica gel but inferior to paper chromatography. Thin-layer chromatography also permits screening for indole auxins and their derivatives in exceptionally auxinrich tissues, such as some developing seeds; otherwise, more sensitive methods are required. The plant material should be extracted by homogenization in a nonreactive, pure solvent that immediately inactivates the enzymes in the extracted tissue. Acetone and methanol are usually preferred, possibly mixed with up to 50% of water if dry tissues, such as seeds, are extracted or if the quantitative extraction of polar auxin metabolites is desired. In some cases, it proved necessary to include an antioxidant, such as diethyldithiocarbamate (95) or butylated hydroxytoluene (94). As the minute amounts of auxins present in vegetative tissues dissolve in water quite as well as in organic solvents, extraction with neutral aqueous buffers has been advocated (96) arguing that: 1) this avoids coextraction of lipids, carotenoids, and chlorophylls and, thus, simplifies purification of the extracted auxin; 2) both the enzymes and their potential substrates are substantially diluted, and the extraction is done at +4 ◦ C when enzymatic reactions are slow. The IAA-ester conjugates present in corn seeds do, however, hydrolyze in neutral buffer at a rate of ca. 3% per h (96). Nevertheless, a number of examples for successful buffer extraction have been published [e.g., (62,97,98)] including a popular compromise approach employing 65 : 35 to 80 : 20 (v/v) mixtures of 2-propanol and a pH 7 imidazole/HCl buffer (99). In many (but not all) plant extracts, it is possible to analyze for overall auxin conjugates (see below) without identifying the individual compounds. Hydrolysis with 1-2 N NaOH for 1 to 2 h at room temperature will set free IAA from its glycosyl and cyclitol esters. Boiling with 7 N NaOH for 3 h will also cleave amino acid and peptide conjugates, as well as indole-3-acetamide. Oxygen must be excluded, in the latter procedure, to avoid IAA formation by chemical oxidation of unknown precursors (100). When the presence of IAA-conjugates insoluble in standard extractants is suspected, alkaline hydrolysis can also be performed with the finely ground plant tissue (100). Identification and quantification of the extracted auxins requires previous sample purification. Analytical methods, which allegedly do not require that step, have been announced in regular intervals, but the accompanying excitement has regularly been short-lived. The optimal protocol for the stepwise elimination of non-auxin plant constituents depends on kind and quantity of the tissue extracted. Generally, one would start with methods with low resolving power, which tolerate high sample loads. Suitable procedures for samples on a 10-gram scale include: evaporation to successively smaller volumes and precipitation of water-soluble plant constituents with organic solvents (alcohols, acetone), solvent partition, and/or column chromatography using sorbents such as silica gel, Sephadex, poly-N-vinylpyrrolidone, anion exchangers, and styrene-divinylbenzene copolymers (e.g., Amberlite XAD-7) (101). For samples on a gram scale, solid-phase extraction in disposable cartridges is more convenient. Sequestration of acidic auxins with an anion

107

exchanger (e.g., aminopropyl- or dimethylaminomethylmodified silica gel) and further purification on a reversedphase cartridge (99) usually eliminates the bulk of nonauxin plant constituents. Microscale procedures suitable for plant samples in the milligram range have also been proposed (98,102). The purification process is completed with methods of high resolving power, such as reversed-phase HPLC and/or capillary GC. Either separation method may be coupled with mass spectrometry for identification and quantitative analysis. Prior to GC, polar compounds need to be converted to volatile derivatives. For IAA and related carboxylic acids, the methyl esters, prepared with diazomethane, have convenient retention times on columns containing moderately polar stationary phases (dimethylsilicone-phenylmethylsilicone mixtures). Further substitution at the indole nitrogen by a trimethylsilyl (36; Fig. 7) or trifluoroacetyl group, or one of their higher-molecular-weight homologues, leads to derivatives that optimally separate on nonpolar stationary phases (dimethylsilicones). Silylation of previously underivatized indole auxins includes both the carboxyl and the indole NH groups. Samples of different origin tend to require different derivatization strategies to shift diagnostically significant ions out of the regions of high background from co-separating contaminants. Purification of an auxin sample until a clean mass spectrum is obtained provides an (almost) error-proof identification, in particular if the exact (four decimal digits) mass of the molecular ion is determined. This parameter, in most cases, unequivocally defines the elemental composition. Auxin quantification is now almost exclusively based on isotope-dilution methods (103) because experience has shown that recoveries during sample purification are small and rarely ever reproducible. A known amount of an isotope-labeled analogue of the target auxin is added as an ‘‘internal standard’’ while the plant material is extracted. After sample purification, the auxin-to-standard ratio is determined. This must be the same as the ratio before purification because recoveries, however low and variable they may be, are identical for the target auxin and its isotopomers. By multiplication of the above ratio with the amount of internal standard added, the amount of auxin in the original sample is obtained (see (79,103) for technical details). For quantification by mass spectroscopy, stable-isotopelabeled internal standards are preferred. The analysis is usually combined with the last purification step, as GC-mass spectrometry or LC-mass spectrometry, using ‘‘single ion monitoring’’ to record the abundance of two to three characteristic ions from the target auxin and the corresponding ions from the internal standard as the sample passes from the chromatographic column into the mass spectrometer. If sample purification was successful, the intensity ratios for the selected corresponding ions from the internal standard and from the auxin of plant origin will be identical and reflect the auxin-to-standard ratio at the time of extraction. The first stable-isotope-labeled standard used in IAA analysis was side-chain deuterated [2 ,2 -2 H2 ]IAA (104).

108


CH2CH2NH2 (45)

CH2COCOOH

CH2CH2OH (46)

(44)

CH2CH

CH2CH COOH

CH2CHO (43)

NH2

(38)

NOSO3− CH2C S-b-D-Glcp (41)

? ?

CH2COOH (1)

CH2CN (40) bacteria only

N H

NOH (42)

CH2CONH2 (39) Figure 8. Pathways of IAA biosynthesis from L-tryptophan. Only the stepwise remodeling of the side chain in the indole 3-position is shown in detail. Enzymes and intermediates are discussed in the text.

The compound can be prepared without special equipment, by base-catalyzed deuterium-protium exchange. Unfortunately, this is reversible, and the deuterium is partially replaced by protium under conditions commonly encountered during sample purification (79). The auxin analyses based on side-chain deuterated IAA as an internal standard, thus, had to be corrected by a factor close to ten (105), when the next generation of stable-isotopelabeled standards became available: [4,5,6,7-2 H4 ]IAA and [2,4,5,6,7-2 H5 ]IAA (79). Their deuterium exchanges so slowly, under the conditions encountered in auxin analysis, that the resulting error may usually be neglected. An even safer internal standard, which is not at all subject to isotope exchange, is [3a,4,5,6,7,7a-13 C6 ]IAA (106). [carbonyl-13 C]IAA is also known (107), and the method for its preparation may deserve consideration if internal standards for the quantification of rarely used substituted IAAs are required. 4-chloro-[2,5,6,7-2 H4 ]IAA was used in the analysis of endogenous 4-chloro-IAA in pea fruit (67), and [2-13 C]IBA was prepared for the quantification of endogenous IBA (108). In studies encompassing a large number of samples of similar origin, it may be possible to simplify auxin analysis, once mass spectroscopy has provided unequivocal identifications and defined the range of concentrations to be expected. A selective detection method may then obviate one or two purification steps. Examples include the conversion of IAA to its pentafluorobenzyl ester, which can be detected by gas chromatography using an electron capture detector (109), the use of a fluorescence detector (indole-specific excitation and fluorescence wavelengths) in connection with HPLC (110), and the indolopyrone fluorescence assay (111). The latter is based on the acidcatalyzed condensation of IAA with acetic acid anhydride to yield the intensely fluorescent 2-methylindolo-α-pyrone (37) (Fig. 7) (112) and has been used with variable success

[e.g., (113,114)]. Accurate results can only be guaranteed with the same degree of sample purification as for GCmass spectrometry, but can be more time-consuming to obtain. Immunochemical methods are promising but should be used with caution in auxin analysis. The target compounds are not immunogenic in mammals unless coupled (as ‘‘haptens’’) to macromolecular carriers. The immediate neighborhood of the coupling site is inaccessible to the immune system; in the remaining part of the hapten, antibodies are fitted to groups of spatially close (but not necessarily linked by chemical bonds) atoms. At first, IAA was coupled via its carboxyl group (115). Some of the resulting monoclonal antibodies (116) are commercially available. IAA extracted from plant samples has to be converted to its methyl ester to react with those antibodies. In an alternative approach, IAA was coupled to a protein carrier via the pyrrole nitrogen using a Schiff-base type linkage (117). To keep both characteristic epitopes of the IAA molecule, the carboxyl and the pyrrole NH groups, freely exposed, it was attempted to construct an antigen starting with 5-hydroxyindole3-acetic acid (118). Technical problems with obtaining a homogeneous protein conjugate were partially solved by selecting monoclonal antibodies complementary to the desired hapten (119). All papers quoted address the affinity and selectivity of the respective antibodies in testtube assays with pure chemicals, but there have been few attempts to compare the accuracy and precision of immunoassays with that of other analytical methods. By using polyclonal antibodies to carboxy-linked IAA (120), the IAA extracted from pine seedlings could only be accurately quantified after multistep sample purification. Essentially, the same conclusion was reached when the corresponding commercial monoclonal antibodies were tested in a variety of plant samples (121). Antibodies


to N-linked IAA accurately quantified free IAA from etiolated corn shoots after purification by a simple solvent partition procedure, but additional clean-up was required for the IAA liberated from ester conjugates by alkaline hydrolysis (122). More successfully, antibodies of this type (123) and monoclonal antibodies made via 5-hydroxyindole-3-acetic acid (119) were used in immunoaffinity sorbents for the isolation of auxins from plant extracts. If auxin quantification is not done by mass spectroscopy, radiolabeled internal standards are obligatory. For accurate results, their specific activity and radiochemical purity must be precisely known. BIOGENESIS As IAA is chemically related to tryptophan (38), this amino acid appeared to be a plausible precursor of native auxin. However, recent evidence reviewed in 124–126 indicates at least one additional pathway. The corn mutant orange pericarp, for instance, is unable to synthesize tryptophan and can only be maintained in the heterozygous state. Homozygous seedlings temporarily survive on the reserve tryptophan derived from the mother plant and, during that period, contain about 50 times higher IAA levels than wild-type seedlings. Feeding the indole precursor, [15 N]anthranilic acid, to the mutant afforded [15 N]IAA, but L-[15 N]tryptophan and L-[2 H5 ]tryptophan were not utilized as auxin precursors (127). This indicates that IAA biosynthesis in this system diverges from the tryptophan pathway before the condensation of indole with serine. Similar results were obtained for a set of Arabidopsis auxotrophs, each of which lacked a different enzyme of the tryptophan pathway (128). Some plants with wild-type metabolism were shown to convert indole to IAA. In corn, this occurs simultaneously: 1) via tryptophan and 2) by a tryptophan-independent route (129,130). Carrot tissues can, at least in vitro, switch back and forth between tryptophan-dependent and tryptophan-independent IAA biosynthesis (131,132). Tryptophan-dependent IAA biosynthesis can follow several routes (Fig. 8). A two-step mechanism involving indole-3-acetamide (39) was first found in the pathogen Pseudomonas syringae pv. savastanoi (133) and eventually explained by the presence of two genes: iaaM, which encodes a tryptophan monooxygenase generating the indole-3-acetamide, and iaaH which encodes a hydrolase converting the amide into IAA (1). These and related genes appear to be widely distributed in bacteria (134) and are used by pathogens to manipulate the auxin metabolism of their host plants at the site of infection, as exemplified by the ‘‘crown galls’’ caused by Agrobacterium tumefaciens. The two genes have also been introduced into dicots, such as tobacco (135) and aspen (136). The transgenic plants exhibit morphogenetic changes, rather than exuberant general vigor. This suggests enhanced levels of IAA in local meristems, even though its bulk concentrations in leaves and internodes are about the same as in the corresponding parts of isogenic wild-type plants. The bulk concentrations of IAA conjugates (see below) are, however, significantly larger in the transgenics (135).

109

In the 1950s indole-3-acetonitrile (40) caused excitement as a ‘‘neutral hormone’’ apparently overturning the rule that all auxins are acids. The compound was first extracted from kohlrabi (137), subsequently found in other representatives of the Cruciferae family, and chemically characterized as ‘‘the first plant hormone isolated in pure form from actively growing plant material’’ (138). Disappointment followed; not only did the nitrile lack auxin activity unless converted to IAA, the original isolate also proved to be an artifact formed from the glucosinolate, glucobrassicin (41), due to inappropriate extraction procedures (139). Crucifers contain hydrolytic enzymes (myrosinases) that are compartmentalized away from glucobrassicin in intact plant tissues but start degrading the glucosinolate as soon as the tissue is damaged. If that happens, 40 is one of the decomposition products formed. As myrosinases have large turn-over numbers and are difficult to inactivate instantly, the roles of 40 and 41 in IAA biogenesis have remained a controversial issue. A plausible intermediate in the formation of both compounds is indole-3-acetaldoxime (42) (140–144). Recent tracer studies performed in roots of axenically grown Arabidopsis plants are most easily interpreted assuming that 40, but not 41, is an intermediate in IAA biosynthesis from tryptophan (145). The last step of this putative metabolic pathway requires a nitrilase, a group of enzymes represented in many, but not all, plant species (146). A set of cloned nitrilases was isolated from Arabidopsis (147–149) and a hydratase forming 39 (see above for its conversion to IAA) was purified from Agrobacterium and Rhizobium (150). At least in vitro, oxime 42 may also be converted to indole-3-acetaldehyde (43) (140,151,152), which is likewise formed in the two remaining pathways shown in Figure 8. The route involving indole-3-pyruvic acid (44) is excellently characterized in microorganisms that feed on tryptophan (134,153) and form IAA as a metabolite, without obvious physiological effects. In seed plants with their strictly controlled, low IAA levels, metabolic flux through this biosynthetic pathway must be significantly less and, thus, difficult to detect, in view of the fact that 44 is chemically unstable at physiological pH. In situ preparation of its pentafluorobenzyl-oxime afforded a stable derivative that was included in mass spectroscopic studies on the kinetics of deuterium incorporation from D2 O into IAA and its putative precursors. Indeed, in the tomato plants used in these experiments, the indole-3-pyruvic acid pathway appears to prevail in IAA biosynthesis (154). Tryptamine (45) and its N-acyl conjugates are widespread plant constituents, and so are the aromatic L-amino acid decarboxylases responsible for its formation from 38 and the amine oxidases which convert 45 into aldehyde 43 (155,156). Both classes of enzymes have been isolated from the same plant source [cucumber seedlings; (157,158)], a necessary, but not a sufficient, condition for an operative tryptamine pathway. Amine 45 is also the precursor of certain indole alkaloids (159) formed, for example, by Catharanthus roseus. The gene for

110


the respective tryptophan decarboxylase (160) is downregulated by auxin (161). This may point to a feedback controlled tryptamine pathway of IAA biosynthesis or merely reflect the fact that the indole alkaloids mostly accumulate in mature tissues that no longer respond to auxin. The IAA-overproducing yucca mutants of Arabidopsis overexpress flavine monooxygenase-like enzymes, which oxidize 45 at the aliphatic nitrogen, thus forming N-(indol-3ylethyl)hydroxylamine which would, by dehydrogenation, afford aldoxime 42 (162). This, in part hypothetic, pathway would link 45 to the biogenesis of 40 and 41 and also provide a novel route to 43. Oxidation of aldehyde 43 completes IAA biogenesis by the indole-3-pyruvic acid and tryptamine pathways and by the putative indole-3-acetaldoxime route. Cucumber (163) appears to contain an indole-3-acetaldehyde oxidase, which is feedback inhibited by synthetic auxin 12 (inhibition by IAA could not be tested because of the assay procedure used). Recent results in Arabidopsis revealed at least four aldehyde oxidase genes, atAO-1 to atAO-4. The atAO-1 protein carries activities towards aldehydes 24 and 43 and, in part, occurs as a homodimer (AO-α) but also associates into a heterodimer (AO-β) with atAO-2, which is more active towards substrates such as naphthalene1-carboxaldehyde (164,165). The fact that AO-α is more abundant in the auxin-overproducing mutant superoot1 (sur1; characterized by increased formation of lateral and adventitious roots) corroborates the involvement of this enzyme in IAA biogenesis (164). Reduction of aldehyde 43 affords indole-3-ethanol (46), a ubiquitous natural product first isolated by Ehrlich (166) as a yeast metabolite. As reviewed in (167), alcohol 46

is subject to reoxidation by enzyme feedback controlled by auxins (168). The compound may, thus, be part of a buffering mechanism, which stabilizes the output of IAA biosynthesis via aldehyde 43. The putative buffering pool can be extended by reversible conjugation. An alkalilabile conjugate of 46, presumably an ester, is stored in pine seeds and utilized as a source of auxin during germination (169). Vegetative plant material converts exogenous 46 to a number of esters and glycosides (170). Of the remaining endogenous indole auxins, IBA (3) appears to originate from IAA, as most thoroughly investigated in seedlings of Arabidopsis thaliana (171) and Zea mays (172,173). The process requires ATP and acetyl Coenzyme A (173). The biogeneses of 4-Cl-IAA and 7Cl-IAA remain to be worked out in detail. In both cases, the corresponding chlorinated tryptophans occur in the same source material and would, thus, be plausible precursors (8,11). However, the Pseudomonas strains, which make 7-Cl-IAA in addition to 7-chloroindole-3pyruvic acid, also form 7-chloroindole and 7-chloroindole3-acetamide (11,174), an observation that may indicate multiple biosynthetic pathways. The bacteria also contain a chloroperoxidase that converts indole to 7chloroindole (175), but may as well act on other endogenous indole derivatives. CONJUGATION Up to 95% of the total auxin pool in vegetative plant tissues is linked, by ester or amide bond, to a diverse array of biomolecules (176,177). Examples for such ‘‘conjugates’’ or

CH2OH

COOH

O O

OCCH2

HCNH

OH N H

HO OH

(CH2)n

O

(50): n = 1; X = H (51): n = 2; X = H

OCCH2

(52): n = 2; X = NH2

HO

N H

OH OH

Figure 9. Examples for IAA conjugates identified in plants by unequivocal methods. Compounds 47 [1-O-(indol-3-ylacetyl)-β-D-glucopyranose] and 48 [2-O-(indol-3-ylacetyl)-myoinositol] and/or its positional isomers are known from many seed plants. Compound 49 [Nε -(indol-3-ylacetyl)-L-lysine] is an IAA metabolite of the microorganism Pseudomonas syringae pv. savastanoi. Conjugates 50 [N-(indol-3-ylacetyl)-L-aspartic acid] and 51 [N-(indol-3-ylacetyl)-L-glutamic acid] are commonly found, except in the grasses; compound 52 [N-(indol-3-ylacetyl)-L-glutamine] was detected in Arabidopsis supplied with exogenous IAA. Conjugate 53 [N-(indol-3-ylacetyl)-L-alanine] is a native constituent of Picea abies shoots.

N H

COOX (47)

HO

OCCH2

COOH HCNH

OCCH2

CH3

OH

N H

(48) (53)

COOH HCNH2 (CH2)4 NH

OCCH2 N H (49)


‘‘bound auxins’’ are shown in Figure 9. Caution is necessary when aliphatic esters are detected because they may be artifacts formed from endogenous indoles when potential alkoxy donors, such as alcohols and ethyl acetate, are used as solvents during sample extraction and workup. 4-Cl-IAA methyl ester is, however, an established constituent of ripening pea seeds (6,7). The most important glucose ‘‘ester’’ of IAA, 1-O-(indole-3-ylacetyl)-β-Dglucopyranose (47) (recommended abbreviation: IAA-Glc), is, strictly speaking, an alkyl acyl acetal because the auxin residue is linked to the hemiacetal function of the glucopyranose moiety. The compound was first detected in Colchicum leaves fed excessive IAA (178) and has since been found many times as a native plant constituent or as a metabolite of exogenous IAA. The enzyme that forms IAA-Glc from IAA and UDP-Glc has been purified and cloned from developing corn kernels (179,180) and Arabidopsis (most abundantly expressed in developing fruit) (181). The IAA residue may then move to positions 2, 4, or 6 at the glucopyranose ring (182), and it may be transferred to myo-inositol to yield, for instance, compound 48. The enzyme catalyzing this reaction has been characterized (183), but it is not known to which hydroxyl group of the cyclitol the acyl moiety is originally transferred; in vitro it migrates between all possible positions. The conjugates formed may be further linked to galactose and arabinose. myo-Inositol may also carry up to three IAA residues. Such complex conjugates have so far only been found in corn; IAA-myo-inositol occurs in other species as well (176,184). An enzyme attaching an additional IAA residue to O-6 of IAA-Glc (47) was recently purified from corn seedlings (185). Sugars, other than glucose, that form conjugates with IAA include rhamnose (186), rutinose (glucosyl-rhamnose) (184), and, most importantly, the sugar moieties of glucans and glycoproteins (176). By in vitro aminolysis, IAA-Glc can also give rise to conjugates of the amide type, but this reaction has not been demonstrated in living plants. The biosynthesis of Nε -(IAA)-L-lysine (49) in the microorganism, Pseudomonas syringae pv. savastanoi is catalyzed by an enzyme that requires ATP and bivalent metal ions as cofactors (187). How seed plants conjugate IAA with amino acids is unknown, even though N-(IAA)-L-aspartic acid (N-(IAA)Asp, 50) (188) and N-(IAA)-L-glutamic acid (N-(IAA)Glu, 51) (189) occur endogenously and as metabolites in feeding experiments (176,190–192). Further examples for amino acid conjugates identified in plant samples by rigorous chemical criteria include N-(IAA)-alanine (53), a native constituent of elongating spruce (Picea abies (L.) Karst.) shoots (193), and N-(IAA)-L-glutamine (52) formed by Arabidopsis thaliana on IAA feeding (194). N-(IAA)polypeptides have been identified in bean seeds (195) and in the prolamine fraction of cereal grains (196). Despite previous tendencies to discuss the significance of the ‘‘bound auxins’’ in global terms, it now appears appropriate to collect specific experimental evidence for the function of each individual conjugate. IAA-myoinositol, for instance, is stored in the seeds of most corn cultivars (176). On germination, the compound is transported into the developing shoot (197,198). Hydrolysis, preferentially at the tip of the coleoptile, is then inferred

111

to supply the IAA involved in its growth and its response to environmental stimuli (199). The hydrolases involved in these reactions have been demonstrated in seedling tissues (200) but were more completely characterized in immature corn kernels (182,183). According to circumstantial evidence, all IAA conjugates (including those not yet found endogenously) with naturally occurring amino acids can be cleaved by plants under the appropriate conditions. A set of group-specific enzymes was recently cloned from Arabidopsis (201,202), as was an N-(IAA)-Asp hydrolase from Enterobacter agglomerans (203). The latter conjugate is of particular interest. Its hydrolysis in growing bean sprouts was painstakingly documented (204) but does apparently not occur in many other plant tissues. Instead, the IAA moiety is oxidized and/or glucosylated to yield metabolites 54–60 shown in Figure 10 (191,205–213). N-(IAA)-L-glutamic acid affords a number of analogous products (191,207), but its metabolism is less well known because of its low abundance. N-(IAA)-Asp and N-(IAA)-Glu are amphipathic molecules and may, thus, be expected to associate with membranes. Indeed, the best studied (in a different context) enzymes that oxidize the indole nucleus at positions 2 and 3 are cytochrome P450 oxidases localized in the membranes of the endoplasmic reticulum (214). This rationalizes the roles of Asp and Glu as tags channeling the IAA moiety into oxidative catabolism but does not reveal the specific role of this pathway in the endogenous control of plant growth and development. Interestingly, a heatsensitive strain of in vitro cultured Hyoscyamus muticus L. cells, which normally die at 33 ◦ C, survived this temperature in the presence of N-(IAA)-Asp (215). This effect was ascribed to the conjugate itself or to its downstream metabolites, as hydrolysis to IAA was not detectable. In accord with this concept, N-(IAA)amino acid conjugates showed specific morphogenetic effects in tomato hypocotyl explants, even if applied together with free IAA (216). The conjugation of native auxins other than IAA has received little attention. 4-Cl-IAA affords an aspartic acid conjugate which is, like the free acid, converted to its methyl ester (6,7,217). Exogenous IBA is converted to its glucose ester (homologous to 47), its aspartic acid conjugate (homologous to 50), and at least two amidetype conjugates with molecular weights in the range of 500–1500 (5). CATABOLISM The degradation of IAA by peroxidases was, during the 1960s and 1970s, at the center of attention of auxin physiologists. The products are, as in the chemical oxidation (84), 3-methyleneoxindole (35), indole3-carboxaldehyde (24, R = H), and indole-3-methanol (25, R = H). Recent evidence favors the involvement of indole-3-methylperoxide and oxindole-3-methanol as intermediates (218). Compounds 24 and 25 (R = H) are known plant constituents (9,219), and further oxidation to indole-3-carboxylic acid and/or glucose conjugation has also been reported (220). However, indole metabolites with a C1 side chain do not necessarily arise via IAA (221). In fact, incubation of pine seedlings with labeled IAA did not

112


H

OH CH2COOH

N H

OGlc CH2CO

O

N H

Asp

CH2CO Asp

O

(61)

N H

(55)

O (56)

H CH2CO

Asp

CH2CO N H

N H

H Asp

O

(50)

CH2CO

HO N H

(54)

O (57)

H CH2CO

Asp N

b Glcp

b Glcp (60)

H CH2CO

N

Asp

Asp

CH2CO

O

N Glcp (b1

(58)

Asp

O

4)b Glcp (59)

Figure 10. Ring-oxidation and glycosylation of N-(indol-3-ylacetyl)-L-aspartic acid, as observed in gymnosperms and dicots. Asp designates an L-aspartic acid residue linked via its amino group. Glc stands for a 1-O-linked glucose residue; the pyranose form (index p) and β-linkage are indicated if known.

yield labeled indole-3-carboxylic acid (222), even though the unlabeled compound occurs endogenously in the same plant material (223). Discussions on the significance of peroxidases in auxin metabolism are further complicated by the existence of a large set of isoenzymes. Detailed kinetic data are only available for commercial horseradish peroxidase (a mixture of isoenzymes), which acts both as a peroxidase proper and as an oxidase. In the peroxidase pathway, the presence of catalytic amounts of a hydroperoxide (recycled in a free radical chain reaction) is sufficient; addition of H2 O2 is not required (224). Many isoperoxidases are localized in the apoplast (e.g., the cell wall) and are released by wounding. Their action may then seriously distort the outcome of experiments that require exogenous application of indole auxins, such as plant tissue culture and metabolic studies with plant sections or tissue slices. In addition to IAA, its ringsubstituted analogues and its conjugates with nonpolar amino acids (225) are sensitive to peroxidase; IPA and IBA are resistant. Even with sensitive compounds, massive degradation can frequently be circumvented by simple treatments. For instance, stem sections used in auxin bioassays are kept in water or buffer for about an hour to rinse off the peroxidases liberated from the cut surfaces. The significance of apoplastic peroxidases in the metabolism of endogenous IAA requires further study. It should not be forgotten that the auxin-binding protein

ABP1 (see below), proposed to be involved in the stem elongation response, is an apoplastic protein. Also, polar IAA transport is mediated by influx and efflux carriers, implying that the transported auxin passes through the apoplast. A convenient way of detecting peroxidase activity is the liberation of [14 C]CO2 from carboxy-labeled IAA. Auxin degradation initiated by peroxidase was, thus, termed the ‘‘decarboxylative pathway.’’ Non-decarboxylative pathways may, as already discussed, involve conjugation with Asp and Glu as the first step, but this is not necessary in all plant tissues. Corn seedlings, for instance, oxidize IAA to oxindole-3-acetic acid [2-oxo-1H-indole-3acetic acid, (61)] (226,227), followed by 7-hydroxylation to 62 and glycosylation to 63 (228) (Fig. 11). Compound 61 is a native constituent of corn seeds (226) and germinating pine seeds (229), and there are earlier reports on its occurrence in rape (Brassica rapa L.) seedlings and in developing currant (Ribes rubrum L.) seeds (227). Ring expansion to 2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid (64) on boiling with acid (230) is highly characteristic and can be exploited for identification. Other ring-hydroxylated oxindole-3-acetic acids (including 2,3-dioxindole-3-acetic acids) with likely, but unverified, metabolic relationships to IAA, and the corresponding quinoline-carboxylic acids formed by acid-catalyzed rearrangement were, for example, isolated from corn and rice (231–233). Evidence


113

H CH2COOH

CH2COOH N H

N H (1)

O (61)

H CH2COOH

CH2OH O

N H

O H CH2COOH

O OH HO

HO

N H

O

OH (63)

(62)

COOH

N H

O

(64)

for nondecarboxylative catabolism of other indole auxins is so far lacking. PERCEPTION AND SIGNAL TRANSDUCTION The signaling pathways involved in auxin action are just being unveiled, and even widely accepted concepts are constantly updated as more complete data become available. Candidates for the auxin receptor were first sought among the proteins, which reversibly bind auxin, with high affinity. It was soon appreciated that the unique combination of an acidic head group and a hydrophobic body containing an aromatic ring system enables IAA to interact with a fair number of proteins that have no obvious function in growth physiology. For instance, a large amount of time was spent on the glutathione-Stransferases (234–238). These enzymes not only bind IAA with high affinity—they are also induced by IAA (239). Yet, no role in IAA physiology and biochemistry could so far be found. A second line of research started with the discovery of reversible in vitro auxin-binding to particulate fractions from corn coleoptiles (240). The affinity of the microsomal fraction (mostly membranes of the endoplasmic reticulum) to a variety of auxins correlated reasonably well with their growth-promoting activities in stem elongation assays (241) and was particularly high for naphthalene-1acetic acid (13), which was, thus, adopted as a standard substrate for binding studies. One of the first results

Figure 11. IAA catabolism in corn (Zea mays). The metabolites shown were also identified as endogenous constituents, and interconnecting pathways were verified by labeling studies. Compound 64 (2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid) is formed by acid treatment of 61. The reaction is useful to distinguish the metabolite from other ring-hydroxylated IAAs.

connecting auxin perception and auxin-binding to the microsomal fraction was obtained in corn seedlings exposed to red light, at a dose that reduces auxin sensitivity in the mesocotyl but not in the coleoptile (242). Indeed, only the mesocotyl responded by reducing the number (but not the affinity) of auxin-binding sites in the microsomal fraction. The ‘‘Auxin Binding Protein’’ (ABP1) from this subcellular fraction was studied in several laboratories (243,244). A highly purified preparation (245) was characterized as an approximately 2 × 20 kDa dimer, with a binding constant (determined at pH 5.5, which is optimal) for 13 of KD = 5.7 × 10−8 . ABP1 is a glycoprotein; cloning of the polypeptide chain (246–248) afforded a refined molecular weight (22 kDa for the monomer) and revealed the presence of a C-terminal signal sequence (KDEL = -Lys-Asp-Glu-Leu-), which would normally keep the protein confined to the lumen of the endoplasmic reticulum. The correctly glycosylated protein was produced in insect cells using the baculovirus expression system (249), but mutation of the -Asp-Glu- sequence in the signal peptide to -Glu-Glnwas required to have the glycoprotein excreted into the culture medium (250). This has secured the quantities of (slightly modified) ABP1 required for structural studies by X-ray crystallography, which are now in progress (251). The results obtained so far are calling for caution with the existing elaborate edifice of indirect evidence on the biochemistry of auxin-binding proteins (243,244,252,253). ABP1 has now been isolated from a number of monocots and dicots (253); it is essential for plant development beyond the stage of the globular embryo (254). In

114


spite of the endoplasmic-reticulum-retention signal, a minor fraction of the protein appears to escape to the plasmalemma, where it is assumed to exert its physiological function. This was first proposed by Löbler ¨ and Klambt (255), based on the fact that anti-ABP immunoglobulin G, which cannot penetrate into the interior of a plant cell, nevertheless inhibits the auxin response of corn coleoptiles. As ABP1 is a soluble protein, it is assumed to communicate with the cell interior via a ‘‘docking protein’’ at the plasmalemma [e.g., (256,257)]. The majority of ABP1, which remains in the endoplasmic reticulum, does not bind IAA to any significant extent because the pH in this cell compartment is well above the optimal 5.5. There is, however, fair evidence that the stem elongation response involves at least one intracellular auxin receptor with a different, so far unknown, structure (258). The signal transduction cascade, by which an auxin stimulus is communicated to the response-executing systems, has been claimed to include, for instance, heterotrimeric G-proteins (‘‘GTP-ases’’), protein phosphorylases, calcium ions, phospholipases A2 and C and their products, lysophospholipids, free fatty acids, and inositol phosphates (257,259), but the existing data do not yet permit unequivocal conclusions. Signal transduction in many, but not all, cases includes gene regulation. Among the 100–150 genes now known to respond to auxins, only the following could so far be identified as primary response genes, which implies activation by auxins through the action of preexisting cell components (i.e., de novo protein synthesis is not required), within a time frame of 2 min to a few hours (260,261). The GST genes encode a family of glutathione S-transferases with unknown functions in auxin physiology. The genes for a number of 1-aminocyclopropanecarboxylic acid synthases (ACS) are likely involved in the coordination of auxin and ethylene effects. The genes of the Aux/IAA family encode shortlived (half-lives 6 to 8 min) transcription regulators. Their function is mediated by four conserved domains, numbered I–IV starting from the N-terminal end (see below). The SAUR genes were mostly studied on the mRNA level (Small Auxin Upregulated RNAs with turnover times of 40 to 50 min). The function of the 9 to 10 kDa proteins they encode is unknown because their abundance in plant tissues is below the detection limit of existing analytical methods. The GH3 genes encode cytoplasmic proteins with molecular weights around 70 kDa, which are more abundant than the Aux/IAA and SAUR proteins. As far as analyzed, the promoters of ACS, SAUR, Aux/IAA, and GH3 genes contain the nucleotide sequence TGTCnC (optimally n = T) as an auxin-responsive module that represents one of the components of composite auxin-responsive elements. The nature of the additional modules is incompletely known, but artificial composite elements have been constructed from appropriately spaced direct or inverted repeats, or palindromes, of the TGTCnC sequence. Such synthetic polynucleotides showed high affinity to a 74 kDa protein with an Nterminal DNA-binding domain: the first isolated auxinresponsive transcription factor, ARF1 (262). Eight more members of this protein family are now known, and

all but one (ARF3) at their C-terminal ends contain the same domains III and IV, which are found in the smaller (20 to 35 kDa) Aux/IAA proteins. These domains appear to contain amphipathic α-helices, which tend to aggregate. Several ARF proteins may, thus, associate via their domains III and IV, and the aggregates may line up, like the coaches of a train, along the promoter of an auxin-responsive gene (hence the need for composite auxin-responsive elements constructed from appropriately spaced modules) (260). The number and kind of transcription factors clustered in this way would determine the degree of gene activation or repression. If present in excess, the Aux/IAA proteins, which also contain domains III and IV, would bind to the ARF proteins, prevent the latter from aggregating, and, thus, affect the regulation of auxin-responsive genes. STRUCTURE–ACTIVITY CORRELATIONS Structure–activity studies are undertaken to reveal the molecular features that determine the relative efficiencies of compounds with similar physiological or pharmacological properties. Chemical entities with molecular weights similar to those of indole auxins tend to exert their biological effects by interacting (as ‘‘substrates’’ or ‘‘ligands’’) with proteins: enzymes or receptors. If the complexes formed are stable enough, they may be studied in detail by X-ray crystallography. The information obtained can then be used to design novel compounds that fit into the active site of a receptor to trigger, or to obstruct, its signaling function. The process of ligand (or substrate) recognition was first rationalized by a key-andlock model (263). If the polypeptide is flexible, induced fit or the hand-glove-model (264) is more appropriate. The recently developed approach of molecular docking analyzes the detailed conformational dynamics for both the protein and the binding ligand and includes the two classical models. As discussed above, no auxin receptor has so far been characterized in a truly unequivocal fashion, and even the number of putative auxin receptors is difficult to define. Other phytohormones, such as ethylene (265), are known to have multiple receptors, and physiological data strongly suggest an analogous situation for the auxins for which, moreover, several endogenous representatives exist. For instance, the highly specific role of IBA in the root system (5,23) could be rationalized by postulating a selective IBA-receptor. Growth of the deseeded pea pod is stimulated by 4-Cl-IAA (can be replaced by 4-methyland, to a minor extent, by 4-ethylindole-3-acetic acids), but is not promoted by IAA and its other monochlorinated and monofluorinated derivatives (68,266), all of which are highly active auxins in pea stems. These results call for a specific 4-Cl-IAA receptor in the pea pericarp or at least for a specific signal transduction pathway (267). The discussion to follow will be confined to auxin perception in the stem elongation response. Even in this case, multiple auxin receptors have been postulated (258). If this should be correct, their substrate specificities appear to be so similar that the above concepts of protein-ligand interaction could be exploited to deduce a consistent


model of the hormone-binding site(s) from the molecular properties of the auxins, which can be accommodated (as evidenced by their growth-promoting activity). This approach was introduced by Katekar (268), who termed the result a stochastic model, a procedure comparable to making a shoe that fits the feet of everybody in a small town of more than a thousand inhabitants. The family of ring-substituted IAAs, which have so far been synthesized, is smaller and more homogeneous; sharper structure-activity correlations may, thus, be expected. Data published until the late 1970s were compiled by Jönsson (269) and Katekar (268). Depending to some extent on the bioassay employed and on the method of evaluation, monohalogenated IAAs were usually more active auxins than the parent compound; methyl and methoxy IAAs showed about the same or lower activity than unsubstituted IAA. More recent developments include the evaluation of dichlorinated IAAs (14) and a number of difluorinated IAAs (270), most of which are highly active auxins (exceptions: 4,7-Cl2 IAA, 5,7-Cl2 -IAA, 5,7-F2 -IAA). Also, monofluorinated IAAs were systematically studied (270,271), and substitution with larger (up to butyl) n-alkyl groups was shown to affect the optimal response, rather than the half-optimal concentration (272,273). For most of the above IAA derivatives, studies on molecular topology resulted in detailed ‘‘identity cards’’ based on the results of experimental methods and computer modeling. X-ray diffraction analysis was used to define the molecular structures of the endogenous phytohormones, IAA (274,275) and 4-Cl-IAA (276), and of their synthetic analogues including other ringchlorinated (276), -dichlorinated (277), -fluorinated (271), and -alkylated (272,273) derivatives. Molecular modeling, including ab initio calculations as well as molecular mechanics and molecular dynamics simulations, was employed to evaluate the stability of the conformers observed in vacuo (the undisturbed ground state) and in aqueous medium (simulating physiological conditions) (278–282). Two conformationally distinct states with minimal free energy (i.e., ‘‘local minima’’) were found: 1) planar (P), with the carboxyl group in the plane of the indole ring, and 2) tilted (T), with the CH2 —COOH bond near-perpendicular to the indole ring (Fig. 12). In the crystalline state, the T-conformation was detected in IAA itself (275) and in all chlorinated (276,277) and fluorinated (271) derivatives examined so far. For the ring-alkylated analogues studied, both conformations occur with equal frequency (272,273). This can be explained by the small energy difference between the P and T conformations, as revealed by computational methods (271,276,278–283). Molecular recognition of auxins has been summarized in a number of historical hypotheses proposed before detailed structural information was available (268,284, 285). The charge separation theory, mostly advocated by Thimann, postulates that, in molecules with auxin activity, a ‘‘partial negative charge’’ at the acidic head group is counter-balanced by a ‘‘partial positive charge,’’ ˚ The original at an approximate distance of 5 to 6 A. concept is based on ambiguous definitions. However,

P

115

T

Figure 12. Space-filling models of IAA in its tilted (T) and planar (P) conformations. The atoms are color-coded as follows: green, carbon; white, hydrogen; blue, nitrogen; red, oxygen. See color insert.

reasonable structure-activity correlations (286) result if ‘‘changes in partial positive charge’’ are interpreted to mean just changes in electron density. The influence of molecular charge patterns on auxin activity was also demonstrated by Hansch’s group (287). A conformational change of the auxin molecule from a planar ‘‘recognition conformation’’ to a tilted ‘‘modulation conformation,’’ to occur during the process of binding to the receptor, was postulated by Kaethner (284). These and related hypotheses were incorporated in a concept termed topography analysis (268,285,288). This approach implies complementarity of the receptor active site and the auxin molecule and is based on analysis of its size and shape, lipophilicity, and the orientation of its chemical functionalities. In a more elaborate combined approach, interaction similarity analysis (283) was employed for the systematic classification of auxins. The results of molecular modeling (conformational analysis and energy evaluation for the conformers), molecular alignment, and interaction energies with selected chemical probes (corresponding to functional groups expected to occur in the receptor) permitted the subdivision of a set of about 50 compounds into the following four classes: strong auxins, weak auxins with weak antiauxin properties, inactive compounds, and growth inhibitors. The compounds studied were modeled in their two low-energy conformations, P and T. When the T-conformation was used, the model more clearly distinguished auxins from antiauxins and inactive compounds. This may suggest that this is the active conformation, even though similarity analysis cannot provide an explicit ligand-binding mechanism. Conformational change from P to T, though not necessarily under exactly the same circumstances as hypothesized by Kaethner (284), is facilitated by the low-energy barriers between these two conformers. To illustrate the use of topography analysis (268,285,288), let us return to the dichlorinated IAAs referred to in Figure 2 (277). According to the doseresponse curves presented, 5,6-dichloro-IAA (7) has significantly smaller optimal and half-optimal concentrations and a slightly larger optimal response than IAA, and, thus, is a more active auxin. This difference can be even more pronounced in other bioassays (14). The positional isomer, 4,7-dichloroindole-3-acetic acid (9) has higher optimal and half-optimal concentrations and, most obviously, a much smaller optimal response. In a concept that is difficult to

116


Figure 13. Deduced orientations of 5,6-dichloroindole-3-acetic acid (7) (bottom panel) and 4,7-dichloroindole-3-acetic acid (9) (top panel) in the compartment of the auxin receptor that accommodates the aromatic ring system. The presentation is based on a model proposed by Katekar (268). The ‘‘shoe’’ loosely fits the indole nucleus except for two tight spots indicated in red: between ring-positions 4 and 5 (‘‘the heel’’) and along positions 1 to 7 (‘‘the back part of the sole’’). The carboxyl group is assumed to interact with a separate compartment of the auxin receptor.

track back to its origin, but was most explicitly formulated by Katekar (268), the active site of the topologically defined auxin receptor was subdivided to include separate compartments for the acidic head group and for the planar body. The latter compartment was visualized in the shape of a shoe, which loosely fits the indole nucleus, except for tight sections between the ‘‘sole’’ and the ‘‘heel.’’ If this concept is correct, then chlorine substitution at positions 5 and 6 can only push the heterocyclic ring system further towards the spacious part of the respective compartment (Fig. 13). The substituents themselves are also well accommodated and have been postulated to be the target of binding interactions, in addition to those involving the aromatic ring system (268). In contrast, for the weak auxin 9, both substituents are forced into the tightly fitting part of ‘‘Katekar’s shoe’’ (Fig. 13). This results in a less favorable energy balance for binding to the auxin receptor and, thus, decreases growth-promoting activity. AGRICULTURAL APPLICATIONS At present, indole auxins are less widely used agrochemicals than their non-indolic analogues. However, IBA has

remained the most popular root-inducing agent for cuttings (289,290) and, in 1999 alone, Current Contents listed close to 50 papers describing its use for the induction of roots on adventive or axillary shoots obtained by in vitro propagation. Optimal concentrations and methods of application vary within wide limits, and every species (and each of its cultivars, if any) requires individual attention. Exceptionally, IBA may even inhibit root induction, as was reported for stem cuttings of Salix planifolia, which root without auxin treatment (291). A number of other indole auxins have been patented for agricultural use (see listing above) but, with regard to their cost, their application can so far only be justified in the production of high value crops. Thus, for example, 5,6-Cl2 -IAA was used for maintaining in vitro cultures that produce tropane alkaloids (16,17). N(IAA) amino acids (292–294) as well as indole-3-ethanol and its glycosides (295), all of which can be metabolized to IAA, were evaluated as slow-release sources of auxin for in vitro propagation. IBA conjugates, such as N-(IBA)-Lalanine (296) and N-(IBA)-L-aspartic acid (297) were more efficient in the rooting of cuttings than the free acid. A number of the above conjugates are commercially available, and they can be easily prepared using inexpensive equipment and chemicals (292–294,296,297). When the substrate-binding properties of the enzymes and receptors involved in auxin biochemistry and physiology are more completely understood, this will provide a rational base for constructing plant growth regulators (indole-based and others), which are as environmentally safe as the natural auxins, at least as active and selective as their synthetic analogues now in use, and no more costly to produce. Manipulating the genes encoding the proteins involved in IAA biogenesis, metabolism, perception, signal transduction, and the response mechanisms may permit plant growth regulation with even less, or no, chemical intervention. In addition to bacterial genes of IAA biogenesis and metabolism (see above), corn IAA-Glc synthase has now been cloned and inserted into transgenic plants, both in the sense and the antisense orientation (177). These are encouraging results, but it must be realized that we have to learn more about the molecular complexities of auxin physiology to use gene manipulation with optimal efficiency. Consumers now tend to avoid ‘‘gene food,’’ but they will balance obvious advantages against possible hazards, as plant biologists provide the necessary background information. Future agricultural technology will, thus, employ less invasive and, in many cases, less expensive (considering the complete cost to society) strategies to produce food of higher quality and ornamentals of higher esthetic value, while interfering as little as possible with biological and ecological diversity. Acknowledgments The authors thank Drs. Jennifer Normanly and Robert S. Bandurski for valuable suggestions and helpful comments on the manuscript.

BIBLIOGRAPHY 1. H. Söding, in W. Ruhland, ed., Encyclopedia of Plant Physiology, Vol. 14, Springer-Verlag, Berlin, 1961, pp. 450–484.


117

2. E. A. Schneider, C. W. Kazakoff, and F. Wightman, Planta 165: 232–241 (1985).

29. M. Böttger, K. C. Engvild, and H. Soll, Planta 140: 89–92 (1978).

3. L. M. Segal and F. Wightman, Physiol. Plant. 56: 367–370 (1982).

30. D. H. McRae and J. Bonner, Plant Physiol. 27: 834–838 (1952).

4. P. W. Zimmerman and F. Wilcoxon, Contrib. Boyce Thompson Inst. 7: 209–229 (1935).

31. T. Hatano, Y. Kato, M. Katayama, and S. Marumo, Experientia 45: 400–402 (1989).

¨ 5. E. Epstein and J. Ludwig-Muller, Physiol. Plant. 88: 382–389 (1993).

32. H. Burström, Physiol. Plant. 3: 277–292 (1950).

6. J.-C. Gandar and C. Nitsch, C. R. Acad. Sci. (Paris), Ser. D 265: 1795–1798 (1967). 7. S. Marumo, H. Hattori, H. Abe, and K. Munakata, Nature 219: 959–960 (1968). 8. D. M. Reinecke, Plant Growth Regul. 27: 3–13 (1999). 9. A. Ernstsen and G. Sandberg, Physiol. Plant. 68: 511–518 (1986). 10. O. Salcher and F. Lingens, Tetrahedron Lett. 34: 3101–3102 (1978).

33. A. Hager, H. Menzel, and A. Krauss, Planta 100: 47–75 (1971). 34. D. L. Rayle and R. E. Cleland, Plant Physiol. 99: 1271–1274 (1992). 35. T. Fr´ıas et al., Plant Cell 8: 1533–1544 (1996). 36. F. Masson, W. Szponarski, and M. Rossignol, Plant Growth Regul. 18: 15–21 (1996). 37. D. P. Briskin and J. B. Hanson, J. Exp. Bot. 43: 269–289 (1992). 38. D. J. Cosgrove, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 391–417 (1999).

¨ 11. C. Lubbe, K.-H. van Pée, O. Salcher, and F. Lingens, HoppeSeyler’s Z. Physiol. Chem. 364: 447–453 (1983).

39. F. W. Went, Rev. Trav. Bot. Néerl. 25: 1–116 (1929).

12. U.S. Pat. 5,994,135 (Nov. 30, 1999), J.-J. Lin, J. Lan, and N. Assad-Garcia, (to Life Technologies, Inc.).

¨ 41. L. Galweiler et al., Science 282: 2226–2230 (1998).

13. J. Slovin, Plant Growth Regul. 21: 215–221 (1997).

¨ 42. A. Muller et al., EMBO J. 17: 6903–6911 (1998).

14. T. Hatano, M. Katayama, and S. Marumo, Experientia 43: 1237–1239 (1987).

43. J. D. Cohen, B. G. Baldi, and K. Bialek, Plant Physiol. 77: 195–199 (1985).

15. U.S. Pat. 4,806,143 (Feb. 21, 1989), S. Marumo, M. Katayama, F. Futatsuya, and M. Saito, (To Nippon Kayaku Kahushiki Kaisha).

44. V. Imhoff, P. Muller, J. Guern, and A. Delbarre, Planta 210: 580–588 (2000).

16. Jpn. Kokai Tokkyo Koho JP 63,216,491 [88,216,491] (Sep. 8, 1988), H. Kamata and S. Marumo (to Nippon Kayaku Co., Ltd.). 17. K. Yoshimatsu et al., Phytochemistry 29: 3525–3528 (1990). 18. K. Kato et al., J. Ferment. Bioeng. 76: 178–183 (1993). 19. U.S. Pat. 5,578,552 (Nov. 26, 1996), M. Katayama, S. Fujii, H. Kimoto, and K. Kato, (to Agency of Industrial Science & Technology and Ministry of International Trade & Industry).

40. K. Okada et al., Plant Cell 3: 677–684 (1991).

45. E. P. Maher and S. J. Martindale, Biochem. Genet. 18: 1041–1053 (1980). 46. M. J. Bennett et al., Science 273: 948–950 (1996). 47. A. Marchant et al., EMBO J. 18: 2066–2073 (1999). 48. G. F. Katekar, Phytochemistry 15: 1421–1424 (1976). 49. M. G. Bures, C. Black-Schaefer, and G. Gardner, J. Comp.Aided Mol. Design, 5: 323–334 (1991). 50. G. Schneider, Annu. Rev. Plant Physiol. 21: 499–536 (1970). 51. M. Jacobs and P. Rubery, Science 241: 346–349 (1988).

20. U.S. Pat. 5,496,794 (Mar. 5, 1996), M. Katayama, S. Fujii, H. Kimoto, and K. Kato (to Agency of Industrial Science & Technology and Ministry of International Trade & Industry).

52. D. E. Brown et al., Plant Physiol. 126: 524–535 (2001).

21. M. Katayama and R. K. Gautam, Biosci. Biotech. Biochem. 60: 755–759 (1996).

54. R. Zettl et al., Proc. Natl. Acad. Sci. USA 89: 480–484 (1992).

22. M. Katayama and R. K. Gautam, J. Pestic. Sci. 22: 331–337 (1997).

56. C. Garbers et al., EMBO J. 15: 2115–2124 (1996).

¨ 23. M. Kaldorf and J. Ludwig-Muller, Physiol. Plant. 109: 58–67 (2000).

57. P. Champagnat, in W. Ruhland, ed., Encyclopedia of Plant Physiology, Vol. 14, Springer-Verlag, Berlin, 1961, pp. 872– 908.

24. T. Ciesielski, Beitr. Biol. Pflanz. 1: 1–30 (1872). 25. C. Darwin, The Power of Movement in Plants, D. Appleton and Company, New York, 1881. 26. P. Larsen, in W. Ruhland, ed., Encyclopedia of Plant Physiology, Vol. 14, Springer-Verlag, Berlin, 1961, pp. 521–582. 27. J. W. Mitchell and G. A. Livingston, Methods of Studying Plant Hormones and Growth-Regulating Substances, Agriculture Handbook No. 336, Agricultural Research Service, United States Department of Agriculture, Washington, D.C., 1968. 28. R. J. Foster, D. H. McRae, and J. Bonner, Proc. Natl. Acad. Sci. U.S.A. 38: 1014–1022 (1952).

53. M. Jacobs and S. F. Gilbert, Science 220: 1297–1300 (1983).

55. M. Ruegger et al., Plant Cell 9: 745–757 (1997).

58. H. E. Dolk, Recl. Trav. Bot. Néerl. 33: 509–585 (1933). 59. R. S. Bandurski, A. Schulze, P. Dayanandan, and P. Kaufman, Plant Physiol. 74: 284–288 (1984). 60. B. F. Wilson, Am. J. Bot. 87: 601–607 (2000). 61. H. Tuominen, L. Puech, S. Fink, and B. Sundberg, Plant Physiol. 115: 577–585 (1997). 62. C. Uggla, T. Moritz, G. Sandberg, and B. Sundberg, Proc. Natl. Acad. Sci. USA 93: 9282–9286 (1996). 63. C. Uggla, E. J. Mellerowicz, and B. Sundberg, Plant Physiol. 117: 113–121 (1998). 64. T. Steinmann et al., Science 286: 316–318 (1999).

118


65. C. Liu, Z. Xu, and N.-H. Chua, Plant Cell 5: 621–630 (1993). 66. T. J. Cooke, R. H. Racusen, and J. D. Cohen, Plant Cell 5: 1494–1495 (1993). 67. V. Magnus et al., Phytochemistry 46: 675–681 (1997). 68. D. M. Reinecke, J. A. Ozga, and V. Magnus, Phytochemistry 40: 1361–1366 (1995).

95. A. Ernstsen, G. Sandberg, and A. Crozier, Physiol. Plant. 68: 519–522 (1986). 96. B. Sundberg, Physiol. Plant. 78: 293–297 (1990). 97. B. Sundberg, C. H. A. Little, R. T. Riding, and G. Sandberg, Physiol. Plant. 71: 163–170 (1987). 98. A. Edlund et al., Plant Physiol. 108: 1043–1047 (1995).

¨ 69. J. Ludwig-Muller, B. Schubert, and K. Pieper, J. Exp. Bot. 46: 423–432 (1995).

99. K.-H. Chen, A. N. Miller, G. W. Patterson, and J. D. Cohen, Plant Physiol. 86: 822–825 (1988).

¨ 70. J. Ludwig-Muller, M. Kaldorf, E. G. Sutter, and E. Epstein, Plant Sci. 125: 153–162 (1997).

100. K. Bialek and J. D. Cohen, Plant Physiol. 90: 398–400 (1989).

71. Brit. Pat. 1,031,880 (June 2, 1966), L. B. Shagalov and N. N. Suvorov (to All-Union Scientific-Research ChemicalPharmaceutical Institute).

101. G. Sandberg, A. Crozier, and A. Ernstsen, in L. Rivier and A. Crozier, eds., The Principles and Practice of Plant Hormone Analysis, Vol. 2, Academic Press, London, 1987, pp. 170–301.

72. H. E. Johnson and D. G. Crosby, J. Org. Chem. 28: 1246–1248 (1963). 73. W. A. Remers, in W. J. Houlihan, ed., Indoles, Part 1, WileyInterscience, New York, 1972, pp. 1–226. 74. K. C. Engvild, Acta Chem. Scand. B31: 338–339 (1977). 75. A. P. Kozikowski and H. Ishida, Heterocycles 14: 55–58 (1980). 76. R. M. Silverstein, E. E. Ryskiewicz, and E. E. Chaikin, J. Am. Chem. Soc. 76: 4485–4486 (1954). 77. C. Runti and G. Orlando, Ann. Chim. (Rome) 43: 308–314 (1953). 78. A. D. Batcho and W. Leimgruber, Org. Synth. 63: 214–225 (1985). 79. V. Magnus, R. S. Bandurski, and A. Schulze, Plant Physiol. 66: 775–781 (1980). 80. R. R. Phillips, Org. React. 10: 143–178 (1959). 81. R. K. Brown, in W. J. Houlihan, ed., Indoles, Part 1, WileyInterscience, New York, 1972, pp. 227–558. 82. R. W. Jackson and R. H. Manske, J. Am.Chem. Soc. 52: 5029 (1930). 83. A. W. Galston and W. S. Hillman, in W. Ruhland, ed., Encyclopedia of Plant Physiology, Vol. 14, Springer-Verlag, Berlin, 1961, pp. 647–670. 84. R. L. Hinman and J. Lang, Biochemistry 4: 144–158 (1965). 85. R. L. Hinman and C. P. Bauman, J. Org. Chem. 29: 2431– 2437 (1964). 86. B. B. Stowe, Anal. Biochem. 5: 107–115 (1963).

102. D. M. Ribnicky, T. J. Cooke, and J. D. Cohen, Planta 204: 1–7 (1998). 103. D. Rittenberg and D. L. Foster, J. Biol. Chem. 133: 737–744 (1940). 104. J. L. Caruso et al., Plant Physiol. 62: 841–845 (1978). 105. J. L. Caruso and C. S. Zeisler, Phytochemistry 22: 589–590 (1983). 106. J. D. Cohen, B. G. Baldi, and J. P. Slovin, Plant Physiol. 80: 14–19 (1986). ˇ Horvat, and G. Sandberg, J. 107. A. Jakas, V. Magnus, S. Labelled Compd. Radiopharm. 33: 933–939 (1993). 108. E. G. Sutter and J. D. Cohen, Plant Physiol. 99: 1719–1722 (1992). 109. E. Epstein and J. D. Cohen, J. Chromatogr. 209: 413–420 (1981). 110. G. Sandberg, B. Andersson, and A. Dunberg, J. Chromatogr. 205: 125–137 (1981). 111. A. Stoessl and M. A. Venis, Anal. Biochem. 34: 344–351 (1970). ¨ 112. H. Plieninger, W. Muller, and K. Weinerth, Chem. Ber. 97: 667–681 (1964). 113. M. Iino, R. S.-T. Yu, and D. J. Carr, Plant Physiol. 66: 1099–1105 (1980). 114. G. Sandberg and A. Dunberg, Physiol. Plant. 55: 315–322 (1982). 115. S. Fuchs and Y. Fuchs, Biochim. Biophys. Acta 192: 528–530 (1969).

87. E. Epstein, J. D. Cohen, and R. S. Bandurski, Plant Physiol. 65: 415–421 (1980).

116. E. W. Weiler, Annu. Rev. Plant Physiol. 35: 85–95 (1984).

88. J. Nowacki, J. D. Cohen, and R. S. Bandurski, J. Labelled Compd. Radiopharm. 15: 325–329 (1978).

118. J. Marcussen, P. Ulvskov, C. E. Olsen, and R. Rajagopal, Plant Physiol. 89: 1071–1078 (1989).

89. J. R. Dunlap, S. Kresovich, and R. E. McGee, Plant Physiol. 81: 934–936 (1986).

119. P. Ulvskov, J. Marcussen, P. Seiden, Planta 188: 182–189 (1992).

90. J. R. Dunlap and K. M. Robacker, Plant Physiol. 88: 379–382 (1988).

120. G. Sandberg, K. Ljung, and P. Alm, Phytochemistry 24: 1439–1442 (1985).

91. T. C. Stasinopoulos and R. P. Hangarter, Plant Physiol. 93: 1365–1369 (1990).

121. J. D. Cohen et al., Plant Physiol. 84: 982–986 (1987).

92. H. Kaldewey, in E. Stahl, ed., Thin-Layer Chromatography. A Laboratory Handbook, 2nd ed., Springer-Verlag, Berlin, 1969, pp. 471–493. 93. A. Ehmann, J. Chromatogr. 132: 267–276 (1977). 94. J. D. Mann and E. G. Jaworski, Planta 92: 285–291 (1970).

117. W. Pengelly and F. Meins, Jr., Planta 136: 173–180 (1977).

and C. E. Olsen,

122. W. L. Pengelly, R. S. Bandurski, and A. Schulze, Plant Physiol. 68: 96–98 (1981). 123. B. Sundberg, G. Sandberg, and A. Crozier, Phytochemistry 25: 295–298 (1986). 124. J. Normanly, J. P. Slovin, and J. D. Cohen, Plant Physiol. 107: 323–329 (1995).

AUXINS, INDOLE AUXINS 125. B. Bartel, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 51–66 (1997). 126. J. Normanly and B. Bartel, Curr. Opin. Plant Biol. 2: 207–213 (1999). 127. A. W. Wright et al., Science 254: 998–1000 (1991). 128. J. Normanly, J. D. Cohen, and G. R. Fink, Proc. Natl. Acad. Sci. USA 90: 10355–10359 (1993). 129. N. I. Rekoslavskaya and R. S. Bandurski, Phytochemistry 35: 905–909 (1994). 130. N. Ilić, Isotopic Methods for Elucidating Indoleacetic Acid Biosynthetic Pathways in Plants, Ph.D. Thesis, University of Maryland, College Park, 1999.

119

156. T. A. Smith, in L. Reinhold, J. B. Harborne, and T. Swain, eds., Progress in Phytochemistry, Vol. 4, Pergamon Press, Oxford, 1977, pp. 26–81. 157. J. E. Sherwin, Plant Cell Physiol. 11: 865–872 (1970). 158. F. W. Percival and W. K. Purves, Plant Physiol. 54: 601–607 (1974). 159. R. J. Parry, in W. J. Houlihan, ed., Indoles, Part 2, WileyInterscience, New York, 1972, pp. 1–64. 160. V. De Luca, C. Marineau, and N. Brisson, Proc. Natl. Acad. Sci. USA 86: 2582–2586 (1989). 161. G. Pasquali et al., Plant Mol. Biol. 18: 1121–1131 (1992). 162. Y. Zhao et al., Science 291: 306–309 (2001).

131. L. Michalczuk, D. M. Ribnicky, T. J. Cooke, and J. D. Cohen, Plant Physiol. 100: 1346–1353 (1992).

163. P. J. Bower, H. M. Brown, and W. K. Purves, Plant Physiol. 61: 107–110 (1978).

132. D. M. Ribnicky, N. Ilić, J. D. Cohen, and T. J. Cooke, Plant Physiol. 112: 549–558 (1996).

164. M. Seo et al., Plant Physiol. 116: 687–693 (1998).

133. A. R. Magie, E. E. Wilson, and T. Kosuge, Science 141: 1281–1282 (1963).

166. F. Ehrlich, Ber. Dtsch. Chem. Ges. 45: 883–889 (1912).

134. C. L. Patten and B. R. Glick, Can. J. Microbiol. 42: 207–220 (1996).

167. V. Magnus and G. Laćan, in R. P. Pharis and S. B. Rood, eds., Plant Growth Substances 1988, Springer Verlag, Berlin, 1990, pp. 360–366.

135. F. Sitbon, B. Sundberg, O. Olsson, and G. Sandberg, Plant Physiol. 95: 480–485 (1991). 136. H. Tuominen et al., Plant Physiol. 109: 1179–1189 (1995).

165. S. Akaba et al., J. Biochem. 126: 395–401 (1999).

168. F. W. Percival, W. K. Purves, and L. E. Vickery, Plant Physiol. 51: 739–743 (1973).

137. H. Linser, Planta 29: 392–408 (1939).

169. G. Sandberg, A. Ernstsen, and M. Hamnede, Physiol. Plant. 71: 411–418 (1987).

138. E. R. H. Jones, H. B. Henbest, G. F. Smith, and J. A. Bentley, Nature 169: 485–487 (1952).

170. G. Laćan et al., Plant Physiol. 78: 447–454 (1985).

139. R. Gmelin and A. I. Virtanen, Ann. Acad. Sci. Fennicae, Ser. A, II. Chemica 107: 1–25 (1961). 140. J. Helmlinger, T. Rausch, and W. Hilgenberg, Phytochemistry 24: 2497–2502 (1985). ¨ 141. J. Ludwig-Muller and W. Hilgenberg, Physiol. Plant. 79: 311–318 (1990). 142. R. Bennett et al., Planta 196: 239–244 (1995). 143. A. K. Hull, R. Vij, and J. L. Celenza, Proc. Natl. Acad. Sci. USA 97: 2379–2384 (2000). 144. P. S. Shukla and S. Mahadevan, Arch. Biochem. Biophys. 137: 166–174 (1970). ¨ 145. A. Muller, H. Hillebrand, and E. W. Weiler, Planta 206: 362–369 (1998). 146. K. V. Thimann and S. Mahadevan, Arch. Biochem. Biophys. 105: 133–141 (1964). 147. J. Normanly, P. Grisafi, G. R. Fink, and B. Bartel, Plant Cell 9: 1781–1790 (1997). 148. B. Bartel and G. R. Fink, Proc. Natl. Acad. Sci. USA 91: 6649–6653 (1994). 149. D. Bartling, M. Seedorf, R. C. Schmidt, and E. W. Weiler, Proc. Natl. Acad. Sci. USA 91: 6021–6025 (1994). 150. M. Kobayashi et al., Proc. Natl. Acad. Sci. USA 92: 714–718 (1995). 151. J. Helmlinger, T. Rausch, and W. Hilgenberg, Phytochemistry 26: 615–618 (1987). 152. R. Rajagopal and P. Larsen, Planta 103: 45–54 (1972). 153. J. Koga, Biochem. Biophys. Acta 1249: 1–13 (1995). 154. T. P. Cooney and H. M. Nonhebel, Planta 184: 368–376 (1991). 155. T. A. Smith, Phytochemistry 16: 171–175 (1977).

¨ 171. J. Ludwig-Muller and E. Epstein, Plant Growth Regul. 14: 7–14 (1994). ¨ 172. J. Ludwig-Muller and W. Hilgenberg, Physiol. Plant. 94: 651–660 (1995). ¨ 173. J. Ludwig-Muller, W. Hilgenberg, and E. Epstein, Phytochemistry 40: 61–68 (1995). 174. O. Salcher, K.-H. van Pée, and F. Lingens, Z. Naturforsch, C. Biosci. 35: 340–341 (1980). 175. W. Wiesner, K.-H. van Pée, and F. Lingens, FEBS Lett. 209: 321–324 (1986). 176. J. D. Cohen and R. S. Bandurski, Annu. Rev. Plant Physiol. 33: 403–430 (1982). 177. U.S. Pat. 5,919,998 (Jul. 6, 1999), R. S. Bandurski, J. B. Szerszen, and K. Szczyglowski (to Michigan State University Board of Trustees). 178. M. H. Zenk, Nature 191: 493–494 (1961). 179. S. Kowalczyk and R. S. Bandurski, Biochem. J. 279: 509– 514 (1991). 180. J. B. Szerszen, K. Szczyglowski, and R. S. Bandurski, Science 265: 1699–1701 (1994). 181. R. G. Jackson et al., J. Biol. Chem. 276: 4350–4356 (2001). 182. S. Kowalczyk and R. S. Bandurski, Plant Physiol. 94: 4–12 (1990). 183. J. M. Ke¸ sy and R. S. Bandurski, Plant Physiol. 94: 1598– 1604 (1990). 184. W. Domagalski, A. Schulze, and R. S. Bandurski, Plant Physiol. 84: 1107–1113 (1987). 185. A. Szmidt-Jaworska, J. Ke¸ sy, and Biochim. Pol. 44: 215–220 (1997).

J. Kopcewicz,

Acta

186. T. Ganguly, S. N. Ganguly, P. K. Sircar, and S. M. Sircar, Physiol. Plant. 31: 330–332 (1974).

120


187. O. Hutzinger and T. Kosuge, in F. Wightman and G. Setterfield, eds., Biochemistry and Physiology of Plant Growth Substances, Runge Press, Ottawa, Canada, 1968, pp. 183–194. 188. J. D. Cohen, Plant Physiol. 70: 749–783 (1982).

218. S. Kobayashi et al., Biochemistry 23: 4589–4597 (1984). 219. B. Sundberg, G. Sandberg, and E. Jensen, Plant Physiol. 77: 952–955 (1985).

189. E. Epstein, B. G. Baldi, and J. D. Cohen, Plant Physiol. 80: 256–258 (1986).

220. G. Wiese and H. J. Grambow, Phytochemistry 25: 2451–2455 (1986). ˇ Simaga, ˇ 221. V. Magnus, S. S. Iskrić, and S. Kveder, Plant

190. W. Andreae and N. E. Good, Plant Physiol. 30: 380–382 (1955). ¨ 191. A. Ostin et al., Plant Physiol. 100: 63–68 (1992).

222. G. Sandberg, Planta 161: 398–403 (1984).

192. F. Sitbon et al., Plant Physiol. 101: 313–320 (1993). ¨ 193. A. Ostin, T. Moritz, and G. Sandberg, Biol. Mass Spectrom. 21: 292–298 (1992). 194. N. M. Barratt et al., Physiol. Plant. 105: 207–217 (1999). 195. K. Bialek and J. D. Cohen, Plant Physiol. 80: 99–104 (1986). 196. L. A. Leverone, T. L. Stroup, and J. L. Caruso, Plant Physiol. 96: 1076–1078 (1991).

Physiol. 69: 853–858 (1982).

223. G. Sandberg, E. Jensen, and A. Crozier, Phytochemistry 23: 99–102 (1984). 224. S. N. Krylov and H. B. Dunford, J. Phys. Chem. 100: 913–920 (1996). 225. R. D. Park and C. K. Park, Plant Physiol. 84: 826–829 (1987). 226. D. M. Reinecke and R. S. Bandurski, Plant Physiol. 71: 226–213 (1983). ¨ 227. H. D. Klambt, Naturwissenschaften 46: 649 (1959).

197. J. Nowacki and R. S. Bandurski, Plant Physiol. 65: 422–427 (1980).

228. H. M. Nonhebel and R. S. Bandurski, Plant Physiol. 76: 979–983 (1984).

198. J. R. Chisnell, Plant Physiol. 74: 278–283 (1984).

229. A. Ernstsen, G. Sandberg, and K. Lundström, Planta 172: 47–52 (1987).

199. R. S. Bandurski, A. Schulze, and J. D. Cohen, Biochem. Biophys. Res. Commun. 79: 1219–1223 (1977). 200. P. J. Hall and R. S. Bandurski, Plant Physiol. 80: 374–377 (1986). 201. B. Bartel and G. R. Fink, Science 268: 1745–1748 (1995). 202. R. T. Davies et al., Plant Cell 11: 365–376 (1999).

230. P. L. Julian, H. C. Printy, R. Ketcham, and R. Doone, J. Am. Chem. Soc. 75: 5305–5309 (1953). 231. H. Kinashi, Y. Suzuki, S. Takeuchi, and A. Kawarada, Agr. Biol. Chem. 40: 2465–2470 (1976). 232. H. Matsushima, H. Fukumi, and K. Arima, Agr. Biol. Chem. 37: 1865–1871 (1973).

203. J.-C. Chou, W. W. Mulbry, and J. D. Cohen, Mol. Gen. Genet. 259: 172–178 (1998).

233. Y. Sahashi, Biochem. Z. 182: 69–72 (1926).

204. K. Bialek, W. J. Meudt, and J. D. Cohen, Plant Physiol. 73: 130–134 (1983).

234. J. Bilang, H. MacDonald, P. J. King, and A. Sturm, Plant Physiol. 102: 29–34 (1993).

205. C. Catala´ et al., Plant Physiol. 100: 1457–1463 (1992).

235. J. Bilang and A. Sturm, Plant Physiol. 109: 253–260 (1995).

¨ 206. R. Pluss, T. Jenny, and H. Meier, Physiol. Plant. 75: 89–96 (1989). ¨ 207. K. Ljung, A. Ostin, L. Lioussanne, and G. Sandberg, Plant

236. D. A. M. van der Kop et al., Plant Mol. Biol. 30: 839–844 (1996).

Physiol. 125: 464–475 (2001). ¨ 208. A. Ostin, M. Kowalczyk, and R. P. Bhalerao, Plant Physiol. 118: 285–296 (1998). ¨ ´ J. Chamarro, and G. Sandberg, J. Mass 209. A. Ostin, C. Catala, Spectrom. 30: 1007–1017 (1995). 210. J. Riov and F. Bangerth, Plant Physiol. 100: 1396–1402 (1992). ¨ 211. H. Tuominen, A. Ostin, G. Sandberg, and B. Sundberg, Plant Physiol. 106: 1511–1520 (1994).

237. F. N. J. Droog, P. J. J. Hooykaas, and B. J. van der Zaal, Plant Physiol. 107: 1139–1146 (1995). 238. R. Zettl, J. Schell, and K. Palme, Proc. Natl. Acad. Sci. USA 91: 689–693 (1994). 239. K. A. Marrs, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 127–158 (1996). 240. R. Hertel, K. S. Thomson, and V. E. A. Russo, Planta 107: 325–340 (1972). 241. P. M. Ray, U. Dohrmann, and R. Hertel, Plant Physiol. 60: 585–591 (1977).

212. S. Tsurumi and S. Wada, Plant Cell Physiol. 27: 559–562 (1986).

242. J. D. Walton and P. M. Ray, Plant Physiol. 68: 1334–1338 (1981).

213. S. Tsurumi and S. Wada, Plant Cell Physiol. 27: 1513–1522 (1986).

243. A. M. Jones, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 393–420 (1994).

¨ M. Frey, and A. Gierl, Phytochem214. E. Glawischnig, S. Grun, istry 50: 925–930 (1999).

244. M. A. Venis and R. M. Napier, Crit. Rev. Plant Sci. 14: 27–47 (1995).

215. J. H. Oetiker and G. Aeschbacher, Plant Physiol. 114: 1385–1395 (1997).

¨ 245. M. Löbler and D. Klambt, J. Biol. Chem. 260: 9848–9853 (1985).

216. V. Magnus, R. P. Hangarter, and N. E. Good, J. Plant Growth Regul. 11: 67–75 (1992).

246. N. Inohara, S. Shimomura, T. Fukui, and M. Futai, Proc. Natl. Acad. Sci. USA 86: 3564–3568 (1989).

217. H. Hattori and S. Marumo, Planta 102: 85–90 (1972).

247. U. Tillmann et al., EMBO J. 8: 2463–2467 (1989).

AUXINS, INDOLE AUXINS 248. T. Hesse et al., EMBO J. 8: 2453–2461 (1989). 249. H. Macdonald (1994).

et al.,

Plant

Physiol.

105:

1049–1057

250. J. Henderson et al., Cell Biol. Intern. 20: 413–422 (1996). 251. R. M. Napier et al., EMBO Workshop on Auxins (Calcatoggio, France, May 13 to 19, 2000), Book of Abstracts, pp. 58–59. 252. H. Barbier-Brygoo, Crit. Rev. Plant Sci. 14: 1–25 (1995). 253. R. M. Napier and M. A. Venis, New Phytol. 129: 167–201 (1995). 254. J. G. Chen et al., Genes Dev. 15: 902–911 (2001). ¨ 255. M. Löbler and D. Klambt, J. Biol. Chem. 260: 9854–9859 (1985). ¨ 256. D. Klambt, Plant Mol. Biol. 14: 1045–1050 (1990). 257. H. Macdonald, Physiol. Plant. 100: 423–430 (1997). ¨ 258. M. Claussen, H. Luthen, and M. Böttger, Physiol. Plant. 98: 861–867 (1996). 259. S. D. Verhey and T. L. Lomax, J. Plant Growth Regul. 12: 179–195 (1993). 260. T. J. Guilfoyle, G. Hagen, T. Ulmasov, and J. Murfett, Plant Physiol. 118: 341–347 (1998). 261. S. Abel and A. Theologis, Plant Physiol. 111: 9–17 (1996). 262. T. Ulmasov, G. Hagen, and T. J. Guilfoyle, Science 276: 1865–1868 (1997).

121

281. M. Ramek and S. Tomić, Intern. J. Quantum Chem. 70: 1169–1175 (1998). 282. S. Tomić, M. Ramek, and B. Kojić-Prodić, Croat. Chem. Acta 71: 511–525 (1998). 283. S. Tomić, R. R. Gabdoulline, B. Kojić-Prodić, and R. C. Wade, J. Computer-Aided Mol. Design 12: 63–79 (1998). 284. J. M. Kaethner, Nature 267: 19–23 (1977). 285. A. B. Rakhaminova, E. E. Khavkin, and L. S. Yaguzhinsky, Biokhimiya 43: 806–823 (1978). 286. W. L. Porter and K. V. Thimann, Phytochemistry 4: 229–243 (1965). 287. C. Hansch et al., J. Am. Chem. Soc. 85: 2817–2824 (1963). 288. M. D. Edgerton, A. Tropsha, and A. M. Jones, Phytochemistry 35: 1111–1123 (1994). 289. H. T. Hartmann and D. E. Kester, Plant Propagation. Principles and Practices, 3rd ed., Prentice Hall, Englewood Cliffs, N.J., 1975. 290. K. V. Thimann and J. Behnke-Rogers, The Use of Auxins in the Rooting of Woody Cuttings. Maria Moors Cabot Foundation, Publication no. 1 (publ. under the auspices of the Harvard Forest), Petersham, M.A., 1950. 291. G. Houle and P. Babeux, Can. J. Bot. 76: 1687–1692 (1998). 292. C. S. Feung, R. H. Hamilton, and R. O. Mumma, Plant Physiol. 59: 91–93 (1977).

263. E. Fischer, Ber. Dtsch. Chem. Ges. 27: 2985–2993 (1894).

293. R. P. Hangarter, M. D. Peterson, and N. E. Good, Plant Physiol. 65: 761–767 (1980).

264. D. Koshland, Jr., Angew. Chem. Int. Ed. Engl. 33: 2375–2378 (1994).

294. V. Magnus, B. Nigović, R. P. Hangarter, and N. E. Good, J. Plant Growth Regul. 11: 19–28 (1992).

265. A. Theologis, Curr. Biol. 8: R875–R878 (1998).

295. S. Jelaska, V. Magnus, M. Seretin, and G. Laćan, Physiol. Plant. 64: 237–242 (1985).

266. D. M. Reinecke et al., Plant Growth Regul. 27: 39–48 (1999). 267. R. van Huizen et al., Plant Physiol. 109: 1213–1217 (1995). 268. G. F. Katekar, Phytochemistry 18: 223–233 (1979). ˚ . Jönsson, in W. Ruhland, ed., Encyclopedia of Plant Phys269. A iology, Vol. 14, Springer-Verlag, Berlin, 1961, pp. 958–1006.

296. E. Epstein and Z. Wiesman, Olea 18: 35–38 (1987). 297. Z. Wiesman, J. Riov, and E. Epstein, Plant Physiol. 91: 1080–1084 (1989).

FURTHER READING

270. M. Katayama et al., J. Pestic. Sci. 23: 289–295 (1998). 271. S. Antolić et al., Acta Crystallogr., Section B 52: 651–661 (1996). 272. B. Kojić-Prodić et al., Acta Crystallogr., Sect. B: Struct. Sci. 47: 1010–1019 (1991). 273. B. Nigović et al., Acta Crystallogr., Sect. B: Struct. Sci. 56: 94–111 (2000). 274. I. L. Karle, K. Britts, and P. Gum, Acta Crystallogr. 17: 496–499 (1964). 275. K. Chandrasekhar and S. Raghunathan, Acta Crystallogr., Sect. B: Struct. Sci. 38: 2534–2535 (1982). 276. B. Nigović et al., Acta Crystallogr., Sect. B: Struct. Sci. 52: 332–343 (1996). 277. S. Antolić et al., Plant Growth Regul. 27: 21–31 (1999). 278. M. Ramek, S. Tomić, and B. Kojić-Prodić, Intern. J. Quantum Chem. Suppl. 22: 75–81 (1995). 279. M. Ramek, S. Tomić, and B. Kojić-Prodić, Intern. J. Quantum Chem. 60: 3–9 (1996). 280. M. Ramek and S. Tomić, J. Mol. Struct. (Theochem.) 454: 167–173 (1998).

Thimann, K. V., Hormone Action in the Whole Life of Plants, The University of Massachusetts Press, Amherst, 1977. A general overview written by one of the founders of auxin biochemistry. Davies, P. J., ed., Plant Hormones: Physiology, Biochemistry, and Molecular Biology, Kluwer Academic Publishers, Dordrecht, 1995. A multiauthor textbook. Letham, D. S., Goodwin, P. B., and Higgins, T. J. V., ed., Phytohormones and Related Compounds—A Comprehensive Treatise, Vol. 2, Elsevier, Amsterdam, 1978. A detailed overview that includes several articles covering classical research on auxin biochemistry. MacMillan, J., ed., Hormonal Regulation of Development I. Molecular Aspects of Plant Hormones, Encyclopedia of Plant Physiology, New Series, Vol. 9, Springer-Verlag, Berlin, 1980. Contains chapters on auxin biochemistry that complement those in the Treatise by Letham et al. Hoykaas, P. J. J., Hall, M. A., and Libbenga, K. R., eds., Biochemistry and Molecular Biology of Plant Hormones, Elsevier, Amsterdam, 1999. Covers analytical methods, structure-activity relationships, biogenesis and metabolism, perception, and signal transduction (including gene regulation) for auxins and other phytohormones.

122

AVIAN REPELLENTS

AVIAN REPELLENTS MICHAEL L. AVERY National Wildlife Research Center Gainesville, Florida

The use of repellents to protect crops from birds has a long history. Early European settlers in eastern North America observed that native Americans used an extract of Veratrum spp. to protect corn seeds from avian depredators: ‘‘Then when the starlings, crows, or other birds, pick up or pluck out the grains of corn, their heads grow delirious, and they fall, which so frightens the rest that they never venture on the field again. When those which have tasted the grains recover, they leave the field, and are no more tempted to visit it again’’ (1). Repellents move birds from one place to the next. After successful application of a bird repellent, the overall amount damage will probably not decrease, but it will be distributed differently. Some persons are philosophically opposed to repellents because they do not reduce damage overall, but instead shift the problem to a neighbor’s field or vineyard. However, by definition, repellents are nonlethal and as such they represent a very appealing approach to the management of bird damage in crops (2). Bird damage is usually highly skewed among sites, with most producers incurring little damage and few suffering high, economically important levels of damage (3). Realistically, the goal of bird damage management is not to eliminate losses, but to reduce them to an acceptable, manageable level. To the extent that a repellent can help redistribute the economic impact among producers, and especially provide relief at the few high-damage sites, it will be a successful component of bird damage management plans. FORAGING THEORY AND CHEMICAL REPELLENTS Birds attack crops because they are readily accessible sources of abundant food obtainable with low expenditure of effort. This is especially important to young birds that are not experienced foragers. In the late summer and fall, newly fledged birds constitute a large portion of many depredating flocks. Because of the availability of large quantities of food, crop fields, vineyards, and orchards provide ideal feeding situations for young birds just learning to fend for themselves. At other times of year, sources of the birds’ natural food may be limited or lacking altogether so that the cultivated crop becomes an essential component of diet (4). The continuing alteration of the natural landscape to accommodate human population expansion will no doubt make it increasingly difficult for birds to find natural sources of food. Given this situation, it is easy to appreciate why agricultural crops are powerful attractions to bird and why depredating birds are not easily dissuaded. With the potential benefits of feeding on the crop so great, there must be a commensurately high potential cost in order to discourage bird use of the protected food.

To be effective, a chemical repellent must affect the way that the bird perceives the crop. For most depredating birds, the benefits to feeding on the crop far outweigh the costs. The challenge is to alter that balance so that either the benefits are greatly reduced or the costs are greatly increased. Basically, increasing the cost to the birds means increasing the amount of time and energy required to feed on the crop. The more time the bird has to spend acquiring the requisite nutritional resources, the less time it can spend on other essential activities such as territorial defense, nest building and mate acquisition, feather maintenance, predator vigilance, and so on. There is therefore substantial pressure on the bird to feed efficiently. In most applications of optimal foraging theory, it is assumed that the animal is maximizing its rate of energy intake (5). Caloric gain is not the only nutritional requirement a bird has, but it seems to be a pervasive one. If it becomes difficult for the bird to maintain a certain rate of energy intake by feeding on the crop, then optimal foraging theory predicts the bird will look for other sources of food. Thus, the net effect of applying a chemical repellent to the crop will be to lower the value of the crop to the bird by reducing its rate of energy intake. This can be accomplished by making the preferred food more difficult to find, more difficult to handle, or more difficult to digest. More Difficult to Find It is not possible to hide the crop from the birds. Nor is it likely that the crop can be disguised so that it looks like something inedible. It is possible, however, to apply the concepts of mimicry theory to crop protection and combine edible, untreated parts of the crop with chemically protected, but visually identical portions of the crop (6,7). This can be accomplished by applying the chemical repellent to some of a seed crop, mixing it with an equal amount of untreated seed, and then broadcasting the mixture on the field (8). Alternatively, certain rows or individual plants in an orchard or vineyard can be sprayed with chemical repellent and the rest left untreated (9). This approach relies on the assumption that treated and untreated food items are not visually distinct. If birds are reliably able to select the untreated food, then there is no advantage to partial treatment. Also, the cost to the bird of making a mistake and selecting a treated food item must be high. Otherwise, there is no reason for the bird to avoid testing and evaluating the alternatives. The repellent treatment should cause the bird to delay its decision long enough so that the energy gained per time spent recognizing, identifying, and selecting the food item declines to where it is no longer profitable. At that point, the bird will move to other locations or search for other types of food. More Difficult to Handle Once the food item is selected and acquired, manipulation of the food item can constitute an important commitment of time and effort by the foraging bird. Intuitively, the more potentially valuable a food item is, in terms of caloric value or nutrient content, the more time the bird should be

AVIAN REPELLENTS

willing to spend to manipulate and consume it. Generally, as the size of the food item increases, the handling time increases as well. Although the bird might be able to eat the larger food item, the longer it takes to handle it, the greater the chances for inadvertently dropping it. Small seeds or small fruits can be ingested with virtually no manipulation. Thus, cedar waxwings (Bombycilla cedrorum) prefer to eat small blueberries because almost always the birds ingest the berry in seconds, whereas larger fruit that potentially yields greater caloric rewards take longer to manipulate and are often dropped and lost (10). The rate of caloric intake is greatest with the smallest size berry. As a rule, red-winged blackbirds (Agelaius phoeniceus) can eat rice seed at a rate of 6–8 seeds/min. The rice seed can be coated, however, with a nontoxic clay-based treatment that greatly increases the time interval between seeds taken by the blackbird (11). The sticky coating on the seed causes the bird to spend time wiping and cleaning its bill so that feeding rates are greatly reduced. As a consequence, the rate of caloric intake declines to the point that birds avoid the clay-coated rice seed (12). More Difficult to Digest After it is recognized, selected, manipulated, and ingested, the food item still has to be digested and assimilated if it is to benefit the bird. Modification of the food item so that it is rendered more difficult to digest will reduce its attractiveness to depredating birds. Certain phenolic compounds, generally referred to as tannins, are effective digestive inhibitors because they form insoluble precipitates with proteins, including various digestive enzymes. The resulting reduced activity of the digestive enzymes causes weight loss and other detrimental physiological effects (13). In some cereal crops such as sorghum and millet, high tannin varieties have been developed specifically for bird deterrence (14). Some frugivorous bird species, including those that cause crop damage, such as the American robin Turdus migratorius and the European starling Sturnus vulgaris, possess a physiological constraint that makes it impossible for them to digest sucrose, a common constitute of many fruits (15). These bird species lack the intestinal enzyme sucrase that hydrolyzes the 12-carbon sucrose molecule, that cannot be assimilated, into the 6-carbon sugars glucose and fructose, which are assimilable. Means of exploiting this digestive constraint so that small cultivated fruits will be less susceptible to bird damage include using sucrose as a spray on ripening fruit (16) and manipulating sugar composition of ripening fruit to produce elevated, bird-resistant levels of sucrose (17). Alternative Food Sources Reducing the value of the crop is one key component to repellent use. The other crucial factor is the availability of alternative sources of food. A bird with no alternatives will tolerate much greater discomfort than will one that has access to other food sources. Thus, chemical repellents will function more effectively with alternative food sources available than with no alternative. The disparity in

123

attractiveness between the crop and potential alternative foods will determine how strong the repellent must be to protect the crop. If the foraging efficiency in the alternative is close to that in the crop, then it will be relatively easy to effect a change in the birds’ behavior. Often, wild seeds or fruits are available in fields or meadows adjacent to the crop, but a number of factors reduce the relative attractiveness of an alternative food source: 1) the birds’ efficiency in feeding on the wild food sources might be less than when they feed in the crop, 2) their risk of being preyed on might be higher than in the crop, 3) the intrinsic quality of the food items (for example, caloric content) might be lower than that of the crop, and 4) competition with other animals for the alternative food might be greater than in the crop. Any of these factors, individually or in combination, might be sufficient to encourage the depredating birds to prefer the crop to the alternatives. Whatever steps that can be taken to increase the birds’ rate of energy intake feeding on the alternative food will likely promote more effective repellent use. One possible tactic that could constitute a part of a long-term management scheme is to provide alternative food patches specifically for avian depredators. In this way, a grower could assure that the alternative food is comparable in quality and abundant enough to satisfy the birds’ requirements. Establishment of feeding sites specifically for pest birds is probably not intuitively pleasing to most producers, and the effectiveness of this management approach needs to be experimentally tested.

CATEGORIES OF CHEMICAL REPELLENTS In general, repellents can be divided into two broad categories based on their modes of action. Primary repellents are painful or irritating upon contact, and the bird responds reflexively without needing to acquire an avoidance response. Extensive research into the nature and characteristics of dozens of primary repellents lead Clark (18) to the conclusion that chemesthesis (pain or irritation) is responsible for avoidance responses produced by these compounds. Many of these compounds have ecological significance in interactions between birds and their natural food items, and one primary repellent compound, methyl anthranilate, is registered as an avian feeding deterrent. Many primary repellents are toxic, but because the compounds are aversive, birds do not ingest enough to cause them harm. Secondary repellents are not aversive immediately but produce illness or discomfort sometime after ingestion. The effectiveness of these compounds is based on the concept of conditioned food avoidance (19). The bird associates the adverse postingestional consequences with the food or with some sensory attribute of the food (e.g., color or taste) and thereby learns to avoid it. The avoidance response produced by a secondary repellent is likely more robust than that from a primary repellent (20,21). Secondary repellents are toxic, and for some compounds, the difference between a repellent dose and a lethal dose may be slight.

124

AVIAN REPELLENTS

AGRICULTURE USES FOR BIRD REPELLENTS Avian repellent compounds have potentially two general uses in agriculture: to reduce bird depredations to crops and to reduce hazards to birds posed by potentially toxic pesticides. For crop protection, both primary and secondary repellents are applicable. The situations in which one or the other will be more appropriate will vary according to a number of factors. A primary repellent will be advantageous when the birds are not resident in the area or where the population of depredating birds is not constant but changes frequently. A primary repellent requires no learning period before the effectiveness of the treatment takes effect. Birds immediately sense the chemical when they eat treated food, and they respond to the sensory irritation. Even though primary repellents require no learning to be effective, birds might tend to test the protected crop, and so additional damage may accumulate even after the same birds have been exposed to the treatment. This is especially true if the primary repellent does not produce sufficiently potent punishment to discourage bird use of a highly preferred food item such as the protected crop. There is temporary irritation from the primary repellent, but no incapacitation; so the risk to the bird is relatively minor, and it tends to continue to try the treated food items. Because a secondary repellent produces no immediate negative consequence to the bird, there will be some continued feeding until the association is made between the treated crop and the discomfort. In field applications, the effectiveness of a secondary repellent will be determined in part by the residency status of the depredating bird population. If the birds are sedentary, then a secondary repellent will most likely be effective because the birds will be in the area a sufficient length of time to acquire the avoidance response and learn to avoid the treated crop. If, however, the birds are mostly transient, the application of a secondary repellent will not be as useful because the birds will be present just a short time. Depending on the time needed to acquire the avoidance response, the affected birds could have departed and been replaced by a different group of birds, which in turn will have to acquire the avoidance response. Damage will occur and accumulate as each new group of birds learns to avoid the repellent-treated crop. AVAILABILITY OF BIRD REPELLENTS Currently, crop damage reduction with chemical repellents is limited to a few registered products (22). The lack

of registered bird-repellent compounds is not due to a lack of potentially useful chemicals. In recent years, many compounds have been identified as bird-repellent (Table 1). In addition to those listed in Table 1, Clark (23) has generated repellency data on dozens of other compounds. New screening methods using structureactivity modeling and tissue culture mean that candidate repellent compounds can be identified more systematically than before (24). The main reasons for the paucity of useful birdrepellent agricultural products are lack of economic incentive and restrictions imposed by regulatory agencies. Increasingly, there is concern for the human health and environmental safety of agricultural chemicals. These concerns have resulted in more extensive and stringent testing requirements, which have elevated the costs of chemical registration considerably. In most cases, the potential market for a bird-repellent compound is relatively small, and the lack of potential sales plus the upfront outlay of funds necessary to obtain the registration combine to discourage economic development of these types of chemicals. There is, therefore, little variety in the chemical bird repellents that are available for agricultural uses. As a result, management options for growers are limited, and in fact for many crops, no repellent is available. The future development of repellent chemicals for crop protection probably lies in expanding the few labels that do exist to cover additional use patterns, rather than registration of new repellent compounds. BIRD-REPELLENT COMPOUNDS Methyl anthranilate (MA) is a naturally occurring compound that is used extensively in the food industry to impart grape or fruity flavor to candy, gum, soft drinks, and other consumables. As such, it is one of a number of compounds generally regarded as safe (GRAS-listed) by the U.S. Food and Drug Administration. Even though MA is palatable to humans, it is an irritant to birds. The bird-repellent properties of MA and related compounds were discovered in the late 1950s (25). The mode of action is via the trigeminal nerve. Thus, all avian species tested so far perceive MA as an irritant, not as a taste repellent per se. The repellency and mode of action of MA have been demonstrated experimentally through behavioral trials with intact and nerve-cut birds (26). Birds consistently reject food and water treated with MA at the appropriate level. This is a reflex response that does

Table 1. Compounds Recently Identified with Bird-Repellent Properties Compound Cinnamamide Coniferyl benzoate Cucurbitacin Imidacloprid Methyl cinnamate Ortho-aminoacetophenone Pulegone

Principal Species Tested

References

Rock dove, Rook, Chaffinch Ruffed grouse, European starling Red-winged blackbird Red-winged blackbird, Brown-headed cowbird Red-winged blackbird European starling European starling, Red-winged blackbird

72,73 74,75 56 76,77 78 65,79 80,81

AVIAN REPELLENTS

not have to be learned. Rejection of tainted food varies, however, according to the motivational state of the bird. With no alternative food, or with a relatively unattractive alternative food available, birds will persist and eat the MA-treated food. If, however, MA-treated food is offered with the same food type available, but untreated, rejection of treated food occurs at much lower treatment levels (27). Because the irritation caused by MA may not be a very strong aversive stimulus, birds tend to return and test the treated food so that loss can accumulate even though the repellent is in place. The strong grapelike odor of MA is not aversive to birds (28). Birds have to contact the MAtreated food with their mouths in order to feel the effects of the compound. In the United States, MA is the active ingredient in various formulated products marketed under the trade names of Bird Shield and ReJeX-iT. These products are registered as bird repellents for use on cherries, blueberries, and grapes. In addition, other formulations are registered for use on turf and water to control geese and other grazing birds. (Information obtained from web sites, www.bsrc.com and www.nei2000.com, as of 1 December 1999.) In using MA-based formulations, it is important to keep in mind several characteristics of MA. 1) This is a volatile compound that dissipates rapidly. The rapid dissipation is exacerbated by degradation due to ultraviolet radiation and due to microbial activity (24). To some extent, the life of the treatment can be extended through encapsulation of the active ingredient and incorporation of ultraviolet protectors and anti-microbial agents in the formulation. 2) Rapid dissipation or degradation of MA can be a mixed blessing. Even though the effectiveness of the treatment will not persist very long, rapid loss of the compound will remove the grapelike flavor of MA so that the taste of the picked fruit is not tainted. The prevention of flavoring of fruit for fresh markets is especially important as these commodities are not washed after picking. The fruit goes directly into containers for shipping to stores. 3) The volatility and reactivity of MA can cause phytotoxic effects on sprayed vegetation (29). Appropriate formulation can ameliorate this problem; so in most cases phytotoxicity of MA should not be a concern. Controlled field evaluations of the efficacy of MA as a bird repellent on fruit crops are few. In New York, bird damage to MA-treated blueberry plots did not differ from that in untreated plots (30). There was, however, some reduction in damage achieved in test plots in grapes and cherries. A large-scale field trial at several sites in Michigan, Oregon, and Washington did not demonstrate reduced bird use of MA-treated blueberry plots (31). Recent field trials suggest that aerial application of MA to corn and sunflower can discourage depredations by flocks of blackbirds (32). When applied to grass, turf, and winter wheat MA reduces grazing by geese and other species at golf courses, parks, and crop fields (33,34). Furthermore, the uses of MA continue to expand. In addition to the turf crop and turf applications, the current registered uses include fogging the compound to disperse nuisance roosts and flocks of birds at airports, applying it to landfills to

125

reduce the numbers of gulls and other bird species, and treating temporary pools and non–fish-bearing bodies of water to discourage use by waterfowl around airports and residential communities. Recent experiments suggest that MA could possibly be used as a secondary repellent (35). The challenge is to encapsulate the MA so that birds ingest it without feeling pain or irritation. Once the repellent is in the gut, irritation by the chemical produces an emetic reaction leading to the formation of a learned avoidance response (35). Anthraquinone In the United States, the use of 9,10-anthraquinone as a bird repellent dates at least from the early 1940s when the first patent for this use was issued (36). Subsequent development and testing of the compound centered on seed treatments, particularly for pine seeds and for rice. Anthraquinone was not registered in the United States, but it was registered in Europe and continues to be used as a seed treatment there. In recent years, however, anthraquinone has resurfaced as a bird repellent in the United States under the brand name Flight Control, and it is now registered as a treatment to repel birds from turf and grass and as a repellent for roosting birds. Additional bird-repellent applications for athraquinone are being developed, including rice and corn seed treatments (37), and aerial application to ripening rice (38,39). Anthraquinone is a secondary repellent and affects birds by causing post-ingestional distress (40). Sometimes, ingestion of anthraquinone-treated food produces vomiting, but often vomiting does not occur and the bird just sits quietly until the discomfort passes. Unlike methiocarb, anthraquinone doe not affect the bird’s nervous system and does not immobilize affected birds. Presumably, the emetic response is produced through irritation of the gut lining, but the actual mechanism is unclear. It is clear, however, that anthraquinone is not a taste repellent or contact irritant. Birds do not hesitate to eat treated food, and they exhibit no sign that treated food is unpalatable to them. The post-ingestional discomfort that results from eating anthraquinone-treated food produces a conditioned aversion to that food type. Birds need to experience the adverse consequences before learning to avoid the protected food. Thus, it is not reasonable to expect losses to cease immediately upon application of the repellent. There will be some level of loss in the crop as the depredating birds acquire the learned avoidance response. Anthraquinone is a stable compound that is virtually insoluble in water. It is not phytotoxic and does not inhibit germination of rice seeds or growth of sprouts. It has very low toxicity to birds and mammals, and it appears to be innocuous to insects as well. There is no known hazard to nontarget species from repellent applications of Flight Control. Another potential aspect to the effectiveness of Flight Control as a bird repellent is the fact that its reflectance spectrum peaks in the near-ultraviolet wavelengths. This part of the spectrum is also where the visual sensitivity of bird species such as the red-winged blackbird is maximal (41). What, if any, role ultraviolet reflectance plays in the repellent nature of Flight Control is

126

AVIAN REPELLENTS

conjectural. Possibly, the ultraviolet reflectance enhances the bird’s ability to associate the appearance of treated food with the adverse post-ingestional consequences and thereby learn more rapidly to avoid the treated food. Methiocarb (3,5-dimethyl-4-[methylthio]phenyl methylcarbamate) This compound was originally developed by Bayer as an insecticide. The bird-repellent properties of the compound were quickly recognized, however, and a number of applications for bird damage management followed (42). Methiocarb is a carbamate, and its mode of action is via the inhibition of acetylcholinesterase at synapses in the nervous system. Unlike many cholinesterase-inhibiting compounds, however, the effects of methiocarb are rapidly reversible, and the animal experiences only transitory disruption. Affected birds exhibit a range of symptoms, including retching, vomiting, and temporary paralysis. The time to onset of symptoms, and the severity of those symptoms, is dependent on the dose received. Typically, vomiting begins within 10 minutes of ingestion of treated food. An affected bird can become immobilized within 30 minutes of ingesting an appropriate dose, and it will recover fully in another 30 minutes. Birds that feed on methiocarb-treated food exhibit no sign that the chemical tastes bad. Treated food is readily accepted, and feeding slows only when the bird begins to detect physiological effects of the chemical. Methiocarb is a secondary repellent, and repellency occurs through aversive conditioning, by which birds that feed on treated food become sick and associate either the food or characteristics of the food with the discomfort (21). As a result, affected birds learn to avoid that food item. Often the avoidance response is locationdependent. For example, common ravens (Corvus corax) that learn not to eat eggs at one site will still feed on eggs at a different location (43). The avoidance response is also affected by various other factors such as the bird’s prior experience with the food item, the strength of the post-ingestional discomfort, and the availability of alternative food. Anthraquinone would likely be similar in these respects. Methiocarb is classified as ‘‘extremely toxic’’ because of its low acute oral rat median lethal dose (LD50 ), 15–35 mg/kg (44). This is important for human health and safety, but it is misleading when considering the effects to birds. Applied properly, methiocarb is very safe with regard to target and nontarget species (45). Although the LD50 is low, free-feeding birds acquire a repellent dose and stop feeding long before a lethal dose is ingested. In North America, methiocarb has been tested extensively in many agricultural applications. It has been used to protect newly seeded and sprouted crops, ripening grain crops, and soft fruits. It was commercially sold as Mesurol and for several years was registered in the United States as a bird repellent on cherries, grapes, and blueberries and as a treatment for corn seed. The registrations lapsed in 1989, however, when the registrant declined to meet additional data requirements specified by the U.S. Environmental Protection Agency. In the United States, methiocarb is

now used as a molluscicide on ornamental plants. Methiocarb is registered as a bird-repellent seed treatment for rice in Uruguay, where the product is known as Draza. The rights to methiocarb were recently acquired from Bayer by Gowan Company (Yuma, AZ). Despite the company’s interest in methiocarb as a bird repellent, the outlook for obtaining agricultural registrations in the United States is bleak given the current regulatory climate and increasingly strict laws protecting human health, such as the 1996 Food Quality Protection Act. Methiocarb has also proved effective as a bird repellent to deter grazing by geese on turf (46) and is as a nonlethal means to reduce avian predation on eggs of endangered species (43) labeled with the USEPA. Avitrol (4-aminopyridine) Avitrol is considered by some to be a ‘‘behavioral repellent.’’ It is highly toxic to birds and mammals. In the United States, there are several registrations for the control of blackbirds, pigeons, and various other bird species. Avitrol repels birds by poisoning some members of the feeding flock, causing them to become agitated and hyperactive. The distress calls emitted by the fatally poisoned birds frighten the other members of the flock so that they leave the area. Presumably, after one such experience, the birds do not return to the site. In experimental evaluations of Avitrol in corn and sunflower fields, however, the compound has not proven consistently effective (47,48). Fungicides Although not designed to be used as bird repellents, a number of fungicides have been shown to reduce feeding activity of various bird species. Thiram (tetramethylthiuram disulfide) is used as a seed treatment. The chemical depresses central nervous system activity but has low oral toxicity (43). There have been several studies that have documented the repellency of thiram to birds (49,50). Ziram (zinc bis[dimethyldithiocarbamate]) has shown potential usefulness as a repellent to protect orchids and other valuable flowers (51). Several copper compounds are used widely as fungicides, and at least two of them, copper oxychloride and copper hydroxide, have been tested for bird repellency (50,52). Each of these compounds reduced consumption of treated food. Copper ingestion can lead to post-ingestional distress, and these compounds probably act as secondary repellents by irritation of the gut lining, although the mode of action is not clear. Panoctine (guazitine triacetate) is used widely throughout the world as a seed treatment, but it is not available in North America. Where it is used, Panoctine is considered a repellent to various bird species. Feeding trials with captive red-winged blackbirds demonstrated repellency in choice tests but not in tests where birds had no source of untreated alternative food (50). Other Compounds Several substances that have offensive properties to humans are marketed as bird repellents. RoPel is marketed as a spray and in granular form as a

AVIAN REPELLENTS

repellent for geese, ducks, and woodpeckers on lawns and around structures. The active ingredients are denatonium saccharide and thymol, neither of which is known to be particularly offensive to birds. Bye-Bye Birdie is sold in granular form as a repellent to deter starlings, pigeons, sparrows, and other birds from structures. It contains 100% naphthalene, which has been shown to be inoffensive to birds (53,54). In Australia, Duck Off is used as a turf treatment sprayed to deter ducks and other species from golf courses and other areas. The active ingredient is aluminum ammonium sulfate, a very astringent compound. Previously, this compound, synergized with sucrose octa-acetate, was sold as a bird repellent in the U.K. as Curb. Field trials of the same compound in Africa showed that it protected ripening cereal grains for several weeks from depredating flocks of birds (55). At least some bird species are sensitive to bitter compounds (56), so it certainly is possible bitter or astringent compounds can be formulated to produce safe, effective bird repellents. There is a persistent impression that capsaicin, the active principle in hot capsicum peppers, is an effective bird repellent. Various products are routinely marketed to deter birds from crops, structures, and for other uses. This is despite the fact that there is well-documented evidence that birds are relatively insensitive to capsaicin, and in fact, seeds of capsicum peppers are dispersed by birds (57). There are fundamental differences between the avian and mammalian chemosensory worlds, and just because a compound is irritating or offensive to mammals does not mean that birds will respond similarly (58). There is evidence that derivatives of the neem plant (Azadirachta indica) have bird-repellent properties (59,60). Recent studies suggest that the degree of avian repellency of neem compounds is determined by the concentration of azadirachtin (61), a compound that inhibits insect growth and development. Lindane (Isotox), an organochlorine compound, was formerly used as a seed treatment. It is no longer manufactured in the United States, and most agriculture uses have been canceled by the U.S. Environmental Protection Agency because it is considered a potential carcinogen. REPELLENTS TO REDUCE INGESTION OF GRANULAR PESTICIDES As a normal part of their dietary habits, birds regularly ingest small particles of grit that serve to grind hard food items in the birds’s gizzard. Grit ingestion has become an important topic in recent years because birds sometimes ingest granular pesticides as they search for grit particles. Many of the granular pesticides are very toxic, and as a result of accidental ingestion by birds, there have been a number of documented bird kills. Many aspects of the granular particle, such as size, shape, texture, and color, can potentially be manipulated to make the granular less appealing or less likely to be taken by a foraging bird (62,63).

127

A potentially useful application of an aversive primary repellent is as a constituent of granular pesticide formulations (64). Because many granular pesticides are very toxic, birds cannot afford to learn to avoid the granules. Thus, a secondary repellent is not appropriate. Primary repellent chemicals may be useful, however, provided such a repellent will be sufficiently irritating to cause a bird to drop the granule immediately. Methyl anthranilate might be a candidate for such a use, but ideally to ensure that the toxic granule is not ingested, a more aversive compound should be sought. Other compounds more aversive than MA have been identified (65), but definitive tests of whether these materials would actually reduce granule ingestion by birds have yet to be performed. Furthermore, compatibility of the repellent with the pesticide formulation would have to be determined.

SUMMARY AND FUTURE DEVELOPMENTS Although there are numerous potential applications for avian repellents, such compounds are not the answer to every crop damage situation. Understanding the specifics of bird-crop interaction is essential to successful use of chemical bird repellents. This is illustrated by the situation in northern California where blackbird damage to wild rice is an ongoing concern. Blackbirds consume seed during the milk, dough, and mature stages, and further damage results from bird movements within the crop that causes seed heads in the mature stage to shatter. Estimated losses range from $121 to $309/ha (66). Control of damage relies on the use of frightening techniques (shotguns, propane cannons, etc.), which have only limited effectiveness. When the bird-repellent Flight Control was applied to ripening plots of wild rice, there was no observed effect on the blackbirds feeding in those plots despite the fact that similar rates of application did reduce blackbird numbers in plots of ripening white rice in Louisiana (67). This result was surprising until it became clear that blackbirds were doing more in the wild rice than just feeding. Blackbirds use wild rice crops for loafing and escape cover, as nighttime roosts, and for nest sites. Thus, unlike fields of white rice, wild rice provides the same resources to blackbirds as their natural habitats. By applying a feeding deterrent, we did not address the other reasons for the birds being there, thus, had little impact on the birds’ activity. The deficiency was not in the repellent, but in the way in which it was used. Even the most successful repellent will not eliminate damage by birds. The only way to accomplish that is to employ netting or some other means of exclusion, an option that in most cases is not economical or practical. Repellents are tools or methods that are best viewed as components of integrated management plans, rather than as solutions by themselves. By combining techniques, it is possible to attack many sensory modalities at once and thereby increase the likelihood of creating an uncomfortable foraging environment for the depredating birds. The effectiveness of various combinations of methods for bird

128

AVIAN REPELLENTS

damage management is an area of research that is largely unexplored. In a similar vein, a promising area of future research is the investigation of various combinations of repellents themselves. Using mixtures of primary repellents and secondary repellents with a color as a visual deterrent creates opportunities for improved repellency with lessactive compound used (67,68). Although many naturally occurring compounds are avian feeding deterrents (6,69), few of these have been evaluated as potential repellents for agricultural use. There is a vast amount of information on chemical ecology and interactions between arthropods and avian predators that could potentially be applied to crop protection. This field is ripe for research and may result in new, improved repellents of the future, although just because a compound occurs naturally is no assurance that it is safe (18).

22. J. R. Mason and L. Clark, Proc. Vertebrate Pest Conf. 15: 115–129 (1992). 23. L. Clark, in J. R. Mason, ed., Repellents in Wildlife Management, Colorado State University Press, Fort Collins, 1997, pp. 343–352. 24. L. Clark, Proc. Vertebr. Pest Conf. 18: 330–337 (1998a). 25. U.S. Patent Office, Patent 2,967,128, 1961, M. Kare, inventor. Bird repellent. 26. J. R. Mason, M. A. Adams, and L. Clark, J. Wildl. Manage. 53: 55–64 (1989). 27. M. L. Avery et al., J. Wildl. Manage. 59: 50–56 (1995). 28. L. Clark, Wilson Bull. 108: 36–52 (1996). 29. M. L. Avery, Proc. Vertebr. Pest Conf. 15: 130–133 (1992). 30. P. D. Curtis, I. A. Merwin, M. P. Pritts, and D. V. Peterson, HortScience 29: 1151–1155 (1994). 31. M. L. Avery et al., J. Wildl. Manage. 60: 929–934 (1996).

BIBLIOGRAPHY 1. A. B. Benson, ed., Peter Kalm’s Travels in North America, Vol. 1, Dover Publications, Inc., New York, 1966. 2. C. A. Liss, in J. R. Mason, ed., Repellents in Wildlife Management, Colorado State University Press, Fort Collins, 1997, pp. 429–433. 3. R. L. Hothem, R. W. DeHaven, and S. D. Fairaizl, Bird Damage to Sunflower in North Dakota, South Dakota, and Minnesota, 1979–1981, Fish and Wildlife Technical Report 15, U.S. Fish and Wildlife Service, Washington, D.C., 1988.

32. L. R. Askham, Proc. Vertebr. Pest Conf. 19: 22–25 (2000). 33. J. L. Cummings, P. A. Pochop, J. E. Davis, Jr., and H. W. Krupa, J. Wildl. Manage. 59: 47–50 (1995). 34. J. R. Mason and L. Clark, Crop Protect. 15: 97–100 (1996). 35. R. Sayre and L. Clark, Am. Chem Soc. Pesticides and Wildlife Symposium, 1999. 36. Protection of seeds against birds, U.S. Patent Office, Patent 2,339,335, 1944, F. Heckmanns and M. Meisenheimer, inventors.

4. S. T. Skeate, Ecology 68: 297–309 (1987).

37. B. F. Blackwell, D. A. Helon, and R. A. Dolbeer, Crop Protect. 20: 65–68 (2001).

5. G. H. Pyke, H. R. Pulliam, and E. L. Charnov, Q. Rev. Biol. 52: 137–154 (1977).

38. M. L. Avery, D. G. Decker, and J. S. Humphrey, Proc. Vertebr Pest Conf. 18: 354–358 (1998).

6. L. P. Brower, Sci. Am. 220: 22–29 (1969).

39. M. L. Avery, E. A. Tillman, and C. C. Laukert, Int. J. Pest Manage. 47: 311–314 (2001).

7. M. L. Avery, J. Wildl. Manage. 49: 1116–1121 (1985). 8. M. L. Avery, J. Appl. Ecol. 26: 433–439 (1989). 9. M. E. Tobin, R. A. Dolbeer, and C. M. Webster, Crop Protect. 8: 461–465 (1989). 10. M. L. Avery, K. J. Goocher, and M. A. Cone, Wilson Bull. 105: 604–611 (1993). 11. D. Daneke and D. G. Decker, Proc. Vertebr. Pest Conf. 13: 287–292 (1988).

40. M. L. Avery, J. S. Humphrey, and D. G. Decker, J. Wildl. Manage. 61: 1359–1365 (1997). 41. J. W. Parrish, J. A. Ptacek, and K. L. Will, Auk 101: 53–58 (1984). 42. G. Hermann and W. Kolbe, Pflanzenschutz. Nachr. Bayer 24: 279–320 (1971). 43. M. L. Avery et al., Colonial Waterbirds 18: 131–138 (1995).

13. R. G. Elkin, J. C. Rogler, and T. W. Sullivan, Poultry Sci. 69: 1685–1693 (1990).

44. G. J. Smith, Pesticide Use and Toxicology in Relation to Wildlife: Organophosphorus and Carbamate Compounds, U.S. Fish and Wildlife Service Resource Publ. 170, Washington, D.C., 1987, pp. 171.

14. R. W. Bullard and J. O. York, Crop Protect. 15: 159–165 (1996).

45. R. A. Dolbeer, M. L. Avery, and M. E. Tobin, Pestic. Sci. 40: 147–161 (1994).

15. C. Mart´ınez del Rio, Physiol. Zool. 63: 987–1011 (1990).

46. M. R. Conover, J. Wildl. Manage. 49: 631–636 (1985).

16. A. M. Socci, M. P. Pritts, and M. J. Kelly, HortTechnology 7: 250–253 (1997).

47. M. R. Conover, J. Wildl. Manage. 48: 109–116 (1984).

12. D. G. Decker, M. L. Avery, and M. O. Way, Proc. Vertebr. Pest Conf. 14: 327–331 (1990).

17. R. L. Darnell, R. Cano-Medrano, K. E. Koch, and M. L. Avery, Physiol. Plant. 92: 336–342 (1994).

48. C. E. Knittle, J. L. Cummings, G. M. Linz, and J. F. Besser, Proc. Vertebrate Pest Conf. 13: 248–253 (1988).

18. L. Clark, Current Ornithol. 14: 1–37 (1998).

49. P. S. Sandhu, M. S. Dhindsa, and H. S. Toor, Trop. Pest Manage. 33: 370–372 (1987).

19. J. R. Garcia, R. Kovner, and K. F. Green, Psychonomic Sci. 20: 313–314 (1966).

50. M. L. Avery and D. G. Decker, J. Wildl. Manage. 55: 327–334 (1991).

20. J. Alcock, Anim. Behav. 18: 595–599 (1970).

51. J. L. Cummings et al., Wildl. Soc. Bull. 22: 633–638 (1994).

21. J. G. Rogers, Jr., J. Wildl. Manage. 38: 418–423 (1974).

52. T. H. Babu, Pavo 26: 17–23 (1988).

AVIAN SPECIES 53. J. R. Mason, Ro-Pel efficacy: Evaluation of Active Ingredients under Optimal Conditions with Red-winged Blackbirds (Agelaius phoeniceus), U.S. Department of Agriculture Bird Damage Research Report 384, Denver, Colorado. 1987, 10 pp. 54. R. A. Dolbeer, M. A. Link, and P. P. Woronecki, Wildl. Soc. Bull. 16: 62–64 (1988). 55. R. L. Bruggers, Vertebrate Pest Control and Management Materials, ASTM STP 680, American Society for Testing and Materials, 1979, pp. 188–197. 56. J. R. Mason and T. Turpin, J. Wildl. Manage. 54: 672–676 (1990).

FURTHER READING Clark, L., Proc. Vertebr. Pest Conf. 18: 330–337 (1998). Clark, L., Current Ornithol. 14: 1–37 (1998). Mason, J. R., ed., Repellents in Wildlife Management, Colorado State University Press, Fort Collins, 1997. Mason, J. R. and Clark, L., Proc. Vertebrate Pest Conf. 15: 115–129 (1992).

AVIAN SPECIES

57. J. R. Mason and L. Clark, Wilson Bull. 107: 165–169 (1995).

PIERRE MINEAU

58. D. L. Nolte, J. R. Mason, and L. Clark, J. Chem. Ecol. 19: 2019–2027 (1993).

National Wildlife Research Centre Canadian Wildlife Service Ottawa, Ontario, Canada

59. N. Shivanarayan and M. A. Rao, Pavo 26: 49–52 (1988). 60. J. R. Mason and D. N. Matthew, Int. J. Pest Manage. 42: 47–49 (1996). 61. R. M. Poche, Proc. Vertebr. Pest Conf. 20: (2002) in press. 62. L. B. Best and D. L. Fischer, Environ. Toxicol. Chem. 11: 1495–1508 (1992).

129

WHY BIRDS?

73. R. W. Watkins, E. L. Gill, and J. D. Bishop, Pestic. Sci. 44: 335–340 (1995).

Birds are an important and visible part of our environment. They have been used for many years as sentinels of general environmental quality, and a large body of literature exists on avian toxicology. Birds are extremely mobile, and it is therefore more difficult to exclude them from areas that have been treated with pesticides. The first comprehensive institutional review of agrochemical use in the United States—otherwise known as the Mrak Commission—concluded that: ‘‘Much of the significant evidence on the worldwide effects of insecticides have been provided by birds’’ (1). In North America, most bird species are federally protected from unlicensed taking or kill, to the level of the individual. Groups such as raptors (hawks, eagles, and owls) are often brought to clinics for rehabilitation, and these clinics can become a valuable source of information and samples. Some bird species are attracted to agricultural pests, and many are economically important for insect pest control (2). Finally, birds, as a group, are particularly sensitive to some of the more toxic classes of pesticides such as the organophosphorus and carbamate insecticides, and their reproduction has been found to be vulnerable to a wide range of pesticides. New pesticides developed in part for their relative safety to humans have been found to be especially toxic to birds.

74. W. J. Jakubas and G. W. Gullion, J. Chem. Ecol. 16: 1077– 1087 (1990).

MAIN PESTICIDES OF CONCERN

75. W. J. Jakubas and J. R. Mason, J. Chem. Ecol. 17: 2213– 2221 (1991).

Early Pesticides

63. J. P. Gionfriddo and L. B. Best, J. Wildl. Manage. 60: 836–842 (1996). 64. J. R. Mason, Pestic. Outlook 5: 33–35 (1994). 65. J. R. Mason, L. Clark, and P. S. Shah, J. Wildl. Manage. 55: 334–340 (1991). 66. D. B. Marcum and W. P. Gorenzel, Proc. Vertebrate Pest Conf. 16: 243–249 (1994). 67. M. L. Avery, D. A. Whisson, and D. B. Marcum. Proc. Vertebr. Pest Conf. 19: 26–30 (2000). 68. L. Clark, B. Bryant, and I. Mezine. J. Chem. Ecol. 26: 1219–1234 (2000). 69. J. R. Mason, J. Wildl. Manage. 53: 836–840 (1989). 70. M. L. Avery and J. R. Mason, Crop Protect. 16: 159–164 (1997). 71. J. R. Mason, J. Neal, J. E. Oliver, and W. R. Lusby, Ecol. Applic. 1: 226–230 (1991). 72. D. R. Crocker and K. Reid, Wildl. Soc. Bull. 12: 456–460 (1993).

76. M. L. Avery, D. G. Decker, D. L. Fischer, and T. R. Stafford, J. Wildl. Manage. 57: 652–656 (1993). 77. M. L. Avery, D. G. Decker, and D. L. Fischer, Crop Protect. 13: 535–540 (1994). 78. M. L. Avery and D. G. Decker, J. Wildl. Manage. 56: 799–804 (1992). 79. L. Clark and P. Shah, J. Chem. Ecol. 20: 321–339 (1994). 80. J. R. Mason, J. Wildl. Manage. 54: 130–135 (1990). 81. M. L. Avery, D. G. Decker, J. S. Humphrey, and C. C. Laukert, Crop Protect. 17: 461–464 (1996).

As reviewed by Brown (3), birds were among the first casualties recorded in the course of our earliest attempts to control pests on a broad scale. Application of 50 kg/ha of a dust containing 40% calcium arsenate to German forests in the mid 1920s resulted in extensive mortality of woodlarks (Lullula arborea) and whitethroats (Sylvia communis). However, applications of a dust of lower concentration or at reduced rates was reported to be safe. Cramp (4) reports kills of songbirds and pheasants foraging in crops treated with the insecticide 4,6-dinitro-o-cresol (DNOC) at rates as low as 1 kg a.i. (active ingredient)/ha in the 1950s.

130

AVIAN SPECIES

Pesticides That Bioaccumulate Most reviews of the impact of pesticides on birds place most (or all) of their emphasis on those pesticides that bioaccumulate, notably, the lipophilic organochlorine insecticides. This has led some authors (and much of the general public) to mistakenly conclude that these are the only pesticides to have a significant impact on birds. The persistent organochlorine insecticides include DDT and its analogs; the cyclodienes such as dieldrin, endrin, heptachlor, and chlordane; hexachlorocyclohexane (HCH, mistakenly referred to as benzene hexachloride or BHC in some texts) and its gamma isomer lindane; the complex chlorinated camphene mixture toxaphene; hexachlorobenzene (HCB), as well as the ‘‘cage-like’’ molecule mirex. Other minor organochlorine compounds are discussed in more in-depth reviews (5). The characteristics persistent organochlorines have in common are their high solubility in fats and their relatively long environmental persistence. In order for them to bioaccumulate, they also need to be resistant to metabolism (or have breakdown products that are slowly metabolized). Impacts are often but not exclusively seen in top carnivores—frequently birds of prey or fish-eating bird species. The classic and oft-cited example of bioaccumulation is that of the use of DDD (a DDT analog) in Clear Lake, California, for the control of a nonbiting gnat species. Three applications of DDD were made to the lake between 1949 and 1957 (6). Some Western grebes (Aechmorphorus occidentalis) died after the second and third application of the insecticide, and the entire colony of 1000 birds stopped breeding. Residues in grebe fat reached levels about 300-fold higher than in the average plankton sample and 80,000-fold higher than in water samples. Because birds are part of the human food chain, bioaccumulation of persistent lipophilic pesticides is of concern whether or not the birds themselves are affected. In 1970, for example, DDT levels in the breast muscle of woodcocks (Scolopax minor) ranged as high as 771 ppm with a mean of 60 ppm (expressed on a lipid weight basis) in areas of New Brunswick, where the insecticide had been used against the eastern spruce budworm (7). At the time, the highest food tolerance for DDT in Canada was 7 ppm (lipid weight). The hunting season was therefore closed. Lethal impacts are most frequent with the cyclodiene insecticides (see below). These are the most acutely toxic to birds of the persistent organochlorine compounds (Table 1). However, even the less acutely toxic organochlorine insecticides, especially those that are refractory to metabolism, can reach lethal levels in brain tissue, typically during periods of environmentally induced fasting or high energy demand, causing a mobilization of fat reserves. Body burdens of persistent organochlorine insecticides interact with a number of physiological processes. The most widespread response is the stimulation of liver microsomal enzyme activity. Although a normal detoxification mechanism, this constant induction of the liver results in enhanced hydroxylation and clearance of natural molecules such as steroids. This is one of the mechanisms through which persistent organochlorine pesticides exert their activity on the endocrine system of

Table 1. Acute Oral Toxicity of Organochlorine Insecticides to Birds∗ Insecticide

No. Species Tested

HD5 (mg/kg Body Weight)

LD50 (mg/kg Body Weight)

12 4 2 5 3 16 6 11 1

1.15 0.09 26.4 123 72.4 4.15 9.53 0.75 12.5

19.8 62.3 220 1330 681 35.1 52.4 1.78 N/A

7 11 4 11

3.47 10.5 292 10.4

125 90.8 N/A 70.7

Aldrin Chlordane Chlordecone DDT Dicofol Dieldrin Endosulfan Endrin HCH (technical mixture) Heptachlor Lindane Methoxychlor Toxaphene

N/A—Not available. ∗ The table gives the number of bird species tested as well as the median LD50 value and the HD5 . This is the value calculated (with 50% confidence) to be at the 5% lower tail of the distribution of LD50 values for birds between 20 g and 1 kg after Mineau and colleagues (40). A wide spread between median LD50 and HD5 indicates uncertainty, usually because of wide species-to-species differences in sensitivity.

birds. Other more specific modes of action have also been uncovered (see below). DDT and Analogs The short-term lethal impacts of forestry, orchard, and shade tree applications of DDT have been reviewed extensively (3,8). Acute poisoning of many songbird species, especially American robins (Turdus migratorius), yellowrumped warblers (Dendroica coronata), and tree swallows (Tachycineta bicolor) were observed after spraying of urban shade trees for the vector of Dutch elm disease. Heavy mortality was recorded at rates as low as 2 kg a.i./ha, although rates applied were often higher. American robins were hit particularly hard as a result of the contamination of earthworms in the soil surrounding the trees. This lethal contamination resulted both from the pesticide falling back to the ground during application as well as from the composting of contaminated leaves after leaf fall. In forestry, application rates of 3.3 kg/ha and above were seen to give rise to immediate mortality of either adult or nestling songbirds or both. Applications of 550–1100 g a.i./ha were deemed to be devoid of acute effects in birds. The 1100-g a.i./ha rate was the rate most commonly used in the course of the extensive spraying programs in eastern North America for control of the spruce budworm (Choristoneura fumiferana) (9). There is evidence that these applications, which lasted from 1945 to 1968, in fact did depress forest songbird numbers (10). Eggshell Thinning The best documented effect of DDT on birds, however, is undoubtedly the effect of its major breakdown product DDE on the avian eggshell. This effect was initially discovered in the field by the British researcher

AVIAN SPECIES

Ratcliffe (11) and confirmed through a series of laboratory feeding experiments conducted at the Patuxent Wildlife Research Center in Maryland (12,13). There are many written accounts and reviews of the way field observations and experimental captive studies were combined to unravel the mystery of declining raptor populations linked to a greater or lesser degree to the use of DDT. Complicating the overall story are a few cases of apparent ‘‘poor fit’’ among DDE residue levels, eggshell thinning, and population declines (14). Many of the early analyses of DDT from environmental samples were made difficult by varying levels of interference by polychlorinated biphenyls (PCBs) and related widespread industrial contaminants. It is widely accepted that DDT acts directly on calcium transport in the avian shell gland, although the details are still being debated. Not all species of birds are affected equally; chickens and other species of the order galliformes are among the least affected. This fact is important given the traditionally heavy reliance on these species for testing purposes. The most severe impacts from eggshell thinning were found in peregrine falcons (Falco peregrinus), brown pelicans (Pelecanus occidentalis), double-crested cormorant (Phalacrocorax auritus), Osprey (Pandion haliaetus), and bald eagle (Haliaetus leucocephalus), as reviewed by Blus (5). Reductions in eggshell thickness of about 18% or more generally result in reduced hatching success (15,16). Current Status of DDT and Analogs The use of DDT was banned in most of the world, although this was probably as much because of widespread contamination of the human population and putative health effects than because of DDT’s impact on bird populations (17). The continued use of DDT against vectorborne human diseases in some parts of the world remains controversial. It continues to affect birds (both raptorial species and smaller insectivores) locally where it is used (18), although these authors point out that these impacts maybe more rapidly reversible in tropical areas. There is continuing concern over local and migratory birds being exposed to continued use of DDT in areas such as South America, Africa, and the Indian subcontinent or feeding on prey items that have migrated to these areas. There is also evidence of continuing exposure of birds in areas of previous heavy use such as orchards (19). Dicofol is an acaricide structurally very similar to DDT. Like DDE, it gives rise to egg-shell thinning in feeding studies. However, its short persistence in the environment means that exposure levels are lower, even in orchards where it is used (20). It is still currently allowed in the United States, provided that DDT-related impurities are kept below 0.1%, a standard that has been emulated by other countries. Methoxychlor is another related organochlorine, although for less persistent, still registered in many countries. There are well-documented aquatic impacts from methoxychlor use, but no direct avian impacts have been recorded. Cyclodiene Insecticides The bird kills seen following the use of DDT were more than matched by the lethal impacts documented following

131

the use of aldrin, dieldrin, and heptachlor seed dressings in Europe (4). Hundreds of kills were recorded involving some 50 bird and 10 mammal species. As reviewed by Brown (3), heptachlor used for fire ant control in the United States was found to cause extensive bird mortality at rates as low as 275 g a.i./ha. Quail populations still had not recovered after 3 years from a 2.2-kg a.i./ha application. Use of heptachlor on wheat in the United States also gave rise to extensive mortality of geese and effects on several raptor species in the United States [see review by Fleming et al. (21)]. There is now an increasing consensus that declines of most raptor species in predominantly terrestrial food webs (e.g., the sparrowhawk Accipiter nisus in Britain) were principally caused by cyclodiene insecticides, especially dieldrin, used as a seed dressing as well as a soil treatment and sheep dip (22–24). It was estimated that at its peak, aldrin (which, in the environment, rapidly breaks down to dieldrin) was used on half of the total U.S. corn acreage requiring a soil insecticide (1). That the relative roles of DDT and cyclodiene insecticides are still debated today (especially with regard to peregrine falcon and bald eagle declines) indicates the extent to which residues of all of these insecticides were correlated in most areas. Exposure of birds to the turf and termite insecticide chlordane results in the accumulation of the same toxic metabolite as exposure to heptachlor, namely, heptachlor epoxide. The toxicity of heptachlor epoxide and that of oxychlordane, the other major chlordane metabolite are considered to be additive (5). From 1986 to 1990, or 15–20 years after cancellation of the use of chlordane in turf, it was estimated that about 17% of songbird and American kestrel (Falco sparverius) mortality reported from suburban New York State were the result of poisoning by cyclodiene insecticides, especially chlordane. The high exposure documented was thought to be through resistant insect populations, especially scarab pests of turf (25). Even more recently (26), the mass mortality of songbirds as well as that of predatory Cooper’s hawk (Accipiter cooperi) in New Jersey suggested that mortality of birds from old chlordane residues may be more frequent and widespread than currently believed. Chlordane continued to be used for termite control until very recently, raising the specter of continued, if more localized, impacts on birds. Endrin is probably the cyclodiene insecticide most readily metabolized and excreted by birds (27). Nevertheless, its high toxicity resulted in massive die-offs of Brown Pelicans when used as a seed treatment on rice (see review by Blus (5)). Its use as a rodenticide in orchards also killed large numbers of birds of prey (28) although this use pattern had been declared environmentally safe by agriculture extension specialists (3). Endrin-poisoned grain was used to control bird pests in Israel, which resulted in secondary kills of birds of prey migrating through this critical area (29). For a period of time in the United States, an endrin-containing toxic perch (Rid-a-Bird) was marketed for the control of nuisance birds such as starlings (Sturnus vulgaris) and pigeons (Columba livia). Subsequently, endrin was replaced by the organophosphorus insecticide fenthion,

132

AVIAN SPECIES

another poor choice from the point of view of secondary poisoning (see below). In the early 1980s, there were threats of a closure of the western waterfowl hunt in the United States and Canada following the use of endrin against cutworm in wheat. Despite its relatively high clearance (at least compared with other organochlorines), both waterfowl and upland game birds had residue concentrations that exceeded allowable tolerances for poultry products intended for human consumption [see review by Fleming et al. (21)].

quail following a 3-month dosing period with 20-ppm HCB. Product registrations in Canada at the time allowed up to 1000 ppm on various cereal seeds. In the early 1970s, levels in the range of 3–4 ppm (fresh weight basis) were seen in eggs of fish-eating birds of the Great Lakes and likely contributed to the high levels of embryonic mortality seen (34). However, because HCB is also an intermediate in the manufacture of several chemicals, industrial pollution rather than use of the chemical on farm fields could have been the source of the contamination.

Hexachlorocyclohexane Isomers Lindane (the gamma isomer of HCH) replaced aldrin and heptachlor as insecticidal seed treatment (at least in North America) after the latter proved to have unacceptable impacts on birds. Immediate improvements (cessation of mortality, enhanced reproduction) were seen in a Canada goose (Branta canadensis) population previously contaminated by heptachlor (30). Initially, heavily contaminated by the more environmentally persistent alpha and beta isomers but now purified to 99%, gamma HCH or lindane continues to be an important seed-treatment chemical in many countries, including the United States and Canada. However, concerns over widespread contamination of the arctic environment, especially by lindane and other HCH isomers, have triggered a reassessment and calls for its phase out. A crude HCH mixture (technical HCH) with a high proportion of the alpha and beta isomers is still used heavily in some parts of the world, notably, the Indian subcontinent. There are few reports of bird intoxications with lindane, but comprehensive studies of its potential reproductive effects in the wild are lacking. Such effects have been reported from poultry studies (31). Toxaphene At its peak in 1980, toxaphene, a complex mixture of chlorinated camphenes, was the most heavily used insecticide in the United States, with its main use in cotton. The chemical was also used as a piscicide as well as for grasshopper control. Mortality of fisheating birds was recorded following its agricultural use in an area surrounding a wildlife refuge, although the presence of DDT could have been a confounding factor (5). The agricultural use of toxaphene led to widespread contamination of the environment, although the difficulty of analyzing for it in environmental matrices meant that it was long ignored as a global contaminant. Toxaphene is not routinely reported from bird samples. HCB Hexachlorobenzene (or perchlorobenzene) is still listed as a current-use fungicide (32). It was registered as a seed dressing for cereals in several countries, but its use in Northern Europe and North America seems to have declined along with the persistent organochlorines. There are no reports of avian casualties, although raptors found dead in The Netherlands had substantial levels of HCB in their livers along with cyclodiene and DDE residues (33). The same authors reported porphyria in

Mirex Mirex replaced dieldrin and heptachlor in the southern United States for the control of imported fire ants. Although no acute effects on birds have been reported, mirex was found to bioaccumulate extensively in biota (35). Nowhere was this more apparent than in Lake Ontario, where the entire food web and several species of fish-eating birds became contaminated with mirex and photomirex (a photodegradate) following contamination by a manufacturing plant (36). Organophosphorus and Carbamate Insecticides Byproducts of the German WWII effort, organophosphorus (OP) insecticides gradually came to replace the persistent organochlorine insecticides. Carbamate (CB) insecticides were soon to follow. To this date, they have remained a mainstay of insect, mite, and nematode control chemicals with minor uses as rodenticides and avicides (see below). Most are relatively short-lived in the environment (although there are notable examples to the contrary), and they are readily metabolized by birds. Therefore, they do not tend to bioaccumulate to any great extent in avian species. Two aspects make these pesticides problematical to birds: 1) their extreme toxicity and 2) the ubiquity and importance of their target sites (cholinergic synapses in the central nervous system, autonomous nervous system, and neuromuscular junctions), which guarantees a plethora of behavioral and physiological effects in exposed individuals. Some of the first insecticides to be introduced such as TEPP (tetraethyl pyrophosphate), parathion (ethyl parathion), and monocrotophos were renowned to cause massive dieoffs of wild birds and mammals in treated crops as well as in neighboring areas affected by pesticide drift. Kill reports were coming in from areas as widely separated as the United Kingdom, the United States, Japan, and South Africa soon after their introduction on the market [see Brown (3) for review, also Mellanby (37)]. As a testament to the fact that birds are often considered expendable in agricultural systems, many of those early products are still widely registered today, although some countries have moved to ban or severely restrict their use. Sensitivity of Birds to ChE Inhibitors As a rule of thumb, birds tend to be more sensitive than mammals to cholinesterase-inhibiting insecticides, sometimes by a wide margin. This is thought to be because birds have generally lower levels of hepatic metabolizing

AVIAN SPECIES

enzymes (38). There are numerous sources of data on the toxicity of OPs and carbamates to birds [see Smith (39) for a useful compilation], but the extreme variation among species (more than 200-fold interspecies differences in susceptibility for well-tested products) as well as the uneven extent of testing among different pesticides makes any comparison difficult a priori. Recent approaches to avian interspecific variation in susceptibility have been the fitting of acute toxicity data to a frequency distribution as well as incorporation of body mass as a scaling factor (40). This approach allows for an unbiased estimate of avian toxicity and a fair comparison among various pesticides. The basis of comparison was set arbitrarily as the mean estimate of the LD50 oral toxicity value to a bird species situated at the 5% tail of the sensitivity distribution for all bird species (referred to as the HD5 or Hazardous Dose5 ). Expressed differently, there is a 50% probability that 95% of bird species will have an LD50 value higher than the stated value. The LD50 or the dose given by gavage, which results in the death of half of the tested individuals (usually within a 14-day period of observation) is used in preference to other measures of lethality for reasons given below (see preregistration assessment below). This approach was used in Table 1 to present the toxicity of persistent organochlorine pesticides to birds. Table 2 lists the avian toxicity of organophosphorus and carbamate pesticides mentioned here. It is immediately apparent that, as a group, these cholinesterase-inhibiting pesticides are far more toxic acutely than are the organochlorine insecticides they replaced. For those unaccustomed to working with LD50 values, the following is provided for illustrative purposes. The toxicity of the carbamate insecticide carbofuran to mallard ducks (Anas platyrhynchos) has been well characterized. The LD50 of an adult mallard is about 0.4 mg/kg body weight. At the rate of application to a U.S. alfalfa crop (1.1 kg a.i./ha), the median lethal dose for an adult mallard of a kilogram body weight would be deposited within an area of approximately 6 cm × 6 cm of crop. Given the ability of ducks to graze such an area in a very short time period, it is not surprising that kills of waterfowl in treated alfalfa crops have been large and frequent (41). Reported Bird Kills Kills of birds from several of the more toxic cholinesteraseinhibiting insecticides are frequent. Diagnosis of those kills is frequently through the measurement of cholinesterase levels in brain tissue (see post-registration monitoring below). In Great Britain which, arguably, has had the longest-running and most comprehensive system in the world for the reporting of wildlife poisoning incidents from pesticides, between one-quarter and one-half of all diagnosed cases from 1975 to the early 1990s resulted from cholinesterase inhibitors (based on annual reports from the Ministry of Agriculture, Fisheries and Food). In The Netherlands, between 1975 and 1988, 82% of poisoned birds were poisoned by cholinesterase inhibitors. This represents 17% of all mortality reported from all causes (42). In North America, it is estimated that since 1965, about 3% of bald eagles examined by the U.S. Fish and Wildlife Service were poisoned by cholinesterase

133

Table 2. Acute Oral Toxicity of Selected Organophosphorous and Carbamate Insecticides to Birds∗ Insecticide Acephate Aldicarb Bendiocarb Carbaryl Carbofuran Carbophenothion Chlorfenvinphos Diazinon Dimethoate Disulfoton Famphur Fenitrothion Fensulfothion Fenthion Fonofos Furathiocarb(a) Methamidophos Methiocarb Methyl parathion Mevinphos Monocrotophos Parathion (ethyl) Phorate Phosphamidon Temephos Terbufos Triazophos

No. Birds Tested

HD5 (mg/kg Body Weight)

LD50 (mg/kg Body Weight)

7 10 4 7 18 9 15 14 10 7 3 12 14 23 10 1 3 33 10 13 23 19 8 15 14 5 5

18.52 0.43 0.72 30.05 0.21 2.00 2.73 0.59 5.78 0.81 0.45 3.37 0.13 0.87 3.86 2.41 1.70 1.06 2.13 0.70 0.42 0.40 0.34 1.08 8.68 0.16 1.68

146.00 2.82 16.24 1870.50 1.65 56.8 23.70 5.25 29.50 11.90 2.70 63.43 0.73 5.62 23.50 — 15.82 7.50 10.80 3.80 2.51 5.62 7.06 4.24 65.60 9.48 9.47

∗

Because the toxicity test was carried out with ‘‘stabilized’’ insecticide, preventing the birds from metabolizing the insecticide to carbofuran, the value for carbofuran may be closer to what would be expected in the field.

inhibitors (43). This is a known underestimate, because the measurement of cholinesterase levels as a diagnostic tool did not begin until the early 1980s in that laboratory. The 12% calculated for red-tailed hawks between 1975 and 1992 (44) is probably more typical of the current situation for either species. It should be noted that, in most diagnostic centers involved in assessing wildlife incidents, the investigation seldom goes further than establishing the primary cause of death. The possible contribution of sublethal pesticide intoxication to the ultimate cause of death (often trauma from collision with structures or vehicles) is rarely investigated, although the link between the two has been demonstrated (45). On a regional level, the importance of poisonings caused by cholinesterase inhibitors can be substantial. For the bald eagle population in the lower Fraser estuary near Vancouver, up to 50% of all bald eagles received at a rehabilitation center between 1990 and 1995 (N = 84) had been exposed to cholinesterase inhibitors just prior to admission (45). Likewise, the use of organophosphorus insecticides applied in dormant oils in California was found to be the main cause of incapacitation of buteo hawks throughout the 1980s and early 1990s (45). Cancellation of parathion for this use pattern resulted in a marked decline in intoxication cases, although exposure to replacement chemicals among resident and migrating red-tailed hawks

134

AVIAN SPECIES

(Buteo jamaicensis) is still very prevalent (46). As first observed by Grue et al. (47), one can predict rather well which cholinesterase-inhibiting insecticides will cause avian mortality by looking at their acute toxicity and extent of use. Although this seems to go against the most basic of toxicology’s principle—that the dose makes the poison—it is an acknowledgment of the fact that many of the cholinesterase-inhibiting insecticides registered today are so acutely toxic to birds that it is difficult to use them without incurring bird mortality. The high proportion of poisonings occurring after labeled uses of cholinesteraseinhibiting insecticides (as opposed to gross abuses of the label) in the United States and Canada is a direct reflection of the continued use in those two countries of OPs and carbamates of extreme toxicity to birds. Treatment of Poisoned Birds Treatment of poisoned wildlife generally follows a fourpronged approach. Atropine is considered to be the most effective antidote for both OP and CB intoxication. By effectively competing with acetylcholine for the same cellular receptors, it prevents overstimulation of the autonomous parasympathetic system. Most importantly, it helps prevent asphixia, the main cause of death. In human subjects, it is customary to constantly infuse atropine in order to maintain optimal concentration throughout recovery from the ‘‘cholinergic crisis.’’ In wildlife rehabilitation, this is impractical and subjects need to be repeatedly injected with atropine. The second prong consists of the administration of chemicals that hasten the release of acetylcholinesterase by the pesticide. This strategy is effective only where intoxication results from an OP pesticide and is recent. [Porter (48) gives 24 hours as a guideline, but this will vary from pesticide to pesticide]. The most frequently used chemical for this purpose is 2-pralidoxime chloride (2-PAM). The third prong in the approach is the provision of supportive symptomatic care, especially positive ventilation in case of respiratory arrest. Finally, it is important to eliminate the source of the exposure. Gastric lavage may be performed where there is evidence of a large food bolus. Excision of the crop was found to be more effective and less stressful than forced regurgitation in the case of bald eagles having scavenged contaminated waterfowl (49). Pesticides absorbed into the subepidermal tissues of birds or mammals can be slowly released over time and result in prolonged re-exposure (50). Where dermal exposure is suspected, a vigorous rinsing of the feet may help limit pesticide entry. The rinsings can be kept for chemical analysis where feasible and warranted. The exact dosages of atropine and 2-PAM to be administered are currently a matter of debate. The ‘‘traditional’’ approach (48), which is based on levels found to be effective in humans is the injection of 0.5-mg/kg atropine IM (or with one-quarter of the total dose given IV). This is repeated after 15 min if no decline in signs is observed. According to the same source, the recommended dosage of 2-PAM is 20 mg/kg IM. A far more ‘‘aggressive’’ approach was recently recommended by Shlosberg and colleagues (51). Their experiments in chickens led them to

recommend 25-mg/kg atropine and 50-mg/kg 2-PAM as the best treatment for an unknown cholinesterase inhibitor. These dosages were established empirically; the highest doses not causing obvious toxicity in normal chickens were retained. These authors point out that each species is different, in particular, the ease with which it breaks down atropine. Ideally, maximum tolerated dosages of both atropine or, better still, an atropine + 2-PAM cocktail should be established for those species commonly treated at rehabilitation centers. Other possibilities for treatment exist, but they have not been systematically investigated in wildlife. Injections of glucose and of vitamin C have afforded some protection to small mammals experimentally dosed with various OPs [see Gallo and Lawryk (52) for review]. The ready availability of both make them obvious candidates for further experimentation in poisoned wildlife. New Chemistry Insecticides The synthetic pyrethroids constituted the third generation of insecticides (assuming organochlorines and cholinesterase-inhibiting insecticides represented the first and second respectively). Although exceedingly toxic to beneficial arthropods and in aquatic systems, pyrethroids are generally of low acute toxicity, either in birds or in mammals. Primary effects on birds are therefore less likely and have only been seen rarely. There have been reports of problems associated with the embryotoxicity of xylenecontaining formulations of synthetic pyrethroids (53), but the possible toxicity of any ‘‘inert’’ formulation component or spray diluant is an issue with any pesticide. A number of recent introductions are worth mentioning. Possibly the most interesting from the point of view of birds is chlorfenapyr, the first of a new class of pyrrole insecticides. This insecticide is the first to be denied registration in the United States largely for bird toxicity issues. It has very high acute toxicity (on par with some of the more toxic OPs and CBs), although mortality is typically delayed for a few days after exposure. It also causes important effects on reproduction at extremely low levels of exposure (54). Its long persistence and high aquatic toxicity were undoubtedly factors that contributed to its regulatory refusal. It is of concern to wildlife authorities that chlorfenapyr is registered in a number of countries, including much of the wintering range of North American bird species. Kills of pigeons and game birds have also been reported in France with two other new insecticides used as seed dressings: imidacloprid and fipronil (annual reports from l’Office National de la Chasse). It is not clear how extensive or important these problems will be. Both of these insecticides have an acute toxicity to birds, which is lower than many of the cholinesterase-inhibiting pesticides they are meant to replace. Rodenticides—Anticoagulants and Other Products Massive kills of birds of prey were recorded in Israel when the organophosphate monocrotophos was used in alfalfa fields to kill voles (55,56). This use pattern was not legally sanctioned but well entrenched in the area’s

AVIAN SPECIES

grower community. Recent attempts to replace the use of rodenticides through manipulation of the water levels in the fields as well as the use of box-nesting barn owls have met with considerable success. The organochlorine insecticide endrin was used extensively in orchards, where it was found to cause a great deal of secondary poisoning of raptors (28). Thallium sulfate was likewise responsible for massive declines in some raptor numbers when used as a rodenticide, and impacts on birds have been recorded also with fluoroacetamide, glucochloralose, and alphachloralose rodent baits (29). Most rodenticides in current use, however, are anticoagulants. Evidence for an impact on birds from firstgeneration products (coumarins such as warfarin and indandiones such as chlorophacinone and diphacinone) is equivocal. Of course, poisoning of birds is likely where baits are widely available and attractive to birds (e.g., grain-based baits). Secondary poisoning is certainly possible but thought to require feeding on contaminated carcasses for several continuous days. Impacts on birds of prey have been seen in the course of massive poisoning campaigns such as the one for vole control in France (57). On the other hand, there is increasing evidence of extensive contamination and mortality of birds of prey from secondary poisoning following the use of ‘‘secondgeneration’’ single feed coumarin products such as difenacoum, brodifacoum, difethialone, bromadiolone, and flocoumafen. An increasing number of raptors are being diagnosed with anticoagulant poisoning in New York State (58) and California (59). Also worrisome is the fact that residues are frequent in birds struck by cars or succumbing from other causes (60). Residues of secondgeneration coumarin compounds are extremely long-lived in livers of exposed wildlife, leading to a high frequency of detection. One important question is whether the presence of bound residues in the liver makes the birds more susceptible to frank anticoagulation following re-exposure. Also, very little is known of potential sublethal effects from such contamination. Treatment of any bird found poisoned by anticoagulant rodenticides is possible with vitamin K as in mammals. Avicides Many of the more toxic organophosphorus or carbamate insecticides or other chemicals such as alphachloralose or strychnine are commonly used criminally to kill birds. Birds are also killed accidentally when chemical baits are used to kill large predators such as wolves (Canis lupus) or coyotes (Canis latrans). However, several legally registered avicides take a heavy toll on nontarget bird species, primarily raptors. Notable are the uses of fenthion as a spray to control red-billed quelea in Sahel Africa and the use of the same organophosphate insecticide in toxic perches (Rid-a-Bird perches) (45,61). The latter have now been banned in the United States and Canada. Fenthion continues to be used for quelea control failing the availability of a cost-effective alternative.

135

Sweden in the mid-1950s (62). The kills as well as declines of raptorial species were blamed on alkyl mercury compounds such as methyl mercury dicyandiamide (DDM) and, to a lesser extent, ethyl mercury halide. Following the Swedish results, surveys were conducted in the Canadian Prairies (beginning in 1968) showing widespread contamination of seed-eating birds, rodents, and their avian predators such as prairie falcons (Falco mexicanus) by DDM and other alkyl mercury compounds (63). Residue levels in eggs of the latter were judged to be high enough to cause hatching failures, but the impact of alkyl mercury fungicides was difficult to tease out from the impact of cyclodiene insecticidal seed treatments used concomitantly. Game birds such as ring-necked pheasant (Phasianus colchicus) and Hungarian partridge (Perdrix perdrix) were found to be contaminated, thereby forcing a closure of the hunting season. Mercury residue levels in the birds exceeded by at least two-fold the tolerance levels for human consumption proposed by FAO (Food and Agriculture Organization) and WHO (World Health Organization) at the time. Alkoxyalkyl mercury dressings were used in France, and, there also, wildlife mortality was reported (62). In the United Kingdom, phenyl mercury dressings continued to be used long after methyl or ethyl mercury dressings were banned in Northern Europe and North America. This use apparently did not result in acute effects in birds, but the extent to which they contributed to a general contamination of the agroenvironment by mercury is not known. Dithiocarbamate fungicides are not acutely toxic to birds but have been shown to cause reproductive effects in the laboratory (see below). Herbicides Few herbicides have a sufficiently high acute toxicity to affect birds directly in any meaningful number. One possible exception may be sodium monochloroacetate, which has been responsible for a few kills in the United Kingdom, including an estimated loss of 300 greenfinches (Carduelis choris), linnets (Acanthis cannabina), goldfinches (Carduelis carduelis), and house sparrows (Passer domesticus) (64). Birds were thought to have been exposed through puddles (see below). More importantly, herbicides are believed to have contributed to decreases in several species of farmland birds in the United Kingdom and other European countries (see indirect effects below). A dramatic loss of avifauna was apparently one of the consequences of the massive defoliation program conducted during the Vietnam War [Westing 71 in Brown (3)]. Similar fears have been expressed regarding the U.S.backed cocaine eradication program in Colombia. As with any other pesticides, herbicides may also cause more subtle and difficult-to-predict reproductive effects in birds (65). HOW PESTICIDES CAN AFFECT BIRDS

Fungicides

Lethality

Extensive mortality of seed-eating birds from mercurycontaining fungicidal seed dressings first came to light in

Because birds can rapidly move into pesticide-treated areas, they run the risk of being exposed to pesticides

136

AVIAN SPECIES

simply by being in the wrong place at the wrong time. Not surprisingly, bird mortality in and around fields and forests treated with agrochemicals is not unusual. Of course, with persistent organochlorine pesticides, poisoning often occurred during periods of fat remobilization, possibly well away from sources of exposure. Mortality is also delayed in the case of other pesticides such as anticoagulant rodenticides and some of the newer pesticides such as chlorfenapyr. Persistent Organochlorine Pesticides Kills of American robins and other species following DDT use were described above. A ‘‘syndrome’’ of typical DDT acute intoxication is described by Mrak (1): ‘‘Birds can fly poorly or flutter along the ground, then become totally disabled, undergo convulsions and die in a very stiff position with legs extended.’’ In fact, this ‘‘syndrome’’ is fairly nonspecific and could also apply to other persistent organochlorine insecticides as well as to many other acutely toxic pesticides. Diagnosis of mortality from persistent organochlorine pesticides typically relies on the measurement of residues in tissues, usually liver, fat, eggs or egg follicles, or brain. The most accurate diagnosis of pesticide-induced lethality is brain concentration. Lethal brain residues have been estimated for several pesticides by Dr. Lucille Stickel and colleagues at the Patuxent Wildlife Research Center through a combination of kill investigation and experimental dosing (Table 3). Organophosphorus and Carbamate Pesticides Typically, but not always, birds poisoned with ChE inhibitors die of anoxia resulting from respiratory failure. This results from one or a combination of factors, notably, excessive secretion in the respiratory tract, bronchoconstriction, failure of the muscles required for respiration, and failure of the respiration center [see Gallo and Lawryk (52) for a review]. Although there are well-described clinical signs that are typical of poisoning by a ChE inhibitor, clinical signs can be so variable as to obscure correct diagnosis. In the somatic nervous system, which controls voluntary muscle movement, overstimulation resulting from pooled acetylcholine at synapses typically gives rise to tremors, muscle twitches, piloerection, occasionally convulsions, and,

more commonly, paralysis, resulting in ataxia. Cholinergic tracts are also important to both the parasympathetic and sympathetic autonomous nervous systems but especially to the former, where they conduct impulses from the neural ganglia to a multitude of effector organs, such as the heart, various glands, the viscera, and so on. Because the autonomous nervous system is subject to constant adjustment through feedback mechanisms, intoxication with a cholinesterase inhibitor sends the intoxicated bird into a veritable ‘‘roller-coaster’’ ride. For example, individuals may show alternating constriction or dilation of the pupils, speeding up or slowing down of the heartbeat, and so on. Also, because the somatic and autonomous systems react to different levels of cholinergic stimulation, some doses of an anticholinesterase may produce apparently opposite signs, e.g., contraction of the striated muscles involved in locomotion and simultaneous relaxation of the smooth musculature, leading to a flaccid gut and food impaction. The rate at which the individual was exposed to the pesticide is as important as the dose. Typically, gradual exposure allows the individual to compensate and tolerate a higher dose than if the exposure was acute. Finally, different cholinesterase inhibitors have different properties that may dictate which clinical signs are expressed. Some pesticides are directly active on synapses, others need to be metabolized to the active molecule; some pass readily into the brain (with obvious effects on the central nervous system), and others have difficulty crossing the blood-brain barrier and therefore show more ‘‘peripheral’’ effects. Porter (48) cautions that many of the ‘‘classic signs’’ of parasympathetic stimulation reported from standard toxicology texts may not be seen in poisoned raptors—certainly not with any consistency. Where an exposure is not lethal, speed of recovery of intoxicated birds varies tremendously. All else being equal, recovery from a carbamate intoxication is generally more rapid than from an organophosphorous pesticide. A bird sublethally exposed to a carbamate pesticide such as the avian ‘‘repellent’’ methiocarb may recover from paralysis in less than an hour. On the other hand, there are examples of raptors that have required as much as 10 months of supportive care and rehabilitation following an acute exposure to a cholinesterase inhibitor. In the absence of re-exposure, the recovery of brain cholinesterase levels following acute exposure to an

Table 3. Lethal Brain Residues for Organochlorine Insecticides Based on the Work of L. Stickel and Colleagues at the Patuxent Wildlife Research Center and Summarized by Blus (5) Insecticide

Brain Levels Diagnostic of Lethality (Fresh Weight Basis)

DDT and metabolites

>20 DDT equivalents where: DDT equivalents = ppm DDE/15 + ppm DDD/5 + ppm DDT

Chlordane

Heptachlor epoxide + oxychlordane > 4 ppm

Or. . .

chlordane > 5 ppm

Heptachlor

Heptachlor epoxide > 9 ppm

Endrin

Endrin > 0.8 ppm

Dieldrin

Dieldrin > 3 ppm

AVIAN SPECIES

organophosphorous pesticide is typically in the order of several weeks to over a month. Because of the far-reaching importance of cholinergic tracts, poisoned individuals may be seriously compromised physiologically even if they initially survive the pesticide exposure. Some of the factors that may contribute to delayed mortality are as follows: • Trauma or other mishaps in the course of intoxication. It has been shown that birds sublethally exposed to cholinesterase inhibitors are more susceptible to predation (66,67). There is also reason to believe that sublethal exposure to cholinesterase inhibitors makes birds more vulnerable to collision with moving (e.g., vehicles) or stationary (e.g., powerlines, fences, buildings, and so on) objects. The evidence for a frequent pesticide involvement in ‘‘trauma cases’’ is twofold: 1) anecdotal evidence from rehabilitation centers where cholinesterase measurements are made on a routine basis [e.g., Porter (48)], and 2) the wealth of human evidence about the various visual and motor effects that affect the safety of workers following exposure to OPs and CBs [see Gallo and Lawryk (52) for review]. Blurred vision is a common complaint; unequal miosis also can lead to a phenomenon called the Pulfrich stereo effect, where depth perception and the ability to compute trajectories are affected. Any of these effects in a flying bird would be expected to lead to higher rates of ‘‘mishap.’’ • An adverse energetic status resulting from anorexia, a reduced ability to thermoregulate, and disruption of the normal circadian patterns. The laboratory evidence for such effects has been reviewed extensively by Grue et al. (68,69). The anorexic response is particularly severe and complex. There is evidence that two separate mechanisms are at play: a physical inability to feed resulting from gastro-intestinal distress as well as a conditioned aversion response where birds are able to continue feeding but shun the food source they believed made them sick. There are some indications of adverse field outcomes as a result of these physiological effects. An inability to feed and severe weight loss were shown in captive songbirds exposed dermally to fenitrothion (70). It has often been observed that the species with the smallest body size (e.g., kinglets, family Regulidae) are the most severely affected in forest spraying operations (9). In the course of red-billed quelea control with the OP fenthion, it is believed that most birds die of starvation and exposure to adverse weather rather than of direct symptoms associated with exposure (71). Exposure of American robins to turf sprayed with diazinon became lethal when night-time temperatures dropped to near freezing even though control birds were not affected (72). A dose that was sublethal in the laboratory rapidly proved lethal in ducklings placed in outdoor ponds (73). Several particularly large mortality incidents involved migrating birds. Of course, the large flock of dying birds increased the probability that the incident would be seen, but the weakened state of the birds and their reduced fat

137

stores may have predisposed them to a lethal intoxication. Having birds form a conditioned aversion to a food source that makes them ill is generally considered favorable by most pesticide specialists because it is seen as a way by which birds can avoid a lethal intake of pesticide-treated foodstuffs or will avoid feeding the contaminated food items to their progeny [e.g., McKay et al. (74)]. Paradoxically, it has been suggested by Nicolaus and Lee (75) that the establishment of conditioned aversion as a result of sublethal pesticide intoxication is a serious impact in itself because it impairs feeding efficiency, which may disrupt reproduction. This would be especially serious where spurious conditioned aversions to critical food sources are established concomitant to a pesticide application. The ability of birds to take insect pests (2), or weed seeds and, hence, their economic value, would also be reduced. • Muscular necrosis as a result of transient anoxia. In humans, this is part of what has been termed an ‘‘intermediate syndrome,’’ which typically presents itself as cardiac or other muscular failure several days after return to normal cholinesterase titers. Neural effects leading to long-term behavioral changes have been reported in humans also. This has not been reported in birds, but it is unlikely it would ever be detected. • Delayed neurotoxicity. This syndrome is the irreversible dying back of neurons as a result of the inhibition of another enzyme, neuropathy target esterase (NTE) by a select group of organophosphorous insecticides. It is noteworthy that the chicken is the usual test organism for this syndrome, but effects are often seen at dosing levels that would be lethal were the animal not antidoted for cholinesterase inhibition effects. This syndrome has not been reported in wild birds, although we might expect to see it first in animals subject to intensive rehabilitation efforts. Mercury Fungicides Borg et al. (62) measured residue levels and examined a number of seed-eating birds, including pheasants, partridges, pigeons, corvids, and finches, as well as their predators and scavengers found dead after the use of mercury seed dressings. Pheasants and partridge were generally found alone, and the other species were frequently in small flocks of tens. Birds were frequently reported to be in poor flesh, indicating a more prolonged death. Enteritis and erosion of the gastric mucosa as well as congestion and fatty degeneration of parenchymatous organs were reported frequently as was myelin degeneration of the peripheral nerves. A few hawks and owls showed traumatic injuries as well as mercury contamination, a situation that may be analogous to the increased vulnerability of birds to mishap following exposure to cholinesteraseinhibiting pesticides. Clinical signs reported from the field included loss of balance, a more or less pronounced ability to use legs and wings, and occasionally tremors and spasms.

138

AVIAN SPECIES

Anticoagulants Birds dying from anticoagulant pesticides display varying degrees of internal (intravisceral, joints, subdermal, etc.) or external bleeding. Because bleeding in the joints is considered to be particularly painful, euthanasia of affected birds should be considered. Birds that are exposed to anticoagulants over a certain threshold may develop bleeding for many reasons related to the day-to-day stresses they are subjected to. Stone et al. (58) describes a great horned owl and several red-tailed hawks bleeding out as a result of minor scratches or foot wounds inflicted by their struggling prey. The dose-response curve for second-generation anticoagulants is extremely steep beyond a threshold of exposure. In practice, this means one bird may be asymptomatic while another with an identical residue load may be fatally hemorrhaging. As described by Mineau (76), many environmental factors of a stochastic nature may contribute to the eventual lethality of a pesticide exposure, which is initially sublethal. The ability to maintain core temperature may not be a critical factor unless the weather is inclement; an increased vulnerability to predation will become critical only in the presence of a predator; and a rodenticideexposed raptor may survive for a long time until it is scratched or bumps itself. Effects on the Endocrine System The ability of persistent organochlorine insecticides to increase the rate of steroid metabolism through induction of the liver microsomal enzyme system was recognized a long time ago [e.g., Mrak (1)]. However, it is only recently (77) that a discussion of this and other modes of action of pesticides on the endocrine system has spilled into the public arena. The political response, in turn, has led to a general re-evaluation of pesticides and of the methods used to assess their safety to birds and other wildlife [see Feyk and Giesy (78) for a review]. Most of the discussion surrounding endocrine effects in birds has been limited to persistent organochlorine contaminants. A limited amount of work, however, has shown that exposure to cholinesterase-inhibiting pesticides can have an effect on reproductive and other hormones. Plasma corticosterone and glucose levels were elevated by parathion or methyl parathion given orally or injected in captive birds (79,80). Parathion in the diet resulted in reduced levels of luteinizing hormone (LH) relative to pair-fed controls (79). Unfortunately, the importance of these effects in a wild situation has not been established. Grue et al. (69) in their review of the literature on cholinesterase-inhibiting pesticides argued that the anorexia that follows exposure to these classes of pesticides may result, in part, from a toxic-mediated endocrine imbalance. The bis-dithiocarbamate fungicide maneb, administered for a 5-week period at the rate at which it is available on treated seed causes testicular atrophy and thyroid hyperplasia [reviewed by Burgat-Sacaze et al. (81)]. Because these effects disappear under an intermittent dosing regime, it is unlikely such dramatic effects would be seen following normal field exposure, even in the case of a seed treatment.

Impacts on the Immune System Immunotoxic properties of pesticides in birds were reviewed by Fairbrother (82). Few studies have been carried out. However, the limited avian data as well as mammalian toxicology suggest that several classes of pesticides can modulate the immune response of birds. This is of potential concern given the importance of disease outbreaks in birds (e.g., fowl cholera, West Nile virus, etc.). Recently, Bishop et al. (83,84) showed immune system stimulation and delayed thymic involution in tree swallow chicks nesting in orchards exposed to mixtures of insecticidal and fungicidal sprays. Impacts on Reproduction Mechanistically, the effects of persistent organochlorine contaminants on avian reproduction are complex and most can be related to endocrine effects. Other than the specific effects of DDE on calcium metabolism leading to eggshell thinning, several studies have also documented delays in laying readiness, a decrease in the number of eggs laid, higher embryonic mortality, and higher perinatal mortality [reviewed by Burgat-Sacaze (81)]. Alkyl mercury fungicides are known to be embryotoxic. At lower doses, they can cause developmental anomalies of the neural system and behavioral deficits (85,86). Different dithiocarbamate fungicide products in laboratory feeding studies have led to cessation of egg laying, early embryonic death, and teratogenesis [reviewed by Burgat-Sacaze (81)]. Other cases of reproductive failure are less well understood. For example, woodcock breeding success was found to be affected by DDT spraying for the eastern spruce budworm in New Brunswick forests (Canada), although eggshell thinning was not the cause of the problem (87,88). Busby et al. (89) documented lowered reproduction in white-throated sparrows (Zonotrichia albicollis) exposed to an overspray of the organophosphorous budworm insecticide fenitrothion. It maybe a moot point to explain exactly why nesting failures were recorded: At that application rate, there was the possibility of lethal effects, serious sublethal effects causing nest abandonment, lack of feeding opportunities, and so on. Eastern bluebirds (Sialia sialis) hatching is still influenced by old DDE residues found in orchard soils (19,90). In orchard-nesting bluebirds, but especially tree swallows, egg survival and other reproductive parameters were inversely correlated with the acute oral toxicity of the pesticide mixtures that were applied (90). Similar results were obtained in American robins and mourning doves (Zenaida macroura) in apple orchards and northern mockingbird (Mimus polyglottus), northern cardinal (Cardinalis cardinalis), and brown thrasher (Toxostoma rufum) in pecan orchards (91,92). In all of these cases, organophosphorous and carbamate insecticides were clearly the most acutely toxic of the products used, although the mechanism for the reduced reproductive performance of the birds was not established. Also, all of the studies showed year-to-year differences in the magnitude of the reproductive effects, suggesting the importance of stochastic variables such as weather. A high proportion of pesticides currently registered have the potential to affect the reproductive process at

AVIAN SPECIES

levels that are not parentally toxic (65). This conclusion is based on the review of standard reproduction studies in the mallard and northern bobwhite (Colinus virginianus) submitted for pesticide registration purposes in many countries. The current study design calls for a long prelaying exposure period of the pesticide in food (10 weeks) followed by another 10 weeks of egg-laying while also being dosed. The extent to which these impacts would also be seen in the course of the much shorter exposure periods more typical of current pesticides is a burning question. Several pesticides have the potential of causing embryonic mortality when eggs are immersed briefly in solutions formulated to represent spray solutions (93). Several herbicides, including paraquat, trifluralin, propanil, diclofopmethyl, as well as a bromoxynil-MCPA mixture were found to be particularly embryotoxic. In some cases, pesticide formulation components have been to blame rather than the active ingredient. In the wild, eggs in open nests could conceivably be contaminated directly by pesticide spray or residues could be carried onto egg surfaces by incubating birds. Effects on Behavior There have been several reviews of the behavioral toxicology of pesticides, especially organometallic products (such as mercury or lead-based products) and the cholinesterase inhibitors (organophosphorous and carbamate compounds). Some of the ongoing debates have been whether behavioral impairments are sensitive effects expected at low levels of exposure (94) and whether these subtle (or not so subtle) effects contribute to reduced survival and should be factored more than they currently are into the assessment of pesticide safety to birds (95,96). The multifaceted impact of cholinesterase-inhibiting compounds on the central and peripheral nervous systems as well as on all neuromuscular junctions gives rise to a plethora of deficits (68,69,94,97) but greatly complicates the exact definition of what constitutes a ‘‘behavioral’’ effect. Many documented behavioral effects are in fact impairments of an individual’s ability to perform. For example, when subtle postural effects are seen at relatively low dosing levels, are these to be considered behavioral effects or as an inability to maintain balance? Similarly, should the reduced singing and activity of exposed birds be considered a behavioral deficit or part of a broader syndrome of general incapacitation? Cholinergic systems are so heavily present in sensory systems also that behavioral deficits may represent a failure of the organism to properly receive the test stimulus (e.g., the perception of a mild electric shock used in many behavioral conditioning studies) rather than a change in its behavioral repertoire. Of the many effects that have been documented, the following are worth noting because of the obvious central nervous system involvement: • Increased aggression between paired individuals has been reported in quail exposed to the carbamate carbaryl (98). Reductions in cooperation between mated individuals would be expected to have serious

139

repercussions on reproductive outcomes. Increased male–male aggression has been reported. • Brunet and Cyr (99) documented that a 1-day exposure to the organophosphorous insecticide dimethoate could disrupt circadian patterns for as long as 12 days in ‘‘free-running’’ songbirds. The authors argued that such long-term disruptions could have serious consequences in the wild. • Vyas et al. (100) showed that a prolonged dietary exposure to the organophosphorous insecticide acephate disrupted the memory for migratory orientation in adult white-throated sparrows (Zonothichia albicollis). However, contrary to expectation, memory for food caches in black-capped chickadees (Poecile atricapillus) were not harmed by substantial exposures to the forestry insecticide fenitrothion (101). • The conditioned aversion aspect of food avoidance, which was discussed earlier (whether one considers it to be a benefit or a liability for exposed birds). Indirect Effects The impacts of pesticides on plants and invertebrates, whether target or nontarget, may secondarily affect birds. The potential for these indirect effects is, at least in North America, seldom considered at time of registration. Yet, evidence abounds that the indirect effects of pesticides on birds can be important. In the most general way, the increased use of herbicides has reduced the need for crop rotation and allowed for larger fields and extensive monoculture. The drift of herbicides into field margins has directly affected the quantity and availability of nesting habitat as well as the capacity of that habitat to support a rich insect fauna critical for birds. Some herbicides, fungicides, and especially insecticides can further reduce the insect biomass available to birds. Reduction in Available Food Supply The most intensive studies have been carried out in the United Kingdom. Of note is the unique study of the common partridge (Perdrix perdrix) carried out by the Game Conservancy Trust from 1968 to the present in the cereal farming area of West Sussex (102,103). By 1970, herbicide use was already well implanted in the area and had already resulted in a 50% reduction in the densities of insects reported from cereal fields in the 1950s. This loss of invertebrate biomass in turn affected the growth and development of partridge chicks and increased their vulnerability to predation and inclement weather by forcing them to forage further afield in the critical early weeks of life. A recent reanalysis of the 30+ years of data highlighted also the importance of insecticides and fungicides in reducing densities of the invertebrate classes most important as chick food (103). Densities of some of the favored food items such as sawflies had dropped more then 10-fold, and the effect was shown to persist into the year after spray. A direct link between insecticide use and chick survival had already been demonstrated by Potts, who documented a halving of chick survival on a farm where dimethoate was used as an aphicide. Despite the relatively high toxicity

140

AVIAN SPECIES

of dimethoate to galliformes, the impact was thought to be largely through insect removal because it fit exactly models relating chick survival to insect density. The indirect nature of the impact was confirmed when similar (although slightly less dramatic—treated farm with survival 65% of that on untreated farms) results were obtained on farms treated with insecticides of very low vertebrate toxicity such as synthetic pyrethroids. Although the partridge has been the ‘‘flagship’’ species in studies of pesticides in British agriculture, it is also likely that a number of other bird species of arable land, notably, corn buntings (Miliaria calandra) and skylarks (Alauda arvensis), have been affected for the same reasons. The use of herbicides has also resulted in a reduced availability of weed seeds in arable crops, and this is thought to be behind the decline of other species such as the linnet (Carduelis cannabina) (104). Interestingly, the continued use of herbicides in Sussex from 1970 to the present has actually led to an increase in the occurrence of several weed species hitherto uncommon such as poppies and goosefoot (103). A further irony is that aphid numbers are thought to have increased dramatically as a result of herbicide use, thus, necessitating the more frequent use of aphicides (105). Whether or not these authors are correct (they failed to consider that a concomitant increase in fertilizer use or new crop varieties might also have been factors), they demonstrated through simple but elegant feeding experiments that aphids represented inferior quality insect food for growing partridge chicks. Of course, impacts of insect control are likely to be felt by those species directly dependent on the pest species. This may be the case for some species of North American woodwarblers (e.g., bay-breasted Dendroica castanea and Tennessee Vermivora peregrina), whose populations explode in relation to outbreaks of the most heavily sprayed forest pest species, the eastern spruce budworm (Choristoneura fumiferana). However, the impact of insect removal on breeding forest songbird species has been variable. One of the earliest studies to show extensive multiyear impact was that of Moulding (106), who studied birds in the middle of a very large block of forest sprayed with carbaryl, a carbamate insecticide of low acute toxicity to birds. Other studies with insecticides of low acute toxicity to birds or higher specificity for the pest (e.g., the biological insecticide Bacillus thuringiensis kurstaki, which affects lepidoptera only) have yielded more variable results. Spray blocks are often small, which allows birds to feed in unsprayed areas, and in any case, it is thought the uneven deposit of the spray allows for insect refugia and continued feeding opportunities after spray [e.g., Holmes (107)]. Furthermore, because forest spraying takes place at a time when the pest, at least, is at epidemic levels, the food supply for some bird species may be super-abundant (108). However, birds have been found to make fewer feeding visits to the nest (107) or to switch prey preferences to more abundant species (108) in response to spray. This could increase the vulnerability of insectivorous birds to other stresses such as inclement weather. Based on the above, we can predict that the highest impacts on forest songbirds should be recorded

where 1) spray blocks are large, 2) spray deposits are good and spray coverage thorough, 3) the insecticide is toxic to many insect types, and 4) the pest is a species poorly utilized by birds, e.g., gypsy moths (Lymantria dispar). Finally, it is notable that none of the forestry studies have been able to study those species thought to be most at risk from forest spray programs—small-sized high canopy species such as kinglets (Regulus sp.). By virtue of their size and high metabolic rates, these species ‘‘walk an energetic tighrope’’ that makes them most vulnerable to reductions in prey biomass. They were the species most affected by organophosphorous insecticide treatments (9), but the effects were likely to have been direct as well as food-mediated. Evidence for an indirect food-mediated effect of pesticides in North American farmland is not as good as in the United Kingdom or other European countries. The long-term quantitative data sets relating bird numbers to agricultural changes are in short supply on the North American continent. Although the use of herbicides and fungicides is typically less per cropped hectare in North America, there are several extensive insect control programs, notably, in corn (for rootworm, European corn borer), oilseed rape or canola (flea beetle, diamond-back moth), wheat (grasshoppers, Russian wheat midge), potatoes (Colorado potato beetle), and others. It was argued by Sheehan and colleagues (109,110) that the loss of aquatic invertebrate prey from small prairie wetlands following aerial overspray of insecticides could have a substantial impact on broods of dabbling ducks by forcing them to move more often, thereby exposing them to higher predation levels. The higher reliance in North America on insecticides of very high acute toxicity to birds [compared with the United Kingdom, for example—Mineau et al. (45)] has meant that authorities and researchers in Canada and the United States have typically been more concerned about the direct impacts of pesticides rather than the indirect ones. There is no reason to think that North American bird species similarly dependent on weed seeds and on the invertebrate food supply in arable lands are not being affected as are their ecological equivalents in Europe. Reduction in Cover Herbicides may further indirectly affect birds by reducing the vegetal cover in and around cropped fields. An early study in Canada (111) showed that the use of 2,4-D in dense nesting waterfowl cover resulted in a reduction of nests established. Based on this study and on the documented cover requirements of waterfowl, Sheehan et al. (109) calculated that the cover-mediated loss of waterfowl production resulting from herbicide use in the Canadian prairies might be of the same magnitude as the total harvest by shooting. Unfortunately, it has not been possible to confirm experimentally these crude estimates. The loss of cover in field margins is likely to lead to higher levels of predation, but this has not been shown experimentally for operational herbicide use. In most agricultural landscapes, available cover is in the form of hedgerows, windbreaks, fencelines, and such linear habitats. Management of these habitats for

AVIAN SPECIES

invertebrate species and for birds is an accepted practice in several European countries but in its infancy in North America (112). HOW BIRDS ARE EXPOSED TO PESTICIDES General Risk Factors Birds ingest pesticides through their food or through preening or grooming. Despite being feathered, they absorb pesticides through their skin, encountering droplets directly or by rubbing against foliage and other contaminated surfaces. Driver and colleagues (113) compared routes of exposure for methyl parathion in quail in a wind tunnel and found that dermal exposure was the most important, in part as a result of the birds dust bathing in the contaminated soil. Mineau et al. (70) showed that birds exposed to fenitrothion in a wind tunnel could receive a lethal dose without any food exposure. Birds are also exposed through their feet. Available data suggest that pesticides absorbed through the feet are slowly released over time and this can result in prolonged intoxication (50). Finally, birds have a very high ventilation rate and inhale vapor and fine droplets. The ecology of the species (i.e., feeding preferences, behavior) along with the characteristics of the chemical (i.e., its persistence, tendency to bioaccumulate, toxicology) and the intended use go a long way in determining the nature and the scope of the impacts on wildlife. A few specific high exposure situations are now described. The evidence for these cases comes from the poisoning literature. Although birds may be exposed to nonlethal pesticides by the same routes and under similar conditions, the absence of reliable biomarkers makes this exposure difficult to quantify. Abuse and Misuse Deliberate attempts to poison wildlife, or abuse, usually involve baits of some kind. The only limit is the imagination of the perpetrator. Typically, liquid insecticides are poured or injected and applied to seed, bread, meat, and so on. Granules can be sprinkled or mixed into a paste. The choice of chemical reflects availability and toxicity. Cholinesterase-inhibiting pesticides typically used in deliberate poisoning attempts have included carbofuran, aldicarb, monocrotophos, parathion, mevinphos, diazinon, and fenthion. There have also been cases with strychnine, chloralose, and anticoagulant rodenticides. The main problem of course is that the baits are often indiscriminate in the species that they kill. Secondary poisoning is also frequent when predators or scavengers take dead or debilitated prey with a highly concentrated bait in their gut. In the United Kingdom, as well as in several European countries, officials estimate that deliberate bird kills due to pesticides outnumber cases where label instructions were strictly followed. Between 1978 and 1986, officials in the United Kingdom estimate that, on average, 71% of incidents were the result of abuse. For birds of prey alone, over 90% of cases recorded between 1985 and 1994 in the United Kingdom were abuse cases. For raptors in the United States during the same period, kills involving

141

labeled uses of pesticides were almost as frequent as were abuse cases (45). This difference appears to be wholly attributable to the high toxicity of insecticides used in the United States, especially the insecticide carbofuran in liquid or granular formulations. In Canada, the proportion of raptor kills resulting from labeled uses seems to be higher still. However, the Canadian tally is heavily biased toward one region where there is a problematic overlap between high wintering populations of bald eagles and intensive agriculture. The term misuse refers to a pesticide application that is not exactly as specified by the label. This may be an application at a rate that is higher than specified, or to a crop or pest other than those listed. Pesticide misuse is difficult to establish, especially after the fact. Also, in some cases, it becomes very difficult to distinguish a misuse from a normal agronomic use when the label contains instructions that are difficult or impossible to follow. Examples are labels that warn against using a product in ‘‘areas frequented by wildlife’’ or labels that require that no granular insecticides be ‘‘left on the soil surface.’’ Grazing Birds Grazing birds are particularly vulnerable to spray applications of pesticides. Kills have been recorded with several pesticide sprays, e.g., diazinon, carbofuran, dimethoate, and triazophos. Grazers typically include geese, ducks, and coots. These birds eat large quantities of foliage because they do not digest cellulose. Fertilized areas are particularly attractive to grazing species because grazers can detect the high nitrogen levels. Golf courses attract grazers because the turf is cut frequently, watered, and fertilized, and courses often have other attractions such as ponds and drainage streams. Over 100 cases of waterfowl mortality were recorded due to the use of diazinon on turf before the pesticide was withdrawn from golf courses and sod farms in the United States (114). Other well-documented problems are kills of ducks and geese in alfalfa fields treated with carbofuran (41) and of sage grouse (Centrocercus urophasianus) feeding on alfalfa crops treated with dimethoate or on potato foliage and weeds in potato fields sprayed with methamidophos (115). Crop Pest Specialists Bird species that feed on agricultural pests such as grasshoppers, leatherjackets (larvae of the crane fly), grubs, and cutworms are at high risk of poisoning. Kills of these species is all the more tragic because they are beneficial to agriculture (2). Some species are particularly vulnerable because they specialize in insect outbreaks. These birds take advantage of pest control operations that result in insects becoming either debilitated or more visible, as when soil organisms come to the surface following treatment. The high toxicity of carbamates for earthworm and the violent coiling behavior of poisoned earthworms has resulted in several cases of poisoning with the insecticide carbofuran (116,117). In a recent case in Argentina, approximately 20,000 Swainson’s hawks were

142

AVIAN SPECIES

poisoned within the span of a few weeks after feeding on grasshoppers sprayed with monocrotophos. Fortunately, that situation was corrected following the intervention of the Argentine government, the manufacturers, and several other cooperators (118). However, a review of the available data on monocrotophos indicates that this insecticide is responsible for frequent and unavoidable poisoning cases where it is still in use, especially in developing countries. As with carbofuran, the extreme toxicity of this product means that it is difficult to find use patterns that do not result in bird kills. Worldwide, grasshopper or locust control operations are likely to result in widespread bird impacts where pesticides of moderate to high toxicity to birds are used. This is because orthoptera are an important ecological component of grassland (and now cropland) systems and several species have evolved to take advantage of this resource. Poisoning incidents that involve the larger flocking bird species such as gulls, partridges, grouse, tinamous, hawks, owls, ibises, egrets, and herons will be easier to detect. More difficult to find will be the small insectivorous birds that may be feeding on the same insects, or feeding them to their young. Granular Insecticides Granular insecticides were designed for convenience and the safety of the person applying the product and to provide timed release of the chemical; yet for birds, many have proven to be disastrous. Several granular products are sufficiently toxic that one to a few granules can kill birds. Kills have been recorded following the use of aldicarb, parathion, carbofuran, fensulfothion, phorate, terbufos, fonofos, disulfoton, diazinon, and bendiocarb. Granular insecticides come up time and time again as a source of wildlife pesticide mortality. The high risk associated with granulars is a result of 1) the high toxicity of several registered products, 2) current agricultural machinery that ensures that granulars are left exposed on the soil surface, and 3) the attractiveness of granules to birds. Despite a lot of research, we still do not completely understand what exactly attracts birds to granules and under what conditions. The similarity between pesticide granules and avian grit has been extensively reviewed (119), and the selection of granules as grit by birds is known to occur. Normally, grit consists of sand or small rocks swallowed by birds, especially seed-eaters, to help in digestion. Granules made of the dried and granulated cob of corn (maize), or other organic substances, are probably taken as food or mistaken for waste grain. It follows that the most dangerous granules are those made of sand (silica) or dried corn. Somewhat less dangerous are clay, gypsum, or coal granules. There is insufficient information on paper granules, but, like corn, they float, which may present a problem in the case of puddling. Granules that are friable and break down quickly in or on the soil are best for birds, but they are the products least convenient to farmers. Regardless of the type of carrier, a pesticide granule is likely to be a problem if a lethal dose can be obtained in a few granules only.

No one, however, has been able to work out what ‘‘few’’ actually is. To date no agricultural machinery or application technique can achieve complete incorporation of the granules into the soil. Birds have also been known to probe the soil for granules or to pull up germinating seeds with granules attached. The worst applications are those made above the soil surface and in a band (a wide strip over the seed furrow) rather than in the seed furrow. In carefully controlled engineering trials, between 6% and 40% of applied granules were left on the soil surface. Side dressing (when granules are applied to either side of the seed furrow after germination) also leaves most granules exposed. There is a great variability in the types of applicators and how they are used. The same equipment can achieve radically different soil incorporation when used by different individuals under different conditions, and grower performance in this respect is significantly worse than that of engineers conducting calibrated trials. Exposure can also occur via invertebrates, especially earthworms, or secondarily through predators and scavengers that eat their prey whole or ingest their gastrointestinal tract contents. In Canada and the United States, we have seen cases of poisoning of waterfowl foraging in puddles in fields as well as kills of their scavengers more than 6 months after pesticide application [e.g., Elliott et al. (49)]. Enhanced granule persistence is more likely to happen in acid, waterlogged soils. Ducks find the granules by sifting through sediments when fields flood, giving rise to ‘out-of-season’ poisoning cases [e.g., Littrell (120)]. Treated Seed Several bird species make heavy use of waste or planted grain in fields. The exposure associated with treated seed is therefore always high. The size and type of seed dictate which bird species are at risk. Treated seed present a similar engineering problem to that of granular formulations: A proportion always remains on the soil surface. Also, Dutch researchers have shown that approximately half of the surface seed results from small spills throughout the field area (121). Historically, seed dressings were one of the main sources of bird exposure to organochlorine and mercurial compounds. Poisoning incidents with seed dressings are still relatively frequent, especially in Europe, where there is a heavy use of cholinesteraseinhibiting pesticides for this purpose (122). Kills have been recorded with carbophenothion, chlorfenvinphos, bendiocarb, furathiocarb, and, to a lesser extent, fonofos. Lindane is the main seed dressing chemical in North America, a situation that is quickly changing as it is gradually phased out. Some kills have been recorded with very new insecticides as well, e.g., imidacloprid, although it is not yet known how serious or frequent a problem this is. The acceptability of any chemical as a seed treatment depends on a consideration of the seed type, the planting equipment, conditions under which the seeds are planted and the species likely to be interested in the seed. As with granules, more seeds are left on the surface in turn areas at field borders. Spills can occur anywhere depending on topography and soil conditions. The number of seeds will diminish over time as they are consumed.

AVIAN SPECIES

The speed of disappearance may provide some indication of the risk of exposure of wildlife species. Systemic insecticides (those that are taken up and translocated within the crop plant) applied either in granules or treated seed can be present in sufficient quantity in germinating plants to lead to primary or secondary toxicity. In a well-documented case in the United States, Swainson’s hawks have died after eating insects that were feeding on cotton seedlings, the seed having been treated with the insecticide disulfoton (45). Puddling, Irrigation It is well known that agricultural development and crop irrigation in particular can attract birds from surrounding areas or entice migrating birds to stop over. Less well recognized is the potential exposure of birds to contaminated water in fields. Large kills of songbirds, raptors, and gamebirds were seen in California vineyards when the irrigation water was spiked with carbofuran for phylloxera control (45). Exposure through puddling of spray solution or foliar washoff need not be restricted to arid areas, however. Puddling can be a significant source of exposure, even in typically wet areas. Kills have been associated with puddling of heavy poorly drained soils used for turnip cultivation in Canada. Several kills of finches have been recorded in Germany when the birds drank spray solution from the leaf whorls of cabbage and other cole crops (123). Even pesticides of relatively low acute toxicity such as the herbicide monochloroacetate killed over 300 finches in a single British incident when the birds drank spray solution from the ground (64).

143

Rodent Control Rodenticides as a rule are not specific to their intended targets and cause direct impacts to nontarget species. Only a detailed knowledge of the habits of the target species and use of specific baiting locations or specialized bait holders can reduce kills of nontarget species. More problematic is secondary poisoning. Unfortunately, the trend has been for the more recent, more efficacious ‘‘single-feed’’ anticoagulants to present a greater hazard to predators than the older products. Compounds such as difenacoum, difethialone, brodifacoum, bromadialone, flocoumafen, difethialone, and other similar ‘‘super’’ coumarin–type products should not be used in a situation in which the target species is likely to be predated or scavenged. Generally, this means that the use of these products should be restricted to human dwellings and grain storage areas. Even then, there is recent evidence (58,59) that there is extensive exposure of raptors to rodenticides restricted to commensal rodent control. There is also evidence from the United Kingdom that second-generation anticoagulants, despite being restricted to bait stations and commensal rodent control, have resulted in a fairly broad contamination of some species such as the barn owl (60). The use of thallium and endrin to control rodents has also been shown to have disastrous consequences on raptors (see earlier). For humane reasons, strychnine is being abandoned as a rodenticide. Although it can certainly lead to secondary poisoning, a moderate degree of safety has been attributed to the fact that some predators discard the gastrointestinal tract of their prey before consuming them (127).

Mosquito and Biting Fly Control

Bird Control

The organophosphorus insecticide fenthion has been associated with large-scale avian mortality when used in mosquito abatement. Seabloom et al. (124) estimated that between 5,000 and 25,000 birds were killed (primarily warblers—37 species were represented in the sample retrieved) when 600 ha of residential and park area in North Dakota were sprayed with 110 g a.i./ha. DeWeese et al. (125) recorded waterfowl, shorebirds, and songbirds killed and debilitated by an application of 47 g a.i./ha to wet meadows in Wyoming. Currently, there is ongoing concern over the use of fenthion for mosquito control in Florida because of the documented mortality of shorebirds. The fact that bird mortality is recorded at such low rates of product application suggests that fogging (delivery of very small insecticide droplets) represents a high exposure situation for birds, presumably through inhalation of spray droplets or impaction and dermal penetration. Kills of shorebirds following the use of the mosquito larvicide temephos has strengthened the belief that this group of birds may be especially sensitive to OP pesticides. Despite the moderate toxicity of this pesticide to the usual test bird species, there have been sporadic kills recorded in Canada, and in one Australian incident of note, 240 shorebirds, primarily red-necked stints (Calidris ruficollis) were killed from a single application in Western Australia (126).

By definition, this involves the sanctioned used of pesticides to kill pest bird species. The acceptability of a chemical for pest bird control generally hinges on the risk to nontarget species. Fenthion, which is used to control pest birds in Africa (Quelea species) and in North America (e.g., house sparrows—Passer domesticus— by means of the Rid-a-Bird perch system), has given rise to frequent secondary poisoning (61). Secondary poisoning is also very likely following the use of toxic organophosphorous or carbamate products in grease for the control of monk parakeets (Myiopsitta monach), as practiced in some South American countries. To a greater or lesser extent, the genesis of pesticide abuse has its roots in various government-sanctioned programs of poisoning wildlife species deemed to be undesirable for whatever reason. For example, given that as recently as the late 1980s, woodpigeons (Columba palumbus) in Britain were being actively poisoned because of their impact on brassica crops with seeds laced with alphachloralose (37), is it surprising that this chemical is now used for unsanctioned (hence, illegal) bird poisonings today? Veterinary Drugs Several poisoning cases have resulted from the use of organophosphorus pesticides for the treatment of ectoparasites and endoparasites in livestock. The most

144

AVIAN SPECIES

interesting case is that of famphur. Famphur remains one of the leading causes of eagle poisonings in the American Southwest. Used as a ‘‘pour-on’’ warble fly treatment, it persists on the hair of cattle up to 100 days after treatment (128). Magpies are poisoned when they eat the hair, and eagles when they scavenge the magpies. Fenthion can also give rise to problems when used this way. Medicated feed at livestock feed yards is another high exposure situation. Sparrows, starlings, and other birds pick up the feed and subsequently are scavenged by hawks and eagles. Finally, waterbodies can be contaminated by treated animals. Forestry Insecticides Insecticides applied to large tracts of forest for the control of defoliators, if at all toxic to wildlife species, are bound to be problematical. In a forestry situation, critical wildlife habitat is sprayed directly, and a large number of individuals of many species are exposed to the chemical. When declines are seen, it is the small canopy-dwelling species that are most affected (9). For that reason, forestry products should be more stringently reviewed than agricultural products. In Canada, the forestry insecticides phosphamidon and fenitrothion were canceled after impacts on birds were judged unacceptable. Although fenitrothion is not as acutely toxic as a number of other anticholinesterase insecticides used in agriculture, its use in forestry leads to severe and widespread inhibition of brain acetylcholinesterase in a number of songbird species. Similar levels of inhibition have been associated with serious sublethal effects as well as mortality (129). Research suggests that fenitrothion is readily absorbed through the skin, and this may help explain the impact on birds when it is applied as a fine aerosol. Concerns have also been raised because of the prolonged cholinesterase inhibition in birds exposed to another OP, acephate, following forest treatments (130). Secondary Poisoning Secondary poisoning occurs when predators, such as hawks or owls, consume prey contaminated by pesticides. Such predators are few because of their position at the top of the food chain. Therefore, the death of one predator may constitute a significant reduction in the local population of that species. Furthermore, predatory birds are important agents of control for a number of species considered to be pests, such as many rodents. Historically, researchers have associated secondary poisoning with persistent organochlorine insecticides and alkyl mercury fungicides that are not readily metabolized and therefore accumulate in tissue. We now know that other currently registered pesticides, even those that are readily metabolized, can cause secondary poisoning under the right conditions—namely, when the predator encounters the pesticide in a high concentration on the surface or in the gastrointestinal tract of its prey (45). Exposure is further enhanced because predators capture birds debilitated by insecticides much more easily than unexposed prey (131).

PRE-REGISTRATION ASSESSMENTS OF PESTICIDE RISK TO BIRDS An Historical Overview The regulatory approval process for pesticides consumes much energy and is the source of continuous debate and never-ending reassessments, conferences, expert meetings, and so on. One ongoing debate centers on the relative merits of field testing vs. risk estimates based largely on data extrapolated from the laboratory. Secondarily, there has also been a debate on the relative merits of extensive vs. intensive field testing. Currently, most of the discussion is on the methods required to deal with the uncertainty in pesticide risk assessments, especially those based on laboratory toxicity data. Unless one has an understanding of the historical basis for these debates, it is difficult to see why they have taken the form that they have. By the late 1960s, there were already mechanisms in place in the United Kingdom and in Canada (and perhaps other countries as well) for advice on avian safety to be funneled through to the regulatory authorities charged with pesticide registration decisions. Systematic review of pesticide applications for their risk to birds began in 1972 in the United States [see Touart and Maciorowski (132], although a framework and formal test guidelines were not proposed until 1978 and adopted in 1982. In the early 1980s, there was the realization within the U.S. Environmental Protection Agency (EPA) that the provisions for triggered reevaluation of registered pesticides and higher tier progression as allowed under U.S. legislation (FIFRA—the Federal Insecticide, Fungicide and Rodenticide Act) were not being utilized. This progression to higher tiers of scrutiny allowed risk assessors not satisfied with the safety of a pesticide, based on laboratory (tier 1 or tier 2) data, to direct the proponent to conduct higher tier tests such as fullfledged field studies. Because the trigger for higher tier testing was based on possible field lethality, there ensued, throughout the 1980s and early 1990s, a number of field trials conducted on the most toxic pesticides registered in the United States. The design of these field trials was heavily influenced by the success of early field tests carried out on products such as granular carbofuran. The combination of extreme toxicity to birds, a short time to death, and the attractiveness of the granules to birds—hence, considerable exposure—ensured that carcasses were relatively numerous and easy to find. Therefore, the basic U.S. field study design (133), although allowing for a diversity of approaches, emphasized lethality and the finding of bird carcasses. Acknowledging the stochasticity of pesticide bird kills, the U.S. EPA proposed a strategy of monitoring a number of fields for the presence or absence of bird casualties (the extensive approach). Meanwhile, based on some early work of their own, the U.K. authorities in conjunction with the British Agrochemical Association and other industry and conservation partners were leaning more to the intensive approach where one (or a few) site would be intensively monitored using a variety of approaches such as mark-recapture, breeding success studies, biochemical markers, and so on. A series of meetings was organized to

AVIAN SPECIES

debate the relative merits of the different approaches, the main two leading to symposium proceedings (134,135) as well as an issue paper published in 1988. In 1992, came a major shift in U.S. policy regarding the value of field testing (132). U.S. EPA management decided to deemphasize field testing. Reasons for a move away from field studies included excessive cost to pesticide registrants, the long time period required, as well as insufficient guidance and lack of standardization on design and conduct. More importantly, most of the studies were inadequate to provide a quantification of the bird impact, although a large proportion did show evidence of a problem (136). The U.S. EPA decided instead to base registration decisions on a more in-depth analysis of the laboratory data and of the uncertainties inherent to those data (the so-called ‘‘probabilistic’’ approach). At the same time, the EPA mandated that ecological risks of pesticides should be characterized with less uncertainty, which, to many, appeared to be at odds with the move away from field studies (137). A new framework for avian risk assessment was developed (ECOFRAM) and is currently being implemented. Expert groups were also held under the auspices of such para-governmental bodies as the OECD (Organization for Economic Cooperation and Development) and scientific societies such as SETAC (Society for Environmental Toxicology and Chemistry) in order to provide coordinated input into the process. Because of the importance of the U.S. pesticide market and because a U.S. registration is highly desirable to pesticide manufacturers, data mandated by the United States are usually available for most of the pesticides currently registered worldwide. With but minor modifications, Canada currently accepts all data submissions pertaining to environmental toxicology generated to U.S. specifications, as do a number of other countries. The OECD also promulgates its own guidelines, although, up to this point, the avian study guidelines under OECD are virtual copies of the U.S. EPA guidelines. This may be changing in the near future because, following a key meeting of experts in 1994 (138), there was a clear wish from the scientific community to revisit the existing avian testing guidelines and make them more suited to current pesticide chemistry and issues of concern. Expert groups are currently reworking all existing guidelines. Since the advent of the EU, there has also been a great deal of activity to standardize avian test guidelines as well as the framework under which the test guidelines are to be considered (139). The EU has drawn heavily on the work of an industry-led stakeholder group, the European Plant Protection Organization or EPPO as well as on the OECD, which regroups most countries of western Europe. Avian Toxicity Testing Choice of Test Species The mallard duck and northern bobwhite are the two main bird species tested under current U.S. guidelines. The Japanese quail (Coturnix coturnix) is often substituted for the bobwhite in Europe, and there is currently an OECD proposal to favor the Japanese quail for reproductive toxicity testing because of its ubiquitous nature and short maturity time. The Japanese quail is

145

also a good species on which to base an extrapolation factor (a factor applied to a toxicity endpoint in order to account for interspecific differences in susceptibility) because of its apparent ‘‘stability’’ with respect to its relative position in species’ sensitivity distributions for different pesticides (40). Other species frequently tested (especially acutely) have included the rock dove, redwinged blackbird, and house sparrow, all birds that have been considered pests at some point. Evaluators assessing the safety of pesticides to humans have the benefit of being able to study several surrogate species in order to extrapolate to the single species of interest. By contrast, wildlife evaluators are constrained to look at a few species and predict impacts on a diverse fauna. There are an estimated 9000 species of birds living in the world today—interspecies extrapolation is therefore the rule. Phylogenetically related birds do not necessarily show a similar sensitivity to any given pesticide, although there are family relationships for some groups of pesticides. Some species do appear to show an inherent susceptibility or resistance to a wide range of environmental toxicants. One cannot rely on the toxicity values obtained in the mallard or bobwhite and assume these values are representative of all bird species. When possible, toxicity values are fit to a distribution and a defined point in the distribution (e.g., the 5% tail with the highest sensitivity) is calculated for all pesticides (40). Where there are insufficient data to draw such a distribution, extrapolation factors are now available that can be applied to those data. Two main strategies have been developed for these factors: universal factors that do not take into account the species that were tested but assume they were chosen at random with regard to sensitivity (140) or the approach of using species-specific extrapolation factors—a different factor being applied depending on the species used as a starting point (40). The main toxicity tests mandated in most jurisdictions fall into four main groups: Acute Toxicity These tests are modeled on the similar mammalian studies. They consist in gavaging birds with the pesticide of interest and calculating the median lethal dose. The test has been criticized for animal welfare reasons, although replacements have not yet been found. Main uncertainties with the test have to do with the method of gavage and the fact that some birds may regurgitate part of the dose (141). Also, uptake from the gut may be different (higher or lower) under gavage conditions. Current thinking is that the determination of an approximate lethal toxicity is sufficient and this can be achieved with a small number of test individuals through such techniques as the ‘‘up and down’’ method. It has also been argued (142) that, given the wide interspecific variation in pesticide sensitivity, it would be preferable to test a variety of bird species rather than just one, as currently mandated by the United States. A recent expert group re-emphasized this need where the toxicity of a pesticide is such that interspecies differences in susceptibility are likely to place some species at risk (139).

146

AVIAN SPECIES

It has been shown that acute toxicity in birds scales to body weight but in a direction opposite to that normally assumed in mammals (143). For the majority of pesticides studied, the smaller the bird the more sensitive it is relative to its body weight. A possible explanation for this is that smaller birds are less likely to survive the disruption in feeding brought about by dosing levels that would otherwise not be lethal. Dietary Toxicity This test was created specifically to better address the risk of pesticides to birds because traditional thinking has been that birds are primarily exposed through contaminated food. Groups of young (circa 5–8 day) quail chicks or ducklings are provided ad libitum with graded concentrations of the test substance mixed into their diet. The measured endpoint is the median lethal concentration of the pesticide in food expressed as milligram pesticide/kilogram feed. Unfortunately, the test is unreliable and the measured endpoint virtually meaningless with any pesticide capable of inducing avoidance behavior in the test birds (144). This includes most carbamate and organophosphorus insecticides, which represent the bulk of chemicals for which pesticide toxicity is an issue. There is currently a proposal to increase the length of this test from the current 5-day exposure duration and to use adult or subadult birds that are singly caged in order to better determine feed consumption. There are also suggestions to impose moderate food stresses on the test birds and to reduce the caloric value of their feed in order to make conditions more similar to those encountered by birds in the wild. Reproductive Toxicity It has long been recognized that chemical effects on reproduction are potentially of the highest ecological relevance and that the detection of such effects should be a high priority for regulatory bodies. A test for reproductive effects in birds is currently part of the regulatory ecotoxicology requirements for pesticides in many countries. Of the three tests currently mandated in birds (the LD50 and dietary LC50 tests being the others), the reproduction test is the most time consuming and most expensive. The test is the only standardized one that focuses on toxicity endpoints other than death and that requires subchronic dosing of the test individuals. The EPA protocol for the avian reproduction test (employing the northern bobwhite and mallard) has been the ‘‘industry standard’’ for such tests. Essentially, the same protocol was subsequently adopted by the OECD and recommended by ASTM (the American Society for Testing and Materials). Over the years, there have been concerns expressed over the continuing relevance of the test. It was designed principally to detect egg-shell thinning and other impacts resulting from the bioaccumulation of persistent organochlorine insecticides. Pesticides are therefore given to the birds over a lengthy period to allow for bioaccumulation to occur, an exposure profile that does not correspond to the persistence characteristics of most

modern pesticides. The test also suffers from having a very low statistical power of problem detection. A proposal for a totally redesigned test using the Japanese quail as test species was formally submitted by Germany for consideration by the OECD in March 1993. In December 1994, a group of technical experts was assembled under the auspices of the OECD and SETAC to assess and, if necessary, redesign avian testing procedures. A working group was set up specifically to look at the reproduction test. That group recommended that the Japanese quail should be considered as a first-tier test species and a protocol for a modified bobwhite/Japanese quail test was drafted and submitted to OECD member countries for comment in late 1998/early 1999. Funds are currently being sought for validation of this new test protocol. Avoidance This test is not currently mandated by the United States but has gained some popularity in the EU. It consists in offering treated and untreated feed in various proportions to test birds and looking at their ability and desire to avoid the treated feed. Current protocols have been developed in Germany. Unfortunately, there is some concern that the test as designed may overestimate the extent to which birds in the wild are able to avoid a toxicologically harmful dose of pesticides (138). An expert task group has been trying to improve on these protocols. Dermal or Inhalation Toxicity Traditionally, ingestion of contaminated food has been identified as the most likely route of exposure for wild birds, and this is still the only route that is commonly assessed in standardized hazard assessment procedures. It is now known that, under the right circumstances, dermal exposure can be more important than can the oral route for birds in treated fields or forests (70,113). The lack of nonoral toxicity tests has been criticized on numerous occasions by expert groups (138,139). Unlike the situation in mammalian testing, there are no standard protocols for testing dermal or inhalation toxicity in birds. Other Tests Relevant to Avian Risk Assessment Several other tests routinely carried out by industry in support of their product for registration purposes are useful in assessing the potential risk to birds. Physicochemical data allow for an assessment of the likelihood of movement of the pesticide away from the area of application as well as the persistence of either the parent compound or breakdown products over time. Several studies, some of which may form part of the ‘‘efficacy’’ package submitted in some countries may provide residue data for estimating exposure in foodstuffs. A very important study, where available, is the chicken metabolism study. This is typically used to assess human safety where crop residues containing the pesticide of interest are to be fed to poultry. The study provides a measure of residues in meat and, most importantly, measures partitioning into eggs. Because metabolism is very different in birds than in mammals, an avian metabolism study is critical in explaining some of

AVIAN SPECIES

the avian toxicology, especially reproductive effects. Tests conducted on terrestrial and aquatic invertebrates are useful in assessing the possible indirect effects of the pesticide. Likewise, effects on nontarget plants provide data on the spectrum of activity of new herbicides and indicate whether wildlife habitat in proximity to farm fields is at risk. The area of nontarget plant assessment is in considerable flux. Guidelines exist, but improvements have been proposed (145). The OECD is currently reviewing this area as well. Exposure Assessment The way in which avian exposure is estimated in the United States and elsewhere is currently under revision. The tendency is to incorporate uncertainty into the measurements and to use Monte Carlo analysis to carry these uncertainties through several risk scenarios. Indirect impacts through habitat modification are not assessed at this time. A wide variety of factors can affect the exposure of birds to pesticides. Application Rates and Expected Residue Levels Under operational conditions, considerable variation in application rates of pesticides can be expected. This is generally not recognized as a problem by the user of pesticides, because all pesticides benefit from a relatively wide margin of safety in terms of both efficacy and crop phytotoxicity. However, the margin of safety to birds and other nontarget organisms is often slight to nonexistent. Exact application of a pesticide according to label instructions requires accurate measuring out of the various tank mix components, perfect condition and calibration of the equipment being used, faultless technique on the part of the applicator, and finally, ideal weather and terrain. Even under the highly regulated and mechanized conditions in effect in industrialized countries, these requirements cannot be met. Rather, it is more reasonable to expect that the rate of the pesticide delivered to the crop follows a broad distribution about the desired application rate. This is especially true for pesticide use in parts of the world where the bulk of the spraying is done by means of backpack sprayers and, hence, is even more vulnerable to human error. There are other situations that give rise to a higher-than-intended rate of application of a pesticide. Drift can be a major problem with either ground or aerial application. One of the important aspects of drift from the point of view of hazard assessment is that, following multiple-swath applications of a pesticide, the additive nature of droplet drift associated with each swath can give rise to high application levels in the downwind parts of the field and beyond. Exposure of wildlife species, if primarily through the consumption of contaminated foods, is only approximately related to the amount of pesticide delivered to the crop and to nearby noncrop areas. In currently accepted risk assessment procedures, residue levels on foodstuffs are estimated on the basis of standard factors, which assume that the rate of application and the area of the impacted surfaces are the only factors having a bearing on the resulting residue levels (146,147). An analysis

147

of residue data on turf grass prompted by widespread mortality of waterfowl caused by the use of the insecticide diazinon showed just how difficult it is to accurately predict residue levels on plant surfaces. Residue values on grass blades following a 1.1-kg active ingredient per hectare mechanized application of diazinon ranged between 17 ppm and 181 ppm over the course of six different studies (96). Yet, well-tended turf is as uniform and structurally simple a ‘‘crop’’ as one is ever likely to encounter in any hazard evaluation. This high degree of variability typical of most pesticide use situations is a serious problem for acutely toxic products with low margins of safety. Field Persistence of Modern Pesticides Most non-organochlorine pesticides are relatively short lived, at least in plant and animal tissue. They are more likely to persist in abiotic components of the environment such as soil, aquatic sediments, or groundwater. The rapid disappearance and lack of bioaccumulation of organophosphorus and carbamate insecticides, for example, is what made these classes of insecticides so attractive when the problems associated with organochlorine insecticides became widely known. However, there are site-specific examples of long environmental half lives so that, here again, generalizations are not always adequate. Granular formulations of some pesticides have been found to be long lived, especially in acid, waterlogged soils. Waterfowl and scavenger mortality has been recorded more than 6 months after the application of some granular products such as fensulfothion, carbofuran, phorate, terbufos, and fonofos (45). Formulation-Specific Concerns It is usually unclear to what extent birds are exposed to the technical pesticide and to what extent the various elements of a pesticide formulation remain with the active ingredient over time. Some components of pesticide formulations are known to enhance the toxicity of the product, whereas others may have the opposite effect. Generally speaking, granular formulations of organophosphate and carbamate insecticides are as toxic or less so than the equivalent technical grade pesticide (148). Unfortunately, the availability and attractiveness of these formulations to birds more than makes up for any reduced toxicity. Liquid formulations, on the other hand, are typically of higher oral toxicity than are the parent material, although this is seldom considered in avian risk assessment (149). It is reasonable to suspect that pesticides formulated to better penetrate insect cuticles or plant cell walls may be more readily absorbed by bird’s skin or gut lining than is the active ingredient alone. The Species at Risk and Their Propensity to Forage in Treated Areas The first step in a pesticide evaluation is a knowledge of the bird species present and therefore potentially at risk in the area of pesticide use. In a large country with several distinct physiographic regions or for countries

148

AVIAN SPECIES

with incomplete faunal surveys, this can be a formidable challenge in itself. The extent to which bird species use cropland varies tremendously with the species, crop, time of the year, and prior pesticide use. Species may change their food habits in relation to an overabundant supply such as during an insect outbreak, and therefore, even an intimate knowledge of the ‘‘normal’’ ecology of a species may not suffice. Flocking species can be very mobile, and their use of any given field is highly variable. Also, the extent to which the use of the pesticide modifies the propensity of wildlife to use the field or adjoining areas for feeding is another question that has not received much attention [but see Kilbride et al. (150)]. The presence of a given array of species in the general area of pesticide use does not necessarily mean that those species will be exposed. On the other hand, because of the nature of droplet drift in both ground and aerial applications of pesticides, bird species need not enter the cultivated field proper to be exposed to pesticides. Methods to quantify the use of fields by birds have been proposed (151). An approach made necessary by the complexity of ecosystems has been to focus the evaluation on a few indicator species. These should be chosen not so much for their inherent physiological susceptibility to the pesticide (this is not usually known) but for the likelihood that their life habits will lead to maximum exposure. Unfortunately, the choice of indicator species has often been made on the grounds of cost, logistics, and overall feasibility rather than on the grounds of more scientifically desirable criteria. In agricultural landscapes heavily treated with pesticides, there is a valid argument to be made that the most vulnerable species are those that are present in the least number or that, perhaps, have been extirpated already. Concentrating all of the assessment and research efforts on a few abundant species may provide pesticide regulators with a false sense of security. Assessing Dietary Exposure In current pesticide assessments, survey data are frequently used to estimate the fraction of food items obtained from treated areas, although it is recognized that this is a first approximation only. Work is underway to better document bird foraging in farm fields through radio-telemetry and observation. Other potentially valuable sources of information include the very large literature describing the consumption of insect crop pests by birds [see Kirk et al. (2) for a review]. Finally, pesticide field trials and recorded bird mortality events offer insight into which species are most at risk. It is a well-known fact that the food intake of a small organism is greater than that of a larger one when expressed as a ratio of its body weight, and allometric equations are available to help estimate food intake. It therefore follows that smaller species tend to be more vulnerable to ingesting a lethal dose of pesticide. Also, as a rule, small birds have an inherently higher susceptibility to acute dosing, as seen earlier. Depending on the time of the year, wildlife species may have higher energy requirements and, hence, food

intakes than at other times. For example, a bird feeding young at the nest or on migration may be expected to have energetic requirements far and above its normal needs at rest. Climatic conditions and factors, such as nutritional status, disease, and parasite load, also exert an influence directly on the toxicity of the pesticides to the organism and indirectly through their influence on food consumption. These are yet additional reasons why predicted exposure and risk may be in error. FIELD ASSESSMENT AND POST-REGISTRATION MONITORING OF THE IMPACT OF PESTICIDES ON WILDLIFE When a pesticide is initially submitted for registration, rarely has it been subjected to much field testing to investigate its potential impacts on birds. Thus, hazard is frequently estimated on the basis of toxicity values to a few test species and the projected use of the product. This assessment often follows a ‘‘quotient’’ method, in which the levels that cause toxicity or mortality of test species are compared with predicted levels of exposure. In theory, safety factors are introduced in this calculation to allow for errors in estimation or extrapolation. Also, as outlined earlier, a recent trend has been to incorporate uncertainties into the quotient by way of Monte Carlo simulations or other probabilistic methods. In practice, however, the level of uncertainty is so high that most of the acutely toxic pesticides, such as organophosphorus and carbamate insecticides, cannot be assessed adequately without field testing. If done at all, either as part of the regulatory process or following a regulatory decision, field testing will follow one of two directions: 1) active monitoring or directed studies where the experimental conditions are controlled and the questions asked are very specific, or 2) passive monitoring, also referred to as incident monitoring, where the intent is to put in place a network of competent observers in order to be able to investigate reported problems or to carry out spot checks of operational pesticide use. It is not always feasible to investigate the effects of a single pesticide on wildlife. In a number of cropping situations, several pesticides are used in quick succession, making the identification of compound-specific impacts difficult. Often, the mosaic of treated fields can be so complex as to make it difficult to assess exposure to any one pesticide. Two approaches then suggest themselves: 1) treated sites or landscapes are compared with nontreated areas provided those can be found and 2) the ‘‘severity’’ of treatment (the a priori expectation of toxicity) for any given site is used as a variable against which a number of different parameters (such as reproductive success) are regressed [e.g., see Bishop et al. (90)]. Great care must be taken in comparing treated with nontreated areas because they are likely to differ in ways other than their pesticide use patterns. Active Monitoring As reviewed above, field studies can be extensive (several sites, often little more than carcass searches) or intensive (one or a few sites, several investigation methods deployed)

AVIAN SPECIES

in scope. In either case, the field study generally consists of the surveillance of individual birds or of a local population prior to, during, and after the application of the pesticide, according to label instructions. A few selected approaches that are used in avian field assessments are discussed below. No single strategy is adequate for all situations. Rather, it is often best to start with testable hypotheses and then devise ways that will allow one to prove or disprove these hypotheses. In probabilistic terms, type I errors (or concluding that there has been an effect from a pesticide when in fact there have been no effects) are uncommon. For example, when one encounters instances of wildlife mortality in a treated field, the probability that this mortality is unrelated to the application is small for the simple reason that it is uncommon to witness random wildlife mortality under normal circumstances. On the other hand, the probability of type II errors (or failure to detect an occurring problem) is much higher. It is easy to delude oneself that one would see an impact if it occurred. A good example of this is carcass searches conducted on treated fields when it has not been ascertained whether affected wildlife are likely to die locally or whether they are likely to leave the area and die further afield. Field studies of a pesticide may rely on controlled application of the product by the experimenters or they may rely on normal operational use of the product, either with or without the knowledge of the user. Again, the question being asked should dictate which strategy is followed. Of course, considerations of cost and logistics often weigh heavily in this consideration. The answer obtained will be interpretable in different ways depending on whether the application was done under controlled or operational conditions. Monitoring Residue Levels Ever since the bioaccumulation propensity of some pesticides was discovered, the quantification of residues in various animal tissues has been a popular method of monitoring for pesticide ‘‘impacts’’ in birds. Birds were often chosen for this purpose, being visible, abundant, and relatively easy to capture. In North America, and to a lesser extent in other OECD countries, efforts were made to standardize the collection of birds at regular intervals in order to chart the extent and the time trends of organochlorine contamination. The birds selected for this purpose were the European starling (Sturnus vulgaris—now introduced worldwide), black duck (Anas rubripes), and mallard, and, in North America at least, the bald eagle. Individuals and research institutes around the world continue to monitor levels of OCs in avian species. Although tissues of choice for persistent organochlorine determination included liver, fat, egg yolk, and brain, pesticide levels have been measured nondestructively also in blood as well as in the chorioallantoic membranes of eggs. Measuring pesticide levels in tissue has not been as useful for replacement insecticides because they generally are readily metabolized and do not bioaccumulate. However, second-generation coumarin anticoagulants resemble OCs in that residues may be very long lived, at least in the liver through specific

149

binding sites. Also, metabolites of organophosphorus pesticides have been detected successfully in bird feces (152) and extracted from the washings of bird’s feet in orchards sprayed with dormant oils laced with organophosphorus insecticides (153). Residues contained in the gastrointestinal tracts of dead birds are often used, in combination with cholinesterase titers, to establish cause and effect in poisoning cases [e.g., Greig-Smith 1991 (154)]. Use of Biomarkers For organophosphorus and carbamate pesticides especially, the main field monitoring strategy has been the measure of cholinesterase titers, either in brains of poisoned or sacrificed individuals or in blood. Cholinesterase inhibition proved to be the ‘‘golden standard’’ of biomarkers because of its ease of measuring, and the fact that brain titers at least can be related directly to morbidity and mortality (76,155). A growing concern of those active in monitoring field impacts of pesticides on birds is that there are not yet any biomarkers to detect the presence of some of the recently introduced pesticides. One must then turn to expensive and time-consuming residue determinations, assuming that validated methods for wildlife even exist. The lower application rates of some new products will undoubtedly add to the difficulty of detection. This is already the case with whole classes of herbicides (e.g., sulfonyl ureas), which are effective against plants at levels that are below detection. The environmentally sound approach would be to make the development of biomarkers mandatory before registrations are granted, especially in the case of products that are likely to give rise to direct impacts. Carcass Searching Because of the extent to which several studies have relied on finding bird carcasses to confirm an impact, it is important to discuss the limitations of this field technique. Carcasses and poisoned wildlife can be very difficult to find for several reasons: 1) The majority of kills consist of very few widely dispersed small birds; 2) many species are cryptically colored and most are difficult to find especially if ‘‘weathered’’; 3) poisoning may be delayed and occur away from the site of intoxication; 4) poisoned individuals will often find cover; and 5) carcasses quickly disappear because of scavengers. There is a large difference between casual searching of fields and a well-organized, intensive search of an area by well-trained and, above all, motivated individuals. The success of the search effort can be affected by uncontrollable variables such as weather and the number of scavenger species present. Because of the very low probability of detection for widely dispersed bird carcasses, it is clear that every carcass counts. Very few carcasses can indicate a sizable impact. Standard Ecological Techniques Most of the methods employed in avian pesticide studies are the standard ‘‘tools of the trade’’ for field ornithology, namely, various survey techniques, banding, mark/recapture, radio-telemetry, and monitoring of nests, either from ‘‘natural’’ populations or populations augmented through the use of constructed nest structures (usually

150

AVIAN SPECIES

for box-nesting species). All shortcomings, biases, and pitfalls attached to these methods apply to pesticide studies.

PRACTICAL CONSIDERATIONS FOR REDUCING THE IMPACT OF PESTICIDES ON BIRDS

Passive Monitoring (Incident Monitoring)

The following are general recommendations to make pest control programs more compatible with bird preservation. These could be incorporated into any communication material intended for farmers, landowners, and pesticide applicators:

Even when a pesticide has been studied extensively under controlled conditions, unforeseen problems and situations can arise following the commercialization of the product. Incident monitoring refers to the capacity of competent authorities to investigate reported problems or to conduct spot checks of use conditions. An absence of incident reports does not necessarily mean there are no problems but, conversely, well-investigated incidents can reveal unforeseen aspects of a pesticide or reinforce a suspicion that arose in the course of field testing [e.g., see Blus and Henny (137) for examples]. An incident monitoring scheme will require a network of individuals trained in carrying out pesticide investigations and in proper handling of carcasses and tissue samples, as well as access to a laboratory equipped to do chemical and biochemical analyses. The usefulness of an incident monitoring scheme will grow as the quality, reliability, and coverage of reports increase. Incident monitoring data can be used to: • Verify whether registration decisions were appropriate, i.e., confirm a risk predicted from laboratory data, or identify a risk not predicted from laboratory data. • Trigger more systematic field studies. • Improve label directions. • Allow recommendations on the ‘‘best’’ product to use under some circumstances. • Trigger a regulatory review. • Ensure that products are being used correctly. • Provide data for potential regulatory action. Undoubtedly, the best system currently is that found in the United Kingdom. The main reason for its success is its stable funding from a tax on pesticide sales. Also, the scheme was designed specifically to capture pesticide incidents, unlike other schemes that began as an effort to track wildlife diseases, e.g., Canada, France, the Netherlands, and other countries. As they moved away from active field testing, U.S. authorities expressed the desire to increase their capacity to detect and record pesticide incidents (136), although this is just barely getting underway at present. There are biases in any reporting system, and it is important to understand and recognize those biases. The biases will depend on how the incident monitoring system is set up and on the persons/organizations responsible. Some biases can be reduced over time, but others are unavoidable. Common biases relate to body size and color of the casualties, numbers and density of the species in any given area, ‘‘status’’ of the species, as well as individual and institutional interests and sensitivities. Most schemes have a strong bias toward the detection of pesticide abuse such as intentional attempts to poison birds, mammalian predators, or real and perceived vertebrate pests (45).

• Use pesticides that are as target-specific as possible. Establish buffer zones around any wetlands, woodlots, drainage ditches, fencelines, hedgerows, or even small rock piles. Do not allow pesticide spray to drift onto these habitats. Leave field edges untreated where economically feasible—this is where the bird activity is the most intense. • Use the least toxic and the least persistent product available for the use required. Assume that the product is as toxic or more to birds and other wildlife than it is to humans. Consider whether spraying is absolutely essential. Consider alternatives to spraying. • Avoid using products that are known to move away from the area of application through vapor drift or runoff. • If you have a choice, use a spraying time that does not coincide with the breeding season for wildlife species in your area. Avoid spraying near nests, dens, or burrows. Avoid also applying pesticides when large flocks of migrant birds are present in the area. • Follow label instructions scrupulously. Take heed of any special warnings concerning fish or wildlife, and abide by specified buffer zones. • Avoid the use of granular formulations of acutely toxic insecticides. If you must use these products and are applying them with mechanized equipment, shut off delivery before you reach the ends of the rows if you can and avoid any spills over bumps, in turn areas, and at loading sites. Cover any visible spills. If you are applying pesticides by hand, ensure that a mechanism is in place to ensure the best possible incorporation of the granules into the soil. • Where it can be done safely, inspect your field and field edges carefully after application. Avoid the repeat use of any product that causes any wildlife mortality. Experience shows that this is only the ‘‘tip of the iceberg.’’ • Treat and dispose of empty containers as directed. Where the necessary programs are available to you, recycle them. • Avoid contamination of any body of water, whether permanent or temporary in nature. Never wash spray equipment in lakes, ponds, or rivers. If drawing water from these areas, use backflow devices. • If carrying out a vertebrate poisoning program, ensure that bait placement minimizes exposure to nontarget species. Locate and remove all carcasses so as to avoid scavenging. Avoid using products of high secondary toxicity (such as single-feed

AVIAN SPECIES

anticoagulants) if it is at all likely that the target species will be at some risk of predation or scavenging. • Protect all species of raptors. These often fall prey to landowners who hold the misguided views that they represent a threat to their livestock. Their benefits to agriculture almost always outweigh their occasional taking of livestock. • Report any incident of wildlife mortality to competent authorities. Only through such feedback will it be possible to minimize wildlife impacts in the future. ARE BIRD POPULATIONS AT RISK FROM PESTICIDE USE? It is clear that the use of persistent organochlorine insecticides had serious consequences on populations of birds of prey and fish-eating bird species. For some bird of prey populations (e.g., peregrine falcons), it was the combined effect of DDE-induced eggshell thinning and dieldrininduced mortality of breeding adults that provided the ‘‘one-two punch’’ that caused them to plummet. Top carnivores were most at risk from persistent organochlorine pesticides and, being slower to reach maturity (i.e., being K-selected species in ecological parlance), they were less able than were more rapidly reproducing species (rselected species) to recover from the pesticide impact. DDE-induced eggshell thinning was probably sufficient to induce declines in the Northern gannet (Morus bassanus) population in the Gulf of St. Lawrence following the use of DDT for forest spraying. Eggshell thinning was also thought to be the primary reason for the collapse of the double-crested cormorant in the Great Lakes and the brown pelican, as reviewed earlier. It may have been Borg and colleagues (156) who first likened the finding of dead birds by mercury seed dressing as the exposed tip of an iceberg where, for every bird detected, there are several with various degrees of latent and manifest intoxications that are hidden below the surface. They believed that the seed dressings were affecting various populations of seed eaters and especially the owls and hawks that predated or scavenged them. As was the case with cyclodiene seed dressings, data suggested that mercury resulted in both acute lethal effects and lowered reproduction. An important question is whether current pesticide use has the potential to reduce bird populations to the same extent. Before we can attempt to answer this question, however, we need to define it carefully. What do we mean by a population-level effect? Are we interested in safekeeping populations at the continental level only or are local population declines of concern. Defining the scale (or size of landscape) at which population attributes are functioning is one of the most difficult tasks facing attempts to measure the impact of pesticides on birds (157). As reviewed by Emlen (158) and O’Connor (157), some species will respond primarily to reductions in early recruitment, and others to subadult or adult mortality. Different species will show varying degrees of density dependence in their reproductive

151

potential or survival. Acute pesticide impacts likely represent additive density independent mortality, whereas indirect effects may reduce carrying capacity and, therefore, increase density dependence. The legal standard for the protection of birds is very high in some countries. In North America, for example, birds are protected at the level of the individual through a Migratory Bird Treaty Act (or Convention in Canada) between the United States and Canada and between the United States and Mexico. Several cases of bird mortality have gone to court, and applicators have been found guilty and fined for killing migratory birds in the United States, even though they were using approved pesticides. The internal standard applied in those cases was an initial warning to the individual or company followed by a legal suit in the case of repeat offenses (the applicator knew that the use of the pesticide was likely to kill migratory birds based on prior experience). Clearly, current pesticide use, even in developed countries such as the United States does not meet the criterion of individual bird protection set under federal treaty law. A somewhat less stringent criterion was proposed by the Administrator of the EPA in the case of the large number of bird kills that resulted from the use of the organophosphorus insecticide diazinon on turf. The standard of unacceptability proposed by the Administrator in his final decision was that ‘‘Absent some countervailing benefit of continued use, as a matter of policy an unnecessary risk of regularly repeated bird kills will not be tolerated’’ (159). It has long been recognized that such a standard can only be applied unevenly. ‘‘Regularly repeated’’ mortality is not likely to be detected unless the birds in question are large and highly visible and they happen to die in large groups in areas of high public visibility—the case of waterfowl killed by diazinon. Most of the ongoing avian mortality is likely to be of small, highly cryptic, and widely dispersed species. Throughout the 1980s, estimates of the yearly bird mortality in the United States from a single formulation of the carbamate insecticide carbofuran were in the millions of birds (160). For many species, these kills might have been sustainable, although the data to make this determination are lacking. Data from regional or national surveys of wildlife population levels are rarely adequate to demonstrate whether impacts from pesticide use have occurred. The activities of a few individual farmers or pesticide applicators can have disproportionate impacts on birds aggregated on staging or wintering grounds. For example, an estimated 20,000 Swainson’s hawks (Buteo swainsoni) were killed in a small area of the Argentine pampas (118); an estimated 10,000 American robins were killed in two small Florida potato fields (161); several thousand migrants of 37 species were killed by a single mosquito control application of fenthion (124). Yet, losses of dispersed breeding birds on territory, although invisible to all but the most determined researchers, may have a more serious impact on the population. In temperate countries, breeding birds are those that have managed to survive the usual high mortality associated with winter or migration. Also, losses of those individuals affect the reproductive potential of that year’s cohort. Recently, it

152

AVIAN SPECIES

has been determined that the population of the globally threatened population of Eleonora’s falcon (Falco eleonorae) in Crete has been declining by about 15% per year, and this decline is blamed on the insecticide methomyl, which is used abusively to kill cats, rats, martens, and other vertebrates deemed to be undesirable or injurious to crops (162). The insecticide is offered in water, which is in short supply in the arid climate of Crete. Abuse of pesticides has been linked to population declines of rare raptor species in such widely dispersed countries as the United Kingdom, South Africa, and Greece [see review by Mineau and colleagues (45)]. Systematic bird survey data are not available in many countries. Where feasible, local lore and knowledge of the local fauna by the growers and field hands should not be overlooked. The bulk of pesticide application is by hand in developing countries, and field hands develop an intimate knowledge of the field and surrounding areas. Field workers should be encouraged to report any mortality, abnormal behavior, or disappearance of wildlife species. Thought should be given to the setting up of standardized surveys to estimate wildlife abundance and diversity in intensively farmed landscapes. Rare, vulnerable, or ecological keystone species should be used as indicator species where relevant and feasible. The absence of bird carcasses is not necessarily a good indication that any given pesticide use pattern is safe to birds. Despite the years of spraying for eastern spruce budworm in eastern Canada, and a reasonably intensive monitoring effort, only a little more than 100 bird carcasses have been recovered in total (163). This is despite indications that severe impacts were taking place, at least in some years of the program [e.g., Pearce et al. (164) estimated that several million songbirds were killed in New Brunswick in the 1975 spray season]. Also, as seen above, some of the more dramatic effects of pesticides on birds are thought to be through loss of insect food at critical periods of the year. One important point is that populations and ranges of bird species inhabiting farmland are declining globally (e.g., 165,166). Long-term surveys in both northern Europe and North America indicate that many species are showing long-term decreases [e.g., Canada: (166,167); the United States: (168,169); Britain: (170,171); Germany: (172); The Netherlands: (173); and Sweden: (174)]. Most notable is the fact that even common and abundant species such as the house sparrow and eurasian skylark (Alauda arvensis) have been declining. Although it is known that agriculture is responsible for these population declines, it is difficult to isolate specific factors for individual species (171). Many far-reaching changes have taken place in agricultural landscapes over the last 50 years aside from the increase in the use of pesticides. Yet, comparisons in Britain and elsewhere between conventional farms and organic farms (matched by habitat availability) have generally found higher species diversity or abundance on the latter. In some cases, reproductive success has also been found to be higher on organic farms (e.g., 175,176). The decline in the gray partridge and other species can be linked to specific pesticide use patterns, as outlined earlier. These results strongly

implicate agricultural practices and pesticide use, in particular, in the decline of farm bird populations. Finally, we have argued before that, at least in the case of acute poisonings in birds of prey, a few pesticides are responsible for most of the problems (45). Whether or not the pesticides in question cause raptor populations to decline is secondary to the fact that the problems can often be solved at minimal cost to agriculture or to the grower community. BIBLIOGRAPHY 1. E. M. Mrak, Report of the Secretary’s Commission on Pesticides and their Relationship to Environmental Health, U.S. Department of Health, Education and Welfare, Washington, DC, 1969, pp. 1–677. 2. D. A. Kirk, M. D. Evenden, and P. Mineau, Current Ornithology 5: 175–269 (1996). 3. A. W. A. Brown, Ecology of Pesticides, John Wiley and Sons, New York, 1978. 4. S. Cramp, Br. Vet. J. 129: 315–323 (1973). 5. L. J. Blus, in D. J. Hoffman, B. A. Rattner, G. A. Burton, and J. Cairns, eds., Handbook of Ecotoxicology, Lewis Publishers, Boca Raton, 1995, pp. 275–300. 6. R. L. Rudd, Pesticides and the Living Landscape, University of Wisconsin Press, Madison, 1964, pp. 1–320. 7. P. A. Pearce and J. C. Baird, Can. Field-Nat. 85: 82–82 (1970). 8. D. Pimentel, Ecological Effects of Pesticides on Non-Target Species, Office of Science and Technology, Executive Office of the President, Washington, DC, 1971, pp. 1–220. 9. D. B. Peakall and J. R. Bart, CRC Crit. Rev. Environ. Control 13: 117–165 (1983). 10. A. J. Erskine, Atlas of Breeding Birds of the Maritime Provinces, Nimbus/Nova Scotia Museum Program, Halifax, 1992, pp. 1–270. 11. D. A. Ratcliffe, Nature 215: 208–210 (1967). 12. R. G. Heath, J. W. Spann, and J. F. Kreitzer, Nature 224: 47–47 (1969). 13. S. N. Wiemeyer and R. D. Porter, Nature 227: 737 (1970). 14. F. Moriarty, A. A. Bell, and H. Hanson, Environ. Pollut. Ser. A 40: 257–286 (1986). 15. J. J. Hickey and D. W. Anderson, Science 162: 271 (1968). 16. D. B. Peakall, Environ. Rev. 1: 13–20 (1993). 17. T. R. Dunlap, DDT: Scientists, Citizens and Public Policy, Princeton University Press, Princeton, NJ, 1981, pp. 1–318. 18. R. J. Douthwaite and C. C. D. Tingle, DDT in the Tropics—The Impact on Wildlife in Zimbabwe of GroundSpraying for Tsetse Fly Control, Natural Resources Institute, Chatham, UK, 1994, pp. 1–195. 19. M. L. Harris et al., Arch. Environ. Contam. Toxicol. 39: 205– 220 (2000). 20. D. R. Clark, Jr., Dicofol (Kelthane) as an Environmental Contaminant: A Review, United States Fish and Wildlife Service Technical Report No. 29, 1990, pp. 1–37. 21. W. J. Fleming, D. R. Clark, Jr., and C. J. Henny, N. Am. Wildl. Conf. 48: 186–199 (1984).

AVIAN SPECIES

153

22. I. Newton and J. Bogan, Nature 249: 582 (1974).

45. P. Mineau et al., J. Raptor Res. 33: 1–37 (1999).

23. I. Newton, in The Sparrowhawk, T. & A.D. Poyser, Calton, UK, 1986, pp. 1–396.

46. R. Hosea, Impacts from Organophosphate Dormant Sprays on Wintering Raptors in the Central Valley, California, California Department of Fish and Game Report No. 96–5, 1996, pp. 1–22.

24. R. M. Sibly, I. Newton, and C. H. Walker, J. Appl. Ecol. 37: 540–546 (2000). 25. J. C. Okoniewski and E. Novesky, J. Wildl. Manage. 57: 631–639 (1993). 26. W. Stansley and D. E. Roscoe, Environ. Toxicol. Chem. 18: 2095–2099 (1999). 27. G. H. Heinz and R. W. Johnson, Toxicology 12: 189–196 (1979). 28. L. J. Blus, C. J. Henny, T. E. Kaiser, and R. A. Grove, Effects on Wildlife From Use of Endrin in Washington State Orchards, Transactions of the 48th N. American Wildlife & Natural Resources Conference, 1983, pp. 1–15. 29. H. Mendelssohn, Effect of Toxic Chemicals on Bird Life—The Impact of Pesticides on Bird Life in Israel, International Council for Bird Preservation XI Bulletin, 1972, pp. 75–104. 30. L. J. Blus, C. J. Henny, D. J. Lenhart, and T. E. Kaiser, J. Wildl. Manage. 48: 1097–1111 (1984). 31. E. A. Sauter and E. E. Steele, Poultry Sci. 51: 71–76 (1972). 32. C. D. S. Tomlin, The Pesticide Manual, 11th ed., British Crop Protection Council, Binfield, Berks, UK, 1997.

47. C. E. Grue, W. J. Fleming, D. G. Busby, and E. F. Hill, Assessing Hazards of Organophosphate Pesticides to Wildlife, Transactions of the 48th N. Amer. Wildl. & Nat. Res. Conf., Washington, DC, 1983, pp. 200–220. 48. S. L. Porter, in Raptor Biomedicine, University of Minnesota Press, Minneapolis, 1993, pp. 239–245. 49. J. E. Elliott et al., Ecotoxicology 6: 219–231 (1997). 50. J. D. Henderson et al., Bull. Environ. Contam. Toxicol. 52: 641–648 (1994). 51. A. Shlosberg, M. Bellaiche, V. Hanji, and E. Ershov, Vet. Hum. Toxicol. 39: 347–350 (1997). 52. M. A. Gallo and N. J. Lawryk, in W. J. Hayes and E. R. Jr. Laws, eds., Handbook of Pesticide Toxicology, Academic Press, San Diego, 1991, pp. 917–1123. 53. D. David, Arch. Anat. Hist. Embr. Norm. et Exp. 65: 99–110 (1982). 54. U.S. Environmental Protection Agency (EPA) 2000. Available at: http://www.epa.gov/pesticides/reg assessment/. 55. H. Mendelssohn and U. Paz, Biol. Conserv. 11: 163–170 (1977). 56. H. Mendelssohn, P. Schlueter, and Y. Aderet, International Council for Bird Preservation 13: 124–129 (1979).

33. J. G. Vos, H. A. Breeman, and H. Benschop, Mededelingen van de Rijksfacateit Landbouwwetenschoppen te Gent 33: 1263–1269 (1968).

57. P. Berny et al., Chemosphere 35: 1817–1829 (1997).

34. D. C. Boersma, J. A. Ellenton, and A. Yagminas, Environ. Toxicol. Chem. 5: 309–318 (1986).

58. W. B. Stone, J. C. Okoniewski, and J. R. Stedelin, J. Wildl. Dis. 35: 187–193 (1999).

35. R. Eisler, Mirex Hazards to Fish, Wildlife, and Invertebrates: A Synoptic Review, Contaminant Hazard Reviews Report No. 1, U.S.D.I. Fish and Wildlife Service, 1985, pp. 1–42.

59. R. Hosea, in T. P. Salmon and A. C. Crabb, eds., Proceedings of the 19th Vertebrate Pest Conference, University of California Davis, Davis, 2000.

36. P. Mineau et al., in J. O. Nriagu and M. S. Simmons, eds., Toxic Contaminants in the Great Lakes, John Wiley and Sons, Inc., New York, 1984, pp. 426–452.

60. I. Newton, I. Wyllie, and P. Freestone, Environ. Pollut. 68: 101–117 (1990). 61. J. O. Keith and R. L. Bruggers, J. Raptor Res. 32: 151–158 (1998).

37. K. Mellanby, Farming and Wildlife, William Collins Sons & Co., London, 1981, pp. 1–178.

62. K. Borg, H. Wanntorp, K. Erne, and E. Hanko, Viltrevy Swedish Wildlife 6: 301–379 (1969).

38. C. H. Walker, Drug Metabolism Reviews 7: 295–323 (1978).

63. N. Fimreite, R. W. Fyfe, and J. A. Keith, The Canadian Field-Naturalist 84: 269–276 (1970).

39. G. J. Smith, Pesticide Use and Toxicology in Relation to Wildlife: Organophosphorus and Carbamate Compounds, U.S. Fish and Wildlife Service Report, 1987.

64. P. W. Greig-Smith, in R. J. Kendall and T. E. Lacher, eds., Wildlife Toxicology and Population Modeling, Lewis Publishers, Boca Raton, FL, 1994, pp. 301–319.

40. P. Mineau et al., Rev. Environ. Contam. Toxicol. 170: 13–74 (2001).

65. P. Mineau, D. C. Boersma, and B. Collins, Ecotox. Environ. Safety 29: 304–329 (1994).

41. P. Mineau, The Hazard of Carbofuran to Birds and Other Vertebrate Wildlife, Canadian Wildlife Service Technical Report No. 177, Environment Canada, Ottawa, 1993, pp. 1–96.

66. J. C. Galindo, R. J. Kendall, C. J. Driver, and T. E. Lacher, Jr., Behav. Neural. Biol. 43: 21–36 (1985).

42. Th. J. Spierenburg, P. E. F. Zoun, and T. Smit, Poisoning of Wild Birds by Pesticides, Netherlands Society for the Protection of Birds, Central Veterinary Institute, 1989, pp. 23–30.

68. C. E. Grue, A. D. M. Hart, and P. Mineau, in P. Mineau, ed., Cholinesterase-Inhibiting Insecticides: Their Impact on Wildlife and the Environment, Elsevier Science Publishers B. V., Amsterdam, 1991, pp. 151–210.

43. J. C. Franson, L. Sileo, and N. J. Thomas, Causes of Eagle Deaths, National Biological Service, Washington, DC, 1995.

69. C. E. Grue, P. L. Gibert, and M. E. Seeley, Amer. Zool. 37: 369–388 (1997).

44. J. C. Franson et al., J. Raptor Res. 30: 7–14 (1996).

67. K. A. Hunt, D. M. Bird, P. Mineau, and L. Shutt, Anim. Behav. 43: 971–976 (1992).

70. P. Mineau et al., J. Environ. Sci. Health B25: 105–135 (1990).

154

AVIAN SPECIES

71. G. G. Pope and P. Ward, Pestic. Sci. 3: 197–205 (1972).

98. C. T. DeRosa, D. H. Taylor, M. P. Farrell, and S. K. Seilkop, Poultry Sci. 55: 2133–2141 (1976).

72. R. Brunet and A. Cyr, Etude de l’effet du chlorpyrifos sur les niveaux d’acetylcholinesterase et la mortalite chez le merle d’amerique (Turdus migratorius) en captivite, CWS Technical Report No. 105-Quebec region, Environment Canada, 1990, pp. 55–80.

100. N. B. Vyas, W. J. Kuenzel, E. F. Hill, and J. R. Sauer, Environ. Toxicol. Chem. 14: 1961–1965 (1995).

73. A. Fairbrother, S. M. Meyers, and R. S. Bennett, Environ. Toxicol. Chem. 7: 499–503 (1988).

101. P. Mineau, P. T. Boag, and R. J. Beninger, Environ. Toxicol. Chem. 13: 281–290 (1994).

74. H. V. McKay et al., J. Appl. Ecol. 36: 283–296 (1999). 75. L. K. Nicolaus and H. Lee, Ecol. Appl. 9: 1039–1049 (1999).

102. G. R. Potts, The Partridge: Pesticides, Predation and Conservation, William Collins Sons and Co., London, UK, 1986.

76. P. Mineau, Chemicals in Agriculture—CholinesteraseInhibiting Insecticides: Their Impact on Wildlife and the Environment, Elsevier Science Publishing Company Inc., New York, 1991, pp. 2–342.

103. J. A. Ewald and N. J. Aebischer, Pesticide Use, Avian Food Resources and Bird Densities in Sussex, Report No. 296, Joint Nature Conservation Committee, Peterborough, UK, 1999, pp. 1–103.

77. T. Colborn, D. Dumanoski, and J. P. Myers, Our Stolen Future, Penguin, New York, 1996, pp. 1–306.

104. R. J. O’Connor and M. Shrubb, Farming and Birds, Cambridge University Press, Cambridge, 1986.

78. L. L. Feyk and J. P. Giesy, in Principles and Processes for Evaluating Endocrine Function in Birds, SETAC Press, Pensacola, FL, 1998, pp. 121–140.

105. C. Borg and S. Toft, J. Appl. Ecol. 37: 557–563 (2000).

79. B. A. Rattner, L. Sileo, and C. G. Scanes, J. Reprod. Fert. 66: 147–155 (1982). 80. B. A. Rattner and J. C. Franson, Can. J. Physiol. Pharmacol. 62: 787–792 (1984). 81. V. Burgat-Sacaze, A. G. Rico, and C. Petit, in Relations entre les traitements phytosanitaires et la reproduction des animaux, ANPP Annales, Paris, 1990, pp. 187–204. 82. A. Fairbrother, in R. J. Kendall and T. E. Lacher, Jr., eds., Wildlife Toxicology and Population Modeling-Integrated Studies of Agroecosystems, Lewis, Boca Raton, 1994, pp. 251–261. 83. C. A. Bishop et al., J. Toxicology and Environmental Health, Part A 55: 561–581 (1998). 84. C. A. Bishop et al., J. Toxicology and Environmental Health, Part A 55: 531–559 (1998). 85. G. Heinz, Bull. Environ. Contam. Toxicol. 131: 554–564 (1975).

99. R. Brunet and A. Cyr, Agric. Ecosys. Environ. 41: 327–336 (1992).

106. J. D. Moulding, The Auk 93: 692–708 (1976). 107. S. B. Holmes, J. Appl. Ecol. 35: 185–194. 1998. 108. N. L. Rodenhouse and R. T. Holmes, Ecology 73: 357–372 (1992). 109. P. J. Sheehan et al., The Impact of Pesticides on the Ecology of Prairie-Nesting Ducks, Canadian Wildlife Service Technical Report No. 19, Environment Canada, Hull, Québec, 1987, pp. 1–696. 110. P. J. Sheehan, A. Baril, P. Mineau, and D. J. Paustenbach, in G. M. Rand, ed., Fundamentals of Aquatic Toxicology: Effects, Environmental Fate and Risk Assessment, Taylor and Francis, Washington, DC, 1995, pp. 827–857. 111. L. W. Dwernychuk and D. A. Boag, Can. Field-Nat. 87: 155–165 (1973). 112. P. Mineau and A. McLaughlin, Journal of Agricultural and Environmental Ethics 9: 93–113 (1996). 113. C. J. Driver et al., Environ. Toxicol. Chem. 10: 21–33 (1991).

86. G. Heinz, J. Wildl. Manage. 40: 710–715 (1976).

114. W. B. Stone, Direct Testimony of Ward Stone in the Matter of Ciba-Geigy Corp et al., U.S. EPA FIFRA Docket Nos. 562 EPA Exhibit W-5, 1987.

87. B. S. Wright, J. Wildl. Manage. 29: 172–185 (1965).

115. L. J. Blus et al., J. Wildl. Manage. 53: 1139–1146 (1989).

88. T. G. Dilworth, J. A. Keith, P. A. Pearce, and L. M. Reynolds, J. Wildl. Manage. 36: 1186–1193 (1972).

116. R. Balcomb, J. Wildl. Manage. 47: 1129–1132 (1983).

89. D. G. Busby, L. M. White, and P. A. Pearce, J. Appl. Ecol. 27: 743–755 (1990). 90. C. A. Bishop et al., Environ. Toxicol. Chem. 19: 588–599 (2000). 91. K. M. Fluetch and D. W. Sparling, Environ. Toxicol. Chem. 13: 1651–1659 (1994). 92. K. A. Patnode and D. H. White, Environ. Toxicol. Chem. 10: 1479–1487 (1991). 93. D. J. Hoffman and P. H. Albers, Arch. Environ. Contam. Toxicol. 13: 15–27 (1984). 94. D. B. Peakall, Residue Reviews 96: 45–77 (1985). 95. H. G. Heinz, Environ. Toxicol. Chem. 8: 463–464 (1989). 96. P. Mineau, in P. Mineau, ed., Cholinesterase Inhibiting Insecticides, Elsevier Science Publishers B.V., Amsterdam, 1991, pp. 277–299. 97. A. D. M. Hart, Environ. Toxicol. Chem. 12: 321–336 (1993).

117. D. R. Dietrich et al., Arch. Environ. Contam. Toxicol. 29: 140–145 (1995). 118. M. J. Hooper et al., Pesticide Outlook 10: 97–102 (1999). 119. L. B. Best, Am. Midl. Nat. 128: 126–138 (1992). 120. E. E. Littrell, Calif. Fish Game 74: 226–231 (1988). 121. J. de Leeuw et al., Risks of Granules and Treated Seeds to Birds on Arable Fields, CML Report No. 118, 1995, pp. 1–131. 122. P. W. Greig-Smith, Hazards to Wildlife From Pesticide Seed Treatments, British Crop Protection Council Monograph No. 39, 1987, pp. 127–134. ¨ 123. V. M. Hommes, W. Buchs, G. Joermann, and J. Siebers, Nachrichtenbl. Deut. Pflanzenschutzd. 42: 113–117 (1990). 124. R. W. Seabloom, G. L. Pearson, L. W. Oring, and J. R. Reilly, J. Wildl. Dis. 9: 18–20 (1973). 125. L. R. DeWeese, L. C. McEwen, L. A. Settimi, and R. D. Deblinger, J. Econ. Entomol. 76: 906–911 (1983).

AVIAN SPECIES 126. S. Keeling, Environment Western Australia 6: 6–8 (1984). 127. P. C. James, G. A. Fox, and T. J. Ethier, J. Raptor Res. 24: 120–123 (1990). 128. C. J. Henny, L. J. Blus, E. J. Kolbe, and R. E. Fitzner, J. Wildl. Manage. 49: 648–658 (1985). 129. B. D. Pauli, S. B. Holmes, R. J. Sebastien, and G. P. Rawn, Fenitrothion Risk Assessment, Technical Report No. 163, Canadian Wildlife Service, Environment Canada, Ottawa, 1993, pp. 1–75.

155

146. F. D. Hoerger and E. E. Kenaga, Environmental Quality, Academic Press, New York, 1972, pp. 9–28. 147. J. S. Fletcher, J. E. Nellessen, and T. G. Pfleeger, Environ. Toxicol. Chem. 13: 1383–1391 (1994). 148. E. F. Hill and M. B. Camardese, Ecotoxicol. Environ. Saf. 8: 551–563 (1984). 149. E. F. Hill, Caution: Standardized Acute Toxicity Data May Mislead, Report No. 6-86, Research Information Bulletin, 1986, pp. 1–2.

130. J. G. Zinkl, C. J. Henny, and L. R. DeWeese, Bull. Environ. Contam. Toxicol. 17: 379–386 (1977).

150. K. M. Kilbride, J. A. Crawford, and B. A. Williams, Env. Tox. Chem. 11: 1337–1343 (1992).

131. K. A. Hunt, D. M. Bird, P. Mineau, and L. Shutt, Arch. Environ. Contam. Toxicol. 21: 84–90 (1991).

151. M. R. Fletcher and P. W. Greig-Smith, The Use of Direct Observations in Assessing Pesticide Hazards to Birds, British Crop Protection Council Monograph No. 40: Environmental effects of pesticides, 1988, pp. 47–55.

132. L. W. Touart and A. F. Maciorowski, Ecol. Appl. 7: 1086– 1093 (1997). 133. E. C. Fite et al., Hazard Evaluation Division, Standard Evaluation Procedure: Guidance Document for Conducting Terrestrial Field Studies, Report No. EPA/540/09–88/109, Environmental Protection Agency, Office of Pesticide Programs, Washington, DC, 1988, pp. 1–66. 134. M. P. Greaves, B. D. Smith, and P. W. Greig-Smith, eds., Field Methods for the Study of Environmental Effects of Pesticides, BCPC, Thornton Heath, UK, 1988, pp. 1–370. 135. L. Somerville and C. H. Walker, eds,, Pesticide Effects on Terrestrial Wildlife, Taylor & Francis, London, 1990, pp. 1–395. 136. D. J. Urban, in L. W. Brewer and K. A. Fagerstone, eds., Radiotelemetry Applications for Wildlife Toxicology Field Studies, SETAC, Pensacola, FL, 1998, pp. 1–10. 137. L. J. Blus and C. J. Henny, Ecol. Appl. 7: 1132 (1997). 138. OECD, Report of the SETAC/OECD Workshop on Avian Toxicity Testing, Report No. 5, OECD, Paris, 1996, pp. 1–185. 139. A. D. M. Hart et al., eds., Avian Effects Assessment: A Framework for Contaminant Studies, SETAC Press, Pensacola, FL, 2001, pp. 1–193. 140. R. Luttik and T. Aldenberg, Environ. Toxicol. Chem. 16: 1785–1788 (1997). 141. J. A. Pascual, A. D. M. Hart, and S. L. Fryday, Environ. Toxicol. Chem. 18: 247–253 (1999). 142. A. Baril, B. Jobin, P. Mineau, and B. T. Collins, A Consideration of Inter-species Variability in the Use of the Median Lethal Dose (LD50 ) in Avian Risk Assessment, Canadian Wildlife Service Technical Report No. 216, Canadian Wildlife Service (headquarters) Environment Canada, Ottawa, 1994, pp. 1–12. 143. P. Mineau, B. T. Collins, and A. Baril, Regul. Toxicol. Pharmacol. 24: 24–29 (1996). 144. P. Mineau, B. Jobin, and A. Baril, A Critique of the Avian 5-Day Dietary Test (LC50 ) as the Basis of Avian Risk Assessment, Canadian Wildlife Service Technical Report No. 215 Canadian Wildlife Service (headquarters), Environment Canada, Ottawa, 1994, pp. 1–23. 145. C. Boutin, K. E. Freemark, and C. J. Keddy, Proposed Guidelines for Registration of Chemical Pesticides: Nontarget Plant Testing and Evaluation, Canadian Wildlife Service Technical Report No. 145, Canadian Wildlife Service (headquarters), Environment Canada, Ottawa, 1993, pp. 1–91.

152. G. P. Cobb and M. J. Hooper, in R. J. Kendall and T. E. Lacher, Jr., eds., Wildlife Toxicology and Population Modeling—Integrated Studies of Agroecosystems, Lewis, Boca Raton, 1994, pp. 35–46. 153. D. M. Fry, et al., in L. W. Brewer and K. A. Fagerstone, eds., Radiotelemetry Applications for Wildlife Toxicology Field Studies, SETAC, Pensacola, FL, 1998, pp. 67–84. 154. P. W. Greig-Smith, in P. Mineau, ed., Cholinesterase-Inhibiting Insecticides. Their Impact on Wildlife and the Environment, Elsevier Science Publishers B.V., Amsterdam, 1991, pp. 127–150. 155. D. B. Peakall, Animal Biomarkers as Pollution Indicators, Chapman and Hall, London, UK, 1992. 156. K. Borg, K. Erne, E. Hanko, and H. Wanntorp, Environ. Pollut. 1: 91–104 (1970). 157. R. J. O’Connor, in R. J. Kendall and T. E. Lacher, Jr., eds., Wildlife Toxicology and Population Modeling—Integrated Studies of Agroecosystems, Lewis, Boca Raton, 1994, pp. 283–300. 158. J. M. Emlen, Environ. Toxicol. Chem. 8: 831–842 (1989). 159. U.S. Environmental Protection Agency (EPA), Remand Decision in the Matter of: Ciba-Geigy et al. Docket Nos.562 et al., Administrator, USEPA, Washington, DC, 1990. 160. U.S. Environmental Protection Agency (EPA), Carbofuran: Special Review Technical Support Document, Office of Pesticides and Toxic Substances, USEPA, Washington DC, 1989. 161. C. Lee, The Florida Naturalist 45: 60–61 (1972). 162. D. Ristow, International Hawkwatcher 3: 10–17 (2001). 163. D. G. Busby, L. M. White, P. A. Pearce, and P. Mineau, in W. R. Ernst, P. A. Pearce, and T. L. Pollock, eds., Environmental Effects of Fenitrothion Use in Forestry. Impacts on Insect Pollinators, Songbirds and Aquatic Organisms, Environment Canada, Atlantic Region, Dartmouth, 1989, pp. 43–108. 164. P. A. Pearce, D. B. Peakall, and A. J. Erskine, Impact on Forest Birds on the 1975 Spruce Budworm Spray Operation in New Brunswick, Canadian Wildife Service Progress Report No. 62, Environment Canada, Ottawa, 1976, pp. 1–7. 165. D. J. Pain and M. Pienkowski, Farming and Birds in Europe: The Common Agricultural Policy and Its Implications for Bird Conservation, Academic Press, San Diego, 1997. 166. E. H. Dunn, Bird Trends 6: 2–10 (1998).

156

AVICIDE

167. C. M. Downes and B. T. Collins, The Canadian Breeding Bird Survey, 1966–1994, Canadian Wildlife Service Report No. 210, Canadian Wildlife Service, Environment Canada, Hull, Québec, Canada, 1996. pp. 1–36. 168. F. L. Knopf, Declining Grassland Birds, U.S. Department of the Interior, National Biological Service, Washington, DC, 1995, pp. 296–298. 169. R. E. Warner, Cons. Biol. 8: 147–156 (1994). 170. R. J. Fuller et al., Cons. Biol. 9: 1425–1441 (1995). 171. G. Sirawardena et al., J. Appl. Ecol. 35: 24–43 (1998). 172. M. Flade and K. Steiof, Population Trends of Common North-German Breeding Birds, 1950–1985: An Analysis of More Than 1400 Census Plots, Proceedings 100th International Meeting, Deutsche Ornithologen-Gesellschaft, Bonn, 1990. 173. F. A. Saris et al., in W. Hagemmeijer and T. Verstrael, eds., Bird Numbers 1992: Distribution, Monitoring and Ecological

Aspects, Proceedings 12th International Conference of IBCC and EOAC. SOVON, Beek-Ubbergen, The Netherlands, 1994, pp. 75–85. 174. J. G. Robertson and C. Berg, Ornis Sveccia 2: 119–130 (1992). 175. J. D. Wilson, J. Evans, S. J. Browne, and J. R. King, J. Appl. Ecol. 34: 1462–1478 (1997). 176. N. Elmegaard, P. N. Andersen, P. Odderskaer, and A. Prang, in N. J. Adams, and R. H. Slotow, eds., Birdlife South Africa, Proceedings of the 22nd International Ornithological Congress, Durban, South Africa, 1999, pp. 1058–1069.

AVICIDE A chemical for killing birds (CIPAC).

B BACTERICIDE

bacteria. The incorporation of mancozeb or maneb with a copper bactericide has been reported to be effective against pathogen isolates classified as copper resistant or tolerant (4,12,13). Streptomycin is the most commonly utilized bactericidal antibiotic for the management of plant pathogens and has been used most frequently on apple, pear, sweet pepper, and ornamental trees. Following the introduction of streptomycin in the early 1950s, several reports appeared of the effectiveness of this compound in managing pathogen populations and disease incidence (14). However, continued usage of streptomycin has often resulted in disease control failures due to the onset of streptomycin resistance in pathogen populations. In contrast to the situation with copper, streptomycin is typically ineffective in reducing populations of streptomycin-resistant bacterial pathogens (15,16), resulting in the need to utilize additional compounds (in many cases, another antibiotic, oxytetracycline) in disease management programs.

GEORGE W. SUNDIN Michigan State University East Lansing, Michigan

Bactericides, compounds that are bactericidal or capable of killing bacteria, have been used for several decades in plant disease management programs. On most agricultural crops, copper-containing compounds are the most commonly utilized bactericide, with antibiotics, most notably streptomycin, applied in situations in which phytotoxicity problems occur with copper usage or in which disease suppression is insufficient when copper compounds are used alone. Of minor importance, the compound 2-bromo-2-nitropropane-1,3-diol (bronopol), a bactericidal preservative used in cosmetic products, has also been reported in the treatment of cotton seeds to control bacterial blight (1), and to control blotch disease of mushroom caused by Pseudomonas tolaasii (2). An additional indirect bactericidal method that could have important future application is the use of chemicals or biotic agents involved in the activation of induced systemic resistance (ISR) in the plant host. The activation of ISR in cucumber by plant growth-promoting rhizobacteria or the synthetic elicitor acibenzolar-S-methyl (ActigardTM ) has been reported to significantly reduce angular leaf spot caused by P. syringae pv. lachrymans in field trials (3). There are a large number of copper-containing bactericides available, including CuSO4 , CuSO4 + lime, Cu(OH)2 , and Cu(NH3 )2 + CuCO3 . Free Cu2+ ion is the form of copper toxic to bacteria; however, copper readily forms complexes with amino acids, carbohydrates, and other organic materials, which can significantly reduce the bactericidal activity of this element (4). Once applied to leaf surfaces, copper compounds can be solubilized, presumably by interacting with organic compounds leached from leaves (5); however, very few bactericidal free Cu2+ ions are present (6). Also the amount of solubilized copper and free Cu2+ ions present is dependent upon leaf surface chemistry, a factor that differs among plant species (6). Additional measures can be utilized to increase the effectiveness of copper bactericides; coppercontaining compounds can be combined with copperchelating fungicides such as mancozeb or maneb, or with iron (FeCl3 · 6H2 O) to increase the availability of free Cu2+ ions (7). Iron also alters the physiology of the target pathogen, making the pathogen more susceptible to copper toxicity (8). Field trials with copper-containing bactericides generally yield variable results (9–11). This may be due to environmental and plant species factors affecting the availibility of copper ions on leaves, and also due to the presence of pathogen isolates differing in sensitivity to copper. Indeed, copper-resistant isolates, which can tolerate two to five times more copper than sensitive strains, commonly occur in field populations of plant-pathogenic

BIBLIOGRAPHY 1. B. Honervogt and H. Lehmanndanzinger, J. Phytopathol. 134: 103–109 (1992). 2. W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 58: 275–281 (1985). 3. G. S. Raupach and 1158–1164 (1998).

J. W. Kloepper,

Phytopathology

88:

4. O. Menkissoglu and S. E. Lindow, Phytopathology 81: 1258– 1263 (1991). 5. P. Arman and R. L. Wain, Ann. Appl. Biol. 46: 366–374 (1958). 6. O. Menkissoglu and S. E. Lindow, Phytopathology 81: 1263– 1270 (1991). 7. G. M. Marco and R. E. Stall, Plant Dis. 67: 779–781 (1983). 8. Y.-A. Lee et al., Phytopathology 83: 1460–1465 (1993). 9. B. D. Olson and A. L. Jones, Phytopathology 73: 1520–1525 (1983). 10. S. M. McCarter, Plant Dis. 76: 1042–1045 (1992). 11. K. A. Garrett and H. F. Schwartz, Plant Dis. 82: 30–35 (1998). 12. C. S. Kousik, D. C. Sanders, and D. F. Ritchie, Phytopathology 86: 502–508 (1996). 13. H. J. Scheck and J. W. Pscheidt, Plant Dis. 82: 397–406 (1998). 14. W. J. Zaumeyer, in Proceedings of First International Conference on the Use of Antibiotics in Agriculture, National Academy of Sciences—National Research Council, Washington, D.C., 1956, pp. 171–187. 15. P. S. McManus and A. L. Jones, Phytopathology 84: 627–633 (1994). 16. G. W. Sundin and C. L. Bender, Can. J. Microbiol. 40: 289–295 (1994). 157

158

BACTERIOCINS

BACTERIOCINS ANNE K. VIDAVER University of Nebraska Lincoln, Nebraska

Bacteriocins, or ‘‘bacteria-killers,’’ are nonreplicating substances distinguished principally by their relatively narrow range of specificity against strains of the same or related species. They are thus specialized antibiotics, principally protein in nature. Inhibitory activity can range from a single or a few related strains to many strains of different taxa (1). Many, if not all, phytopathogenic and plant-associated bacteria produce bacteriocins (2,3). Relatively few have been studied, but producing species have been reported in Agrobacterium, Burkholderia, Clavibacter, Curtobacterium, Erwinia, Pseudomonas, Ralstonia, Rathayibacter, and Xanthomonas. The substances are usually given common names based on the producing genus or species and strain, for example, agrocins produced by Agrobacterium (4) and syringacins produced by P. syringae (5). The composition of most bacteriocins to date appears to be protein, and some resemble bacteriophages (1). Such structures are considered defective phage particles. These high molecular weight particles are generally resistant to trypsin (protease) and the heat labile, in contrast to low molecular weight bacteriocins, which are usually inactivated by trypsin, the heat stable, and nonsedimentable by centrifugation. In Agrobacterium radiobacter (A. rhizogenes or nontumorigenic A. tumefaciens) K84, the major specific bacteriocin produced is a ‘‘fraudulent’’ or adenine-substituted nucleotide (6); a second bacteriocin produced by the same strain and a transfer-deficient derivative appears to be a di-substituted cytidine nucleoside (4). Bacteriocins are secreted by producing bacteria under different conditions, often physical depending on the growth stage of the producer. Usually only a small number of cells in a culture are producers, as production is usually lethal to the producing cell. Factors such as UV light, mitomycin C, temperature, and media components can affect production. The effects of such agents currently cannot be well predicted. Producers may secrete more than one bacteriocin depending upon the environment or condition of growth, as well as genetic capability. In some cases producers may become sensitive to their own bacteriocin in late stages of growth, but immunity is the norm. Detection of bacteriocins is usually done in vitro, with agar-grown or liquid-grown potential producers assayed against potential susceptible strains on agar plates. If liquid cultures are used, both cells and supernatants from centrifuged suspensions should be tested for bacteriocin production. Generally cells in late-log phase or stationary phase are the best producers. Bacteriocin production and sensitivity may be used to differentiate strains that are otherwise indistinguishable; such bacteriocin typing has been used in epidemiological studies.

Synthesis of bacteriocins may be inherent to the producer or conferred by genetic exchange. Genes for production may be plasmid borne (7) or on a chromosome. If genes are on plasmids, they may be referred to as bacteriocinogenic factors. Few bacteriocins of plant-pathogenic and plantassociated bacteria have been isolated biochemically and characterized (3,4,6,8). Thus their respective modes of action are not well known. Those studied from other groups of bacteria have different modes of action: some inhibit protein synthesis, others affect DNA replication. A single molecule or particle of bacteriocin may be sufficient to kill a sensitive cell. All appear to require a specific receptor site on the cell wall, membrane, or in the periplasmic space. Resistance may occur in populations of sensitive cells. Resistance may be present in a small fraction of cells, or mutation to resistance may occur. Due to the difficulty of the task, few studies of bacteriocin production and sensitivity have been done on or in plants or in the environment. Indeed, it is not known whether the highly efficacious commercially available biocontrol strains A. radiobacter K84 and K1026 (7), produce bacteriocins in soil or on plant roots. In vitro, the strains are inhibitory against many strains of A. tumefaciens and A. rhizogenes of different geographic origin. These strains are the only current examples of bacteriocin producers widely used in plant pathology. Bacteriocins and producers are finding use in food microbiology, notably nisin from Lactococcus lactis, used by 45 countries in pasteurized cheese spreads and other products to inhibit clostridia (9). Due to both widespread production and sensitivity among many bacteria studied, it is assumed that such substances have a role in niche competition (10).

BIBLIOGRAPHY 1. M. A. Daw and Falkiner, Micron 27: 467–479 (1966). 2. D. C. Gross and A. K. Vidaver, in Z. Klement, K. Rudolph, and D. C. Sands, eds., Methods in Phytopathology, Akademiai Kiado, Budapest, Hungary, 1990, pp. 245–249. 3. D. C. Sigee, Bacteral Plant Pathology: Cell and Molecular Aspects, Cambridge University Press, l993, pp. 287–292. 4. S. C. Donner et al., Physiol. Mol. Plant Pathol. 42: 185–194 (1993). 5. M. L. Smidt and A. K. Vidaver, Can. J. Microbiol. 28: 600– 604 (1982). 6. W. P. Roberts, M. E. Tate, and A. Kerr, Nature 265: 379–381 (1977). 7. N. C. McClure, A.-R. Ahmadi, and B. G. Clare, Appl. Env. Microbiol. 64: 3977–3982 (1998). 8. A. K. Vidaver, Plant Dis. 67: 471–475 (1983). 9. J. Raloff, Science News 153: 89,90 (1998). 10. M. A. Riley and D. M. Gordon, Trends Microbiol. 7: 129–133 (1999).

BIOASSAYS: PHYTOTOXICITY TO SUCCEEDING CROPS

BAND APPLICATION Application of pesticide to a continuous restricted area such as a crop row.

BATCH A specific quantity or lot of a test or reference substance produced during a defined cycle of manufacture in such a way that it could be expected to be of a uniform character and should be designated as such (OECD).

BBA Biologisches Bundesanstalt Abteilung (German Regulatory authority)

BEER-LAMBERT LAW This describes the relationship between the incident light and the light absorbed by a molecular species. It may be expressed in the form: D = 1/Aλ = εcl, where D is the optical density and Aλ is the absorbance (defined as log I0 /It , where It is the intensity of the light transmitted through the sample and I0 is the intensity of the incident beam at a fixed wavelength λ). ε λ is the molar absorptivity (or molar extinction coefficient) of the substance, c is its molar concentration and l is the path length through the cell containing the substance.

BIOACCUMULATION Process of accumulation of a pesticide residue in an organism due to both direct uptake from the environmental matrix (bioconcentration) and uptake from food (biomagnification) (IUPAC).

BIOASSAYS: PHYTOTOXICITY TO SUCCEEDING CROPS WILFRIED PESTEMER Federal Biological Research Centre of Agriculture and Forestry Berlin, Germany

Bioassays in general are defined as experiments for estimating the potency of a chemical by analysis of the reaction that follows its application to living organisms (1). Within the scope of this chapter, a quantitative bioassay can be defined as an assay in which an unknown amount of a herbicide or other phytotoxic pesticide is measured by comparing the reaction that follows its application with the reaction caused by known standard rates of the same compound.

159

Bioassay can be classified as an indirect method to determine herbicide concentration, which possesses some limitations. Only phytotoxic compounds can be detected; therefore, they are mainly applicable to potentially phytotoxic compounds, mainly herbicides. However, this can be detrimental, if the amount of the compound is overestimated due to phytotoxic metabolites, or if a mixture of chemicals is to be analyzed. Most bioassay methods require a time of at least some days to show an adequate response, so that dissipation of the herbicide during the test period and the resulting steady change of the concentration must be taken into account. The range of sensitivity is often limited, because a close relationship between dose and response occurs only within a comparatively small range of concentrations. Because of these limitations, bioassays have been regarded as insensitive, time-consuming, and inconsistent during the period of fast improvement of chemical methods. Bioassays can provide low-cost and sensitive analyses, especially for highly biologically active compounds, for screening purposes and during the development of herbicides, whereas physical–chemical methods of analysis are not yet available. AUTHORIZATION OF PLANT PROTECTION PRODUCTS (PPP) When considering risks, a distinction has to be made between the risks to crop production or the other purposes of plant protection and to users, consumers, or the natural balance. A report on risk reduction in the field of plant protection products (PPP) in Germany was published recently by the Federal Ministry of Food, Agriculture and Forestry (2), and some considerations relevant to this chapter are summarized in the following. The legal basis for the authorization of PPP in the Federal Republic of Germany lies in the Plant Protection Act (3). Authorization and marketing of plant protection products in the Federal Republic of Germany are subject to very strict guidelines. To eliminate risks from plant protection products to the greatest possible extent, they may only be marketed once they have been authorized by the Federal Biological Research Centre for Agriculture and Forestry (BBA). The BBA determines that authorization of a PPP may only be granted if, among other things, examination of the PPP shows that: 1. The PPP is sufficiently effective in the light of scientific knowledge and technique. 2. The requirements for the protection of the health of humans and animals when handling hazardous materials do not conflict with the authorization. 3. The PPP, when used for its intended purpose and in the proper manner, or as a result of such use: a. Does not have any harmful impact on human and animal health or on groundwater. b. Does not have any other impact, particularly with regard to ‘‘adverse ecological effects,’’ which in the light of the present state of scientific knowledge is not justifiable.

160


The criteria used by the Federal Biological Research Centre (BBA) to test whether the plant protection product fulfils authorization requirements are determined according to BBA directives, which encompass internationally recognized directives of the Food and Agricultural Organization (FAO), Organization of Economic Development and Cooperation (OECD), and World Health Organization (WHO). They can be found in the pamphlet ‘‘Criteria for assessment of plant protection products in the registration procedure’’ (4) and are practically in agreement with Directives of the European Union (EU). The requirement for authorization is proof that the product is sufficiently efficacious according to the current state of scientific knowledge and techniques. No claims of efficacy, which involve unfavorable practical conditions, (although it is possible to control a harmful organism or achieve a protective purpose under unfavorable practical conditions), can be accepted. Another part of the strategy of risk reduction is to deny authorization of a PPP, which, although efficient, causes plant damage, reduction in quality, or any other disadvantageous impact (such as changes in flavor, etc.). This also applies to phytotoxic damage in succeeding crops. It must be shown on the basis of experiments with graded dosages that the absolute minimum amount of the product necessary (‘‘marginal amount’’) is not exceeded. Under Commission Directive 93/71/EEC, applicants must provide evidence on a range of efficacy-related issues, including phytotoxicity and unintended sideeffects to succeeding crops. It is also a requirement that trials submitted in support of an authorization must be conducted by an ‘official’ or ‘officially recognised’ testing organization.

has a temporary impact on the agro-ecosystem. In this chapter, an overview will be given of the potential phytotoxic effects of herbicides on higher plants, especially those on succeeding crops. Phytotoxicology is defined as a field of study concerned with effects arising from chemicals taken up by plants via air or soil either unintentionally or intentionally. The presence of xenobiotics—like other growth factors—does not influence higher plants adversely until a substantial excess of the natural exposure level occurs. Positive effects of low exposure doses are occasionally observed, but they do not affect crop yields significantly. The activity remains detectable for a certain period depending on environmental conditions. To estimate the potential phytotoxicity, it is necessary to have sufficient knowledge of the dynamics of xenobiotic substances. Different factors and processes, like climate, soil moisture, and temperature; physical–chemical behavior of chemicals; soil parameters; and so on, influence the distribution of a substance within the compartments of an agro-ecosystem as shown in Table 1. Xenobiotics reach the soil either intentionally, by direct application, or unintentionally through spillage. In both cases, undesirable side effects may occur, just as with many pollutants of the environment. Harmful substances are removed by different factors, thus cleansing the environment. The extent of any harmful effects depends on the rate of breakdown, as well as the leaching pattern of the substance, which is influenced by the soil characteristics, such as variations in organic carbon and water content or pH-value varying in the soil profile.

HERBICIDE BEHAVIOR CONNECTED WITH POTENTIAL PHYTOTOXICITY

PLANT AVAILABILITY AND UPTAKE OF HERBICIDES FROM SOIL

Environmental chemicals are substances produced or introduced into the environment by human activities and may be potentially hazardous to biota and humans. In this case, biota means all animals, plants, and microorganisms (5). Herbicides are an essential component of modern techniques used in plant and food production by agricultural and horticultural systems. Every cropping system

The availability of herbicides in the soil to plants depends on many factors, including the physicochemical properties of the compound, climatic and soil conditions, the activities of microflora and fauna in the soil and the method of application, all of which influence the fate of the active compound. They all exist simultaneously and influence each other, resulting in a complex dynamic behavior.

Table 1. Important Compartments of Ecosystems and Xenobiotic Dynamics Compartment

Important Influences

Important Factors and Processes

Atmosphere

Movement of air, rainfall, temperature

Climatic factors, photolysis, volatilization, drift, emission, deposition, radiation

Standing crop

Microclimate

Ecoclimate, evapotranspiration, plant cover, nutrient supply, plant diseases

Tillage zone

Soil moisture and soil temperature

Edaphon (soil organisms), degradation, metabolism, sorption, convection, dispersion, diffusion, erosion

Root zone (unsaturated zone)

Moisture content, water movement

Edaphon, rooting, macropores, leaching

Saturated zone

Groundwater table, ground water stream

Sorption, convection, dispersion


Herbicides must first be taken up into the plant before becoming active. Depending on the type of application, different sites of uptake are possible. From post-emergence application the compound penetrates into the plant via the shoot and the leaves, but root uptake might also occur under these conditions. After pre-sowing or pre-emergence application, i.e., after application of the herbicide onto or into the soil, the active compound may be taken up via the seed, the embryo, the seedling, or the adult plant. The herbicide may be taken up actively or may passively penetrate into the plant. The key processes, adsorption, desorption, and degradation, determine the availability of chemicals to plants in the soil. These processes are time dependent, as shown in Figure 1. The figure illustrates that only a part of the amount of herbicide originally applied to the soil surface is available to the plant. Soil is the focus of interest, because all chemicals used by humans contaminate the upper soil layer to some extent. The soil is acting as a filter, a buffer, and a transformation system in close connection to differing soil contaminations by discharge of substances through garbage, waste water, or air pollution and the intensive and concentrated land use, e.g., by agriculture, human habitations, and recreation. The importance attached to soil is also shown by the diversity of governmental responsibilities. Damages to plants arise from Inorganic and organic pollutants, phytotoxic gas, and ionizing radiation taken up from the air Inorganic and organic pollutants [e.g., agrochemicals, environmental chemicals (xenobiotics), heavy metals] taken up via soil Every year hundreds of new chemical substances are created. Because many of these substances differ significantly from natural biochemicals, detailed studies have been designed to discover potential harmful effects. The qualitative and quantitative assessment of xenobiotic pollution in the different compartments of the environment (soil, water, air, plants, animals, and humans) is the basis for conducting specific remedial measures. Virtually all

161

treatment measures have side effects that accompany the desired protection of plants. It is up to research to assess and evaluate the benefit-risk ratio. Furthermore, trace level contamination of crop plants may also be detected arising from a variety of sources (e.g., industry and motor vehicle emissions, households, horticulture, agriculture, and radiation). A prerequisite for obtaining reliable data is the use of modern analytical technology, e.g., gas chromatography with specific detectors and in combination with mass spectrometry, high performance liquid chromatography, atomic absorption, and emission spectrometry. All xenobiotics, like pesticides and environmental chemicals, are dispersed in the natural environment. To estimate the exposure, accumulation, and toxicity to organisms, the order of magnitude of transfer processes among soil, water, and air and the resulting concentrations in the respective compartments have to be assessed. Figures 2 and 3 shows the main factors influencing the soil-plant-transfer of chemicals and the mechanisms of plant contamination by xenobiotics. The activity remains detectable for a certain period depending on environmental conditions. To estimate the potential phytotoxicity, it is necessary to have sufficient knowledge of the dynamics of the substance in question. Inactivation, distribution, and their availability to plants (uptake) are important processes, which can occur simultaneously and may influence each other. In this connection, it is worth mentioning that symptoms of chemical damage are similar to damages caused by frost, heat, nutrient deficiency, or excess, as shown in Table 2. In this connection, it is necessary to consider that plants show only a restricted pattern of reactions. The assignment of damage symptoms is therefore very rarely clear. Furthermore, a universally valid statement or a prediction based on expected effects is not possible, because a combination of effects may occur, leading to unexpected and confusing symptoms. Another important factor in the risk assessment of chemicals is their availability to plants. This affects not only activity in plants, but also degradation and the potential duration of effects. Additionally, data on degradation rates, adsorption, and solubility in

Herbicide application Soil surface Time Availability:

Herbicide not adsorbed plant available

Herbicide reversibly adsorbed

Herbicide irreversibly adsorbed

Behavior:

Herbicide not leached not volatilized not degraded

Metabolites not and/or reversibly adsorbed

Metabolites irreversibly adsorbed

Activity:

Herbicide + Metabolites plant available biologically active

Metabolites biologically non-active

Figure 1. Main factors determining plant availability of herbicides.

162

BIOASSAYS: PHYTOTOXICITY TO SUCCEEDING CROPS Table 2. Damage Symptoms of Potential Phytotoxic Substances Symptom

Effects Caused By

Chlorosis

Pb; Co; Ni; Zn; deficiency of Mg, N and S; Fe; Cu; Mo; phytotoxins, radiation; K; SO2 ; O3 ; NO2 and certain herbicides

Necrosis

O3 ; Co; Zn; deficiency of N, K, P and S; Mg; Fe; B; phytotoxins; radiation; frost; Cu; Cr; Zn; Cd; Co; Ni; HF; B; Ni; Cl; and certain herbicides

Disturbance of growth

Phytotoxins; frost; radiation and certain herbicides

Foliage shed

Ethylene; Al; As and certain herbicides

Reduced growth

Peroxyacetylnitrate (PAN); Zn; PCB; deficiency of N; phytotoxins and certain herbicides

Climatic factors (Rainfall, temperature etc.)

Direct deposition onto grain

Period, season (Long-term behaviour of xenobiotics in soil)

Plant properties (Plant species, plant metabolism, treated part of plants etc.) Growing conditions (Manuring ploughing depth etc.)

Soil properties (Clay-, humus contents, pH-value etc.)

Figure 2. Main factors influencing the soil–plant transfer of xenobiotics.

water, obtained by preliminary investigations, must be taken into account. GENERAL STATEMENTS FOR THE APPLICATION OF BIOASSAYS Bioassay results are appropriate for comparison of the effects of different potentially phytotoxic xenobiotics on several plant species or the influence of one compound on several crops. They can be extrapolated to field conditions only in their relation to each other, but can nevertheless often replace expensive field experiments. The simulated ‘‘worst case’’ conditions in bioassay experiments result in a considerable safety margin, because the optimum uptake conditions of the bioassay are not found in the field under normal conditions. Laboratory bioassay methods have a good reproducibility and can therefore be substituted for field experiments in many cases as ‘‘realistic worst-case’’ scenarios (6). Bioassays may be conducted in situ, in vivo, or in vitro and are used to a great extent for three key

Deposition onto leaves and translocation to grain

Resuspension

Deposition onto soil surface

Root uptake and translocation into plant

Figure 3. Mechanisms of plant contamination by xenobiotics.

factors: the development of herbicide (biological activity and selectivity), for the quantitative detection of herbicides mainly in the soil (environmental fate), and for herbicide research in general. The following methods may be used: 1. Bioassays in water culture (nutrient solutions or soil extracts) Rapid tests (e.g., isolated chloroplasts, isolated mitochondria, Chlorella algae, leaf disk sinking test; CO2 - or O2 -measurements either manometrically or polarographically, CO2 measurements by infrared gas analysis) Growth tests—visual evaluation only (no special equipment required), quantitative measurements 2. Bioassays in hydroponic culture (nutrient solutions or soil extracts) using growth test as above. See point 1.


163

Table 3. Overview of Bioassay Methods and their Applications for Main Groups of Herbicides with Different Sites of Action (7) Type of Assay

Response Parameter

Site of Action

Algae or photoautotrophic cell culture

O2 -development, cell counts, cell density, fluorescence

Photosynthesis phosphorylation cell division

E.g., triazines, ureas, triazinones, pyrimidines, nitriles, chloroacetanilides, carbamates, diphenyl ethers, phenols

5 min–48 h

Heterotrophic cell culture

Conductivity of growth medium

Nucleic acid metabolism

Sulfonylureas, imidazolinones

8 d

Other short term assays

Leaf disk buoyancy

Photosynthesis phosphorylation radical damage

E.g., triazines, ureas, triazinones, pyrimidines, bipyridinium

30 min–24 h

Chlorophyll fluorescence


Compounds triazines, ureas, triazinones, pyrimidines, bipyridinium

8–48 h

Tetrazolium reduction

Phosphorylation, fatty acid synthesis

Compounds alkanoic acid esters, diphenyl ethers, oximes

2 h

Growth test—shoot uptake (floating or submerged)

Visual assessments, weight or length of whole plant or plant parts, chlorophyll-content area covered (Lemna)


E.g., triazines, ureas, triazinones, pyrimidines, bipyridinium compounds

10 min–72 h

Germination test

Plant counts, radicle length, shoot length

Growth regulation, cell division

E.g., alkanoic acids, amides, carbamates, chloracetanilides, dinitroanilines, oximes, thiocarbamates

2–4 d

Shoot or radicle elongation

Shoot length, radicle length

Growth regulation, cell division and elongation, nucleic acid metabolism

E.g., alkanoic acids, amides, carbamates, chloracetanilides, dinitroanilines, oximes, thiocarbamates, imidazolinones, sulfonylureas

2–6 d

Growth test—root uptake

Visual assessments, weight or length of whole plant or plant parts, chlorophyll-content

Photosynthesis, phosphorylation, radical damage

E.g., phenols, triazines, triazinones, ureas, pyrimidines, bipyridinium

8–21 d

Nucleic acid metabolism

Compounds, sulfonylureas, imidazolinones

3. Bioassays in soils, which may be Rapid tests (employing soil extracts for the tests as in 1a) Growth tests as above. See point 2. A great variety of methods (see Table 3) have been described, depending on the purpose of the test as described above. The majority of the tests have been used for the determination of residual phytotoxicity in soil (carryover of residues and effects to succeeding or following crops), resulting in the documentation of numerous assays for the quantitative determination of herbicides in soil. GENERAL CONDITIONS FOR THE USE OF QUANTITATIVE BIOASSAYS Test Species A suitable test species should show a close relationship between the amount of herbicide applied and the reaction

Chemical Classes

Duration

of the plant, with the required sensitivity. A rapid development of fresh weight and a manifestation of treatment effects at an early growth stage shortens the duration of the assay and keeps dissipation of the test compound to a minimum. A low inherent variability between replications and consistency between repeated assays even under slightly varying conditions will provide more reliable results. Weeds, although very sensitive, are often too variable in their reactions due to the wide range of genotypes within the species. The use of crop plants results in a markedly lower variability as shown in Figures 4 and 5. Careful selection of uniform pregerminated seeds or seedlings can also lower variability considerably. Gathering results, e.g., plant counts, determination of fresh weight, or leaf length, is important, and easy handling of the species is an additional criterion for selection of the test species. A great number of higher plant species have been used for herbicide bioassays. Table 4 shows a number of selected test species, which are used at the Institute for Ecological Chemistry at present.

164


Test species (EDP-code) Avena fatua (AVEFA) Zea mays (ZEAMX) Glyzine max (GLXMA) Setaria italica (SETIT) Oryza sativa (ORYSA) Sinapis alba (SINAL) Lolium multiflorum (LOLMU) Bromus secalinus (BROSE) Digitaria sanguinalis (DIGSA) Echinochloa crus-galli (ECHCG) Triticum aestivum (TRZAS) Amaranthus retroflexus (AMARE) Bromus inermis (BROIN) Grossypium hirsutum (GOSHI) Panicum miliacaeum (PANMI) Solanum nigrum (SOLNI) Ipomea ssp. (IPOSS) Veronica persica (VERPE) Matricaria inodora (MATIN) Stellaria media (STEME) Galium aparine (GALAP) Sida spinosa (SIDSP) Chenopodium album (CHEAL) Lolium perenne (LOLPE) Alopecurus myosuroides (ALOMY) Viola arvensis (VIOAR) Agropyron repens (AGRRE) Polygonum convolvulus (POLCO) Setaria faberi (SETFA) Sorghum halapense (SORHA) Abutilon theophrasti (ABUTH) Cassia tora (CASTO) Sesbania exaltata (SEBEX) Setaria veridis (SETVI) Brachiaria plathyphylla (BRAPP) Leptochloa dubia (LEFDU) Cyperusm iria (CYPIR)

100

Germination percentage 80

60

40

g fresh weight (2 weeks after seedling emergence) 20

0

10

20

30

40

50

60

70

Figure 4. The suitability of different crops and weeds as representative species for use as test species for bioassays to assess phytotoxicity.

Comparability of Standard Curves and Unknown Samples Untreated controls and standards with known concentrations of the test compounds have to be included in every quantitative bioassay with unknown samples. Several concentrations ranging from no effect to total damage are necessary to provide sufficient information to establish reliable standard curves. The results are appropriate for comparing the effects of different potentially phytotoxic xenobiotics to a plant species (Fig. 6) or the influence of one compound on several crops, as shown in Figure 7. The number of concentrations and replications per treatment depends on the expected reaction and its variability. If the range of concentrations leading to the desired effects is well known and consistent, fewer different treatments have to be included than for an assay with a compound having unknown or variable effects. Variability of the results will also require a higher number of replications. Values between 90% and 100% of the untreated controls are the most valuable for the determination of the dose-response relationship. Bioassay indicators (especially higher plants) have a very limited response range, making it necessary to use different test species or dilute the samples to achieve reactions in the unknown samples within the valid range of the standard curve. In many cases, e.g., after experimental applications of herbicides, the expected concentrations in the samples can be estimated, and thus, suitable dilutions can be chosen. However, if no information is available about the concentration, a wider range of dilutions or preliminary screening tests have to be carried

out. Sometimes it may be appropriate to use two different species of differing sensitivities to achieve a wider range of concentrations for quantification and to avoid dilutions. It is important to pay attention to possible differences between the unknown samples and the standard curve, especially where field samples are involved. Organic material and nutrient content have a great influence on herbicide activity (11). It may be necessary to equalize the nutrient status by supplying concentrated nutrients to each sample. Standards made up from solutions similar to the samples are preferable, e.g., leachates from untreated plots or soil extracts rather than pure nutrient solution for the evaluation of leaching samples. As for the standard curve, the material used for dilutions should be similar to the samples. Growth Conditions The growth conditions should be close to the optimum for the test species. Fast growth provides a short duration of the assay as well as a higher sensitivity due to a higher intake of herbicides, because most herbicides are taken up by the plant passively with the water stream. During winter, additional lighting may be necessary in glasshouses, but the results will vary still more throughout the year than in growth chambers. For some plant species, e.g., Lemna, the day length is important for a vigorous growth, especially for stock cultures. The pH of the nutrient solution may not only influence the growth, but also the uptake of the pesticide by the plants and thus the response.


= coefficient of variation (V%)

Test species (EDP-code)

0

10

5

25

20

15

HELAN TRZAW AVESA HORVW TRZAS VICFX ZEAMS SINAL BEAVA BROIN IPOSS BRSNW SOLNI AVEFA TRFPR LOLMU GLXMA POAAN SETIT ALOMY SIDSP VERPE GOSHI MYOAR ORYSA GALAP ANTAR

(Standard deviation)

0

5

10

15

20

25

g fresh weight (2 weeks after seedling emergence) Figure 5. The variability of different crops and weeds used as test species for bioassays to assess phytotoxicity.

Table 4. Average Duration of Germination and Number of Plants per Pot for Important Test Species (8), Complemented, EDP Codes (9) Scientific Name Allium cepa Avena sativa Beta vulgaris var. altissima Brassica napus Brassica rapa ssp. rapa Cucumis sativus Daucus carota Glycine max Gossypium hirsutum Helianthus annuus Hordeum vulgare Lactuca sativa Lens culinaris Lepidium sativum Linum usitatissimum Lolium multiflorum Lolium perenne Secale cereale Sinapis alba Solanum nigrum Solanum tuberosum∗ Sorghum vulgare Trifolium incarnatum Trifolium pratense Triticale Triticum aestivum Vicia faba Zea mays ∗

30

Common Name Onion Oats Sugar beet Winter rape Turnip Cucumber Carrot Soybean Cotton Sunflower Summer barley Head lettuce Lentil Garden cress Flax Italian ryegrass Perennial ryegrass Winter rye White mustard Black nightshade Potato Common sorghum Carnation clover Red clover Triticale Winter wheat Field bean Maize

Two top cuttings, rooting over 14 days.

EDP Code ALLCE AVESA BEAVA BRSNW BRSRR CUMSA DAUCS GLXMA GOSHI HELAN HORVS LACSA LENCU LEPSA LINUT LOLMU LOLPE SECCW SINAL SOLNI SOLTU SORVU TRFIN TRFPR TTLSS TRZAW VICFX ZEAMX

Plants/Pot

Germination in Days

7 5 5 7 7 4 7 4 4 3 5 5 5 7 7 7 7 5 7 7 2 7 7 7 5 5 3 5

7 5 7 6 6 10 8 6 10 6 5 7 10 5 5 5 5 5 5 5 14 6 5 5 5 5 6 7

165

166


% fresh weight 100

ED10

90 80

ED30

70 60

ED50

Isoproturon

50

Pendimethalin

40

Glyphosate 30

Chlorotoluron

20

Metamitron

10 0 0.0001

0.001

0.01

0.1

1

10

100

1000

10000 100000

[g a.i/ha] Figure 6. Comparison of logistic dose-response curves for foxtail millet (Setaria italica) and five herbicides applied post-emergence [from (10)].

100

ED10

% fresh weight

90 80

ED30

70

SINAL

60

ED50

BRSNW

50

VERPE

40

SOLNI

30

SIDSP IPOSS

20

GOSHI

10

GLXMA

0 0.01

0.1

1

10 [g Glyphosate/ha]

100

1000 Max. dosage

Figure 7. Comparison of logistic dose-response curves for selected plants and the herbicide glyphosate applied post emergence [from (10)].

Statistical Evaluation The relationship between plant response and herbicide concentration can be described as an S-shaped curve, which is almost linear around the point of inflection. A typical dose-response curve for the relationship between herbicide concentration and the growth of a test species is shown in an idealized form in Figure 8. The statistical treatment of the data should provide a reliable conclusion from the standard curve to the concentration in the samples; thus, the application of regression procedures to the standards is the most appropriate (13). Detailed descriptions of the mathematical procedures are given elsewhere (14); only a

brief general overview of possible methods in general is given here. Several approaches for the mathematical description of the relationship have been used, including transformations to achieve linearity (15,16) and nonlinear regressions (13,17,18). The concentrations in the unknown samples are then quantified by comparing the data with the standard curve, the central part being the most reliable base. If the reaction is expressed as a percentage of untreated controls, the part of the standard curve between 30% and 70% of the controls (11) may be used. Below and above these values, effect and concentration are not closely related.


167

Stimulation Response (% of untreated)

Untreated

ED10

NEL / NOEL no (observable) effect level

ED30

Reduction

ED50 ED70 Total damage Log dose

Figure 8. General shape of the dose-response curve between test plants and phytotoxic compounds [from (12), modified].

PRACTICAL USE OF BIOASSAY METHODS FOR SCREENING AND QUANTITATIVE DETERMINATION OF EFFECTS TO SUCCEEDING CROPS

STANDARDIZED BIOASSAY FOR THE DETERMINATION OF ED10 -(NOEL) AND ED50 -VALUES FOR HERBICIDES AND SELECTED FOLLOWING CROPS IN SOIL Introduction and Objectives

Within the scope of the national and international registration procedures for plant protection products (PPP) in several regions, tests regarding the effects of PPP on higher terrestrial plants are required. For the EU member states, the data requirements are laid down in Directive 91/414/EEC concerning the placing of PPP on the market. In the annexes II and III of this directive, special results of plant tests are not only required for assessing the efficacy and phytotoxicity of the PPPs or for justifying the recommended dose, but also for assessing the effects on succeeding crops, on the evolution of resistance within the plant populations and on nontarget plants. Except for efficacy and phytotoxicity testing, a tiered approach is practiced or recommended for all of these plant tests, in which the first tier is based on bioassays under controlled conditions. Because the results of such bioassays trigger tests in a higher tier, such a system must show a high level of standardization and reproducibility. One internationally harmonized bioassay protocol is the OECD test guideline 208 Terrestrial Plants Growth Test (19). Further information is given in the ISO International Standard 11269-2 on soil quality—Determination of the effects of pollutants on soil flora, Part 2. Effects of chemicals on the emergence and growth of higher plants (ISO, 1995). Furthermore standards are being set up by the European and Mediterranean Plant Protection Organization (EPPO), namely, Guidelines for the ‘‘Efficacy evaluation of PPPs.’’ Four general guidelines describe 1) phytotoxicity assessment (20), 2) design and analysis of efficacy evaluation trials (21), 3) conduct and reporting of efficacy evaluation trials (22), and last but not least 4) effects on succeeding crops (23). Figure 9 shows the decision-making scheme of this guideline for estimating effects on succeeding crops.

The requirements, which are laid down in the abovementioned EPPO- or OECD-guidelines for the biological evaluation of pesticides ‘‘effects on succeeding crops,’’ are not defined in detail. In relation to the German ¨ ¨ von Herguideline VI/13-1 ‘‘Prufung der Phytotoxizitat biziden auf nachgebaute Kulturen—Tolerance of Plant Protection Products in Subsequent Crops’’ biological detection procedures (bioassays) have been carried out at the Weed Research Institute and the Institute for Ecological Chemistry of the Federal Biological Research Centre for Agriculture and Forestry (BBA). A set of about 30 monocotyledonous and dicotyledonous test species have been used to assess the residual behavior of phytotoxic compounds (8,24). About 300 crop/herbicide combination dose-response relationships, which show the sensitivity of different crop plants, have been determined and were integrated into a database (25–27). The bioassay method described here is used for the determination of characteristic values regarding the phytotoxicity of herbicides to rotational crops. Two aspects are to be particularly considered here: 1. Selectivity between the treated main crop and the potential rotational crops. This is used as the decision criterion for the performance of field experiments. 2. Sensitivity of rotational crops to residues of herbicides remaining in soil. The selectivity index is calculated by comparison of the ED50 -values (ED = Effective Dose) for mainand succeeding crops. The ED50 is the mean effective concentration, which reduces the fresh weight to 50% of the untreated control plants. It can be obtained from the dose-response curve for each crop, which is tested using the method described here. An example is given in Figure 10,

168


1. Test a range of representative crop types for biological activity in soil of the active substance and on significant soil metabolites

2. Any biological activity in soil?

No

Yes 3. Obtain results on the fate and behavior of the active substance in soil, including examination of any biologically active metabolites

4. Do the fate and the behavior studies indicate that a biologically active level of active substance or metabolites will be present when sensitive crops are planted?

No 5. No further testing required

Yes 6. Conduct field trials on large plots treated in the previous crop. Use limited replication and plant two or more crops when they would normally be planted in the rotation. Assess effects visually. Examine the effects of cultivation where appropriate

7. Are significant effects seen on the sensitive crops?

No

Yes 8. Is there any management practise that can be recommended on the label to remove the risk to sensitive crops?

Yes

No 9. Is it still wished to plant a sensitive crop at the usual time in the rotation? Yes 10. Examine the effect on yield of the crop in detailed trials. These must be fully replicated and plot size large enough so that yield may be taken

No

11. Add a label warning concerning the level of effects seen or, if unacceptable, specify the interval before the sensitive crop may be planted

Figure 9. Decision-making scheme on the extent of testing needed to examine effects on succeeding crops and on the consequent recommendations [from (23)].

where the selectivity factors for four test plants and three herbicides were compared. The vertical assessment between compounds with lowest and highest activity is between a factor of 6.8 with SOLNI (Solanum nigrum, black nightshade) and the herbicides metamitron and isoproturon and a factor of about 121 with SINAL (Sinapis alba, white mustard) and the herbicides metamitron and glyphosate. This means that glyphosate has a 121 times greater effect than does metamitron on the test species white mustard. The horizontal comparison of the tested plants shows selectivity factors between the most sensitive and most insensitive species of 16.6 in the case of SINAL (Sinapis alba, white mustard) and SOLNI (Solanum nigrum, black nightshade) and a selectivity factor of 84.5

with AVESA (Avena sativa, oats) and SOLNI (Solanum nigrum, black nightshade). Another example is given in Figure 11. Within different varieties of one crop (Triticum aestivum, winter wheat), a great range of sensitivity occurs. The horizontal comparison of the tested winter wheat varieties showed sensitivity factors between the most susceptible (‘ZENTOS’) and the most insensitive variety (‘KANZLER’) of about 100. The sensitivity of following crops to residues in soil is measured using the NOEL (no-observable-effect level). ¨ According to Pestemer and Gunther (12), it can also be read from the dose-response curve as the concentration that causes a reduction of 10% compared with the untreated control (ED10 , i.e., 90% of weight is reached).


169

g a.i./ha

Isoproturon (I) Glyphosate (G) Metamitron (M)

3000 2500 2000

2537 1500 1000

970

866

16

0

SOLNI [M/I = 6.8]

AVESA/SOLNI = 23.3

676

109 36

500

93

AVESA/SINAL = 84.5

8 265

50 LOLMU [M/I = 17.3]

Selectivity factor between most sensitive and most insensitive test species

SINAL [M/G = 121.3]

SINAL/SOLNI = 16.6

223 AVESA [M/I = 11.4]

Test species [selectivity factors between herbicides with lowest and highest activity]

Figure 10. Vertical and horizontal assessment by comparing ED50 values of three herbicides and four test species (EDP codes; see Table 4).

100 2.5

90

70 60

1.5

50 40

1

30

Sensitivity factor

ED 50-value (mg/kg soil)

80 2

20

0.5

10 0

0 os nt Ze ö d ce Al ast r nt Ko al p m Ko s o id Al b lu C k lis be O os n re Bo r lu Pa a in eg R s ra Ta s ira M r o kt Fa ro i am R n iko it

M

z Fa

Reference variety “Kanzler”

Winter wheat (varieties)

Using this value, the most sensitive rotational crops or inactivation times for a compound in soil may be determined. The necessity of bioassay or field experiments is derived according to a decision scheme (Fig. 12), depending on the effectiveness and degradation characteristics of the active ingredient (28). For compounds showing only or mainly soil activity, standardized bioassays with the main crop and representative rotational crops have to be carried out. Only for chemicals that show a wide selectivity index (>100) and a slow degradation in soil (DT50 > 60 d), field experiments are also required. The bioassay method used here allows standardized conditions (soil, climate) for the test plants. Due to optimum uptake conditions, the plants are steadily

Figure 11. Sensitivity of selected varieties of winter wheat [ED50 (reference variety)/ED50 (other varieties)] to the herbicide chlorotoluron.

exposed to ‘‘chemical stress,’’ because the amount of chemical dissolved in the soil solution is at its maximum under the high soil water content in the range of maximum water holding capacity (worst case). Moreover, the plants are forced to take up the active ingredient, because in opposition to field conditions, the roots cannot grow out of the treated soil layer. The results are suitable for comparison of the effects of different herbicides on several plant species or the influence of one compound on several crops. They can be extrapolated to field conditions only in their relation to each other. Nevertheless, the procedure may often replace expensive field experiments. The simulated ‘‘worst case’’ conditions result in a considerable safety margin, because the optimum uptake conditions of the bioassay are not found in the field

170


Test plant

Herbicide

Pot (6 cm diameter) No

60 ml substrate (e.g., soil)

Effect completely or partly via soil

Glass fibre wick Petri dish with black lid

Yes Bioassay with a main crop and 5 representative rotational crops estimation of the sensivity index

Nutrient solution Figure 13. Bioassay pot with self-watering system.

Yes

DT90 70 ED 30 - ED 50 (severe damage)

60

ED50 -> 50 >ED 50 (total damage)

40 30

Red clover

20

Winter rape

10

Sugar beet

0 10

100 1000 Log dose [mg a.i./kg soil] in 0–10 cm soil layer

Figure 17. General shape of the dose-response curve between plants and phytotoxic compounds. Comparison of logistic dose-response curves for selected plants [mod. after (12)].


175

Crop Carrot Sugarbeet Spring wheat Spinach Maize Head lettuce Garden pea Oat French bean Cornsalad Field pea Field bean Common flax 500

400 300 200 100 ED10-value [µg Chlorotoluron/kg soil]

0

10

20 30 40 50 Time of inactivation [weeks]

60

Figure 18. Predicted time of inactivation [2.1-kg chlorotoluron/ha–10-cm soil layer] for different crops (parameter for the simulation of degradation using PEMOSYS: Application: 1.5.95; Soil: loamy sand, 1% org. C; weather station: Berlin).

Crop Field bean Maize Winter wheat Winter barley Winter rye Oat Winter rape Red clover Perennial ryegrass Sugarbeet White mustard Phacelia Sunflower 16

14 12 10 8 6 4 2 ED10-value [µg Prosulfuron/kg soil]

0

10

20 30 40 50 Time of inactivation [days]

60

Figure 19. Predicted time of inactivation [15-g prosulfuron/ha–10-cm soil layer] for different crops (parameter for the simulation of degradation using PEMOSYS: Application: 1.5.95; Soil: loamy sand, 1% org. C; weather station: Berlin).

Nevertheless, bioassays may be used for the quantitative determination of residues and give results comparable to physical or chemical methods. They are often sufficiently sensitive, and they are particularly suitable in screening procedures for new compounds where no physical or chemical methods have been developed yet. In this chapter, examples of different methods described in the literature are given, and a standardized bioassay procedure is described in detail. For the highly biologically active groups like sulfonylureas or imidazolinones, bioassays are still a standard method of quantitative determination. An advantage of

using a bioassay for estimating residues of these herbicides is the extreme sensitivity of a number of plant species. For these herbicides, it is significant that bioassays can provide the quantitative determination as well as the risk assessment for succeeding crops within a one-step procedure. Bioassays for quantitative analysis are also an option, where the expensive laboratory equipment and highly skilled personnel are not available, but suitable growth conditions for a bioassay can be provided. As environmental concerns over pesticide use continue to gain more and more importance, bioassays will still be necessary in future, because additional information is

176


needed on possible impacts on the environment as well as on total residual amounts. Unlike bioassays, instrumental methods give no indications of possible hazards, whether it be for crops or nontarget organisms. BIBLIOGRAPHY 1. J. C. Streibig, Quantitative Assessment of Herbicide Phytotoxicity with Dilution Assay, Thesis, Royal Veterinary and Agricultural University, Copenhagen, 1992. 2. Risk reduction in the field of plant protection products in Germany. Federal Ministry of Food, Agriculture and Forestry (FMFAF) (1998). Available at http://www.bml.de/englisch/pflanzenschutz/risk3-1.htm 3. Act Governing the Protection of Crop Plants (Plant Protection Act) of 15 September 1986 (Fed. Law Gazette I P. 1505), last amended by Article 10 of the Act of 27 June 1994 (Fed. Law Gazette, I, P. 1440). 4. Criteria for assessment of plant protection products in the registration procedure, Mitteilungen aus der Biologischen Bundesanstalt fur ¨ Land- und Forstwirtschaft, Berlin-Dahlem, No. 285, Kommissionsverlag Paul Parey, Berlin und Hamburg, 1992. ¨ 5. P. Gunther and W. Pestemer, Environmental Management 14: 381–388 (1990). ¨ 6. W. Pestemer and P. Pucelik-Gunther, Standardized Bioassay for the Determination of ED10 -(NOEL) and ED50 -values for Herbicides and Selected Following Crops in Soil, Reports from the Federal Biological Research Centre for Agriculture and Forestry, Vol. 29, ISSN-No. 0947-8809, 1997, p. 26. ¨ 7. W. Pestemer and P. Pucelik-Gunther, in H.-J. Stan, ed., Analysis of Pesticides in Ground and Surface Water I—Progress in Basic Multi-Residue Methods, Chemistry of Plant Protection (Editor-in-Chief: W. Ebing), Vol. 11, Springer-Verlag, New York, 1995, pp. 219–231. ¨ 8. P. Gunther, Biotest mit höheren Pflanzen zur Untersuchung und Bewertung des Verhaltens von SulfonylharnstoffHerbiziden und anderen Xenobiotika im Boden, Dissertation, ¨ Hannover, 1991. Universitat 9. Anonymous, Important Crops of the World and their Weeds (Scientific and Common Names, Synonyms, and WSSA/WSSJ Approved Computer Codes), 2nd edition, publ. by Business Group Crop Protection, BAYER AG, Leverkusen, Germany, 1992. 10. W. Pestemer and P. Zwerger, in A. A. M. Del Re et al., eds., Human and Environmental Exposure to Xenobiotics, XI. Symposium Pesticide Chemistry, Cremona, Italy, 11–15 Sept. 1999, pp. 763–770. 11. W. Pestemer, Weed Research 16: 357–363 (1976). ¨ 12. W. Pestemer and P. Gunther, in J. C. Streibig and P. Kudsk, eds., Herbicide Bioassays, CRC Press, FL, 1993, pp. 137–152. 13. J. C. Streibig, Weed Research 28: 479 (1988). 14. J. C. Streibig, M. Rudemo, and J. E. Jensen, in J. C. Streibig and P. Kudsk, eds., Herbicide Bioassays, CRC Press, FL, 1993, pp. 29–55.

18. J. C. Streibig, Acta Agriculturae Scandinavica 30: 59–64 (1980). 19. OECD Guideline for the Testing of Chemicals—Proposal for Updating Guideline 208—[Terrestrial (Non-Target) Plant Test: 208 A: Seedling Emergence and Seedling Growth Test; 208 B: Vegetative Vigour Test], Draft Document July 2000. 20. EPPO Standards (Guidelines for the efficacy evaluation of plant protection products), PP 1/135(2), Phytotoxicity assessment, 1999. 21. EPPO Standards (Guidelines for the efficacy evaluation of plant protection products), PP 1/52(2), Design and analysis of efficacy evaluation trials, 1999. 22. EPPO Standards (Guidelines for the efficacy evaluation of plant protection products), PP 1/182(2), Conduct and reporting of efficacy evaluation trials, 1999. 23. EPPO Standards (Guidelines for the efficacy evaluation of plant protection products), PP 1/207(1), Effects on succeeding crops, 1999. 24. W. Pestemer and B. Auspurg, Nachrichtenblatt des Deutschen Pflanzenschutzdienstes (Braunschweig) 38: 120–125 (1986). ¨ 25. P. Gunther, W. Pestemer, T. K. James, and A. Rahman, 8th EWRS-Symposium Quantitative Approaches in Weed and Herbicide Research and Their Practical Application, Vol. 2, Braunschweig, Germany, June 1993, pp. 777–784. 26. P. T. Holland et al., 9th International Congress of Pesticide Chemistry, Vol. 2, Book of Abstracts, London, July 31–Aug. 3, 1998, pp. 6B–023. ¨ 27. W. Pestemer and P. Pucelik-Gunther, Nachrichtenblatt des Deutschen Pflanzenschutzdienstes (Braunschweig) 51: 32–37 (1999). 28. W. Pestemer, P. Zwerger, and G. Heidler, Annual Report of the BBA, 1996, pp. 170–171. 29. W. Pestemer, L. Stalder, and C. A. Potter, Berichte Fachgebiet Herbologie, Universitat ¨ Hohenheim 24: 53–61 (1983). 30. W. Pestemer, Berichte Fachgebiet Herbologie Universitat ¨ Hohenheim, 24: 85–96 (1983). ¨ 31. J.-E. Garcia-G., W. Pestemer, and P. Gunther, Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 44: 105–108 (1992). ¨ 32. P. Gunther, M. Heiermann, G. Maas, and W. Pestemer, Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 46: 10–15 (1994). ¨ 33. P. Gunther, W. Pestemer, and E.-P. Thies, Mitteilungen aus der Biologischen Bundesanstalt fur ¨ Land- und Forstwirtschaft (Berlin-Dahlem), H 301: 493 (1994). 34. L. Stalder and W. Pestemer, Weed Research 20: 341–347 (1980). ¨ 35. P. Gunther, A. Rahman, and W. Pestemer, Weed Research 29: 141–146 (1989). 36. W. Pestemer, L. Stalder, and L. Eckert, Weed Research 20: 349–353 (1980).

15. A. Nyffeler et al., Weed Research 22: 213–222 (1982).

¨ 37. P. Gunther and W. Pestemer, in J. E. Hall, D. R. Sauerbeck, and P. L’Hermite, eds., Effects of Organic Contaminants in Sewage Sludge on Soil Fertility, Plants and Animals, ECSC¨ EEC-EAEC, Brussel, Belgium, 1992, pp. 103–111.

16. W. Pestemer, Berichte Fachgebiet Herbologie Universitat ¨ Hohenheim, 24: 85–96 (1983).

38. G. Edwards-Jones et al., Proc. Brighton Crop Protection Conference—Weeds 561–566 (1989).

¨ 17. P. Gunther, A. Rahman, and W. Pestemer, Weed Research 29: 141–146 (1989).

39. J. G. Ferris, T. C. Frecker, B. M. Haigh, and S. Durrant, Computers & Electronics in Agriculture 6: 295–317 (1990).

BIODEGRADABILITY: ASSESSMENT ¨ 40. B. Gottesburen, Konzept, Enwicklung und Validierung des wissensbasierten Herbizid-Beratungssystems HERBASYS, ¨ Hannover, 1991. Dissertation, Universitat 41. W. Pestemer et al., Zeitschrift fur ¨ Pflanzenkrankheiten und Pflanzenschutz special ed. XII 179–190 (1990). 42. J. Zhao et al., in A. Barth et al., eds., Anwendungen der ¨ Kunstliche ¨ Kunstlichen ¨ Intelligenz-18, Fachtagung fur Intel¨ ligenz, Saarbrucken, Sept. 1994, pp. 185–199. ¨ 43. B. Gottesburen et al., Zeitschrift fur ¨ Pflanzenkrankheiten und Pflanzenschutz 97: 394–415 (1990).

BIOAVAILABILITY Extent to which a pesticide residue can be taken up into an organism from the total present in its food and environment (IUPAC).

BIOCIDES Substances that kill living organisms (USEPA).

BIOCONCENTRATION Process by which an organism accumulates a pesticide residue directly from its environment as the net result of processes whereby the rate of uptake exceeds that of elimination (IUPAC).

177

mercury away from the vicinity of the cells), 3) enzymatic attack that is of no detectable benefit to the microorganism (e.g., cometabolic reactions in which a physiologically useful primary substrate induces the production of enzymes that fortuitously alter the molecular structure of another compound), and 4) nonenzymatic reactions stemming from by-products of microbial physiology that cause geochemical change (e.g., consumption of oxygen, production of fermentation by-products, or an alteration in pH). ‘‘Biodegradation’’ of organic compounds is the partial simplification or complete destruction of their molecular structure by physiological reactions catalyzed by microorganisms (2–7). Biodegradation is routinely measured by applying chemical and physiological assays to laboratory incubations of flasks containing pure cultures of microorganisms, mixed cultures, or environmental samples (e.g., soil, water, sediment, or industrial sludges). When attempting to measure biodegradation or judge the biodegradability of substances, the investigator must define the environmental context so that potential reactants and products can be identified. Microorganisms can catalyze only reactions that are thermodynamically possible. Furthermore, reaction mechanisms are largely constrained by precedents set during the evolution of physiological and biochemical functions. Because of ongoing microbial evolution and biochemical research, our understanding of mechanisms by which microorganisms degrade substrates continues to expand. LABORATORY METHODS Principles for Measuring Biodegradability in the Laboratory

BIODEGRADABILITY: ASSESSMENT EUGENE L. MADSEN Cornell University Ithaca, New York

Herein are described basic principles pertinent to the design, implementation, and interpretation of both laboratory and field determinations of biodegradability. The information is applicable to both organic and inorganic contamination problems whose solutions can be addressed microbiologically. However, the focus here is primarily on assessing the biodegradability of organic compounds. DEFINITIONS OF BIODEGRADABILITY AND BIODEGRADATION ‘‘Biodegradability’’ embodies qualities representing the susceptibility of substances to alteration by microbial processes (1). The substances may be organic or inorganic. The alteration may be brought about by 1) intra- or extracellular enzymatic attack that is essential for the growth of the microorganism(s) (e.g., the attacked substances are used as a source of carbon, energy, nitrogen, or other nutrients or as a final electron acceptor), 2) enzymatic attack that is beneficial because it serves some protective purpose (e.g., mobilization of toxic

Biodegradation methodologies are designed to confirm, demonstrate, and explore both the net chemical changes and the associated intracellular details pertinent to how microorganisms influence the fate of contaminants. The procedures span a broad range of disciplines and sophisticated protocols. Figure 1 provides an overview of the variety of objectives, disciplines, and protocols that play key roles in biodegradation research. The two phases that serve as main divisions in Figure 1 result from the degree to which scientific detail is pursued. Phase 1 procedures treat samples of soil, sediments, water, or industrial effluents simply as ‘‘black boxes’’ that do or do not make contaminant compounds disappear, as judged by analytical chemical criteria. Phase 2 begins with the isolation of pure cultures of contaminant-degrading microorganisms. Once these have been obtained, refined physiological, enzymatic, and molecular biological assays may then be performed. As DNA sequences of genes that code for metabolic pathways become increasingly available, molecular procedures will continue to gain prominence in biodegradation protocols. One of the final goals of the procedures shown in phase 2 is understanding the molecular basis for gene expression and regulation. Design and Implementation of Biodegradation Assays Using Environmental Samples and Pure Cultures The traditional black-box approach to biodegradation assays asks the question, ‘‘Are microorganisms within

178

BIODEGRADABILITY: ASSESSMENT

Phase 1: Laboratory enrichment and demonstration of net metabolic activity 1. Soil, sediment, water, or industrial effluent in field site 2. Aseptically remove, contain, transport to laboratory 3. Divide into replicate live and abiotic treatments 4. If appropriate, add radiolabeled or unlabeled organic compound of interest 5. Use tools of analytical chemistry or physiology to periodically measure consumption of parent compound and coreactants or production of metabolites or physiological endproducts 6. Compare time-course data obtained from live and abiotic treatments 7. Interpret and consider more reductionistic procedures shown in Phase 2. Phase 2: Isolation of pure cultures and examination of physiological, biochemical, and molecular basis of pollutant metabolism 8. Isolate pure cultures capable of expressing metabolic activity determined in Phase 1 9. Characterize growth, cell yield, sequential induction, and other physiological characteristics of the microorganisms during pollutant metabolism 10. Extract and identify metabolites, enzymes, and cofactors associated with pollutant metabolism 11. Cell-free examination of metabolites, enzymes, and cofactors 12. Determine portion of genomic or plasmid DNA that codes for pollutant metabolism by screening a cloned DNA library, by transposon mutagenesis, or other procedures 13. Conduct hybridization, restriction mapping, and sequencing DNA analyses seeking open reading frames, homology with similar genes, and other key insights 14. Elucidate details of gene expression and regulation via a variety of genetic and molecular techniques that include transposon mutagenesis, construction of expression clones, insertional inactivation, and inducer/reporter experiments. Figure 1. Two phases of procedures for understanding biodegradation processes. Phase 1 begins with environmental samples. Phase 2 proceeds through biochemical and molecular aspects of pollutant metabolism by single microorganisms (from Ref. 1).

this complex microbial community (e.g., derived from soil, water, sediment, or industrial sludge) able to metabolize the compound of interest?’’ To answer this question, one aseptically gathers samples from a given field location, dispenses known weights or volumes of the samples to replicated vessels, handles the samples in a variety of ways that include a treatment that has been either sterilized or poisoned, incubates the test samples under laboratory conditions, and employs over time both chemical and physiological assays that monitor the fate of the test compound within experimental vessels (Fig. 1; phase 1). The objective of this general experimental design for biodegradation procedures using environmental samples or pure cultures is remarkably simple, yet there is a substantial series of obstacles that must be overcome before one obtains clear data that truly test a given set of specific hypotheses. Every design parameter selected for inclusion in a biodegradation assay can influence the resultant data. Therefore, decisions made in implementing biodegradation assays should be well reasoned. Table 1 summarizes many of the practical and theoretical decisions that must be made in developing biodegradation protocols. Step 1, a background issue

considering information use, is fundamental to all related experimental decisions. The degree to which experimental minutiae of a given testing protocol must be initially considered is commensurate with the scrutiny that the final data will undergo. Artifacts and biases in data are virtually unavoidable in biodegradation assays (see later); thus, it may be wise to simply accept methodological limitations rather than worry about initial potential technical design flaws that may later have no practical impact. Once the reason for conducting the biodegradation assay has been put in perspective (Table 1, step 1), another background issue, that of physiological conditions, should be confronted. Step 2 appears in Table 1 to acknowledge the fact that biodegradation is only a small portion of the perhaps thousands of physiological reactions occurring simultaneously when both pure cultures and mixed microbial populations in environmental samples are incubated in the laboratory. These physiological processes feed one another, interact in complex ways, and can be governed by many of the sometimes inadvertent physical and chemical manipulations made while preparing, incubating, and sampling assay vessels. Uncertainties become particularly


179

Table 1. Steps and Decisions Essential for Implementing Biodegradation Assays (After Ref. 7) Step

Decisions

1. Background: Determine how the resultant data will be interpreted and used

Objectives range from information about crude ‘‘biodegradation potential’’ to tests of specific hypotheses about physiological or biochemical factors governing biodegradation processes

2. Background: Select the physiological conditions under which pollutant metabolism is to be measured

The pivotal physiological concern is defining the mechanism by which the compound(s) is metabolized. Of primary importance is discriminating among such possibilities as cometabolic reactions, its use as an electron acceptor, and its use as a carbon and energy source. Other concerns address conditions in experimental flasks such as nutrient sufficiency, which final electron acceptor regimen should dominate, what pollutant concentration ranges should be examined, and if conditions should change (batch culture) or remain constant (continuous culture) during the assay

3. Practice: Select and aseptically prepare or sample the microorganisms whose physiological activity is of interest

For assays using environmental or industrial samples, aseptic sampling techniques involve use of tools (such as flame-sterilized scoops, spatulas, and knives) and sample placement within sterilized glass or plastic containers. For assays using pure cultures of microorganisms, the microorganisms must be aseptically grown under conditions that carefully define the cell physiological status (e.g., stage of growth, cell numbers, induced enzyme systems, nutritional state) desired by the investigator

4. Practice: Select the physical apparatus and hence the physiological setting for biodegradation reactions to occur

Glass (or plastic) vessels must be assembled. These contain the test compound(s), the microorganisms being studied, and any accompanying components of soils, sediments, sludges, and water in various ratios. Experimental hardware may be fitted with a variety of gas and water exchange assemblies for maintaining physiological conditions and assaying reaction progress

5. Practice: Select a metabolic activity assay that is sensitive, effective, convenient, inexpensive, and compatible with experimental objectives

The general assay categories are physiological assays (e.g., respirometry or growth) and chemical assays (which include gas chromatography, gas chromatography-mass spectrometry, high-performance liquid chromatography, and radiotracer techniques)

6. Practice: Aseptically prepare stock solutions of 14 C-labeled organic compounds. Check radiopurity

The validity of the results from biodegradation assays using 14 C-labeled substrates is dependent on substrate radiopurity and aseptic preparation of stock solutions

7. Practice: Complete the experimental design parameters for the assay vessels and the assays themselves

a. Concentration of the test substrate(s) b. Number of replicated flasks per treatment c. Whether flasks can be sampled repeatedly or if they require sacrifice at each sampling period d. Frequency of sampling e. Method of preparing abiotic controls f. Methods for separating radioactive parent and product compounds from one another

striking when one is attempting to troubleshoot failed attempts to demonstrate biodegradation activity. The interplay between fundamental knowledge of physiology and experimental design parameters demands that a variety of issues be confronted: 1) The mechanism by which the compound is metabolized (e.g., as a carbon source, as a nitrogen source, or as a cometabolic substrate whose transformation will occur only when another compound is supplied), 2) inclusion versus exclusion of potential growth-limiting vitamins and minerals, 3) inclusion versus exclusion of air in the headspace of the reaction vessel, 4) the solid-to-liquid ratios used in test vessels containing

soil, sediments, or sludges, 5) the multiple roles of compounds in physiological reactions (for instance, nitrate can serve as both a nitrogen source and a final electron acceptor), and 6) the fact that the compound whose biodegradation is being tested may be toxic at high concentrations or fall below some minimum threshold value for uptake and cell growth at low concentrations. Background considerations raised in steps 1 and 2 guide most of the practical steps needed for completing the implementation of the biodegradation assays (Table 1, steps 3 to 7). Detailed considerations pertinent to steps 3 to 7 (Table 1) have been discussed previously (1,7,8).

180


FIELD METHODS Assessing Biodegradation in the Field There is a fundamental paradigm for verifying that the biodegradation processes we hope are occurring are actually occurring in field sites. The paradigm begins by modestly admitting that both microorganisms and their habitats are incomplete puzzles. Our task is to relentlessly find new ways to create the puzzle pieces describing microbiological processes and to assemble them logically. The scientific disciplines that contribute information and techniques toward creating the puzzle pieces include microbiology, geochemistry, hydrology, biochemistry, soil science, physiology, molecular biology, analytical chemistry, computer modeling, and both environmental and chemical engineering. It must be recognized that each of these disciplines is actively being advanced and therefore, contributes a dynamic spectrum of expertise to bioremediation, ranging from theoretical and basic knowledge to applied and practical instrumentation. Verifying field biodegradation is perhaps best achieved in two mutually supportive ways (9): 1) succinctly using common sense and 2) using elaborate reasoning and analyses (see later). The succinct answer is ‘‘We know that bioremediation is taking place when all of the available information congeals as a coherent picture (it makes sense).’’ There needs to be consistency, redundancy, and convergence of all types of evidence from as many of the appropriate scientific disciplines as are available. Because the key players in bioremediation are microorganisms, it is essential that the process makes sense to the microorganisms themselves, in the physiological and thermodynamic contexts where they reside. Contexts for bioremediation processes range from a variety of field sites in which organic contaminants have spilled accidentally (e.g., marine coastlines, desert soils, freshwater streams, or anaerobic deep subsurface sediments) to various aerobic or anaerobic engineered stirred and staged bioreactor systems. Regardless of the particular context, each must be scrutinized as a habitat for microbial metabolism in which individual cells can develop into populations and complex ecological communities whose fundamental physiological needs for adenosine triphosphate generation, carbon assimilation, terminal electron acceptors, other inorganic and organic nutrients, and dynamic intercellular interactions (competition, synergism, interspecies hydrogen transfer, commensalism, predation, parasitism, etc.) demand constantly improving sets of hypotheses aimed at refining our understanding of bioremediation. Once the fundamental thermodynamic, nutritional, and ecological bases for the sought metabolic functions are initially conjectured, a series of hypotheses will naturally unfold that provide a means for documenting the bioremediation process of interest on a site-specific and case-specific basis. Table 2 contains four examples of contaminants in field sites whose physiological contexts dictate how microorganisms can metabolize the offending organic compounds. Knowledge from laboratory-based (using environmental samples, mixed cultures, and/or pure cultures) assays provides the biochemical basis for

mechanisms operating in the field. Answers to key questions, such as ‘‘Are the contaminants suitable carbon and electron sources?,’’ ‘‘Which physiological electron acceptors (oxygen, NO3 − , Fe3+ , Mn4+ , SO4 2− , carbon dioxide) are required coreactants?,’’ ‘‘Are the contaminants, themselves, final electron acceptors?,’’ and ‘‘What competing reactions may slow or prevent the sought biodegradation?,’’ provide a framework that launches a broad array of possible assays that can argue for or against the successful establishment of a given biodegradation process (1,7,8). The example contaminated sites of Table 2 range from aerobic soil to aerobic and anaerobic aquifers. The assays range from field-based oxygen probes, to counts of contaminant-degrading bacteria, to laboratory biodegradation assays, and to molecular biological assays of DNA and ribonucleic acid. The following sections of this article elaborate on the reasoning and detailed analyses required for the generation and testing of hypotheses that allow bioremediation technology to be verified. Related aspects of bioremediation and biodegradation have been reviewed in several recent books (2,21–29). A Three-Step Strategy for Verifying Bioremediation The reasons for establishing sound scientific criteria for microbiological involvement in contaminant loss are 1) biodegradation processes are often unique in their capacity to break intramolecular bonds of contaminant compounds; thus, contaminants can be destroyed and not simply transferred from one location to another, as is the case in many other pollution control technologies; 2) when the mechanism of pollutant destruction is certain, key site management decisions about process enhancement can be made; and 3) for bioremediation to meet pollutioncontrol needs of the society, the industry must adopt some standards for uniformity and quality control so that credibility and reliability can be attained (25–27). However, the question remains: What is an adequate proof of bioremediation? The legal system of the United States provides a variety of categories of certainty in interpreting evidence. The categories depend on the type of the case and the significance of the issues. Among the different burdens of proof are 1) proof beyond reasonable doubt, 2) proof in a clear and convincing manner, and 3) proof beyond a preponderance of doubt. This article neither intends, nor is able, to dictate to regulatory or legal agencies what level of proof should be deemed adequate for bioremediation technology practitioners. Nonetheless, approaches are discussed here that can be used to distinguish biotic and abiotic reactions affecting contaminants at field sites in which bioremediation technology is being applied (for additional discussion see Refs. 1,6,7,11,12,25,26,30–37). The consensus of a National Research Council (NRC) (25) committee in recommending criteria proving in situ bioremediation is as follows: 1. Develop historical records documenting loss of contaminants from field sites. 2. Perform laboratory assays unequivocally showing that microorganisms in site-derived samples have

Table 2. Examples of Contaminated Sites, Hypothesized Key Bioremediation Processes, and the Corresponding Field and Laboratory Measurements That Allow Site-Specific and Case-Specific Verification of Microbiological Destruction of Contaminants (After Ref. 9) Example Sites Aerobic soil contaminated with petroleum products

Hypothesized Key Bioremediation Processes Heterotrophic microorganisms are growing using petroleum components as carbon and energy sources (10,11,12). Metabolism in this context relies on oxygen, both in the attack of aliphatic and aromatic compounds, and as a final electron acceptor in respiratory chains

Supportive Field and Laboratory Measurements • Coincident depletion of petroleum components and oxygen in the field • Corresponding production of carbon dioxide • High numbers of petroleum-degrading aerobic heterotrophs inside but not outside the contaminated areas • If petroleum has a distinctive 13 C/12 C ratio, this should be reflected in the carbon dioxide • Adding nitrogen or phosphorus fertilizer to replicated plots may relieve nutrient limitation, hence enhance loss from field plots compared to unfertilized controls • Genes involved in the catabolism of petroleum components should be expressed in high abundance inside but not outside the contaminated zone

Anaerobic aquifer contaminated with perchloroethylene

Dehalorespiring bacteria are using chlorinated aliphatic compounds as final electron acceptors (13,14). Dechlorination reactions are governed by: complex microbial and chemical interactions that generate physiological electron sources (especially hydrogen gas); the presence of alternative electron acceptors (NO3 − , Mn4+ , Fe3+ , SO4 2− , carbon dioxide) in site sediments and waters; and ecological and physiological competition among the microorganisms carrying out the metabolism that links electron donors and acceptors (15,16)

Aerobic aquifer contaminated with TCE

TCE destruction is achieved cometabolically by aerobic microorganisms supplied with a primary carbon source. Oxygenase enzymes (involved in metabolizing primary substrates such as methane, propane, toluene, and phenol) fortuitously convert TCE to unstable compounds that spontaneously hydrolyze to nontoxic and/or readily biodegradable components (17–19)

Anaerobic aquifer contaminated with jet fuel

Aromatic fuel components, especially toluene, serve as growth substrates for anaerobic microorganisms that utilize sulfate as a final electron acceptor

• Dechlorinated daughter products, trichloroethene (TCE), dichloroethenes (DCE), vinylchloride (VC) within contaminant plume • Products of complete detoxification, such as ethene, should be inside and not outside the plume • Adaptation of site microorganisms to dehalorespiration can be documented by finding dechlorination activity in site samples from inside but not outside the contaminant plume • Immunological or polymerase chain reaction–based data demonstrating the presence of dehalorespiring enzymes, genes, and characteristic bacteria inside but not outside the plume • Microcosms prepared with site samples consume TCE only when supplied with both oxygen and the primary substrate • In recirculating field site waters, TCE loss is enhanced only when both oxygen and the primary substrate are supplied • Assays for oxygenase enzymes, genes, and appropriate metabolites (e.g., trans-dichloro-ethylene oxide) reveal high abundances inside but not outside the contaminated zone • Microcosms containing site sediments incubated under sulfate reducing conditions produce 14 CO2 from 14 C-labeled toluene and benzene (20) • Sulfate is depleted along the groundwater flow path in the field site • Dissolved inorganic carbon (e.g., carbon dioxide) increases along the flow path in field sites (20) • Contaminant plume has ceased advancing despite a constantly dissolving reservoir of jet fuel (20) • A solute transport model accounts for dispersion, flow velocity, and adsorption, and produces biodegradation rate estimates that are consistent with microcosm estimates (20)

181

182


the potential to metabolize the contaminants under expected site conditions. 3. Demonstrate that the metabolic potential measured under criterion 2 is actually expressed in the field. To achieve this, microbiological mechanisms of contaminant attenuation must be distinguished from abiotic ones. Evidence deemed suitable for these purposes will vary according to the contaminants and conditions found at each site. Implementing Step One: Site Monitoring to Understand Site Biogeochemistry and Establish Historical Trends of Contaminant Behavior. It must be recognized that virtually all locations in the biosphere (from the poles to the equator, contaminated or pristine sites, engineered bioreactors, or the deep sea) are inhabited by microorganisms. Furthermore, whenever physiological resources are available, microbial metabolic activity will occur. Thus, site characterization is designed to assess the resources and guide the documentation of their exploitation by microorganisms. There is a critical need to relate results of geochemical measurements performed on field samples directly back to in situ processes and conditions. For details of completing in situ analyses, avoiding site-sampling artifacts, and understanding site biogeochemistry see References 7,38–40. All site characterization data must be interpreted in terms of the physiological processes that produce and consume geochemical constituents. Final electron acceptors that dominate the physiological reactions of field sites (or discrete zones therein) provide useful criteria for categorizing biogeochemical regimes as aerobic, denitrifying, iron reducing, manganese reducing, fermentative, dehalogenating, sulfate reducing, or methanogenic (41–43). These physiological regimes are often separated in space and/or time in field sites and largely determine the mechanisms of contaminant biodegradation. Information establishing the physiological regime(s) operating at particular field sites is provided by field measurements of the contaminants themselves, of concentration gradients of coreactant final electron acceptors (e.g., oxygen, NO3 − , Fe3+ Mn4+ , SO4 2− , halogenated compounds), and of end products of microbial metabolism (e.g., carbon dioxide, Fe2+ , Mn2+ , S2− , N2 O, NH4 + , organic acids, reductive dehalogenation daughter products, and methane) along site transects. In this regard, Lovley and coworkers (44) and Chapelle and coworkers (45) have devised a gas sampling bulb protocol for anaerobic groundwaters in the field, which, in combination with hydrogen gas determinations and Winkler titrations for oxygen (40), provides definitive information on dominant anaerobic redox couples. The goal of establishing site-specific historical records of the behavior of contaminants, coreactants, and metabolic products is a simple one. Theoretically, compilation of such field data documents the effects of contaminantattenuating processes over time. In conjunction with other assays (see later), field data can be interpreted in ways that may implicate biodegradation as a cause of pollutant losses. However, as in understanding in situ physiological regimes, obtaining robust, interpretable field-monitoring data may be an elusive goal if contaminant characteristics

and site conditions are complex. The overall objective is to establish a site-monitoring regime using a network of consistent sampling locations, which affords the acquisition of contaminant concentration and other measurements that are comparable over time. If the distribution of contaminants at the site and factors influencing contaminant transport (e.g., climate, hydrology, commercial, or industrial activities) are erratic, then the pertinent database on contaminant behavior may be so noisy as to mask any trends. However, in many sites the type of contaminant monitoring protocols required by concerned regulatory agencies can be integrated over time and can sometimes produce a clear historical record of diminishing contaminant concentrations from year to year. When such data exist, they assist in meeting the first criterion for proving in situ bioremediation. Implementing Step Two: Laboratory Assays Demonstrating That Microorganisms in Site Samples Have the Potential to Transform the Contaminants Under Expected Site Conditions. The biodegradation assays discussed in this article are designed to ask the question: ‘‘Are microorganisms within samples of soil, water, or sediment microbial communities able to metabolize the compound(s) of interest under conditions that are relevant to the specific field site of interest?’’ To answer this question, one gathers samples aseptically from a given field site or bioreactor, dispenses known weights or volumes of the samples to replicated vessels, handles the samples in a variety of ways that include a treatment that has been either sterilized or poisoned, incubates the test samples under laboratory conditions, and employs both chemical and physiological assays that monitor the fate of the test compound within experimental vessels over time (Fig. 1, phase 1). These procedures have been described earlier. Implementing Step Three: Evidence for Field Expression of Biodegradation Potential. Three sources of uncertainty must be confronted and overcome when demonstrating that microorganisms are the active agents of pollutant loss in bioremediation projects. 1. We must acknowledge that extrapolation from laboratory-based metabolic activity assays to the field is usually unwise because of the propensity of microorganisms in field samples to respond to laboratory-imposed physiological conditions that are unlikely to match those in the field perfectly (6,46). 2. The spatial heterogeneity of field sites may impede or completely prevent trends in the behavior of environmental contaminants from being discerned (12,20). 3. The action of a multitude of both abiotic and biotic processes may contribute simultaneously to pollutant attenuation (6,20). To contend with these challenges, several strategies have been developed for verifying the success of field bioremediation efforts in truly activating pollutant-destroying microbial processes in field sites and bioreactors. These (comprehensively codified in Table 3) are simple, logical expressions of the fundamental paradigm for verifying

Table 3. Strategies for Obtaining Evidence for Field Expression and Biodegradation Processes (After Ref. 9) Type of Strategy

Principles and Examples

References

Internal conservative tracers

Assess loss of certain compounds relative to the persistence of less-biodegradable, but similarly transported, compounds. Examples include using ratios of straight-to-branched chain alkanes (C17 /pristane, C18 /phytane) and ratios of other compounds to hopane in crude oil; ratios of lower to higher chlorinated congeners in PCB mixtures (trimethyl benzene can serve as a conservative tracer in benzene-toluene-ethylbenzene-xylene (BTEX) plumes); ratios of nonchlorinated to chlorinated aromatics in mixed solvents; and selective metabolism of one stereoisomer of particular pesticides (e.g., σ -chlorocyclohexane)

Added conservative tracers

In some field sites, contaminant mixtures may lack internal tracers but be amenable to the addition of materials that provide a baseline measure of various transport processes. Examples include helium to assess oxygen loss or carbon dioxide production in groundwater, propane to assess toluene loss from a stream, and bromide to assess groundwater flow

25,58,59

Added radioactive tracers

In rare instances, regulatory authorities have allowed intentional field release of radioactive (e.g., 14 C-labeled) pollutants in field sites. Subsequent recovery of 14 CO2 , 14 C-metabolites, and 14 C-parent compounds provide, definitive proof of metabolic and other field processes

60–63

Added stable isotopic tracers

Pollutant compounds that are nonradioactive, but isotopically labeled with deuterium or 13 C, have been released in field sites. Subsequent stable isotopic analyses of field samples for labeled CO2 , metabolites, and/or the parent compound provide proof of metabolic and other field processes

64,65

Stable isotopic fractionation patterns

CO2 has different 13 C/12 C ratios depending on the 13 C/12 C signature of the substrates respired and the 13 C-enriching process of methanogenesis. When site-specific signatures of both inorganic and organic carbon reservoirs have been characterized, the relative contribution of pollutant biodegradation to the pool of CO2 can be discerned. The radioactive (14 C) component of CO2 is also revealing because petroleum contaminants contain no 14 C

49,66–70

Detection of intermediary metabolites

When sufficient biochemical knowledge of pollutant biodegradation has accrued, particular metabolites can be targeted using a combination of careful sampling and analytical chemistry. Detection of stable (dead-end) metabolites and transient metabolites (indicative of ‘‘real-time’’ biodegradation) has been reported. The metabolites include trans- dichloroethylene oxide, dihydrodiols of aromatic compounds, DDE, and hydroxylated pesticides

71–77

Replicated field plots

Some relatively homogeneous field sites are amenable to randomized block designs of treatments that stimulate microbiological activity. Comparing the loss of pollutants from plots with and without nutrients and/or inocula can demonstrate effectively the role of microorganisms in field biodegradation

47,53,78–81

Microbial metabolic adaptation

Naturally occurring microbial communities that grow in response to pollutant exposure have predictable characteristics relative to adjacent unexposed communities. Adaptation is reflected in laboratory or field measurement of: qualitative pollutant metabolisn or rates of pollutant metabolism; numbers of specific pollutant degraders; and enhanced concentrations of protozoan predators of bacteria inside but not outside contaminant plumes

16,82

Molecular biological indicators

Based on molecular biological characterization of pure cultures capable of pollutant metabolism, a variety of assays consistent with established genetic sequences and their expression can be devised. These include polymerase chain reaction (PCR) amplification of structural genes, messenger ribonucleic acid (mRNA) extracted from field sites, reverse-transcriptase PCR detection of mRNAs, nucleic acid sequencing, immunodetection of enzymes and metabolites, and 16S ribosomal RNA analysis of the composition of microbial communities

83–90

Gradients of coreactants and/or products

Ongoing in situ metabolism of pollutants consumes physiological final electron acceptors and generates metabolic endproducts that reflect site-specific pollutant metabolism. Chemical gradients in field sites should be apparent using measures that include oxygen, NO3 − , Mn4+ , Fe3+ , SO4 2− , CO2 , NO2 − , N2 O, Mn2+ , Fe2+ , methane, hydrogen, pH, and alkalinity

In situ rates of respiration

A subset of the previous entry that has been effectively applied to engineered bioremediation of subsurface sites involves cessation of an oxygen (or air) sparging regime, followed by insertion of an oxygen probe that documents real-time oxygen consumption. This respiratory activity should be high inside but not outside the contaminated area. The conserved gas, helium, can be included in the sparging step to account for diffusional O2 loss

47–57

20,91,92

93,94


183

184


Table 3. (Continued) Type of Strategy

Principles and Examples

Mass balances of contaminants, coreactants, and products (total expressed assimilative capacity)

Under well-defined hydrogeologic regimes, fluxes of water contaminants and physiological electron donors or acceptors can be quantified in a cross-sectional analysis of site sampling stations. The stoichiometry of all appropriate aerobic, anaerobic, isotopic fractionation, and inorganic equilibria reactions can serve to predict and distinguish biotic from abiotic processes and to identify contributions from a variety of microbiological groups

Computer modeling that incorporates transport and reaction stoichiometries of electron donors and acceptors

This approach considers quantitative aspects of fluid flow, dilution, sorption, volatilization, mixing, microbial growth, and metabolic reaction stoichiometries to achieve an integrated and predictive tool for understanding all processes influencing the fate of pollutant compounds. This approach resembles the previous entry, but is implemented on a larger scale and uses more sophisticated computations

bioremediation introduced previously and in Table 1. The strategies that appear in Table 3 are firmly based on the physiological principles that distinguish between biotic and abiotic contaminant attenuation processes. Four of the strategies involve tracers (internal conservative, added conservative, added radioactive, and added stable isotopic) that either account for or circumvent problems arising from abiotic changes in field concentrations of contaminants and related metabolites. Six of the strategies in Table 3 rely on detailed prior knowledge of specific microbiological processes (stable isotope fractionation, detection of intermediary metabolites, stimulation of microbial activity in replicated field plots, metabolic adaptation, in situ respiration, and gradients of coreactants and/or products) that are manifest as observable geochemical changes in the field. The molecular biological strategy in Table 3 is an elegant, emerging approach that is constantly strengthened by genetic links that are forged between information from pure cultures and real-world mixed microbial communities. The linkages are limited by the relatively small database of genetic sequences pertinent to pollutant metabolism and the uncertain metabolic diversity that may arise when genes of unrelated lineage may have converged on the same metabolic function. The final two strategies in Table 3 (computer modeling and mass balances of contaminants, reactants, and products) attempt to account quantitatively for both transport and metabolic processes within entire field sites or along distinct transects therein. CONCLUSION Understanding and proving biodegradation processes under laboratory and field conditions is a science of ongoing discovery. This discovery requires a close dialog among many disciplines. It must be recognized that only under relatively rare circumstances is a proof of field bioremediation unequivocal when a single piece of evidence is relied on. In the majority of cases, the complexities of contaminant mixtures, their hydrogeochemical settings, and accounting for competing abiotic mechanisms of contaminant loss make it a challenge to document biodegradation processes. Unlike controlled laboratory experimentation wherein measurements can usually be interpreted easily, cause-and-effect relationships are often very difficult to

References 56,92,95–97

92,98–101

establish at field sites. Furthermore, certain bioremediation data that may be convincing for some authorities may not be convincing for others. Thus, in documenting bioremediation, the several approaches described previously should be independently pursued: a consistent, logical case relying on convergent lines of independent evidence should be built. The three-step strategy for verifying bioremediation described previously has been augmented recently by that of a new NRC Committee (26). What might be considered a fourth step and an overarching goal is assuring the public that bioremediation in specific sites will be reliable, sustainable, and quantitatively complete. BIBLIOGRAPHY 1. E. L. Madsen, in C. J. Hurst et al., eds., Manual of Environmental Microbiology, ASM Press, Washington, D.C., 1997, pp. 709–720. 2. M. Alexander, Biodegradation and Bioremediation, Academic Press, New York, 1999. 3. L. P. Wackett and C. D. Hershberger, Biocatalysis and Biodegradation, ASM Press, Washington, D.C., 2001. 4. R. M. Atlas, Curr. Opin. Biotechnol. 3: 220–223 (1991). 5. D. T. Gibson, ed., Microbial Degradation of Organic Compounds, Marcel Dekker, New York, 1984. 6. E. L. Madsen, Environ. Sci. Technol. 25: 1662–1673 (1991). 7. E. L. Madsen, in R. Burlage et al., eds., Techniques in Microbial Ecology, Oxford University Press, New York, 1998, pp. 354–407. 8. D. D. Focht, in R. W. Weaver et al., eds., Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties, Soil Science Society of America, Madison, Wis., 1994, pp. 407–426. 9. E. L. Madsen, in J. J. Valdes, ed., Bioremediation, Kluwer Academic Publishers, Netherlands, 2000, pp. 101–122. 10. R. M. Atlas and C. E. Cerniglia, Bioscience 45: 332–338 (1995). 11. H. S. Rifai et al., in R. E. Hinchee et al., eds., Intrinsic Bioremediation, Battelle Press, Columbus, Ohio, 1995, pp. 1–29. 12. J. T. Wilson and M. D. Jawson, in H. D. Skipper and R. F. Turco, eds., Bioremediation Science and Applications, Soil Science Society of America, Inc., Madison, Wis., 1995, pp. 293–303.

BIODEGRADABILITY: ASSESSMENT 13. X. Maymo-Gatell et al., Science 276: 1568–1571 (1997). 14. P. L. McCarty, Science 276: 1521–1522 (1997). 15. D. E. Fennell et al., Environ. Sci. Technol. 31: 918–926 (1997). 16. R. M. Yager et al., Environ. Sci. Technol. 31: 3138–3147 (1997). 17. G. D. Hopkins et al., Environ. Sci. Technol. 27: 2542–2547 (1993). 18. S. M. Pfiffner et al., J. Ind. Microbiol. Biotechnol. 18: 204–212 (1997). 19. L. Semprini and P. L. McCarty, Ground Water 30: 37–44 (1992). 20. F. H. Chapelle et al., Ground Water 34: 691–698 (1996). 21. H. D. Skipper and R. F. Turco, eds., Bioremediation Science and Applications, Soil Science Society of America, Inc., Madison, Wis., 1995. 22. R. L. Crawford and D. L. Crawford, eds., Bioremediation: Principles and Applications, Cambridge University Press, New York, 1996. 23. P. E. Flathman et al., Bioremediation: Field Experience, Lewis Publishers, Boca Raton, Fla., 1994. 24. R. D. Norris et al., Handbook of Bioremediation, Lewis Publishers, Boca Raton, Fla., 1994. 25. National Research Council, In Situ Bioremediation: When Does It Work? National Academy Press, Washington, D.C., 1993. 26. National Research Council, Natural Attenuation for Groundwater Remediation, National Academy Press, Washington, D.C., 2000. 27. B. E. Rittmann et al., In Situ Bioremediation, 2nd, ed., Noyes Publications, Park Ridge, Neb., 1994. 28. L. Y. Young and C. E. Cerniglia, eds., Microbial Transformation and Degradation of Toxic Organic Chemicals, WileyLiss, Inc., New York, 1995. 29. I. M. Head, Microbiology 144: 599–608 (1998). 30. A. Heitzer and G. S. Sayler, TIB Tech. 11: 334–343 (1993). 31. J. M. Tiedje, In Situ Bioremediation: When Does It Work?, National Academy Press, Washington, D.C., 1993, pp. 110–120. 32. J. A. McDonald and B. E. Rittmann, Environ. Sci. Technol. 27: 1974–1979 (1993). 33. J. F. Quensen III and J. M. Tiedje, in D. Sheehan, ed., Methods in Biotechnology, 2. Bioremediation Protocols, Humana Press Inc., Totowa, N.J., 1997, pp. 241–253. 34. J. P. Salanitro, Ground Water Monit. Remediation 13: 150–161 (1993). 35. M. J. R. Shannon and R. Unterman, Annu. Rev. Microbiol. 47: 715–738 (1993). 36. P. J. Swannell et al., Microbiol. Rev. 60: 342–365 (1996). 37. R. Atlas and R. Unterman, in A. L. Demain and J. E. Davies, eds., Manual of Industrial Microbiology, 2nd ed., ASM Press, Washington, D.C., 1999, pp. 666–681.

185

40. American Public Health Association (APHA), in A. E. Greenberg et al., eds., Standard Methods for the Examination of Water and Wastewater, 18th, ed., APHA Washington, D.C., 1992. 41. E. J. Bouwer, in R. D. Norris et al., eds., Handbook of Bioremediation, Lewis Publishers, Boca Raton, Fla., 1994, pp. 149–175. 42. H. F. Hemond and E. J. Fechner, Chemical Fate and Transport in the Environment, 2nd, ed., Academic Press, New York, 2000. 43. J. J. Stumm and W. Morgan, Aquatic Chemistry, 3rd, ed., John Wiley and Sons, New York, 1995. 44. D. R. Lovley et al., Environ. Sci. Technol. 28: 1205–1210 (1994). 45. F. H. Chapelle et al., Environ. Sci. Technol. 31: 2873–2877 (1997). 46. E. L. Madsen, in G. Stotzky and J.-M. Bollag, eds., Soil Biochemistry, vol. 9, Marcel Dekker, Inc., New York, 1996, pp. 287–370. 47. J. R. Bragg et al., Nat. (Lond.) 368: 413–418 (1994). 48. M. R. Harkness et al., Science 259: 503–507 (1993). 49. A. W. Jackson et al., Environ. Sci. Technol. 30: 1139–1144 (1996). 50. D. H. Kampbell et al., J. Hazard. Mater. 49: 197–204 (1996). 51. P. Ludwig et al., Mar. Chem. 38: 13–23 (1992). 52. R. C. Prince et al., Environ. Sci. Technol. 28: 142–145 (1994). 53. P. H. Pritchard and C. F. Costa, Environ. Sci. Technol. 25: 372–379 (1991). 54. V. A. Tett et al., FEMS Microbiol. Ecol. 14: 191–199 (1994). 55. T. H. Wiedemeier et al., in R. E. Hinchee et al., eds., Intrinsic Bioremediation, Battelle Press, Columbus, Ohio, 1995, pp. 31–51. 56. R. A. Williams et al., J. Ind. Microbiol. Biotechnol. 18: 177–188 (1997). 57. C. Zipper et al., Appl. Environ. Microbiol. 62: 4318–4322 (1996). 58. H. Kim et al., Environ. Sci. Technol. 29: 108–116 (1995). 59. W. D. Robertson, Environ. Toxicol. Chem. 13: 183–191 (1994). ¨ 60. F. Fuhr et al., in H. Frehse, ed., Pesticide Chemistry— Advances in Industrial Research, Development, and Legislation. Proceedings of the Seventh International Congress of Pesticide Chemistry (IUPAC), VCH Publishers, Weinheim, Germany, 1990, pp. 37–48. ¨ 61. F. Fuhr et al., eds., The Lysimeter Concept: Environmental Behavior of Pesticides, American Chemical Society, Washington, D.C., 1998. 62. K. Lee et al., Microb. Ecol. 11: 337–351 (1985). 63. W. G. MacIntyre et al., Water Resour. Res. 29: 4045–4051 (1993). 64. J. Thierrin et al., Ground Water 33: 469–475 (1995).

38. E. R. Weiner, Applications of Environmental Chemistry: A Practical Guide for Environmental Professionals, Lewis Publishers, Boca Raton, Fla., 2000.

65. J. Thierrin et al., Hydrol. Sci. J. 38: 309–322 (1993).

39. L. H. Keith, Environmental Sampling and Analysis: A Practical Guide, Lewis Publishers, Boca Raton, Fla., 1991.

67. M. E. Conrad et al., Environ. Sci. Technol. 31: 1463–1469 (1997).

66. P. K. Aggarwal and R. E. Hinchee, Environ. Sci. Technol. 25: 1178–1180 (1991).

186

BIODEGRADATION IN SOIL

68. E. L. Grossman, in D. J. Hurst et al., eds., Manual of Environmental Microbiology, ASM Press, Washington, D.C., 1997, pp. 565–576.

94. D. X. Li, in R. E. Hinchee et al., eds., Monitoring and Verification of Bioremediation 3(5), Battelle Press, Columbus, Ohio, 1995, pp. 115–126.

69. D. Hunkeler et al., Environ. Sci. Technol. 33: 2733–2738 (1999).

95. P. Höhener et al., J. Microbiol. Methods 32: 179–192 (1998).

70. J. E. Landemeyer et al., Environ. Sci. Technol. 30: 1120– 1128 (1996).

97. D. A. Wolfe et al., Environ. Sci. Technol. 28: 561A–568A (1994).

71. H. R. Beller and A. M. Spormann, J. Bacteriol. 179: 670–676 (1997).

98. B. A. Bekins et al., J. Contam. Hydrol. 14: 279–294 (1993).

72. H. R. Beller et al., Environ. Sci. Technol. 29: 2864–2870 (1995). 73. W. P. Flanagan and R. J. May, Environ. Sci. Technol. 27: 2207–2212 (1993). 74. R. N. Lerch et al., Environ. Sci. Technol. 29: 2759–2768 (1995). 75. L. Semprini et al., Ground Water 28: 715–727 (1990).

96. L. Semprini et al., Water Resour. Res. 31: 1051–1062 (1995).

99. C. Y. Chiang et al., Ground Water 27: 823–834 (1989). 100. M. M. Lang et al., Ground Water 35: 565–573 (1997). 101. L. Semprini and P. L. McCarty, Ground Water 29: 365–374 (1991).


76. L. Semprini et al., Water Resour. Res. 31: 1051–1062 (1995).

RON TURCO

77. M. S. Wilson and E. L. Madsen, Environ. Sci. Technol. 30: 2099–2103 (1996).

Purdue University West Lafayette, Indiana

78. J. A. Glaser and R. T. Lama, in H. D. Skipper and R. F. Turco, eds., Bioremediation of Science and Applications, Soil Science Society of America, Inc., Madison, Wis., 1995, pp. 117–133. 79. R. T. Lamar et al., Soil Biol. Biochem. 26: 1603–1611 (1994). 80. R. L. Raymond et al., Appl. Environ. Microbiol. 31: 522–535 (1976). 81. R. K. Steffan et al., Environ. Sci. Technol. 33: 2771–2781 (1999). 82. E. L. Madsen et al., Science 252: 820–833 (1991). 83. F. J. Brockman, Mol. Ecol. 4: 567–578 (1995). 84. R. S. Burlage, in D. J. Hurst et al., eds., Manual of Environmental Microbiology, ASM Press, Washington, D.C., 1997, pp. 115–123. 85. J. T. Fleming et al., Environ. Sci. Technol. 27: 1068–1074 (1993). 86. A. Ogram et al., Appl. Environ. Microbiol. 61: 763–768 (1995). 87. S. Selvaratnam et al., 3981–3985 (1995).

Appl.

Environ.

Microbiol.

61:

88. M. S. Shields and S. C. Francesconi, in R. L. Crawford and D. L. Crawford, eds., Bioremediation: Principles and Applications, Cambridge University Press, New York, 1996, pp. 341–390. 89. M. S. Wilson et al., Appl. Environ. Microbiol. 65: 80–87 (1999). 90. J. Zhou et al., Appl. Environ. Microbiol. 63: 2384–2390 (1997). 91. B. C. Alleman and A. Leeson, eds., Natural Attenuation of Chlorinated Solvents, Petroleum Hydrocarbons, and Other Organic Compounds, Battelle Press, Columbus, Ohio, 1999. 92. H. S. Rifai et al., in R. E. Hinchee et al., eds., Intrinsic Bioremediation, Battelle Press, Columbus, Ohio, 1995, pp. 53–58. 93. G. B. Davis et al., in R. E. Hinchee et al., eds., Monitoring and Verification of Bioremediation 3(5), Battelle Press, Columbus, Ohio, 1995, pp. 193–201.

While the production of food and fiber is directly dependent on soil, the importance of microorganism to the formation and stability of soil has only been fully realized in the last 100 years. The contributions of the soil organisms to the function of soil can be viewed as being the biochemical engine that drives most of the important soil processes. The microorganisms are responsible for the breakdown of dead plant and animal materials, effectively ridding the earth of millions of tons of organic materials each year. It is during this process that the microorganisms provide the organic building blocks that are used to establish soil structure, helping to form a stable land surface. A well-structured soil contributes to good plant growth and water infiltration, which diminish the potential for runoff events and protects water quality. It is during the plant and animal degradation process that microorganisms begin a recycling process, which converts organic forms of nutrients to inorganic and plant available forms. Microorganisms have been playing an even more important role. It is now clearly established that microorganisms not only degrade natural organic materials but they are responsible for degradation of many materials of human origin. These materials can include oils, pesticides, gasoline, and organic solvents. The soil microbial populations have been shown to be responsible for the conversion of contaminants to less dangerous materials, although toxic products are possible. This article’s focus is on the factors that affect the functioning of the microbial component of soil. We will address this by providing information describing the resident status of organisms in soil, and then describing the factors that control their activity and finally, describe how microbial population responds to pressures from humans and how we harness their inherent abilities. In this regard, we will show how inputs from humans can both suppress and maximize the functioning of soil microbial populations. A clear understanding of these factors is needed if we are to successfully manage soil to maintain both its inherent functions and to meet secondary goals such as allowing environmental cleanup.


THE SOIL STRUCTURE

187

Aggregate Formation

Defining the Microbial Habitat Soil particles, sand, silt, clay (i.e., the components of soil texture) are the base units of soil structure and it is the arrangement of these particles that results in soil that are typically viewed in the landscape. Soil particles range in size from sand particles as large as 2 mm to tiny clay particles (i.e., very coarse sand 2.0 to 1.0 mm, coarse sand 1.0 to 0.5 mm, medium sand 0.5 to 0.25 mm, fine sand 0.25 to 0.10 mm, very fine sand 0.10 to 0.05 mm, silt 0.05 to 0.002 mm, clay less than 0.002 mm). These particles rarely exist as independent units. Soil particles are more typically found bound together as aggregates with the linkage between particles resulting from organic bridges and binding agents often of microbial origin. In turn, it is the size and stability of all of the aggregates that determine the size and arrangement of soil pores. It is the soil pores that control the flow of air and water in soil (Fig. 1). Although it was pointed out in 1927 (1), we are only now fully appreciating the importance of the soil aggregate as a habitat for soil microorganisms. Because of the microorganism’s size (typically 1 µM or less), a tendency toward sessile growth, the soil’s low intrinsic nutrient status (2), and their resulting slow growth rates, soil microorganisms are generally required to function under multiple limitations. In response to these multiple stresses, a typical terrestrial bacterium is found to colonize mineral surfaces often within a soil aggregate. In turn, they become indirectly responsible for improving the stability of the soil aggregates as the microcolony of cells grows and produces polysaccharides and other agents that bind the cells to the surface and also bind smaller soil particles together.

Organic phase

Water film KDOC-soil DOC

Colony KD-DOC

Soil mineral phase

KD-soil

Organic

Soil organic phase Figure 1. Diagram depicting the relationship between organic contaminants and the microbial populations. The distribution of contaminants between the soil surface and the soil solution is indicated by the KD . The value KDOC indicates the distribution of soil organic matter between surface and solution, and KD-DOC indicates the reaction between contaminants and the dissolved organic matter in soil. In the case of KD and KDOC , a lager value indicates more solid-phase retention and a smaller dissolved solution content for the organic matter. In the case of KD-DOC , a larger value indicates more interaction between the dissolved organic matter than the contaminant and less contaminant in solution. The shaded areas represent water films.

The exact process of aggregate formation is speculative but is thought to follow a sequence in which a few particles become bound into a microaggregate. The microaggregates are then bridged or combined with other microaggregates to form larger aggregates (3). Both biological and physical processes aid in the formation of soil aggregates. Soil freeze and thawing cycles, soil drying, and fungal hyphae growth tend to push particles together. Microbial decomposition of organic residues (plant materials) releases many organic materials that act as bridge and then stabilize the adjoining particles. These materials become the core of what will be soil organic matter. Recent work has shown the organic materials released by the microorganisms to be biophysically complex with molecular weights exceeding 7,000 gmol−1 (4). It is generally thought that stable aggregates are created largely by microbial processes as the binding agents, which hold the aggregate together, are the materials released by cells during the degradation of plant materials or resident soil organic matter. Burrowing soil animals such as worms and insects and the growth of plant roots also push the smaller aggregates together and the reoriented surfaces act as locations for microbial colonization and additional fungal growth. Fungi have been shown to release compounds such as glomalin (a glycoprotein), which further stabilizes the matrix (5,6). Singh and Singh (7) demonstrated differences in the portion of microaggregates and macroaggregates as a function of soil use. Forest soil, with little disturbance, showed the greatest number of macroaggregates, whereas cropland soils were formed primarily from microaggregates. During aggregate formation, soil organic matter begins to buildup, as it was made unavailable within the newly formed microaggregates. Soils that have high levels of contamination (metals or organics) tend to be reduced in structure (8), possibly associated with lower microbial activity and impact from anthropogenic actions. Clearly, significant changes in microbial biomass and metabolism along with soil management will alter the soil structure (9). The distribution of aggregate sizes within a unit of soil will alter the resulting pore size distribution in that soil tillage or other disturbance greatly reduces the aggregate size and liberates entrained (trapped) carbon that became resident in the formation process (10). These changes can affect subsequent plant growth (11). A high-quality soil will have a range of aggregate sizes from small to large; correlated to this is a range of pore space sizes. From the microbial perspective, aggregate geometry and size along with the resulting pore spaces are critical to their function both directly from the perspective of water and air infiltration and indirectly from the perspective of the improved plant growth, which becomes a source of carbon (11,12). The net effect is that biological processes form the aggregates and the arrangement of both the sizes of aggregates will create pore spaces because pore spaces are found within and between both small and large aggregates. The pore spaces inside the aggregates are sometimes referred to as occurring within the soil matrix because they occur inside a soil structural unit (13). These matrix

188


pores may connect to the larger pore spaces that occur between the stable soil aggregates. However, matrix pores may occur within an aggregate without being connected to the larger aggregate pore spaces. As a result, a typical soil is intermixed with small and large holes and connected and unconnected channels. The soil microbial community is associated with both types of pore spaces as the population is found both in and outside the aggregate. However, most microbial populations tend to be found on the inside of pore spaces within aggregates. Transport of air and water within soil is governed by the three-dimensional arrangement of pores. Thus, the proximity to pores strongly influences functioning of the soil microorganisms inside the aggregate. Therefore, soil aggregate stability is a key soil property affecting the functioning of the soil (14). The stability of the aggregate reflects both microbial and nonmicrobial processes. At many contaminated or deteriorated sites, the soil structure has been lost, which then limits the soil’s ability to function. Soil Microorganism—Living in an Aquatic World Even in what seems to be a dry soil, soil bacteria function best in an aquatic environment. They function best when covered by a water film. This water film fills small soil pores and acting as transport system for nutrients and dissolved oxygen. Soil water is described using units of megapascals (MPa). These are tension values and indicate the amount of energy needed to extract free water. Different soil types (i.e., clay, silt, or sand) will have different contents (percentage moisture) at the same water potential. For example, at 20% moisture, a clay soil has a greater ability to hold water (lower water potential) than a sandy soil. A saturated soil has a water content equivalent to 0 mPa, whereas at a water potential of −1.5 mPa, little water is available and the soil is dry. Soil microbial populations function best at water contents (%) equivalent to −0.01 MPa of water potential pressure (15). At the −0.01 MPa the water potential–content combination, the soil tends to have an optimum arrangement in terms of both water film thickness and open space for the exchange of oxygen from the air (Fig. 1). Soil microbial activity will decline as the soil dries (moves toward more negative water potentials) because without water, the bacteria will dehydrate and lose function and they are unable to receive nutrients. Conversely, too much water will suppress aerobic metabolic activity because the soil water near the cells cannot be replenished with oxygen at a rate corresponding to microbial consumption. As a result, interactions between oxygen and water within the soil are complex because the gas that enters the soil in the open portion of the pore must dissolve in water before the bacteria use it. The solubility of oxygen in water is 0.028 mL O2 mL−1 H2 O atm−1 (16) or as the more common expression 8 mg O2 L−1 (70 ◦ C). The situation is further complicated by the diffusion rate of O2 in water which is about 1 × 104 of the diffusion of oxygen in air [values of diffusion coefficients are 2.5 × 105 cm2 sec−1 in water versus 0.189 cm2 sec−1 for air (17)]. As a result, the level of oxygen tends to decrease with soil depth as both poor water solubility and low diffusion rates limit

oxygen transport. The decline in oxygen levels down the soil profile reflects both respiration by organisms near the soil surface and restriction on flow related to decreasing pore size and increasing water content with depth in the profile. However, the larger pore spaces near the soil surface generally allow for oxygen diffusion for the upper portions of the profile keeping it aerobic. A somewhat analogous situation occurs at a smaller scale within soil aggregates. The population near the aggregate surface first uses the available oxygen. As was found with the soil profile, free water in the soil pores limits the inward flow of oxygen. However, in the case of the aggregate, the restrictions on the flow of oxygen into the aggregate are even more significant because the oxygen must diffuse down very small pore spaces. As a result, an aggregate within an otherwise aerobic soil can develop an anaerobic core. Smith and Arah in 1986 (18) and others (19) demonstrated the presence of anaerobic microsites or conditions in otherwise aerobic soil. MICROORGANISMS IN SOIL Diversity and Cell Density in Soil Trevors (20) has described soil as a ‘‘virtually limitless pool of genetic information contained in bacteria.’’ This conclusion reflects the fact that surface soil can contain some 4,000 different microbial genotypes with as many as 109 (one billion) cells in 1 g (21,22). Others have suggested the number could be as high as 40,000 bacterial species in 1 g. Subsurface soils tend to have lower population levels but can exceed 107 cells g−1 (23,24). Surprisingly, given the sheer numbers of microorganisms, the microbial population comprises less than 3% of the soils organic carbon and occupy only 0.001% of the soil’s volume (25). Therefore, microorganisms are not densely packed but rather live as ‘‘islands’’ or microcolonies on soil surfaces in aggregates or embedded in decaying organic matter (26). Hissett and Gray (27) have shown that microorganisms reside on less than 0.17% of the surface of the organic matter of the soil and less than 0.02% of the mineral surfaces of the soil. This pattern of distribution reflects the lager surface area of the soil. For example, the specific surface area of soil clay can range from 5 to more than 750 m2 g−1 . Movement of Cells in Soil Microorganisms generally adhere to soil surfaces by electrostatic interactions, London-van der Waals forces, and hydrophobic interactions (28,29). The overall movement of bacteria in soil is controlled by their tendency to undergo sorption onto soil particles or their transport and trapping in small soil pores (30). Most movement of bacteria in soil occurs by passive transport where the cells are moved with flowing water. On the other hand, active transport that is facilitated through the expenditure of energy is fairly limited (31–33). Tan and coworkers (34) found that passive movement was retarded by the adsorption of bacteria onto surfaces. Generally, passive movement will carry bacteria greater distances, over several centimeters, than active movement (35), which carries then over a few millimeters.


The predominant factors affecting bacterial transport are the ionic strength of the suspending solutions, which affects the charge properties of the cell and the surface, the soil surface properties, and the flux rate of water through the system. Trevors and coworkers (36) found that the movement of a genetically engineered strain of Pseudomonas through a soil column was a function of water flow rate and the number of times the column was flushed. This is supported by the work of Gagliardi and Karns (37) who showed for Escherichia coli that high levels of bacterial penetration into the subsurface could be seen when high rates of water input occurred along with the application of the bacteria. Cho and Kim (38) demonstrated using Salmonella typhi that bacteria introduced with manure could survive in both viable and viable but nonculturable (resting) states. Longer-term survival was not addressed in either study but Gagliardi and Karns (37) showed that significant growth could be seen even in the short periods (18 days) following application of bacteria and manure. They reported a 15-fold increase in the number of bacteria going in and leaving the system, when compared with the numbers inoculated into the system. Cell Retention on Surfaces Cell retention in soil is a function of the type of bacterium and the soil surfaces. It has been reported that the migration of bacteria with a hydrophobic exterior was two to three times slower compared with a similar strain with hydrophilic surface properties (31); authors were able to correlate cell retention with the adhesion of the hydrophobic strains. Other work has shown that the contribution of flagella to movement declines as the soil dries and the water film thickness decreases. As opposed to increasing the potential for movement, it is suggested that presence of flagella may enhance cell sorption onto soil surfaces (32). The movement of motile bacteria, especially ones with large numbers of flagella, may be impaired as the flagella increase the cell’s overall volume preventing its movement through small pore necks (32,33).

189

oxygen and nutrients. Pores between areas colonized by bacteria and areas colonized by predators such as protozoa also affect the long-term survival of the bacteria. They are most often found within pore spaces connected to adjoining interaggregate spaces through a channel with a pore neck sized between 0.25 and 6 µm in diameter (45,46) (Figs. 1 and 2). A pore with a 0.25 µm diameter limits entry of bacteria, whereas sizes in excess of 6 µm allows entry of protozoa. By colonizing pore spaces connected with a pore neck size of less than 6 µm, the bacteria can be protected from protozoa, which require a larger pore diameter to invade the space (40,46–49). It has also been shown that protozoan activity and predation often coincide with high bacterial numbers and activity (50). Therefore, the feeding activities of protozoa are effective in controlling the size of active population of soil bacteria and may control the establishment of inoculated species sometimes used in remediation situations or as seed inoculants (46,51). Microorganisms colonize the soil matrix as the soil is forming and have also been reported to occur in the microaggregate soil fractions between 2 and 53 µM in size (50). Recent work has suggested soil bacteria to be mainly associated with the clay and silt fractions and fungi to be associated with the organic materials and coarse sand fractions (52). A ready supply of carbon and electron acceptors (oxygen, Mn4+ , Fe3+ , SO4 2− ) is thought to be a controlling factor for the active colonization of an area (53,54). Gram-positive bacteria are often found on the outside surface. In contrast, gram-negative bacteria are generally found within the aggregates and pore spaces (47). This distribution may reflect a selective pressure applied by changes in water availability because gram-positive bacteria are generally more resistant to drying than are gram-negative bacteria (15). The internal regions of the aggregate will have more stable water content because the small pores spaces are slower to dry when compared with the outside that is exposed to the pore space air. The slower drying rate reflects the smaller pore necks and a lower flux of water. On the other hand, the wetter conditions can also lead to anaerobic conditions. These findings

Surface and Pore Neck Colonization The location of bacterial colonies within soil is influenced by soil structure, the location of nutrients, and pore size (39–42). The exact factors that lead to colonization of a given pore or surface region of a pore space are unresolved. However, detailed analysis of soil structure has shown that greater than 80% of the cells found in soil are located within the smaller aggregate fractions of the soil (43) rather than on outer surfaces. Work by Fisk and coworkers (44) has shown that for introduced bacteria, a prime location for colonization is along the intergrain area where mineral and organic materials meet. They also observed that most of the resident bacteria are located along the same interfacial areas. At the microscale, the arrangement of cells in soil aggregates is the predominant factor influencing cell behavior. Soil bacteria are sessile and become physically resident on soil surface. The size of the actual pore space in which bacteria reside governs the amount of available

Pore space Organic matter

O2 Organic contaminant Colony

CO2 Water film Mineral phase

Figure 2. Diagram depicting the relationship of a resident soil microbial populations to the open pore space, organic matter, and organic pollution. Water film thickness is variable as is the distribution of both natural organic matter and the contaminant.

190


support the conclusion that the soil microbial population is discontinuous, not forming a biofilm as is found in the typical wastewater biotreatment facilities (44). Feeding the Microbial Community Once attached to soil, soil microorganisms remain relatively immobile for the majority of their existence. As a result, organic materials must be transported to the microorganisms to be used. Therefore, the arrangement of micropores, which controls the flow of water in turn controls, the flow of nutrients, will influence the locations where colonies develop. The discontinuous arrangement of microbial population structure in and out of the pore spaces is a key factor affecting transformations of organic compounds. While the residency of the bacterial population inside small pore necks protects them from predation, small pore neck sizes also limit access to nutrients, which must diffuse inside the soil aggregate to be used. This diffusion-linked pattern of growth accounts for the microorganism’s occurrence along interfacial areas between regions of soil organic matter and mineral surfaces. The mineral surface gives the bacteria a point of attachment, access to potential electron acceptors other than oxygen, whereas the adjacent organic materials provide a long-term source of nutrients over a short diffusion pathway (Fig. 2). The active portion of the soil microbial community makes use of carbon and nitrogen derived from/during plant decomposition or the mineralization of native soil organic matter (55). A strong correlation between plant productivity and active soil microbial population has been shown (56). Others have shown a strong correlation between microbial biomass and available soil carbon (57). Subsurface microorganisms are dependent on carbon leached from surface materials or materials delivered from adjacent aquifers and the capillary rise of groundwater. Patterns of microbial activity reflect the pattern of available carbon. In particular, long-term undisturbed sites tend to have higher carbon and activity levels (enzymes, respiration) deeper in the soil profile than do sites that are routinely tilled (58). Mixing of the active soil population with residues and the inclusion of air and water that enhances the degradation processes. Of particular note is the finding that manure, which is high in dissolved organic carbon (DOC), can significantly increase the size of soil microbial biomass (59) and can influence subsequent degradation rates of chemicals (60). Work has shown that soil microbial diversity is also affected during the composting of manure in the soil (61). These findings suggest that the microorganisms in soil and the subsurface are generally faced with a poor nutrient availability and that increasing the nutrient availability will alter the composition of the population. Because of the lack of available nutrients, the majority of the organisms in soil are in a ‘‘resting stage’’; they are alive but are maintaining themselves in a low level of activity (25,62). In fact, less than 1% of the soil bacteria are typically recovered using typical laboratory isolation procedures (63). Studies to compare soils have confirmed this finding. Direct isolation of DNA from soil shows a higher phylogenetic diversity

than does isolation of individual colonies (64). Indeed, the inactive biomass constitutes the major portion of the ‘‘limitless pool of genetic resource’’ described by Trevors (20). Environmental stress or other situations (e.g., an input of nutrients or chemicals) can cause part of these inactive members to respond and become active, whereas other parts of the active population may become inactive. This switching allows a staged response to an outside perturbation and avoids the need for all members of the population to maintain themselves at high levels of activity under all conditions. This is a critical consideration because most soil and subsurface material has limited available nutrients. Shift ability is most clearly demonstrated in an example of microbial response between acttive and inactive states in a special region near the roots called the rhizosphere. This rhizosphere region forms near a growing root (approximately 1 mm away) when microbial populations are consuming carbon, and other materials, released from the root. Recent studies (65) have confirmed earlier reports and shown that the types of active microorganisms near the root are responding to the types of available nutrients in the rhizosphere. Before these studies, most rhizosphere work indicated the number of cells increased in the rhizosphere, but a clear correlation to changes in the population structure (types of organisms) had not been made (66). Like the situation occurring for surface soil, the subsurface is composed of diverse microbial population capable of many biochemical processes. The subsurface tend to have smaller pore spaces as the geologic materials are more tightly packed and as a result, the size of the biomass and its activity tends to decrease with increasing depth. Small pore neck size, high bulk densities [low nutrients, poor water availability, and few dissolved electron acceptors (especially oxygen)]. The literature on the subsurface has shown it to be both complex and heterogeneous (67,68). SOIL MICROBIOLOGY AND CONTAMINATION Transport and Delivery of Contamination in Soil Influences Biodegradation Contaminant metabolism (or any metabolism) is governed by transport and the availability of substrates. As Andersson and Henrysson (69) point out, contaminants such as polycyclic aromatic hydrocarbons (PAH) are limited in their availability to indigenous microflora and this makes degradation or remediation difficult. ¨ Malina and coworkers (70) and Welp and Brummer 1999 (71) describe three factors as limiting hydrocarbon biodegradation in the field. These are the inherent toxic nature of the material and metabolites that may form, the net availability of the chemicals as modulated by surface sorption, solubility, and speciation, and changes that may occur in the microbial population in response to the chemical. Ogram and coworkers (72) showed the organic molecule 2,4-D once sorbed to soil was recalcitrant to biodegradation and Shelton and Parkin (73) reported that desorption of carbofuran in soil limited biodegradation. Moreover, Scribner and coworkers (74) reported that the biodegradation of simazine was limited


by desorption. These studies indicate that sorptiondesorption processes play a major role in biodegradation by affecting bioavailability of contaminants in the soil. This is because soil is a surface-dominated environment and sorption is the main mechanism controlling the level of the chemical in solution. In general, the level of organic carbon and pH of the system, which affects both the chemical and the surface chemistry, control sorption processes. The toxicity of some materials is modulated by the interactions with a surface that retains some fraction of the material lessening the solution concentration. The majority of studies investigating sorption and transformation of chemicals in soil have been conducted at relatively low chemical concentrations. The dominant mechanisms responsible for the retention of chemicals in soil and subsurface environments are hydrophobic partitioning (in organic matter), hydrogen bonding, and dipolar interactions. Soil organic matter has been consistently implicated as a major component in controlling the sorption in soil. The early work of Talbert and Fletchall (75) showed strong positive correlation between the chemical retention by soil and organic matter. Others have confirmed this (76–78). In studies of sorption using Atlantic coastal plain soils, Johnson and Sims (76) observed that retention of several chemicals correlated strongly with organic matter content and exchangeable acidity. These data suggest that although hydrophobic partitioning of chemicals into soil organic matter is an important mechanism, electrostatic and pH effects are also important (79,78). These mechanisms do not act independently, however, and the relative contribution of one mechanism over another will depend strongly on the amount of soil organic matter, pH, and clay content. The sorption of dissolved, nonpolar organic compounds in soil and sediment is a widely studied phenomenon (80,81). This process is described as an equilibrium distribution of the nonpolar organic solute between the aqueous phase and the organic matter in the soil. The mechanism of retention is thought to be a partitioning phenomenon, similar to the partitioning of hydrophobic organic compounds between

191

an organic solvent phase and aqueous phase in a biphasic solvent system. This process is often characterized by linear sorption isotherms in which the sorbed concentration is directly proportional to the solution phase concentration. However, there have been a number of other studies to show that surface hydrolysis, hydrogen bonding, and surface-mediated chemisorption can occur and will affect the retention of the material (82–84). High concentrations of chemicals pose a significant nonpoint source pollutant contamination potential at contaminated sites (24,85). Sorption as identified by studies at dilute aqueous solution may not be a predominant mechanism controlling the retention of concentrated chemicals typically encountered at contaminated sites. This is because at contaminated sites, bulk chemical trapping in pore spaces and matrix creates a chemical ‘‘source zone’’ because organic compounds in pore spaces diffuse into soil aggregates (86). Bulk chemical trapping retains a higher fraction of the total load. Whereas sorption and trapping in the smallest pores may hold a fraction of the material that is more difficult to degrade. Microbial degradation of source zones tends to be from the outside of the zone inward. This reflects the fact that chemicals are often toxic in the source zone and that microorganisms may be limited by the supply of oxygen or secondary nutrients (Fig. 3). Overall distribution and transport of chemicals to a location in the profile is limited by the flux of water through the system and retention. Vapor transport of chemicals may also occur. The retention reactions for either can include surface sorption and trapping in small pores and spaces. In terms of biodegradation of introduced materials, Sawhney and coworkers (87) and Steinberg and coworkers (88) showed that 1,2-dibromoethane (EDB), a water-soluble, biodegradable, and weakly sorbed organic molecule, persisted in soil for long periods. This is in contrast to controlled laboratory studies in which it was shown that the material could be rapidly degraded in culture. In soil, the chemical appears to become entrapped in the soil matrix passing down soil pores (possibly smaller

Slope/water movement

Leaking pipe

Soil surface

Source zone Anaerobic zone

Dense layer glacial till

Aerobic zone

Active degradation

Figure 3. The zones typically encountered near a hydrocarbon leak or spill. The source zone is surrounded by a zone of oxygen depletion, an aerobic zone, and zone of active degradation. The zone of active aerobic degradation is influenced by both the availability of oxygen and the availability of secondary nutrients, primarily nitrogen, and phosphorus. Movement of the hydrocarbon is often influenced by the presence of dense subsurface layers.

192


than 0.25 µm) where it is protected from degradation. Others, Borchers and Perry (89) and Powlson (90), have shown that in soil, there are pools of physically protected nitrogen and carbon, which act as a slow-release source of the nutrient. Again, it seems the pore neck size may limit diffusion of nutrients to microorganisms and the soil solution. Roberston and coworkers (168) found that there was a significant patterning of NO3 − release across a landscape indicating localized differences in microbial ability and the formation of NO3 − . Studies to address the temporal and spatial variability associated with the field-scale distribution of microorganism and their abilities to degrade recalcitrant organic compounds are limited (91). Evaluation of field-scale variability in a soil microbial populations has found that major differences in a Rhizobium population are evident at sampling distances of less than 0.2 µm (92) and that there is a spatial variability in the degradation of pesticides in soil (41). Others have shown similar results and confirm that the distribution of bacteria in soil is not uniform, but that the distribution reflects both the soil structure and available nutrient supply (42). Coresuil and Weber (93) suggest that a correlation between the size of the standing biomass and the onset of hydrocarbon degradation exists and as a result, a critical population size is needed before rapid degradation becomes possible. This has also been shown for the degradation of propylene glycol (85). PHYSIOLOGICAL PRINCIPLES AND THE UNDERLYING BIODEGRADATION REACTIONS Using Soil Organisms for Biodegradation Biodegradation describes the processes microorganisms use to catabolize (i.e., break down and simplify) a variety of compounds (chemicals, soil organic matter, and plant and animal residues) that would otherwise persist in the environment. In general, these simplification processes are used by the resident soil microflora as a means of obtaining both nutrients (carbon, nitrogen, phosphorus) and energy (ATP). Biodegradation describes a fundamental set of processes in which microorganisms are converting

materials to recover energy and materials for building new cells. Madsen (94) has defined bioremediation as ‘‘a managed or spontaneous process in which biological, especially microbial, catalysis acts on pollutant compounds, thereby remedying or eliminating environmental contamination.’’ Therefore, bioremediation is a direct utilization of inherent microbial abilities and can take many forms (Fig. 4). Three factors, namely, the microorganisms, the contaminant, and the environmental setting function to modulate the field-scale biodegradation and bioremediation process (94–98). The contaminant’s chemical structure (i.e., the number of carbon rings, side chains, halogens, and bonding arrangement), concentration (ngg−1 to gg−1 ), physical placement (either the soil surface or in solution), the presence of electron donors or acceptors, and the physiological abilities of the microorganisms, either resident or introduced, interact to control the success of biodegradation or bioremediation. If any of these components are less than optimum, microbial-mediated degradation will be slow or not take place. In fact, it has been suggested that the long-term residency of contaminants in soil and the subsurface indicate the presence of poor or suboptimal conditions and the suboptimal condition has caused the contaminated site to form in the first place. The operational goal of a successful bioremediation program is to overcome the suboptimal conditions at the site by maximizing the functioning of the resident or applied microbial population to achieve a biodegradation of the materials. Removing the rate-limiting factors and promoting the activity of the microbial populations should allow the community to destroy the pollutant. In essence, the key question in bioremediation becomes: what chemical or biological feature of the site has prevented the microbial population from removing the pollutant? Successful bioremediation of surface and subsurface environments can result from a manipulation of the contaminated system to encourage the destruction of the contaminant by the microorganisms. This approach is bolstered by the fact that many laboratory studies have shown that biodegradation of pollutant chemicals can occur. However, the transformation rates estimated in the laboratory studies tend to be much higher than that found in the field. It is suggested that this difference reflects a

Compound properties Concentration Solubility Retention (Kd) Volatility (KH) Other chemicals Chemical structure Halogens (Cl, Br, F, I) Ring content (number) Saturation/substitutions

Figure 4. A diagram relating the three major components in site remediation, the compounds chemistry, the site properties, and the abiliies of the microbial populations.

Potential for remediation

Interaction

Site characteristics Degree of heterogeneity Pore structure/compaction Water availability O2 availability /redox Site chemistry pH Organic matter Available nutrients (N, P, K)

Site/soil biology Types of microorganisms Biochemical ability


spatial variability that hampers the microbial population. Therefore, the microbial community’s ability to respond to the chemical is lessened in the field, as compared with the response at the laboratory scale (69). Microbial Processing of Contamination Long and coworkers (24) showed that exposure to high-level (mg kg−1 ) petroleum contamination alters the characteristics of a subsurface microbial community. These results indicate that high levels of petroleum contaminants can exert toxic effects on microorganisms, but at low concentrations it is possible to enrich specific degraders. Contamination of a forest soil with pentachlorophenol was shown to reduce both soil microbial biomass and the numbers of collembolans, enchytraeids, and fungal-feeding nematodes. Therefore, it was felt that the diversity of soil fauna was reduced with the high-level contamination (99). Low levels of contaminants (a few µg kg−1 ) can result in the development of chemical-adapted microbial communities in the contaminated samples. This illustrates an outcome of Trevors (20) concept of ‘‘limitless genetic resource’’ as portions of the population are able to adapt to the situation. However, in highly contaminated materials, containing metals, or organics, or both, total microbial biomass is generally lower (52,24) indicating that a toxic effect has occurred. It has been shown that the chemical 1,2-dichlorobenzene reduces the fungal population size and can selectively reduce bacterial numbers (100). Function can also be impacted. Contamination of soil with copper has been shown to alter nitrification rates (101). These findings, in part, explain why some contamination can persist as it impacts basic microbial functions. Soil microbial populations have been shown to adopt to materials such as aircraft deicing fluids (ADF)—Propylene Glycol, provided the population is not exposed to extremely high levels at the outset (85). With exposure to low levels of ADF (5 to 10%) the onset of degradation was delayed, but the overall rate of degradation was high. They found that high levels of ADF (40%) inhibited all degradation, a situation that is analogous to that described by Long and coworkers (24). It is also clear that mixed contamination can have differential effects on the response of the population. Gasoline, which is composed of many organic molecules, will degrade in soil. However, the presence of high levels of 2-ethyltoluene and trimethylbenzene will inhibit the degradation of other fractions of the gasoline, again showing the interrelatedness of the response (102). Assimilatory and Dissimilatory Reactions and the Removal of Contamination—Aerobic What is clear from the available wealth of reaction data is that bioremediation can make use of the four features of an active population, that is, their need for assimilatory nutrients such as carbon, nitrogen, sulfur, phosphorus, their need for a source of electrons, their enzymatic nonspecificity (co-oxidation), and their need for terminal electron acceptors. A detailed assessment of the redox and energy needs of microorganisms set in the context of biodegradation of contaminants in soil and water can be found

193

in Harris and Arnold (103). They state that growth of an organism is a function of both assimilatory and dissimilatory steps, whereas maintenance of the organisms is primarily dissimilatory. Assimilatory reactions gather the required carbon, nitrogen, sulfur, and phosphorus for biomass (cell) production. Dissimilatory reactions provide the energy for assimilatory reactions and any subsequent maintenance processes. In general, the utilization of nitrogen and phosphorus is to satisfy the assimilatory aspects of the degradation process and the oxidation of material is to release reducing power from the substrate to generate ATP. Most efforts at bioremediation presumed that microbial populations respond to the targeted material as a source of carbon under aerobic conditions. Only recently has the potential of anaerobic processes been exploited. For assimilatory processes the ratio of carbon to nitrogen should approach 30 : 1 (104) if optimum microbial biomass production is to occur. However, Dibble and Bartha (105) have shown oil degradation to be most rapid at C : N rations of 60 : 1, whereas others (106) have shown that degradation of propane and butane quickly becomes nitrogen-limited and that in response, the population may begin to fix gaseous nitrogen. The application of nitrogen sources should be done with care. Wrenn and coworkers (107) showed that in poorly buffered sea salt solutions, application of NH4 + to aid in the degradation of crude oil reduced the pH and degradation rates as compared with other nitrogen sources. O’Connor and Young (108) showed that nitrogen additions to phenol-contaminated sites would enhance degradation of the organic as much as twofold. They pointed out that the effectiveness of the nitrogen source was related to the type and position of the substitution on the phenol. Zhou and Crawford (109) provided an evaluation of nutrient application and the kinetics of BTEX degradation. They showed that all types of nitrogen (NH4 + as vapor, NH4 NO3 ) were equal in their effectiveness as nitrogen source for BTEX degradation. One of the interesting findings of their work was that very low C : N ratios (1.8 : 1) suppressed degradation as compared with a ratio of 50 : 1. They also showed that in subsurface samples, an optimum oxygen addition giving a 10% enrichment was the most effective in stimulating degradation. The importance of adding phosphorous to the bioremediation system was also noted. Mills and Frankenberger (110) have shown that the addition of K2 HPO4 at up to 500 mg kg−1 would enhance degradation of diesel fuel. Rasiah and coworkers (111) have also indicated the importance of added phosphorous sources in oil degradation. They found an effect from the type of nitrogen source added. In general, they rated the effectiveness of the nitrogen sources as Ca(NO3 )2 > NaNO3 > KNO3 > NH4 NO3 > NH4 Cl. Others have indicated that for bioremediation, the ratio should be in the range of 120 : 10 : 1, C : N : P, respectively (112). Pope and Matthews (113) point out that nutrient requirements for enhancing biodegradation in the field have not been thoroughly studied. Finklea and Fontenot (114) showed that typical field cultivation (to enhance air and water entry) combined with an addition of nitrogen, phosphorus, and potassium (13-13-13) was effective in stimulating the degradation of atrazine in

194


contaminated soil. Applications of manures were shown to stimulate the degradation of atrazine and 2,4-D in soil. It is suggested that the DOC in the manure stimulated the general soil growth of the population. However, other work has shown atrazine to be a ready source of nitrogen (115) and the addition of soluble carbon could have stimulated a demand for nitrogen resulting in the degradation of the chemical. A large-scale use of fertilizer was made in efforts to remediate the Exxon Valdez oil spill in Prince William Sound, Alaska (116). Work by Ilyinsky and coworkers (117) had indicated that fertilizer materials coated in oil or an oil membrane would have improved penetration into oils in the environment. The fertilizer addition gave higher hexadecane and phenanthrene mineralization than was found in the untreated soil (118). Mineral nutrients have been effective in stimulating the degradation of petroleum wastes in surface soil (111) and p-nitrophenol in aquifer solids (119). In contrast, addition of nitrogen of up to 80 mg kg−1 did not affect the mineralization of added atrazine in a surface soil (115). Pothuluri and coworkers (120) were able to increase the degradation of alachlor in a subsurface soil 15 to 35% by adding a mixture of glucose and hydrolyzed casein. In contrast, glucose reduced the degradation of several xenobiotic compounds (119,121,122) in lake water and aquifer solids. A consideration of the importance of minor or trace elements is lacking for bioremediation systems. Semprini and coworkers (123–126) have shown that it is possible to use substrate co-oxidation to degrade groundwater resident contaminants, in particular, chlorinated aliphatic hydrocarbons (CAH). The system is based on the co-oxidation, or the fortuitous transformation, of an organic compound by microorganisms. The microorganism receives no direct benefit from the fortuitous transformation of the CAH material. In the case of CAHs, few with more than two chlorine substitutions have been shown to support aerobic microbial growth as a carbon source. Semprini (123–126) has shown that methane monooxygenase used to oxidize methane by methanotrophic bacteria is able to concurrently oxidize CAHs. When the contaminated site is flushed with methane and oxygen to stimulate the methanogens, the co-oxidation of the CAHs may also occur. In other studies, they have shown a similar approach using the co-oxidation CAHs with phenol as the primary substrate in a cometabolizing system (127). A co-oxidation approach has been used by Aziz and coworkers (128) in the construction of a hollow-fiber flow-through system that couples a CH4 oxidation by a methanotrophic bacteria with the co-oxidation of a CAH—tricloroethlyene (TCE)—in contaminated groundwater. They were able to show a significant reduction in TCE with co-oxidation. Regardless of the types of available carbon and nitrogen, microorganisms must use an electron donor/acceptor couple to capture energy derived from dissimilatory reactions. This approach is universal and used in fermentative, anaerobic or aerobic reactions. For aerobic processes, the cell’s biochemical system is routed to capture the generated energy (electrons released) during oxidation by reducing NAD+ (nicotinamide adenine dinucleotide) to NADH. ATP is generated while the NADH is being reoxidized to

NAD+. In aerobic systems, the electrons are passed down the electron transport chain, across the cell membrane, and this creates an energy gradient. Under aerobic conditions, the electron acceptor is oxygen. However, other substances such as NO3 − and SO4 − are also selectively used. ATP synthase enzymes capture the electrical and chemical potential of the gradient and the overall process is referred to as oxidative phosphorylation as it results in the formation of ATP. In aerobic systems, the combined dissimilatory and assimilatory reactions using oxygen as an electron acceptor are described by the following reaction: Substrate + Nutrients + O2 −−−→ Biomass + CO2 + H2 O + Metabolites + Energy An example reaction (disregarding biomass production) to illustrate the process is the oxidation of glucose: C6 H12 O6 + 6O2 −−−→ 6CO2 + 6H2 O + energy. This translates to two moles of oxygen per mole of carbon utilized. The stoichiometry for toluene oxidation is: C6 H5 − CH3 + 9O2 −−−→ 7CO2 + 4H2 O + energy. This translates to 2.57 moles oxygen per mole of carbon oxidized to carbon dioxide. If we consider benzene (and disregard biomass production), we find the following reaction: C6 H6 + 7.5O2 −−−→ 6CO2 + 3H2 O The transfer of electrons demands 7.5 moles or 240 g of oxygen per mole of benzene when an aerobic system is in place. This translates to 3.15 g of oxygen per gram of benzene. A 10,000 kg hydrocarbon plume contained in soil would require 3.15 × 104 kg of oxygen for complete mineralization. Air contains 21% oxygen by volume; to complete the degradation, a total of about 1.5 × 105 kg of air would be required. Assuming the contaminated soil has a bulk density of 1.33 Mg m−3 , it would contain 50% pore space and 50% solids. Assuming half of the pore space is filled with water, we would have an available air supply of about 0.33 Mg or 3,300 kg of air. Under these conditions it would take some 45 replacement volumes of air to supply enough oxygen to allow mineralization of the plume. This assumes that all of the material is available for degradation, the material is evenly distributed within the site, the bulk density is uniform throughout the profile, the oxygen that is available instantaneously and other nutrients (nitrogen and potassium) that are not limiting, and that the chemical levels are not toxic. Few of the constraints aforementioned are commonly found to occur at a contaminated site. For example, the level of oxygen within the soil profile will tend to decrease with depth and the secondary nutrients tend to be limited. Moreover, as the biomass builds up, it will require a significant level of oxygen to maintain itself. When the concentration of oxygen in the profile is decreased by 5%, reflecting oxygen utilization at the surface, the volume of air needed to


resupply the degradation processes in the subsurface is increased by 150% to 3.8 × 105 kg. It stands to reason that the aerobic degradation rate may become a function of oxygen resupply to the regions undergoing degradation processes. The resupply is a function of transfer into and through both the soil profile and the water films. Some have suggested the use of hydrogen peroxide (47% oxygen), and not soil, as a soluble form of oxygen for groundwater supplementation. Oxygen is rapidly released from the hydrogen peroxide as the liquid encounters enzymes and metals. However, hydrogen peroxide is toxic to microorganisms at concentrations above 100 mgL−1 and successful use has been limited (129,130). Prosen and coworkers (131) suggested a system in which pure oxygen is generated on-site and this, instead of air, is pumped into the subsurface. Because air is 21% oxygen, use of pure oxygen would increase the efficiency fivefold. In all cases, the introduction of air or oxygen into anoxic subsurface conditions can be problematic because the oxygen can undergo abiotic reactions with reduced mineral surfaces and can be removed from the solution before it can serve as a terminal electron acceptor for microbial processes (132). Assimilatory and Dissimilatory Reactions and the Removal of Contamination—Anaerobic Modeling efforts for subsurface systems support the finding that for xenobiotic compounds, lack of oxygen is a limitation for degradation (133). Under aerobic conditions, pyridine and hydroxylated pyridines were more rapidly transformed than under anaerobic conditions (134). They tested surface and subsurface soil that had been exposed to these compounds for several decades. All the pyridine derivatives tested were degraded within two weeks in the presence of oxygen. Under anaerobic conditions, however, longer time periods were required. Jet fuel in the subsurface environments can have a long-term residence as a result of a lack of available oxygen (135). A similar situation is suggested for propylene glycol because oxidative degradation was enhanced by the presence of oxygen (85). Remediation sites can have a further complication because of the presence of a second material such as oil that can inhibit the flow of air or water. Oil perched on water changes the diffusion profile and acts as a barrier for oxygen transfer into water (136) providing a diffusivity of about 2 × 103 cm2 s−1 for oxygen moving in oil (137). Moreover, the film thickness will change the transfer rate; films thicker than 100 µm will diminish the overall transfer velocity by as much as half. Under oxygen-limited conditions, strict and facultative anaerobic bacteria are able to metabolize and grow if supplied with one of a number of alternative electron acceptors. The use of anaerobic organisms offers possibilities in bioremediation applications (95,138). In contrast to aerobic respiration, anaerobic organisms make use of a number of electron acceptors. A general reaction scheme for various electron acceptors typically used by microorganism is given by: Substrate + {NO3 − , Mn4+ , Fe3+ , SO4 2− , CO2 } −−−→ Biomass + CO2 + {N2 , Mn2+ Fe2+ S2− CH4 }

195

The type of electron acceptor favored will reflect the redox status of the system. Therefore, the sequence of reduction, NO3 − , Mn4+ , Fe3+ , SO4 2− , CO2 reflects the oxidizing capacity of the chemical half-reaction (139). Kazumi and coworkers (140) showed that 3-chlorobenzoate could be degraded with NO3 − , Fe3+ , or SO4 2− acting as the electron acceptor. McFarland and Sims (141) have developed a conceptual framework for interpreting the thermodynamics of PAH degradation in the environment. They extended their model to not only oxygen as an electron acceptor, but also NO3 − , Mn4+ , Fe3+ , SO4 2− , CO2 . They pointed out that thermodynamically the reduction of Mn4+ may be favored in groundwater and subsurface systems given its widespread availability and that the ◦

G for the reduction of Mn4+ gives a free energy change similar to that found with oxygen. However, the usefulness of Mn+4 as an electron acceptor is limited by the low solubility of the mineral (141). At the center of a subsurface hydrocarbon plume, the conditions may be anoxic and highly reduced (142). At the edges of the plume, the conditions are more aerobic and better oxygenated because the contaminated water mixes with noncontaminated water. Others (143) have used the subsurface formation of methane, an indicator of extremely reduced conditions, to map plume migration in soil and aquifers. More recently, Lovely and coworkers (144) have demonstrated the use of dissolved hydrogen to describe the predominant terminal electron accepting processes (TEAPs) occurring in an aquifer. This approach allows a better description of the redox chemistry and contaminant transformation processes occurring in anoxic groundwater systems. The TEAPs method is more robust than other signature processes because it relies on the occurrence of characteristic concentrations of hydrogen. Hydrogen values are less ambiguous than CH4 or SO4 because hydrogen is quickly cycled, is poorly reactive with mineral surfaces, and has a short halflife (145). Chapelle and coworkers (145) used the TEAPs concept to describe the distribution of redox states within a hydrocarbon-contaminated aquifer. Nitrogen as NO3 − can be introduced to serve as an electron acceptor in moderately reduced or hypoxic (low oxygen) conditions. For example, it has been shown that for toluene, the following reaction is possible: C6 H5 − C6 + 6NO3 − −−−→ 7CO2 + 4H2 O + 3N2 + energy The advantage of NO3 − as an electron acceptor is that it is more soluble than oxygen (660 g NO3 − L−1 H2 O), allowing it to be distributed throughout a contaminated aquifer or subsurface material. Dissolved NO3 − will move with water through flow paths and into micropores and spaces. NO3 − will serve as an electron acceptor in the remediation of contaminated aquifers in which BTEX is the primary contaminant (132,146,147). Gersberg and coworkers (148) used NO3 − as an electron acceptor in the remediation of BTEX in an oxygenpoor aquifer. This NO3 − approach was also used by Burland and Edwards to degrade benzene under anaerobic conditions and supported other studies that had shown that anaerobic removal of benzene was possible (149).

196


Kazumi and coworkers (140) showed that the utilization of monochlorobenzoate isomers (2-,3-, and 4-chlorobenzoate) by microbial consortia in river sediments was possible under denitrifying conditions. They were also able to show that a loss of 3-chlorobenzoate would occur under ironand sulfate-reducing conditions and under methanogenic conditions. For a given hydrocarbon, the NO3 /N2 couple is energetically similar to the O2 /H2 O couple. The standardstate reduction potential for oxygen is 1.22 volts, whereas NO3 − reduction to nitrogen gas an E0h of 1.24 volts. This shows the relative energy available in the redox couples is quite similar. Not all bacteria capable of aerobic degradation of a given aromatic contaminant and capable of NO3 − reduction are automatically capable of degradation of the same contaminant when using NO3 − as the electron acceptor. It is unclear as to the exact pathway used by facultative microorganisms when degrading aromatic compounds under NO3 − -reduction conditions. Downs and coworkers (150) has used NO3 − in an approach similar to that of Hutchins and coworkers (132) to degrade BTEX, but they implicated that aerobic step was needed in the degradation of benzene. Others have reported a similar finding (132,152). Rate constants developed for the comparative studies of BEXT removal under either aerobic or denitrifying conditions indicated that reactions occurring under denitrifying conditions are somewhat slower than that reported for aerobic processes (132). Leahy and coworkers (152) demonstrated the potential of toluene-oxidizing bacteria to degrade trichloroethylene (TCE) under hypoxic conditions when NO3 − was present. They also demonstrated that TCE could act as its own inducer suggesting the possibility that concurrent introductions of toluene into TCE-contaminated sites is not necessary to achieve degradation. Anaerobic processes are particularly effective in removing halogens from haloorganic compounds; however, the rates are relatively slow. Nozawa and Maruyama (153) demonstrated that the anaerobic metabolism of phthalate and other aromatic compounds can be conducted by the denitrifying soil bacterium Pseudomonas sp. strain P136. In addition, dinoseb (an insecticide) that is not degraded in contaminated soil under aerobic conditions can be degraded when anaerobic conditions are established (155,156). In this study, anaerobiosis was developed by treating the soil with starchy potato-processing waste materials and allowing the aerobic population to deplete the available oxygen. The anaerobic microbial consortium degraded dinoseb completely, and the formation of polymerization products produced aerobically was avoided. This approach has been applied to the removal of munition-contaminated soil. Funk and coworkers (157) reported that a wide spectrum of explosives materials, including 2,4,6-trinitrotoluene, could be rapidly reduced and then degraded in soil systems when anaerobic conditions were established. The anaerobic transformation of polychlorinated biphenyls, DDT, and percholoroethylene has also been reported (158). Moreover, the nematicide 1,2-dibromo-3-chloropropane (DBCP), which persists in groundwater and soil, has been shown to be converted to

organic products and carbon dioxide when soil suspensions are placed under anaerobic conditions (159).

CONCLUSION Over the last 100 years, the study of soil microbiology has revealed that a wide array of inherent biochemical abilities exist in each gram of soil. Trevors’ (20) statement describing soil as a virtual limitless pool of genetic information contained in bacteria can be coupled to other work that has shown microorganisms to be distributed in almost every type of soil and terrestrial location. The importance of the soil organisms to the ecosystem stability is clear. In fact, the study of soil microorganisms was hampered for years by our inability to separate the cells from their growth environment. This makes the study of soil ecology unique because few other areas of ecological investigation have such difficulty in separating the studied organisms from their habitat. Therefore, the structure and function of soil reflects the interrelatedness of the organism and their habitat. This habitat is constantly changing in response to the actions of the population of cells that are present. This is somewhat surprising, given the wide distribution of cells across a volume of soil, but illustrates the importance of their biochemical processing abilities. Our use of soil is dependent on the microbial population and their ability to reduce the waste and recycle these stored nutrients. In recent years, our dependence on soil microorganisms has expanded as we now rely on them to remove pollutant materials. BIBLIOGRAPHY 1. S. A. Waksman, Principles of Soil Microbiology, Williams & Wilkins, Baltimore, Md., 1927. 2. D. R. Zak et al., Ecology 75: 2333–2347 (1994). 3. J. Six et al., Soil Sci. Soc. Am. J. 64: 681–689 (2000). 4. H.-R. Schulten and P. Leinweber, Biol. Fertil. Soils 30: 399–432 (2000). 5. J. D. Jastrow et al., Soil Biol. Biochem. 30: 905–916 (1998). 6. S. F. Wright et al., Soil Sci. Soc. Am. J. 63: 1825–1829 (1999). 7. S. Singh and J. S. Singh, Biol. Fertil. Soils 22: 76–82 (1996). 8. C. Y. Jan, Environ. Manage. 22: 683–695 (1998). 9. I. C. Mendes et al., Soil Sci. Am. J. 63: 873–881 (1999). 10. J. Six et al., Soil Sci. Soc. Am. J. 63: 1350–1358 (1999). 11. P. L. de Freitas et al., Crop Sci. 39: 725–730 (1999). 12. J. Postma et al., Appl. Environ. Mircobiol. 56: 495–502 (1990). 13. P. J. Kuikman et al., Soil Biol. Biochem. 22: 1063–1073 (1990). 14. E. Amezketa, J. Sustainable Agric. 14: 83–151 (1999). 15. R. F. Harris, in J. F. Parr et al., eds., Water Potential Relations in Soil Microbiology, ASA, Madison, Wis., 1981, pp. 23–95.


197

16. D. J. Greenwood, Minnesota Agric. Fish Food Tech. Bull. 29: 261–390 (1975).

46. D. A. Wright et al., Appl. Environ. Microbiol. 61: 3537–3543 (1995).

17. R. I. Papendick and G. S. Campbell, in L. F. Elliott et al., eds., Water Potential Relations in Soil Microbiology, Soil Science Society of America, Madison, Wis., 1981, pp. 1–22.

47. G. Kilbertus et al., Soil Biol. Biochem. 11: 109–114 (1980).

18. K. A. Smith and J. R. M. Arah, Fed. Eur. Microbiol. Soc. (FEMS) 33: 247–261 (1986).

49. R. C. Foster, Biol. Fertil. Soils 6: 189–203 (1988).

19. J. C. Gottschal, FEMS Symp. Fed. Eur. Microbiol. Soc. 33: 263–273 (1986).

51. L. S. England et al., Soil Biol. Biochem. 25: 525–531 (1993).

20. J. T. Trevors, Water Air Soil Pollut. 101: 45–67 (1998). 21. V. Torsvik et al., Appl. Environ. Microbiol. 56: 782–787 (1990a). 22. V. Torsvik et al., Appl. Environ. Microbiol. 56: 776–781 (1990b).

48. J. K. Martin and R. C. Foster, Soil Biol. Biochem. 17: 261–269 (1985).

50. A. Winding et al., Biol. Fertil. Soil. 24: 133–140 (1997).

52. E. Kandeler et al., Biol. Fertil. Soils 32: 390–400 (2000). 53. D. R. Lovely, Adv. Agron. 54: 175–231 (1995). 54. J. Wu et al., Soil. Sci. Soc. Am. J. 52: 295–296 (1988). 55. D. C. Coleman et al., Adv. Ecol. Res. 13: 1–55 (1983). 56. L. C. Broughton and K. L. Gross, Oecologia 125: 420–427 (2000).

23. A. Konopka and R. F. Turco, Appl. Environ. Microbiol. 57: 2260–2268 (1991).

57. H. Yao et al., Microb. Ecol. 40: 223–237 (2000).

24. S. C. Long et al., Microbiol. Ecol. 30: 297–307 (1995).

58. R. J. Haynes, Biol. Fertil. Soils 30: 210–216 (1999).

25. G. P. Sparling, in D. Vaughan and R. E. Malcolm, eds., Soil Organic Matter and Biological Activity, Martinus Nijhoff Junk, Dordrecht, The Netherlands, 1985, pp. 223–262.

59. H. P. Collins et al., Soil Sci. Soc. Am. J. 56: 783–788 (1992).

26. M. J. Sadowsky and M. Schortemeyer, Global Change Biol. 3: 217–244 (1997). 27. R. Hissett, and T. R. G. Gray, in J. M. Anderson and A. MacFadyen, eds., The Role of Terrestrial and Aquatic Organisms in Decomposition Processes, Blackwell, Oxford, U.K., 1976, pp. 23–39. 28. M. C. van Loosdrecht et al., Appl. Environ. Microbiol. 53: 1898–1901 (1987). 29. M. C. van Loosdrecht et al., Microbiol. Rev. 54: 75–87 (1990). 30. M. S. Smith et al., J. Environ. Qual. 14: 87–91 (1985). 31. F. Huysman and W. Verstraete, Soil Biol. Biochem. 25: 83–90 (1993). 32. S. Issa et al., Soil Biol. Biochem. 25: 959–965 (1993a). 33. S. Issa et al., Soil Biol. Biochem. 25: 951–958 (1993b). 34. Y. Tan et al., Soil Sci. Soc. Am. J. 56: 1331–1340 (1992). 35. E. L. Madsen and M. Alexander, Soil Sci. Soc. Am. J. 46: 557–560 (1982). 36. J. T. Trevors et al., Appl. Environ. Microbiol. 56: 401–408 (1990). 37. J. V. Gagliardi and J. S. Karns, Appl. Environ. Microbiol. 66: 877–883 (2000). 38. J. C. Cho and S. J. Kim, J. Microbiol. Methods 36: 227–235 (1999). 39. T. Hattori and R. Hattori, Crit. Rev. Mircobiol. 4: 423–461 (1976). 40. K. Killham et al., Soil Biol. Biochem. 25: 57–62 (1993). 41. P. S. C. Rao et al., in W. Y. Garner et al., eds., Evaluation of Pesticides in Groundwater, American Chemical Society, Washington, D.C., 1986, pp. 100–115. 42. A. Richaume et al., Soil Biol. Biochem. 25: 641–643 (1993). 43. L. Ranjard et al., Microb. Ecol. 39: 263–272 (2000). 44. A. C. Fisk et al., Biol. Fertil. Soils 28: 111–116 (1999). 45. C. E. Heijnen and J. A. van Veen, FEMS Microbiol. Ecol. 85: 73–80 (1991).

60. J. A. Entry and W. H. Emmingham, Can. J. Soil Sci. 75: 379–383 (1995). 61. H. Insam et al., Microb. Ecol. 31: 77–87 (1996). 62. T.-H. Anderson and K. H. Domsch, Biol. Fertil. Soils 1: 81–89 (1985). 63. A. Fægri et al., Soil Biol. Biochem. 28: 55–63 (1977). 64. J. S. Dunbar et al., Appl. Environ. Microbiol. 65: 1662–1669 (1999). 65. J. Steer and J. A. Harris, Soil Biol. Biochem. 32: 869–878 (2000). 66. S. J. Grayston and C. D. Campbell, Tree Physiol. 16: 1031–1038 (1996). 67. W. C. Ghiorse and J. T. Wilson, Adv. Appl. Microbiol. 33: 107–172 (1988). 68. D. C. Dobbins et al., Crit. Rev. Environ. Control 22: 67–136 (1992). 69. B. E. Anfersson and T. Henrysson, Appl. Microbiol. Biotechnol. 46: 647–652 (1996). 70. G. Malina et al., Environ. Technol. 19: 977–991 (1998). ¨ 71. G. Welp and G. W. Brummer, Ecotoxicol. Environ. Safety 43: 83–90 (1999). 72. A. V. Ogram et al., Appl. Environ. Microbiol. 49: 582–587 (1985). 73. D. R. Shelton and T. B. Parkin, J. Environ. Qual. 21: 115–120 (1991). 74. S. L. Scribner et al., J. Environ. Qual. 21: 115–120 (1991). 75. R. E. Talbert and O. H. Fletchall, Weeds 13: 46–52 (1965). 76. R. M. Johnson and J. T. Sims, Soil Sci. 155: 339–348 (1993). 77. J. J. Pignatello et al., Environ. Sci. Technol. 27: 1563–1571 (1993). 78. A. B. Paya-Perez et al., Chemosphere 25: 887–898 (1992). 79. G. Basile et al., Agrochimica 34: 181–192 (1990). 80. S. W. Karickhoff et al., Water Res. 13: 241–248 (1979). 81. C. T. Chiou, in B. L. Sawhney and K. Brown, eds., Reaction and Movement of Organic Chemicals in Soils, SSSA Special Publ. 22, Soil Science Society of America, Madison, Wis., 1989, pp. 1–30.

198


82. G. J. Welhouse and W. F. Bleam, Environ. Sci. Technol. 26: 959–964 (1992). 83. G. Welhouse and W. F. Bleam, Environ. Sci. Technol. 27: 494–500 (1993a). 84. G. J. Welhouse and W. F. Bleam, Environ. Sci. Technol. 27: 500–505 (1993b). 85. D. S. Bausmith and R. D. Neufeld, Water Environ. Res. 71: 459–464 (1999).

112. R. C. Sims, Bioremediation of Hazardous Waste Sites: Practical Approaches to Implementation, U.S. EPA. EPA/600/K93/002, Washington, D.C., 1993, pp. 3.1–3.8. 113. D. F. Pope and J. E. Matthews, Bioremediation of Hazardous Waste Sites: Practical Approaches to Implementation, U.S. EPA. EPA/600/K-93/002, Washington, D.C., 1993, pp. 7.1–7.5.

86. K. M. Scow and M. Alexander, Soil Sci. Soc. Am. J. 56: 128–134 (1992).

114. H. C. Finklea and M. F. Fontenot Jr., in H. D Skipper and R. F Turco, eds., Bioremediation: Science and Applications, SSSA Special Publ. 43, ASA, Madison, Wis., 1995, pp. 221–235.

87. B. L. Sawhney et al., J. Environ. Qual. 17: 149–152 (1988).

115. N. A. Assaf and R. F. Turco, Pestic. Sci. 41: 47 (1994).

88. S. M. Steinberg et al., Environ. Sci. Technol. 21: 1201–1208 (1987).

116. S. M. Hinton, in H. D. Skipper and R. F. Turco, eds., Bioremediation: Science and Applications, SSSA Special Publ. 43, ASA, Madison, Wis., 1995, pp. 211–219.

89. J. G. Borchers and D. A. Perry, Can. J. Forest Res. 22: 298–305 (1992). 90. D. S. Powlson, J. Soil Sci. 31: 77–85 (1980). 91. T. B. Parkin and D. R. Shelton, J. Environ. Qual. 21: 672–678 (1992). 92. A. G. Wollum and D. K. Cassel, Soil. Sci. Soc. Am. J. 48: 1082–1087 (1984). 93. H. X. Coresuil and W. J. Weber Jr., Water Res. 28: 1415– 1423 (1994). 94. E. L. Madsen, Environ. Sci. Technol. 25: 1663–1673 (1991). 95. H. F. Stroo, J. Environ. Qual. 21: 167–175 (1992). 96. J. M. Tiedje, In Situ Bioremdiation: When Does It Work, National Academy Press, Washington, D.C., 1993.

117. V. V. Ilyinsky et al., Biol. Sci. Bull. 46: 62–65 1991. 118. J. E. Lindstrom et al., 2514–2522 (1991).

Appl.

Environ.

Microbiol.

57:

119. C. M. Swindoll et al., Appl. Environ. Microbiol. 54: 212–217 (1988). 120. J. V. Pothuluri et al., J. Environ. Qual. 19: 525–530 (1990). 121. Y. Wang et al., J. Agric. Food Chem. 33: 495–499 (1985). 122. C. M. Aelion et al., Environ. Toxicol. Chem. 8: 75–86 (1989). 123. P. V. Roberts et al., Ground Water 28: 591–604 (1990). 124. L. Semprini et al., Ground Water 28: 715–727 (1990). 125. L. Semprini et al., Ground Water 29: 239–250 (1991).

97. N. R. Adrian et al., Appl. Environ. Microbiol. 60: 3632–3639 (1994).

126. L. Semprini and P. L. McCarty, Ground Water 29: 265–374 (1991).

98. A.-S. Allard and A. H. Neilson, Int. Biodet. Biodegr. 39: 253–285 (1997).

127. G. D. Hopkins et al., Appl. Environ. Microbiol. 59: 2277– 2285 (1993).

99. J. Salminen and J. Haimi, Biol. Fertil. Soils 23: 182–188 (1996).

128. C. E. Aziz et al., Environ. Sci. Technol. 29: 2574–2583 (1995).

100. I. P. Thompson et al., Environ. Toxicol. Chem. 18: 1891– 1898 (1999). 101. O. Kostov and O. V. Cleemput, Biol. Fertil. Soils 33: 10–16 (2001).

129. B. C. Lawes, in R. E. Hinchee and R. F. Olfenbuttel, eds., In situ Bioreclamation: Application and Investigation for Hydrocarbons and Contaminated Site Remediation, Butterworth-Heinemann, Woburn, Mass., 1991, pp. 143–156.

102. F. R. Solano-Serena et al., Biodegradation 9: 319–326 (1998).

130. P. Morgan and R. J. Watkinson, Water Res. 26: 73–78 (1992).

103. R. F. Harris and S. M. Arnold, in H. D. Skipper and R. F. Turco, eds., Bioremediation: Science and Applications, SSSA Special Publ. 43, ASA, Madison, Wis., 1995, pp. 54–87.

131. B. J. Prosen et al., in R. E. Hinchee and R. F. Olfenbuttel, eds., In situ Bioreclamation: Application and Investigation for Hydrocarbons and Contaminated Site Remediation, Butterworth-Heinemann, Woburn, Mass., 1991, pp. 523–528.

104. E. Paul and F. E. Clark, Soil Microbiology and Biochemistry, Academic Press, New York, 1996. 105. J. T. Dibble and R. Bartha, Appl. Environ. Microbiol. 37: 729–739 (1979).

132. S. R. Hutchins, Appl. Environ. Mircrobiol. 57: 2403–2407 (1991). 133. S. Dhawan et al., Groundwater 31: 271–284 (1993).

106. P. L. Toccalino et al., Appl. Environ. Microbiol. 59: 2977– 2983 (1993).

134. J. P. Kaiser and J. M. Bollag, Soil Biol. Biochem. 24: 351–357 (1992).

107. B. A. Wrenn et al., J. Ind. Microbiol. 13: 279–286 (1994).

135. H. G. Song and R. Bartha, Appl. Environ. Microbiol. 56: 646–651 (1990).

108. O. A. O’Connor and L. Y. Young, Arch. Environ. Contam. Toxicol. 25: 285–291 (1993). 109. E. Zhou and R. L. Crawford, Biodegradation 6: 127–140 (1995). 110. S. A. Mills and W. T. Frankenberger Jr., Bull. Environ. Contam. Toxicol. 53: 280–284 (1994). 111. V. Rasiah et al., Water Air Soil Pollut. 65: 143–151 (1992).

136. A. L. Dowing and G. A. Truesdale, J. Appl. Chem. 5: 570–581 (1955). 137. R. P. Schwarzenbach et al., Environmental Organic Chemistry, John Wiley & Sons, New York, 1993. 138. C. S. Carr and J. P. Hughes, Envrion. Sci. Technol. 32: 1817–1824 (1998).

BIODEGRADATION OF XENOBIOTICS BY ENGINEERED MICROBES 139. A. J. B. Zehnder and W. Strumm, Biology of Anaerobic Microorganism, John Wiley & Sons, New York, 1988. 140. J. Kazumi et al., Appl. Microbiol. Biotechnol. 43: 929–936 (1995). 141. M. J. McFarland and R. C. Sims, Ground Water 29: 885–896 (1991). 142. B. H. Wilson et al., Geomicrobiol. J. 8: 225–240 (1990). 143. T. B. Payne and P. B. Dugin, Proceedings of 2nd Outdoor Action Conference on Aquifer Restoration, Ground Water Monitoring and Geophysical Methods, National Well Water Association, Dublin, Ohio., 1988, pp. 1173–1188. 144. D. R. Lovely et al., Environ. Sci. Technol. 28: 1205–1210 (1994). 145. F. H. Chapelle et al., Water Resour. Res. 31: 359–371 (1995). 146. P. J. Evans et al., Appl. Environ. Microbiol. 57: 450–454 (1991a). 147. P. J. Evans et al., Appl. Environ. Microbiol. 57: 1139–1145 (1991b). 148. R. M. Gersberg et al., World J. Microbiol. Biotechnol. 11: 549–558 (1995). 149. S. M. Burland and E. A. Edwards, Appl. Environ. Microbiol. 65: 529–533. 150. M. Nales et al., Bioremediation 2: 125–144 (1998). 151. W. C. Downs et al., in P. E. Flathman et al., eds., Bioremediation: Field Experience, Lewis, Boca Raton, Fla., 1994, pp. 361–379. 152. J. Flyvbjerg et al., J. Contam. Hydrol. 12: 133–150 (1993). 153. J. G. Leahy et al., Appl. Environ. Microbiol. 62: 825–833 (1996). 154. T. Nozawa and Y. Maruyama, J. Bacteriol. 170: 5778–5784 (1988). 155. T. O. Stevens et al., Biodegradation 2: 1–13 (1991). 156. R. H. Kaake et al., Appl. Environ. Microbiol. 58: 1683–1689 (1992). 157. S. B. Funk et al., Appl. Environ. Microbiol. 59: 2171–2177 (1993). 158. J. M. Suflita et al., Science 218: 1115–1117 (1982). 159. R. A. Bloom and M. Alexander, J. Environ. Qual. 19: 722–726 (1990).

BIODEGRADATION OF XENOBIOTICS BY ENGINEERED MICROBES ESTRELLA DUQUE ANTONIO CABALLERO ˜ ABRAHAM ESTEVE-NU´ NEZ ´ CARMEN MICHAN MANUEL ESPINOSA-URBEL ANA SEGURA ´ -ISABEL RAMOS-GONZALEZ ´ MARIA JUAN L. RAMOS Estación Experimental del Zaid´ın Granada, Spain

The mineralization of organic molecules by microbes is essential for the carbon cycle to operate. The massive

199

mobilization of compounds stored in natural resources or the introduction of xenobiotics into the biosphere produces unidirectional fluxes that result in the persistence of many chemicals in the biosphere, where they are a source of pollution. Molecular biology offers the tools to optimize the biodegradative capacities of microorganisms, accelerate the evolution of new metabolic activities, and construct totally new pathways through the assemblage of catabolic segments from different microbes. The number of genetically engineered microbes (GEMs) for potential use in biodegradation has increased significantly in the last few years and a number of catabolic modules for the specific evolution of new pathways are being constructed so that a number of new recombinant microorganisms will soon be available. The behavior and functioning of some of the already available recombinant microbes have been tested in microcosms in which the survival and fate of recombinant microbes in different niches under laboratory conditions have been found to be similar to those of unmodified parental strains. Recombinant DNA (rDNA), both on plasmids and on the host chromosome, is usually stably inherited by GEMs. The potential lateral transfer of rDNA from GEMs to other microbes is significantly diminished, although not totally inhibited, when rDNA is incorporated on the host chromosome. KEY CONCEPTS A large proportion of organic compounds of biological and chemical origin are ultimately mineralized (degraded to carbon dioxide, water, and other inorganic compounds), predominantly by microorganisms, as part of the continuous cycling of carbon between inorganic and organic states. The mineralization of a compound involves its structural alteration and the formation of metabolic intermediates that serve either as carbon skeletons for cellular constituents or as fuels for energy generation. Mineralization of organic compounds is a central feature of the carbon cycle and is a process critical to the maintenance of life on this planet. Most naturally occurring molecules are easily mineralized, as are industrial chemicals with structures similar to organic compounds of biological origin. However, many xenobiotics (compounds that exhibit structural elements not found in natural ones) are not readily mineralized, and persist in the biosphere. This is because the structural elements of such compounds are chemically very stable, have novel substituents that are not generally found in organic molecules of biological origin, are toxic for microorganisms, or inhibit degradative enzymatic attack. Some organic compounds in nature undergo partial degradation; however, this is not necessarily beneficial: microbes may not gain energy for growth, and more recalcitrant toxic compounds or highly reactive products, which subsequently undergo chemical changes such as polymerization, may be formed. Xenobiotics introduced in natural resources are not readily integrated into the natural nutrient cycles. As a consequence of the progressive accumulation of pollutants in the biosphere, environments hostile to biological systems, that is, those polluted by aliphatic

200

BIODEGRADATION OF XENOBIOTICS BY ENGINEERED MICROBES

and aromatic hydrocarbons, chlorinated compounds, nitrotoluenes, dyes, explosives, organic solvents, and others, are appearing with increasing frequency (1). Effective steps should be taken to protect environments from pollution. A variety of alternative treatments, including physical, chemical, and biological approaches, have been developed to reduce or eliminate contamination by hazardous compounds (1–3). Physical treatments comprise, among other methods, adsorption to activated carbon, filtration, and incineration. Chemical approaches involve solvent extraction or surfactant precipitation. These treatments are expensive, and may in some cases generate unwanted products. The biological approach, including continuous and batch treatments of liquid wastes, composting in situ and on-site soil treatments, and so on is usually cheaper and involves less risk to human health or the environment (3–8). The most promising approach is to optimize the biodegradative capacities of microorganisms, accelerate the evolution of new activities, and exploit them to eliminate these pollutants (3,8). The introduction of some chemicals into the environment exerts selective pressure for the evolution of the corresponding catabolic activities. For example, the herbicide 2,4-dichlorophenoxyacetic acid, insecticides such as DDT and parathion, and explosives such as nitrate ester derivatives can be mineralized by single microbes or communities of microbes (7–15). However, other chemicals such as certain organic solvents, polychlorinated aromatic compounds, dioxins, and dibenzofurans are highly recalcitrant. In such cases, the evolution of new activities in the laboratory may be helpful because the frequency and types of genetic events needed (mutation, alteration of gene expression, gene dosage, gene transfer, and so on) may be carefully controlled under selective conditions (3,8,16–18). The experimental evolution of catabolic pathways offers considerable potential for accelerating the evolution of bacteria that are able to degrade toxic industrial chemicals, and this may be useful for reducing environmental pollution. Several strategies have been successfully applied to construct bacteria that are able to eliminate a wide range of organic solvents such as toluene derivatives, a number of chlorinated aromatic compounds arising from human industrial activities (chlorobenzoates, chlorosalicylates, and chlorophenols), and the more recalcitrant polychlorinated biphenyls (PCBs) and explosives such as 2,4,6trinitrotoluene (TNT) (3,8,17–23). Many of these bacteria belong to the genus Pseudomonas, a group of microorganisms that exhibit a wide range of metabolic activities against natural and xenobiotic compounds. The so-called fluorescent Pseudomonas group includes strains whose biochemical, physiological, and genetic characteristics have been well characterized (24,25). A number of genetic tools—wide host range cloning plasmids and cosmids, transposons and mini transposons, gene markers and probes, reverse genetics, and so on—have made it possible to design recombinant derivatives of this group of bacteria with increased biodegradative properties (26–31). Some of

these constructions are summarized in the following chapter. Furthermore, some of these recombinant bacteria and their parental wild-type strains have been selectively introduced in polluted and nonpolluted environments, and their survival, performance, and ability to transfer recombinant DNA have been monitored. HYBRID PATHWAYS FOR METABOLISM OF CHLOROORGANIC COMPOUNDS Hybrid Pathways for Chlorobenzoates and Chlorotoluenes Pseudomonas sp. B13 exhibits two ortho-cleavage pathways, one for the metabolism of benzoate and the other for the catabolism of m-chlorobenzoate (23). These pathways do not allow the mineralization of other chlorobenzoates or any alkylbenzoates (23,32). The chromosomal ortho-cleavage pathway for benzoate seems to be similar to other catabolic pathways for the metabolism of benzoate by Pseudomonads. The m-chlorobenzoate pathway is partially chromosomeencoded and partially plasmid-encoded. In this pathway, m-chlorobenzoate is first oxidized to m-chlorocatechol by chromosome-encoded genes; then m-chlorocatechol undergoes ring cleavage and chlorine elimination, a process involving four plasmid-encoded enzymes—catechol 1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase. The products resulting from the action of the plasmid enzymes are further metabolized by chromosomally encoded enzymes. In Pseudomonas strains growing on m-chlorobenzoate, the plasmid-encoded genes involved in the degradation of this compound were duplicated; this duplication ensured an increase in protein dosage (33,34). Pseudomonas sp. B13 cannot grow on p-chlorobenzoate or mixtures of alkylbenzoates and halobenzoates. In contrast, Pseudomonas sp. FR1 (pFRC20P), a derivative of B13 that was constructed through the recruitment of genes from different microorganisms, is able to grow on p-chlorobenzoate and mixtures of alkylaromatic and chloroaromatic compounds (17). Its construction involved chromosomal integration of the genes for the metabolism of p-chlorobenzoate and alkylbenzoates to p-chlorocatechol and alkylcatechols, respectively. The construct was completed by introducing a mobilizable plasmid (pFRC20P) carrying a key gene for the metabolism of alkyl-lactones. The genes encoding the TOL plasmid pWW0 toluate dioxygenase (xylXYZ) and the next enzyme in the pathway (a dehydrogenase encoded by xylL), together with the positive regulator xylS, were cloned into Tn5 and delivered into the host chromosome (Fig. 1). This allowed the recombinant bacteria to grow on p-chlorobenzoate and also allowed the metabolism of p-methylbenzoate to γ -methyl-lactone (Fig. 1). The resulting bacterium was called Pseudomonas sp. FR1. Plasmid pFRC20P carries the gene that encodes for an isomerase from Alcaligenes that allowed the conversion of γ -methyl-lactone into β-methyl-lactone (17) and subsequently allowed mineralization of the alkylbenzoate. Furthermore, this isolate grew and simultaneously assimilated mixtures of alkylbenzoates and halobenzoates.


Recruitment benzoate 1,2-dioxygenase

B13

3CB

3CC

4CB

4MB

201

Further recruitment of γ-lactone isomerase

3CB

4CB

4MB

3CB

4CB

4MB

3CC

4CC

4MC

3CC

4CC

4MC γ -L

γ -L OAA

OAA

OAA

OAA

OAA

β -L

TCA

TCA

TCA

TCA

TCA

TCA

Figure 1. Expansion of the range of chloroaromatic compounds degraded by Pseudomonas sp. B13. Details of the pathway expansion strategies are as described in the text.

Hybrid strains that mineralized p-chlorobenzoate (17,22), although able to transform 3,5-dichlorobenzoate, did not grow on this compound because the XylS regulator was not activatable by 3,5-dichlorobenzoate. By recruiting a XylS mutant regulator that is activated by this compound, a derivative mineralizing 3,5-dichlorobenzoate was constructed. Recruitment into Pseudomonas sp. B13 of the upper pathway enzymes (xylene monooxygenase, together with benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase) encoded by the TOL plasmid pWW0 (34–36) resulted in strains that are able to mineralize m-chlorotoluene (19,37,38). Haro and de Lorenzo have investigated the degradation of o-chlorotoluene, a compound for which microbes with biodegradative activities have not been found. They took advantage of the relaxed substrate activity of toluene dioxygenase of P. putida F1, which functions as a monooxygenase with 2-chlorotoluene and converts this into o-chlorobenzyl alcohol (39). It was suggested that further metabolism of the latter to o-chlorobenzoate may be mediated by the TOL pathway benzyl alcohol dehydrogenase (XylB) and benzaldehyde dehydrogenase (XylC) (39). A gene cassette has been engineered so that the chromosomal todABC genes encoding toluene dioxygenase and the xylBC are expressed from the Pu promoter for the upper pathway of the TOL plasmid. This construct is now available for the transformation of o-chlorotoluene to o-chlorobenzoate (39). Once this module is transferred to an o-chlorobenzoate degrading strain, mineralization of o-chlorotoluene will be achieved.

of chlorobenzoates or their metabolic products has been observed in the Pseudomonas testosteroni strain B356, with m-chlorobenzoate being the most effective inhibitor (45). In the case of 3-chlorobiphenyl, the rapid formation of m-chlorocatechol from m-chlorobenzoate led to toxicity, manifested as a decrease in viable cells during substrate utilization (46). This toxicity was because of meta cleavage of m-chlorocatechol, which might produce a reactive acyl chloride intermediate. A similar mechanism for the interference of m-chlorocatechol with the utilization of biphenyl and monochlorobiphenyl is inactivation by m-chlorocatechol of 2,3-dihydroxybiphenyl dioxygenase, which is necessary for biphenyl metabolism. Two approaches have been tested to overcome chlorobenzoatemediated inhibition of PCB degradation: the use of mixed cultures consisting of PCB and chlorobenzoate degraders and the in vivo and in vitro combination of PCB and chlorobenzoate pathways (46–51). The Burkholderia cepacia strain JHR222 is a hybrid strain that was able to mineralize: 2-, 3-, and 4-chlorobiphenyl and o-, m-, p-chloro-, and 3,5-dichlorobenzoate, but not other isomers such as 2,3-, 2,5-, 2,6-, and 3,4-dichlorobenzoate. A problem related to the mineralization of complex mixtures of chlorobiphenyls by this strain is that certain dichlorobenzoates inhibited the metabolism of monochloro-substituted biphenyls (53). Further developments in this area are foreseen via the use of sequential anaerobic treatments to achieve dechlorination of PCBs and further aerobic metabolism of low chloro-substituted biphenyls (54).

Self-Inhibiting Metabolic Routes: The Case of Chlorobiphenyl Metabolism

A Hybrid Enzyme for Trichloroethylene Cometabolism

A number of biphenyl-degrading microorganisms that have been isolated cometabolize a variety of polychlorinated biphenyls (PCBs) (39–44). The degradation of biphenyl and chlorinated analogs is initiated by dioxygenation at the 2,3-position. The 2,3-dihydro2,3-dihydroxybiphenyl formed is dehydrogenated to 2,3-dihydroxybiphenyl, which then undergoes meta cleavage. Benzoates are produced by hydrolysis of the ring cleavage product. In most cases, biphenyl-degrading organisms are not able to mineralize the chlorobenzoates formed from chlorobiphenyls, although some strains cometabolize chlorobenzoates. An inhibitory effect

Trichloroethylene (TCE) has been recognized as one of the most significant environmental pollutants in soil and groundwater (1). This and related compounds persist in the environment and are suspected carcinogens. Wackett and Gibson (55) showed that toluene dioxygenase plays a role in the cometabolic elimination of TCE. Later, Suyama and coworkers (56) constructed a hybrid strain in which the bphA1 gene coding for biphenyl dioxygenase was replaced by the todC1 gene, which codes for toluene dioxygenase of P. putida F1 within the chromosomal biphenyl-catabolic bph gene cluster. This hybrid strain efficiently removed trichloroethylene and cis 1,2-dichloroethylene.

202


MODIFIED PATHWAYS FOR ALKYLAROMATICS

HYBRID PATHWAYS FOR NITROAROMATICS

Expansion of the Catabolic Potential of the TOL Plasmid Catabolic Pathways for Metabolism of Alkylaromatics

Mineralization of Mononitrotoluenes

Pseudomonas putida KT2440, harboring the selftransmissible TOL plasmid pWW0, grows on a variety of aromatic hydrocarbons used as solvents, including toluene, m- and p-xylene, and m-ethyltoluene (36). The lateral alkyl chain of these aromatic compounds is oxidized to yield alkylbenzoates, which are further metabolized to Krebs cycle intermediates via catechol and alkylcatechols. In this strain, the metabolism of p-ethylbenzoate is blocked (21). By introducing a series of mutations in the TOL plasmid, the recombinant TOL plasmid pWW0-EB62 that allows the host microbe to grow on p-ethylbenzoate while maintaining its ability to grow on benzoate and m-methylbenzoate and p-methylbenzoate (21) was constructed. The strategy involved the isolation of several mutants, which are detailed later. XylSArg45Thr is a mutant protein that allows the induction of the metacleavage pathway in response to p-ethylbenzoate as an effector. When the mutant regulator was introduced in Pseudomonas bearing the TOL plasmid, p-ethylbenzoate was oxidized to p-ethylcatechol, which inactivated the wild-type xylE-encoded catechol 2,3-dioxygenase. Ramos and coworkers (21) then isolated a mutant xylE gene that encoded an enzyme resistant to inactivation by its substrate p-ethylcatechol. This mutant catechol, 2,3-dioxygenase, exhibited a single amino acid substitution (threonine 253 isoleucine) (57). The xylS and xylE mutations were recombined in the TOL plasmid pWW0EB62 so that bacteria grew on p-ethylbenzoate as the sole C-source. This microbe has been extensively studied in microcosms (see following text). de Lorenzo and coworkers (58) used the upper TOL operon of plasmid pWW0, together with its regulator xylR, to construct a cassette that would allow the bioconversion of toluene and a number of derivatives to the corresponding benzoates. This cassette can be transferred to microorganisms that use the housekeeping ortho cleavage pathway of catechol for the metabolism of toluene and its derivatives. Mineralization of Mixtures of Several Aromatics The construction of a hybrid strain that is able to mineralize components of a benzene, toluene, and p-xylene mixture was achieved by redesigning two metabolic pathways of P. putida for toluene metabolism. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture (59). A hybrid strain was constructed by cloning the todC1C2BA gene encoding toluene dioxygenase on plasmid pRSF1010 and introducing the resulting plasmid into P. putida bearing the TOL plasmid pWW0. The hybrid strain, called P. putida TB105, was able to mineralize a benzene, toluene, and p-xylene mixture without accumulating any metabolic intermediates.

Apparently, the mononitrotoluene that can more easily be mineralized by microbes is p-nitrotoluene (60–62), because o-nitrotoluene and m-nitrotoluene are more recalcitrant, although a Pseudomonas strain that is able to metabolize o-nitrotoluene has been described (62). To date no microbe that is able to deal with m-nitrotoluene has been described. Two independent reports originally documented the mineralization of pnitrotoluene by bacteria belonging to the genus Pseudomonas (10,60). Nitrotoluenes may be regarded as structural analogs of toluenes and xylenes, the original substrates of the TOL plasmid-encoded pathway. In fact, the degradative pathway found in p-nitrotoluene-mineralizing microbes involves stepwise oxidation of the methyl substituent with p-nitrobenzoate as the intermediate (10,60). Further metabolism of p-nitrobenzoate occurred via p-hydroxylaminobenzoate, which was then transformed into protocatechuate, a central intermediate of the metabolism of aromatic compounds (61). In contrast to p-nitrotoluene, o-nitrotoluene and m-nitrotoluene are more recalcitrant, and until recently only one Pseudomonas strain had been isolated because of its capability of metabolizing o-nitrotoluene (62). Delgado and coworkers (63) examined the ability of nitrosubstituted compounds to serve as substrates for the TOL pathway enzymes toluene monooxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. All three enzymes were able to transform the substituted nitroaromatic substrates, when the nitro group was located at the meta or para position of the aromatic ring, revealing that enzymes of the TOL catabolic pathway can metabolize m-nitrotoluene and p-nitrotoluene to the corresponding m-nitrobenzoate and p-nitrobenzoate. The XylR regulator controls transcription of the genes of these enzymes from the Pu promoter. Its effector specificity is such that it recognized o-nitrotoluene and p-nitrotoluene as effectors, but was not activated by m-nitrotoluene (64). Therefore, the potential of the TOL upper pathway seems to be limited to the degradation of p-nitrotoluene as it is the only compound that acts as both effector and substrate. Transfer of the TOL upper pathway module to a p-nitrobenzoatedegrading Pseudomonas sp. strain resulted in the expansion of its catabolic potential to include p-nitrotoluene (65; Fig. 2). A mutant XylR regulator that gained the ability to recognize m-nitrotoluene without losing its ability to recognize the other effectors was selected (64). This mutant regulator, together with the TOL upper catabolic pathway, provides the potential for the biotransformation of m-nitrotoluene to m-nitrobenzoate. This module can be transferred to a Pseudomonas strain that is able to mineralize m-nitrobenzoate (66). This was achieved after cloning the xylR7 mutant allele and the upper genes enzymes into a mini Tn5. The mini Tn5 was transferred to strain JS51, and mineralization of m-nitrotoluene was achieved (67).


P. putida 2440 (pWW0∆Pm)

P. fluorescens 410P

P. fluorescens 410PRif (pWW0∆Pm)

CH3

COOH

CH3

COOH

NO2

NO2

NO2

NO2

CH2OH

COOH

CH2OH

COOH

NO2

NHOH

NO2

NHOH

X

NH3

NH3

CHO

COOH

NO2

OH

CHO

COOH

NO2

OH

OH

OH

Ring cleavage

COOH

203

CO2+H2O

NO2

Ring cleavage

COOH

CO2+H2O

NO2

Figure 2. A catabolic pathway for the metabolism of 4-nitrotoluene. The hybrid pathway was as constructed by in vivo mating between strains with different catabolic potentials (35).

REMOVAL OF ORGANOMERCURIALS Mercury (Hg) is a toxic metal that has been released into the environment in substantial quantities. Its toxicity results from the capacity of Hg(II) to bind sulfhydryl, thioether, and imidazole groups and thereby inactivate enzymes (68). Organic species of mercury (both alkyl and aromatic derivatives) accumulate in tissues of higher organisms, causing serious health problems (69). The merTPABD genes of transposon Tn501, which encodes the enzymes involved in organomercury resistance (69), were cloned into a mini Tn5 that was used to insert the genes into the genome of P. putida. Transconjugants that constitutively expressed the mer genes (high resistance to phenylmercury) were selected (69). This allowed the engineered bacteria to cleave Hg from an organic moiety and reduce the Hg(II) released to Hg0 . These properties were also combined with benzene and toluene catabolism of several Pseudomonas strains,

allowing the degradation of the aromatic moiety of the organomercurial as well as the detoxification of the metal component. IMPROVED REMOVAL OF POLLUTANTS IN THE PLANT RHIZOSPHERE The Bioremediation of sites polluted by compounds at very low concentrations may be favored by microbes that are able to grow in plant rhizospheres, in which the nutrient supplied by the plant may help to colonize soil sites that are poor in available substrates and ultimately enhance the elimination of pollutants (70–76). This process is known as plant-assisted microbial bioremediation or rhizoremediation. Pseudomonas fluorescens sp. F113 is an isolate from the sugar beet rhizosphere, which is an excellent root colonizer and shows potential for serving as an instrument of biological control (75). This microbe has been subjected to pathway expansion to include biphenyl and

204


certain monochlorinated biphenyls (20). The bph genes were cloned into a mini-Tn5 operon encoding resistance to a herbicide in the suicide plasmid pDDPCB (20) and then delivered into the host chromosome through this vector system. Pseudomonas sp. F113-PCB is a derivative that carries the bphABCD genes for the conversion of biphenyl into benzoate. Plant compounds that induced biphenyl metabolism by bacteria have been identified. For example, l-carvone induced Arthrobacter sp. strain B13 to cometabolize Aroclor 1242 (a mixture of PCBs), resulting in significant degradation of 26 isomers in the mixture. Several compounds structurally related to l-carvone, including limonene, p-cymene, and isoprene, also induced cometabolism of PCBs by certain bacteria. These results suggest that in the plant rhizosphere gratuitous inducers may facilitate the activation of catabolic pathways and the removal of xenobiotic compounds. In the plant rhizosphere, the metabolism of alkylbenzoates was stimulated so that degradation was enhanced and the remaining levels of these aromatics were lower than those found in soil without plants (76). A similar observation was seen for removal of TNT in the plant rhizo˜ and J. L. Ramos, unpublished). sphere (A. Esteve-Nu´ nez TRACKING GEMs IN MICROCOSMS AND BEHAVIOR OF RECOMBINANT MICROBES Tools for Tracking GEMs in the Environment Most of the recombinant bacteria constructed for biodegradation bear antibiotic-resistance markers. Although these markers are extremely useful in molecular genetics and microbial ecology, their use in uncontained applications is not very important (77). To avoid the use of antibiotic-resistance selection markers in GEMs designed for environmental applications, a series of nonantibioticresistance markers were developed. These markers include spontaneous resistance to phosphinothricin, bialaphos, heavy metal ions such as mercury, arsenate, or tellurite (28,29,77–79), and nutritional markers such as the genes that enable the organism to grow on lactose as the sole C-source (80). Some of these markers were introduced into the host microorganisms via mini-Tn5 derivatives (28). Another approach, which may ultimately solve the problem of the presence of selectable markers, is the use of a selectable marker, that is, kanamycin resistance, flanked by two tandem res (resolution) sites of plasmid RP4, which can be provided as a cassette within a mini transposon (81). The res site is a short sequence that, once recognized by the resolvase of the ParA system, undergoes site-specific recombination that results in the deletion of the intervening DNA sequences (82). ParA can be provided in a suicide replicon. An alternative is the use of genes such as lux and luc, which encode for light emission (83). This system now has been used to mark and track Pseudomonas strains that are able to colonize a number of plant roots (71,73). An alternative to the introduction of a selectable marker in the target strain is to use tools that specifically recognize

the target microbe. Monoclonal antibodies (mAbs) are powerful tools for tracking microorganisms because they can recognize epitopes on the surface of bacteria and thus serve as in situ identifiers. A series of mAbs were produced against whole cells of Pseudomonas putida KT2440 (84). One was shown to recognize the O-antigen of P. putida LPS. In the laboratory, this mAb specifically recognized the strain when grown in different culture media and at different growth stages. The mAb was used to track the strain after its release in a mesocosm established in Plussee Lake in northern Germany (85). Another approach used in our laboratory has been to introduce new epitopes on bacterial surface proteins. For example, a mammalian coronavirus epitope (86) has been cloned into the OprL protein, a surface protein of P. putida, and is now being used as a reporter to track bacteria bearing rDNA (87). The use of direct gene probes and PCR is a powerful approach to detect microorganisms without prior cultivation (88,89). For example, amplification of chromosomal genes from a highly specialized subpopulation of the total microbial community from the top layer sediment of the Elbe River in Germany made it possible to identify aerobic microbes that are able to degrade biphenyl (90). Survival, Propagation, and Stability of GEMs in Their Target Ecosystems and Their Effects on Indigenous Microorganisms A series of features that are crucial for the safe and effective functioning of some of the earlier recombinant and wild-type bacteria have been examined in soil microcosms with and without plants and in sewage microcosms. Microcosms offer a suitable approach to evaluate the survival and functioning of GEMs; however, it should be recalled that the samples are taken from nature and introduced into the laboratory. This imposes some limitations as a number of parameters are closely controlled, that is, incubation temperature, light/dark cycles, and so on. The survival of some of the recombinant microbes described earlier was assayed by introducing both parental and recombinant strains in the edaphic and the sewage water microcosms. These recombinant bacteria were designed to eliminate pollutants and were added to polluted soils and aquatic microcosms in relatively high numbers, for example, 106 –108 colony forming units (CFU) per gram of soil or milliliter of sewage water. Most studies have focused on the behavior of these microbes during the initial period after introduction into the microcosms, usually between four and eight weeks. It was generally observed that both the recombinant microbes and their parental strains were able to establish in soil, rhizosphere, and aquatic microcosms. In each ecosystem, the number of bacteria tended to reach the microcosm’s carrying capacity, so that in microcosms rich in organic matter, the number of microbes was usually higher than that in microcosms poor in nutrients (72–74,88–96). Recombinant bacteria survived better than parental strains only when the former were introduced in soil or aquatic microcosms where up to 0.1% (wt/wt or wt/vol) pollutant had been added, that is, under conditions strongly favorable to the GEM. For example, the introduction of P. putida EEZ15


(pWW0-EB62) or Pseudomonas sp. FR1 (pFRC20) in soils supplemented with 0.1% (wt/wt) p-ethylbenzoate and pmethylbenzoate allowed the strain to become established at at least one order of magnitude higher levels than in soils without the supplement (63,78,93). A similar finding was obtained when these bacteria were introduced into microcosms consisting of wastewater from a sewage treatment plant (91,92). Although survival of parental and recombinant bacteria approached 100% in shortterm experiments, in the long term, a steady decline was observed in CFU per gram of soil until numbers in some cases fell below the detection limits (93). Recombinant and parental bacteria survive in soils, and in certain cases, the total number of CFU increased. For example, when a low number (104 CFU per gram of soil) of P. putida (pWW0-EB62) was introduced in nonsterile soils, the strain multiplied to a density of 106 –107 CFU per gram of soil (78). The stability of the genetic information introduced into Pseudomonas sp. FR1 (pFRC20P), Pseudomonas sp. F113-PCB, and P. putida EEZ15 (pWW0-EB62) has been studied (63,75,78). These strains can be considered genetically stable bacteria as the phenotype acquired through genetic manipulation was maintained under laboratory culture conditions in the absence of selective pressure. Furthermore, some natural properties of these strains that are not related to the recombinant phenotype, that is, biocontrol traits, growth rates, and pigment production, remained unaltered. The recombinant trait did not affect the competitive ability of the Pseudomonas sp. F113-PCB and P. putida strains in colonization assays in nonsterile soil microcosms on sugar beet, corn, tomato, and spinach-seedling roots (72–75). Furthermore, these strains have been shown to be stable in soil; 100% of the bacteria recovered after prolonged incubation in soil retained the ability to use the aromatic compounds that they were designed to deal with. The effect of the introduction of wild-type or recombinant microbes into the indigenous microbiota has been studied by estimating the ‘‘total’’ number of culturable microbes in relatively rich medium (i.e., peptone agar) or by counting the indigenous population that is able to use a certain compound as the sole C-source, (i.e., p-hydroxyphenylacetic acid degraders), the denitrifying bacteria, or the ‘‘heterotrophic’’ population (74,89–95). Neither the wild-type nor the recombinant microbe affected indigenous microbiota, which suggests that natural environments have a certain buffering capacity against the introduced microbes. The earlier cases suggest that the possible risks from the use of recombinant microbes in bioremediation are similar to those posed by the parental nonmodified strains. It also seems that the physicochemical and biological parameters of the microcosms affect the parental and recombinant microbes equally. Functionality of Recombinant Microbes in Target Microcosms The introduction of bacterial strains into the environment for in situ bioremediation will usually require that microorganisms be able to survive in high numbers and express

205

the desired catabolic phenotype. For pollutants to be efficiently mineralized by natural and genetically modified microbes, the degrading microbe must not only become established in the polluted sites but also must express catabolic genes in response to the pollutant, even in the presence of other compounds. Soils, river sediments, and sewage treatment plants are complex environments where gene expression can be inhibited or stimulated. Pseudomonas putida harboring either the wild-type pWW0 or the recombinant TOL plasmid were able to mineralize 14 Clabelled substrates (p-methyl-14 C-benzoate) for at least a month (95,96). In soils, mineralization was monitored as the evolution of 14 CO2 , whereas in aquatic microcosms the metabolism of alkylaromatic and chloroaromatic compounds was monitored chromatographically by measuring the disappearance of the target chemical from the polluted site (92). It was shown that Pseudomonas sp. FR1 (pFRC20P) enhanced the rate of degradation of a mixture of m-chlorobenzoate and p-methylbenzoate that had been added to the water column of sediment cores made of intact-layered sediments from the Plussee Lake and the Rhine River (92). Pseudomonas sp. FR1 bearing the bph genes inserted on the chromosome was able to remove up to 100 ppm of p-chlorobiphenyl per gram of sediment slurry in five days (20). In these assays, recombinant bacteria established at close to 108 CFU per gram of sediment. Transfer of Recombinant DNA from Pseudomonas to Other Microorganisms Gene transfer from recombinant microbes has been analyzed under optimum laboratory conditions and in soil and aquatic microcosms. The genetic information introduced into Pseudomonas sp. F113-PCB was inserted into the host chromosome via a mini Tn5 lacking the transposase gene. In Pseudomonas sp. FR1 (pFRC20P), the information was located partially on the bacterial chromosome (Tn5:xylXYZ, xylS) and partially on a mobilizable broad host-range plasmid (pFRC20P). In P. putida (pWW0-EB62), the recombinant information was on a self-transmissible TOL plasmid. The recombinant DNA in these three strains was stably maintained under laboratory growth conditions and in bacteria introduced into soil and aquatic microcosms. In the laboratory, there was no transfer of the recombinant DNA from the chromosome of Pseudomonas sp. F113PCB or Pseudomonas sp. FR1 (pFRC20P) to other Pseudomonas or to indigenous bacteria in soil and aquatic microcosms (63,75,91). In contrast, plasmid pFRC20P was transferred to other microbes, but only if a helper plasmid was supplied in the mating mixture (93). In these cases, transfer of the plasmid led to about 10−5 transconjugants per recipient. Transfer of the wildtype TOL plasmid or the recombinant pWW0-EB62 was restricted to microorganisms belonging to members of the Pseudomonad rRNA group I (P. putida, P. fluorescens, P. stutzeri, and so on) and some enterobacteriaceae. In matings on plates, the rate of TOL transfer was on the order of 1 to 10−2 transconjugants per recipient, and the rate of intergeneric transfer ranged from 10−4 to 10−8 transconjugants per recipient (97,98).

206


In soils, the TOL plasmid was transferred from P. putida to other strains of Pseudomonas only at high cell densities (higher than 106 CFU/g soil). Transfer was influenced by the type of soil used, the incubation temperature, the initial inoculum size, and the presence of chemicals that affected the survival of donor or recipient bacteria. Maximal transfer was observed in soils incubated at 15–17 ◦ C, when the donor and recipient loads were about 108 CFU per gram of soil (97). In conclusion, recombinant DNA can be transferred between microorganisms. Transfer is limited by the vector used to introduce the recombinant DNA into the host microbe and by the nature of the recipient microorganism. Chromosomal information is less likely to be transferred than is information on mobilizable plasmids. The latter is less likely to be transferred than recombinant DNA on self-transmissible plasmids. Not all locations on the chromosome are similar in terms of expression, physical structure, or mobilization. ´ This is particularly true in Pseudomonas. Ramos-Gonzalez and coworkers (98) labeled the chromosome of P. putida randomly at 34 independent positions with the same marker, a mini-Tn5-Km. They found that the TOL plasmid was able to mobilize these insertions on the host chromosome at a rate between 10−4 and 10−8 transconjugants per recipient. Furthermore, mobilization of the host chromosome by the TOL plasmid occurred when the TOL plasmid and the marked chromosome were located in the two independent microbes, a phenomenon called retrotransfer (98–100). Retrotransfer involves the movement of a self-transmissible replicon to a recipient organism, the capture of DNA, and its return to the original donor (100). It follows that although chromosomal insertions are less likely to be mobilized than rDNA on plasmids, they can nonetheless be transferred potentially. The deliberate release of GEMs in the environment raises a series of scientific and public concerns, and an active biological containment can provide a means to increase the predictability of the behavior and fate of recombinant microbes (101). Such biologically active containment systems have been shown to control the survival of GEMs and to inhibit lateral gene transfer (95,101,102).

the formation and accumulation of toxic intermediates. An example of this is the newly identified catechol 2,3dioxygenase that is able to deal with chlorosubstituted substrates (105–107). This, in turn, can increase the catalytic potential and efficiency of microorganisms for the degradation of xenobiotics and can enhance their survival in environmental settings. Specifically designed pathways can also be introduced into microorganisms derived from contaminated sites of interest and hence, adapted to prevailing environmental conditions. As information accumulates on the genetic determinants of the characteristics that are important for treatment processes, such as tolerance or resistance to the toxic effects of solvents and other pollutants, surfactant production, and so on, our ability to generate more effective biocatalysts will increase (108–117). In recent years, a number of new catabolic pathways for toxic and recalcitrant compounds have been elucidated. These include pathways for the metabolism of dibenzodioxins and new pathways for chloroaromatic and polar and apolar nitroaromatic compounds such as nitrobenzoate, monosubstituted, and polysubstituted nitrotoluenes (6,10,12,65,118–136). Equally important is the fact that many enzymes are extremely relaxed in their substrate specificity and are thus able to deal with multiple substrates. Some dioxygenases for onering aromatic compounds function also as monoxygenases and attack compounds with two or three aromatic rings and heterocycles (131–134). It is therefore expected that new catabolic pathways will be constructed on the basis of these catabolic modules, and as a consequence, the number of microbes able to mineralize halo-substituted and nitro-substituted aliphatic and aromatics, organic solvents, insecticides, and other xenobiotics should increase steadily. Bioremediation research in simulated environments does not accurately represent real environmental conditions, and therefore, such experiments cannot test how prospective bioremediation schemes respond to the changes in the weather and to the movements of materials that may typify polluted sites. For these reasons, and to more fully address concerns regarding the safety and reliability of the approaches that are now being developed, carefully controlled field studies for bioremediation purposes need to be performed.

CONCLUSION Bringing about in vitro evolution of the catabolic pathways usually requires thorough knowledge of the biochemistry of the reactions involved in the degradation of a compound and of the genetics of the pathway. More specifically, intimate awareness of the organization of the genes that encode the enzymes and of the operation of the regulatory circuits that govern the activation of gene expression, including alteration of substrate and effector specificity by DNA shuffling (18,102,103), is required. These regulators can be useful for the design of new biosensors (104). The judicious combination of segments from different metabolic pathways in the appropriate bacteria can provide complete catabolic routes for recalcitrant xenobiotic compounds, and can circumvent certain problems related to substrate incompatibilities, and to

Acknowledgments The work, carried out in the authors’ laboratories, was supported mainly by a biotechnology grants from the European Commission (QLK3-CT-2000-00170 and QLK-CT-2001-0435) and a grant from the Spanish CICYT (BIO2000-0964). We thank M. M. Fandila and C. Lorente for secretarial assistance.

BIBLIOGRAPHY 1. L. H. Keith and W. A. Telliard, Environ. Sci. Technol. 13: 416–423 (1979). 2. D. L. Kaplan, Biotransformation pathways of hazardous energetic organo-nitro compounds, in D. Kamely, A. Chakrabarty, and G. Omenn, eds., Biotechnology and Biodegradation, Advances in Applied Biotechnology Series, vol. 4, Portfolio Publ. Co., Houston, Tex., 1989, pp. 155–181.


207

3. J. L. Ramos and K. N. Timmis, Microbiol. Sci. 4: 228–237 (1987).

36. M. J. Worsey and P. A. William, J. Bacteriol. 124: 7–13 (1974).

4. S. B. Funk et al., Appl. Environ. Microbiol. 59: 2171–2177 (1993).

37. U. Brinkmann and W. Reineke, FEMS Microbiol. Lett. 75: 81–87 (1992).

5. D. L. Kaplan and A. M. Kaplan, Appl. Environ. Microbiol. 44: 757–760 (1982).

38. J. B. Robertson et al., 2643–2648 (1992).

˜ et al., Biotechnol. Bioeng. 58: 510–514 (1998). 6. G. Pinar

39. A. Haro, PhD dissertation. Universidad Autónoma de Madrid, Madrid, Spain, 1996.

7. J. L. Ramos et al., Nature/Biotechnology 14: 320–322 (1996). 8. J. L. Ramos et al., Bio/Technology 12: 1349–1356 (1994). 9. E. Duque et al., J. Bacteriol. 175: 2278–2283 (1993). 10. B. E. Haigler and J. C. Spain, Appl. Environ. Microbiol. 59: 2239–2243 (1993). 11. J. M. Pemberton and P. R. Fisher, Nature 268: 732–733 (1977). 12. A. Preuss, J. Fimpel, and G. Diekert, Arch. Microbiol. 159: 345–353 (1993). 13. J. H. Slater and A. T. Bull, Phil. Trans. R. Soc. London Ser. B 297: 575–597 (1982). 14. R. V. Subba-Rao and M. Alexander, Appl. Environ. Microbiol. 49: 509–516 (1985). 15. G. F. White and J. R. Snape, J. Gen. Microbiol. 139: 1947–1957 (1993). 16. J. L. Ramos et al., Proc. Natl. Acad. Sci. U.S.A. 83: 8467–8471 (1986). 17. F. Rojo et al., Science 238: 1395–1398 (1987). 18. K. N. Timmis, R. J. Steffan, and R. Unterman, Ann. Rev. Microbiol. 48: 525–557 (1994). 19. M. A. Abril et al., J. Bacteriol. 171: 6782–6790 (1989). 20. D. N. Dowling, R. Pipke, and D. F. Dwyer, FEMS Microbiol. Lett. 113: 149–154 (1993). 21. J. L. Ramos et al., Science 235: 593–596 (1987). 22. P. R. Lehrbach et al., J. Bacteriol. 158: 1025–1032 (1984). 23. W. Reineke and H.-J. Knackmuss, Nature 277: 385–386 (1979). 24. N. Mermod et al., Gene Cloning and Manipulation in Pseudomonas, in J. R. Sokatch, ed., The Bacteria, vol. 10, Academic Press, New York, 1986, pp. 325–355. 25. J. Sokacht, ed., The Biology of Pseudomonas, The Bacteria, vol. 10, Academic Press, New York, 1986. 26. J. M. Blatny et al., Appl. Environ. Microbiol. 63: 370–379 (1997). 27. V. de Lorenzo et al., J. Bacteriol. 172: 6568–6572 (1990). 28. V. de Lorenzo and K. N. Timmis, Methods Enzymol. 235: 386–405 (1994). 29. M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172: 5557–6567 (1990). 30. J. J. Rodr´ıguez-Herva and J. L. Ramos, J. Bacteriol. 178: 5836–5840 (1996). 31. H. P. Schweizer, Methods Mol. Biol. 62: 17–27 (1997). 32. E. Dorn et al., Arch. Microbiol. 99: 61–70 (1974). 33. V. M. Rangnekar, J. Bacteriol. 170: 1907–1912 (1988). 34. J. R. van der Meer et al., Microbiol. Rev. 56: 677–694 (1992). 35. S. Molin et al., Annu. Rev. Microbiol. 47: 139–166 (1993).

Appl.

Environ.

Microbiol.

58:

40. D. Ahmad, R. Masse, and M. Sylvestre, Gene 86: 53–61 (1990). 41. M. Ahmed and D. D. Focht, Can. J. Microbiol. 19: 47–52 (1973). 42. D. L. Bedard et al., Appl. Environ. Microbiol. 53: 1094– 1102 (1987). 43. L. H. Bopp, J. Ind. Microbiol. 1: 23–29 (1986). 44. K. Furukawa et al., J. Bacteriol. 171: 5467–5472 (1989). 45. M. Sondossi, M. Sylvestre, and D. Ahmad, Appl. Environ. Microbiol. 58: 485–495 (1992). 46. J. J. Arensdorf and D. D. Focht, Appl. Environ. Microbiol. 60: 2884–2889 (1994). 47. R. H. Adams et al., Appl. Environ. Microbiol. 58: 647–654 (1992). 48. J. Havel and W. Reineke, Appl. Environ. Microbiol. 58: 2706–2712 (1993). 49. J. Havel and W. Reineke, FEMS Microbiol. Lett. 108: 211–217 (1993). 50. W. J. Hickey, D. B. Searles, and D. D. Focht, Appl. Environ. Microbiol. 59: 1194–1200 (1993). 51. W. J. Hickey, V. Brenner, and D. D. Focht, FEMS Microbiol. Lett. 98: 175–180 (1992). 52. Y. Hrywna et al., Appl. Environ. Microbiol. 65: 2163–2169 (1999). 53. J. Stratford et al., Arch. Microbiol. 165: 213–218 (1996). 54. S. Ripp et al., Environ. Sci. Technol. 34: 846–854 (2000). 55. L. Wackett and D. T. Gibson, Appl. Environ. Microbiol. 54: 1075–1078 (1988). 56. A. Suyama et al., J. Bacteriol. 178: 4039–4046 (1996). 57. A. Wasserfallen, PhD Disertation, University of Geneva, Geneva, Switzerland, 1989. ´ 58. S. Panke, J. M. Sanchez-Romero, and V. de Lorenzo, Appl. Environ. Microbiol. 64: 748–752 (1998). 59. J. Y. Lee et al., J. Bacteriol. 61: 2211–2217 (1995). 60. W. Rhys-Williams, S. C. Taylor, and P. A. Williams, J. Gen. Microbiol. 139: 1967–1972 (1993). 61. P. E. J. Groenewegen 1599–1605 (1992).

et al.,

J.

Gen.

Microbiol.

138:

62. B. E. Haigler, W. H. Wallace, and J. C. Spain, Appl. Environ. Microbiol. 60: 3466–3469 (1994). 63. A. Delgado et al., Appl. Environ. Microbiol. 58: 415–417 (1992). 64. A. Delgado and J. L. Ramos, J. Biol. Chem. 269: 8059–8062 (1994). ´ et al., J. Bacteriol. 179: 3036–3038 (1997). 65. C. Michan 66. L. J. Nadeau and J. C. Spain, Appl. Environ. Microbiol. 61: 840–843 (1995).

208


˜ et al., submitted (2002). 67. A. Esteve-Nu´ nez 68. E. Niebor and D. H. S. Richardson, Environ. Pollut. Ser. B 1: 3–26 (1980). 69. J. M. Horn et al., Appl. Environ. Microbiol. 60: 357–362 (1994). 70. P. A. Lund and N. L. Brown, J. Mol. Biol. 205: 343–353 (1989). 71. S. Alvey and D. E. Crowley, Environ. Sci. Technol. 30: 1596–1603 (1996). 72. S. D. Cunningham and D. W. Ow, Plant Physiol. 110: 715–719 (1996).

96. E. Duque, S. Marqués, and J. L. Ramos, Microb. Releases 2: 175–177 (1993). ´ 97. M. I. Ramos-Gonzalez, E. Duque, and J. L. Ramos, Appl. Environ. Microbiol. 57: 3020–3027 (1991). ´ 98. M. I. Ramos-Gonzalez, M. A. Ramos-D´ıaz, and J. L. Ramos, J. Bacteriol. 176: 4635–4641 (1994). 99. M. C. Ronchel, M. A. Ramos-D´ıaz, and J. L. Ramos, Environ. Microbiol. in press (2000). 100. M. Mergeay et al., Mol. Gen. Genet. 209: 61–70 (1987). 101. E. D´ıaz et al., Mol. Microbiol. 13: 855–861 (1994).

73. C. Ramos et al., FEMS Microbiol. Ecol. 34: 91–102 (2000).

102. J. L. Ramos, S. Marqués, and K. N. Timmis, Ann. Rev. Microbiol. 51: 341–373 (1997).

74. L. Molina et al., Soil Biol. Biochem. 32: 315–321 (2000).

¨ 103. E. Skarfstad et al., J. Bacteriol. 182: 3008–3016 (2000).

75. G. M. Brazil et al., Appl. Environ. Microbiol. 61: 1946– 1952 (1995).

104. S. K. van Bergen et al., Environ. Sci. Technol. 34: 704–709 (2000).

76. C. Ronchel et al., Appl. Environ. Microbiol. 65: 4904–4911 (1998).

105. A. E. Mars et al., J. Bacteriol. 181: 1309–1318 (1999).

77. J. M. Tiedje et al., Ecology 70: 298–315 (1989). ´ 78. J. L. Ramos, E. Duque, and M. I. Ramos-Gonzalez, Appl. Environ. Microbiol. 57: 260–266 (1991). ´ 79. J. M. SanchezRomero, R. D´ıaz, and V. de Lorenzo, Appl. Environ. Microbiol. 64: 4040–4046 (1998). 80. D. J. Drahos et al., Prerelease Testing Procedures: U.S. Field Test of a lacZY-Engineered Soil Bacterium, in M. Sussman et al., eds., The Release of Genetically Engineered Microorganisms, Academic Press, London, U.K., 1988, pp. 181–191. 81. C. S. Kristensen et al., J. Bacteriol. 177: 52–58 (1995). 82. L. Eberl et al., Mol. Microbiol. 12: 131–141 (1994).

106. A. E. Mars et al., J. Bacteriol. 179: 4530–4537 (1997). 107. S. Kaschabek et al., J. Bacteriol. 180: 296–302 (1998). 108. J. L. Ramos et al., J. Bateriol. 177: 3911–3916 (1995). 109. A. Inoue and K. Horikoshi, Nature 338: 264–266 (1989). 110. A. Segura et al., Environ. Microbiol. 1: 191–198 (1999). 111. J. Sikkema, J. A. M. de Bont, and B. Poolman, Microbiol. Rev. 59: 201–222 (1994). 112. J. L. Ramos et al., J. Bacteriol. 180: 3323–3329 (1998). 113. R. A. Kanaly and S. Harayama, J. Bacteriol. 182: 2059– 2967 (2000). 114. A. Kahnert et al., J. Bacteriol. 182: 2869–2878 (2000).

83. J. J. Prosser, Microbiology 140: 5–17 (1994).

115. D. H. Pieper et al., FEMS Microbiol. Lett. 29: 63–67 (1985).

´ 84. M. I. Ramos-Gonzalez et al., J. Bacteriol. 174: 2978–2985 (1992).

˜ and J. L. Ramos, Environ. Sci. Technol. 32: 116. A. Esteve-Nu´ nez 3802–3808 (1998).

85. I. Brettar et al., Microb. Ecol. 27: 99–122 (1994).

˜ et al., J. Bacteriol. 182: 1352–1355 (2000). 117. A. Esteve-Nu´ nez

86. F. Gebauer et al., Virology 183: 225–238 (1991).

118. J. Havel and W. Reineke, FEMS Microbiol. Lett. 62: 163–169 (1991).

87. J. J. Rodr´ıguez Herva et al., Environ. Microbiol. 1: 479–488 (2000). 88. R. M. Atlas, Biodegradation 3: 137–146 (1992).

119. E. Andujar et al., J. Bacteriol. 182: 789–795 (2000). 120. J. Armengaud, K. N. Timmis, and R. M. Wittich, J. Bacteriol. 181: 3452–3461 (1995).

89. R. W. Erb and I. Wagner-Döbler, Appl. Environ. Microbiol. 59: 4065–4073 (1993).

121. B. Happe et al., J. Bacteriol. 173: 7313–7320 (1993).

90. I. Wagner-Döbler et al., Appl. Environ. Microbiol. 58: 1249–1258 (1992).

122. K. Valli, H. Wariishi, and M. H. Gold, J. Bacteriol. 181: 3105–3113 (1999).

91. K. Nusslein et al., Appl. Environ. Microbiol. 58: 3380–3386 (1992).

123. S. Beil et al., J. Bacteriol. 180: 5520–5528 (1998).

92. R. Pipke et al., Appl. Environ. Microbiol. 58: 1259–1265 (1992). 93. E. Duque et al., Genetically Engineered Pseudomonas Strains for Mineralization of Aromatics: Survival, Performance, Gene Transfer, and Biological Containment, in E. Gally, S. Silver, and B. Withold, eds., Pseudomonas: Molecular Biology and Biotechnology, American Society for Microbiology, Washington, D.C., 1992, pp. 429–437. 94. M. C. Ronchel et al., Appl. Environ. Microbiol. 61: 2990– 2994 (1995). 95. M. C. Ronchel et al., Appl. Environ Microbiol. 64: 4904– 4911 (1998).

124. V. Riegert et al., J. Bacteriol. 181: 4812–4817 (1999). 125. A. Schenzle et al., J. Bacteriol. 181: 1444–1450 (1999). 126. J. Parales et al., J. Bacteriol. 180: 1194–1199 (1998). 127. P. G. Rieger et al., J. Bacteriol. 181: 1189–1195 (1999). 128. S. S. Park and H. S. Kim, J. Bacteriol. 182: 573–580 (2000). 129. J. Hawari et al., Appl. Environ. Microbiol. 64: 2200–2206 (2000). 130. D. An, D. T. Gibson, and J. C. Spain, J. Bacteriol. 176: 7462–7467 (1994). 131. R. E. Parales et al., J. Bacteriol. 182: 1641–1649 (2000).

BIOLOGICAL CONTROL OF PLANT DISEASES 132. K. Lee, S. M. Resnick, and D. T. Gibson, Appl. Environ. Microbiol. 63: 2067–2070 (1997). 133. S. M. Resnick and D. T. Gibson, Appl. Environ. Microbiol. 62: 4073–4080 (1996). 134. J. B. Robertson et al., 2643–2648 (1992).

Appl.

Environ.

Microbiol.

58:

135. M. C. M. Jasper et al., J. Bacterial 183: 270–279 (2001). 136. D. H. N. Pieper et al., J. Bacterial 184: 1466–1470 (2002).

BIOLOGICAL CONTROL OF PLANT DISEASES CAROLEE T. BULL USDA–ARS Salinas, California

Biological control of plant disease has been described for many crops and diseases but development of these into commercially viable disease control options is rare. Biological control has yet to achieve the consistent levels of control and use that have been obtained by other methods (1–4). THE NATURE OF BIOLOGICAL CONTROL Although the concept of biological control has been used in the field of plant pathology since 1914 (5), consensus on a single definition of biological control has eluded researchers. The term biological control was first used by entomologists. For insect pests, biological control is a component of an integrated pest management strategy. It is the ‘‘use of natural enemies, including pathogens, to control pests’’ (6). Reduction in insect pest numbers is a key factor in biological control. Several types of biological control exist, including natural, classic, augmentative, and inundative. Natural biological control occurs through organisms and environmental factors, with no human input. Classic biological control typically involves an active human role. For example, when an insect pest is accidentally introduced into a new geographic area without its associated natural enemies, the subsequent introduction of its natural enemies (by humans) to an area is classic biological control. Augmentative biological control involves actions taken to increase the populations of biological control agents. This is in contrast to inundative biological control, which is the direct release of biological control agents to control a pest (6). Entomologists use biological control as part of an integrated pest management strategy. Biological control of plant pathogens also has its greatest potential as part of an integrated strategy. But unlike the situation for insect pests, reduction in disease is the goal of plant pathologists and reduction in the pathogen population may not achieve this goal. Though early research showed that fungal species were able to parisitize fungal pathogens (1,7,8), parasitism is not the only way that biological control can occur. Plant pathologists have defined biological control more broadly because disease control may occur in the absence of pathogen reduction.

209

Plant disease is the result of the interaction of the environment, the host plant, and the pathogen, and this relationship is referred to as the disease triangle (Fig. 1a). Biological control agents are organisms that interact with the components of the disease triangle and result in reduced disease. By separating the biological control agents from the environmental component of the disease triangle, the concept of the biological control pyramid is formed (Fig. 1b). The biological control pyramid allows us to visualize the complex interactions between the biological control agent, the environment, the host plant, and the pathogen that result in disease control. This model helps to conceptualize factors that affect the success of this disease-control strategy. Some plant pathologists prefer a more inclusive definition, whereas others prefer a more restrictive definition (1,9–11) (Table 1). Biological control can be a catch-all phrase that lumps together control by plant disease resistance, application of biologically derived pesticides, crop rotation, and other more specific disease control strategies (1). It may be more useful to consider each of these strategies separately. Here, we reserve the term biological control for those situations in which it is clear that a biological agent other than man is interacting with the disease triangle and results in disease control. Biological control is therefore disease control–mediated by an additional organism(s), which changes the outcome of the interaction between the environment, a pathogen, and the host plant. Biological control is also used by plant pathologists to refer to the control of weeds using plant pathogens.

Pathogen

(a)

Host (b)

Environment Pathogen

Biological agent(s)

Host Environment Figure 1. Biological control disease pyramid.

210

BIOLOGICAL CONTROL OF PLANT DISEASES Table 1. Effect of Definition on the Character of Biological Control Definition Characteristic

A

B

C

D

E

• Predation of organisms on a pathogen resulting in inoculum reduction but no disease reduction

X

• Predation of organisms on a pathogen resulting in inoculum and disease reduction

X

X

X

X

X

• The use of naturally occurring organisms whose gene products result in inoculum and disease reduction

X

X

X

X

X

X

X

X

X

• The use of genetically modified organisms which produce new gene products resulting in inoculum reduction • The use of genetically modified organisms which produce new gene products resulting in inoculum and disease reduction • Application of a compound (antibiotics, toxins) derived from a biological control agent which reduce disease

X

• Disease control through engineering plants for resistance to plant pathogens

X

X

• Disease control using a killed culture of an antagonist

X

X

A. ‘‘Use of natural enemies, including pathogens to control pests’’ (6). B. ‘‘Total or partial destruction of pathogen populations by other organisms’’ (10). C. A biological phenomenon mediated by an organism(s) (outside of the disease triangle and other than man) which results in disease control (this manuscript). D. ‘‘The control of a disease by an organism other than man’’ (1). E. ‘‘1) The reduction of the amount of inoculum or disease producing activity of a pathogen accomplished by or through one or more organisms other than man. 2) Use of natural or modified organisms, genes or gene products to reduce the effects of undesirable organisms (pests) and to favor desirable organisms such as crops, trees, animals, and beneficial insects and microorganisms’’ (11).

Although many of the same problems need to be overcome for these control strategies to be useful, they are conceptually very different (12). Additionally, the term biological control is generally not used in relation to viral diseases; instead cross protection with mild viral strains or control of insect vectors are important biological strategies (13). For plant pathology, the closest analogy to natural biological control in entomology is the phenomenon of suppressive soils. Suppressive soils have been defined as ‘‘soils in which the pathogens cannot establish, they establish but fail to produce disease, or they establish and cause disease at first but diminish with continued culture of the crop’’ (14). Suppressiveness is mediated by a biological phenomenon and has been documented for soils worldwide (14,15). DEVELOPMENT OF BIOLOGICAL CONTROL AS A DISEASE CONTROL STRATEGY Biological control was first used as a term in plant ¨ pathology in 1914 (Biologische bekampfung) (5). Since then many scientific publications have been generated in the field of biological control of plant pathogens (Table 2). The majority of publications report the phenomenon of biological control in a particular crop or cropping system. Although there have been approximately 6,000 articles published on biological control, there are only 22 biological products registered for use against plant pathogens by the U.S. Environmental Protection

Table 2. Publications on Biological Control of Plant Pathogens Number of Publicationsa Years 1970–1974 1975–1979 1980–1984 1985–1989 1990–1994 1995–1999d

CAB 37b (61) 151 307 1052 2125 2178(2420)

Agricola 121c (202) 278 433 656 936 829(921)

a Number of publication during the given years with the keywords biological control, or biocontrol in relation to plant pathogens. b CAB database only goes back to 1972 therefore the 1970–1974 time period is an underestimate. The number in parentheses are estimates based on the three year figures. c Agricola did not have plant disease codes in 1970 and 1971 for searching therefore the 1970–1974 time period is an underestimate. The number in parentheses are estimates based on the three year figures. d The 1995–1999 time period only includes papers up to July of 1999 and is an underestimate of the number of papers. The number in parentheses are estimates based on the three year figures.

Agency (USEPA) and these products are made from formulations of only 19 active ingredients (USEPA; http://www.apsnet.org/online/feature/biocontrol/). Although many agents have been described, few are developed into commercially available biological control

BIOLOGICAL CONTROL OF PLANT DISEASES

products. Using products registered by the US-EPA underestimates the importance of biological control to plant production since many agents may be registered as general soil inoculants, or are being used in other countries (16). Additionally, it is difficult to estimate the role of natural biological control because it is difficult to quantify this information from individual farms or regions. A clear analysis of the impact of biological control on commercial agriculture is needed. Biological control of crown gall is an example of successful biological control. Since 1979 Agrobacterium radiobacter strain K84 has been registered by the USEPA and is still being used as a biological control against crown gall on stone fruit rootstocks caused by Agrobacterium tumefaciens. Several products are available for use in the United States and worldwide (17). Biological control of crown gall involves the production of a bacteriocin (agricin 84) that inhibits infection by A. tumefaciens (18). Although bacteriocins are produced by many plantassociated bacteria, successful control-based on production of these compounds appears to be unique to this agent (19). MANY ORGANISMS ARE RESPONSIBLE FOR CONTROLLING PLANT DISEASES Organisms including yeast, bacteria, fungi, actinomycetes, and avirulent pathogens have been reported to control disease (1). Although nonculturable organisms may suppress disease, culturable organisms are most frequently studied, because of their potential for commercialization and the ease with which they can be studied. Although single isolates or types of organisms have been used in biological control studies in the past, a more ecologically sound approach involving a mixture of organisms is currently being investigated by a number of groups (20–23). Scientists are evaluating mixtures based on the abilities of individuals in the mixtures to occupy unique niches (21) or to function by different mechanisms (22). One group is selecting effective mixtures, and later removing individual strains that do not contribute to the groups effectiveness rather than selecting individuals first (20). Mixtures may increase efficacy because particular organisms may mediate disease control under different conditions. MANY TYPES OF DISEASES ARE CONTROLLED BIOLOGICALLY Moderate achievements have been made using biological control for the control of foliar, fruit, stem (1,9), soilborne (1,4,24), and postharvest pathogens (25,26). Biological control has been reported for fungal (27–29), and bacterial pathogens (17,30,31), viral diseases (13), and nematodes (32). Biological control strategies have even been reported for hydroponic systems (33). Strategies for biological control differ with the type of pathogen and the infection court. BIOLOGICAL CONTROL OF PLANT DISEASES ARE MEDIATED BY A NUMBER OF DIFFERENT MECHANISMS Because biological control in field situations has been variable, there has been an emphasis on understanding

211

how biological control operates (2–4,34,35). Understanding how biological control is achieved may improve the consistency of control either by improving the mechanism or by using the biological control agent in situations where it is predicted to be most successful. Conclusive evidence for involvement of a particular factor in biological control is demonstrated by the strict correlation between the appearance of the factor and biological control (4). Testing of this strict correlation has been called ‘‘Koch’s postulates for biological control mechanisms.’’ Molecular genetic tools have been used effectively to create near isogenic strains for these tests. Antibiosis Plant pathologists use the term antibiosis to describe the production of compounds by a biological control agent that directly inhibit the growth or normal activity of a pathogen (2,4,35). These inhibitory compounds can be antibiotics or other secondary metabolites (2,35). Secondary metabolites produced by bacterial antagonists have been shown to inhibit plant pathogens in vitro and in vivo (36,37). Many different compounds produced by bacterial and fungal antagonists have been shown to be important in disease control. Some of the compounds implicated in disease control are: anthranilate, 2,4-diacetylphloroglucinol, gliotoxin, gliovirin, hydrogen cyanide, kanosmine, oomycin A, phenazines, pyoluteorin, pyrrolnitrin, and zwittermicin A (35,38,39). Production of secondary metabolites by antagonists of soilborne pathogens is regulated by environmental signals and genetic regulators (40–42). Nutrient requirements for the production of secondary metabolites are specific. Minor changes in laboratory media often result in changes in the quality and quantity of secondary metabolites produced (43). Genetic engineering has been used as an approach to improve disease control. Strains have been engineered to produce inhibitory secondary metabolites that formerly did not produce (44) increased quantities of the antibiotics to increase disease control (45). Parasitism Parasitism is an interaction involving physical contact between antagonistic microbes and plant pathogens, which reduces the pathogen’s inoculum density (46). Parasitism by the antagonist Trichoderma harzianum has been well studied in the last decade. The role of enzymes involved in parasitism by this antagonist is being researched (47). Several lines of evidence indicate that parasitism may be involved in biological control: 1) reduction in pathogen inoculum levels, 2) observation of direct contact between the antagonist and the pathogen, 3) damage to the pathogen at points of direct contact, 4) isolation of cell-wall degrading enzymes from the antagonist, and 5) demonstration that enzymes purified from the biological control agents damage the pathogen or reduce disease (4). Parasitism has been discussed widely as a mechanism by which antagonists control soilborne fungal diseases, and in some cases in the biological control of postharvest (48) and phyllosphere diseases (49).

212


Stimulation of Plant Defense Mechanisms Plant resistance to virulent pathogens can be increased by ‘‘immunizing’’ the plant through inoculation with lessvirulent or nonpathogenic fungi, bacteria, or viruses. Through chemical interactions the plant’s own defenses are stimulated to prevent disease (50). Induced resistance by biological control agents have been implicated in disease control (29,50). Nutrient Competition Nutrient competition is difficult to demonstrate because enzymes involved in nutrient use and accumulation may be important to the survival and growth of the organism (34). Iron competition is the most well-studied example of nutrient competition because siderophore production is amenable to molecular genetic analysis. Siderophores are high-affinity iron chelators, which are produced by microbes under iron-limiting conditions (51). Biologically available iron is a limiting factor in most soils. Utilization of the limited iron by antagonists that produce siderophores may effect the ability of a pathogen to cause disease by further limiting the iron available to the pathogen (51,52). Competition for other nutrients is very difficult to study. In many cases it is the mechanism that is suggested by default due to the lack of evidence for other mechanisms (4,34). FACTORS INFLUENCING DISEASE CONTROL Using the biological control pyramid as a conceptual model allows us to see that all of the components of the pyramid (host, environment, pathogen, and antagonist) can influence the success of biological control. Optimizing and integrating findings about characteristics of each component is the current challenge that scientists working on biological control face. Host An interesting discussion of the role of the host in biological control has been written (53). Plant genotype has a selective effect on the organisms which colonize the rhizosphere (54). Significantly, differences in biological control efficacy have been documented among cultivars of the same plants, suggesting that genetic variation within the host may influence biological control (53). Plant hosts have different levels of resistance to the pathogens and different levels to which they can support biological control (53). Quantitative Trait Loci (QTL) mapping has been used to identify genetic regions in tomato which are associated with increase disease suppression by biological control agents (55). In this example more than one host factor appears to be involved. Understanding how to maximize these host factors to increase biological control should lead to improvements in biological control in the future. Pathogen Just as the host can have different levels of resistance to a given pathogen, the pathogen population may also vary in both virulence and its susceptibility to biological control. Plant pathology literature is full of

examples that demonstrate that isolates of a pathogen can differ in virulence. Few studies have demonstrated variation in factors that could alter the pathogen’s amenability to biological control. For example, populations of Erwinia carotovora subsp. carotovora differ with regard to the quantity and type of siderophore that they produce (56,57). If competition for iron is the primary mechanism by which this pathogen is controlled, we can expect inconsistencies in disease control to coincide with variability in siderophore production by the pathogen (57). Pathogen insensitivity to chemicals has been documented frequently (58) and it is logical to expect that pathogens can also differ in their sensitivity to biochemicals produced by antagonists. For example, there are differences in sensitivity among strains of the take-all pathogen, Gaumannomyces graminis var tritici, to antibiotics important in biological control of this pathogen (23). Additionally, pathogens can have a direct negative impact on biological control agents and the mechanisms by which they control disease. For example, fusaric acid production by Fusarium oxysporum f. sp. radicis-lycopersici negatively impacted both the biological control agent and production of critical antibiotics by this antagonist (59). The amount of the pathogen will also impact biological control. Current dose-response models suggest that plant disease is a function of the density of the pathogen and the biological control agent (55,60). In general, biological control is more successful when populations of the pathogen are lower. Biological Control Agent(s) The population level of the biological control agent in the infection court is likewise important to achieve biological control. For years colonization of biological control agents was studied because it was assumed that increased colonization would lead to increased biological control; and in 1992 this relationship was demonstrated (61). There is a limit at which higher populations of the antagonist will no longer confer increased benefit (60). As we understand more about how biological control agents achieve biological control, items that are important to success become apparent. Many of these factors relate to the mechanism by which the agents control disease. Maximizing the timing and quantity of secondary metabolite production on plant surfaces is one promising area of research (40–42,45). Environment A large body of data demonstrates that the environment directly affects hosts and pathogens and subsequently influences disease expression. The environment also influences biological control but only a few illustrations are given here. Many factors have been shown to influence colonization of bacterial agents, which is an important characteristic of antagonists applied as seed treatments for control of root diseases (3). Soil physical and chemical properties also have been shown to influence the ecology of biological control agents and in some cases disease control (62,63). In an innovative study, soil minerals have been shown to influence the production of critical compounds by an antagonist (59).


INTEGRATING WHAT WE KNOW It is clear that improvements in the efficacy of biological control are needed for it to become commercially viable. To date, work to improve biological control has centered on improving microorganisms released in an inundative approach. The effect of cropping practices on beneficial microorganisms has received less attention (64). Currently there are a number of laboratories with renewed interest in integrated cropping systems as an approach to enhance biological control. Some laboratories are concentrating on optimizing organic amendments as nutritional bases for introduced microorganisms (65), while others are concentrating on developing new cropping systems involving biological agents (66–68). Studies by these laboratories should help develop optimal environmental, host, and antagonist combinations that can be delivered to agriculturists. An integrated approach along with an understanding of the pathogen population being targeted should allow greater success of this control strategy. BIBLIOGRAPHY

213

to Crop Disease Control, CRC Press, Boca Raton, Fla., 1997, pp. 95–109. 17. A. Kerr and M. E. Tate, Microbiol. Sci. 1: 1–14 (1984). 18. D. A. Cooksey and L. W. Moore, Phytopathology 72: 919–921 (1982). 19. A. K. Vidaver, Plant Dis. 67: 471–475 (1983). 20. R. Fukui, H. Fukui, and A. M. Alvarez, Phytopathology 89: 366–373 (1999). 21. W. Janisiewicz, Phytopathology 86: 473–479 (1996). 22. C. Dunne et al., Plant Pathol. 47: 299–307 (1998). 23. M. Mazzola, D. K. Fujimoto, L. S. Thomashow, and R. J. Cook, Appl. Environ. Microbiol. 61: 2554–2559 (1995). 24. T. C. Paulitz and W. G. D. Fernando, in R. S. Utkhede and V. K. Gupta, eds., Management of Soilborne Diseases, Kalyani Publishers, India, 1996, pp. 185–217. 25. C. T. Bull, in Inderjit, K. M. M. Dakshini, and C. L. Foy, eds., Principles and Practices in Chemical Ecology, CRC Press, Boca Raton, Fla., 1999, pp. 517–530. 26. S. Droby and E. Chalutz, in C. L. Wilson and M. E. Wisniewski, eds., Biological Control of Posthavest Diseases, CRC Press, Boca Raton, Fla., 1994, pp. 63–75. 27. I. Chet and J. Inbar, Appl. Biochem. and Biotechnol. 48: 37–43 (1994).

1. R. J. Cook and K. F. Baker, The Nature and Practice of Biological Control of Plant Pathogens, APS Press, St. Paul, Minn., 1983.

28. F. N. Martin and J. E. Loper, Crit. Rev. Plant Sci. 18: 111–181 (1999).

2. D. R. Fravel, Annu. Rev. Phytopathol. 26: 75–91 (1988).

29. Y. Elad, Eur. J. Plant Pathol. 102: 719–732 (1996).

3. D. M. Weller, Annu. Rev. Phytopathol. 26: 379–407 (1988).

30. R. H. Scherff, Phytopathology 63: 400–402 (1973).

4. J. Handelsman and J. L. Parke, in T. Kosuge and E. W. Nester, eds., Plant–Microbe Interactions, vol. 3, Molecular and Genetic Perspectives, McGraw-Hill, New York, 1989, pp. 27–61.

31. K. B. Johnson and V. O. Stockwell, Annu. Rev. Phytopathol. 36: 227–248 (1998). 32. Z. A. Siddiqui and I. Mahmood, Bioresour. Technol. 69: 167–179 (1999).

5. C. von Tubeuf, Naturwissenschaftliche Zeitschrift fur ¨ Forstund Landwirtschaft 12: 11–19 (1914).

33. T. C. Paulitz, Hort. Sci. 32: 193–196 (1997).

6. J. R. Coulson et al., 110 years of biological control research and development in the United States Department of Agriculture USDA/ARS Admin Report 2000-1.

34. T. C. Paulitz, in R. R. Baker and P. E. Dunn, eds., New Directions in Biological Control: Alternatives for Suppressing Agricultural Pests and Dieseases, Alan R. Liss, New York, 1990, pp. 713–724.

7. H. L. Barnett and V. G. Lilly, Mycologia 54: 72–77 (1962). 8. R. Weindling, Phytopathology 22: 837–845 (1932).

35. J. Handelsman and E. V. Stabb, Plant Cell 8: 1855–1869 (1996).

9. J. H. Andrews, Can. J. Plant Pathol. 12: 300–307 (1990).

36. C. Keel et al., Mol. Plant–Microbe Interact. 5: 4–13 (1992).

10. G. N. Agrios, Plant Pathology, Academic Press, San Diego, Calif., 1997.

37. L. S. Thomashow, D. M. Weller, R. F. Bonsall, and L. S. Pierson, III, App. Environ. Microbiol. 56: 908–912 (1990).

11. J. E. Loper and L. J. Stowell, Biological Control of Plant Diseases, APS Press Slide Collection, APS Press, St. Paul, Minn., 1992.

38. V. Anjaiah et al., Mol. Plant–Microbe Interact. 11: 847–854 (1998).

12. E. N. Rosskopf, R. Charudattan, and J. B. Kadir, in T. S. Bellows and T. W. Fisher, eds., Handbook of Biological Control: Principles and Applications of Biological Control, Academic Press, San Diego, Calif., 1999, pp. 891–918. 13. C. M. Rush and J. L. Sherwood, in N. A. Rechcigl and J. E. Rechcigl, eds., Environmentally Safe Approaches to Crop Disease Control, CRC Press, Boca Raton, Fla., 1997, pp. 139–159. 14. K. F. Baker and R. J. Cook, Biological Control of Plant Pathogens, W.H. Freeman, San Francisco, Calif., 1974.

39. W. Howie and T. Suslow, Phytopathology 76: 1069 (1986). 40. N. Corbell and J. E. Loper, J. Bacteriol, 177: 6230–6236 (1995). 41. A. Sarniguet et al., Proc. 12255–12259 (1995).

Natl.

Acad.

Sci.

USA

92:

42. L. S. Pierson, III, D. W. Wood, and E. A. Pierson, Annu. Rev. Phytopathol. 36: 207–226 (1998). 43. D. W. James and N. I. Gutterson, Appl. Environ. Microbiol. 52: 1183–1189 (1986).

15. D. Hornby, Annu. Rev. Phytopathol. 21: 65–85 (1983).

44. A. M. Fenton et al., Appl. Environ. Microbiol. 58: 3873–3878 (1992).

16. P. A. Backman, M. Wilson, and J. F. Murphy, in N. A. Rechcigl and J. E. Rechcigl, eds., Environmentally Safe Approaches

46. R. Baker, Can. J. Plant Pathol. 9: 370–379 (1987).

45. U. Schnider et al., J. Bacteriol. 177: 5387–5392 (1995).

214

BIOLOGICAL CONTROL OF WEEDS

47. V. Lorito et al., J. Bacteriol. 178: 6382–6385 (1996). 48. C. L. Wilson et al., Crop Protection 10: 172–177 (1991). 49. J. Kranz, in J. P. Blakeman, ed., Microbial Ecology of the Phylloplane, Academic Press, London, 1981, pp. 327–352. 50. L. C. van Loon, P. A. H. M. Bakker, and C. M. J. Pieterse, Annu. Rev. Phytopathol. 36: 453–483 (1998). 51. J. E. Loper and J. S. Buyer, Mol. Plant–Microbe Interact. 4: 5–13 (1991). 52. C. A. Ishimaru and J. E. Loper, in L. L. Barton and B. C. Hemming, eds., Iron Chelation in Plants and Soil Microorganisms Systems, Academic Press, San Diego, Calif., pp. 27–73. 53. K. P. Smith and R. M. Goodman, Annu. Rev. Phytopathol. 37: 473–491 (1999).

herbicides account for 60% of the agricultural pesticides applied (1). Chemical herbicides, although effective, have problems associated with their use. There is concern over contamination of water and problems caused by overapplication and persistence in soil of many herbicides. Herbicide resistance has been noted in more than two hundred weed species (2). These factors, coupled with the banning of many pesticides, high cost of registration and stringent regulation of weed control pesticides have opened avenues for use of plant pathogens as biological control agents for weeds. This process involves the use of a pathogen to suppress or control a weed population through the intentional creation of a plant disease epidemic.

54. V. Edel, C. Steinberg, N. Gautheron, and C. Alabouvette, Phytopathology 87: 693–697 (1997).

BIOLOGICAL CONTROL STRATEGIES

55. K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96: 4786–4790 (1999).

Strategies for weed biocontrol are grouped into the classical approach, consisting of a single, inoculative introduction of the weed biological control agent; augmentative, consisting of periodic releases of the pathogen; and the inundative, or biopesticide tactic. The classical approach is primarily directed toward invasive weeds. This approach is also appropriate where cost of control is a limiting factor, such as in pasture or rangelands. Organisms used in the classic approach require little or no manipulation once they are released in the field. An example of classical biological control is the use of Puccinia chondrillina for the control of skeleton weed, Chondrilla juncea. Chondrilla juncea was a major problem in wheat-growing areas of Australia (3). The fungus was also released in the western United States, where it has been effective against this weed (4). Another rust fungus, Phragmidium violaceum, was established in Chile in 1973 and controlled Rubus constrictus effectively and R. ulmifolius to a lesser extent (3). Phragmidium violaceum acts by causing the plants to become more sensitive to frost damage. This allows for the invasion of secondary pathogens. Phragmidium violaceum was also released into Australia (5). M. J. Morris (6) has established another highly successful classic biological control program in South Africa, where the gall-forming rust, Uromycladium tepperianum, was introduced to control the invasive tree species, Acacia saligna. The fungus causes extensive gall formation on branches and twigs, accompanied by a significant energy loss. Heavily infected trees are eventually killed. The fungus was introduced from Australia into the Western Cape Province between 1987 and 1989, and within eight years the disease had become widespread and the tree density has decreased by as much as 80% in rust-established sites. A comprehensive review of agents used in the classic approach was provided by A. K. Watson (7). Inundative application of a pathogen (bioherbicide) utilizes agents in a manner that is similar to applying chemical herbicides. The active ingredient, the fungal or bacterial propagule, is formulated and applied as needed. These agents are not expected to become established, rather, target weed populations may be treated with multiple applications. Six bioherbicides have been registered in the United States, Canada, Japan, and South Africa (Table 1). DeVine , composed of Phytophthora palmivora to control Morrenia odorata

56. C. T. Bull, C. A. Ishimaru, and J. E. Loper, Appl. Environ. Microbiol. 60: 662–669 (1994). 57. C. T. Bull, Genetic Analysis of Catechol Siderophore Production by Erwinia carotovora, Oregon State University, Corvallis, Ore., 1993. 58. C. J. Delp, Plant Dis. 64: 652–657 (1980). 59. B. K. Duffy and G. Défago, Phytopathology 87: 1250–1257 (1997). 60. K. B. Johnson, Phytopathology 84: 780–784 (1994). 61. C. T. Bull, D. M. Weller, and L. S. Thomashow, Phytopathology 81: 954–959 (1991). 62. B. K. Duffy, B. H. Ownley, and D. M. Weller, Phytopathology 87: 1118–1124 (1997). 63. D. H. Kim and I. J. Misaghi, Phytopathology 86: 1238–1241 (1996). 64. B. Schippers, A. W. Bakker, and P. A. H. M. Bakker, Annu. Rev. Phytopathol. 25: 339–358 (1987). 65. H. A. J. Hoitink and M. J. Boehm, Annu. Rev. Phytopathol. 37: 427–446 (1999). 66. C. T. Bull, Proceedings of the Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, San Diego, Calif., November 1–4, 1999, pp. 7. 67. N. Kokalis-Burelle et al., Proceedings of the Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, San Diego, Calif., November 1–4, 1999, pp. 83. 68. C. D. Hoynes, J. A. Lewis, R. D. Lumsden, and G. A. Bean, J. Phytopathology 147: 175–182 (1999).

BIOLOGICAL CONTROL OF WEEDS ERIN N. ROSSKOPF USDA–ARS, USHRL Fort Pierce, Florida

Weeds are one of the most costly and limiting factors in crop production, causing losses in yield by competing with crop plants for light, water, nutrients, heat energy, carbon dioxide, and space. In the United States chemical

Table 1. A List of Registered and Unregistered Bioherbicides or Agents Under Development (17) Weed

Pathogen (Registered or Trademark Name)

Countrya

Target Crop(s)

Statusb

Abutilon theophrasti

Colletotrichum coccodes

Soybean

Canada

4∗

Acacia mearnsii

Cylindrobasidium laeve (Stumpout )

Tree plantations

South Africa

5

Aeschynomene virginica

Colletotrichum gloeosporioides f. sp. aeschynomene (Collego )

Rice and soybean

USA, Arkansas

5

Amaranthus spp.

Phomopsis amaranthicola

Vegetables

USA, Florida

3

Chenopodium album

Ascochyta caulina

Various

Holland

4

Cuscuta spp.

Alternaria destruens

Cranberries

USA, Florida

4

Cyperus spp.

Dactylaria higginsii

Various

USA, Florida

3

C. esculentus

Puccinia canaliculata (Dr. BioSedge )

Various

USA, Georgia

5∗

C. rotundus

Cercospora caricis

Various

Brazil; Israel

3

Cytisus scoparius

Fusarium tumidum

Tree plantations

New Zealand

3

Canada

3

Echinochloa spp.

Exserohilum monoceras

Rice

Eichhornia crassipes

Cercospora rodmanii

Aquatic habitats

Alternaria eichhorniae

Australia; Japan

3

Philippines–Canada

3

USA, Florida;

4∗

South Africa

3

Egypt; India; SE Asia

3

Euphorbia heterophylla

Helminthosporium sp.

Various

Brazil

3

Grass weeds

Dreschlera spp. and Exserohilum spp.

Cereals

Vietnam–Australia

2

Citrus

USA, Florida

3

Hakea sericea

Colletotrichum gloeosporioides

Tree plantations

South Africa

4

Imperata cylindrica

Colletotrichum caudatum

Various

Malaysia

1

Malva pusilla

Colletotrichum gloeosporioides f. sp. malvae (BioMal )

Various

Canada

5∗

Mikania micrantha

Cercospora mikaniicola

Plantation crops

Malaysia

2

(DeVine )

Morrenia odorata

Phytophthora palmivora

Citrus

USA, Florida

5

Poa annua

Xanthomonas campestris pv. poae (Camperico )

Turf grass

Japan

4

Pteridium aquilinum

Ascochyta pteridis

Pastures

Scotland, UK

3

Rottboellia chochinchinensis

Sporisorium ophiuri

Cereals

Thailand–UK

3

Colletotrichum sp. nov. nr. graminicola

Cereals

Thailand–UK

3

Sagittaria spp.

Rhynchosporium alismatis

Rice

Australia

2

Senecio vulgaris

Puccinia lagenophorae

Various

UK–Switzerland

4

Senna obtusifolia

Alternaria cassiae

Soybean

Brazil

3

Sesbania exaltata

Colletotrichum truncatum

Soybean and rice

USA, Mississippi

4

Solanum viarum

Ralstonia solanacearum

Citrus and sod

USA, Florida

3

Brazil–USA, Florida

2

Philippines

2

Colletotrichum spp. Sphenoclea zeylanica

Alternaria alternata f.sp. sphenocleae

Rice

Colletotrichum gloeosporioides

Rice

Malaysia

2

Striga hermonthica

Fusarium nygamai

Various

Sudan–Germany

3

Fusarium oxysporum

Cereals

West Africa–Canada

3

Fusarium semitectum var. majus

Sorghum

Sudan–Germany

3

Taraxacum officinale

Sclerotinia sclerotiorum

Lawn and garden

Canada

4

Ulex europaeus

Fusarium tumidum

Plantation crops

New Zealand

3 (continued overleaf )

215

216

BIOLOGICAL CONTROL OF WEEDS

Table 1. (Continued) Weed

Pathogen (Registered or Trademark Name)

Countrya

Target Crop(s)

Statusb

Various annual weeds

Myrothecium verrucaria

Various

USA, Maryland

3

Various broad-leaved trees

Chondrostereum purpureum

Tree plantations

Holland

4

Canada

5

(BiochonTM ) (ECOclearTM ) Various grasses; including Imperata cylindrica and Panicum maximum

Bipolaris sacchari, Dreschlera gigantea, Exserohilum longistratum, E. rostratum

Various

USA

3

Various composite weeds

Pseudomonas syringae pv. tagetis

Various

USA; Canada

4

Xanthium spp.

Colletotrichum orbiculare

Various

Australia

4∗

Note: Compiled from published and unpublished reports. a Countries joined by a hyphen are engaged in a cooperative project to develop the bioherbicide agent; countries separated by a semicolon are engaged in independent work on the said weed-pathogen system. b Status: 1 = in exploratory phase; 2 = laboratory and/or greenhouse testing underway; 3 = field trials in progress; 4 = under early commercial or practical development; 4∗ = commercial development tried but registration uncertain; 5 = available for commercial or practical use; and 5∗ = registered as a microbial herbicide but currently unavailable for use due to economic reasons.

in Florida citrus has been extremely successful. This was the first commercially available mycoherbicide (8,9). The term mycoherbicide refers to a fungal-based product that is applied inundatively for weed management. DeVine is available by special order through Abbott Laboratories (Abbott Park, IL). Collego , Colletotrichum gloeosporioides f. sp. aeschynomene, is registered for the control of northern jointvetch (Aeschynomene virginica) in rice and soybeans. Collego was developed by scientists of the University of Arkansas, the U.S. Department of Agriculture, Agricultural Research Service, and the Upjohn Company (10). Collego is now available from Encore Technologies (Minnetonka, MN). Two products based on the fungus Chondrostereum purpureum, a wound-invading pathogen of broad-leaved trees, are currently in use or under development as bioherbicides. BiochonTM is used for the control of Prunus serotina in Dutch forests and ECO-clearTM as a stump application in Canadian forests (11). A bacterial pathogen, Xanthomonas campestris pv. poae, is the most recently registered bioherbicide. Japan Tobacco, Inc. has registered this bacterial agent under the name Camperico to control annual bluegrass (Poa annua) in Bermudagrass, zoysia grass, and Kentucky bluegrass (12). A list of bioherbicides, including the presently registered agents, and those that are said to be used as unregistered, local-use agents, and experimental candidates is provided in Table 1. DELETERIOUS RHIZOBACTERIA AND WEED CONTROL Deleterious rhizobacteria are nonparasitic bacteria that colonize plant roots and reduce plant growth. These bacteria are used like fungal bioherbicides, in that they are applied inundatively, to soil before weed emergence. This method does not result in eradication of the target weed, but depresses its competitive ability (13). The use of these agents is also being pursued in Canada, where more than 2,000 isolates of rhizobacteria obtained from prairie soils are under investigation (14).

DEVELOPMENT Whether the pathogen to be used as a weed biological control agent is a bacterium or a fungus, some basic developmental steps are generally followed. Efficacy of the agent must be demonstrated and optimal environmental conditions determined. The host range of the biological control agent must be sufficiently narrow as to prevent any impact on nontarget plants. Fermentation and solid substrate methods are evaluated for production of the agent (15) and formulations are developed. A considerable amount of research has been devoted to formulation of agents. A review of these approaches is provided by S. Green (16). Additional information on weed biological control can be found in E.N. Rosskopf et al. (17). BIBLIOGRAPHY 1. A. L. Aspelin, Pesticide Industry Sales and Usage—1992 and 1993 Market Estimates, U.S. Environmental Protection Agency, Washington, D.C., 1994. 2. I. Heap, International Survey of Herbicide Resistant Weeds, Online, Internet, January 18, 2000. Available www.weedscience.com, 1999. 3. S. Hasan, in K. G. Mukerji and K. L. Garg, eds., Biocontrol of Plant Diseases, CRC Press, Boca Raton, Fla., 1988, pp. 129–151. 4. D. M. Supkoff, D. B. Joley, and J. J. Marois, J. Appl. Ecol. 25: 1089–1095 (1988). 5. E. Bruzzese and R. P. Field, in E. S. Delfosse, ed., Proceedings of the Sixth International Symposium on Biological Control of Weeds, Agriculture Canada, Ottawa, 1985, pp. 609–612. 6. M. J. Morris, Biol. Control. 10: 75–82 (1997). 7. A. K. Watson, in D. O. TeBeest, ed., Microbial Control of Weeds, Chapman & Hall, New York, 1991, pp. 2–23. 8. R. Charudattan, in M. N. Burge, ed., Fungi in Biological Control Systems, Manchester University Press, Manchester, U.K., 1989, pp. 86–110.

BIOLOGICAL CONTROL, SURVEY 9. R. Charudattan, in D. O. TeBeest, ed., Microbial Control of Weeds, Chapman & Hall, New York, 1991, pp. 24–57. 10. R. C. Bowers, Weed Science 34: 24–25 (1986). 11. S. F. Shamoun and W. E. Hintz, Proceedings of the Fourth International Bioherbicide Workshop Programme, University of Strathclyde, Glasgow, Scotland, 1998, p. 14. 12. S. Imaizumi et al., Biol. Control. 8: 7–14 (1997). 13. R. J. Kremer and A. C. Kennedy, Weed Technol. 10: 601–609 (1996). 14. S. M. Boyetchko, in K. G. Mukerji, B. P. Chamola, and R. K. Upadhyay, eds., Biotechnological Approaches in Biocontrol of Plant Pathogens, Plenum Publishing, London, 1999, pp. 73–97. 15. B. W. Churchill, in R. Charudattan and H. L. Walker, eds., Biological Control of Weeds with Plant Pathogens, John Wiley & Sons, New York, 1982, pp. 139–156. 16. S. Green et al., in G. J. Boland and D. Kuykendall, eds., Plant–Microbe Interactions and Biological Control, Marcel Dekker, New York, 1998, pp. 249–281. 17. E. N. Rosskopf, R. Charudattan, and J. B. Kadir, in T. S. Bellows and T. W. Fisher, eds., Handbook of Biological Control, Academic Press, San Diego, Calif., 1999, pp. 891–918.

BIOLOGICAL CONTROL, SURVEY LESLIE C. LEWIS Agricultural Research Service USDA, Ankeny, Iowa and Iowa State University Ames, Iowa

INTRODUCTION Biological control is the action of natural enemies in maintaining another organism’s population density at a lower level than would otherwise occur. In this article, however, the traditional definition has been expanded to include chemicals that duplicate or mimic natural ones. The major effort in biological control has been focused on the suppression of populations of pest insects. However, biological control of weeds and plant pathogens is a very active area of research. Biological control techniques also have been used successfully to suppress populations of animals other than insects. Biological control is a global endeavor; specialists in this discipline have developed a worldwide organization, International Organization for Biological Control of Noxious Animals and Plants (IOBC). More information on IOBC may be obtained by contacting: Secretary-General, G. Mathys, 1, rue Le Nôtre, F-75016 Paris. Classical biological control involves: 1) discovery, 2) importation, 3) release, and 4) establishment of exotic species of natural enemies. Most pest insects, especially in the United States, are pests because they were introduced accidentally, or on purpose, leaving their complex of natural enemies behind. For example, several thousand species of insects have been introduced into the United States in attempts to control a few species of insect pests.

217

Applied biological control is sometimes implemented when a natural enemy does not become established. This involves: 1) augmentation and 2) conservation. Augmentation is the manipulation of a natural enemy, and conservation is the manipulation of the environment, both to enhance the impact of the natural enemy on the pest population. Examples of augmentation are colonization and periodic release of natural enemies and improvement of the efficiency of a natural enemy through selection. Conservation involves, e.g., using different cultural practices that serve to protect the natural enemy in the environment. Natural enemies are: 1) parasitoids, 2) predators, and 3) pathogens. A parasitoid (parasite) is an insect that lays an egg on, or in, a single host insect. The parasitoid develops internally, killing the host. Predators are freeliving organisms that kill and consume their prey immediately or within a short period of time. Predatory insects kill and consume more than one host or prey during their development. Predators are found in all major orders of insects. Parasitoids and predators are referred to as entomophagous insects. Pathogens are microorganisms that cause diseases in insects. Most insect diseases are caused by bacteria, fungi, protozoa, microspora, and viruses. These organisms are collectively referred to as entomopathogens. The phylum Nematoda consists of a group of round worms called nematodes. Some nematodes are natural enemies of insects; some nematodes behave like parasitoids, whereas others behave like insect pathogens. These nematodes are referred to as entomophagous nematodes. In the biological control of weeds, natural enemies usually are phytophagous insects and microorganisms that cause diseases of plants. Natural enemies of plant pathogens include predacious insects that feed on the fruiting bodies of pathogenic fungi and microorganisms that are parasitic on other microorganisms.

History Several references are made to early examples of natural enemies being used to control pests. For example, in the year 1200, Chinese citrus growers purchased nests of the predacious ant, Oecophylla smaragdina, and placed them in citrus trees to control a caterpillar, Tessarotoma papillosa. Many other examples of successful biological control are recorded, including the introduction to California of the vedalia beetle, Rodolia cardinalis, in 1888 to control the cottony cushion scale, Icerya purchasi (24). This project saved the citrus industry of California and was the impetus for additional biological control projects against insect pests. Widespread adoption of host-specific biological control techniques was slowed by the development of relatively inexpensive synthetic chemical insecticides that had a broad spectrum of insect targets. These compounds were envisioned to be the permanent solution for problems with insect pests. The public concern for environmental conservation that has surfaced over the past 25 years has again stimulated interest in, and financial support for, biological control programs. During this time, intensive research has been

218

BIOLOGICAL CONTROL, SURVEY

conducted in the area of insect biochemistry; specifically, insect hormones and semiochemicals. Hormones are chemicals that are produced in certain body tissues, are transported within the body by the circulatory system, and elicit a response in another part of the body. Semiochemicals also are produced within the body but are emitted externally to elicit a response. The hormones of interest in biological control are those that regulate growth and molting of the insect; the semiochemicals are those that elicit a sexual response or attract an insect to a point source. The use of biochemicals, such as hormones and semiochemicals, is not a method of biological control in the strict sense. These chemicals, plus male-sterile techniques, host plant resistance, and genetic manipulation, are merely other biological methods used to control insects. In another recent and important development, entomologists have determined the economic threshold or economic injury levels for many important insect pests on many agricultural crops. These data allow a grower to determine how many insects can be tolerated by a crop before control is economically feasible. In developing this information, researchers have become aware that complete control or elimination of a pest is not necessary and that measures less drastic than a chemical insecticide, such as biological control, can be effective in protecting crops. This has brought about the present-day approach to pest control known as integrated pest management where a holistic approach is taken in suppressing pest populations. Briefly, this involves the assessment of a pest problem and the judicious use of two or more methods of control (parasitoids, chemical insecticides, predators, pathogens, hormones, semiochemicals, plant resistance, and cultural practices) in a compatible system that will reduce the pest population to an acceptable level. At all times, attention must be paid to using sound ecological principles so as not to disturb the beneficial regulatory factors present in nature.

Figure 1. Example of an ecosystem.

greatly disturb the host insect. Later, as the parasitoid reaches maturity, the host insect becomes immobile, at which time the parasitoid will leave the host’s body and pupate. Shortly afterward, the host usually dies. There are many variations in the parasitoids as to developmental stage of host most desirable for parasitization. Some parasitoids prefer young larvae, some older larvae, and some even the pupal stage. Other parasitoids parasitize only insect eggs. An example of a ‘‘generalized’’ hymenopteran parasitoid is Bathyplectes curculionis, imported from Italy to assist in controlling the alfalfa weevil, Hypera postica. Some parasitoids deposit several eggs in a host insect. These eggs hatch, and then at maturity several hymenopterous pupae are seen attached to the surface of the host insect. Trichogramma spp. are well-known as egg parasitoids. The females of these minute insects deposit one or more eggs within the eggs of different species of insects (Fig. 2). There is sufficient nutritive material present within the egg to support the development of this small parasitoid.

BIOLOGICAL CONTROL OF INSECTS (1–24) Entomophagous Insects An ecosystem has a relationship between a host (plant, animal, or commodity), insects feeding on the host, and organisms feeding on the insects. An example of this relationship is illustrated in Figure 1. Parasitoids The great majority of parasitoids are found in the insect orders of Strepsiptera, Coleoptera, Lepidoptera, Hymenoptera, and Diptera, with by far the most being classified as Hymenoptera and Diptera. To be successful, a parasitoid should have the following attributes: 1) good searching ability, 2) high biotic potential, and 3) ability to operate effectively throughout the entire geographic range of its host. A typical female parasitoid selects a suitable host, deposits a single egg either on or within it, and leaves in search of other suitable hosts. The parasitoid egg hatches and develops within the host insect. During the early stages of development, the parasitoid does not

Figure 2. Trichogramma wasp preparing to oviposit in an insect egg. ∗ Courtesy of Gerald R. Carner, Clemson University.


Some Hymenoptera possess a biological modification known as polyembryony, the development of several individuals from a single egg. In this situation, an adult female, e.g., Macrocentrus grandii (Fig. 3), a parasitoid of the European corn borer, Ostrinia nubilalis, deposits a single egg in a second-stage corn borer larva. The primary embryonic germ divides by fission to form two secondary germs which split into a variable number of morulae, each of which develops into an embryo. Dipterous parasitoids exhibiting a general developmental cycle are represented by Winthemia quadripustulata, a parasitoid of the armyworm, Psuedaletia punctata. The adult female deposits an egg directly behind the head capsule of a suitable host. The parasitoid larva hatches from the egg and chews its way into the body tissue of the host, develops to a mature larva, chews its way out of the larva, and pupates. A dipterous parasitoid with a different biology is Blepharipa prantensis, a parasitoid of the gypsy moth, Lymantria dispar. The female parasitoid deposits its egg on the foliage where gypsy moth larvae will feed. The parasitoid is fully developed within the egg, and the slight pressure exerted by the mandibles during feeding causes the parasitoid to break out of the egg shell. Bonnetia comta (Fig. 4), a parasitoid of several cutworms, deposits eggs on host fecal material. These eggs immediately hatch, and the neonates or planidia rest on their freshly cast egg chorion waiting for their host to move. Still another unique modification is the larvipositing phenomenon exhibited by Lydella thompsoni, a parasitoid of O. nubilalis. The adult female searches for entrance holes that tunneling larvae of the European corn borer have made and deposits live larvae in the fecal material voided by the feeding corn borer larva. The parasitoid

Figure 3. Adult female Macrocentrus grandii, parasitoid of the European corn borer, Ostrinia nubilalis.

Figure 4. Adult female, Bonnetia comta, parasitoid of several cutworm larvae.

219

larvae actively search for and enter the host by chewing through the insect cuticle. These few examples of parasitoids give only a brief overview of the fascinating and sometimes complex biology of insect parasitoids. Insect Predators Predacious insects differ from parasitoids in that a predator consumes several insect forms during its life cycle. Also, in many instances, both the immature and adult stages of the insect are active predators. Most orders of insects contain at least one family of predators. The scope of this article does not allow a treatment of examples from all orders of insects; only a few predators that a home gardener or general naturalist would most likely recognize are described. The order Coleoptera (beetles) contains by far the greatest number of predacious species. The ubiquitous lady beetle (Fig. 5) has been observed by many. The female lady beetle lays its eggs in clusters in a habitat near a food source. The newly hatched larva is a voracious feeder, consuming insect eggs, aphids, and small immature insects. The adult lady beetle also is predacious, devouring a general variety of prey. However, some of these coccinelid beetles are very specific in their prey, i.e., the vedalia beetle, Rodolia cardinalis, feeds only on the cottony cushion scale, Icerya purchasi. The ground beetles are another group of ubiquitous Coleoptera that are general predators, feeding on insect eggs, caterpillars, and even the adult moths of some species. There also are many predacious aquatic beetles that prey on almost any aquatic form of life that can be captured. Dragonflies and damselflies (order Odonata) frequent country ponds in the summer. The immature forms of these insects are aquatic and are predators of pond life. The adults capture their prey in flight. Depending on the size of the predators, the prey ranges from mosquitoes to adults of the orders Lepidoptera and Hymenoptera. Another common predator found near water is the robber fly (order Diptera). The larvae feed on pest insects in the soil, whereas the adults capture flying insects in flight. Their prey consists mainly of mosquitoes and midges that are readily found in an aquatic environment. Syrphid larvae, also in the order Diptera, feed on aphids. Most members of the order Neuroptera are predators. A representative is the green lacewing. The larvae of this insect feed on aphids, mealybugs, thrips, and other small insects. These larvae also are known to prey on the eggs of

Figure 5. Adult lady beetle, Coleomegilla maculata.

220


several lepidopterans. The adult lacewings prey on aphids and mealybugs if they feed on insects at all. The praying mantid is predacious in both the immature and adult stages. These insects prey on almost any insect that they can overcome. They frequent flowers and capture insects visiting the flowers for nectar. The Chinese mantid, Tenodera aridifolia sinensis, is frequently made available commercially for the home gardener. Other Predators Insects are a main ingredient in diets of several birds, rodents, fish, amphibians, reptiles, and arachnids. For example, several birds, including the downy woodpecker, crow, robin, red-winged blackbird, purple grackle, chickadee, starling, and ring-necked pheasant, are known predators of the European corn borer (25). Entomopathogens Insect pathogens (entomopathogens) are microorganisms that cause diseases in insects. These pathogens can cause diseases in beneficial insects as well as pest insects. In this article, only diseases of pest insects will be addressed. Bacteria Entomopathogenic bacteria can be divided into three groups—those that fit the description of classical biological control (see Chap. 13.2), those that fit the description of applied biological control, and those that fit into either group. Regardless of the category in which these bacteria are classified, they have one thing in common, they can be grown in an insect and/or in a bacteriological medium. The most prominent bacteria in biological control are in the genus Bacillus, the group of gram-positive, mobile, spore-forming rods. Bacillus thuringiensis is the most widely known and researched bacterium within this group and is differentiated from other spore-forming bacilli by the presence of a parasporal body that is formed within the sporangium during sporogenesis. The parasporal body is a high-molecular-mass protein crystal that is referred to as crystalline protein, δ-endotoxin, as well as a parasporal body. This protein moiety possesses some of the insecticidal properties of the bacterium (26). Certain subspecies of B. thuringiensis under specific growing conditions produce several other toxins with insecticidal activity: α-exotoxin, β-exotoxin, and γ -exotoxin. The δendotoxin, however, is the most important toxin relative to insecticidal activity and thus is the one most studied. The δ-endotoxin is produced commercially in submerged culture under the conditions described in the patent (27). Fermentation medium is crucial to successful production. It primarily contains an energy source (carbohydrate) and a nitrogen source. Common sources of carbohydrate are hydrolyzed corn products, starch, dextrose, and molasses. Sources of nitrogen are fishmeals, cottonseed flour, corn steep liquor, soybeans, autolyzed yeast, and casein. Trace minerals, namely Mg2+ , Mn2+ , Fe2+ , Zn2+ , and Ca2+ , are usually added to the medium. Bacillus thuringiensis is primarily a pathogen of lepidopterans. A general infection cycle is illustrated

Figure 6. Infection cycle of the bacterium Bacillus thuringiensis.

in Figure 6. To elicit its effect, the crystalline protein must be consumed by a susceptible insect larva. The protein crystal is actually a protoxin that is hydrolyzed by enzymes in the gut of susceptible insects, releasing the pure toxin. The toxin causes paralysis of the gut. The insect either starves to death, or the midgut epithelial cells are damaged, allowing the gut contents, including B. thuringiensis spores, to enter the hemocoel. At this time, a general infection occurs. Most Lepidoptera are susceptible to the δ-endotoxin, but there are basic differences in the pathological responses of insects to this toxin. Researchers have divided lepidopterous larvae into four groups according to response (28). The first group, designed Type I, shows a rapid general gut paralysis resulting in death 1–7 h after ingestion of the crystal toxin. Type II larvae are characterized by midgut paralysis a few minutes after ingestion of crystals and by cessation of feeding, but not by general paralysis. Type III larvae have to ingest both spores and crystals, do not exhibit either general or gut paralysis, but do stop feeding. Affected larvae enter a morbid state of varying duration with a fairly slow bacterial multiplication and finally, with a lethal infection. Type IV larvae are not susceptible to the crystal toxin or spores but are susceptible to the thermostable exotoxin(s). Within these categories, the definitive response is dependent on the age of the insect. For example, the European corn borer is a Type III larva responding to a combination of spores and crystals. However, pure crystals are highly toxic to neonate larvae and moderately toxic to 6-d-old larvae. A combination of spores and crystals is necessary to cause maximum larval mortality (29). Not only are the insects categorized relative to their response, but also isolates are divided into subspecies based on the antigenic properties of the flagella that are present during the vegetative stage of growth (30,31). Some subspecies are further divided by the serology of the crystalline protein (32). With use of one or both of these techniques, >20 subspecies of B. thuringiensis have been identified. Insects susceptible to B. thuringiensis do not necessarily respond the same to all subspecies of B. thuringiensis. Likewise, a subspecies of B. thuringiensis does not have the same virulency to all susceptible insects. A bioassay


technique is used to quantify the toxicity or potency of the many isolates of the several subspecies. Bacillus thuringiensis subspecies israelensis was isolated from a mosquito breeding site in Israel (33). The discovery of this subspecies provided researchers with an isolate with consistently high toxicity against larvae of mosquitoes and blackflies (order Diptera). All other subspecies are predominantly toxic for larvae of Lepidoptera. This subspecies also contains a parasporal crystal protein. However, this crystal is irregular in size and instead of a single crystal, there are 3–5 crystals. The pathology caused by subspecies israelensis is similar to that of other subspecies of B. thuringiensis in susceptible Lepidoptera. The pathology in black flies is most likely very similar. Bacillus thuringiensis subspecies israelensis is also produced by submerged culture. β-Exotoxin [23526-02-5], the other toxin produced by B. thuringiensis, is being emphasized as an insecticide.βExotoxin OH HOOC HO HO P O O

OH COOH

NH2

O

N

OH O

N

O

HO OH

N N

O

OH OH

β-Exotoxin is a nucleotide produced during the vegetative stage of bacterial growth (34). It is composed of adenine, ribose, glucose, and allaric acid with a phosphate group. β-Exotoxin is heat stable and is produced by subspecies thuringiensis, kenyae, morrisoni, tolworthi, and darmstadiensis. Recent work (35) has shown that these subspecies produce different quantities of exotoxin, depending on the culture medium. Also, indications are that some subspecies produce more than one exotoxin. Commercial production of β-exotoxin is carried out under patent US 3758383 (36). Bacillus popilliae causes a ‘‘milky disease’’ in some beetles in the family Scarabaeidae, but most noted in the Japanese beetle, Popillia japonica. Bacillus popilliae is eaten by the grub; the spore germinates and penetrates the gut wall. Once in the hemocoel, the bacterium readily multiplies (septicemia), giving the grub a whitish appearance visible through the cuticle, thus the name milky disease. Bacillus popilliae readily develops in larvae of the beetle, but microbiological techniques have not been developed to induce B. popilliae to sporulate in vitro; thus, all production is carried out by inoculating Japanese beetle larvae and harvesting the infected larvae. This technique has a drawback because the Japanese beetle is not easily grown in the laboratory. Bacillus sphaericus is a spore-forming rod that is readily found in nature. It has been isolated from mosquito larvae, and much research has been conducted on several strains of this bacterium that are insecticidal to several species of mosquitoes. Bacillus sphaericus does not produce a parasporal body or β-exotoxin as does B.

221

thuringiensis. However, there is a proteinaceous toxin located in the spore coat. Susceptible mosquito larvae consume the spore and partly digest the spore wall, releasing a toxin. This toxin causes feeding inhibition, swelling of the midgut cells, body tremors, and then death. Death can occur as soon as 0.5 h after consumption of the spores. Like B. thuringiensis, B. sphaericus is produced in submerged culture. Pathogens with Protozoal Characteristics

Microspora. The phylum Protozoa recently has been split into seven distinct phyla (37). This phylum contains the order Microsporida, intercellular parasites found essentially in all orders of insects, with new species being described and host records being published frequently. Pathologically, microsporidia differ greatly from other disease-causing organisms in that they generally do not cause immediate death of the host. A microsporidian infection usually is chronic. There are no specific symptoms to describe insects infected with a microsporidium. The infected insect usually is lethargic, has reduced feeding, is small, has morphological deformities, and fails to molt or pupate. Although microsporidia are widely distributed in the insect world, most microsporidia infect a single, or at most, a very few hosts. Nosema pyrausta is a microsporidium of this type and is a classical biological control agent. Nosema pyrausta predominantly infects larvae of the European corn borer, Ostrinia nubilalis. A schematic illustration of the relationship between N. pyrausta and O. nubilalis is presented in Figure 7. Nosema pyrausta has a life cycle typical of the order Microsporida. A corn borer larva eats a spore (the resting stage N. pyrausta); the spore extrudes its polar filament, injecting a sporoplasm into a midgut cell. Some development occurs in the midgut, whereas other sporoplasms migrate through the midgut into the hemocoel and eventually infect other tissues. In the European corn borer, infections predominantly take place in the Malpighian tubules and in the reproductive tissues of the female insect. At this point, two routes are possible for the microsporidium (both routes occur in all cases of infection). The infection can develop in the midgut, causing these cells and spores to slough

Figure 7. Infection cycles of the microsporidium Nosema pyrausta.

222


into the lumen and pass from the body in the fecal material. The fecal material then becomes a reservoir of N. pyrausta spores available to infect other corn borer larvae feeding in the same plant. This is known as horizontal transmission, i.e., transmission within a generation. This route readily occurs in nature and is an effective means of disease transmission (38). If the insect in which the reproductive tissues became infected was a female, the spores either are disseminated within the developing insect egg and infect the embryo during development (transovarial transmission), or they are passed on the egg shell and are consumed by the larvae at eclosion (transovum transmission). Transovarial and transovum transmission are collectively known as vertical transmission, i.e., transmission to a subsequent generation. Horizontal and vertical transmission are two very effective means of transmitting and maintaining an organism within an ecosystem. The impact of N. pyrausta occurs in several ways. If the infection is intense enough (a tremendous number of spores per insect larva), the infected insect will die. If the insect develops to the adult with this infection, the impact of N. pyrausta is elicited by a shortened life span, reduction in the number of eggs laid, and a reduction in the number of eggs hatching and developing to maturity. Vairimorpha necatrix is a microsporidium that infects a great number of insects, all being phytophagous Lepidoptera. This microsporidium also can cause an acute pathology, resulting in death of the susceptible larvae within several hours, and is an example of an applied biological control agent. If a newly hatched or relatively early stage larva consumes an excessive number of spores, the midgut cells are damaged, allowing entry of the midgut contents into the hemocoel, and the larva dies from bacterial septicemia. If the larva does not die from septicemia, the spore enters the hemocoel, and an acute infection of the fat body occurs. This infection becomes very intense, and the host usually dies before pupation or during the pupal period. The European corn borer is not as susceptible to V. necatrix as are several larvae in the family Noctuidae (cutworms, armyworms, etc.), and a few insects develop to adults. However, because of the intense infections at this time, the production of offspring is unlikely. The acute pathology caused by V. necatrix makes it improbable that any horizontal or vertical transmission will occur. If transmission does occur, it is likely by an insect larva feeding on infected cadavers, by cannibalism, or by insects feeding on foliage contaminated by infected insects that ruptured after death (horizontal transmission) (39).

Apicomplexa. The gregarines and some coccidia with potential as biological control agents are representatives of this phylum. An infection usually occurs by consumption of spores. The development cycle involves schizogeny, which increases the number of spores tremendously, destroying tissues and depleting energy reserves. This cycle takes 1–6 weeks, depending on the specific organism. Mattesia grandis, a pathogen of the boll weevil, Anthonomus grandis, is an example of this group and contributes to overwintering mortality of this

insect. Mattesia trogodermae, a pathogen of a storedgrain beetle, Trogoderma glabrum, has great potential for controlling these beetles in warehouses. Adult males can be contaminated with spores and can transmit these spores to females during copulation (40). Commercial application of pathogens with protozoal characteristics is limited because these organisms must be produced in vivo, in their respective hosts. Insect Viruses There are two general types of insect viruses, the occluded and nonoccluded viruses. The occluded-type viruses are characterized by having the infective units, the virions, enclosed by a protein capsule (occlusion body). This is the most emphasized group relative to biological control and includes the nuclear polyhedrosis viruses (NPV), the cytoplasmic polyhedrosis viruses (CPV), the granulosis viruses (GV), and the entomopox viruses or insect pox viruses. The iridescent viruses, parvoviruses and picornaviruses are representatives of the nonoccluded viruses. The occlusion body of an NPV is many-sided and encloses several virions. The virions are rod-shaped, and replication occurs within the cell nucleus. A generalized cycle, using a nuclear polyhedrosis virus, is illustrated in Figure 8. The inclusion bodies (occlusion bodies) are consumed by the insect and the proteinaceous capsule is hydrolyzed in the alkaline gut, releasing the virions. The virions enter the midgut cell nucleus, at which point they replicate and destroy the cell, or else the virions pass through the cell into the hemocoel and infect cell nuclei of other tissues, replicate, encapsulate, and rupture the cell. Insects infected with NPV exhibit sluggish retarded growth and a behavioral change characterized by moving to the top of a host plant where they die, and the integument usually ruptures. The primary tissues infected with a nuclear polyhedrosis virus are the midgut, tracheal matrix, fat body, and hypodermis. A viral infection in these tissues with eventual rupturing of the cells gives rise to a classical viral death where the internal organs have liquefied, and the cuticle of the insect is a mere ‘‘bag’’ of viral occlusion bodies. Once this ‘‘bag’’ breaks, the occlusion bodies contaminate the surface to which the

Figure 8. Infection cycle of a nuclear polyhedrosis virus.


insect was attached and become available for consumption by other susceptible insects. Insects infected with a CPV display discoloration of the midgut (visible through the integument), experience retarded growth, and eventually die. The integument does not rupture. The shape of the occlusion body varies, and several virions, icosahedral in shape, are within each occlusion body. The infection occurs within the cytoplasm of the cell. A GV infection is relatively nondescript but is generally characterized by retarded growth of the insect and a paling of color of the integument. In some infections, the occlusion bodies are passed into the hemolymph, giving a milky color. The occlusion bodies of this virus are formed within the cell nucleus and usually contain a single rod-shaped virion. Insects infected with a pox virus are sluggish and have an extended developmental stage. The occlusion bodies develop predominantly in the fat body, both in the nucleus and in the cytoplasm of the cell. The occlusion body contains many ovoid infective units. Insect viruses were believed to be host specific (only infecting a single species of insect), but research has revealed that some nuclear polyhedrosis viruses will infect more than one species. For example, the nuclear polyhedrosis virus from the alfalfa looper, Autographa californica, infects several alternate hosts (41), including some from which a virus has never been isolated, i.e., the European corn borer and the black cutworm (42,43). This aspect of a wider host range for a virus increases the feasibility of using viruses in applied biological control. Insect viruses can be produced only in respective hosts. Fungi Most entomopathogenic fungi belong to the class Deuteromycetes (Fungi Imperfecti), with some very important entomopathogenic fungi in other classes. Insects infected with a fungus exhibit general lethargy, slowed growth, cessation of feeding, and changes in coloration of the integument. The difference between fungi and other pathogens is that the former do not have to be eaten by the insect to cause disease, but instead grow through the insect’s skin. A general infection cycle for fungi is illustrated with Beauveria bassiana as an example (Fig. 9). The resting stage or conidium comes in contact with the insect cuticle. The conidium germinates and enzymatic action partly digests the cuticle, allowing the hyphae to penetrate the cuticle. The hyphae develop, forming a network of mycelia within the body cavity. Death usually occurs after this mycelial growth has spread throughout the body cavity. At this time, the mycelia grow out of the body, forming conidiophores and conidia. These formations are unique and definitive in some fungi. For example, B. bassiana conidia cover the infected insect, transforming it into a white covered mummy. This fungus is referred to as the white muscardine. Metarrhizium anisopliae turns an insect into a green-covered mummy and is called the green muscardine fungus. Both these fungi infect a wide range of insects. Nomuraea rileyi also coats insects with a green covering but is not as ‘‘fuzzy’’ as M. anisopliae. The conidia

223

Figure 9. Infection cycle of the fungus, Beauveria bassiana.

of these fungi become airborne and eventually infect other insects. Some insects exhibit a typical behavior pattern once infected. Diptera (flies) infected with Entomophthora musca climb to the top of vegetation, wrap their legs around a grass stem, for example, and die; then mycelia grow on the external surface. A similar response occurs in grasshoppers infected with Entomophaga grylli. These fungi forcibly discharge the conidia from the conidiophores. Both these fungi are members of the class Zygomycetes. The class Oomycetes contains the species Lagenidium giganteum, a pathogen of several species of mosquitoes. Entomopathogenic fungi are omnipresent; however, their effectiveness as a biological control agent is dependent on temperature, moisture, and wind. Each species has certain requirements relative to these environmental variables that must be met for the fungus to be effective. Fungi can be produced on submerged culture or on agar plates. Nematodes Nematodes (phylum Nematoda) are elongated cylindrical worms that are parasitic in animals or plants, or are freeliving in soil or water. Nematodes are of importance in the biological control of insects. Several families of nematodes have members parasitic to insects. In a recent review, nine families are listed that show potential as biological control agents (14). In this article the families Mermithidae and Steinernematidae are be mentioned as examples of insect parasitic forms. The mermithids have both aquatic and terrestrial species. Romanomermis culicivorax is an aquatic species that is parasitic to several species of mosquitoes. Romanomermis culicivorax hatches from an egg and actively seeks a mosquito larva and penetrates the larval integument by use of a stylet and enzymatic secretions. The nematode completes its development within the insect hemocoel and then leaves the host by boring through the integument. At this time, the host usually dies. Once in the aquatic environment, the nematode develops to adulthood and mates. The female deposits its eggs to continue the cycle. Mermis nigrescens, a representative of a terrestrial mermithid, is mainly a parasite of grasshoppers, but will

224


occasionally parasitize other insects, including beetles. Adult M. nigrescens live in the soil. The female climbs vegetation, lays her eggs, and returns to the soil. An insect eats an egg. Once in the gut, the egg hatches, and the nematode penetrates the midgut, enters the hemocoel, develops within for several weeks, and then leaves the host (the host dies at this time); the nematode enters the soil and develops to adulthood. Mating may or may not occur. Reproduction can take place parthenogenetically. The steinernematids are represented by Neoaplectana carpocapsae, which parasitizes terrestrial insects within several orders. Infective nematode larvae actively enter a host through the mouth, the anus, or through a spiracle, and proceed to the hemocoel, where they begin development. The steinernematids have a symbiotic relationship with a bacterium. As the N. carpocapsae larvae develop, a bacterium, Achromobacter nematophilus, is released through the anus. This bacterium creates a desirable environment in which N. carpocapsae develops. The nematodes develop to maturity and mate, and the female deposits her eggs. These eggs hatch, and the larvae develop to infective stage and then leave the host, ready to start the cycle again. Nematodes exist in nature. However, some nematodes, including both the aforementioned ones, are produced commercially and, therefore, are available as applied biological control agents. Methods have been developed for in vitro production of nematodes. A high-protein source free from bacterial contamination is a necessity (14). BIOLOGICAL CONTROL OF WEEDS The biological control of weeds theoretically involves the use of any organism that uses the weed as a food source or as a host. In general, most successful programs in biological control of weeds involve the use of phytophagous insects or insects that feed on these weeds. There are several programs throughout the world that have been successful in controlling weeds. For illustrative purposes, a few of the more prominent ones will be emphasized. Phytophagous Insects Control of the prickly pear cactus in Australia in the 1920s was one of the earliest successes. Larvae of the moth Cactoblastis cactorum were imported from Argentina and destroyed populations of this group of cacti. In the United States, these cacti have been substantially controlled by a cochineal insect, Dactylopius opuntiae, imported from Hawaii. The Klamath weed, Hypericum perforatum, was introduced to northwestern North America in the early 1900s and has been successfully suppressed by two beetles, Chrysolina hyperici and C. quadrigemina, which were imported from Europe via Australia. Musk thistle, a weed in range and pasture land in many areas, is host to a weevil, Rhinocyllus conicus, that lays its eggs in the seed head of the thistle. Rhinocyllus conicus was introduced into western Canada and into the United States (Montana, Nebraska, and more recently Iowa) and has a definite negative impact on this weed. These three plants are examples of control of terrestrial weeds. Successful control of an aquatic plant, the alligator

weed Alternanthera philoxeroides, has been demonstrated in inland waterways in Florida. Control has been obtained with a beetle, Agasicles hygrophila, and a moth larva, Vogtia malloi. Plant Pathogens The use of plant pathogens for control of weeds is a relatively new approach. Most of the emphasis has been with plant pathogenic fungi. Basic research is performed under carefully controlled conditions to assure the specificity of pathogens. Phytopthora palmivora is a fungus that is specific for the milkweed vine. Some of these plant pathogenic fungi maintain themselves in the soil and thus are excellent biological control agents. Alternaria cassiae is a fungus that shows potential for control of sicklepod. Several other fungi are being extensively researched for biological control of weeds. This discipline will have a tremendous impact on weed control in the future. Higher Animals Tansy ragwort, Senecio jacobaea, is a biennial weed containing pyrrolizidine alkaloids. These alkaloids cause liver damage in livestock that consume these weeds. However, recent research has shown that sheep are seemingly immune to this alkaloid. Therefore, they can graze this plant and keep it from producing seeds, thus reducing the plant population (44). Biological control of weeds is a very active area of research. More and more phytophagous insects and plant pathogens are being evaluated as potential biological control agents. Once the basic research has been completed, several of these organisms will most likely be used to aid in suppression of weeds. When selecting weedy plants as potential targets for biological control, one must be certain that the weed is classified as a weed in all areas of the country where it occurs. In some parts of a country, a certain plant might be a weed. However, in other locations, the plant might be beneficial as food source, habitat for wildlife, or for ground cover. The host range of phytophagous insects and plant pathogens also must be extensively researched to be absolutely sure that desirable plants that are closely related and might become an alternative host, do not exist in the country of introduction. BIOLOGICAL CONTROL OF PLANT PATHOGENS Plant pathogens are a group of pests that cause tremendous loss of plant life every year and are primarily suppressed or controlled by chemical pesticides. However, there are biological control agents that use these microorganisms as sources of food or as hosts. Macroorganisms Fungi (smuts, rusts, and mildews) are food sources for several insects, but applied biocontrol of plant pathogens by insects presently has not been encouraging. Microorganisms The complex interrelationships between plant pathogens and microorganisms that have the potential of controlling them currently are being studied. Cucumber powdery


mildew has been experimentally controlled by application of the fungus Ampelomyces quisqualis (45). Ampelomyces quisqualis is a hyperparasite that obtains nutrients from the mildew. The fungus Trichoderma harzianum has been shown to degrade plant pathogens, such as Schlerotium rolfsii, Rhizoctonia solani, and Pythium aphanidermatum. Plant nematodes, Criconemella xenoplax, have been suppressed by the fungus Hirsutella rhossiliensis. Such relationships have been known for many years and recently have received attention from researchers. Here again, researchers are faced with a very complicated biological system in which a tremendous amount of basic research must be conducted before biological control of plant pathogens can be realized to its full potential. BIBLIOGRAPHY 1. C. B. Huffaker, P. S. Messenger (ed.): Theory and Practice of Biological Control, Academic Press, New York, 1976. 2. P. Debach: Biological Control by Natural Enemies, Cambridge University Press, New York, 1974. 3. C. P. Clausen: Entomophagous Insects, McGraw-Hill, New York, 1940. 4. E. Kurstak (ed.): Microbial and Viral Pesticides, Marcel Dekker, New York, 1982.

225

20. R. J. Cook, K. F. Baker: The Nature and Practice of Biological Control of Plant Pathogens, The American Phytopathology Society, St. Paul 1983. 21. R. Van den Bosch, P. S. Messenger, A. P. Gutierrez: An Introduction to Biological Control, Plenum Publishing, New York 1982. 22. R. M. Anderson, E. U. Canning, A. E. R. Taylor, R. Muller (ed.): Parasites as Biological Control Agents, Symposia of the British Society for Parasitology, vol. 19, Cambridge University Press, Cambridge 1982. 23. H. G. Miltenburger (ed.): Safety Aspects of Baculoviruses as ¨ Forschung Biological Insecticides, Bundesministerium fur und Technologie, Bonn 1980. 24. Reference 1, F. J. Simmonds, J. M. Franz, R. I. Sailer: History of Biological Control, Chap. 2. 25. W. A. Baker, W. G. Bradley, C. A. Clark: Biological Control of the European Corn Borer in the United States, USDA Technical Bulletin No. 983, 1948, pp. 180–181. 26. T. A. Angus, Nature (London) 173: 545–546 (1954). 27. Bioferm Corp., US 3073749, 1963 (J. C. Megna). 28. Reference 6, A. M. Heimpel, T. A. Angus: Diseases Caused by Certain Sporeforming Bacteria, vol. 2, Chap. 2. 29. M. B. Mohd-Salleh, L. C. Lewis, J. Invertebr. Pathol. 39: (1982) 290–297. 30. H. deBarjac, A. Bonnefoi, Entomophaga 7: 5–31 (1962).

5. G. E. Cantwell (ed.): Insect Diseases, vol. 1 and 2, Marcel Dekker, New York, 1974.

31. H. deBarjac, A. Bonnefoi, Entomophaga 18: 5–17 (1973).

6. E. A. Steinhaus (ed.): Insect Pathology: An Advanced Treatise, vol. 1 and 2, Academic Press, New York, 1963.

32. J. Krywienczyk, H. T. Dulmage, P. G. Fast, J. Invertebr. Pathol. 31: 372–375 (1978).

7. E. W. Davidson (ed.): Pathogenesis of Invertebrate Microbial Diseases, Allanhead, Osmun Publ., Totawa, N.J., 1982.

33. L. J. Goldberg, J. Margalit, Mosq. News 37: 355–358 (1977).

8. Ullmann, 4th ed., 13: 238. 9. H. D. Burges, N. W. Hussey (ed.): Microbial Control of Insects and Mites, Academic Press, New York, 1971. 10. H. D. Burges (ed.): Microbial Control of Pests and Plant Diseases 1970–1980, Academic Press, New York, 1981.

34. J. Farkas, K. Sebesta, K. Horska, Z. Samek, et al., Collect. Czech. Chem. Commun. 34: 1118–1119 (1969). 35. M. B. Mohd-Salleh, C. C. Beegle, L. C. Lewis, J. Invertebr. Pathol. 35: 75–83 (1980). 36. International Mineral & Chemicals Corp., US 3758383, 1973 (T. R. Shieh, M. H.Rogoff). 37. N. D. Levine, J. O. Corliss, F. E. G. Cox, G. Deroux et al., J. Protozool. 27: 37–58 (1980).

11. L. A. Bulla, Jr., T. C. Cheng (ed.): Comparative Pathobiology, Biology of the Microsporidia, vol. 1, Plenum Publishing, New York, 1976.

38. L. C. Lewis, Can. Entomol. 110: 897–900 (1978).

12. K. M. Smith: Virus-Insect Relationships, Longman Group Ltd., London, 1976.

39. L. C. Lewis, R. D. Gunnarson, J. E. Cossentine, Can. Entomol. 114: 599–603 (1982).

13. D. C. Kelly, J. Gen. Virol. 63: (1982) 13.

40. T. J. Shapas, W. E. Burkholder, G. M. Boush, J. Econ. Entomol. 70: 469–474 (1977).

14. G. O. Poinar, Jr.: Nematodes for Biological Control of Insects, CRC Press, Boca Raton, Fla., 1979. 15. J. J. Menn, M. Beroza (ed.): Insect Juvenile Hormones: Chemistry and Action, Academic Press, New York, 1972. 16. D. A. Nordlund, R. L. Jones, W. J. Lewis (ed.): Semiochemicals: Their Role in Pest Control, J. Wiley & Sons, New York, 1981. 17. M. Beroza (ed.): Pest Management with Insect Sex Attractants and Other Behavior-Controlling Chemicals, Amer. Chem. Soc., Washington, D.C., 1976.

41. P. V. Vail, D. L. Jay, J. Invertebr. Pathol. 21: 198–204 (1973). 42. L. C. Lewis, R. E. Lynch, J. J. Jackson, Environ. Entomol. 6: 535–538 (1977). 43. L. C. Lewis, J. R. Adams, J. Invertebr. Pathol. 33: 253–256 (1979). 44. S. H. Sharrow, W. D. Mosher, J. Range 480–482 (1982).

Manage. 35:

45. L. Sundheim, Plant Pathol. 31: 209–214 (1982).

18. V. B. Wigglesworth: Insect Hormones, W. H. Freeman & Co., San Francisco, 1970.

FURTHER READING

19. Biological Control of Pests in China, USDA, Office of International Cooperation and Development, Scientific and Technical Exchange Division, China Program, Washington, D.C., 1982.

Adams, E. B., Line, R. F., Phytopathology 74: 745–748 (1984). Bajan, C., Bilewicz-Pawinska, T., Fedorka, A., Kmitova, K.: Report 8th International Plant Protection Congress, Sec. 5, Moscow, 1975, pp. 33–40.

226


Bari, M. A., Kaya, H. K., J. Econ. Entomol. 77: 225–229 (1984). Bay, E. C., Berg, C. O., Chapman, H. C., Legner, E. F. in C. B. Huffaker, P. S. Messenger (ed.): Theory and Practice of Biological Control, Academic Press, New York 1976, Chapter 18. Beegle, C. C., Lewis, L. C., Yamamoto, T.: Bacteria, in C. M. Ignoffo (ed.): Handbook of Naturally Occurring Pesticides: Microbial Insecticides, CRC Press, Boca Raton, Fla., (in press). Bestman, H. J., Stereoselective Synthesis of Pheromones via Phosphonium Ylides, in P. Doyle, T. Fujita (ed.): Pesticide Chemistry: Human Welfare and the Environment, Synthesis and Structure Activity Relationships, vol. 1, Oxford 1982, pp. 77–82. Bowers, W. S., Fales, H. M., Thompson, M. J., Uebel, E. C., Science 154: 1020–1021 (1966). Bowers, W. S., Ohta, T., Cleere, J. S., Marsella, P. A., Science 193: 542–547 (1976). Bowers, W. S.: Toxicology of Precocene, in J. Coats (ed.): Insecticide Mode of Action, Academic Press, New York 1982, 403–427. Brand, J. M., Young, J. C., Silverstein, R. M.: Insect Pheromones: A Critical Review of Recent Advances in Their Chemistry, Biology, and Application, Prog. Chem. Org. Nat’l. Prod. 37: 1–190 (1979). Burges, H. D., Huber, J., Croizier, G., Entomophaga 25: 341–348 (1980). Burges, H. D., Krieg, A., Luthy, P., deBarjac, H., Entomophaga 27: 225–236 (1982). Burges, H. D., J. Invertebr. Pathol. 28: 217–222 (1976). Campbell, W. C., Fisher, M. H., Stapley, E. O., Alberts-Schönberg, G. et al., Science 221: 823–828 (1983). Clausen, C. P. (ed.): Introduced Parasites and Predators of Arthropod Pests and Weeds: A World Review, Agriculture Handbook No. 480, U.S. Agriculture Research Service, USDA, Washington, D.C., 1978. Cornelius, S. J., Godfray, H. C. J., Entomophaga 29: 341–345 (1984). Cross, J. V., Plant Pathol. 33: 417–423 (1984). Reference 4, Cunningham, J. C., pp. 335–386. Dybas, R. A.: Avermectins: Their Chemistry and Pesticidal Activities, in P. Doyle, T. Fujita (ed.): Pesticide Chemistry: Human Welfare and the Environment, Synthesis and Structure Activity Relationships, vol. 1, Pergamon Press, Oxford 1982, pp. 83–90. Forrer, H. R., J. Werder: The Influence of Antagonistic Fungi on ¨ the Spore-Formation of Rust Fungi, in H. Geissbuhler (ed.): Advances in Pesticide Science, Pergamon Press, Oxford 1978, Part 2, pp. 383–388. Gaaboub, I. A., Hayes, D. K., Environ. Entomol. 13: 803–812 (1984). Gaugler, R., J. Nematol. 13: 241–249 (1981). Gotlieb, A. R., Brosseau, M. H., Watson, A. K.: Abstracts of 1984 Weed Science of America Meeting, Miami, Fla., 1984, p. 68. Haas, R., Pal, R., Bull. Entomol. Soc. Am. 30: 17–25 (1984). Hendrick, C. A.: Juvenile Hormone Analogs: Structure-Activity Relationships, in J. Coats (ed.): Insecticide Mode of Action, Academic Press, New York 1982, pp. 315–402. Henry, J. E., Onsager, J. A., Entomophaga 27: 197–201 (1982) . Henry, J. E., Ann. Rev. Entomol. 26: 49–73 (1981). Hussey, N. W., Bravenboer, L.: Control of Pests in Glasshouse Culture by the Introduction of Natural Enemies, in C. B. Huffaker (ed.): Biological Control, Plenum Publishing, New York, 1971, pp. 195–216.

Juss, A.: Natural Pesticides from the Neem Tree (Azadirachta indica), in H. Schmutterer, K. R. S. Ascher,H. Rembold (ed.): Proceedings of the First International Neem Conference, German Agency for Technical Cooperation, Eschborn, Federal Republic of Germany, 1981. Klassen, W., Ridgway, R. L., Inscoe, M.: Chemical Attractants in Integrated Pest Management Programs, in A. F. Kydonieus,M. Beroza (ed.): Insect Suppression with Controlled Release Pheromone Systems, vol. 1, CRC Press, Boca Raton, Fla., 1982. Klun, J. A., Plimmer, J. R., Bierl-Leonhardt, B. A., Sparks A. N., J. Chem. Ecol. 6: 165–175, 177–183 (1980). Klun, J. A., Plimmer, J. R., Bierl-Leonhardt, B. A., Sparks A. N., et al., Science 204: 1328–1330 (1979). Klun, J. A.: Insect Sex Pheromones, in J. L. Hilton (ed.): Agricultural Chemicals of the Future, Proc. Beltsville Agric. Symp. 8, Rowman & Allanheld, Totowa, N.J., 1985, pp. 381–386. Ladd, T. L., Jr., Warthen, J. D., Jr., Klein, M. G., J. Econ. Entomol. 77: 903–905 (1984). Lavie, D., Glotter, E.: The Cucurbitanes, a Group of Tetracyclic Triterpenes, inProgress in the Chemistry of Organic Products, vol. 29, Springer-Verlag, Wien, New York 1971, pp. 308–362. Lynch, R. E., Klun, J. A., Leonhardt, B. A., Schwarz, M., Environ. Entomol. 13: 121–126 (1984). Lynch, R. E., Lewis, L. C., Berry, E. C., J. Econ. Entomol. 73: 4–7 (1980). Majori, G., Ali, A., J. Invertebr. Pathol. 43: 316–323 (1984). Martin, D. F., Laster, M. L., Proshold, F. I., Lindgren, P. D. et al., Environ. Entomol. 13: 701–707 (1984). McCoy, C. W., Couch, T. L., Fla. Entomol. 65: 116–126 (1982). Metcalf, R. L., Rhodes, A. M., Metcalf, R. A., Ferguson, J. et al., Environ. Entomol. 11: 931–937 (1982). Metcalf, R. L., Bull. Entomol. Soc. Am. 25: 30–35 (1979). K. Mori: Synthetic Chemistry of Insect Pheromones and Juvenile Hormones, in Recent Developments in the Chemistry of Natural Carbon Compounds, vol. 9, Publishing House of the Hungarian Academy of Sciences, Budapest 1979, pp. 11–209. Nakanishi, K.: Steroids, in K. Nakanishi, T. Goto, S. Ito, S. Natori et al.: Natural Products, Chemistry, vol. 1, New York-London 1974, p. 535. Reference 47, S. Nozoe: Mono- and Sesquiterpenes, p. 39. Pistrang, L. A., Burger, J. F., Can. Entomol. 116: 975–981 (1984). Podgwaite, J. D., Rush, P., Hall, D., Walton, G. S., J. Econ. Entomol. 77: 525–528 (1984). Raina, A. K., Klun, J. A., Science 225: 531–532 (1984). Reimann, R., Miltenburger, H. G., Entomophaga 27: 267–276 (1982). Rhodes, A. M., Metcalf, R. L., Metcalf, E. R., J. Am. Soc. Hortic. Sci. 105: 838–842 (1980). Riechert, S. E.: Spiders as Biological Control Agents, Ann. Rev. Entomol. 29: 299–320 (1984). Robertson, J. L., Kimball, R. A., Can. Entomol. 111: 1361– 1368 (1979). Robertson, J. L., Kimball, R. A., Can. Entomol. 111: 1369– 1380 (1979). Shands, W. A., Simpson, G. W., Storch, R. H., J. Econ. Entomol. 65: 799–805 (1972). Siddall J. B., Cross, A. D., Fried, J. H., J. Am. Chem. Soc. 88: 862–863 (1966). Standfast, H. A., Muller, M. J., Wilson, D. D., J. Econ. Entomol. 77: 419–421 (1984). Stengel, M., Entomophaga 27(no◦ H. S.) 105–114 (1982).

BIOMASS: SOIL MICROBIAL BIOMASS Stevens, L. M., Steinhauer, A. I., Coulson, J. R., Environ. Entomol. 4: 947–952 (1975). Stirling, G. R., Phytopathology 74: 55–60 (1984). Sundheim L., Plant Pathol. 31: 209–214 (1982). Van De Veire, M., Vacante, V., Entomophaga 29: 303–310 (1984). Wakabayashi, N., Waters, R. M.: Juvenile Hormones and Analogs, in E. D. Morgan, B. Mandava (ed.): Handbook on Natural Pesticides: Insects, vol. 1, Boca Raton, Fla., (in press). Warthen, J. D., Jr., USDA, Sci. and Educ. Admin., Agricultural Research Results, 1979, no. 1. Wisdon, C. S., Smiley, J. T., Rodriguez, E., J. Econ. Entomol. 76: 993–998 (1983). Wright, J. E., J. Econ. Entomol. 77: 1029–1032 (1984). Wyatt, I. J.: Progress Towards Biological Control Under Glass, in Jones, D. P., M. E. Solomon (ed.): Biology in Pest and Disease Control, Blackwell Scientific Publ., London 1974, pp. 293–301. Reference 4, Yearian, W. C., Young, S. Y., pp. 387–423.

BIOMAGNIFICATION Bioaccumulation of a pesticide through an ecological food chain by transfer of residues from the diet to body tissues. The tissue concentration increases at each trophic level when there is rapid uptake and slow elimination (IUPAC).

BIOMASS: SOIL MICROBIAL BIOMASS WILLIAM R. HORWATH University of California Davis, California

Soil microbial biomass is an important component of soil that regulates many processes associated with energy transfers and nutrient cycling. These functions are critical to maintaining ecosystem productivity at all levels of the food web. The soil biomass is composed of a wide range of microorganisms including viruses, bacteria, fungi, microfauna, and macrofauna. The soil microbial biomass expresses functions to take advantage of the multitude of soil niches composed of different habitats and substrates. These functions range from pathogenesis, symbiosis, and heterotrophic to chemoautotrophic activities. The soil microbial biomass is a component of the soil that is considered to be part of the active fraction. The active fraction includes the microbial biomass, recently deposited plant residues, root exudates, and easily degradable portions of the soil organic matter such as light fraction, which are thought to play a prominent role in nutrient cycling and major energy transfers (1). In most soils, the soil microbial biomass comprises about 5% of total soil carbon and about 1% of total soil nitrogen (2). The microbial biomass is most active in the surface soil where most of the recent plant and litter inputs, mainly from above and belowground production and turnover, provide substrate (food) for microbial activity. Deeper soil horizons contain less plant input, and therefore a corresponding decrease in microbial population size and activity. However, in most soils, microbial biomass is present at all soils depths to the depth of the bedrock including deep sediments of 1,000 feet or more (3).

227

The most important function of the soil microbial biomass is decomposition of organic material. During decomposition, the microbial biomass releases nutrients from plant litter and gains energy for metabolic processes. Without this function, dead plant material would accumulate and limited nutrients would be available for plant uptake. Depending on the quality of litter, the microbial biomass either utilizes the nutrients (immobilize) or if there are excess of nutrients, they become available (mineralize) for other organisms or plant uptake. In this respect, the soil microbial biomass acts as a source and sink for nutrients in the soil. For these reasons, the soil microbial biomass is the main regulator of nutrient cycling in the soil, and therefore, regulates net primary production of ecosystems. The importance of the soil microbial biomass goes beyond nutrient cycling. One of the important by-products of decomposition is the formation of stable organic matter. The soil organic matter, through its interaction with minerals, serves many functions that increase soil quality through enhancement of physical, chemical, and biological characteristics of the soil matrix. An important consequence of an increase in soil organic matter is the storage of vital plant nutrients, such as nitrogen, phosphorus, sulfur, and trace metal elements. The diversity of soil microbial biomass leads to other important biogeochemical processes, which regulate gaseous flux of carbon, nitrogen, and other nutrients. In contrast, other functions of the soil microbial biomass, such as the production of growth regulators, can serve to be detrimental or enhance plant growth. The amazing diversity of the soil microbial biomass is the foundation of a complex ecosystem component that regulates the productivity of the earth’s biomes. This article will concentrate on the soil microbial biomass, which is composed mainly of bacteria and fungi and will emphasize carbon and nitrogen cycling in soil. HABITAT OF THE SOIL MICROBIAL BIOMASS Soils are formed through the dissolution of primary minerals and subsequent reformation of secondary minerals, such as clays and sesquioxides. The secondary minerals are rich in ion exchange activity and form stable complexes with organic matter. The interaction of minerals and soil organic matter lead to the formation of soil structure through the creation of aggregates and porosity. This complex three-dimensional matrix produces a wide variety of habitats in which the soil microbial biomass exist. The three-dimensional matrix is composed of solid, liquid, and gas phases. Characteristically, a well-developed soil contains 50% solids and 50% pores. The pores in the soil matrix contain the soil solution and air. The ionic exchange capacity of the minerals act to adsorb nutrients required for the growth of the soil microbial biomass and act as a surface to exist on. The wide array of habits in soil creates a complex predator-prey interaction, which is unrivaled compared to aboveground ecosystems (Fig. 1). Soil microorganisms inhabiting larger pore areas are subject to grazing by larger organisms, such as protozoa and nematodes. However, soil organisms living in larger spaces normally have access to a greater food supply through the movement of the soil solution and exploration

228

BIOMASS: SOIL MICROBIAL BIOMASS

by plant roots. Microorganisms inhabiting smaller areas, such as capillary spaces, are protected from predation by larger organisms. However in these confined spaces, microorganisms are often subject to oxygen limitations and substrate availability. The variety of habitats is directly responsible for influencing the immense diversity of the soil microbial biomass. Through long-term adaptive strategies microorganisms have adapted to these specialized niches in the soil. The ecological theory that examines the distribution of species based on substrate availability and growth involves the concept of r and K selection (4). A soil microorganism adapted to bountiful energy and nutrient sources are designated as r-selected. Microorganisms existing under low energy and nutrient deprived conditions are termed K-selected. Selection pressures and physical environment would be unique to r- and K-selected organisms leading to diversity of function (5). The K-selected organisms would strive to produce a high growth- rate per unit of substrate because food supply would be erratic in the smaller capillary pores or protected spaces in the soil. The r-selected organisms on the other hand would put much energy into competitiveness to be able to survive the predation pressures, which exist in the larger soil pore spaces. This interesting ecological hierarchy has led to the notion of protected and nonprotected soil microorganisms (1). The concept has been widely used in simulation efforts to describe microbial activity in soil. It is believed that K-selected or protected organisms live mainly dormant existences until exposed to a new food supply. Predation pressure, substrate availability, and specialized habitats lead to selection of microorganisms with specific functions creating a complex nutrient cycle for all of the essential elements in soil. THE COMPOSITION OF THE SOIL MICROBIAL BIOMASS The soil microbial biomass is composed of a large number species that vary widely in their function. The soil microbial biomass is generally considered to be composed primarily of bacteria and fungi. Bacteria and fungi normally compose over 70% of the total soil biomass (Table 1). Faunal components make up a significant portion of the total soil biomass and also contribute to carbon cycling and other biogeochemical processes. The importance of

Figure 1. Depiction of the soil habitat showing soil structure and the interaction of organisms inhabiting various niches. The trophic level interactions depict protected and nonprotected regions of the soil matrix.

the microbial and faunal components of the soil biomass to soil processes can often lead to a contentious debate among scientists. The size of soil microbial biomass and all of its major components is described in Table 1. Because they represent the majority of the soil microbial biomass, bacteria, and fungi are considered responsible for the majority of the energy flow and nutrient cycling that occur in soil. However, the faunal component of the soil biomass is responsible for influencing the size of the soil microbial biomass through predation thus influencing their ability to process plant litter and soil organic matter. Soil fauna also increases microbial substrate availability by physically burying plant litter in the soil. Larger soil fauna are also responsible for reducing the size of plant litter thus increasing its surface area, making it more accessible to the soil microbial biomass. The functional diversity of the soil microbial biomass is required to be able to take advantage of the wide array of plant materials and habitats found in the soil. The soil represents an oligotrophic environment, becoming more nutritionally limited as the depth of soil increases. In the soil surface, soil aggregates also represent oligotrophic environments. For example, the interior of an aggregate may have extremes in pH, aeration, and redox potential. The soil has a wide variety of these niches, sometimes called hot spots, producing an array of microhabitats, which maintain the immense diversity of the soil microbial biomass.

Table 1. The Mass of Soil Biomass Components in Soil Soil Biomass Component

Tonnes Per Hectare

Bacteria Fungi Actinomycetes Protozoa Nematodes Earthworms Other fauna (collembola, mites, arthopods etc.)

1 to 2 2 to 5 1 to 2 Up to 0.5 Up to 0.2 0 to 2.5 Up to 0.5

Source: K. Killham, Soil Ecology, Cambridge University Press, New York, 1994.


DISTRIBUTION OF THE SOIL MICROBIAL BIOMASS The soil microbial biomass requires energy, nutrients, and habitat to exist. The soil horizon containing the most organic matter, nutrients, and plant influence normally contains the most soil microbial biomass. Soils are typically composed of distinct layers or horizons formed from the depositional, eluvial, and illuvial processes. The surface soil horizon, called the A horizon, is generally rich in organic matter as a result of plant litter deposition and root turnover. Eluvial and illuvial processes and plant root exploration deposits organic matter and nutrients in deeper soil horizons. Deep sediments and buried soils can also contain significant amounts of organic matter, however the bulk of the organic matter is usually located in the A horizon. Beneath the A horizon is the B horizon, which often contains appreciable amounts of eluviated clay or other amorphous minerals, depending on soil age. Beneath the B horizon is the C horizon containing unweathered parent material. Having additional or not well-defined horizons can often complicate soil horizon determination. The A horizon, because of the large organic matter input from the above- and belowground plant production, forms a friable structure because of a wider range of aggregates sizes promoting good aeration and moisture holding capacity. For these reasons, the greatest number of microorganisms exist in the A horizon (Fig. 2). There are a number of exceptions in which the number of microorganisms can be influenced by soil characteristics such as the type of mineralogy, ecosystem, and water regime. A young or highly weathered old soil may influence the number of microorganisms because of low organic matter content. An example of this type of soil is a tropical soil, which is highly weathered and contains most of its organic matter in a quickly decomposed litter layer. Conifers forests and bogs, often produce acidic soil affecting the number of microorganisms in the upper soil layers. Extreme climatic conditions produce soils with prolonged dry (arid) or wet (aquic) regimes. Arid soils can accumulate salt and/or sometimes sodium causing high electrolyte potential in the soil solution thus affecting the number of microorganisms. On the other hand, in wet climates the landscape often contains areas with frequent or sustained high water tables leading to lower aeration and thus a decrease in the activity of microbial biomass.

Number of soil microrganisms per gram soil 6

Soil depth (meters)

10 0 0.5 1

107

108

109

Soil horizon A B C

1.5 2

Figure 2. Numbers of soil microorganisms shown as a function of soil depth.

229

The rhizosphere effect or plant root effect dramatically affects the number of microorganisms distributed throughout the soil profile. In the vicinity of plant roots, high microbial activity exists due to increased deposition of compounds, such as amino acids and carbohydrates. The rhizosphere affect exists wherever plant roots explore different soil horizons. The rhizosphere effect is temporary and is maintained as long as the plant root is alive and immediately following its death during decomposition processes. METHODS TO ASSESS MICROBIAL BIOMASS IN SOIL The size of the soil microbial biomass is primarily dependent on soil type and ecosystems productivity. Finer textured soils and soils with more silt and clay tend to have a larger soil microbial biomass. Finer textured soils have more surface area and structure leading to greater number of niches for the soil microbial biomass to inhabit. Plant detrital input from aboveground production and root turnover regulates the size of the soil microbial biomass through substrate (food) availability. Other factors, such as soil temperature and moisture, also regulate the soil microbial biomass but manifest themselves more in regulating microbial activity and turnover. The following discussion will focus on methods to determine soil microbial biomass carbon and nitrogen. These two elements have been examined extensively especially in their relationship to nutrient cycling in soil. A multitude of other methods exist to describe specific microbial components. Collection of Soil Samples The appropriate selection of soil samples dictates the kind of information needed to assess soil microbial biomass size and activity. Soils are not uniform and often vary dramatically on scales of less than a meter. Table 2 lists the physical, chemical, and biological factors, which influence the distribution in size of the soil microbial biomass. Table 2. Soil, Environmental, and Organismal Factors Affecting the Distribution and Activity of the Soil Microbial Biomass Soil Factor

Environmental Factor

Mineralogy Parent material

Rainfall Temperature

Soil age Topography

Rain shadow Exposure (north vs. south) Elevation Landscape depression Water springs History of burning

Soil pH Water-holding capacity Water infiltration Particle size (sand, silt, and clay) Bulk density (g/cc) Soil organic matter content Fertility Erosion

Cultivation Riparian influence Pollution

Organismal Factor Plant cover Net primary production Vegetation history Animal (grazing) Human influence Animal (burrowing)

230


One of the most important considerations when taking soil samples is the representativeness of the sample. Nontypical areas, such as low spots, steep slopes, eroded areas and so on should be avoided if they represent only a small fraction of the landscape of interest. Statistically speaking, soil sample collection should strive for reducing the error of the measurement to less than 20% and preferably 10% of the sample population mean. Depending on the uniformity of a landscape, 4 to 10 samples are required to determine statistical significance when comparing treatments or other experimental variables. Compositing a number of soil samples to produce up to five individual samples may often reduce the error of the measurement. Heterogeneous landscapes may require a form of geostatistics called ‘‘Kriging’’ to determine the dependence of soil properties on microbial biomass size or activity. Soil samples taken under field conditions should be stored at 4 ◦ C until analyzed. If samples cannot be immediately transported to the laboratory, they should be stored at field soil temperatures taking care to avoid exposure to direct sunlight. Soils samples should be analyzed within one week after sampling and preferably in less than three days (6). Microscopy The oldest method used to examine soil microbial biomass size is microscopic examination. Dutch merchant A. Van Leeuwenhock first saw microorganisms through a microscope in the seventeenth century. The examination of a dispersed soil suspension is the most common procedure for counting soil microorganisms with a microscope. The advent of fluorescence microscopy utilizing ultraviolet light combined with flurogenic vital stains, which fluoresce under ultraviolet light enables the observation of living microorganisms in a soil dispersion preparation (7). Many stains have been developed to assist in identifying microorganisms in the dark background of a soil dispersion. Dyes that bind DNA (acridine orange) sulfhydryl groups in protein (5-(4,6-dichlorotriazin-2-yl) aminofluorescein) and helical DNA (4,6-diamidino-2-phenylindole2-HCl) are available. Other dyes can probe metabolic activities, such as electron transfer, with the reduction of sensitive dye tetrazolium chloride. Immunofluorescent techniques using specific antigens are also available to count specific bacteria or fungal groups or species. The use of dyes and microscopic counting permits counting of both bacteria and fungi to estimate their mass in soil. Many of the methods to determine microbial biomass size and carbon and nitrogen content are calibrated using the microscopic counting techniques. The volume of carbon and nitrogen and their content in the soil microbial biomass can be estimated using microscopic counts of bacteria and fungal hyphal length. Bacteria and fungal volumes and mass can be derived using average cell lengths and diameter. The number of microorganisms and their approximate carbon and nitrogen content can be determined as shown in the formulas presented in Table 3. The carbon content of bacteria and fungi is assumed to be constant using the factors shown in Table 2. The nitrogen content of bacteria and fungi can be estimated assuming a carbon to nitrogen ratio of six to one for

Table 3. Factors Used in Estimating the Carbon and Nitrogen Content of the Soil Microbial Biomass Using Microscopy Bacterial Numbers A v W d N = nf F V D N = number of bacteria per gram soil n = bacteria per field of view A = smear of filter area F = counting field area v = volume of sample applied to smear or filter V = volume of dispersed soil d = dilution factor W = wet weight of soil D = dry weight of soil Bacterial Carbon Content B = NUeSC (10−6 ) B = bacterial biomass carbon (microgram (µg) per gram soil) N = number of bacteria per gram soil U = average bacterial volume (µm3 ) r2 L; r = bacterial radius, L = bacterial length e = bacterial density (1.1 × 10−3 ) S = bacterial solids content (0.3 in soil) C = % bacterial carbon (0.45) Fungal Biomass Carbon F = π r2 LeSC (1010 ) F = fungal biomass carbon (microgram (µg) per gram soil) r = average hyphal radius (1.13 µm) L = hyphal length cm/g soil e = density in soil (1.3) S = solids content (0.3 in soil) Source: (E. A. Paul and F. E. Clark, Soil Microbiology and Biochemistry, Academic Press, New York, 1996.)

bacteria and eight to one for fungal biomass. The carbon to nitrogen ratio for bacteria and fungi were developed from pure culture studies. The carbon to nitrogen ratio under field conditions may vary substantially depending on substrate availability and species, leading to some uncertainty in the exact estimation of microbial carbon and nitrogen. Besides estimating true carbon and nitrogen contents, other limitations of microscopic counting include the tedious nature of counting microorganisms and soil colloidal interference. These shortcomings often lead to nonstandard approaches among users of microscopic counting techniques. Chemical analysis of soil microbial biomass carbon and nitrogen can avoid some of these problems; however, it must be stressed that these methods are calibrated with the microscopic counting technique. Chloroform Fumigation Incubation Method to Estimate Soil Microbial Biomass A number of chemical techniques have been used to estimate soil microbial biomass and activity. The most popular approach to estimate soil microbial biomass is the use of chloroform vapor followed by incubation.


Other methods utilizing chloroform with direct extraction techniques and metabolic approaches have also been utilized (6). Exposing the soil microbial biomass to chloroform vapor dissolves lipids in their cell walls allowing the cytoplasmic constituents to leak into the soil. For the determination of microbial carbon and nitrogen, the soil can be incubated for 10 days following exposure to chloroform vapor and the amount of carbon dioxide and ammonium determined (8). Because not all microorganisms are killed during the chloroformfumigation procedure, the surviving biomass quickly consumes the labile components of the dead biomass mineralizing both carbon and ammonium. The fraction of the mineralized carbon and ammonium is used to estimate the original standing microbial biomass (9,10). Soil microbial biomass carbon is calculated as follows: FC − UFC BC = 0.41 Where BC is soil microbial biomass carbon, FC is the carbon dioxide produced from the chloroform treated soils after 10 days of incubation and UFC is the carbon dioxide produced in an untreated control sample during a period of 10 days following a preincubation of 10 days (8). The proportion (0.41) used to convert the measured carbon dioxide into standing microbial biomass carbon was determined using radioactively labeled microbial biomass (9). The use of the 10- to 20-day control often leads to small or negative microbial biomass carbon values leading to the following equation, which subtracts a partial control: (FC − FC K1 ) − (UFC K2 ) 0.41 Values for K1 and K2 are 0.29 and 0.23, respectively. The use of this formula strongly correlated to soil microbial biomass estimations obtained by microscopic counting (11). The preceding equation corrects for control soils having high background carbon dioxide production rates. These types of soils often have high organic matter content or recently added plant residues. The determination of soil microbial biomass nitrogen is calculated with the following equation: BC =

FN − UFN 0.54 Where BN is soil microbial biomass N, FN is the ammonium mineralized in the chloroform treated soils and UFN is the ammonium mineralized in an untreated control sample incubated for 10 days in conjunction with the chloroformfumigated sample. The proportion (0.54) used to convert the measured ammonium into standing microbial biomass nitrogen was proposed for samples with a FC to FN ratio of 700 K and Equation 5 applies otherwise. The MPBPVP version is slightly enhanced relative to the Stein and Brown method as a result of including several new groups (e.g., thiophoshorus [P = S]; quaternary ammonium) and correction factors (e.g., for amino acids and phosphates). Lyman (9) summarizes basic features and claimed accuracy of several additional methods that are broadly applicable to organic compounds. Lai et al. (10) developed a nonlinear group contribution method that seems to be the most accurate method currently available (Table 2), as indicated by the low average absolute error of only 1.3%, despite the relatively large training set (1,169 compounds). However, it is complicated, and as a result, users must obtain the Fortran-based DOS program (called NBP) from the authors to perform estimations. This certainly puts the method at a practical disadvantage, considering that MPBPVP is Windows-based, available for free download at the previously mentioned EPA website, and it requires only chemical structure entry as either Chemical Abstracts Service (CAS) number or the substance’s SMILES notation (Simplified Molecular Information and Line Entry System) (11). ESTIMATING VAPOR PRESSURE Vapor pressure is a saturation property and has many uses in exposure and risk assessment. In consumer and occupational settings where direct exposure to a substance can occur (for example, during application of a product or as a result of spills), the substance’s vapor pressure is a principal determinant of exposure via inhalation. In environmental assessment, vapor pressure serves as a crude measure of the tendency of a substance to partition from water bodies to air (i.e., to volatilize), as does boiling point. More importantly, it can be

used to estimate Henry’s Law constant, which is the relevant property that expresses this tendency. The ratio of the two saturation properties, vapor pressure and water solubility, serves as an estimate of the air/water partition coefficient KAW or dimensionless Henry’s law constant (H), which is generally assumed to reflect relative air–water partitioning tendency at the more dilute levels characteristic of environmental situations. Volatilization from soil is arguably of even greater importance than is volatilization from water for agricultural chemicals. Volatilization from soil is controlled in part by vapor pressure, with water solubility and sorption to soil components also playing a major role. In the so-called Dow method for estimating half-life for volatilization from soil, for example, this half-life is estimated by Equation 7: t 1 (in days) = 1.58 × 10−8 2

Koc S Pb

(7)

where Koc is the soil sorption coefficient normalized to organic carbon content, S is the water solubility in milligrams/liter, and Pb is the vapor pressure in millimeters of mercury. Note that the term in parentheses above is essentially the ratio of soil/water and air/water partition coefficients and thus is equivalent to an inverse air/soil partition coefficient. Finally, as noted above, vapor pressure controls adsorption and transport via atmospheric aerosols. Generally, the most accurate estimates of vapor pressure at environmentally relevant temperatures are derived when measured values exist for critical temperature and pressure, heat of vaporization, and vapor pressure at some reference temperature, such as the boiling point (for solids, the melting point is also required). However, such data are often unavailable even for relatively common substances. In principle, it is preferable in this situation to use structural information alone to estimate vapor pressure directly, rather than via the process of first estimating boiling/melting points. Indeed, methods based on group/interactive coefficients (UNIFAC) (12) and

288

CHEMICAL PROPERTIES ESTIMATION Table 3. Broadly Applicable Estimation Methods for Vapor Pressure Method

Methodology

Performance Statistics

UNIFAC (12)

Group and interactive coefficients

Training: n = 320 values (number of chemicals not stated); average difference between calculated and measured = 0.01–8.73 kPa (varies for difference chemical classes) Validation: n = 13 chemicals (number of values not stated); average difference = 0.84–13.6 kPa

Banerjee et al. (13)

Two methods: Solvatochromic parameters (method 1); UNIFAC coefficients (method 2); need melting point for both

Validation (method 1): n = 53; r2 = 0.96 Validation (method 2): n = 118; r2 = 0.90

MPBPVP (74)

Antoine and modified Grain methods; from estimated boiling point and melting point (14)

Validation: n = 805; r2 = 0.941; sd = 0.717 log (mm Hg); me = 0.476 log (mm Hg) (using only estimated boiling point and melting point)

Mackay et al. (18)

Melting point and boiling point

Validation: n = 72 (only hydrocarbons and halocarbons); me = 0.096 ln (Pa); average percent error = 10

Abbreviations as in Table 1.

solvatochromic parameters (13) have been developed, but they are not widely used because of the limited availability of coefficients and parameter values, and much less extensive validation (Table 3). Thus, the recommended estimation methods for vapor pressure continue to be the venerable Antoine equation (Equation 8) for liquids that boil below 200 ◦ C and have vapor pressures >10−2 kPa at 25 ◦ C, and for higher boiling, less volatile substances, the Grain–Watson method (14,15). The Antoine Equation (16) is empirical and has the general form: ln

Pl Pb

=B

1 (T − C)

−

1 (Tb − C)

(8)

where Pl is the vapor pressure of the liquid at the reference temperature T, Pb is the vapor pressure at the boiling point Tb , and B and C are fitted constants. In order to apply this equation, it is necessary to know the boiling point Tb and to have values for the constants B and C. B and C can be determined from equations and lookup tables, as detailed in Sage and Sage (15). If the normal boiling temperature is not known, as is often the case, it must be estimated using one of the methods in the preceding section, or another method. The Antoine equation was developed for liquids and gases and provides an adequate fit in the range of 1–100 kPa, but it is not recommended for higher boiling substances (vapor pressures < 1 kPa). For such substances, the Grain–Watson method is preferred. This method (14,15) has a theoretical basis in the Clausius–Clapeyron equation, which places certain constraints on the shape of the P-T curve (Fig. 1). Both methods estimate vapor pressure for the liquid, or subcooled (supercooled) liquid in the case of substances that are solids at room temperature.

As indicated above, the subcooled liquid vapor pressure is the more relevant parameter for environmental assessment, but the vapor pressure of the solid can be calculated using the procedure of Grain, as detailed in Sage and Sage (15) and Lyman (14). It can also be estimated from the fugacity ratio F, which is PSS /PSL , where the numerator and denominator are the saturation solidand liquid-phase vapor pressures, respectively (17). F can be estimated from melting point after making certain assumptions: Tm F = exp −6.79 (9) T−1 where T is the temperature of interest, usually 298 K (25 ◦ C). The value of F calculated thusly for a given substance is also the same as the ratio of solid- and liquid-phase water solubilities, i.e., SSS /SSL . Again using naphthalene as an example, because naphthalene melts at 80 ◦ C (i.e., Tm = 353 K), at 25 ◦ C (T = 298 K), the value of F is 0.286 (Equation 9), which means that the solidphase vapor pressure is 0.286 times the subcooled liquid vapor pressure. The estimation program MPBPVP uses the Antoine and Grain–Watson methods, as well as a third method, that of Mackay et al. (18). The latter also estimates vapor pressure from boiling point (and melting point, for solids), using the following equation: Tb Tb − 0.803 ln ln P = −(4.4 + ln Tb ) 1.803 T−1 T Tm (10) − 6.8 T−1 The melting point term above is ignored for liquids, i.e. when Tm < T (generally an environmentally relevant temperature, such as 25 ◦ C). This equation was derived using

CHEMICAL PROPERTIES ESTIMATION

two adjustable parameters, one based on comparing predicted and measured vapor pressures for hydrocarbons and halogenated compounds. Lyman (14) has suggested that this equation should still provide reasonable estimates for other classes of chemicals, but because it has not been as thoroughly evaluated, MPBPVP reports the estimate but does not use it when selecting a recommended value (which for solids is the Grain–Watson estimate, and for liquids and gases is the average of Antoine and Grain–Watson). Table 3 summarizes performance statistics for the estimation methods discussed in this section. Whichever method is used, the estimator should bear in mind that accuracy can be enhanced considerably if measured values are entered for boiling point and melting point (if applicable). Further, estimation errors increase rapidly at vapor pressures below one Pascal (10−5 atm). At much lower vapor pressures, e.g., in the range of 10−3 or 10−4 Pa, which is common for organic substances, estimation accuracy may be no better than an order of magnitude.

ESTIMATING n-OCTANOL/WATER PARTITION COEFFICIENT The usefulness of the n-octanol/water partition coefficient as a measure of partitioning and correlate of other properties like water solubility has been discussed. This ratio is generally called KOW but may also be referred to as Poct , especially in the pharmaceutical literature. It is usually expressed on a logarithmic basis (i.e., as log KOW ) because measured values span 12 or more orders of magnitude, and because the logarithmic form is better suited to use in predictive equations for other properties such as those based on linear free energy relationships. The utility of log KOW has long been recognized, and as a result, many methods for estimating this parameter from chemical structure have been developed, especially in the last 10 years. It is impossible to discuss or even mention all of these in the space of this article. Table 4 lists and provides basic information on the methodological

Table 4. Broadly Applicable Estimation Methods for n-Octanol/Water Partition Coefficient Method

289

Methodology


Meylan and Howard (24); KOWWIN (75)

140 Fragments + 260 correction factors

Total: n = 11,285; r2 = 0.954; sd = 0.390; me = 0.290 Training: n = 2,430; r2 = 0.981; sd = 0.219; me = 0.161 Validation: n = 8,855; r2 = 0.95; sd = 0.427; me = 0.327

Hansch and Leo (25,26); PC-CLOGP3 (76)

Fragments + Correction factors

Total: n = 9,740a ; r2 = 0.89; sd = 0.63; me = 0.41

Hansch and Leo (25,26); CLOGP UNIX (77)


Total: n = 7,250; r2 = 0.96; sd = 0.3 (using equation: Log P = 0.914 CLOGP + 0.184)b

CLOGP for Windows v1.0; Oct 1995 (78)


Total: n = 8,942; r2 = 0.917; sd = 0.482 (using equation: Log P = 0.876 CLOGP + 0.307)

Rekker and de Kort (27)


Training: n = 1,054; r2 = 0.99 Validationc : n = 20; r2 = 0.89; sd = 0.53; me = 0.40

Niemi et al. (22)

Molecular connectivity indices (MCI) + algorithmically derived variables

Training: n = 2,039; r2 = 0.77 Validation: n = 2,037; r2 = 0.49

Klopman et al. (79)

98 Fragments + correction factors

Training: n = 1,663; r2 = 0.928; sd = 0.38

Suzuki and Kudo (80)

424 Fragments

Total: n = 1,686; me = 0.35 Validation: n = 221; me = 0.49

Ghose et al. (81)

110 Fragments

Training: n = 830; r2 = 0.93; sd = 0.47 Validation: n = 125; r2 = 0.87; sd = 0.52

Bodor and Huang (20)

Molecular orbital

Training: n = 302; r2 = 0.96; sd = 0.31; me = 0.24 Validation: n = 128; sd = 0.38

Broto and Vandycke (82)

110 Fragments

Training: n = 1,868; me ∼ 0.4

Devillers et al. (83)

66 Atomic and group contributions from Rekker

Training: n = 800; r2 = 0.96; sd = 0.387

Abbreviations as in Table 1. All standard deviations and mean errors are in log units. a Taken from the Meylan and Howard database (24,75); the difference between the entire database (11,285) and the number used (9,740) is primarily a result of compounds not handled by the PC-CLOGP program due to ‘‘missing fragments.’’ b These statistics were determined after removing large, systematically deviating compounds and other large deviant structures where the underlying difficulty is conformational (77). c Tabulation of 20 drug chemicals from Rekker et al. (84).

290


approach as well as the performance for several methods that are judged to be the most useful for broad application to organic substances. Hand calculations are still possible, but the complexity of the calculations (often involving myriad fragment coefficients and correction factors), together with the widespread availability of convenient software, have all but eliminated any real need to perform them. Research publications and reviews on log KOW estimation and applications have proliferated in concert with the estimation methods. One of the more useful recent reviews is that by Leo (19), who offers insightful commentary on the strengths and weaknesses of various approaches to log KOW estimation as well as the more familiar software. Also addressed are techniques and pitfalls of KOW measurement. Leo (19) divides log KOW estimation methods into several classes depending on the methodological approach. Methods based on the familiar atom/fragment contribution approach are well established and have a long record of successful application. In contrast, many of the more recent methods (20,21) use parameters of the entire solute molecule such as charge densities, electrostatic potential, and molecular surface area, volume, weight, and shape and, therefore, may be referred to as whole molecule methods. These methods attempt to overcome various inefficiencies of the fragment contribution approach (oversimplification of steric and conformational effects in complex structures; the need for numerous correction factors; the inability to estimate log KOW for uncorrelated fragments). However, despite promising early results, they still have not been applied to a sufficient number and variety of substances to prove their merit as generally applicable estimation methods. The same can be said of methods based on graph-theoretic variables calculated from molecular structure, such as molecular connectivity indices (22), which can be thought of as a subset of whole molecule methods. Another approach is to calculate log KOW by extension from a measured value for a structurally similar substance. In principle, this approach is preferable to the others because method errors should be minimized when the estimated structure is very close to the measured one, but of course a measured value for a related substance must be available. Fujita et al. (23) first proposed this approach. Experimentally adjusted estimates can be done with the KOWWIN program (see below), but the closest analog has to be selected by the user. Leo (19) further divides atom/fragment contribution methods into ‘‘reductionist’’ versus ‘‘constructionist’’ approaches. The chief distinction is that in the former, fragment coefficients and correction factors are derived statistically, that is, by multiple regression using a database of measured log KOW values, including (preferably) many substances containing the given fragment; whereas in the latter, coefficients and factors are evaluated from carefully measured log KOW values for the simplest compounds in which a given fragment occurs. The suggestion is that constructionist methods are more clearly based on chemical fundamentals. In truth, however, there has been a major infusion of sound chemical reasoning in the development of reductionist methods as well, despite

the implication that statistics rule willy nilly. Two methods have emerged as the most frequently used, with the Meylan and Howard (24) method as the front runner on the reductionist side, and that of Hansch and Leo (25,26) as the only constructionist method in widespread use. The method of Rekker and de Kort (27) has also developed and maintained a steady clientele. Validation statistics (Table 4) show that all three methods just mentioned perform very well for their respective validation sets, but the same can be said of several other methods. Table 5 presents salient information on computer software available for estimating log KOW . The Meylan and Howard method (24) is implemented in LOGKOW (for MS-DOS) and KOWWIN (for Microsoft Windows), and the Hansch and Leo method is implemented in the various forms of CLOGP. Whereas accuracy is certainly an important objective in estimating log KOW as it is with other chemical properties, it is not the only consideration. Many applications do not demand highly accurate log KOW values; in any case, statistics do not really provide an unambiguous measure of accuracy, because there is no standard validation set of substances and agreed measured values. In choosing software, other factors may be as if not more important. Foremost among these are price; operating platform (e.g., UNIX workstation versus Windows-based PC); means of entering chemical structure; coverage relative to the universe of organic (and other) chemical substances; and convenience features. An example of a coverage issue is the inability of CLOGP programs to estimate log KOW for substances with ‘‘missing fragments,’’ which are still common (a generic shortcoming of the ‘‘constructionist’’ approach). KOWWIN does not have this limitation. As examples of convenience features, CLOGP and KOWWIN both have associated databases containing thousands of SMILES notations, plus measured log KOW values for many of these substances. SMILES (11) seems to have emerged as the gold standard for structure entry, replacing Wiswesser Line Notation and other methods, and it is used by many programs including KOWWIN and CLOGP. Free online log KOW calculators are available for both CLOGP and LOGKOW, at the following websites: CLOGP: http://www.daylight.com/daycgi/clogp LOGKOW: http://esc.syrres.com/interkow/kowdemo. htm The LOGKOW program is also available for free download, as part of the EPI Suite (http://www.epa.gov/opptintr/exposure/docs/episuite.htm). ESTIMATING WATER SOLUBILITY The relationship of water solubility to air/water partition coefficient (Henry’s Law constant) has been described, but its importance goes well beyond this. It is a direct measure of the tendency for water to exclude a substance, i.e., the hydrophobicity of the substance. It therefore plays a critical role in such processes as scavenging of atmospheric substances by precipitation (i.e., washout);


291

Table 5. Computer Software for Estimating n-Octanol/Water Partition Coefficient Software

Estimation Method

Comments

LOGKOW; KOWWIN (75)

Meylan and Howard (24)

MS-DOS and MS-Windows operating systems; database of 11,295 experimental log KOW values; structure entry via SMILES (11) notation; database of 60,000 SMILES notations indexed by CAS number; MS-Windows version integrates with other commercial drawing programs and SMILES depiction programs; can import a variety of scientific file formats such as MOLFILEs

PC-CLOGP (76)

Hansch and Leo (25,26)

MS-DOS operating system; structure entry via SMILES notation; database of 19,900 SMILES notations indexed by CAS number

CLOGP (85)


UNIX operating system; older versions available for the VAX operating system; database of experimental log KOW values; structure entry via SMILES notation

MacLOGP (86)


Apple Macintosh operating system; database of 9,000 experimental log KOW values; structure entry via SMILES notation

CLOGP for Windows (78)


MS-Windows operating system; database of 9,000 experimental log KOW values; structure entry via SMILES notation

ACD/LogP (87)

Proprietary fragment constant method

MS-Windows operating systems; database of 5,000 experimental log KOW values for 3,600 compounds; structure entry via drawing module or MOLFILE import

ProLogP (88)

Broto and Vandycke (82); Rekker and de Kort (27)

MS-DOS operating system; structure entry via graphic drawing module, MolFile, or MolNote

ATOMLOGP (89)

Ghose et al. (81)

MS-DOS operating system; database of 4,500 experimental log KOW values; structure entry via SMILES notation

AUTOLOGP

Devillers et al. (83)

IBM-PC

ChemLogP

Suzuki and Kudo (80)

IBM-PC or Power Mac

dissolution of spilled substances in aquatic and terrestrial environments; rate of transport of a substance to and via ground water; uptake and tissue distribution of ingested substances in occupational, consumer and environmental settings; and so forth. Sparingly soluble substances also tend to partition into solid phases and biota, and as a result, water solubility has been successfully correlated with partition coefficients for these other phases, e.g., soil sorption coefficient (Koc ) and bioconcentration factor (BCF) for aquatic organisms. Last, solubility has indirect effects on transformation processes like microbial degradation and hydrolysis, by limiting availability of the substance for these reactions. Although not as voluminous as for log KOW , the literature on and the number of published estimation methods for water solubility are large. The majority of the latter are regression equations that relate solubility and log KOW for various chemical classes and for organic substances as a whole. This is especially true of the older literature. In contrast, recent activity in this field has tended to emphasize other approaches, but especially fragment contribution methods. Table 6 lists some of these and provides salient information on methodology and accuracy. In addition, Yalkowsky and Banerjee (1992; see ‘‘Suggestions for Further Reading’’) have published a book entirely devoted to the topic of water solubility, and the review by Mackay (28) offers a relatively concise

overview with emphasis on the thermodynamic foundation of this property. Yalkowsky and Banerjee (1992) concluded that the most practical means of estimating water solubility for structurally diverse organic substances was by regressionderived correlation with log KOW (including melting point for solids). This reference is somewhat dated, but the recommendation of broadly applicable log KOW based methods is still valid. The general-use equation recommended by Mackay (28) is: log S = −1.25 log KOW − 0.01(Tm − 298) + 1.10

(11)

The EPI Suite (http://www.epa.gov/opptintr/exposure/ docs/episuite.htm) contains a program called WS/KOW that estimates water solubility from log KOW , melting point (only required for solids), molecular weight, and a series of correction factors (Table 6). The other method recommended by Mackay (28) is called AQUAFAC (Aqueous Functional Group Activity Coefficients), and as the name implies, it estimates solubility using a group contribution approach. This method has undergone considerable development and improvement since the Yalkowsky and Banerjee book. It is still more limited in scope than are KOW -based regressions, but it now has sufficiently broad applicability to warrant recommendation. Several other fragment contribution methods are also included in Table 6. Based on published statistics, these methods

292

CHEMICAL PROPERTIES ESTIMATION Table 6. Broadly Applicable Estimation Methods for Water Solubility Method

Methodology


Meylan et al. (90); WS/KOW (91)

Log KOW + MP + MW + 15 correction factors

Training: n = 1,451; r2 = 0.970; sd = 0.409; me = 0.313 Validation: n = 817; r2 = 0.902; sd = 0.615; me = 0.480 Validation set Ia : n = 85; r2 = 0.865; sd = 0.961; me = 0.714

PCCHEM (92)

Three equations: one for log KOW > 0.5; one for log KOW < 0.5; and one for organic acids. All equations include log KOW + MP

Validation in Meylan et al. (90): n = 1,373b ; r2 = 0.940; sd = 0.553; me = 0.417 Validation set Ia : n = 85; r2 = 0.768; sd = 1.263; me = 0.942

AQUAFAC (93)

44 Group parameters for aqueous activity coefficients

Training: n = 970; me = 0.45 log units (tenfold cross validation)

Banerjee (94)

UNIFAC—Activity coefficient—temp. dependent term

Training: n = 549 (113 chemicals); r2 = 0.92; me = 0.42 log units

Kuhne et al. (95)

49 Groups; 6 correction terms; two MP terms

Training: n = 694 (351 liquids; 343 solids); r2 = 0.95; average absolute error = 0.38 log (mol/L)

¨ Schu¨ urman (unpublished)

35 Fragments and 205 correction factors, plus MPc

Training: n = 1,668; r2 = 0.922; sd = 0.589; me = 0.40 Validation: n = 781; r2 = 0.851; sd = 0.865; me = 0.58

SRC Group Contribution Water Solubility (96)

125 Fragments and 32 correction factors; no MP or MW

Training: n = 1,000; r2 = 0.975; sd = 0.336; me = 0.28 Validation: n = 3,923; r2 = 0.860, sd = 0.869; me = 0.70

Klopman et al. (97)

33 Fragments

Training: n = 483; r2 = 0.947; sd = 0.528 Validation I: n = 483; r2 = 0.953; sd = 0.546 (ten fold cross validation) Validation II: n = 21; sd = 1.25 log units (independent validation set)

Abbreviations as in Table 1. All standard deviations and mean errors are in log units. a Same 85 chemicals as Validation Set I from Meylan et al. (90); these chemicals had measured log KOW values but lacked data on melting point or melted with decomposition. b Same chemicals as in the 1,451 training set of Meylan et al. (90) except that log KOW values for 77 compounds could not be estimated by CLOGP. c Similar statistics with the use of MW.

appear promising, but in general, they suffer from inadequate validation and there is insufficient experience with their application. The same can be said of estimation methods based on molecular connectivity indices and linear solvation energy (‘‘solvatochromic’’) parameters, which Mackay (28) suggests may still be worthy of consideration for investigators familiar with their complexities. Lyman (14) offers an important cautionary message, and it is that the intended applications of solubility estimates may imply different conditions from those assumed by the estimation methods. The most obvious effect is temperature. Estimation methods generally calculate solubility in pure water at 25 ◦ C, but water solubility is influenced by temperature, and temperatures in rivers, lakes, ground waters, and the like can cover a very wide range. Water solubility also tends to decrease with increasing salinity via a salting out effect. Decreases in water solubility with decreasing temperature

and increasing salinity (within environmentally relevant ranges) are usually relatively small, but nevertheless may be significant. Moreover, dissolved and colloidal organic matter can increase apparent solubility, especially for substances that tend to associate strongly with these materials. ESTIMATING HENRY’S LAW CONSTANT Henry’s Law constant (H; sometimes Hc or HLC) is an expression of the air/water partitioning behavior of a chemical substance. It is defined as the ratio of a chemical’s concentration in air to its concentration in water, when these two phases are in contact and equilibrium distribution of the chemical is achieved. H is, therefore, a partition coefficient, and this is more explicit when H is expressed as KAW , the air/water partition


coefficient, as in the earlier section on partitioning properties. Substances with high values of KAW tend to be distributed to air in multiphase systems (i.e., they evaporate or volatilize), whereas substances with low values tend to remain in soil and water or be washed out of the atmosphere if released to air. Half-lives for volatilization from soil and water are strongly influenced by KAW , but they are actually a complex function of both chemical and system properties. System properties include water depth, turbulence, presence of modifying materials such as adsorbents and surface-active substances, soil properties (e.g., moisture content), and so forth, and they are not treated here. Despite their age, the reviews by Thomas (29,30) in Lyman’s handbook (see ‘‘Suggestions for Further Reading’’) are excellent overviews of volatilization processes and the many variables that affect rates. It is common to express H as the ratio of vapor pressure and solubility: P (12) H= S However, this is not a definition of H. It is more accurate to view the vapor pressure/solubility ratio as an estimation method derived from theory, but subject to certain limitations that are often (but should not be) ignored. Equation 12 only applies when the concentration in water is fairly low, and it is not applicable to substances

that are miscible in water such as ethanol. As an estimation method, the vapor pressure/water solubility ratio is clearly the preferred approach for sparingly soluble substances, but only if measured values are available for the two input parameters. Because both properties (but especially vapor pressure) are temperature sensitive, the two values must have been measured at or corrected to the same temperature, and they must be for the same physical state, solid or liquid, depending on the melting point. Attention must also be paid to units, because there are several different ways of expressing both vapor pressure and water solubility. This method is not recommended for chemicals soluble in water to more than several percent; according to Mackay et al. (31), results for substances with solubilities in the range of 1–10% should be viewed with caution. Other methods for estimating H are available if the conditions for using the vapor pressure/solubility ratio are not met. These are listed in Table 7, which also includes basic information on methodology and performance. Information in Table 7 is largely from the comprehensive review by Staudinger and Roberts (32). Hine and Mookerjee (33) developed separate models using bond and group contribution approaches, which were successful but are now somewhat dated. Considerably later, Meylan and Howard (34) updated and expanded on the Hine and Mookerjee work, and these newer models

Table 7. Broadly Applicable Estimation Methods for Henry’s Law Constanta Method

293

Methodology


Hine and Mookerjee (33)

Two models: 34 Bond contributions (model 1); 70 group contributions (model 2)

Training: n = 255; r2 = 0.946; sd = 0.400; me = 0.26 (bond) Training: n = 215; r2 = 0.996; sd = 0.108; me = 0.080 (group)

Nirmalakhandan and Speece (36)

Two models: 2 molecular connectivity indices (MCI) plus 11 Polar factors (PF)(model 1); one MCI, 11 PF, and one hydrogen bonding term (model 2)

Training: n = 180; r2 = 0.932; sd = 0.445 (model 1) Training: n = 180; r2 = 0.976; sd = 0.261; me = 0.19 (model 2) Validation: n = 20; r2 = 0.820; sd = 0.332; me = 0.27 (only considered alcohols, esters, and halogenated and non-halogenated hydrocarbons)

Meylan and Howard (34)

Two models: 59 bond contributions (model 1); 59 bond contributions + 15 correction factors (model 2)

Training: n = 345; r2 = 0.940; sd = 0.45; me = 0.30 (model 1) Training: n = 345; r2 = 0.970; sd = 0.343; me = 0.21 (model 2) Validation: n = 74; r2 = 0.965; sd = 0.460; me = 0.31

HENRY (98)

59 Bond contribution (9 revised coefficients) + 35 correction factors

Training: n = 90 (pesticides); r2 = 0.96; sd = 0.44; me = 0.34

Russell et al. (38)

2 Atomic charge terms, 2 charged surface area terms, number of heavy atoms

Training: n = 63; r2 = 0.957; sd = 0.356; me = 0.28 Validation: n = 7; r2 = 0.916; sd = 0.414; me = 0.34

Suzuki et al. (37)

31 Group contributions + one MCI

Training: n = 229; r2 = 0.984; sd = 0.220; me = 0.15

a From Staudinger and Roberts (32). Abbreviations as in Table 1. All standard deviations and mean errors are in log units.

294


have been further improved and incorporated into EPI Suite as the HENRYWIN program for Windows. Besides being broadly applicable, the bond/group contribution methods are straightforward, reasonably well validated, and available free in a convenient format. In addition to the bond/group contribution methods, several methods are available that use other molecular properties or molecular connectivity indices in regression equations for predicting H (Table 7). Brennan et al. (35) found the Meylan and Howard (34) bond contribution method and the molecular connectivity method of Nirmalakhandan and Speece (36) to have comparable accuracy, as determined by a common set of 150 diverse organics. They concluded that either method could be used with confidence. However, a potential disadvantage of the Nirmalakhandan and Speece method is the use of connectivity indices, whose physical meaning is not always obvious and whose calculation requires some effort at least initially. Other models that use connectivity indices are the works by Suzuki et al. (37) and Russell et al. (38), but these have more limited training sets and/or limited or no validation sets (Table 7). Finally, estimation based on activity coefficients (i.e., UNIFAC, Universal Functional Group Activity Coefficients) (31) and solvatochromic parameters (39) has been suggested, but these potentially powerful approaches still suffer from limited availability of necessary input data.

HA + H2 O ===⇒ H3 O+ + A−

where HA is an acidic substance and A− is the ionized species. Values of pKa can span many orders of magnitude. Basic substances seek to acquire rather than donate a proton(s), and their behavior can be expressed analogously: B + H2 O ===⇒ BH+ + OH−

(14)

where B is a base. The equilibrium constant for reaction 14 is Kb and is a measure of basicity. A practice sometimes observed is to express behavior of weak bases in terms of the acidity of the ‘‘conjugate acid,’’ which in this case is BH+ : BH+ + H2 O ===⇒ H3 O+ + B

(15)

This allows acid-base behavior to be expressed on the same basis, i.e., as pKa , for all substances that are acids or bases. However, this practice may lead to confusion if it is not made clear (often the case) which species is being described. Concentrations of the ionized and unionized forms of an acid are equal when pH = pKa , but the ratio of the two forms changes by a factor of 10 for each pH unit above or below the pKa . The exact percentages in the dissociated and undissociated forms can be calculated using the Henderson–Hasselbach equation:

ESTIMATING ACID DISSOCIATION CONSTANT (pKa ) Many substances, and certainly many agricultural chemicals, act as acids or bases at environmentally relevant pH values, and therefore may be charged or uncharged (ionized or unionized) depending on pH. This is important because charged and uncharged molecular species generally exhibit different behavior. Ionization strongly affects environmental partitioning, attributable to effects on water solubility (ionized species have higher solubility) and sorption to solids or colloidal material. Solubility also affects volatility: Ionic substances normally have negligible volatility and are often treated differently from so-called semivolatile substances by modelers. In soils that are near neutral in pH, weak acids will generally be present as anions (i.e., in the dissociated form), and this form will tend to have higher mobility and lower volatility. Weak bases will tend to be uncharged, and this form will exhibit stronger sorption to organic matter than the charged (protonated) form. Biotic and abiotic transformation reactions also may be affected by whether a substance is present in ionic or uncharged form, as can bioaccumulation and toxicity. For these reasons, there is substantial demand for methods to predict relative amounts of ionized/unionized substance as a function of pH, for substances that have not been or for whatever reason cannot be tested. The usual way to express acidity for relevant substances is via the acid dissociation constant, pKa , which is by definition the negative logarithm of the equilibrium constant for the dissociation of an acid, by the following reaction:

(13)

% dissociated = 100

10(pH−pKa ) 1 + 10(pH−pKa )

(16)

As an example, acetic acid has a pKa of 4.75, and the approximate percentages dissociated at various pH values are as follows:

pH 2.75 3.75 4.75 5.75 6.75

% Dissociated 1 10 50 90 99

Because the range of environmentally relevant pH is ca. 5–8, one can see that acetic acid is almost completely ionized at pH values around 7. It also follows that acids with pKa values of 3 or lower can be assumed to behave as charged species (anions) under nearly all environmentally relevant conditions, because they are already 99% ionized at pH 5. Dissociation is affected by ionic strength and temperature, but the effects are generally small compared with the influence of molecular structure; e.g., for typical organic acids, Ka changes by less than 10% between 5 and 60 ◦ C (40). There is a large database of measured pKa values for organic acids and bases (41–43), and this should be consulted before estimation methods are applied. Historically, the latter have mostly been in the form of linear free energy relationships (LFER) of the Hammett/Taft


295

Table 8. Broadly Applicable Estimation Methods for Acid Dissociation Constant (pKa ) Method

Performance Statisticsa

Methodology

Perrin et al. (43)

Linear free energy relationships (LFER)

Statistics vary with particular chemical classes of acids or bases

Klopman and Fercu (99)

22 fragments

Training: n = 2,464 acids Validation: n = 182; r2 = 0.88; concordance between experimental and predicted = 98.8% Validation: n = 214; r2 = 0.70; concordance between experimental and predicted = 90.5%

Hilal et al. (45): SPARC

Blend of LFER, SAR, and perturbed molecular orbital (PMO) methods

Training: n = 775 Validation I: n = 4,000 (3,500 chemicals); rmse in pKa units = 0.35 Validation II: n = 358 (214 azo dyes); rmse in pKa units = 0.62

pKalc 3.1 (44)

Hammett and Taft equations (LFER)

Statistics vary with particular chemical classes of acids or bases

Abbreviations as in Table 1.

type, in which the pKa for a target structure is calculated using regression equations from the known pKa for a parent structure (e.g., benzoic acid for a substituted benzoic acid) plus a substituent constant(s) applicable to the target structure. The monograph by Perrin et al. (43) has an extensive compilation of these data. More recently, other techniques have been applied to prediction of pKa , as summarized in Table 8. The computer program pKalc (44) uses Hammett/Taft correlations and may provide a more convenient mechanism for calculating pKa values than consulting hard copy sources like Perrin et al. (43). The SPARC (SPARC Performs Automated Reasoning in Chemistry) (45) program reportedly performs well (Table 8) and uses a novel approach that is a blend of LFER, structure/reactivity relationships (SAR), and perturbed molecular orbital (PMO) methods. The SPARC user submits a query to the University of Georgia mainframe via the Internet, after which calculations are performed and the results sent back to the submitter. SPARC’s services, which include several other properties in addition to pKa , are available free at http://ibmlc2.chem.uga.edu/sparc/. ESTIMATING VEGETATION/AIR PARTITION COEFFICIENT The vegetation/air partition coefficient KPA describes the equilibrium distribution of a chemical substance between air and aerial vegetation when the two phases are in contact with one another. The importance of uptake by the above-ground (foliar) parts of plants scarcely needs mention in the context of agrochemicals. Moreover, quantitative study of plant uptake is an integral part of efficacy and safety studies for active ingredients in pesticide formulations. But KPA is still relevant in the context of premanufacture screening, and because (as noted earlier in this article) many agricultural chemicals are not subject to the same testing requirements as apply to pesticide active ingredients. KPA is affected by temperature and by properties of both the plant and

chemical substance. The science of plant/air partitioning is young, and for this reason, estimation methods are not well advanced. There are no broadly applicable estimation methods analogous to those for BCF; yet, screening-level predictions of KPA are possible for several plant species. This is an active area of research, and the state of the field has been reviewed recently by McLachlan (46,47). Several methods for estimating KPA are available, but these are all similar. KPA is calculated from the volume fractions for various plant compartments (air, water, lipid) and either KOW and KAW or the ratio of these two substance properties, KOA . An example is the ‘‘linear method,’’ in which KPA is calculated from KOW and KAW as follows: KPA = vA +

n vL KOW vW + KAW KAW

(17)

where n is 1.0 and vA , vW , and vL are the volume fractions for cuticular air, water, and lipid. Other methods are given in McLachlan (47) and have used values of the KOW exponent slightly less than one, and different treatments of the lipid compartment. All methods give similar results. Plant/air partitioning data are currently limited to a few species, such as azalea leaves, ryegrass, and spruce needles. Volume fractions are difficult to define ¨ and vary with plant species, but the values in Muller et al. (48) may be used for screening purposes. These are for ryegrass and are vW = 0.65; vCL = 0.003; vC = 0.004; vCA = 0.078; and vP = 0.039, where the subscripts CL, C, CA, and P stand for cellular lipids, cuticle, structural carbohydrates, and protein, respectively. For purposes of applying Equation 17, vA may be set to 0.1; vL may be calculated as vCL plus vC (i.e., 0.007); and vP and vCA can be set to zero for substances with log KOW > 2. Estimation error should be expected to potentially exceed one order of magnitude. Users should also bear in mind that in any real-world situation, plant exposure may be affected by kinetically limited gaseous or particle-bound

296


deposition of an airborne substance, whereas KPA only reflects equilibrium gas phase/foliage distribution. Recently Hiatt (49) published an estimation method for foliar uptake of volatile organic compounds (VOCs) and PCBs by grass. The regression equation calculates the BCF for grass leaves based solely on a chemical substance’s octanol/air partition coefficient: Log BCF = 0.9728 log KOA − 1.517

(18)

where BCF is defined as the concentration of the substance in leaves on a dry weight basis divided by the concentration in air. The squared correlation coefficient (r2 ) of 0.99 is impressive, but the method is new and untested for substances other than VOCs and PCBs. ESTIMATING SORPTION TO AEROSOL PARTICLES Physical removal of a chemical substance from the atmosphere can occur by wet or dry deposition of gaseous or particle-borne substance. The main factors affecting such removal are known, and deposition processes and their rates therefore can be modeled with varying degrees of sophistication. These factors include the substance properties water solubility and saturation liquid-phase vapor pressure (PSL ), and system properties, chiefly temperature, surface area of particles per unit volume of air (θ ), and intensity and duration of precipitation. With respect to chemical properties, Henry’s Law determines the distribution between the gas phase and atmospheric water droplets, but for airborne particles, vapor pressure is paramount. Henry’s Law may in fact underpredict pesticide concentrations in fog droplets by several orders of magnitude (50). Substances with PSL > 10−2 Pa will exist almost entirely in the vapor phase; whereas if PSL < 10−6 Pa, a substance will be completely sorbed to particulate matter. At intermediate values, the adsorbed fraction can be estimated as shown below. The distribution of a substance between the particulate and gas phases affects not only deposition, but also the substance’s reactivity and, therefore, its propensity for long-range transport and distribution. Several models for predicting the fraction of airborne substance sorbed to particulates (ϕ) are available. The Junge–Pankow model is the most familiar of these and probably still the one most often used: ϕ=

(PSL

cθ + cθ )

(19)

where c is a factor that depends on the excess heat of desorption from the particle surface and the other symbols are as given previously. It is usually assumed that c is 17.2 Pa cm, as given by Bidleman and Harner (51). For substances that are solids at ambient temperatures, it is necessary to use the subcooled liquid vapor pressure as explained in the earlier section on partitioning properties. For screening-level calculations, general estimates of θ can be used (units of cm2 aerosol/cm3 air): 0.42 × 10−6 ; 1.5 × 10−6 ; 3.5 × 10−6 ; and 11 × 10−6 for clean continental background; average background; background plus local

sources; and urban air, respectively. The value 11 × 10−6 for urban air is roughly equivalent to 140 µg/m3 of total suspended particulates. Two alternative methods estimate the particle/gas partition coefficient, Kp , which can then be converted to a value of ϕ using Equation 20: ϕ=

Kp (TSP) [1 + Kp (TSP)]

(20)

where Kp has the units m3 /µg and TSP is the Total Suspended Particle concentration in µg/m3 . The first of the two alternative methods is the Mackay adsorption model (52), and it simplifies to a one-parameter relationship between Kp and PSL : 3 × 10−6 (21) Kp = PSL The second method (51) is based on the octanol/air partition coefficient, KOA , and with certain assumptions simplifies to Equation 22: Log Kp = log KOA − 12.61

(22)

This method is advantageous because it avoids the problem of converting solid to subcooled liquid vapor pressures, and because KOA is directly measurable at ambient temperatures. Experimental values are available for many substances. The article by Bidleman and Harner (51) gives an excellent overview of experimental techniques as well as estimation methods for ϕ and Kp . For a more detailed treatment, the reader should consult Lane (53). ESTIMATING SOIL/SEDIMENT SORPTION COEFFICIENT Sorption coefficients (Kd ) express the extent to which a substance is distributed at equilibrium between some solid phase and the aqueous phase, when the two phases are in contact with each other. In the broadest terms, the solid phase can be any solid of interest, such as in soil, sediment, suspended sediment, or even wastewater (e.g., activated sludge solids). Sorption is one of the most important processes controlling the environmental fate and distribution of agrochemicals. Sorption affects leaching (migration) of chemical substances from surface to deeper soil layers; contamination of ground water; runoff from surface soils; volatilization from soil and water; removal from the water column via suspended sediment or colloidal matter, and subsequent deposition; and availability of the substance for transformation processes like photolysis and biodegradation. Sorption occurs when there is a favorable (negative) change in free energy associated with the interaction between the chemical substance and solid phase. Sorption occurs by a wide variety of mechanisms that can be classified broadly as enthalpy- or entropy-driven. The former include van der Waals interactions, electrostatic interactions, hydrogen bonding, charge transfer, ligand exchange, direct and induced dipole–dipole interactions, and chemisorption (generally means covalent bonding).


Hydrophobic ‘‘bonding’’ (partitioning) is the chief entropydriven process. For soil, the sorption coefficient Kd is expressed as a ratio of concentrations, as follows: Kd =

[X]soil (mg/kg) [X]water (mg/L)

(23)

where X is the solute (sorbate). Generally Kd is in units of liters/kilogram. Kd is a function of soil/sediment and environmental properties as well as the molecular structure of the solute. Soil properties include pH, ionic strength (and salinity), amount/type of organic matter, content/type of clay minerals, particle size distribution and surface area of solids, and concentration of dissolved/colloidal organic matter. Temperature is important; generally, sorption decreases with increasing ambient temperature. Despite this complexity, sorption is dominated by one or two processes for many organic substances, and this makes generalizations (and estimation of sorption coefficients) possible. The sorption of most neutral organics in soils and sediment correlates with the organic matter content of the solid phase. This has enabled what Doucette (54) calls the ‘‘Koc approach’’ to predicting sorption of nonpolar organics. Koc is the soil/sediment sorption coefficient Kd after normalization to the organic carbon (oc) content of the solid phase: Kd Koc = (24) (% oc/100) In theory, if oc is the primary sorbent in soil, Koc should be independent of soil type. Obviously this greatly oversimplifies reality, but experiments do show that variation in Koc is generally much lower than is variation in Kd . Sometimes, especially in the older literature, Kom (om, organic matter) is used instead of Koc . Kom can be converted to Koc (and vice versa) using the generalization that percent organic carbon = organic matter/1.7, which implies that organic matter generally is about 60% organic carbon by weight. The Koc approach is most appropriate for nonpolar substances when the oc content of the soil/sediment is >0.1% and clay content is relatively low. Koc has been correlated with a variety of substance properties, usually as log Koc . Table 9 summarizes salient features of several broadly applicable log Koc estimation methods, but many more equations exist than are mentioned here—see Doucette (54) for a more complete listing. Most broadly applicable estimation methods in current use are based on linear or multiple linear regression against either log KOW , molecular connectivity indices (MCI), group (molecular fragment) contribution factors, or capacity factors generated by reverse phase (usually octadecylsilane) HPLC. Some excellent correlations have been reported for the HPLC methods, but these have not gained much favor because the other methods, by contrast, require only knowledge of the chemical structure. Among methods in this latter category, those based on MCI seem to be the most widely used at present. MCI are molecular descriptors that are calculated by a sophisticated sort of bond counting, and they express topological features of a molecule such as size, branching, unsaturation, and cyclicity. Environmental

297

applications such as Koc estimation were pioneered by Sabljic (55,56) but have expanded considerably. The Koc estimation program PCKOC (Table 9), in EPI Suite, uses this approach in combination with a series of polarity correction factors adapted from Sabljic (56). Hand calculation of the lower order MCI is possible but can be challenging; however, computer programs are now available that accomplish this easily. In PCKOC/EPI Suite, these calculations are transparent to the user. The most recent work on log KOW -based estimation methods includes several improved QSARs from Sabljic et al. (57), and one from Bintein and Devillers (58) that also uses pKa as an input variable for acids and bases (Table 9). The latter study did not include a separate validation step, and the training set was limited, but the improvement in accuracy effected by pKa is consistent with similar effects of certain polarity correction factors in the model of Meylan et al. (59), and it may have a similar rationale, namely, correction for ionization. Correction for acid-base behavior can be considered a desirable feature of estimation programs intended for general application to agricultural chemicals. Using the acetic acid example from the earlier section on pKa , this acid would be expected to exist almost entirely in the anionic form in neutral soils, and thus would be expected to be mobile. But weaker acids (having higher pKa ) might have significant amounts of undissociated substance, especially in more acid soils, and this uncharged species should be more strongly adsorbed to organic matter. The situation for bases is slightly more complicated because soils generally have a net negative charge (clay minerals are negatively charged), and ionic interactions may thus add another sorption mechanism (electrostatic interaction) for bases that are strong enough to be protonated at the soil pH. In general, for organic acids and bases, the Koc approach can be applied without quantitative consideration of pKa if it is known that the neutral form of the substance dominates at the pH of the soil solution. But if both ionic and nonionic forms are present, it is necessary to know the relative amounts and to consider sorption of the two forms separately. The two basic approaches to Koc estimation that are recommended are correlations with log KOW and MCI. Other approaches exist and may work well with specific classes of substances, but for the most part, these are not broadly applicable. The polarity correction factors of Meylan et al. (59) are helpful in estimating Koc for the more polar organics, but for weak acids and bases, the method of Bintein and Devillers (58) is recommended if it can be applied. Seth et al. (60) recently suggested that it is preferable to correlate the ratio Koc /KOW with KOW or some other molecular property, rather than log Koc with log KOW , because this gives a better indication of uncertainty associated with predicted Koc values. The uncertainty is high and is a reflection of the high variability of measured Koc values, which is mainly a result of variability in the composition of organic matter plus experimental difficulties in measuring Koc . They suggested that Koc be expressed as a distribution of values rather than as a single value; specifically, that Koc can be calculated as 0.35 KOW ± a factor of 2.5, which is intended to represent the 95% confidence limits. Other recent work includes a

298

CHEMICAL PROPERTIES ESTIMATION Table 9. Broadly Applicable Estimation Methods for Soil/Sediment Sorption Coefficient (Koc ) Method

Methodology


From log KOW

Validation: n = 202; r2 = 0.621; sd = 0.759; me = 0.653 (Meylan et al. (59) same validation set)

From water solubility

Validation: n = 122; r2 = 0.718; sd = 0.661; me = 0.608 (Meylan et al. (59) same validation set)

Meylan et al. (59): PCKOC (100)

MCI and polar fragment correction factors

Training: n = 189; r2 = 0.955; sd = 0.230; me = 0.182 Validation: n = 205; r2 = 0.856; sd = 0.462; me = 0.344

Bintein and Devillers (58)

From log KOW and pKa (for acids or bases)

Training: n = 53 chemicals (229 values); r2 = 0.933; sd = 0.433 Training: n = 87 chemicals (500 values); figures given but no statistics

Baker et al. (101)

From log KOW

Training: n = 72 chemicals; r2 = 0.91

Sabljic et al. (57)

From log KOW . Two equations: method 1 for hydrophobics; method 2 for nonhydrophobics

Training: n = 81; r2 = 0.887 (method 1) Training: n = 390; r2 = 0.631 (method 2)

Sabljic et al. (57)

MCI; for hydrophobics only

Training: n = 81; r2 = 0.96

Bahnick and Doucette (102)

MCI

Training: n = 56; r2 = 0.94

Tao et al. (61)

74 Fragments and 24 correction factors

Training: n = 592 chemicals; r2 = 0.970; average error = 0.366 log units Validation: range of r2 = 0.968–0.973 for 40-fold cross validation (leave 50 out)

PCCHEM (92)

Abbreviations as in Table 1. All standard deviations and mean errors are in log units.

new model developed by Tao et al. (61), based entirely on group contribution and correction factors (Table 9). More experience is needed with the Tao et al. (61) model as well as with the approach suggested by Seth et al. (60). ESTIMATING BIOCONCENTRATION AND BIOACCUMULATION For chemical substances with certain properties, concentrations in organisms can be achieved that are much higher than levels in the organisms’ environment. This is of potential concern because not only can a substance reach levels such that direct toxicity to an exposed organism is observed, but also consumers of that organism can then become exposed through their diets, even though there may be no direct route of exposure. ‘‘Consumers’’ includes humans as well as wildlife. Most data on bioaccumulation of chemical substances are from aquatic studies, mainly with fish, and existing estimation methods simply mirror the availability of data; i.e., they attempt to predict fish bioaccumulation. Nevertheless, bioaccumulation is of broad concern, and for this reason, estimated values are widely used as a risk factor in chemical screening activities. Prominent among these are numerous efforts underway to identify potential POPs and PBTs (persistent, bioaccumulative and toxic chemicals). The key terms are bioaccumulation, bioconcentration, and biomagnification. For aquatic organisms, bioaccumulation is the process by which a chemical substance reaches

a level that exceeds the concentration of the substance in the water, considering all possible routes of exposure. For fish, this means primarily uptake from diet and transport across respiratory surfaces (gills). Bioaccumulation is the most general of these terms and can be thought of as what takes place under field conditions. Bioconcentration has a more restrictive definition and is the process in which the higher concentration is achieved exclusively as a result of exposure to waterborne substance (diet is not included). Bioconcentration certainly occurs in nature, but for all practical purposes, it can only be measured in the laboratory. This fact has positive and negative consequences. The positive one is that because it is much more easily measured than the other parameters, fish bioconcentration studies are the principal source of data for modeling; without BCF data, there would be no screening-level estimation of bioaccumulation. The negative aspects are discussed more thoroughly below but in essence amount to the fact that BCF greatly oversimplifies bioaccumulation, which is the real parameter of interest. Biomagnification is the process in which the higher concentration is achieved as a result of dietary absorption; in other words, when an exposed organism achieves a higher level of a substance than does the level achieved by the organism’s prey. Bioaccumulation is a combination of bioconcentration and biomagnification. Recent reviews treat this area in much more detail (62,63). By definition, BCF is the ratio of a substance’s concentration in an aquatic organism (wet weight) to its concentration in the surrounding water at equilibrium,


when the exposure is only to waterborne substance. For most organic chemicals, equilibrium partitioning of the substance between cellular lipids and the exposure medium is the primary cause of bioconcentration, a fact that has been known for a long time (64). For this reason, hydrophobicity, expressed as log KOW , correlates fairly well with measured log BCF for many tested substances. Table 10 gives basic information on broadly applicable BCF estimation methods, nearly all of which still use log KOW as the primary or only property calculated from chemical structure. Accuracy is not one of the strong points of BCF estimation methods. The older regression equations (65,66) in essence predict that log BCF increases linearly with log KOW regardless of the log KOW value. However, numerous studies have shown that measured (apparent) BCF values reach a maximum around log KOW = 6–7, after which they decline with log KOW . More recently, models have been developed that mathematically represent both of these phases (67,68). The model of Meylan et al. (68) is the most complicated because it treats ionic and nonionic substances separately, includes a variety of correction factors, and gives special treatment to organotins and mercury compounds. This model affords a convenient basis for screening-level assessment and is available as BCFWIN in EPI Suite (http://www.epa.gov/opptintr/exposure/docs/episuite. htm). BCFWIN has by far the largest training set of any of the models in Table 10. Besides uncertainties associated with the log KOW values, there are several potential problems in predicting BCF from log KOW . First, because of the very low water solubility, it can take a long time for equilibrium to be reached for substances with log KOW > 6; the potential consequence is that some measured BCF values may be erroneous because the studies were not run long enough. Second, lipid content varies in aquatic organisms, and this can result in different degrees of bioconcentration. In principle, BCF can be normalized to lipid content, but these data are not always available; thus, not

299

all estimation methods are based on lipid-normalized BCFs. Third, lack of consideration of metabolism can result in overprediction of bioconcentration potential for certain organisms and substances. An example is benzo(a)pyrene, which is metabolized in fish but not in other aquatic organisms. The broadly applicable BCF estimation methods (Table 10) overpredict fish BCF for this substance because they do not explicitly consider metabolism. In general, at the present time, there is not adequate knowledge to permit development of group contribution-based methods to predict susceptibility to metabolism. Another issues is that partitioning across gill membranes may be restricted for substances with one or more of the following characteristics: MW > approx. 700; effective cross-sectional diameter > 0.95 nm; chain length > 4.3 nm or 25–30 carbons; and lipid solubility < 2 mMol/kg. Opinion is divided as to whether these criteria should be used as presumptive evidence against the potential for bioconcentration. Bioconcentration by definition involves exposure to waterborne substance. It is common to assume that this is the same as freely dissolved substance—i.e., that there is no test substance in the exposure medium that is sorbed to dissolved, colloidal, or particulate organic matter and, therefore, unavailable for uptake. But the dissolved fraction is operationally defined as what passes through a 0.4-µm pore-size membrane; thus, the possibility exists that the exposure medium in many BCF studies actually does contain test substance that is sorbed and not truly dissolved. This possibility increases with log KOW , and it is undoubtedly realized at times because very hydrophobic substances partition strongly to the sorbed state. The decrease in observed BCF with increasing log KOW above values around 6–7 has been attributed to this phenomenon by some researchers, who suggest that true bioconcentration potential continues to increase above this log KOW value, but cannot be effectively measured because of the extremely low water solubility of the test substance and exceedingly long equilibration times required. This

Table 10. Broadly Applicable Estimation Methods for Bioconcentration Factor (BCF) Method

Methodology


Veith et al. (65)

Log KOW

Training: n = 84; r2 = 0.823

Isnard and Lambert (103)

Two models: log KOW (model 1); Water solubility (model 2)

Training: n = 107; r2 = 0.817; sd = 0.51 (model 1) Training: n = 107; r2 = 0.753; sd = 0.59 (model 2)

Veith and Kosian (66)

Log KOW

Training: n = 122; r2 = 0.86

Saito et al. (104)

Three models: log KOW (model 1); i and o a (models 2 and 3)

Training, model 1: n = 107; r2 = 0.811; sd = 0.496 Training, model 2: n = 21; r2 = 0.962; sd = 0.442 Training, model 3: n = 107; r2 = 0.936; sd = 0.735

Bintein et al. (67)

Log KOW (bilinear model)

Training: n = 154; r2 = 0.903; sd = 0.347

SRC BCF program: BCFWIN (68)

Log KOW plus correction factors

Training set for nonionics: n = 614; r2 = 0.72; sd = 0.68; me = 0.49 Training set for ionics: n = 82; r2 = 0.62; sd = 0.40; me = 0.30 Combined training set: n = 696; r2 = 0.74; sd = 0.65; me = 0.47 For comparison: Bintein et al. (67): n = 696; r2 = 0.58; sd = 1.25; me = 0.94 Veith and Kosian (66): n = 696; r2 = 0.32; sd = 1.62; me = 1.12

a b

Abbreviations as in Table 1. All standard deviations and mean errors are in log units. Sum of inorganic and organic character.

300


may appear to call into question the bilinear and BCFWIN estimation models (Table 10) when applied to substances with log KOW > 6–7. The significance of this issue may be more academic than practical because dietary exposure is likely to be far more important than is exposure to dissolved material for such substances. In essence, this implies that no model designed to predict bioconcentration potential is appropriate for substances with log KOW > 6–7, because BCF models do not adequately address bioaccumulation potential, which is more important. There are different ways of dealing with this situation. A conceptually simple and conservative approach is to assume, as with the older log BCF-log KOW correlations, that BCF reflects bioaccumulation potential and is a monotonically increasing function of log KOW . Along with this comes an implied assumption that it is acceptable to overestimate bioaccumulation potential, especially at higher log KOW values, because such substances will be automatically targeted for more detailed (and presumably, mechanistically more accurate) assessment (63). However, this may not always be the case. An alternative approach is to take the apparent decline in BCF with log KOW at log KOW values above 6–7 seriously. The reason is that it is not co clear that this decline unfairly represents bioaccumulation potential. Several recent studies found that uptake of very hydrophobic substances by fish declined above log KOW 6–7 even in careful experiments with exposures exclusively via the dietary route (69–71). On the other hand, there is also recent evidence from studies with eel and zebra mussels (72) that body burdens can be as high for substances with log KOW values of 7–9 as for much less hydrophobic substances; no decline with increasing log KOW was observed in that study. Clearly, bioaccumulation is complex, site-specific, and a topic of intense current research. Generally speaking, the more sophisticated approaches suggested for estimating bioaccumulation factors (BAFs) are not practical for chemical screening because of limited availability of required data. Calculation of BAFs may be practical at higher levels of assessment, especially for the better known and data-rich pesticides. True BAFs reflect both bioconcentration and food chain accumulation and are site-specific parameters whose determination requires field measurements, preferably using animals at or near the top of the food chain. However, determination of BAF may be facilitated if an organism’s trophic level is known and a food chain multiplier (FCM) is available for the substance of interest. The FCM is then multiplied by the measured or estimated BCF to yield the estimated BAF: BAF = BCF × FCM

(25)

FCMs are sparse, but values for a few common pesticides can be found in EPA Report 823-R-00001 (73). Mackay and Fraser (63) suggest a tiered approach to estimating bioaccumulation potential that provides a useful framework for making decisions on how to address this endpoint for specific chemicals. Tier 1 involves screening, and here, BCF estimation methods are appropriate, especially for substances with log KOW < 6. Substances that are suspected of being

bioaccumulative based on partitioning properties can then be subjected to progressively more detailed evaluation. Mechanistic models, analogous to physiologically based pharmacokinetic models (PBPK), are now available for fish and are capable of quantifying all of the important processes, e.g., biomagnification, metabolism, and elimination by fecal egestion. For still more detailed analysis, food web models such as that of Gobas and Morrison (62) can be applied (free download at http://fas/sfu.ca/rem/era/era.html). As models become more elaborate, the data requirements for running them become extensive. CONCLUDING REMARKS Chemical properties frequently used in environmental assessment include melting temperature, boiling temperature, vapor pressure, n-octanol/water partition coefficient, water solubility, acid or base dissociation constant, Henry’s Law constant, sorption coefficient for soils and sediments, and bioconcentration/bioaccumulation factor for aquatic organisms. Broadly applicable estimation methods are available for all of these properties. Most of the newer methods were developed using much larger and more varied training sets; thus, they are more likely to be useful for diverse or structurally complex substances than are older methods. A potential disadvantage of currently available estimation methods is that most are applicable mainly to nonpolar organics, not weak acids/bases and ionic substances. The development of broadly applicable estimation methods has been more successful for some properties than for others. Melting point has been a particular problem because of difficulties in developing a rigorous theoretical basis. However, it is easily measured and accurate experimental values are readily available for thousands of organic compounds. Accuracy is also not one of the strong points of methods for bioaccumulation potential and soil/sediment sorption coefficients, because of the complexity of the underlying phenomena. These are active areas of research. Rapid and convenient chemical property estimation software is now readily available, in some cases for free. Nevertheless, the cardinal rule in estimating chemical properties should be do not do it if reliable measured values are available. A second rule to live by might be ‘‘estimator, know thy substance.’’ Fortunately, there are a substantial number of online databases of measured values, making it much easier to follow rule #1. To discourage irrational exuberance for convenient but potentially inaccurate estimation software, some of the more prominent data resources and their URL addresses are listed in this article. Many are free. URL ADDRESSES FOR INTERNET RESOURCES MENTIONED IN THIS ARTICLE Data Resources Available by Subscription http://www.pharmacy.arizona.edu/outreach/aquasol /index.html http://www.aiche.org/dippr/projects/801.htm


http://www.cas.org/ONLINE/DBSS/dipprss.html http://www.daylight.com/ Data Resources with Free Web Access http://wizard.arsusda.gov/acsl/ppdb.html http://ace.orst.edu/info/nptn/ppdmove.htm http://esc.syrres.com/interkow/physdemo.htm http://webbook.nist.gov/chemistry/ http://solvdb.ncms.org/ http://esc.syrres.com/efdb.htm http://toxnet.nlm.nih.gov/ http://ecdin.etomep.net/ Resources that Specialize in Providing Links to Other Resources http://ace.orst.edu/info/nptn/ http://www.chemfinder.com/ http://www.ilpi.com/msds/ Chemical Property Estimation Software (Free or Free Access) http://www.epa.gov/opptintr/exposure/docs/episuite. htm http://ibmlc2.chem.uga.edu/sparc/ Online Log KOW calculators (Free) http://www.daylight.com/daycgi/clogp http://esc.syrres.com/interkow/kowdemo.htm Bioaccumulation/Biomagnification Food Web Model (Free) http://fas/sfu.ca/rem/era/era.html BIBLIOGRAPHY 1. M. Tesconi and S. H. Yalkowsky, Melting point, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 3–27. 2. J. C. Dearden, The prediction of melting point, in M. Charton and B. I. Charton, eds., Advances in Quantitative Structure-Property Relationships, Vol. 2, JAI, Stamford, CT, 1999, pp. 127–175. 3. P. Simamora and S. Yalkowsky, Group contribution methods for prediction of the melting points and boiling points of aromatic compounds, Ind. Eng. Chem. Res. 33: 1404–1409 (1994). 4. J. Krzyzaniak, P. Myrdal, P. Simamora, and S. Yalkowsky, Boiling point and melting point prediction for aliphatic, non-hydrogen bonding compounds, Ind. Eng. Chem. Res. 34: 2530–2535 (1995).

301

8. P. I. Gold and G. J. Ogle, Estimating thermophysical properties of liquids. Part 4—boiling, freezing and triple-point temperatures, Chem. Eng. 76: 119 (1969). 9. W. J. Lyman, Boiling point, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 29–51. 10. W. Y. Lai, D. H. Chen, and R. N. Maddox, Application of a nonlinear group contribution model to the prediction of physical constants. 1. Predicting normal boiling points with molecular structure, Ind. Eng. Chem. Res. 26: 1072–1079 (1987). 11. D. Weininger, SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules, J. Chem. Inf. Comput. Sci. 28: 31–36 (1988). 12. T. Jensen, A. Fredenslund, and P. Rasmussen, Pure-component vapor pressure using UNIFAC group contribution, Ind. Eng. Chem. Fundam. 20: 239–246 (1982). 13. S. Banerjee, P. H. Howard, and S. S. Lande, General structure-vapor pressure relationships for organics, Chemosphere 21: 1173–1180 (1990). 14. W. J. Lyman, Estimation of physical properties, in W. B. Neely and G. E. Blau, eds., Environmental Exposure from Chemicals, Vol. I, CRC, Boca Raton, FL, 1985, pp. 13–47. 15. M. L. Sage and G. W. Sage, Vapor pressure, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 53–65. 16. C. Antoine, Tensions des vapeurs: Nouvelle relation entre les tensions et les tempe, Compt. Rend. 107: 681–684 (1888). 17. R. S. Boethling and D. Mackay, Introduction, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. vii–xvi. 18. D. Mackay, A. Bobra, D. W. Chan, and W. Y. Shiu, Vapor pressure correlations for low-volatility environmental chemicals, Environ. Sci. Technol. 16: 645–649 (1982). 19. A. Leo, Octanol/water partition coefficients, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 89–114. 20. N. Bodor and M.-J. Huang, An extended version of a novel method for the estimation of partition coefficients, J. Pharm. Sci. 81: 272–281 (1992). 21. Y. Sasaki, H. Kubodera, T. Matuszaki, and H. Umeyama, Prediction of octanol/water partition coefficients using parameters derived from molecular structures, J. Pharmacobio.-Dyn. 14: 207–214 (1991). 22. G. J. Niemi, S. C. Basak, G. D. Veith, and G. Grunwald, Prediction of octanol-water partition coefficient (Kow ) with algorithmically derived variables, Environ. Toxicol. Chem. 11: 893–900 (1992).

5. R. M. Dannenfelser and S. Yalkowsky, Estimation of entropy of melting from molecular structure: A non-group contribution method, Ind. Eng. Chem. Res. 35: 1483–1487 (1996).

23. T. Fujita, J. Iwasa, and C. Hansch, A new substituent constant, pi, derived from partition coefficients, J. Am. Chem. Soc. 86: 5175–5179 (1964).

6. K. G. Joback and R. C. Reid, Estimation of pure-component properties from group contributions, Chem. Eng. Commun. 57: 233–243 (1987).

24. W. M. Meylan and P. H. Howard, Atom/fragment contribution method for estimating octanol-water partition coefficients, J. Pharm. Sci. 84: 83–92 (1995).

7. S. E. Stein and R. L. Brown, Estimation of normal boiling points from group contributions, J. Chem. Inf. Comput. Sci. 34: 581–587 (1994).

25. C. Hansch and A. J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York, NY, 1979.

302


26. C. Hansch and A. Leo, Exploring QSAR: Fundamentals and Applications in Chemistry and Biology, American Chemical Society, Washington, DC, 1995.

44. CompuDrug NA, Inc. 1996. pKalc, Version 3.1 (computer software). Available at: http://www.acs.org/pubgen/software/pkalc.htm.

27. R. F. Rekker and H. M. de Kort, The hydrophobic fragmental constant; an extension to a 1000 data point set, Eur. J. Med. Chem. 14: 479–488 (1979).

45. S. H. Hilal, L. A. Carreira, and S. W. Karickhoff, Estimation of chemical reactivity parameters and physical properties of organic molecules using SPARC, in Quantitative Treatments of Solute/Solvent Interactions: Theoretical and Computational Chemistry, Vol. 1, Elsevier, New York, NY, 1994, pp. 291–353.

28. D. Mackay, Solubility in water, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 125–139. 29. R. G. Thomas, Volatilization from water, in W. J. Lyman, W. F. Reehl, and D. H. Rosenblatt, eds., Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York, NY, 1982, pp. 15-1–15-34. 30. R. G. Thomas, Volatilization from soil, in W. J. Lyman, W. F. Reehl, and D. H. Rosenblatt, eds., Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York, NY, 1982, pp. 16-1–16-50. 31. D. Mackay, W. Y. Shiu, and K. C. Ma, Henry’s Law constant, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 69–87. 32. J. Staudinger and P. V. Roberts, Critical review of Henry’s Law constant for environmental applications, Crit. Rev. Environ. Sci. Technol. 26: 205–297 (1996). 33. J. Hine and P. K. Mookerjee, The intrinsic hydrophilic character of organic compounds correlations in terms of structural contributions, J. Org. Chem. 40: 292–298 (1975). 34. W. M. Meylan and P. H. Howard, Bond contribution method for estimating Henry’s Law constants, Environ. Toxicol. Chem. 10: 1283–1293 (1991). 35. R. A. Brennan, N. Nirmalakhandan, and R. E. Speece, Comparison of predictive methods for Henry’s Law coefficients of organic chemicals, Water Res. 32: 1901–1911 (1998). 36. N. N. Nirmalakhandan and R. E. Speece, QSAR model for predicting Henry’s Law constant, Environ. Sci. Technol. 22: 1349–1357 (1988). 37. T. Suzuki, K. Ohtaguchi, and K. Koide, Application of principal components analysis to calculate Henry’s Law constant from molecular structure, Comput. Chem. 16: 41–52 (1992). 38. C. J. Russell, S. L. Dixon, and P. C. Jurs, Computer-assisted study of the relationship between molecular structure and Henry’s Law constant, Anal. Chem. 64: 1350–1355 (1992). 39. M. H. Abraham et al., Hydrogen bonding 34: The factors that influence the solubility of gases and vapors in water at 298 K, and a new method for its determination, J. Chem. Soc. Perkin. Trans. 2: 1777–1791 (1994). 40. J. C. Harris and M. J. Hayes, Acid dissociation constant, in W. J. Lyman, W. F. Reehl, and D. H. Rosenblatt, eds., Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York, NY, 1982, pp. 6-1–6-28. ¨ 41. G. Kortum, W. Vogel, and K. Andrussow, Dissociation Constants of Organic Acids in Aqueous Solution, Butterworths, London, U.K., 1961.

46. M. S. McLachlan, Framework for interpretation of measurements of SOCs in plants, Environ. Sci. Technol. 33: 1799–1804 (1999). 47. M. S. McLachlan, Vegetation-air partition coefficient, in Handbook of Property Estimation Methods for Chemicals, R. S. Boethling and D. Mackay, eds., Lewis/CRC, Boca Raton, FL, 2000, pp. 115–123. ¨ 48. J. F. Muller, D. W. Hawker, and D. W. Connell, Calculation of bioconcentration factors of persistent hydrophobic compounds in the air/vegetation system, Chemosphere 29: 623–640 (1994). 49. M. H. Hiatt, Leaves as an indicator of exposure to airborne volatile organic compounds, Environ. Sci. Technol. 33: 4126–4133 (1999). 50. D. E. Glotfelty, J. N. Seiber, and L. A. Liljedahl, Pesticides in fog, Nature 325: 602–605 (1987). 51. T. F. Bidleman and T. Harner, Sorption to aerosols, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 233–260. 52. D. Mackay, S. Paterson, and W. H. Schroeder, Model describing the rates of transfer processes of organic chemicals between atmosphere and water, Environ. Sci. Technol. 20: 810–816 (1986). 53. D. A. Lane, Gas and Particle Phase Partition Measurements of Atmospheric Organic Compounds, Gordon and Breach, Reading, Berkshire, U.K., 1999. 54. W. J. Doucette, Soil and sediment sorption coefficients, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 141–188. 55. A. Sabljic, Predictions of the nature and strength of soil sorption of organic pollutants by molecular topology, J. Agric. Food Chem. 32: 243–246 (1984). 56. A. Sabljic, On the prediction of soil sorption coefficients of organic pollutants from molecular structure: Application of molecular topology model, Environ. Sci. Technol. 21: 358–366 (1987). ¨ 57. A. Sabljic, H. Gusten, H. Verhaar, and J. Hermens, QSAR modeling of soil sorption, improvements and systematics of log Koc vs. log Kow correlations, Chemosphere 31: 4489–4514 (1995). 58. S. Bintein and J. Devillers, QSAR for organic chemical sorption in soils and sediments, Chemosphere 28: 1171–1188 (1994).

42. E. P. Serjeant and B. Dempsey, Ionization Constants of Organic Acids in Aqueous Solution, Pergamon, New York, NY, 1979.

59. W. M. Meylan, P. H. Howard, and R. S. Boethling, Molecular topology/fragment contribution method for predicting soil sorption coefficients, Environ. Sci. Technol. 26: 1560–1567 (1992).

43. D. D. Perrin, B. Dempsey, and E. P. Serjeant, pKa Prediction for Organic Acids and Bases, Chapman and Hall, New York, NY, 1981.

60. R. Seth, D. Mackay, and J. Muncke, Estimating the organic carbon partition coefficient and its variability for hydrophobic chemicals, Environ. Sci. Technol. 33: 2390–2394 (1999).

CHEMICAL PROPERTIES ESTIMATION 61. S. Tao et al., Estimation of organic carbon normalized sorption coefficient (Koc ) for soils using the fragment constant method, Environ. Sci. Technol. 33: 2719–2725 (1999). 62. F. A. P. C. Gobas and H. A. Morrison, Bioconcentration and biomagnification in the aquatic environment, in R. S. Boethling and D. Mackay, eds., Handbook of Property Estimation Methods for Chemicals, Lewis/CRC, Boca Raton, FL, 2000, pp. 189–231. 63. D. Mackay and A. Fraser, Bioaccumulation of persistent organic chemicals: Mechanisms and models, Environ. Pollut. 110: 375–391 (2000).

303

77. A. J. Leo, 30 years of calculating Log Poct , QSAR Meeting, Duluth, MN, July 23, 1992. 78. BioByte Corporation, CLOGP for Windows (computer software), BioByte Corp., Claremont, CA, 1995. 79. G. Klopman, J. Y. Li, S. Wang, and M. Dimayuga, Computer automated log P calculations based on an extended group contribution approach, J. Chem. Inf. Comput. Sci. 34: 752–781 (1994). 80. T. Suzuki and Y. Kudo, Automatic log P estimation based on combined additive modeling methods, J. Comput.-Aided Mol. Des. 4: 55–198 (1990).

64. J. L. Hamelink, R. C. Waybrandt, and R. C. Ball, Proposal: Exchange equilibriums control the degree chlorinated hydrocarbons are biologically magnified in lentic environments, Trans. Am. Fish. Soc. 100: 207–214 (1971).

81. A. K. Ghose, A. Pritchett, and G. M. Crippen, Atomic physicochemical parameters for three dimensional structure directed quantitative structure-activity relationships III: Modeling hydrophobic interactions, J. Comput. Chem. 9: 80–90 (1988).

65. G. D. Veith, D. L. DeFoe, and B. V. Bergstedt, Measuring and estimating the bioconcentration factor of chemicals in fish, J. Fish. Res. Bd. Can. 36: 1040–1048 (1979).

82. P. Broto and C. Vandycke, Molecular structures: Perception, autocorrelation descriptor and SAR studies, Eur. J. Med. Chem. 19: 71–78 (1984).

66. G. D. Veith and P. Kosian, Estimating bioconcentration potential from octanol/water partition coefficients, in Physical Behavior of PCBs in the Great Lakes, Ann Arbor Science, Ann Arbor, MI, 1983, pp. 269–282.

83. J. Devillers, D. Domine, and W. Karcher, Estimating n-octanol/water partition coefficients from the autocorrelation method, SAR QSAR Environ. Res. 3: 301–306 (1995).

67. S. Bintein, J. Devillers, and W. Karcher, Nonlinear dependence of fish bioconcentration on n-octanol/water partition coefficient, SAR QSAR Environ. Res. 1: 29–39 (1993).

84. R. F. Rekker, A. M. ter Laak, and R. Mannhold, On the reliability of calculated log P values: Rekker-, Hansch/Leo-, and Suzuki-approach, Quant. Struct.-Act. Relat. 12: 152–157 (1993).

68. W. M. Meylan et al., Improved method for estimating bioconcentration/bioaccumulation factor from octanol/water partition coefficient, Environ. Toxicol. Chem. 18: 664–672 (1999).

85. Daylight Chemical Information Systems (DCIS), CLOGP Program (computer software), DCIS, New Orleans, LA, 1996.

69. A. T. Fisk, R. J. Norstrom, C. D. Cymbalisty, and D. C. G. Muir, Dietary accumulation and depuration of hydrophobic organochlorines: Bioaccumulation parameters and their relationship with the octanol/water partition coefficient, Environ. Toxicol. Chem. 17: 951–961 (1998). 70. K. Kannan et al., Bioaccumulation and toxic potential of extremely hydrophobic polychlorinated biphenyl congeners in biota collected at a Superfund site contaminated with Aroclor 1268, Environ. Sci. Technol. 32: 1214–1221 (1998). 71. K. A. Maruya and R. F. Lee, Aroclor 1268 and toxaphene in fish from a southeastern US estuary, Environ. Sci. Technol. 32: 1069–1075 (1998). 72. A. J. Hendriks, H. Pieters, and J. de Boer, Accumulation of metals, polycyclic (halogenated) aromatic hydrocarbons, and biocides in zebra mussel and eel from the Rhine and Meuse Rivers, Environ. Toxicol. Chem. 17: 1885–1898 (1998). 73. USEPA, Bioaccumulation Testing and Interpretation for the Purpose of Sediment Quality Assessment: Status and Needs, EPA-823-R-00-001, U.S. Environmental Protection Agency, Washington, D.C., 2000. 74. Syracuse Research Corporation (SRC), MPBPVP Program. Estimation of Melting Point, Boiling Point, and Vapor Pressure (computer software for MS-DOS & MS-Windows 3.1, versions 1.35), SRC, Syracuse, NY, 1995. 75. Syracuse Research Corporation (SRC), LOGKOW & KOWWIN Programs. Estimation of Log Octanol/Water Partition Coefficient (computer software for MS-DOS & MSWindowsaˆ 3.1, versions 1.35), SRC, Syracuse, NY, 1995. 76. Pomona Medicinal Chemistry Project, PC-CLOGP Version 3.32, U.S. EPA version 1.2 (computer software), Pomona College, Claremont, CA, 1987.

86. C. Hansch, A. J. Leo, and D. Leo, BioByte Corp. Newsletter, Fall 1994. MacLOGP (computer software for the Macintosh). BioByte Corp., Claremont, CA, 1994. 87. Advanced Chemistry Development (ACD), ACD/LogP, ACD, Toronto, Ontario, Canada, 1995. 88. CompuDrug, ProLogP, Expert System for the Calculation of log P (computer software version 4.2), CompuDrug, Rochester, NY, 1993. 89. GS Corporation, ATOMLOGP (computer software). Produced by General Sciences Corp., available from the American Chemical Society, Washington, D.C., 1994. 90. W. M. Meylan, P. H. Howard, and R. S. Boethling, Improved method for estimating water solubility from octanol/water partition coefficient, Environ. Toxicol. Chem. 15: 100–106 (1996). 91. Syracuse Research Corporation (SRC), WS/KOW Program. Estimation of Water Solubility from Log Octanol/Water Partition Coefficient (computer software for MS-DOS & MSWindows 3.1, versions 1.35), SRC, Syracuse, NY, 1996. 92. GSC, PCGEMS User’s Guide. Report prepared under task 3-10 of USEPA contract no. 68-02-3970. General Sciences Corporation (GSC), Laurel, MD, 1987. 93. P. B. Myrdal, A. M. Manka, and S. H. Yalkowsky, AQUAFAC 3: Aqueous functional group activity coefficients. Application to the estimation of aqueous solubility, Chemosphere 30: 1619–1637 (1995). 94. S. Banerjee, Estimating water solubilities of organics as a function of temperature, Water Res. 30: 2222–2225 (1996). 95. R. Kuhne et al., Group contribution methods to estimate water solubility of organic chemicals, Chemosphere 30: 2061–2077 (1995).

304

CHEMOTHERAPY OF PLANT DISEASES

96. W. M. Meylan and P. H. Howard, Water mation by Base Compound Modification: Prepared for the USEPA under contract Syracuse Research Corp., Environmental Syracuse, NY, 1996.

Solubility EstiCurrent Status. no. 68-D2-0141. Science Center,

97. G. Klopman, S. Wang, and D. M. Balthasar, Estimation of aqueous solubility of organic molecules by the group contribution approach. Application to the study of biodegradation, J. Chem. Inf. Comput. Sci. 32: 474–482 (1992). 98. Syracuse Research Corporation (SRC), HENRY Program. Estimation of Henry’s Law constant (computer software for MS-DOS & MS-Windows 3.1), SRC, Syracuse, NY, 1996. 99. G. Klopman and D. Fercu, Application of the multiple computer automated structure evaluation methodology to a quantitative structure-activity relationship study of acidity, J. Comput. Chem. 15: 1041–1050 (1994). 100. Syracuse Research Corporation (SRC), PCKOC Program. Estimation of Soil Adsorption Coefficient (computer software for MS-DOS & MS-Windows 3.1 versions), SRC, Syracuse, NY, 1995. 101. J. R. Baker, J. R. Mihelcic, D. C. Luehrs, and J. P. Hickey, Evaluation of estimation methods for organic carbon normalized sorption coefficients, Water Environ. Res. 69: 136–144 (1997). 102. D. A. Bahnick and W. J. Doucette, Use of molecular connectivity indices to estimate soil sorption coefficients for organic chemicals, Chemosphere 17: 1703–1715 (1988). 103. P. Isnard and S. Lambert, Estimating bioconcentration factors from octanol-water partition coefficient and aqueous solubility, Chemosphere 17: 21–34 (1988). 104. S. Saito, A. Tanoue, and M. Matsuo, Applicability of the I/Ocharacters to a quantitative description of bioconcentration of organic chemicals in fish, Chemosphere 24: 81–87 (1991).

SUGGESTIONS FOR FURTHER READING Lyman W. J., Reehl W. F., and Rosenblatt D. H., eds., 1982; 1990. Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds, McGrawHill, New York, NY, 1982; American Chemical Society, Washington, D.C., 1990. Boethling R. S. and Mackay D., eds., Handbook of Property Estimation Methods for Chemicals: Environmental and Health Sciences, Lewis/CRC, Boca Raton, FL, 2000. Neely W. B. and Blau G. E., eds., Environmental Exposure from Chemicals, Vol. I. CRC, Boca Raton, FL, 1985. Reed R. C., Prausnitz J. M., and Poling B. E., The Properties of Gases and Liquids, McGraw-Hill, New York, 1987. Yalkowsky S. H., Sinkula A. S., and Valvani S. C., Physical and Chemical Properties of Drugs, Marcel Dekker, New York, 1980. Yalkowsky S. H. and Banerjee S., Aqueous Solubility. Methods of Estimation for Organic Compounds, Marcel Dekker, New York, 1991. D. Calamari, ed., Chemical Exposure Predictions, Lewis, Boca Raton, FL, 1993. Howard P. H. and Meylan W. M., Prediction of physical properties, transport and degradation for environmental fate and ¨ exposure assessments, in F. Chen and G. Schu¨ urmann, eds., Quantitative Structure-Activity Relationships in Environmental Sciences-VII, SETAC, Pensacola, FL, 1997, pp. 185–205.

CHEMOTHERAPY OF PLANT DISEASES OTIS C. MALOY Washington State University Pullman, Washington

ANTON BAUDOIN Virginia Polytechnic Institute and State University Blacksburg, Virginia

Chemotherapy is the treatment or cure of diseased plants by the application of chemical compounds. Chemotherapy differs from chemical protection by killing or inactivating the pathogen after it has infected the plant (1–3). The chemical compound is called a chemotherapeutant and may act locally at the site of infection as a topical therapeutant or may be distributed through the plant system as a systemic therapeutant (2). Topical therapeutants include a number of inorganic compounds such as mercuric chloride and calcium hydroxide (lime-sulfur) as well as some organic compounds (e.g., dodine) that penetrate disease lesions (e.g., apple scab) and kill the invading fungus along with a zone of healthy plant tissue. This is actually a form of chemical excision and these chemicals are more correctly referred to as eradicants rather than therapeutants (2). There are a number of compounds that are not strongly systemic, but that do penetrate into tissue, including dicarboximides, cyprodinil, some ergosterol biosynthesis inhibitors and strobilurins. They have been called locally systemic or local penetrants and can be used in what is called a curative mode, that is, applied within a few days after infection. When applied after infection, limited symptoms, such as chlorotic spots, may develop, but further fungal development and reproduction is inhibited. Systemic therapeutants are translocated to tissues remote from the site of infection (1,2). Plants do not have a circulatory system similar to mammals and therefore most systemic compounds are limited in their movement and distribution within the plant. The most common movement is upward (acropetal) in the xylem (apoplast). When absorbed by the roots, these chemicals are distributed mainly to mature leaves, and only limited amounts go to growing points and fruits. When absorbed by leaves, they travel to leaf tips and margins. Downward (basipetal) movement is limited to compounds that penetrate the cuticle, enter the protoplasm (symplast), and move in the phloem. Movement in both directions (ambimobile) is not common in plants (1,2,4). Topical therapeutants are usually broad-spectrum fungicides toxic to a wide range of fungi and bacteria. Systemic therapeutants are generally more selective; some being active against only bacteria and others against certain groups of fungi. For example, streptomycin is active primarily against bacteria, carboxin against basidiomycetes, and ethirimol against powdery mildew fungi (1). Interest in chemotherapy by injecting or infusing chemicals into plants began in the early 1900s (2). Most of the early attempts were with inorganic compounds such as copper sulfate, iron sulfate, potassium cyanide, lithium

CHIRALITY AND CHIRAL PESTICIDES

salts, mercuric chloride, and arsenic compounds. Some of these materials proved to be too phytotoxic for use. In 1906 Bolley injected fruit trees with iron sulfate and reported reduced attacks by Taphrina, and in 1923 Brooks and Storey poured a solution of 8-quinolinol sulfate into holes bored in branches of plum trees and reported efficacy against Stereum purpureum. Horsfall, Dimond, and others used this same compound in the mid-1930s in attempts to control Dutch elm disease (3). Modern plant chemotherapy was advanced with the discovery of antibiotics. In 1944 Brown and Boyle (5) cured crown gall in several plant species by wrapping galls with penicillin-soaked cotton and making multiple punctures into the gall with a sterile needle. Soon after Anderson and Neinow (6) immersed seedlings of many vegetable plants in streptomycin sulfate solutions and demonstrated that the antibiotic was absorbed through the roots and translocated in the seedlings. They also found that streptomycin was toxic to some plants. Streptomycin became one of the primary materials for the control of fire blight in apples and pears but as a protectant and eradicant rather than a chemotherapeutant. The majority of systemic fungicides are applied primarily as protectants and there have been very few applications as chemotherapeutants against wellestablished diseases since the expense is often prohibitive and the results have been disappointing. Pressurized and nonpressurized injections of benzimidazole fungicides or propiconazole into elms for treatment of Dutch elm disease have extended the life of some trees, but the fungus is rarely killed and treatments must be repeated every few years (7). Injections of oxytetracycline into trunks or scaffold branches of fruit trees has given remission of symptoms of the phytoplasma diseases pear decline (8) and X-disease of peach (9) but, as with Dutch elm disease, the treatments must be repeated annually because the antibiotic is not sufficiently translocated through the trees. Oxytetracycline is also sometimes used against lethal yellowing disease of certain palms, as a ‘‘holding action’’ until resistant replacement palms can be planted and given time to grow up. This is justified only for high-value landscape specimens (10).

BIBLIOGRAPHY 1. J. Dekker, in J. G. Horsfall and E. B. Cowling, eds., Plant Disease, vol. I, Academic Press, New York, 1977, pp. 307–325. 2. R. W. Marsh, ed., Systemic Fungicides, John Wiley & Sons, New York, 1977. 3. J. G. Horsfall and A. E. Dimond, Annu. Rev. Microbiol. 5: 209–222 (1951). 4. St. Neumann and F. Jacob, in H. Lyr, ed., Modern Selective Fungicides, Gustav Fischer Verlag, New York, 1995, pp. 53–73. 5. J. G. Brown and A. M. Boyle, Phytopathology 35: 521–524 (1945). 6. H. W. Anderson and I. Nienow, Phytopathology 37: 1 (1947). 7. R. J. Stipes and R. J. Campana, eds., Compendium of Elm Diseases, APS Press, St. Paul, Minn., 1981.

305

8. A. L. Jones and H. S. Aldwinckle, eds., Compendium of Apple and Pear Diseases, APS Press, St. Paul, Minn., 1990. 9. J. M. Ogawa, ed., Compendium of Stone Fruit Diseases, APS Press, St. Paul, Minn., 1995. 10. R. E. McCoy, B. J. Carroll, C. P. Poucher, and G. H. Gwin, Phytopathology 66: 1148–1150 (1976).

CHIRALITY A carbon atom with four different substituents that lacks a center of symmetry and a molecule containing one or more such asymmetric carbon atoms is termed a chiral molecule. If a molecule has one or more chiral centers, nonsuperimposable structures that are mirror images are termed enantiomers. Molecules that have nonsuperimposable structures that are not mirror images are called diastereoisomers. Diastereoisomers differ in their physical and chemical properties, whereas enantiomers have identical physical and chemical properties and differ only in their ability to rotate the plane of polarized light clockwise or counterclockwise, a mixture containing equal amounts of a pair of enantiomers is called racemate.

CHIRALITY AND CHIRAL PESTICIDES NORIO KURIHARA Kyoto, Japan

INTRODUCTION History and Significance There are numerous chemicals, in particular organic compounds, that have a specific effect on an organism. We call these compounds ‘‘biologically active compounds.’’ When a compound has a complex structure, it tends to be chiral. Chirality may have a considerable influence on the biological activity of a compound. Chirality is the term that indicates an asymmetry in the molecular structure. The term ‘‘chiral’’ comes from a Greek-word ‘‘cheir’’ which means ‘‘hand.’’ The mirror image of a hand is, of course, not superimposable on the original image of the hand. This relationship is called ‘‘chirality.’’ The mirror image of molecular structure of a chiral compound is not superimposable to the image of the original molecule. The significance of molecular chirality in the chemical aspects of life sciences has long been recognized since Pasteur’s resolution of the optical isomers of tartrate by manual separation of crystals of the racemate salt and also the theory of van’t Hoff and LeBel on the tetrahedral orientation of the four carbon bonds in organic compounds. The difference in biological activities of individual enantiopure isomers of natural compounds and synthetic drugs has also attracted the attention of scientists for a long time. Generally, one enantiopure isomer is more active than its counterpart. Sometimes, the other isomer does not have any observable biological activity. This is easily understandable because all organisms

306


constitute a chiral environment. Most enzymatic pathways are stereoselective, and most receptors for various chemical messengers including hormones are also highly stereoselective. The basis of these stereoselectivities or more appropriately enantioselectivities is the chemical structure of all enzymes and receptors that are basically proteins and/or protein oligomeric aggregates. They are chiral and, for the most part, possess a high degree of enantiopurity. There are many examples of the enantiomeric and enantiotopic selectivity of biologically active compounds. One of the popular examples from among human nutrients is the so-called essential amino acids, which have the (S)-structures without exception. None of the (R)counterparts are essential for human nutrient. (S)Glutamate is used as a food flavor enhancer, whereas the (R)-isomer does not have any such properties. When chiral and prochiral (= a compound of symmetric structure that becomes chiral through a simple biotransformation) pharmaceuticals and agrochemicals are introduced into biological systems, they may exhibit high enantioselectivity in their biological effect. In fact, there are many examples among agrochemicals. Classical insecticides of natural origin, such as pyrethrins (pyrethrin, jasmolin, and cinerin) (1), rotenoids [rotenone (2) and deguelin], and nicotine (3) are all optically active compounds and, in each case, one of the enantiopure isomers is the most highly biologically active. Examples of enantioselectivity in biological activity were also recently recognized among synthetic agrochemicals including insecticides, fungicides, herbicides, plant growth regulators, and insect pheromones. These examples are described later in detail. Circumstances The production and development of enantiopure agrochemicals on a commercial scale has been promoted and accelerated by the following situations: 1. Many lead compounds for new agrochemicals may be found among natural products, which are often chiral and enantiopure compounds. It is usually the case that the biological activity may also be specific to only one enantiomeric isomer, and the other isomers may have no activities. At an early stage in efforts towards developing a new biologically active compound starting from a natural model, research workers find it desirable to examine the activity of the enantiopure products in addition to the racemic ones. 2. A subject of high priority among environmental studies is that of finding a way to overcome the burdens of innumerable kinds of chemical residues on our living environment, and the governmental regulations continue to become more strict. The regulation by some national governments of preferential use of enantiopure agrochemicals rather than the racemic preparations is certainly an important factor for manufacturers in deciding if they should develop enantiopure products and release them to

the market. Actually, for aryloxypropanoate herbicides, some countries approve only the enantiopure biologically active isomer as the product for sale (in this connection, for pharmaceuticals, before a racemic preparation is registered, the study of the activity of individual enantiomers is required). This situation accelerates every effort to reduce the amount of chemicals applied to open fields, such as agrochemicals. It encourages the manufacturers to adopt a policy of developing a more biologically active ingredient, which needs a lower application rate, and as a link to this policy, to develop a more biologically active enantiopure isomer rather than a racemic preparation that contains the inactive or less active isomer. 3. Various methods are now available to prepare (synthesize and/or resolve) enantiopure compounds and analyze them with good resolution and sensitivity. In addition, various naturally occurring or synthetic optically active (often enantiopure) starting compounds are currently easily available for synthetic operations. Among them are amino acids and terpenes and optically active catalysts, reagents, protecting groups for functional groups, or chromatographic stationary phase materials. These materials are being developed at an increasing rate. Nowadays, these materials and techniques simplify the task of the manufacturers in developing enantiopure commercial products. Merits: The Benefits of Using Enantiopure Compounds A lower dose of an agrochemical applied to the field when enantiopure isomers are used instead of racemic preparation gives the chemical a better image from the ecological stand point and tends to make the product more highly competitive in the market. There are additional benefits in preparing an enantiopure compound. Due to the lower amount to be treated in the production processes, costs of storage and transport as well as the labor and energies expended for treatment may be reduced. When an appropriate starting compound and/or intermediate is available from among the chiral pool or even obtained by optical resolution of a racemic material, the synthetic routes toward the final product may require less chemical inputs such as reactants and solvents as well as consuming less energy during the reaction and purification. These factors can at least partly counterbalance the additional costs of developing and manufacturing the enantiopure isomer rather than the racemic preparation. Additionally, the selectivity of the compound can be improved when an enantiopure isomer is used compared with the racemate that may include isomers possessing antagonistic activity or showing adverse effects. This will widen the safety margin of the enantiopure product. Patterns of Different Biological Activities Among Isomers Many recently developed agrochemicals have chiral structures because of their complicated structures. They


may show several patterns of enantioselectivities in their biological activities and some enantiopure isomers of them are marketed: 1. An enantiomer may be much more favorably biologically active than its counterpart (thus, than the racemate) as evidenced by many pyrethroids and some organophosphorus insecticides. Fungicides of various types including triazoles and acylanilides as well as many of the herbicides of aryloxypropanoate and other types also show the similar pattern. 2. When an enantiomer is biologically active, its antipode may inhibit similar activity. The pheromone of the gypsy moth (4) is one of the examples of this relationship. 3. An enantiopure isomer may have an adverse effect that is not (or less) observed in the case of other isomers: e.g., the delayed neuropathy effect caused by EPN oxon (5), an organophosphorus insecticide, is higher in the (S)-isomer that is the isomer of lower insecticidal activity. 4. Quite a different type of agrochemical activity may be observed between enantiomeric pairs. Some triazoles are active as fungicidal as well as showing activity of plant growth regulators. In some cases, the isomers showing lower fungicidal activity show a much higher plant growth regulating activity than the others. One of chloroacetanilides that provided a structural lead for metalaxyl fungicide also shows similar enantiomeric structure-activity relationships: one enantiomer shows good fungicidal but lower herbicidal activity, whereas the other isomer with no fungicidal activity exhibits a high phytotoxicity. 5. A mixture of the enantiomeric isomers in a certain ratio may be more biologically active than any mixture in other ratios as observed in some insect pheromones. 6. There are some other patterns of enantiomeric differences in the biological activities shown by some compounds, and the examples will be described later in the sections of individual agrochemicals. Synthesis and Preparation Many examples of enantiomeric differences in biological activities observed have continued to provide challenging targets for the synthetic organic chemists who desire to prepare enantiopure biologically active compounds. To attain this goal, several techniques may be utilized (Table 1). Developing a new type of stereoselective reaction catalyst is one of such examples. Optical resolutions through a formation of classical diastereomeric derivatives followed by fractional crystallizations and chromatographic separations have often been successful for obtaining the intermediate alcohols and acids in some steps of the long course of synthesis of the pyrethroid insecticides. Microbial lipases and animal esterases are sometimes used for hydrolyzing intermediate esters. Enantiopure natural terpenes and amino acids (chiral pools) afford useful starting materials and

307

intermediate molecular building blocks in the course of synthesis of enantiopure pyrethroid and organophosphorus insecticides, such as optically active malathion, and also of the biologically active enantiopure isomer of a herbicide glufosinate. Asymmetric induction techniques employing chiral amines have been successfully used in the synthesis of some optically active phosphinates and phosphates. As an example of hydrogenation catalysts, enantiopure metal-diphosphine catalysts have been developed and successfully used during the course of the sophisticated synthesis of metolachlor, an acylanilide herbicide. Analysis To obtain evidence of the optical purity (enantiopurity), various techniques are available, among which liquid chromatography using a chiral column is one of the most frequently used. When the analyte is a mixture of optical isomers of which each has an appropriate functional group, they can be derivatized to, e.g., a mixture of diastereomeric esters that are efficiently analyzed by means of conventional chromatographic technique. Synthetic intermediates and product hydrolysates of various pyrethroids have been analyzed by these techniques. The same techniques have been applied for several organophosphates, aryloxypropanoates, and insect pheromones. INDIVIDUAL CHIRAL AGROCHEMICALS Pyrethroids (1) Pyrethrins described originally are natural products obtained from chrysanthemum flowers. Pyrethrins are chiral and composed of several optically active isomers. They have been extracted and purified to be used as insectcontrolling agents. Later, many synthetic compounds of simpler structures based on modifications of the natural pyrethrins have been examined for their insecticidal activities, and numerous ‘‘synthetic pyrethroids’’ have been developed and marketed nowadays. Many of them are optically active and enantiopure because, since the initiation of such efforts, strategy of the synthetic chemists for constructing active pyrethroid structures involved extensive utilization of naturally occurring enantiopure pyrethrin molecules. For example, chrysanthemic acid (acid portion of the natural pyrethrin I) was esterified with a simpler alcohol of a structure similar to natural pyrethrolone, etc. (S)-bioallethrin (6) (2), the ester of (1R)trans-chrysanthemic acid with (S)-allethrolone, is one of the earliest examples. In this study, the large difference in biological activity between stereoisomers was clearly indicated. Modification of the structure of the acid portion in the synthetic pyrethroids (esters) produces various highly biologically active insecticides. As an early example, permethrin (7) (3) did not utilize plant-derived materials and has a dichlorovinyl moiety instead of the dimethylvinyl group present in the natural pyrethrins coupled with 3-phenoxybenzyl alcohol. Introduction of an α-cyano-group in the alcohol portion gives a more highly biologically

308


OCH3 H3C

O H H3C

H3CO H

O

CH3

H H R

O

O

H

O

H

O

O

H

CH3

R = CH3: Pyrethrin I R = COOCH3: Pyrethrin II

Natural pyrethroids (1) N

Rotenone (2)

CH3 H N

O

(S)

H (+)-Disparlure (4) (sex pheromone of the gypsy moth)

Nicotine (3)

O H

OC2H5

P O

O

H NO2

(S)-EPN oxon (5) (hen paralysis activity)

O

(S)-Bioallethrin (6)

O

O O

O

H

O

3

Cl Cl

a

1

H

Br

CN O

H

O

H

Br (1R)-cis-(aS)-Deltamethrin (8)

Permethrin (7) O

Cl

O

S

O

1

H

H

O

a

2

O H

O

CN O

H

(2S, aS)-Fenvalerate [= Esfenvalerate] (10)

(1R)-cis-Kadethrin (9) Figure 1

active cypermethrin, and as a logical extension of this development program, deltamethrin (8) (4) has been prepared. Interestingly, its high activity resides in the (1R)-cis [or (1R)-cis-(αS)]-isomer, the stereochemistry different from the highly insecticidal natural pyrethrins. The highest knock-down effect among the kadethrin (9) isomers is exhibited also by its (1R)-cis-isomer (5). Several pyrethroids with no cyclopropane ring in the structure are now being used world wide. Fenvalerate isomers, one of the examples of this category, have been intensively studied to examine biological activities of respective isomers, not only with respect to their insecticidal activities but also

their adverse biological effects. The isomer (S)-α-cyano3-phenoxybenzyl (S)-2-(4-chlorophenyl)-3-methylbutyrate [so-called (2S, αS)-isomer = esfenvalerate (10)] shows the highest insecticidal activity with no adverse effects, whereas one of the other isomers, (2R,αS)-isomer, shows granuloma formation activity in mice liver (6) and essentially no insecticidal activity. Some insecticidal activities and mammalian toxicities with respect to granuloma formation are shown in Table 2. Some other stereochemistry-activity relationships among several pyrethroids are exemplified in Table 3, in which the higher activity of (1R)-isomers to insects is marked.


309

Table 1. Preparation and Synthesis of Enantiopure Agrochemicals Method

Example∗

Procedure

Resolution of diastereomeric derivatives of a racemic alcohol, acid, etc.

Fractional recrystallization of diastereomeric ester, salt, etc. Chromatographic resolution of diastereomeric ester, salt, etc.

(−)-Naphthylethylamine salt of (RS)-trans-chrysanthemic acid; camphor sulfonate of triadimefon. Use of L-proline in the resolution of rac-methamidophos.

Resolution of racemate by enzymatic or microbial reaction

Enantioselective reaction, e.g., hydrolysis, of racemate followed by purification of the product

Hydrolysis of rac-allethrolone acetate with Bacillus subtilis

Enantioselective preparative chromatographic resolution of racemate

Liquid (or gas) chromatography using a chiral column, esp. simulated moving-bed technology (= continuous counter current chromatography)

Salithion with Chiralcel OB etc; isofenphos with Sumipax OA-4,000; acephate with Chiralcel OC.

Synthesis using an enantiopure molecular building block (chiral pool)

Natural compounds, e.g., amino acids, terpenes, and some organic acids as lactic acid and malic acid and their simple derivatives as building blocks

D-Valine

Synthesis using a chiral catalyst or a chiral auxiliary: asymmetric induction

Utilization of a chiral catalyst e.g., catalytic hydrogenation of a CN double bond in the presence of an enantiopure ligand

Iridium-(with a chiral diphosphine ligand)-catalyzed hydrogenation of an imine intermediate for (S)-metolachlor

∗

for the synthesis of fluvalinate; enantiopure pentan-1,2-diol for propiconazol; (S)-2-chloropropanoic acids for (R)-aryloxypropanoate.

These and various other examples are described in the text.

Table 2. Biological Activities of Fenvalerate Isomers Stereochemistry (Configuration)∗1 2RS, αRS 2S, αS∗2 2S, αR 2S, αRS 2R, αRS ∗1 ∗2

Relative Insecticidal Activity Housefly

Mosquito Larva

1.0 3.5 0.05 2.0

1.0 2.7 0.29 1.9

30

590 152 500 3,700

Tetramethrin

(1R)-trans (1R)-cis (12)

84 120

2.43 1,000

Furamethrin

(1R)-trans (1R)-cis (13)

96 143

2.88 26

1,700

Phenothrin

(1R)-trans (1R)-cis (14) (1S)-trans (1S)-cis

0.78 1.10 >30 >30

>5,000 >2,500 >5,000 >5,000

∗

KT50 = time of 50% knockdown.

Structures of some chrysanthemic acid esters O O

(1R)-cis-Resmethrin (11) 1R

O O

O O

(1R)-cis-Tetramethrin (12)

N O O O

(1R)-cis-Furamethrin (13)

O O (1R)-cis-Phenothrin (14)

(1R)-trans and (1S)-trans-isomers of both resmethrin and permethrin undergo much faster hydrolytic metabolism with esterase than the (1R)-cis- and (1S)-cis-isomers do. Differences in the oxidative ester cleavage metabolism are not very large, except for the (1R)-trans-permethrin, where the rate is about 20-fold the rate of the other isomers. Environmental fate differences between stereoisomers are generally small, as reported for the half-lives of optical isomers of several pyrethroids in aerobic soils. Preparation and synthesis of enantiopure pyrethroids may be accomplished by means of conventional fractional crystallizations and/or chromatographic separations of diastereomeric derivatives of racemic synthetic intermediates coupled with appropriate optically active reagent. The structural basis of most pyrethroids is an ester which can be constructed from an acid moiety and alcohol moiety, either (or both) of which may be chiral and can be rather simply converted to some diastereomeric salts and/or esters, which can be resolved into the

O

O

respective optically active pair components. (1RS)-transchrysanthemic acid is resolved to its enantiomers by using (−)-α-naphthylethylamine via a mixture of diastereomeric salts. In this connection, the (1S)-trans-acid, which is not efficiently purified in this procedure, can be racemized (8) to the (RS)-cis, trans-isomeric mixture, which can then be resolved again as above to obtain the (1R)trans-acid (16) for the insecticidally more active ester. Asymmetric hydrolysis of allethrolone acetate was conducted using a bacterium Bacillus subtilis strain (9) and also a Pseudomonas lipase (10). In this process, the (S)allethrolone acetate (17), which may be used to lead to the more active allethrin isomer, is not hydrolyzed. Enantiopure compounds may be utilized as a molecular building block in the synthetic processes towards an optically active pyrethroid. Many of them may be obtained from natural products, such as (+)-car-3-ene (18) (11) and D-valine to construct enantiopure isomers of chrysanthemic acid and the partial structure of fluvalinate (19), respectively. Asymmetric induction using diazoacetate in


H

Cl F3C

311

H

H O

O

H

O

H O

O Flufenprox (15)

(1R,3R)-Chrysanthemic acid (16) [or (1R)-trans-] F3C H

AcO

H H

O (S)-Allethrolone acetate (17)

Cl

O

(+)-Car-3-ene (18)

CN

Cl

H Chiral Cu complex

O

Fluvalinate (19)

Cl3C + N2CHCOOEt

O

N H

H

H

Cl Cl

>>

COOR

H

Cl

COOR

Dichlorovinyl analog of chrysanthemic acid (20) O H3C

O H3C H

P

HO

H3N H

O

O H N

N H

H3C HO

Bilanafos (Bialaphos) (S)-(+)-isomer; (21)

O H3C

79% ee

N

OCH3

N

H3CO

N

P

O

RO

P

(22)

O

(Zeiss, 1987) O

(24)

H3C

(22)

(Minowa et al. 1984, 1987)

OCH3

N

H3CO

Michael addition

OC2H5

(23)

H3CO

H 3N H

Glufosinate (S)-Phosphinothricin (22)

N

+

O

O

HO

P

P

O H CH3

O

O

O H2 / RhCl[(R,R)-norphos]3

OR′′

90.8% ee

P(Ph)2 (22)

(Zeiss, 1991)

NHCOR′

HH P(Ph)2

(R,R)-norphos (25)

Figure 2

the presence of a chiral copper complex was successful in the synthesis of the dichlorovinyl analog (12) (20) of chrysanthemic acid. α-Pinene is also employed by means of enantioselective Favorskii rearrangement to prepare chrysanthemic acid.

Some conventional analytical techniques to differentiate enantiomers have been successfully applied in various pyrethroids and their optically active components. Esterification with octan-2-ol followed by gas chromatography is used to separate chrysanthemic acid isomers (13). By

312


means of a chiral HPLC column, the optical isomers of phenothrin are separated. With a chiral gas chromatograph (GC) column, allethrolone enantiomers are separated. Supercritical fluid chromatography with carbon dioxide as mobile phase separates four fenvalerate isomers. The technique often shows a better resolution performance per unit time than conventional HPLC and requires a temperature range much below that for conventional GC analyses. Organophosphorus Compounds (14) Many of the important organophosphorus agrochemicals are chiral with the phosphorus atom as a chiral center. They may be chiral also, with the carbon atom as a chiral center. They form an important class of compound among insecticides, since the development of highly insecticidal parathion by Schrader. Some organophosphorus compounds are used as fungicides and herbicides. There are many reports on biological activity differences between chiral isomers of these compounds, but none of the optically active and enantiopure phosphorus agrochemicals are marketed except for bilanafos (bialaphos) [the herbicidally active form is (S)-(+)-isomer] (21). It contains an (S)-alanine moiety, which was first isolated from an Actinomycete. The hydrolysate of bilanafos is glufosinate [the herbicidally active form is (S)-phosphinothricin = 4-[hydroxy(methyl)phosphinoyl-(S)-homoalanine] (22). For many chiral organophosphorus agrochemicals, the enantiomeric differences have been reported for in vivo activities, such as insecticidal, fungicidal, and herbicidal activities as well as mouse and rat toxicities, and delayed neurotoxicities to hen. There are also reports on enantiomeric differences for in vitro activities, such as inhibition of acetylcholine esterase, carboxyesterase, acetyl esterase, and oxidative metabolic reactions. Some examples of differences are also found in in vivo metabolic rates. In some cases, reverse relationships of different

biological activities are observed between enantiomers: (R)-EPN oxon is the more highly insecticidal isomer and has no special toxicity against hens, whereas the (S)-enantiomer (5), the lower insecticidal one, causes a significant paralysis (delayed neuropathy) activity. Several asymmetric induction pathways are reported for the synthesis of (S)-phosphinothricin (22), which is chiral on the carbon atom in the amino acid residue. The asymmetric synthesis is achieved by Michael addition of a chiral amine (23), which is derived from α-pinene via 2-hydroxy-3-pinanone and converted to methyl-vinylphosphinate methyl (15), by alkylation of a chiral bislactime (24) with an ethyl group in a phosphinate structure (16), or by asymmetric hydrogenation of α-acylamidoacrylate using an optically active diphosphine ligands [such as (R,R)-norphos (25) (17)] as ligand of the rhodium catalyst RhCl[Ligand]3 (18). (S)glutamic acid, as a chiral pool component, was successfully utilized to synthesize (S)-phosphinothricin in high yield and high optical purity with 98% enantiomeric excess (19). Among the reports on the relationship between the absolute configurations and insecticidal activities, some examples are shown in Table 4 as the ratios of the activities along with those of mouse toxicities (toxicities are measured as LD50 values, and the toxicity ratios here are the reverse ratios of the LD50 values). Notably, almost every (R)-enantiomer (one possessing the (R)configuration at the chiral phosphorus center) is more highly toxic than its (S)-isomer. Some remarkable exceptions are observed: In fonofos oxon, the toxicities of the (S)-enantiomer are much higher than those of the (R)-isomer. In salithion isomers also, the (S)form is about 3 times more insecticidal to housefly than its (R)-counterpart. Some other examples of the comparative toxicities are found in the literatures (see Ref. 14).

Table 4. Comparative Insecticidal Activities and Mouse Toxicities (reverse values of LD50 s) of the Enantiomers of Organophosphorus Insecticides Mouse Enantiomeric Pairs R versus S (R)-(−): (S)-(+)-fonofos (R)-(+): (S)-(−)-fonofos oxon (R)-(+): (S)-(−)-CYP∗1 (31) (R)-(+): (S)-(−)-CYP oxon (R)-(+): (S)-(−)-EPN (R)-(+): (S)-(−)-EPN oxon (5) (R)-(+): (S)-(−)-leptophos (R)-(+): (S)-(−)-salithion (35) (R)-(+): (S)-(−)-acephate (34) (R)-(+): (S)-(−)-methamidophos (33) (R)-(+): (S)-(−)-methyl phosphonothiolate (+): (−)-isofenfos∗2 (+): (−)-diethyl malathion∗2 (+): (−)-methyl parathion∗2

Housefly 3.97 0.083 2.73 — 3.00 1.65 3.89 0.33 5.00 6.25 — 11.2 ca.2

Other Insect Species Mosquito larva Tobacco cutworm Tobacco cutworm

1.8 0.082 31.5 53.2 17.0 7.14 — — — — — — —

Intraperitoneal 1.97 0.38 1.09 — 0.94 — — — — — — — —

Toxicity ratios R vs. S or simply (+) vs. (−) are shown for each of the enantiomeric pairs asymmetric at the chiral phosphorus center. ∗1 CYP = Cyanofenphos (31). ∗2 Configurations are unknown.

Oral 3.37 0.16 — — 0.97 1.63 1.17 — — — 5.0 (rat) — ca. 2 (2S,3S)] [PGR activity (2S,3S) > (2R,3R)]

(R)-(E)-Triapentenol (41) [Fungicidal activity (R)] [PGR activity (S)]

Cl

(2S,4R)-Propiconazole (42) and (2S,4R)-Etaconazole (43) Cl R

O

R

R

S

O

CH2C2H5 and C2H5

N N

H N

Figure 4

fungicidal, but low herbicidal, activity, whereas the (1S)isomer is highly herbicidal, but not fungicidal. Actually, the two (1R)-isomers (in the case that there is a chiral axis, two (R)-isomers exist) contain all the fungicidal activity of the product. Further replacement of chlorine in this compound by a methoxyl group affords the excellent fungicide metalaxyl (44) (27). Among enantiomeric isomers of metalaxyl, the (R)isomer alone possesses fungicidal activity. Because the reduction of the amount of chemical to be applied is advisable from an ecological point of view, various

synthetic approaches towards the large scale production of the (R)-isomer have been developed. Among these approaches, rhodium [= Rh(nbd)2 BF4 ]-catalyzed enantioselective hydrogenation of the enamide intermediate is now known to be superior (the ligand for the catalyst is Me-dupos in this case). The route is shown in Figure 6. Some newer approaches to the enantioselective catalytic hydrogenation are discussed (35). In this report, the related enamide hydrogenation in the synthetic pathway of clozylacon, a fungicide related to metalaxyl, is also discussed.

316


O

OH H2 / RuCl2(R)-binap∗

OH

OH

1-bromoacetyl-2,4-dichlorobenzene H+, ∆

N Br

N

O

N N N

RS

R O Cl

O H R

N

S

O

(Alkali) Cl

Cl

Cl

Propiconazole (42) (2S,4R-isomer is shown.) [Condensate salt with HNO3 is fractionally recrystallized to resolve isomers.]

PPh2

∗ binap =

PPh2

(Atropoisomerism due to the hindered rotation around the C-C axis between two naphthalene rings enables to differentiate the chiral isomers, aR and aS)

Figure 5. Synthesis of propiconazonal by utilizing an enantiomer of pentan = 1,2 = diol.

Morpholine Fungicides (27) Some morpholine derivatives are highly active against cereal fungi. A most important example of such activity in this group is fenpropimorph (47). Among its four stereoisomers, the (S)-cis-isomer is most potent against barley mildew and brown rust of wheat, whereas the (R)cis-isomer is less active to these diseases (36). According to the structure of the assigned sites of action—double bond migration from 8 to 7 in the intermediate steroid during the sterol biosynthesis—several isomers of a morpholine derivative having a partial structure similar to the carbocation intermediate in the double bond migration have been examined for their potency against a barley parasite fungus Erisyphe graminis. The similar partial structure to that of the 8-C = C substrate is important in exhibiting the potency. The compounds examined are, however, much weaker fungicides than fenpropimorph, which remains the most important morpholine fungicide. Camphoric acid treatment followed by fractional crystallizations of the resulting diastereomeric salt is used for the preparation of enantiopure compounds in this series. Fungicidal Compounds of Other Types The synthesis and enantiomeric differences of fungicidal activities have been reported for spiroxamine (48), having a spiroketalamine structure (37). Structurally somewhat similar spirocyclic 3-amino tetrahydofuran (49) is also a fungicide, which inhibits fungal sterol biosynthesis. This has also four stereoisomers consisting of two enantiomeric pairs, and the stereostructure-activity relationship studies are being conducted (38). When the structure of herbicide bromobutide (50) is somewhat modified, rice blasticide activity is revealed by the compound diclocymet (51), which has two chiral centers. Among the four stereoisomers, the [(S)-acid, (R)-amine]-isomer is most active (39)

(Fig. 7). Another new blasticide, which has a somewhat similar structure to diclocymet and possesses cyclopropanecarboxamide structure with a phenethylamine moiety, carpropamid (51a), has also been reported (39a) (Fig. 7). The compound has three chiral centers, one at the phenethylamine structure and two at the cyclopropane ring, thus indicating the existence of eight stereoisomers. However, because the synthesized carpropamid isomers have the E-configuration at the two chiral centers of the cyclopropane ring, four possible stereoisomers have all been isolated and examined on their activity against blast disease. The most active isomer was the one possessing the (R)-configuration at the α-methylbenzylic carbon and of the (+)-optical rotation sign concerning the α-carbon to the amide carbonyl. Chiral Aryloxypropanoates and Other Herbicides (40,41) Currently, aryloxypropanoate herbicides are also available in their enantiopure forms, which are marketed (Fig. 8). 2-Aryloxypropanoic acids and their esters both have good herbicidal and PGR activities. Because position 2 is the chiral center, examination of the herbicidal activity of enantiomers of various aryloxypropanoates has been performed. Many reports indicate the higher herbicidal activity of the (R)- than the (S)-enantiomer. Fluazifop-P-butyl (52) (a selective grass herbicide for rape and other broadleaf crops), trifop-methyl (53) [and napropamide (54), an aryloxyalkanamide], fenoprop (55), and related compounds all show higher herbicidal activities in their (R)-enantiomers. The respective (R)-isomers of dichlorprop-P (56) (Duplosan PP) and mecoprop-P (57) (Duplosan KV), which have higher herbicidal activities than their antipodes, are marketed, and some countries, such as Sweden and the Netherlands, approve only the (R)form, but not the racemic products, of these herbicides for agricultural use. The (R)-form of diclofop-methyl (58) has higher activity against millets and oats in post-emergence

CHIRALITY AND CHIRAL PESTICIDES H

317

OCH3 R

N

O

{Only (R)-isomer is illustrated.}

O X

X = Cl [(R):Fungicidal>Herbicidal; (S):Herbicidal / No Fungicidal] X = OCH3 [Metalaxyl (44). No Phytotoxicity. Fungicidal ( R) > (S).] H aR

OCH3 Ferrocenyldiphosphine ligand (46)

S

N O P Fe

Cl

H PR2 Ph

(R = 3,5-xylyl)

Ph

(aR,1S)-Metolachlor (45) Synthesis of (R)-metalaxyl(44) (route via the enantioselective enamide-hydrogenation) O

OCH3

OCH3 O

NH2

N

O Cl

O

O

OCH3 H2

O

N

[Rh(nbd)2BF4] / (R,R)-Me-duphos∗

(R)-Metalaxyl (44)

O OCH3

∗ nbd = norbornadiene

P

Me-duphos P

(S)-cis-Fenpropimorph (47) (Fungicide)

H N

O

Figure 6

application, but, in pre-emergence application, both enantiomers of this herbicide are equally bioactive against weeds in the rice field. Similarly, the post-emergence herbicidal activity of 2-[(pyridyloxy)phenoxyl]-propanoates is exclusively due to the (R)-enantiomer, but the preemergence activity of the both enantiomers are equivalent. There is a report that the conversion of the (S)- into (R)isomer by soil microorganisms takes place. The finding may explain the above activity relationships. An in vitro study has shown that the (+)-2-(2,4dichlorophenoxy)propanoic acid (56), which has the Rconfiguration, is active, whereas the (−)-isomer is inactive.

For the preparation of biologically active (R)aryloxypropanoates, (S)-2-chloropropanoic acid derived from (R)-lactic acid (e.g., by treating with thionyl chloride) is condensed with the pertinent 4-aryloxyphenol (42). 4-Arylation may be followed after the condensation with hydroquinone. Several minor modifications of the chloropropanoate route have been examined. Optically active diclofop-methyl, quizalofop-ethyl, haloxyfop, and fenoxyprop may be synthesized by these procedures using components of chiral pool. Enzyme-catalyzed hydrolysis of racemic aryloxypropanoate esters is also successful for the enantioselective preparation of fluazifop and diclofop.

318


O ∗

O ∗

∗

O

N

∗

N H Spirocyclic 3-amino tetrahydrofuran (49) (Fungicide)

Spiroxamine (48) (Fungicide) Br

S

H N

Cl

H N

H

O

CN

R

O

Cl

H Bromobutide (50) (Herbicide)

Diclocymet (Rice Blasticide) (51) [Modified from bromobutide] Cl

H H3C ∗

Cl

∗

H N

C2H5 O

∗

Cl

H3C Carpropamid (51a) (Fungicide)

Figure 7

Fluazifop-P-butyl (X = H, Y = CF3, Z = N, W = n-C4H9) (52) Trifop-methyl (X = H, Y = CF3, Z = CH, W = CH3) (53) Diclofop-methyl (X = Y = Cl, Z = CH, W = CH3) (58) O Y

X

Z

W

O ∗

O

O O O ∗

Napropamide (54)

N

O Fenoprop (X = Y = Cl) (55) Dichlorprop (X = Cl, Y = H) (56) Mecoprop (X = CH3, Y = H) (57)

Y

Cl

O ∗

OH

X

Figure 8. Aryloxypropanoates herbicides.

There are several reports of the preparation of an enantiopure acid from racemic acids, by fractional crystallizations of diastereomeric salts with optically active 1phenylethylamine, enantioselective biochemical reactions, or resolution with enzymes. A new type herbicide—hydantocidin (59)—isolated from a culture of Streptomyces hygroscopicus has a

spiro hydantoin structure connected to ribose ring. From D-tartrate as a starting chiral pool, all possible 15 stereoiso-

mers have been synthesized (43). All the corresponding stereoisomers of L-series have also been prepared. Among them, the N1 -β-D-isomer, which has a ribose moiety, exhibits a broad spectrum herbicidal activity, and the N1 -α-D-isomer is much less active. No other stereoisomers have significant herbicidal activities. The above syntheses are not easy to apply to the economic production. After evidence was obtained that the compound acts as a proherbicide of 5 -phosphate by inhibiting adenylsuccinate synthase in de novo purine biosynthesis and the X-ray analysis of the enzyme-inhibitor complex enabled investigators to design molecular models of the inhibitor, a biotin-like phosphonate model compound (60) was synthesized. (44). Another ribose-containing molecule, β-Dribosyl 1,2,4-triazole (61) is found to be herbicidal. The ribose configuration is important for the activity because analogues with some other pentoses instead of ribose attaching to 1,2,4-triazole are inactive (45). Another type of herbicidal antibiotic, the structure of which has a hydroxylated tetrahydropyran moiety, is now reported. A major component of this group, pseudomonic acid A (62) (46) has several chiral centers. Simplification of the structure seems essential for the stereostructure-activity relationship analysis and for inexpensive production of the active ingredient. After some structural modification, a hypnotic that has an imidazole-5-carboxylic acid structure (63) affords, the compound (64) that shows good herbicidal activity, and in which there is significant enantioselectivity in exhibiting


Hydantocidin model (60) O H N H NH H H S HO P OH O

N1-b-D-Hydantocidin (59) (Herbicide) HO

O

H N

O

NH

OH OH

319

O

OH N HO O

N

HO

O

N

O

b-D-Ribosyl H 1,2,4-triazole (61)

OR

O

OH

Pseudomonic acid A (62) [R = (CH2)8COOH]

OH OH

Imidazole-5-carboxylic esters N

N

N

OCH3

N

H

O

OCH3 O

Hypnotic activity (63)

Herbicide (64)

Herbicides derived from antibiotics O

Alk,H O S

OH

∗

Alkenyl, Alk

R

S R

O

H, COR, COOR (R = Alk)

F2HCCON

Alk, Hal O

OH

3-Aryl thiotetronic acid derivatives (Herbicide derived from an antibiotic) (66)

H An analog of the antibiotic thiamphenicol (Herbicide) (65)

Figure 9

activity: the (R)-enantiomer is most active (27). The mode of action studies reveal that the compounds inhibit sterol biosynthesis in weeds. Analysis by means of computerassisted molecular modeling of stereochemical differences among molecules exhibiting activity is an interesting approach. Modification of antibiotics is reported to produce new type of herbicidal compounds: difluoroacetamide analog of thiamphenicol of (R,R)-configuration (65) (47) and thiotetronic acids derived from thiolactomycin (66) (48). Insect Juvenile Hormones and Pheromones (49) Some insect hormones can be applied as agrochemicals. Steroidal α-ecdysone and related moulting hormones are,

however, not popularly utilized as agrochemicals. On the other hand, insect juvenile hormones and several synthetic mimics are being used in practice. Insect juvenile hormones and pheromones are used, generally as enantiopure forms, because they exhibit, in most cases, marked differences in biological activity when the activities of enantiomeric isomers are compared. Sometimes a mixture of a definite ratio of the compound is required for effective pheromone activity (see below). Hormones and pheromones are highly biologically active and are needed in much smaller amounts than conventional insecticide chemicals for controlling insect physiology and behaviors, thus making the manufacturing scale much less than is the case with the majority of other categories

320


O

O OCH3

O (10R,11S)-JH I (67)

OCH3

O

12,000 : 1

(10S,11R)-ent-JH I (68)

JH activity O

O OCH3

O (R)-JH III (69)

OCH3

O 50 : 1

(S)-ent-JH III (70)

JH activity

yeast

O

(R)-JH III (69)

O

HO

O

(S)-ent-JH III (70)

(meso) O OCH3 OsO4 / 5 mol% (DHQ)2-PHAL(71)∗

O

HO

(etc)

OCH3 OH

O

1. MsCl, 2. K 2CO3

OCH3

O (DHQ)2-PHAL (71) ∗

( ∗ 1,4-bis(dihydroquinine)phthalazine)

N

N H

H

N

N

O

O

O

H

H

O

N N Figure 10

of agrochemicals. This is another important reason that these agrochemicals can be manufactured and marketed mostly in enantiopure forms. Among most identified insect juvenile hormones, activity exclusively resides in the enantiomer of natural origin, and other stereoisomers have very little or no

juvenile hormone activity. For example, the natural isomer of JH I (67) having (10R,11S)-configuration shows about 12,000-fold activity of the (10S, 11R)-enantiomer (= entJHI) (68) when tested on the silk worm larval moulting activity (50). Size of the activity differences between enantiomers may be much less than in the above example,


321

Tartrate as a chiral building block -1CH2CH3

COOH HO

H

H

OHCO

H

H

OH

O

OCHO (CH2)3COCH3

COOH

O (+)-exo-Brevicomin (72)

HO H HOOC

OHCO H

COOH O H

OH

D-(−)-Tartaric

H

OCHO

acid

Tartrate as a chiral building block -2H HO HOOC

COOH HO

O

O

H

L-(+)-Tartaric

O

acid

H

O THP

O

O

OH

H

O

(7R,8S)-(+)-Disparlure (4)

Asymmetric epoxidation of a double bond OH

Asymmetric epoxidation∗

H O

OTs

(7R,8S)-(+)-Disparlure (4)

H Asymmetric epoxidation∗ : With (+)-Diethyl tartrate, Ti(O-i-Pr)4, t-BuOOH

Figure 11

such as the 50 times higher activity of JH III (69) than its enantiomer ent-JH III (70). However, the general trend seems to be high stereoselectivity (enantioselectivity) of the juvenile hormone activity. There are several relationships between stereochemistry and activity among the chiral insect pheromones. The following relationships are classified according to Mori’s review (49): 1. Only one enantiomer is active. Its antipode does not inhibit this pheromone activity. 2. Only one enantiomer is active. Its antipode inhibits this pheromone activity. 3. Only one enantiomer is active. Its diastereomer inhibits this pheromone activity. 4. The natural pheromone is a single enantiomer. Its antipode or diastereomer is also active.

5. The natural pheromone is an enantiomeric mixture. Each of the enantiomer is active. The combined effect is additive. 6. The pheromones of several insect species have a common chemical structure, but have different stereochemistry. 7. Both enantiomers of a single compound are necessary for the activity. The racemate is active, but neither of the enantiomer is active. 8. One enantiomer is more active than any other stereoisomers, but an enantiomeric or diastereomeric mixture is more active than the most active isomer (the effect is synergistic). 9. One enantiomer is active on male insects, whereas the antipode is active on females. 10. Only the mesoisomer is active.

322


Asymmetric hydrolysis of meso-diacetate H

H OAc

H OAc

Lipase AK

OAc

O

OH

H

H

(73)

H

O

(3S,4R)

(5R,6S) COOH

HOOC

NH2

OH

(R)-Glutamic acid

(S)-Sulcatol (74)

NH2

OH OH

HO O

(R)-Ipsdienol (75)

(S)-Serine

O

O O

O

(R)-Olean (76)

(S)-Olean NH

HO O

N H

H3C

NH2 O O HO

OH OH

HO

OH

Kasugamycin (77) Figure 12

Therefore, the importance of a highly reliable synthetic method for the preparation of respective enantiopure isomers of pheromones is beyond question because the amount of the pure pheromone isolated from the insect body is generally extremely small and it is normally a volatile liquid. This means that it is very difficult to determine the absolute configuration of the isolated material, and the determination of the configuration of these compounds becomes possible only after the comparison of the synthesized stereoisomers. Also, the most pertinent composition of the pheromone—enantiopure or a mixture of a definite ratio of the enantiopure components—is required for application in the field. Because pheromones and hormones are usually highly effective when only a very small quantity is needed to exhibit their activities, it is profitable to prepare them as commercial products even though they involve a multistep sophisticated synthetic route conducted in a much smaller scale compared with other categories of agrochemicals. Numerous sophisticated synthetic methods have been reported for the enantiopure insect juvenile hormones and pheromones. Earlier syntheses in the 1970s conveniently use some optically resolved intermediates as the building

block of enantiopure molecules. Later, the utilization of enantioselective biochemical reactions, such as yeastcatalyzed reduction of a meso-ketone to an enantiopure alcohol, was also among the successful synthetic methods. Some recent methods of preparation have used chemical asymmetric synthesis such as enantioselective osmium tetroxide dihydroxylation of a C=C double bond in the presence of an ingeniously constructed chiral catalyst (DHQ)2 − PHAL (71), e.g., in a synthesis of (R)-JH III (69) (51) (Fig. 10). For the synthesis of optically active pheromones, natural (and related commercially available) chiral pools are often skillfully used as a starting compound for a synthetic pathway, exemplified by the preparation of (+)-exo-brevicomin (72) from D-tartaric acid (52). The antipode, L-tartaric acid, the natural component, was used also in the synthesis of (7R, 8S)-(+)-disparlure (4), sex pheromone of the gypsy moth (53). An enantioselective epoxidation of a C=C double bond has been utilized in another synthesis of disparlure (54). Enzyme-catalyzed reactions are also used in several syntheses. For example, partial enantioselective hydrolysis of meso-diacetate (73) in the presence of lipase was used successfully for the


synthesis of the aggregation pheromone of the spined citrus bug (55). Amino acids such as glutamic acid and serine have been used for the construction of a chiral building block for the synthesis of some optically active pheromones e.g., (S)-sulcatol (74) (56) and (R)ipsdienol (75) (57). (S)-Malic acid is the key compound for a convenient chiral building block in the synthesis of (R)-olean (76), a pheromone of a spirostructure (58). Some other categories of optically active agrochemicals are being manufactured and marketed. Most of them are microbial products. Gibberellins are very popular and widely used as plant growth regulators. Several antibiotics, such as blasticidini-S and kasugamycin (77), are also widely used in agriculture as fungicides to control diseases, e.g., blasts in rice fields. A microbial product insecticide abamectin(s) is also a notable example. As already described, bilanafos (bialaphos), a peptide phosphonate, is used as herbicide. Many efforts are actively being made to further discover lead compounds of natural origin. Environmental impact due to heavy application of agrochemicals to open fields must be reduced, and the requirement by the general public to reduce the application of agrochemicals has recently become more stringent. For chiral agrochemicals, the use of only the biologically active (or most active) enantiopure isomer should also contribute greatly to the reduction of the chemical inputs into the environment. The IUPAC Commission ‘‘Agrochemicals and the Environment’’ recommends, ‘‘where an enantiopure isomer does not have the desired biological activity, it is preferable to remove the isomer when economically feasible even if it does not pose a significant risk’’ (41). Besides, it is beyond question that, where one or more enantiomers in a mixture pose significant environmental or human health risks, then the isomers should be removed, even where they contribute to the desired biological activity. Studies are needed to better define the mechanisms of toxicity and fates of individual enantiopure isomers of chiral and prochiral agrochemicals in target and nontarget organisms and to find improved methods for production of enantiopure isomers, which will involve applications of biotechnology, asymmetric synthesis, and separation.

BIBLIOGRAPHY

323

7. A. M. Hooper, B. P. S. Khambay, and D. G. Beddie, Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1A–003. 8. G. Suzukamo, M. Fukao, and T. Nagase, Chemistry Letters 1799–1802 (1984). 9. T. Oritani and K. Yamashita Agric. Biol. Chem. 39: 89–96 (1975). 10. S. Mitsuda, S. Nagashima, and H. Hirohara, Appl. Microbiol. Biotechnol. 31: 334–337 (1989). 11. M. Matsui et al., Agric. Biol. Chem. 31: 33–39 (1967). 12. H. Nozaki, H. Takaya, S. Moriuti, and R. Noyori, Tetrahedron 24: 3655–3669 (1968). 13. A. Murano, Agric. Biol. Chem. 36: 2203–2211 (1972). 14. M. Sasaki, in N. Kurihara and J. Miyamoto, eds., Chirality in Agrochemicals, John Wiley & Sons Ltd., Chichester, UK, 1998, pp. 85–139. 15. N. Minowa, M. Hirayama, and S. Fukatsu, Bull. Chem. Soc. Japan 60: 1761–1766 (1987). 16. H. J. Zeiss, Tetrahedron Lett. 28: 1255–1258 (1987). 17. Brunner and Pieronczyk, Angew. Chem. Int. Ed. 18: 630–631 (1979). 18. H. J. Zeiss, J. Org. Chem. 56: 1783–1788 (1991). 19. H. J. Zeiss, Abstract Papers, 7th International Congress of Pesticide Chemistry, Hamburg, 1990, O1B–13, p. 105. 20. H. Yoshikawa, T. Shono, and M. Eto, J. Pestic. Sci. 9: 455–462 (1984). 21. H. Kohsaka, Y. Oguri, M. Sasaki, and K. Mukai, J. Pestic. Sci. 12: 415–419 (1987). 22. K. Nakamura and S. Yamamura, Tetrahedron Lett. 38: 437–438 (1997). 23. H. Ohkawa, N. Mikami, A. Mine, and J. Miyamoto, Agric. Biol. Chem. 39: 2265–2267 (1975). 24. H. Ohkawa, N. Mikami, and J. Miyamoto, Agric. Biol. Chem. 41: 369–376 (1997). 25. A. Hirashima and M. Eto, Agric. Biol. Chem. 47: 2831–2839 (1983); S. Y. Wu and M. Eto, Agric. Biol. Chem. 48: 3071–3080 (1984). 26. F. Spindler and T. Frueh, in N. Kurihara and J. Miyamoto, eds., Chirality in Agrochemicals, John Wiley & Sons Ltd., Chichester, UK, 1998, pp. 141–173. 27. G. D. R. Tombo and D. Bellus, Angew. Chemie Int. Ed. 30: 1193–1215 (1991). 28. W. Koeller, Pestic. Sci. 18: 129–147 (1987).

1. K. Chamberlain, N. Matsuo, H. Kaneko, and B. P. S. Khambay, in N. Kurihara and J. Miyamoto, eds., Chirality in Agrochemicals, John Wiley & Sons Ltd., Chichester, UK, 1998, pp. 9–84. 2. J. R. Tessier, A. P. Teche, and J. P. Demoute, in J. Miyamoto and P. C. Kearney, eds., Proceedings of 5th International Congress on Pesticide Chemistry, Kyoto, Vol. 1, 1983, pp. 95–100.

29. H. Takano, Y. Oguni, and T. Kato, J. Pestic. Sci. 11: 373–378 (1986). 30. Y. Funaki, Y. Ishiguri, T. Kato, and S. Tanaka, in J. Miyamoto and P. C. Kearney, eds., Proceedings of 5th International Congress on Pesticide Chemistry, Kyoto, Vol. 1, 1983, pp. 309–314. 31. M. Kitamura et al., J. Am. Chem. Soc. 110: 629–631 (1988).

3. M. Elliott et al., Nature 246: 169–170 (1973).

32. H. Moser, G. Ryhs, and H. Sauter, Z. Naturforsch. 37b: 451–462 (1982).

4. M. Elliott et al., Nature 248: 710–711 (1974).

33. A. Togni et al., J. Am. Chem. Soc. 116: 4061–4066 (1994).

5. J. Lhoste and F. Rauch Pesticide Sci. 7: 247–250 (1976).

34. F. Spindler et al., Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1A–029.

6. H. Kaneko, M. Matsuo, and J. Miyamoto Toxicol. Appl. Pharmacol. 83: 148–156 (1986).

324

CHITIN BIOSYNTHESIS INHIBITORS

35. F. Spindler, B. Pugin, H. Buser, and H.-U. Blaser, Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1A–028. 36. W. Himmele, E.-H. Pommer, Angew. Chemie Int. Ed. 19: 184–189 (1980). 37. W. Kraemer et al., Abstract papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1B–005. 38. W. Pfrengle, B. Pabst, and J. Polotzek-Neudeck, Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1B–006. 39. Y. Oguri and M. Sasaki, Abstract Papers, 9th International Congress of Pesticide Chemistry, London, 1998, 1B–016. 39a. S. Kagabu and Y. Kurahashi, J. Pestic. Sci. 23: 145–147 (1998).

FURTHER READING Crosby, J., Manufacture of optically active materials: An agrochemical perspective, Pestic. Sci. 46: 11–31 (1996). Kurihara, N. and Miyamoto, J., eds., Chirality in Agrochemicals, John Wiley & Sons Ltd., Chichester, UK, 1998. Kurihara, N. et al., Chirality in synthetic agrochemicals: Bioactivity and safety consideration, Pure and Applied Chem. 69: 2007–2025 (1997). Tombo, G. D. R. and Bellus, D., Chirality and crop protection. Angew. Chemie Int. Ed. 30: 1193–1215 (1991). Williams, A., Opportunities for chiral agrochemicals, Pesticide Sci. 46: 3–9 (1996).


40. T. Haga et al., in N. Kurihara and J. Miyamoto, eds., Chirality in Agrochemicals, John Wiley & Sons Ltd., Chichester, UK, 1998, pp. 175–197.

P. J. JEWESS IACR–Rothamsted Harpenden, Hertfordshire United Kingdom

41. N. Kurihara et al., Pure & Applied. Chem. 69: 2007–2025 (1997). 42. H. J. Nestler and H. Bieringer, Z. Naturforsch. 35b: 366– 371 (1980). 43. S. Sugai et al., Tetrahedron 47: 2111, 2145 (1991). 44. W. Foery and H. Tobler, Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Volume 1, 1998, 1A–031. 45. G. Mitchell, S. M. Ridley, S. K. Vohra, and M. Woods, Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Volume 1, 1998, 1C–020. 46. P. Bellini, J. M. Clough, and G. Hatter, Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1A–032. 47. C. Langevine et al., Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1C–021. 48. M. Dollinger et al., Abstract Papers, 9th International Congress of Pesticide Chemistry, London, Vol. 1, 1998, 1C–004. 49. K. Mori, in N. Kurihara and J. Miyamoto eds., Chirality in Agrochemicals, John Wiley & Sons Ltd., Chichester, UK, 1998, pp. 199–257. 50. S. Sakurai et al., Experientia 46: 220–221 (1990). 51. G. A. Crispino and K. B. Sharpless, Synthesis 777–779 (1993).

Chitin is potentially a very attractive target for insecticides, because it is confined to arthropods (insects, spiders, mites, crustaceans, etc.), mollusks, annelid worms, coelenterates (hydras, coral polyps, etc.), the eggs of nematodes, and certain fungi. Consequently, specific inhibitors of its biosynthesis would be expected to have little effect on major nontarget organisms, such as mammals, birds, or plants. In practice, this expectation is realized and insecticides that act as chitin-biosynthesis inhibitors have extremely low mammalian toxicity, negligible phytotoxicity, and low environmental impact. However, they are mostly slow-acting, and this perceived defect by the farmer or horticulturist has somewhat limited their commercial success. Although certain fungicidal natural products, such as nikkomycin and polyoxin D (1) are very active inhibitors of chitin synthesis and demonstrate insecticidal activity in laboratory bioassays (1), they have not been commercialized. In contrast, two groups of insecticides that interfere with chitin biosynthesis have been discovered by random screening. These are the benzoylureas (Table 1) (also referred to as acylureas or benzoylphenylureas) and the thiadiazinone compound buprofezin (2). These will be treated separately in this article, because they have different detailed modes of action and insecticide uses.

52. K. Mori, Tetrahedron 30: 4223–4227 (1974).

O

53. K. Mori, T. Takigawa, and M. Matsui, Tetrahedron 35: 833–837 (1979). 54. K. Mori (1986).

and

T. Ebata,

Tetrahedron

42:

HO2C

NH

3471–3478

55. K. Mori, M. Amaike, and H. Watanabe, Liebigs Ann. Chem. 1287–1294 (1993). 56. K. Mori, Tetrahedron 31: 3011–3012 (1975).

O H2N

OH

O HO2C

OCH2 OH

NH2

57. K. Mori and H. Takikawa, Tetrahedron 47: 2163–2168 (1991). 58. K. Mori et al., Tetrahedron Lett. 25: 3875–3878 (1984).

N H

N O

OHOH (1)

O


325

Structure–Activity Relationships

O N N

N S (2)

BENZOYLUREAS Developmental History This group of insecticides was discovered during the course of a project that sought to discover novel herbicides. Scientists at Philips-Duphar (now Uniroyal) in the Netherlands combined two herbicides of differing modes of action—dichlobenil [‘‘Casoron’’; (3)] and diuron (4)—to form the benzoylated phenylurea DU19111 (5). This was nonherbicidal but toxic to insects (2). Although without activity on adult insects, it interfered with the molting process of the immature stages (larvae or nymphs) and caused death due to a failure to discard the old larval skin. Further development work at Philips-Duphar eventually led to the commercialization of diflubenzuron (Table 1) in 1974, which is marketed under the product name ‘‘Dimilin.’’ At the time, uptake by the agrochemical industry of this promising new type of insecticide lead was slow, mainly due to industry’s preoccupation with the fast-acting organophosphorus, carbamate, and (later) pyrethroid insecticides. Also, owing to the slow-acting nature of diflubenzuron and other benzoylurea insecticides (affected larvae may continue to feed until the next molt), there was a conception that they would be difficult to market because farmers were used to seeing rapid results in the shape of dead pests shortly after spraying. In addition to other problems, including lack of systemic activity, limited insecticidal spectrum, and formulation difficulties, this ensured that development of benzoylureas would be slow. However, on the positive side, vertebrate toxicity (both acute and chronic) was found to be very low, environmental impact negligible, and toxicity to beneficial insects was also low so that the compounds could be used in integrated pest-management systems. Meanwhile, some of the toxicological and environmental problems with neurotoxic insecticides has led to further efforts on benzoylurea development and the commercialization of a number of other products with improved activities. The discovery of compounds with acaricidal activity, such as flufenoxuron, also led to enhanced interest, particularly because of mite-resurgence problems caused by decline in the population in their natural enemies due to overusage of pyrethroid insecticides. The 12th edition of the Pesticide Manual lists 10 active ingredients that are commercialized or in late-stage development, and they currently comprise some 3% of the total worldwide insecticide market, worth $250 million p.a. in sales. Because of their favorable mammalian toxicity, newer compounds such as fluazuron, lufenuron, and hexaflumuron are being targeted at human and animal health pests (cockroaches, cattle ticks, and fleas) as well as, or instead of, conventional agrochemical uses.

Early work on the synthesis of analogs of DU19111 indicated there was little scope for variation in the substituents on the benzoyl ring, as only derivatives with at least one ortho-substituent group retained insecticidal activity. Substitution at other positions generally had a deleterious effect on activity. Ortho-substituents can be methyl, methoxy, trifluoromethoxy, or pentafluoroethoxy and afford active compounds, although all analogs that have been commercialized have ortho-halogen substituents and the insecticidal activity generally follows the order: 2,6-difluoro > 2-chloro > 2,6-dichloro. Among the 10 benzoylureas currently being commercialized, only one (triflumuron) does not have the 2,6-difluoro substituent pattern. This unusual structure–activity relationship has been interpreted as due to the influence of the ortho-substituents on the bond angles of the acylurea function, implying an optimum conformational requirement of this group for interaction with the binding site (as yet unknown). There is much more scope for variation in the aryl ring, although it must be substituted by electron-withdrawing groups (generally halogen, haloalkyl, or haloalkoxy) for optimum activity. Substitution by a second aryl group such as phenoxy or pyridyloxy (e.g., flufenoxuron, chlorfluazuron, and fluazuron) also generates active structures and has extended the pesticidal spectrum to include mites and ticks. Early studies showed that benzoylureas interfered with the biosynthesis of chitin, which is vital to the integrity of the insect integument (see below), and a good relationship was shown between its inhibition in vitro and insecticidal activity (3).

CN Cl

Cl

(3) O CH3

N

N H

Cl

CH3

Cl (4)

Cl O

O N H

N H

Cl

Cl Cl

(5) Physical Properties and Formulations Benzoylureas have some of the most unusual physical properties of any crop protection chemicals. They are all highly crystalline, lipophilic solids with high melting points (Table 2). Consequently, they have extremely low

326

CHITIN BIOSYNTHESIS INHIBITORS O

Table 1. Structures and Nomenclature of Benzoylurea Insecticides

N H

R

Common name Chlorfluazuron

Trade names Aim, Atabron, Helix, Jupiter

Manufacturer

O N H

Ar

R

Ar

Ishihara Sangyo

Cl 2,6-difluoro

Cl O

CF3 N

Cl

Diflubenzuron

Dimilin

Uniroyal 2,6-difluoro

Fluazuron

Acatak

Cl

Syngenta

Cl

Cl 2,6-difluoro

O

CF3 N

Flucycloxuron

Andalin

Uniroyal 2,6-difluoro

CH2O

N

Cl

Flufenoxuron

Cascade

BASF

F

Cl

2,6-difluoro

Hexaflumuron

Consult, Consol, Recruit, Trueno

O

Dow AgroSciences

CF3

Cl 2,6-difluoro

O

CF2CHF2

O

CF2CHFCF3

O

CF2CHFOCF3

Cl

Lufenuron

Match

Syngenta

Cl 2,6-difluoro Cl

Novaluron

Rimon

Makhteshim

Cl 2,6-difluoro

Teflubenzuron

Dart, Diaract, Nemolt, Nomolt

BASF

F

Cl

2,6-difluoro

F Cl

Triflumuron

Alsystin, Baycidal, Starycide

Bayer

vapor pressure, very low water-solubility, and their solubility in many organic solvents is also low. This results in both advantages and disadvantages for their crop protection uses, toxicology, ease of formulation, and interactions with the environment. Values of Log Ko/w are in the range 3.9 to 6.9; so they are not translocated in

2-chloro

OCF3

plants and have no systemic activity. They are strongly sorbed to soil (Koc 760–30000) so that they do not leach and contaminate groundwater; however, they consequently have no insecticidal activity in the soil. This combination of high Log Ko/w and low water solubility and vapor pressure results in slow rates of loss and degradation in biological


327

Table 2. Physical Properties of Benzoylurea Insecticides and Buprofezin Common Name

Mol. wt.

M.Pt., ◦ C

Log Ko/w

Vapor Press Pa at 25 ◦ C

Water Solubility, mg/l at 25 ◦ C

Koc , est

Chlorfluazuron Diflubenzuron Fluazuron Flucycloxuron Flufenoxuron Hexaflumuron Lufenuron Novaluron Teflubenzuron Triflumuron Buprofezin

540.7 310.7 506.2 483.9 488.8 461.1 511.2 492.7 381.1 358.7 305.5

226.5 (dec) 228 219 (dec) 143.6 (dec) 169–172 202–205 164.7–167.7 176–179 222.5 195 106

5.8 3.9 5.1 6.9 6.6 5.7 5.1 5.3 4.6 4.9 4.3

dichloromethane > diethyl ether > acetonitrile > 1 − butanol > 2-propanol > acetone > dioxane > tetra-hydrofuran > methanol > pyridine > water. Reversed-Phase HPLC By contrast to adsorption HPLC, reversed-phase HPLC (RP-HPLC) employs a nonpolar stationary phase and an apolar mobile phase. It has been estimated that about 80 to 90% of HPLC analyses used RP separation mode. The popularity of RP-HPLC is due to its higher reproducibility, lower equilibration time of the RP stationary phases, and the lower cost, higher optical transparency, and lower toxicity of mobile phases. Moreover, RP mobile phases are less inflammable and cause less environmental pollution. The most typical stationary phase in RP-HPLC is octadecylcoated silica; however, silicas bonded with shorter and longer alkyl chains (C1 , C4 , C6 , C8 , C30 ) have also found application in RP-HPLC (12,13). Like unmodified silica, modified silicas also cannot be used at extreme pHs; therefore, alkyl bonded alumina RP stationary phases have been developed and used for RP-HPLC (14,15).

343

Polymer-based RP stationary phases have also been synthesized and employed in RP-HPLC (16,17). The retention mechanism of RP-HPLC has not been entirely elucidated. It is generally accepted that the hydrophobic interactions between solutes and the apolar ligands on the surface of the stationary phase govern the retention. However, it has been established that adsorption, sterical parameters, and electrostatic interactive forces may contribute to retention. The retention parameters and chromatographic characteristics (capacity factor, separation factor, resolution, theoretical plate number, etc.) can be calculated in the same manner as described above for adsorption HPLC. The order of elution strength of solvents is nearly opposite in RP-HPLC to that in adsorption HPLC. Various aspects of RP-HPLC such as retention models (18–20), separation mechanism (21–23), effect of stationary phase (24,25), and quantitative structure-retention relationship have been previously discussed in detail (26). PRACTICAL HPLC A typical HPLC instrument consists of a separation and a detection unit. Separation is performed on a column with a mobile phase delivery system, samples being introduced by an injection device. The system accounting for the delivery of mobile phase includes one or more pumps with filters, degasser, and transfer tubing. The detection unit contains a detector and a signal output device. The pump delivers the mobile phase into the injector and column. The prerequisites for pumps are a precise and reproducible flow rate of mobile phase with as small a fluctuation as possible. Separation of solutes with similar retention characteristics can be achieved using one mobile phase with fixed composition (isocratic elution mode). Sometimes the retention behavior of solutes is very different and each solute cannot be eluted with one mobile phase. Changing the composition of mobile phase during the separation process overcomes this difficulty (gradient elution). Correct injection of sample into the top of the separation column is one of the most important parts of HPLC analysis. The sample can be injected via septum with a syringe or using valve injection with internal or external loop. Human subjectivity may influence the precision of the injection with a syringe but valve injection is entirely automated and the error of injection can be lower than 0.5%. An ideal detector must comply with the following requirements: sensitivity, highly specific or highly universal response, wide linear dynamic range, minimal extra-column band broadening, and so on. Detectors can be classified according to their selectivity and according to the physicochemical process employed for the detection. UV-VIS spectrometers are the most widely used detectors. They are very sensitive; however, the solute molecule must have UV or VIS absorption to be detectable. Solutes without UV adsorption properties may be derivatized but derivatization may be time consuming and can decrease the reproducibility of the analysis. Multiwavelength detectors (diode array detectors, DAD) can measure the whole spectra of solutes in the on-line, mode, increasing sensitivity when the

344

CHROMATOGRAPHY, HPLC

maximum absorbance of solutes is different, and may help the identification of solutes according to their spectral characteristics. Fluorescence detectors use an Hg or Xe lamp as excitation light source, the emitted light is measured at right angles to the axis of the excitation light beam. The sensitivity of fluorescence detection is 10 to 100 times higher than that of traditional UV-VIS detectors, and the selectivity is better too. The output signal of light scattering detectors is proportional to both the concentration and the molecular mass of solutes; therefore, it can also be used for the determination of the molecular mass of solutes. The refractive index detector measures the refractive index of the mobile phase and the solute. It is a universal detector; however, the sensitivity is markedly lower than that of UV-VIS detectors, and it cannot be used with gradient elution. The amperometric or coulometric detector is very sensitive and specific for solutes that can be reduced or oxidized in an aqueous environment. The electrical conductivity detector can be employed for the detection of ionizable solutes such as acids, bases, and salts. Hyphenated techniques in HPLC expose higher sensitivity and are suitable for the identification of solutes, which is impossible with traditional detector types. Radioactive detection, HPLC-FTIR (Fourier transform infrared), HPLC-MS (mass spectrometry) with atmospheric, electrospray or ionspray, particle beam, and fast atom bombardment ionization provide unique possibilities for the analysis and identification of solutes. A new and promising possibility is the coupling of HPLC to an atomic emission detector.

TROUBLESHOOTING Column care in HPLC is a prerequisite of reliable analytical work. Samples must not contain particulate matter and must not be cloudy because impurities can adsorb on the stationary phase, causing deteriorating column performance. Samples have to be purified before injection by solvent extraction, solid-phase extraction, filtration, or centrifugation, and they have to be dissolved in the mobile phase to avoid solvent peaks. Use of HPLC grade solvents increases column life, sensitivity, and avoids the appearance of ghost peaks on the chromatogram. The mobile phase must be filtered before use, and the column must be guarded by a short precolumn (about 1/20 length of the separation column). When using gradient elution with buffer and organic solvent, it must be verified that the buffer remains soluble, even at the highest concentration of the organic modifier. When the performance of the column deteriorates, it can be regenerated by washing the column with appropriate solvents or solvent mixtures. The volume of solvent may be 20-fold that of the column volume. Columns must be stored in the mobile phase defined by the manufacturer. Mobile phases must be filtered and degassed prior to being poured into the reservoir. The reservoir must be placed at a higher level than the pump. The use of contaminated mobile phases may lead to decreased sensitivity, wandering baselines, and irreproducible retention times, especially

in the gradient elution separation mode. Troubleshooting has been discussed in detail in Jinno et al. (27). HPLC ANALYSIS OF PESTICIDES Sample Preparation Strategies Pesticides and pesticide decomposition products are generally present in very low concentrations in complex matrices such as biological samples, foods and food products, ground- and waste waters, sludges, sediments, soils, and so on. The successful separation and preconcentration of the analyte is a prerequisite of the reliable and reproducible chromatographic process. Traditional liquid–liquid extraction (LLE) and Soxhlet extraction have been frequently used in the analysis of pesticide residues. Because of the considerable amount of organic solvent required for the extraction, the solvent has to be extremely pure to limit the contamination of the extract by other coextracted pollutants in the solvent. Moreover, the relatively large quantity of discharged solvent may increase the environmental burden and may endanger the health of laboratory staff. Numerous alternative preconcentration methods have been developed to overcome the difficulties of the traditional extraction procedures mentioned above. Static and dynamic headspace analysis for volatile solutes, solid-phase extraction (SPE), solid-phase microextraction (SPME), and supercritical fluid extraction (SFE) have found application in up-to-date sample preparation and enrichment procedures. The character of the constituents of the accompanying matrix (organics such as proteins, carbohydrates, lipids, humic acids, or inorganics such as silica, alumina, mixed metal oxides, water, etc.) as well as the physicochemical parameters of the pesticides to be preconcentrated define the technique to be employed. The potentially great variety of combinations of pesticide-matrix pairs makes it impossible to predict safely the optimal extraction method. Unfortunately, this must be found by trial and error and relies on the expertise of the chromatographer. As SPME has been generally combined with gas–liquid chromatography and SPME-GC has found application in the analysis of volatile compounds, it has negligible importance for HPLC analysis of pesticides. Similarly, SFE is usually coupled to methods other than HPLC. Solid-Phase Extraction (SPE) SPE can be applied to preconcentration of analytes present in liquid (both organic or inorganic) matrices. The liquid is passed through a cartridge packed with an appropriate sorbent, which is more or less specific for the pesticides or pesticide decomposition products. Because of the strong attractive interaction pesticides, the decomposition products are selectively adsorbed and concentrated on the sorbent. The cartridge can be washed and dried after finishing the preconcentration step, and then the adsorbed compounds can be eluted from the SPE cartridge with a small volume of strong solvent. The resulting eluate contains the analytes in concentrated form together with some components of the accompanying matrix in lower concentrations.


The binding of solutes to the sorbent phase is governed by hydrophobic and hydrophilic (electrostatic) interactive forces, depending on the character of the sorbent. As the strength and selectivity of such forces is practically unknown in the overwhelming majority of cases, theoretical considerations cannot be applied to the prediction of the SPE behavior of pesticides to be extracted from various matrix types and complex matrices. Basic principles of SPE (28), and the application of SPE for the extraction of environmental matrices (29) have been reviewed, and the use of SPE for the extraction of polar pesticides has been reported (30). Organochlorine Pesticides HPLC methods used for analysis of pesticides are generally multiresidue techniques suitable for simultaneous determination of more than one pesticide. As pesticide mixtures present in the environment contain pesticides belonging to different chemical classes, the division of the following subchapters according to chemical characteristics is an arbitrary one. The best procedure to find an adequate HPLC procedure for a given separation problem is to consult the index system. Because of their high persistence and toxicity, much effort has been devoted to the development of adequate HPLC methods for separation and quantitative determination of organochlorine pesticides and their decomposition products in various matrices. Many HPLC methods have been developed for the analysis of this class of pesticides in water. Thus, residues

345

of endosulfan and other pesticides have been determined in water using LLE and SPE preconcentration followed by RP-HPLC and diode array detection (DAD) (31). Aliquots of 400 mL of filtered water samples were passed through a SPE cartridge filled with octadecylsilica (C18 ) sorbent, and the pesticide residues were eluted with 1 mL of acetonitrile (ACN). The eluate was employed for a HPLC/DAD system without further concentration and prepurification steps. LLE was performed by extracting the same quantity of water with 3 × 100 mL of dichloromethane. The combined extracts were dried, redissolved in 1 mL of ACN : water 1 : 1 v/v, and applied for HPLC. Separation of pesticide residues was carried out on a C18 column (150 × 3 mm I.D., particle size 5 µm). Gradient elution was 2-min isocratic of 56% water, 27% ACN, 17% methanol, in 20 min to 5% water, 5% methanol, 90% ACN. Flow rate was set to 1 mL/min. Mean recoveries and relative standard deviations of SPE and LLE extraction procedures are listed in Tables 1 and 2. The recovery values were highly dependent on the type of pesticides and on the method of extraction; therefore, LLE was employed for extraction of methomyl, dimethoate, chlorpyrifos ethyl, and carbophenothion, whereas other pesticides were extracted with SPE. It was established that the method is simple, rapid, sensitive, accurate, and reproducible, and it can be employed for measurement of pesticides in drinking, ground-, and sea waters. A fully automated HPLC method has been developed for the separation and quantitative determination of dicofol and other pesticides in water. Pesticides have been detected with a combined photodiode array/postcolumn

Table 1. Mean Recoveries (R) and Relative Standard Deviation (RSD, n = 5), Both in % of Tested Compounds Using Off-line C18 SPE Cartridges in Drinking, Ground, and Sea Waters. Spiking Level: 0.1 and 1.0 µg/L. Water Volume: 400 mL Spiking Level 0.1 µg/L Drinking

Spiking Level 1.0 µg/L

Ground

Sea

Drinking

Ground

Sea

Pesticide

R

RSD

R

RSD

R

RSD

R

RSD

R

RSD

R

RSD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

14 39 90 89 101 85 91 113 82 108 96 103 99 89 84 94 102 86 89 95 104

20.1 25.3 10.4 7.9 8.8 8.9 7.8 10.6 9.3 9.1 8.6 6.3 5.5 7.8 8.1 10.1 11.0 6.0 7.7 9.8 10.1

— — 89 99 96 98 105 114 102 91 106 94 106 92 94 105 98 103 96 89 87

— — 9.8 8.3 8.5 9.6 8.4 9.3 8.8 10.7 9.1 8.5 9.9 5.3 7.2 8.9 11.6 9.6 7.8 10.1 11.9

— — 91 94 89 93 92 90 88 88 98 95 101 83 92 84 89 89 92 72 53

— — 7.2 9.9 6.2 4.6 6.3 8.5 6.7 5.9 8.8 6.4 5.5 6.7 9.3 8.5 9.7 5.4 6.6 7.7 5.3

25 41 95 104 99 105 103 107 100 106 101 112 102 106 110 99 105 97 108 99 101

22.3 30.1 9.9 12.3 10.6 9.3 11.7 10.1 10.2 11.3 10.3 9.2 7.6 8.3 9.1 9.8 11.4 9.1 9.6 10.9 11.4

— — 92 103 91 97 99 107 112 105 103 96 98 104 91 104 103 92 107 91 89

— — 10.7 10.8 9.9 10.6 9.2 9.4 9.8 11.9 10.7 10.4 11.8 11.7 9.4 9.5 11.6 11.9 9.9 11.5 10.2

— — 98 99 91 92 98 101 106 97 99 98 99 93 110 95 91 88 97 84 65

— — 8.7 8.2 6.1 5.5 7.2 9.3 6.3 5.4 9.9 5.3 6.1 7.9 10.6 9.1 10.2 9.1 7.4 9.9 7.2

1 = methomyl; 2 = dimethoate; 3 = aldicarb; 4 = diclorvos; 5 = carbofuran; 6 = atrazine; 7 = diuron; 8 = dichloran; 9 = methiocarb; 10 = folpet; 11 = triazophos; 12 = iprodione; 13 = vinclozolin; 14 = chlorfenvinphos; 15 = chlorpyrifos-m; 16 = endosulfan-s; 17 = tetradifon; 18 = ß-endosulfan; 19 = αendosulfan; 20 = chlorpyrifos; 21 = carbophenothion. Reprinted with permission from Parrilla and Vidal (31).

346


Table 2. Mean Recoveries (R) and Relative Standard Deviation (RSD, n = 5), Both in % of Tested Compounds Using LLE with Dichloromethane in Drinking, Ground, and Sea Waters. Spiking Level: 0.1 and 1.0 µg/L. Water Volume: 400 mL Spiking Level 0.1 µg/L Drinking

Spiking Level 1.0 µg/L

Ground

Sea

Drinking

Ground

Sea

Pesticide

R

RSD

R

RSD

R

RSD

R

RSD

R

RSD

R

RSD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

90 76 99 77 117 109 104 101 — 80 122 156 114 127 61 81 98 — 41 131 87

14.6 11.0 11.7 11.5 10.1 12.8 10.2 14.5 — 15.1 16.4 14.2 15.3 10.6 11.6 13.2 14.6 — 15.7 14.1 11.8

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

1 = metomyl; 2 = dimethoate; 3 = aldicarb; 4 = diclorvos; 5 = carbofuran; 6 = atrazine; 7 = diuron; 8 = dichloran; 9 = methiocarb; 10 = folpet; 11 = triazophos; 12 = iprodione; 13 = vinclozolin; 14 = chlorfenvinphos; 15 = chlorpyrifos-m; 16 = endosulfan-s; 17 = tetradifon; 18 = ß-endosulfan; 19 = αendosulfan; 20 = chlorpyrifos; 21 = carbophenothion. Reprinted with permission from Parrilla and Vidal (31).

derivatization/fluorescence detection system (32). Preconcentration of pesticides was performed on 10 × 3-mm SPE cartridges filled with poly(styrene-divinylbenzene) or ethylvinylbenzene-divinylbenzene copolymer sorbents using 100 mL of water acidified to pH 3. Pesticides were separated on a C18 column (150 × 4.6 mm I.D.) using gradient elution: initial conditions were 95% 10 mM aqueous phosphate buffer (pH 3) and 5% ACN : water (90 : 10, v/v). The ratio of buffer was decreased to 0% in 25 min followed by a final hold of 5 min. DAD was operated between 190 and 350 nm; pesticides were hydrolyzed and derivatized by o-phthalaldehyde before fluorescence detection, excitation, and emission wavelengths were set to 330 and 460 nm, respectively. The retention times, recoveries, lowest limit of detection (LOD), and lowest limit of quantitation (LOQ) determined by DAD are compiled in Table 3. It was found that the sensitivity (LOD and LOQ values) of fluorescence detection was superior to DAD detection; therefore, its application has been highly recommended. It was further established that the fully automated SPE-HPLC method can be used for the analysis of a wide variety of pesticides in water. HPLC has been employed not only for the determination of pesticide residues in water, but also in other accompanying matrices. Thus, a combined HPLC-GC method was developed for the determination of endosulfan and other pesticide residues in red wines (33). The scheme of the HPLC-GC system is shown in Figure 1. HPLC separation was performed on a C18 column (10 × 2 mm I.D., particle size 5 µm). GC analysis was carried out on a BGB-5 capillary column (30 m × 0.25 mm

I.D., film thickness 0.25 µm) preceded with a BGB-5 retaining precolumn (1 m × 0.53 mm I.D.). HPLC cleanup resulted in good GC separation of pesticides, as shown in Figure 2. The limits of quantitation varied between 5 µg/L (vinclosolin, α-endosulfan, and β-endosulfan) and 10 µg/L (procymidone, tetradifon). The list of wines and the concentration of pesticides residues are compiled in Table 4. The sensitivity of the automated HPLC-GC method made possible the detection and quantitation of pesticide residues in red wines at very low concentrations, and the procedure has been proposed for the routine monitoring of pesticides in wines. Aldrin, dieldrin, endrin, heptachlor, and p,p -DDT have been separated and quantitated in medicinal plants using a different HPLC method (34). Dried plant samples (100 g) were macerated with 500 mL of n-hexane and then filtered and evaporated to 5 mL in vacuum. Florisil SPE cartridge was conditioned by 5 mL of n-hexane and then the sample was passed through the cartridge. Analytes were eluted with 20 mL of n-hexane-diethyl ether (85 : 15, v/v). The eluate was evaporated to dryness and redissolved in 2 mL of ACN. Separation was carried out on a C18 column (250 × 4 mm, I.D., particle size, 5 µm), and the analytes were detected by DAD at 224 nm. Initial mobile phase composition was ACN : H2 O (70 : 30, v/v) to 85 : 15 in 11 min, 7-min hold, to 100% ACN in 3 min, 5-min final hold. Some validation parameters of the method are compiled in Table 5. Because of its simplicity, reliability, and high sensitivity, this was proposed as a method for the determination of these organochlorine pesticides in medicinal plants and phytotherapeutical preparations.

Table 3. Retention Times (tR ), Recoveries, and LOD/LOQ Values of Pesticides Analyzed by System with Diode Array Detector Mean Recovery ± RSD at Indicated Fortification Level (µg/L) LOD/LOQ Analyte 2-Nitrophenola 4-Nitrophenol 2,4-D 3-OH carbofuran Aldicarb Aldicarb sulfone Aldicarb sulfoxide Ametryn Aminocarb Atrazine Azinphos ethyl Azinphos methyl Bendiocarba Benfluralinc Benfuracarb Bentazone Bifenthrine Biretanolc Bupirimatec Butocarboxim Butoxycarboxim Captan Carbaryl Carbofuran Carbophenothion ethyl Carbosulfan Carboxinc Chlorpyrifos methyl Chloridazona Chlorobenzylate Chloropropylate Chlorothalonilc Chlorotoluronc cis-Permethrin Cycloate Decamethrin Deethylatrazine Deisopropylatrazine Desmedipham Dialifos Dichlofluanid Dicloran Dicofol Diflubenzuronc Dimethoate Dioxacarb Diphenylaminea Diuronc EPTC Ethiofencarb Ethofumesatea Fenamiphosc Fenarimol Fenitrothiona Fenoxycarb Fenobucarb Fenthionc Fenvalerate Flucythrinate Fluometuronc

tR , min 15.50 13.49 14.9 11.37 13.8 8.7 7.93 17.7 7.71 16.84 21.05 19.25 15.63 24.16 23.35 13.93 27.95 20.06 20.83 13.35 9.06 18.76 16.36 15.6 25.13 27.36 16.75 22.78 12.26 22.33 23.38 20.29 16.46 27.48 23.1 26.34 12.03 9.9 18.8 22.86 21.01 18.8 24.52 19.97 12.13 12.1 20.42 17.06 21.17 16.55 19.65 19.49 19.2 20.68 20.11 18.33 21.65 26.44 25.05 16.26

1 105 ± 1 19 ± 3 98 ± 1 87 ± 2 87 ± 1 6±4 ND 97 ± 1 — 103 ± 1 99 ± 2 100 ± 5 97 ± 2 76 ± 6 100 ± 5 97 ± 2 33 ± 5 96 ± 1 101 ± 2 107 ± 3 ND 107 ± 4 107 ± 1 96 ± 3 72 ± 1 — 92 ± 2 92 ± 1 61 ± 4 103 ± 1 102 ± 2 98 ± 2 98 ± 1 29 ± 4 60 ± 7 38 ± 2 72 ± 1 9±1 97 ± 5 84 ± 2 83 ± 4 89 ± 1 64 ± 9 98 ± 1 75 ± 7 76 ± 3 95 ± 1 102 ± 1 116 ± 1 80 ± 1 105 ± 2 100 ± 2 103 ± 5 96 ± 1 92 ± 2 127 ± 9 95 ± 4 31 ± 4 21 ± 7 102 ± 1

347

0.5 84 ± 1 19 ± 2 95 ± 4 81 ± 6 94 ± 3 ND ND 99 ± 1 85 ± 2 98 ± 3 95 ± 1 116 ± 12 100 ± 8 78 ± 4 73 ± 9 40 ± 7 19 ± 7 99 ± 1 99 ± 1 96 ± 6 ND ND 97 ± 2 95 ± 2 58 ± 6 44 ± 2 108 ± 10 90 ± 5 56 ± 3 97 ± 1 126 ± 7 89 ± 6 102 ± 2 27 ± 8 84 ± 3 32 ± 2 52 ± 7 5±2 96 ± 1 84 ± 4 50 ± 7 99 ± 5 54 ± 14 101 ± 2 75 ± 2 105 ± 7 106 ± 2 97 ± 1 93 ± 1 70 ± 10 105 ± 2 100 ± 3 108 ± 6 100 ± 4 85 ± 4 100 ± 5 102 ± 2 34 ± 5 22 ± 8 103 ± 2

0.1 98 ± 2 NDb 97 ± 5 87 ± 13 91 ± 1 ND ND 109 ± 3 94 ± 3 101 ± 3 105 ± 2 116 ± 12 75 ± 3 82 ± 10 ND ND 20 ± 1 97 ± 3 89 ± 7 54 ± 11 ND ND 126 ± 7 90 ± 1 38 ± 11 28 ± 1 75 ± 2 97 ± 8 56 ± 22 89 ± 6 246 ± 10 88 ± 4 102 ± 5 25 ± 7 49 ± 12 ND 61 ± 10 ND 92 ± 4 74 ± 4 29 ± 23 90 ± 5 116 ± 3 96 ± 2 76 ± 7 107 ± 5 101 ± 10 99 ± 1 72 ± 7 66 ± 5 111 ± 4 100 ± 5 134 ± 3 106 ± 3 100 ± 6 103 ± 5 106 ± 3 28 ± 8 24 ± 10 98 ± 4

µg/L 0.05/0.05 0.50/0.50 0.10/0.10 0.10/0.10 0.10/0.10 1.00/1.00 >1.0 0.02/0.05 0.10/0.10 0.02/0.05 0.10/0.10 0.10/0.10 0.10/0.10 0.05/0.05 0.50/0.50 0.20/0.50 0.10/0.10 0.02/0.05 0.10/0.10 0.10/0.20 >1.0 1.00/1.00 0.02/0.02 0.10/0.10 0.05/0.10 0.10/0.10 0.05/0.05 0.10/0.10 0.05/0.05 0.10/0.10 0.10/0.10 0.02/0.05 0.02/0.02 0.10/0.10 0.10/0.10 0.20/0.20 0.02/0.05 0.10/0.20 0.05/0.10 0.05/0.05 0.10/0.20 0.05/0.05 0.05/0.10 0.02/0.05 0.10/0.10 0.05/0.10 0.02/0.02 0.02/0.02 0.10/0.10 0.05/0.10 0.10/0.10 0.10/0.10 0.05/0.05 0.05/0.10 0.10/0.10 0.10/0.10 0.10/0.10 0.10/0.10 0.10/0.10 0.02/0.02

Table 3. (Continued) Mean Recovery ± RSD at Indicated Fortification Level (µg/L) LOD/LOQ Analyte Furathiocarb Hexaflumuron Hydroxy-atrazinec Imazamethabenz Imazaquinc Imidaclopridc Iprodione Isoprocarb Isopropalinec Isoproturonc I-Cyhalothrin Linuronc MCPA Metamitronc Methiocarb Methomyl Methoxychlor Metobromuronc Metolachlor Metolcarb Metoxuronc Mevinphos 1 Mevinphos 2 Molinate Monolinuronb Napropamide Naptalam 1 Naptalam 2 Naptalam 1 + 2 Nuarimol o,p-DDEa o,p-DDT Oxadiazon Oxamyl Oxycarboxinb p,p-DDEa p,p-DDT Paraoxona Paraoxon methyla Parathiona Parathion methyla Penconazole Pendimethalinc Pentachlorophenol Phosmet Pirimicarbc Pirimiphos ethylc Pirimiphos methylc Prochloraz Promecarb Prometryn Propachlor Propanilc Propazine Propoxur Propyzamide Pyrazophos Simazine Simetryne Temephos

tR , min 23.68 22.29 9.9 14.23 14.46 12 21.91 17.1 25.66 16.75 25.6 18.88 15.37 11.71 18.5 9.47 23.54 17.1 20.22 14.74 14.14 11.36 12.69 19.94 16.87 19.94 17.5 19.53 19.53 17.82 27.35 26.74 24.19 9.13 13.98 26.58 26.22 17.34 14.79 21.8 19.53 20.93 24.48 21.66 19.04 14.59 24.6 22.68 22.01 18.77 19.23 17.36 18.23 18.15 15.63 19.84 21.9 14.99 15.54 24.15

1 85 ± 2 80 ± 2 — 105 ± 1 39 ± 5 95 ± 4 100 ± 4 95 ± 8 85 ± 5 101 ± 1 27 ± 5 100 ± 1 13 ± 2 73 ± 1 94 ± 4 13 ± 5 122 ± 7 102 ± 2 112 ± 1 116 ± 2 99 ± 1 88 ± 3 96 ± 2 131 ± 3 97 ± 1 99 ± 1 43 ± 5 119 ± 4 76 ± 5 93 ± 1 51 ± 5 48 ± 5 99 ± 6 16 ± 2 101 ± 1 60 ± 5 49 ± 5 98 ± 1 101 ± 1 101 ± 1 101 ± 1 91 ± 1 102 ± 2 93 ± 1 104 ± 1 101 ± 1 89 ± 2 100 ± 2 82 ± 1 102 ± 3 97 ± 2 104 ± 3 100 ± 1 101 ± 1 140 ± 1 102 ± 2 92 ± 1 100 ± 5 99 ± 1 67 ± 4

0.5 84 ± 3 80 ± 3 57 ± 1 99 ± 1 37 ± 2 92 ± 5 101 ± 5 127 ± 2 65 ± 12 101 ± 2 24 ± 7 100 ± 2 16 ± 6 55 ± 2 97 ± 1 5±1 96 ± 3 105 ± 1 117 ± 1 95 ± 1 102 ± 2 90 ± 7 99 ± 2 98 ± 5 101 ± 1 101 ± 2 54 ± 10 118 ± 6 78 ± 7 96 ± 14 27 ± 4 27 ± 10 102 ± 4 10 ± 9 98 ± 3 39 ± 6 38 ± 10 108 ± 1 98 ± 2 97 ± 5 102 ± 4 123 ± 5 92 ± 2 99 ± 3 96 ± 8 96 ± 3 85 ± 4 93 ± 1 92 ± 1 101 ± 8 86 ± 3 115 ± 2 108 ± 1 98 ± 2 101 ± 4 98 ± 2 102 ± 8 98 ± 6 101 ± 2 36 ± 10

0.1 71 ± 8 101 ± 10 55 ± 4 94 ± 1 31 ± 9 73 ± 4 115 ± 6 98 ± 6 100 ± 15 97 ± 6 25 ± 5 92 ± 6 ND 55 ± 7 91 ± 8 ND 113 ± 9 104 ± 2 147 ± 3 79 ± 11 101 ± 3 95 ± 4 89 ± 6 78 ± 3 94 ± 2 107 ± 4 51 ± 15 113 ± 4 82 ± 15 89 ± 6 25 ± 3 26 ± 10 92 ± 6 ND 88 ± 6 31 ± 4 38 ± 9 98 ± 5 99 ± 2 97 ± 1 98 ± 15 276 ± 5 93 ± 7 92 ± 1 138 ± 15 92 ± 1 83 ± 4 86 ± 3 78 ± 8 103 ± 17 81 ± 43 108 ± 6 109 ± 9 94 ± 6 88 ± 3 101 ± 7 101 ± 4 62 ± 7 86 ± 3 66 ± 3

µg/L 0.10/0.20 0.02/0.05 0.10/0.10 0.05/0.05 0.05/0.05 0.02/0.05 0.05/0.10 0.10/0.10 0.02/0.05 0.02/0.02 0.10/0.10 0.02/0.02 0.50/0.50 0.02/0.05 0.10/0.20 0.50/0.50 0.10/0.10 0.02/0.02 0.10/0.10 0.10/0.10 0.02/0.02 0.10/0.10 0.05/0.10 0.10/0.10 0.02/0.02 0.05/0.05 0.10/0.10 0.02/0.02 0.05/0.05 0.05/0.05 0.10/0.10 0.10/0.10 0.05/0.05 0.20/0.50 0.05/0.10 0.10/0.10 0.10/0.10 0.05/0.05 0.02/0.02 0.05/0.05 0.02/0.05 0.10/0.10 0.10/0.10 0.02/0.02 0.02/0.05 0.05/0.05 0.02/0.02 0.02/0.02 0.10/0.10 0.10/0.10 0.05/0.10 0.10/0.10 0.02/0.02 0.02/0.05 0.10/0.10 0.05/0.05 0.02/0.02 0.02/0.05 0.02/0.05 0.10/0.10


348


349

Table 3. (Continued) Mean Recovery ± RSD at Indicated Fortification Level (µg/L) LOD/LOQ Analyte

tR , min

Terbumeton Terbutylazine Tetradifonc Thiobencarb Thiodicarb Thiofanox Thiophanate ethyla Thiophanate methyla Tolyfluanid Tralkoxydim trans-Permethrin Triasulfuron Trifluraline

16.63 19 23.7 22.54 15.64 16.36 17.8 15.48 21.87 24.48 26.91 16.11 24.39

1

0.5

113 ± 2 105 ± 1 83 ± 4 93 ± 1 91 ± 2 82 ± 8 55 ± 5 63 ± 5 92 ± 2 102 ± 2 33 ± 6 103 ± 8 91 ± 6

83 ± 3 99 ± 2 81 ± 7 88 ± 3 94 ± 5 122 ± 10 51 ± 10 40 ± 3 80 ± 9 107 ± 7 27 ± 3 97 ± 1 83 ± 2

µg/L

0.1 70 ± 33 103 ± 8 86 ± 5 79 ± 7 105 ± 3 81 ± 20 100 ± 25 18 ± 14 88 ± 6 117 ± 11 23 ± 7 99 ± 9 96 ± 1

0.05/0.05 0.02/0.05 0.05/0.05 0.05/0.05 0.10/0.10 0.10/0.20 0.10/0.20 0.10/0.10 0.10/0.10 0.10/0.10 0.10/0.10 0.05/0.05 0.05/0.05

a

Analyte quantitated at 275 nm. ND = below LOD. c Analyte quantitated at 250 nm. Reprinted with permission from Patsias and Papadopoulou-Mourkiduo (32). b

Conditioning: 2min 700 µl/min, 10:90 EtOH: H2O

7

∗ 2

Injection: 0−1 min 700 µl/min, 80:20 MeOH: H2O

1 5

Washing: 1−3 min 700 µl/min, 10:90 EtOH: H2O in backflush mode

3 4

6

LC run: 15 min LC separation: 3−15 min 50 µl/min, 80:20 MeOH: H2O

0

Transfer: 3.6−11.2 min 50µl/min, 80:20 MeoH: H2O T = 49 °C, SVE open, He 600 ml/min GC run: 58 min GC separation: 49 °C (15 min) 125 °C 300 °C (4 min), 25 °C, 3 °C and 25 °C/min, He 2.2 ml/min, SVE closed at 11.28 min

Washing the LC column: 5 min 700 µl/min, 100 MeOH

20

40 Time, min

60

Figure 2. HPLC-GC analysis of a wine sample spiked with the pesticides 100 µg/L, endosulfan isomers 50 µg/L, both. Peaks: 1 = vinclosolin, 2 = quinalphos, 3 = procymidone, 4 = α-endosulfan, 5 = β-endosulfan, 6 = carbophenthion (internal standard) and 7 = tetradifon. Reprinted with permission from ¨ Hyötylainen et al. (33).

Organophosphorus Pesticides

∗

Figure 1. Analytical procedure for analysis of pesticides in red wine. ∗ The steps can be carried out during previous GC program. ¨ Reprinted with permission from Hyötylainen et al. (33).

Many preconcentration techniques have found successful application in the residue analysis of organosphosphorus pesticides. Thus, the use of cloud-point preconcentration is useful for the enrichment of organophosphorus pesticides from water (35). Another procedure applied C18 and styrene-divinylbenzene SPE supports for the same purpose and separated and quantitated the preconcentrated pesticides by HPLC and high flow pneumatically assisted electrospray mass spectrometry (36). Polar organophosphorus pesticides were also

350


Table 4. Red Wines Studied

80 Pesticides Found

USA: Carigan, Barbera, Ruby Cabernet, Carnelian Spain: Garnacha Italy: Corina Veronese, Rondinella, Molinara, Rossignola, Negtata Chile: Cabernet Sauvignon Kypros Hungary: Kékfrankos Bulgaria: Merlot and Pinot Noir Spain: Tempranillo, Garnacha Italy: mixture

France: mixture Italy: Schiava, Merlot, Lambrusco

NF NF NF NF NF Vinclosolin 8 µg/L NF Vinclosolin 10 µg/L Vinclosolin 10 µg/L Tetradifon 25 µg/L Procymidone 36 µg/L Tetradifon 30 µg/L Tetradifon 27 µg/L

NF = no pesticides found. ¨ Reprinted with permission from Hyötylainen et al. (33).

Table 5. Recovery of Aldrin, Dieldrin, Endrin, and Heptachlor, and p,p-DDT, from Blank Spiked Samples Standard

Amounta %

Recovery (Mean)

S.D.

n

Endrin Dieldrin Heptachlor p,p -DDT Aldrin

5.0 5.0 5.0 6.5 5.0

µg µg µg µg µg

94.2 94.9 93.0 101.5 61.2

2.94 4.11 6.88 7.07 3.75

3 4 3 4 4

UV absorbance, 215 nm

Wine: Country and Grape

40

0 4

80

3 1 6 2

40

5

7

8

0 0

10

20 Retention time, min

30

Figure 3. On-line SPE-LC-DAD chromatograms at 215 nm of 100 mL of Llobregat river blank (upper part) and spiked with a mixture of triazine and organophosphorus pesticides at 1 ng/mL (lower part). Peak numbers: 1 = deethylatrazine, 2 = simazine, 3 = atrazine, 4 = propazine, 5 = parathion-methyl, 6 = fenitrothion, 7 = diazinon, and 8 = chlorpyrifos. Reprinted with permission from Lacorte et al. (38).

Limit of Detectionb 0.5 0.6 0.4 0.1 0.8

µg/g µg/g µg/g µg/g µg/g

S.D. = Standard deviation. a Amount of pesticide standard that blank sample was spiked with for recovery experiments. b Limit of detection defined as concentration of analyte that yields a signal-to-noise ratio of 1 : 3 at 224 nm. Reprinted with permission from Grice et al. (34).

determined in water by SPE combined with HPLCionspray spectrometry (37). An automated on-line SPE-HPLC/DAD method was employed for the determination of organophosphorus and other pesticides in river water using a C18 precolumn (10 × 2 mm I.D.) and a sample volume of 100 mL (38). Water samples were filtered twice before analysis. Measurements were carried out on a C18 analytical column (150 × 4.6 mm I.D.). Initial mobile phase composition was ACN : water 5 : 95 (v/v) at a flow rate of 0.1 mL/min. The flow rate was increased to 1 mL/min in 1 min, and the concentration of ACN in the mobile phase was enhanced to 95% vol. in 31 min followed with a final hold of 5 min. Pesticides were detected at 215, 250, and 275 nm, and identified by MS in a separate experiment. Characteristic chromatograms of blank and spiked samples are shown in Figure 3. Some interfering impurities were found on the chromatogram; however, it was assumed that the system is suitable for the measurement of these pesticides at very low concentrations (detection limit 30–100 ng/L). Repeatability and reproducibility of the method using HPLC-grade and river waters are compiled in Table 6.

Organophosphorus pesticides have also been determined in water by an off-line SPE-HPLC-APCI-MS method (39). Analysis was performed on a C8 column (250 × 4.6 mm I.D., particle size 5 µm). Mobile phase consisted of methanol-water acidified with 0.1 M acetic acid, methanol concentration varying from 30% to 90% in 30 min at a flow rate of 1.2 mL/min. A chromatogram of spiked ground water is shown in Figure 4. Some validation parameters of organophosphorus pesticides applying the optimized method are compiled in Table 7. The detection limit of pesticides was lower than the limits defined by the European Union; therefore, the method was proposed for the analysis of these pesticides in water. Off-line SPE combined with HPLC-DAD was also employed for the investigation of the decomposition of organophosphorus pesticides in estuarine, river, and groundwaters (40). The preconcentration capacity of various SPE sorbents such as styrene-divinylbenzene, C18 , and sulfonated styrene-divinylbenzene was compared, and styrene-divinylbenzene was selected because it gave highest recoveries (77–105%). Separation was carried out on a C18 column (150 × 4.6 mm I.D.). Mobile phase components were water (1% acetic acid) and ACN (1% acetic acid). Gradient elution began with 70% water during 10 min, and increased to 100% ACN in 15 min, final hold 5 min. Flow rate was 1 mL/min. HPLC/APCI/MS applied (0.5% acetic acid) and ACN (0.5% acetic acid). Gradient elution began with 70% water and increased to 100% ACN in 30 min, 5 min hold, at a flow rate of 1 mL/min. Chromatograms are shown in Figure 5. The method can be employed for the simultaneous determination of pesticides and their decomposition products in natural waters. The halflives of some organophosphorus pesticides determined in different waters are compiled in Table 8. It was found that photolysis exerted the highest effect on the transformation


351

Table 6. Repeatability and Reproducibility (Expressed as the Coefficient of Variation) of the Method Using 100 mL of HPLC-Grade Water and Llobregat River Water Spiked at 1 ng/mL (n = 5) Milli-Q Water Repeatability Reproducibility

Compound Deethylatrazine Simazine Atrazine Propazine Parathion-methyl Fenitrothion Diazinon Chlorpyrifos

1.1 0.7 2.1 3.1 0.1 7.7 n.c. 2.6

Llobregat River Water Repeatability Reproducibility

9.7 12.9 10.1 6.3 2.5 1.4 2.4 14.2

10.2 23.8 3.8 8.9 4.0 4.6 4.7 4.1

2.3 49 3.5 13.1 5.2 6.3 10.2 12.2

n.c. = not calculated. Reprinted with permission from Lacorte et al. (38).

% 100

Table 7. Percentage Recovery and Standard Deviation of the Studied Pesticides After Preconcentration of 200 mL of Ground Water Spiked at 0.2 µ/L on ENV and LiChrolut Cartridges

4

10

3 1 2

5

7 6

Recovery (%)

11

89 50

0

10 20 Retention time, min

30

Figure 4. HPLC-APCI-MS chromatogram in positive ionization mode that corresponds to spiked groundwater (at a level of 0.2 µg/L) preconcentrated onto LiChrolut EN cartridges. Acquisition was performed in scan mode. Peak identifications: 1 = metamidofos, 2 = acephate, 3 = vamidothion sulfoxide, 4 = vamidothion, 5 = trichlorfon, 6 = paraoxon-methyl, 7 = fensulfothion, 8 = parathion-methyl, 9 = fenitrothion, 10 = azinphos-ethyl, and 11 = fenthion. rt = Retention time (min). Reprinted with permission from Lacorte et al. (39).

rate of pesticides and the chemical structure of pesticides considerably influenced the decomposition rate. The decomposition of chlorpyrifos in water was studied in detail using GC/ECD, GC/MS, and HPLC/MS. Degradation products were preconcentrated on C18 SPE disks before separation steps. The decomposition products 3,5,6-trichloro 2-pyridinol, O-ethyl-O-(3,5,6-trichloro2-pyridyl) phosphorothioate, and O,O-diethyl-O-(3,5,6trichloro-2-pyridinyl) phosphate were identified (41). As the GC-MS libraries cannot be used for LC-MS, the construction of HPLC-API-MS library, including a considerable number of pesticides, has been recently reported (42). Organophosphorus pesticides have also been determined not only in water, but also in food products by HPLC. Thus, a multiresidue technique was developed for the determination of 28 organophosphorus pesticides and three metabolites in crops (43). Pesticides were extracted with ACN, the organic phase was passed through a graphitized carbon black SPE cartridge, and

Compound

m/z

ENV

Acephate Azinphos-ethyl Fenitrothion Fensulfothion Fenthion Metamidophos Naled Paraoxon-methyl Parathion-methyl Trichlorfon Vamidothion Vamidothion sulfoxide

143 160 125 157 231 94 127 234 109 109 146 241

154 ± 6 63 ± 17 58 ± 10 95 ± 5 21 ± 23 31 ± 11 n.d. 56 ± 11 46 ± 6 n.d. 72 ± 10 46 ± 3

LiChrolut 125 ± 15 132 ± 9 76 ± 10 122 ± 5 32 ± 12 24 ± 15 n.d. 69 ± 12 76 ± 5 46 ± 15 83 ± 15 106 ± 3

LOD (pg) 104 50 120 30 200 60 250 180 210 70 110 80

Acquisition was performed in the SIM mode at the mass indicated, and the LODs were calculated from direct injection of a standard. Reprinted with permission from Lacorte et al. (39).

then the pesticide residues were backflushed with 6 mL of methanol-dichloromethane (20 : 80, v/v). The eluate was evaporated to dryness and redissolved in the mobile phase. HPLC/DAD separation was performed on C18 column using gradient elution. Chromatograms of the extract of blank and spiked apple are shown in Figure 6. The recoveries were relatively high (61–96%), and the relative standard deviations low (5–10%). Detection limits varied considerably among the individual pesticide residues (3 to 493 ng/g). Azamethiphos residues were quantitatively determined in salmon tissue by HPLC-fluorescence detection (44). Issues were extracted with ethyl acetate, centrifuged, dried with anhydrous sodium sulfate, redissolved in water, and extracted again with n-hexane. Azamethiphos in the aqueous phase was preconcentrated on a C18 SPE cartridge, eluted with methanol, dried again, and redissolved in water-ACN (90 : 10, v/v). Analysis was carried out on a C18 column (250 × 3.2 mm I.D., particle size, 5 µm) using isocratic elution (ACN-water, 32 : 68, v/v). The excitation and emission wavelengths

352

CHROMATOGRAPHY, HPLC (a)

3

AU

1 4 2

(b)

5

AU Abundance, %

(c)

9

6

8

7

123

5

100

7

8

10 50 11

1 2 34

9

0 0

10

20 Time, min

Figure 5. HPLC chromatograms for the injection of 20 µL of the extract obtained by SPE of 100 mL of ground water spiked at 40 µg/L in target compounds at t = 0 with DAD detection (a) and after 2 weeks with DAD detection (b)) and with APCI-MS detection in SIM mode (c). Detected compounds were 1) parathion-methyl, 2) fenitrothion, 3) parathion-ethyl, 4) pentachlorophenol, 5) 4-nitrophenol, 6) paraoxonmethyl, 7) 3methyl-4-nitrophenol, 8) fenitrooxon, 9) paraoxonethyl, and 10) Smethylisomer of fenitrothion. Reprinted with permission from Castillo et al. (40). Table 8. Half-lives (t1/2 ) in Days of Some Organophosphorus Pesticides and Pentachlorophenol and Selected Transformation Products in Estuarine Water (EW), River Water (RW), and Ground Water (GW) Exposed to Sunlight Using Off-line SPE and HPLC/DAD Parent Compounds Parathion-methyl Fenitrothion Parathion-ethyl Pentachlorophenol Transformation products 4-Nitrophenol 3-Methyl-4-Nitrophenol Paraoxon ethyl Paraoxon methyl

t1/2 in GW

t1/2 in EW

t1/2 in RW

3 1 2 0.07

4 1 2 0.07

4 1 3 0.08

n.q. 0.4 0.5 n.q.

5 3 4 n.q.

5 4 4 n.q.

n.q.: not quantified due to coelution problems. Reprinted with permission from Castillo et al. (40).

were set to 230 and 345 nm, respectively. Recovery values are compiled in Table 9. It was established that the method is simple, rapid, and requires only a small quantity of organic solvents; therefore, its application is highly advocated. Carbamate Pesticides Because of the considerable importance of carbamate derivatives in up-to-date agrochemical practice, numerous

HPLC methods have been developed and employed for their determination in various matrices. Supercritical fluid extraction (SFE) followed by RP-HPLC has been used for the measurement of carbamate pesticides in soils and cereals. The results of SFE were compared with those obtained by traditional liquid–liquid extraction (45). The optimal conditions for SFE extraction of pesticides are compiled in Table 10. The trap contained stainless steel beads. Analysis was carried out on a C18 column (250 × 4.6 mm I.D., particle size, 5 µm) employing gradient elution. Initial composition of the mobile phase was methanol-water (58 : 42, v/v) for 2 min, and methanol concentration was increased in steps of 6, 2, and 8 min with rates of 0.067, 10.8, and 1.25% methanol/min, respectively. Flow rate was 0.75 mL/min. Pesticides were derivatized postcolumn with orthophthalaldehyde (OPA) and detected with a fluorescence detector (excitation and emission wavelengths were 330 and 465 nm, respectively). The method is not suitable for the extraction of aminocarb from soil, but it is appropriate for its extraction from cereals. The recoveries of propoxur, carbaryl, and methiocarb from various soils are compiled in Table 11. The data suggest that the type and composition of soil exert a marked influence on the recovery of carbamate pesticides by SFE. Accelerated solvent extraction (ASE) and RP-HPLC has been applied for the measurement of N-methylcarbamate pesticides in fruits also (46). Samples were homogenized, an aliquot of 5 g was blended with 6 g of Extrelut particles, and the mixture was used for ASE. ASE conditions were extraction temperature, 100 ◦ C; extraction pressure, 2000 psi; preheating period, 5 min; static extraction, 5 min; extraction solvent, 60 mL of acetonitrile; solvent flash, 19.8 mL; nitrogen purge, 60 s. Two grams of NaCl was added to the extract and shaken for 10 min, and then the phases were separated and the organic phase was dried with anhydrous sodium sulfate. N-methylcarbamate pesticides were preconcentrated on a carboxylic acid column (500 mg). Separation was carried out on a C18 column (150 × 4.6 mm I.D.) using a methanol-water gradient (18% methanol between 0 and 10 min; 18% and 70%, 10 and 40 min; 7% and 90%, 40 and 43 min; 90%, 43 and 60 min; 18%, 60 and 75 min). The column was thermostated at 50 ◦ C, and the flow rate was 0.8 m/min. Pesticides were postcolumn derivatized with OPA and detected by a fluorescence detector (excitation and emission wavelengths 340 and 445 nm, respectively). The efficiency of the ASE method was compared with that of traditional methanol extraction (10-g sample extracted with 20 mL of methanol). The efficacy of the mini-column cleanup is shown in Figure 7. The recoveries of N-methylcarbamate pesticides from various matrices are listed in Table 12. The data indicate that the method is very suitable for the determination of N-methylcarbamate pesticides in these selected foods. However, it was established that citrus fruits (grapefruit, lemon, and orange) show a natural fluorescence that interferes with the measurement. This drawback was partly eliminated by changing the parameters of the mini-column cleanup. Recoveries using the method were commensurate with those obtained by the commonly used methanol extraction; that is, ASE can substitute the liquid–liquid extraction


353

Figure 6. Chromatograms of a blank apple sample (a) and the same sample spiked with 27 of the 31 organophosphorus at the following concentration levels:

13 7 5 6 4

3 1 2

0

20

29 1719 10 111216 21 30 18 9 23 14 25 26 27 8 28 31

40

60

Time, min

Table 9. Recoveries of Azamethiphos from Spiked Salmon Samples Recovery, ppb, from Samples Spiked at Indicated Level Set 1 2 3 4 5 6 Mean Rec.% RSD, %

Control NDa ND ND ND ND ND ND

5.2 4.25 4.86 4.05 4.11 4.31 4.97 4.43 85.3 8.1

10.4 —b 8.87 7.90 9.04 8.98 9.48 8.94 86.0 5.7

20.8 19.2 17.9 18.4 16.9 18.7

41.6 37.0 37.3 33.8 36.4 36.7

83.2 69.0 71.7 69.4 71.3 70.6

18.2 87.6 4.8

36.2 87.1 3.9

70.4 84.6 1.7

ND = not detected. b Sample lost in rotary evaporator. Reprinted with permission from Pfenning et al. (44). a

method for the determination of this class of pesticides in foods. The application parameters of HPLC-atmospheric pressure chemical ionization/mass spectrometry, and HPLC-postcolumn fluorometry were compared for the

1 = paraoxon-methyl 500; 2 = malaoxon 6400; 3 = paraoxon-ethyl 800; 4 = methidathion 500; 5 = azinphos-methyl 200; 6 = phosmet 200; 7 = parathion-methyl 800; 8 = malathion 2000; 9 = triazophos 200; 10 = fenitrothion 800; 11 = azinphos-ethyl 200; 12 = chlorfenvinphos 200; 13 = quinalphos 200; 14 = parathion-ethyl 400; 16 = etrimphos 1000; 17 = diazinon 800; 18 = coumaphos 800; 19 = fonofos 200; 21 = phoxim 400; 23 = chlorpyriphos-methyl; 24 = disulfoton 2000; 26 = isofenphos 500; 27 = fenchlorophos 500; 28 = temephos 400; 29 = chlorpyriphos-ethyl 200; 30 = pyrimiphos-ethyl 200; 31 = carbofenthion-ethyl 400. The chromatography was carried out with the ‘‘Alltima’’ column using CH3 CN/H2 O as mobile phase in gradient elution: CH3 CN from 28% to 39% in 20 min, and then from 39% to 88% in 40 min. Reprinted with permission from Lagana et al. (43).

Table 10. SFE Working Conditions CO2 density Pressure CO2 flow-rate Chamber temperature Equilibration time Extraction time Nozzle temperature Trap temperature Rinsing solvent volume Rinsing solvent flow-rate

0.90 g/mL 378 bar 2.0 mL/min 54 ◦ C 1.00 min 30.00 min a. 45 ◦ C b. 35 ◦ C a. 30 ◦ C b. 35 ◦ C 1.4 mL 0.5 mL/min

a. Extraction step. b. Rinsing step. Reprinted with permission from Izquierdo et al. (45).

determination of 11 carbamate pesticides in apple, cauliflower, potato, lettuce, and celery. Samples were extracted with methanol, and the methanol phase were cleaned up on a charcoal-Celite column. Analysis was performed on a C8 column with acetonitrile-water

354

CHROMATOGRAPHY, HPLC Table 11. Recovery of Carbamate Pesticides from Spiked Soil Samples % Recovery ± s.d. Sample Soil 1 Soil 2 Soil 3 Soil 4 Soil 5 Soil 6 Soil 7

Propoxur

Carbaryl

Methiocarb

92 ± 2 58 ± 6 40 ± 8 78 ± 6 77 ± 9 81 ± 9 79 ± 7

92 ± 5 59 ± 9 42 ± 2 68 ± 1 86 ± 5 90 ± 11 80 ± 10

64 ± 14 51 ± 15 48 ± 1 49 ± 3 59 ± 8 84 ± 10 60 ± 11

A: before cleanup A: after cleanup

Reprinted with permission from Izquierdo et al. (45).

gradient. It was established that both detection methods provide similar results; therefore, they are suitable for the measurement of these pesticides in the concentration range of 10–100 ppb. The coefficient of variation of recoveries varied between 11% and 33% for fluorescence detection, and 9% and 26% for MS (47). N-methylcarbamate pesticides have also been measured in the ACN extracts of green pepper using an activated carbon membrane on-line cleanup and RP-HPLC separation with postcolumn derivatization with OPA (48). Separation was carried out on a C18 column with methanol-water gradient elution. It was found that an activated carbon membrane retained the majority of impurities. Various cleanup procedures have also been compared for the preconcentration of N-methylcarbamate insecticides from potato and carrot for RP-HPLC analysis (49). The scheme of the extraction and partitioning processes are shown in Figure 8. Adsorbent columns of 200 × 20 mm I.D. were filled with silica, alumina, Florisil, and silanized Celite-charcoal 4 : 1 w/w. SPE cartridges were reversedphase C8 , C18 , and CN; direct-phase SPE cartridges were silica, CN, and NH2 . Separation was made on a C18 column (125 × 4 mm I.D., particle size, 5 µM). The mobile phase consisted of ACN-water (30 : 70, v/v), and the flow rate was 1.0 mL/min. Analytes were detected at 195 nm. Recoveries of N-methylcarbamate insecticides from potato and carrot employing the most efficient enrichment techniques are compiled in Table 13. The data suggest that each preconcentration and cleanup method can be used for the measurement of N-methylcarbamate insecticides in potato and carrot. Detection limits were between 0.5 and 7.5 ng. HPLC/particle beam/MS has also found application in the assessment of carbaryl, baygon, methiocarb, and methiocarb sulfoxide in lettuce and apple (50). Samples were mixed with dichloromethane and then filtered, evaporated to dryness, redissolved in methanol, filtered again, and used for HPLC. Analysis was performed on a C18 column using methanol-0.05 M sodium acetate (pH adjusted to 5 by acetic acid) (70 : 30, v/v). Recoveries were 53 to 72%, and detection limits in SIM (single ion monitoring) mode varied between 5 and 20 ng. The use of automated in-tube solid-phase microextraction (SPME) prior to HPLC analysis of carbamate pesticides has been recently reported (51). Pesticides were

5

11 14 16

10 7

B: before cleanup B: after cleanup 13 6

0

20 Retention time, min

8

15

9

17

40

Figure 7. Cleaned-up chromatograms of grapefruit fortified with 0.2 µ/g of each pesticide. A, grapefruit fortified group A mixture; B, grapefruit fortified group B mixture. Peak identification: 1, butoxycarboxim; 2, oxamyl; 3, methomyl; 4, dioxacarb; 5, metolcarb; 6, propoxur; 7, bendiocarb; 8, carbofuran; 9, carbaryl; 10, xylylcarb; 11, XMC; 12, pirimicarb; 13, isoprocarb; 14, trimethacarb; 15, fenobucarb; 16, methiocarb; and 17, promecarb. Reprinted with permission from Okihashi et al. (46).

enriched in coated GC capillaries and transferred to a C18 column (100 × 8 mm I.D., particle size, 4 µm). Pesticides were eluted under isocratic conditions (ACN-water, 50 : 50, v/v; flow rate 1.4 mL/min) and were detected at 220 nm. The extracted amounts of pesticides are compiled in Table 14. It has been stated that the method is


355

Table 12. Mean Recoveries of 17 N-methylcarbamate Pesticides in Food with ASE Banana

Pesticide Butoxycarboxim Oxamyl Methomyl Dioxacarb Metolcarb Propoxur Bendiocarb Carbofuran Carbaryl Xylylcarb XMC Pirimicarb Isoprocarb Trimethacarb Fenobucarb Methiocarb Promecarb

Green Beans

Broccoli

Melon

Carrot

Recovery (%)a

RSD (%)

Recovery (%)a

RSD (%)

Recovery (%)a

RSD (%)

Recovery (%)a

RSD (%)

Recovery (%)a

RSD (%)

85.0 87.4 89.0 90.1 86.0 92.5 91.8 94.5 100.5 92.1 90.6 88.6 86.2 95.8 89.9 99.4 97.5

3.4 2.6 2.0 1.7 1.7 1.3 1.3 1.1 1.5 1.2 1.3 2.9 1.5 1.2 1.3 1.1 1.3

67.1 82.6 78.4 98.6 70.7 84.4 76.3 87.0 90.6 78.5 77.0 70.0 78.6 82.6 83.7 80.4 88.9

10.4 6.5 6.0 6.1 16.7 6.9 5.7 6.5 6.5 10.0 10.7 11.3 6.4 7.9 6.4 9.1 7.8

88.8 76.9 100.5 87.4 89.4 74.9 88.2 78.5 80.9 96.4 89.7 77.5 79.5 90.8 79.8 88.7 80.6

1.1 1.3 0.5 0.3 0.8 2.5 0.9 9.1 2.3 2.3 0.4 0.5 0.5 0.5 0.1 0.3 0.2

88.4 90.6 78.4 85.8 61.5 78.1 80.5 87.2 93.8 78.4 74.7 82.2 68.2 82.6 76.0 94.3 83.3

3.8 5.7 9.2 6.9 25.6 6.6 4.1 4.3 6.9 9.1 11.0 2.9 9.7 5.7 7.3 2.6 14.9

87.7 91.7 85.7 71.8 62.1 80.1 71.3 88.1 97.7 78.9 76.7 93.1 70.2 85.2 79.6 87.2 88.7

14.3 10.4 5.1 18.5 24.6 4.4 17.1 5.8 10.1 10.2 11.6 7.8 7.8 6.3 4.1 8.3 5.3

a

Mean of three experiments. Mean of five experiments. Reprinted with permission from Okihashi et al. (46). b

simple, efficient, and selective and can be employed for the analysis of these carbamate derivatives in aqueous samples. Urea Pesticides Supported liquid membranes, new tools for the enrichment of pollutants present in low concentration, have been employed for the preconcentration of urea pesticides. It was reported that supported liquid membranes extracted efficiently phenoxy acids, sulfonylurea herbicides, and triazines from water for HPLC analysis (52). Phenylurea herbicides have also been determined in water using online sorptive enrichment, HPLC, and MS. The detection limit of the electrospray mass spectrometric detection was about 10 ng/L (53). Many HPLC methods have been developed and successfully applied for the residue analysis of urea pesticides in waters. On-line preconcentration of phenylurea and triazine herbicides from water and their subsequent separation has been reported (54). Analysis was performed on a C18 column (10 × 2 mm I.D., particle size 8 µm) combined with atmospheric pressure chemical ionization ion-trap mass spectrometry. The column was preconditioned with methanol and water, and then the water sample was loaded onto the column. After the preconcentration process, the column was washed again and the separation was carried out on the same column by the gradient elution shown in Table 15. It was found that this on-line shortcolumn LC-MS-MS method can be used for the detection of solutes at 0.1–1-µg/L level in 4 mL of river water. C18 SPE cartridges and HPLC/UV have also been employed for the measurement of phenylurea herbicides in drinking water (55). Aliquots of 500-mL volume were passed through the C18 cartridges (flow rate of 9 mL/min), and the pollutants were eluted with 1.5 mL of ACN.

Separation was carried out on a C18 column (250 × 4 mm I.D., particle size 5 µm) using gradient elution using 0.01 M phosphate buffer (pH 7) and ACN. Some validation parameters (recovery, reproducibility, detection limit) of the method are compiled in Table 16. It was concluded from the data in Table 16 that the method is suitable for the determination of phenylurea herbicides in drinking water. Immunosorbents have also found application for the selective on-line preconcentration of phenylurea herbicides from environmental waters (56). Unpurified polyclonal antibodies against isoproturon and chlorotoluron were immobilized on aldehyde-activated porous silica support, and a precolumn of 50 × 4.6 mm I.D. was filled with the mixture of these immunosorbents. Samples were filtered and passed through the precolumn (flow rate of 2 mL/min). Separation was carried out on a C18 column (250 × 4.6 mm I.D., particle size 5 µm) employing gradient elution: from 65% A (0.005 M KH2 PO4 , pH 2 adjusted with acetic acid) and 35% ACN to 25% A in 20 min at a flow rate of 1 mL/min. Solutes were detected at 244 nm. Chromatograms of spiked ground- and river water are shown in Figure 9. The detection limit was found to be between 0.01 and 0.03 µg/L by extracting 10 mL of water. It was stated that the high specificity of the immunosorbent makes it suitable for the enrichment of special pesticides from waters. On-line SPE and HPLC/DAD has been used for the simultaneous detection of triazine and phenylurea herbicides in drinking water (57). Pesticides were enriched on styrene-divinylbenzene copolymer sorbents and separated on a C18 column (150 × 4.6 mm I.D., particle size 3.5 µm) using gradient elution composed of ACN and 1 mM ammonium acetate. The gradient program is compiled in Table 17. The recoveries from tap water

356


Figure 8. Scheme of extraction and partitioning procedures before HPLC-UV analysis of the selected N-methylcarbamate insecticides in potato and carrot samples. Reprinted with permission from Nunes et al. (49).

spiked at 0.090 µg/L are listed in Table 18. It was found that the method reduces run-times required for measurements and increases selectivity compared with other HPLC techniques. The phenylurea herbicide isoproturon was selectively preconcentrated from various matrices such as water,

plasma, and urine by employing an antiserum covalently bonded to aldehyde-activated silica. The herbicide was eluted from the sorbent with ethanol-phosphate buffered saline (pH, 7.2–7.4) 50 : 50 v/v, analyzed on a C18 column under isocratic conditions (mobile phase: methanol-water 70 : 30, v/v). Detection limits were 50 ng/L and 5 ng/L for samples of 50 mL and 1 L of volume (58). Another HPLC method was used for the simultaneous measurement of imidazolinone, sulfonylurea, and sulfonamide herbicides in surface water. Pesticides were enriched on a polystyrene-divinylbenzene sorbent after acidification, eluted with methanol, purified on an anion exchange cartridge, and analyzed on a C8 column. Mobile phases for gradient elution consisted of ACN and water containing 0.15% acetic acid. Solutes were detected with electrospray MS. Recovery values were 70 to 114%, and the relative standard deviation was lower than 13%. The limit of quantitation was as low as 0.1 ppb (ng/mL) (59). A different HPLC procedure has been used (immunosorbents coupled on-line with HPLC/MS) for the analysis of triazine and phenylurea herbicides in natural waters and sediments (60). Sediment samples of 10-g weight were Soxhlet extracted (100 mL of methanol, 12 h), and the methanol phase was preconcentrated, diluted with groundwater, and analyzed as a water sample. Immunosorbent was conditioned with 6 mL of 0.01 M sodium phosphate buffer containing 0.15 M NaCl (pH 7.4) and 0.2% azide and 3 mL of water. Sample aliquots of 20 mL were circulated on the immunosorbent cartridge (80 mg of bonded silica), and then they were eluted with the HPLC mobile phase. Solutes were separated on a C18 column (250 × 4.6 mm I.D., particle size, 5 µm). Mobile phase consisted of ACN-water with 0.01 M ammonium acetate for triazines, and with 0.5% acetic acid for phenylurea derivatives. A chromatogram of phenylurea herbicides is shown in Figure 10. The chromatogram indicates that a sample volume as small as 20 mL is sufficient for the separation and quantitation of these herbicides in sediments and water. The recovery and repeatability values are compiled in Table 19. Except for deisopropylatrazine and diflubenzuron, other herbicides were quantitatively retained on the immunosorbent support. It was reported that the procedure is reliable and can be used for the determination of herbicides in both sediments and water.

Table 13. Recovery of the N-methylcarbamate Insecticides in Potato and Carrot Samples Recovery (%) Potato

Carrot

Compound

DA

SCC

CN

DA

SCC

CN

Methomyl Aldicarb Carbofuran Propoxur Carbaryl

92(5.5) 89(7.0) 85(5.8) 91(6.0) 79(4.2)

91(4.9) 91(4.9) 82(4.5) 87(4.0) 91(2.6)

90(4.5) 90(4.5) 90(5.6) 92(4.7) 89(5.4)

—a —a 93(6.8) —a 80(4.6)

—a —a 92(8.0) —a 90(7.5)

—a —a 91(3.8) —a 87(4.3)

DA, deactivated alumina; SCC, silanized Celite-charcoal; CN, normal phase cyanopropyl cartridge; R.S.D.s (%) in parentheses (n = 3). a Not recommended for protection of carrots. Reprinted with permission from Nunes et al. (49).


357

Table 14. Amounts Extracted with Different Capillaries (ng) After 25 Aspirate/Dispense (a/d) Steps of 25-µL Volume at a Flow Rate of 63 µL/min, n = 6; the Concentration of the Aqueous Sample Was 2000 µg/L for Each Compound GC Capillary Fused silica SPB-1 PTE-5 SPB-5 Supelcowax Omegawax 250

Carbaryl

Propham

Methiocarb

Promecarb

Chlorpropham

Barban

13 ± 1 6±1 21 ± 1 15 ± 1 35 ± 3 173 ± 5

24 ± 1 46 ± 1 42 ± 1 72 ± 3 32 ± 3 159 ± 9

29 ± 2 55 ± 1 57 ± 3 71 ± 4 61 ± 3 248 ± 6

20 ± 1 42 ± 1 35 ± 1 56 ± 5 22 ± 3 114 ± 5

29 ± 1 126 ± 3 112 ± 5 202 ± 6 94 ± 6 323 ± 8

43 ± 1 125 ± 3 197 ± 6 166 ± 6 271 ± 5 468 ± 9

Reprinted with permission from Gou et al. (51).

Table 15. Gradient HPLC Conditions

Triazines

A-B (80 : 20, v/v) to (50 : 50, v/v) in 5 min 0.5

Phenylureas

A-B (95 : 5, v/v) to (50 : 50, v/v) in 5 min, held 2 min. 0.5

a

Eluents: (A) Water-methanol (95 : 5, v/v) and (B) watermethanol (5 : 95, v/v). Reprinted with permission from Hogenboom et al. (54).

0.0025 ∗ Absorbance, 244 nm

Compounds

(a)

Linear Gradient Elution Conditionsa Flow-rate (mL/min)

Table 16. Recovery, Reproducibility, and Detection Limit of Phenylurea Herbicides

Fenuron Monuron Metabenzthiazuron Fluometuron Diuron Chlorbromuron Chloroxuron Neburon Metoxuron Chlorotoluron Isoproturon Buturon

Recovery (%)

Reproducibility LOD (µg/L)

C.V. (%), n=5

32.6 98 98 99 99 93 95 99 100 103 105 150

3.1 3.6 3.4 5.6 2.7 4.7 5.3 5.4 5.3 6.6 7.0 7.7

5.4 3.8 12.4 5.3 5.2 6.6 4.5 43.6 13.8 14.0 9.7 15.6

23 4

5

−0.0025 0

20

10 Time, min

(b) 0.0025

∗

Absorbance, 244 nm

Compound

1

1 23 4

5

Reprinted with permission from Sanchis-Mallols et al. (55).

Concentration of herbicides determined in real samples are compiled in Table 20. Antifouling pesticides have been determined in seawater by off-line SPE followed by HPLC-APCI/MS (61). Pesticides and pesticide byproducts were preconcentrated from 500 mL of water on graphitized carbon black (500 mg), ethylvinylbenzene-divinylbenzene copolymer (200 mg), and styrene-divinylbenzenecopolymer (200 mg) SPE supports. A carbon black cartridge was conditioned with 6 mL of dichloromethane-methanol (8 : 2, v/v), 9 mL of methanol, and 9 mL of HPLC grade water. Polymer cartridges were conditioned with 6 mL of methanol and 6 mL of HPLC grade water. After preconcentration analytes were eluted with methanol and dichloromethanemethanol mixture, evaporated to dryness, and redissolved

−0.0025 0

10

20

Time, min

Figure 9. HPLC-UV traces obtained at 244 nm after on-line immunoextraction of 10 mL of ground (a) and river (b) water spiked with 0.05 µg/L of each phenylurea herbicides. Peak numbers: 1 = chlorotoluron, 2 = isoproturon, 3 = metobromuron, 4 = linuron, and 5 = chlorbromuron. The asterisk indicates an impurity arising from the synthesis of the immunosorbent. Reprinted with permission from Martin-Esteban et al. (56).

in 1 mL of ACN. Pesticides were separated on a C18 column (75 × 4.6 mm I.D.; particle size 4 µm) using a methanol-water gradient (from 30% methanol to 100% in 10 min, final hold 2.5 min). The highest recovery values

358

CHROMATOGRAPHY, HPLC Table 17. HPLC Gradient Program Time (min)

% Acetonitrile

Flow (mL/min)

0.00 30.00 49.00 50.00 52.00 52.50 56.00 57.0 58.00

10.0 30.0 59.0 100.0 100.0 100.0 100.0 100.0 10.0

1.000 1.000 1.000 1.000 1.000 2.000 2.000 1.000 1.000

Reprinted with permission from Mills et al. (57).

Table 18. Recoveries in Tap Water, at Wavelengths Selected for Routine Operation

Atrazine Chlorotoluron Diuron Isoproturon Linuron Methabenzthiazuron Propazine Simazine Terbutryn Trietazine

Wavelength (nm)

% Recovery of 0.090 µ/L Spiked Tap Water

220 240 250 240 250 240 240 220 230 230

101.8 97.5 104.5 111.8 103.8 98.0 100.3 100.8 102.1 108.1

Reprinted with permission from Mills et al. (57).

1

Ion intensity, %

100

2 3 4

5

0 0

10

20

Time, minutes Figure 10. On-line SPE of 20 mL of groundwater sample certified by Aquacheck with a mixture of herbicides through an anti-chlorotoluron immonosorbent followed by LC/APCI/MS in PI mode of operation and under SIM conditions. Peaks: 1) monuron; 2) chlorotoluron; 3) isoproturon; 4) diuron; 5) linuron. Reprinted with permission from Ferrer et al. (60).

were obtained on a graphitized carbon black support. The parameters of the calibration equation and the lowest limit of detection (LOD) are compiled in Table 21. It was assumed that the method could be routinely employed for monitoring antifouling pesticides and their byproducts in seawater samples. The performance of UV and APCI/MS detection has been compared for the determination of phenylurea

Table 19. Recoveries of Extraction (%) and Repeatability (Relative Standard Deviation Among Replicates, n = 5)a Compound Deisopropylatrazine Deethylatrazine Simazine Atrazine Deuterated atrazine Propazine Terbutylazine Irgarol Monuron Chlortoluron Isoproturon Diuron Linuron Diflubenzuron

Recoveries (%)

Repeatability (%)

0 87 89 102 103 97 101 86 80 90 90 91 88 42

10 1 3 4 5 4 11 4 6 8 4 4 2

a Obtained after the percolation of 20 mL of groundwater spiked at 0.2 µg/L with a mixture of triazines and phenylureas through the anti-atrazine and antichlorotoluron immunosorbent, respectively. Reprinted with permission from Ferrer et al. (60).

herbicides in waters (62). Pesticides were extracted on a C18 SPE cartridge, or a large volume injection method was applied (LVI). Separations were carried out either on a single (100 × 4.6 mm. I.D.) or on coupled C18 columns (50 × 4.6 mm.I.D. + 100 × 4.6 mm. I.D.). Particle size was in both instances 3 µm. The mobile phase consisted of methanol-water gradient. The results are compiled in Table 22. The data obtained by the various methods were comparable; however, the higher sensitivity of LVI-LC-LCAPCI-MS made it the preferred method for the analysis of phenylurea herbicides in waters. Urea herbicides have been determined not only in various types of waters, but also in soils. Because of the adsorption of pesticides to the soil particles, sample preparation methods are more complicated than in the case of waters. Microwave-assisted solvent extraction (MASE) followed by RP-HPLC was employed for the measurement of some sulfonylurea herbicides in soil (63). Soil samples of 10 g were extracted with 20 mL of solvents at 60 ◦ C for 10 min. The amount of coextracted impurities was high in the case of 0.1 M NaHCO3 extracting solvent; therefore, MASE was used with 20 mL of dichloromethane-methanol (90 : 10, v/v). After extraction, the organic phase was dried over anhydrous sodium sulfate, evaporated to dryness, and redissolved in 100 µL of ACN and 900 µL of water. Separation was carried out on a C18 column (100 × 4.6 mm I.D., particle size, 3 µm) with an isocratic mobile phase of methanol-0.1% phosphoric acid (45 : 55, v/v), and solutes were detected at 226 nm. The chromatogram of five sulfonylurea herbicides under the experimental RP-HPLC conditions is shown in Figure 11. The recovery values and their relative standard deviations are compiled in Table 23. The data suggested that MASE connected to RPHPLC is a suitable technique for the analysis of a mixture of sulfonylurea herbicides in soil. The advantages of the


359

Table 20. Concentration Values (µg/kg) of the Pesticides Analyzed in Sediment Samples and in Seawater Samples (µg/L) After Analysis by Solid-Phase Immunosorbent Extraction Followed by LC/APCI/MS Compound Samples

Deethylatrazine

Atrazine

Linuron

Diuron

Irgarol

DARa

19.5 6.5 15.4 — — — —

33.6 19.2 39.2 — — — —

—c — — 139.0 59.2 — —

— — — — — 0.04 0.02

— — — — — 0.04 0.03

0.58 0.34 0.39

Sedimentsb 1 Sediment 2 Sediment 3 Sediment 4 Sediment 5 Seawaterd 1 Seawater 2 a

DAR, deethylatrazine-to-atrazine ration. Sediment samples were collected in the Elba Delta during 1990–1991. c —, not detected. d Seawater samples were collected in Masnou area during 1996. Reprinted with permission from Ferrer et al. (60). b

Table 21. HPLC-APCI-MS Calibration Data and LODs of the Analyzed Antifouling Agents in Seawater Samplesa (y = a · x + b) Compound 1-(3,4-Dichlorophenyl) urea Chlorothalonil Demethyldiuron Dichlofluanid Diuron Irgarol Irgarol byproduct Sea-nine 211 TCMTB

Calibration Parameters A B R2

LOD(ng/L)

194659

1933

0.9998

2.0

467260 567728 51248 2.106 68078 97395 3.106 23617

27268 11681 6907.9 19216 6917.6 1776.8 21870 1961

0.9875 0.9995 0.9912 0.9994 0.9918 0.9996 0.9977 0.9983

1.0 1.0 4.0 1.0 4.0 2.0 1.0 20.0

a SPE was carried out by passing 500 mL of seawater sample through the carbon cartridges. Calibration range from 25 to 2000 µg/L. Reprinted with permission from Martinez et al. (61).

method are the short extraction time, the negligible solvent consumption, and a low detection limit (5-µg/kg soil). A slightly different procedure was applied for the analysis of linuron and related compounds in soil (64). It was found that the moisture content of soil, its composition, the type of extracting solvent, and the time of storage equally influence the recovery, as demonstrated by the data in Table 24. It was established that MASE is a suitable extraction technique prior to RP-HPLC and that the combined method can be successfully used for the analysis of phenyl urea herbicides in soil. Phenylurea pesticides have been determined in biological matrices too. Thus, diflubenzuron and its metabolites (2,6-difluorobenzamide, 4-chlorophenylurea, 4-chlorophenylaniline) were measured in pine needles by SPE-HPLC/DAD (65). Pine needles (3 g) were shaken with 25 mL of ACN for 10 min, and then filtered and enriched on a SPE cartridge. Separation was carried out on a C18 column (150 × 4 mm I.D., particle size, 5 µm), and the isocratic mobile phase consisted of ACN-methanolwater (50 : 2 : 48, v/v) at a flow rate of 1.0 mL/min. Detection wavelength varied according to the absorption

maximum of the solute. Typical chromatograms are shown in Figure 12. Recoveries varied according to the type of SPE sorbent, as illustrated by the data in Table 25. The results prove that the highest recoveries can be achieved by using an aminopropyl silica cartridge. The validation parameters of the method are listed in Table 26. They establish the utility of the method and suggest that it can be used for monitoring these pesticides in pine groves. Triazine Derivatives Numerous HPLC methods were developed for the measurement of triazine herbicides and their decomposition products in environmental waters, employing various preconcentration techniques, HPLC systems, and detection procedures. Thus, the successful application of a supported liquid membrane (SLM) technique for the preconcentration of alkylthio-s-triazine herbicides has been reported (66). The scheme of the extraction device is shown in Figure 13. Water samples were buffered to pH 7.0 to contain the triazine derivatives in uncharged form. Uncharged molecules readily diffuse through the hydrophobic liquid membrane into the acceptor liquid containing 0.1 M aqueous H2 SO4 . The efficiency of undecane, dihexyl ether, and their mixture as membrane solvents was compared. Separations were performed on a C18 column (250 × 4.6 mm I.D.) with the isocratic mobile phase, 56% ACN-44% 0.05 M sodium acetate (adjusted to pH 7.0 with 0.5 M sulfuric acid) at a flow rate of 1.0 mL/min. Analytes were detected at 235 nm. Recovery values obtained with the three membrane solvents are compiled in Table 27. The detection limit was 0.03 µg/L at 1-µg/L concentration of pesticides. It was concluded that this technique can be employed for the measurement of this class of pesticides in water. An on-line SPE-HPLC method was developed and used for the determination of pesticides and phenolic compounds in natural waters (67). The extraction efficacy of a carbon black (sorbent A), a functionalized polymeric resin (sorbent B), and a highly cross-linked styrene-divinylbenzene copolymeric resin (sorbent C) was compared. Acidified samples (pH 2.5) were passed through

Table 22. Analysis of Reference Water Samples with Different Methodsa Concentration of Analyte (µg/L)b II III IV

Samplec

Ref.d

Ia

DW-1 DW-2 DW-3 SW-1 SW-2 SW-3 GW-1 GW-2

0.35 n.s. 0.22 n.s. 0.15 0.10 0.15 0.10

0.36 10 (dermal) mg/kg. Aldicarb acts by inhibition of cholinesterase. Aldicarb is a broad-spectrum systemic insecticide used for seed and soil treatment and as a nematicide and an acaricide. It is stable in water with a DT50 of 3240 days (pH 5.5 and 15 ◦ C) (7). The rate of hydrolysis increases under increasingly basic conditions (pH 9) with DT50 of ca. 75 days (8). Aldicarb readily oxidizes to the corresponding sulfone, aldoxycarb [1646-88-4] (mp 99 ◦ C, vp 12 mPa at 25 ◦ C), which is soluble in water to 10 g/L. The rat oral LD50 of aldoxycarb is 27 mg/kg, and it is a registered insecticide in its own right. The degradation of aldicarb in soils, plants, and mammals generates products that are based on the combination of hydrolytic, oxidative and hydration pathways, and the conjugation of products. Initial oxidation of the sulfide moiety is followed by oxidation to the sulfoxide and the sulfone. Hydrolysis of the sulfoxide and the sulfone affords the corresponding oximes and nitriles. These may be further converted to amides or acids. In soil, the major pathway was oxidation to the sulfoxide, and the sulfone was also formed to a lesser extent. These steps were followed by conversion to the oximes and nitriles, which in soils were ultimately mineralized. Pathways are shown in Figure 3. Aldicarb sulfoxide and sulfone occur as soil, mammalian, and plant metabolites. The major metabolic pathways are similar in plants and animals, with rapid oxidation to the sulfoxide and a lesser

CH3

CH3

CH3SCHCCH3

CH3CCHSCH3

NOCONHCH3

NOCONHCH3

E

Z

butocarboxim (25) Technical grade 85% in xylene contains (E) and (Z) comes in ratio (85–90 : 15–10) Butoxycarboxim Butoxycarboxim (26), [34681-23-7], 3-methylsulfonylbutanone O-methylcarbamoyloxime (IUPAC), C7 H14 N2 O4 S, MW 222.3, mp 85 –89 ◦ C, consists of colorless crystals, which are readily soluble in water and polar organic solvents. Butoxycarboxim is obtained by the reaction of 3-methylsulfon-2-butanone with hydroxylamine, followed by treatment with methylisocyanate [1440]. Butoxycarboxim is used on potted ornamentals against similar pests to those controlled by butocarboxim.

CH3

CH3

CH3SO2CHCCH3 NOCONHCH3

CH3CCHSO2CH3 NOCONHCH3

E

Z butoxycarboxim (26)

930

INSECTICIDAL CARBAMATES

O MeS

CH3 MeS

CCH

NOCONHMe

CH3

CH3 CCH

O

CH3

O

CH3

MeS

CCH

NOCONH2

O

CH3

MeS

CCH

O

CH3

O

CH3

MeS

CCH

O

CH3

NOCONHCH2OH

NOCONHMe

CH3

aldicarb

(aldicarb sulfoxide)

(23)

CH3 R

CCH

CH3

NOH

RC

CH3

CN

CH3

R′CHO

NOCONHMe

aldoxycarb (aldicarb sulfone) R′CONH2

R′COOH

(24)

CH3 R′

R

C CH3

R′CH2OH Figure 3. Aldicarb: metabolic and degradative pathways.

Methomyl Methomyl (27) [16752-77-5], S-methyl-N-(methylcarbamoyloxy)thioacetimidate (IUPAC), C5 H10 N2 O2 S, MW 162.2, mp 78–79 ◦ C, consists of colorless crystals, which are fairly soluble in water and highly soluble in methanol, ethanol, acetone, and isopropanol. Methomyl is produced by chlorination of acetaldoxime and conversion of the resulting α-chlorooxime with sodium methylmercaptide. Methomyl controls a wide range of insects and spider mites in fruits, vines, olives, hops, vegetables, and ornamentals.

SCH3 CH3NHCO2N

Oxamyl (announced in 1968) is used for control of chewing and sucking insects, spider mites, and nematodes in ornamentals, vegetables, potatoes, and other crops.

(CH3)2NCOC

NOCONHCH3

SCH3 oxamyl (28)

Thiodicarb

C CH3

methomyl (27) Oxamyl Oxamyl (28) [23135-22-0], N,N-dimethyl-2-methylcarbamoyloxyimino-2-(methylthio)acetamide (IUPAC), C7 H13 N3 O3 S, MW 219.3, mp 100–102 ◦ C, consists of colorless crystals, which are readily soluble in water, methanol, ethanol, acetone, and fairly soluble in toluene. Oxamyl is produced by chlorination of the oxime of methylglycolate, reaction with methanethiol and alkali, and conversion to the carbamate with methyl isocyanate.

Thiodicarb (29) [59669-26-0], 3,7,9,13-tetramethyl-5,11dioxa-2,8,14-trithia-4,7,9,12-tetra-azapentadeca-3,12-di ene-6,10-dione (IUPAC), C10 H18 N4 O4 S3 , MW 354.5, mp 173–174 ◦ C, consists of colorless crystals, which are sparingly soluble in water, readily soluble in dichloromethane, acetone, methanol, and xylene. Thiodicarb is produced by reaction of N,N -thiobis(methylcarbamic acid fluoride) with 2-methylthioacetaldoxim in the presence of a base. Thiodicarb is a carbamate insecticide (cholinesterase inhibition) with molluscicidal properties. It was introduced in France in 1988 and used as a pelleted bait containing 4% active ingredient at 5 kg/ha.

INSECTICIDAL CARBAMATES

action on acetylcholine receptors. The mechanism of interaction with acetylcholinesterase is analogous to the normal three-step hydrolysis of acetylcholine. However, the third reaction step is much slower for the carbamylated enzyme than for the acetylated one. The importance of structural complementarity of the insecticidal carbamates to the active site of acetylcholinesterase is demonstrated by the pronounced difference in activities of D-2-(sec-butylphenyl) methylcarbamate and L-2-(sec-butylphenyl) methylcarbamate (the L isomer is five times more toxic) and of the 2-, 3-, and 4-substituted phenyl methylcarbamates, where the 4-isomers are virtually inactive. Detoxification of carbamate insecticides occurs in vivo through microsomal hydroxylation, N-demethylation of carbamyl nitrogen, side chain oxidation, and ring hydroxylation. Methylenedioxyphenyl synergists prevent oxidation largely by inhibiting the microsomal enzymes.

CH3 CH3NCO2N

C CH3

S CH3 CH3NCO2N

C CH3

Thiodicarb (29) Thiofanox Thiofanox (30) [39196-18-4], 3,3-dimethyl-1-methylthio2-butanone O-methylcarbamoyloxime (IUPAC), C9 H18 N2 O2 S, MW 218.3, mp 56.5–67.5 ◦ C, is a colorless solid, which is moderately soluble in water and is readily soluble in common organic solvents. Thiofanox is obtained by reaction of 3,3-dimethyl-1(methylthio)-2-butanone with hydroxylamine, followed by reaction with isocyanate. Thiofanox is a systemic soil insecticide effective against a wide range of insect pests on many crops.

NEREISTOXIN PRECURSORS (Fig. 4) Cartap

C(CH3)3 CH3NHCO2N

Cartap (31) [15263-52-2], S,S -(2-dimethylaminotrimethylene) bis(thiocarbamate) hydrochloride (IUPAC), C7 H15 ClN3 O2 S2 , MW 273.8, mp 179–181 ◦ C, forms colorless, slightly hygroscopic crystals that are readily soluble in water, are slightly soluble in methanol and ethanol, and are insoluble in acetone, diethyl ether, ethyl acetate, and benzene. Cartap is obtained by hydrolyzing 1,1-dithiocyanato2-dimethylaminopropane with hydrochloric acid. Cartap is the pro-insecticide of the natural toxin nereistoxin. It is used for the control of chewing and sucking insects,

CCH2SCH3

thiofanox (30) MODE OF ACTION The insecticidal carbamates are cholinergic. Poisoned insects and animals exhibit violent convulsions and other neuromuscular disturbances. These insecticides carbamylate acetylcholinesterase and may have a direct

H3C

S

N

SCONH2

H3C

O

H3C

SCONH2

N

S

H3C

cartap

nereistoxin monoxide

(31)

(33)

H3C

H3C

S

N

SSO2C6H5

N

SSO2C6H5

S H3C

H3C nereistoxin

bensultap

(32)

(35) S

H3C

N

931

S S

CH3 thiocyclam (34) Insecticidal precursors of nereistoxin

Figure 4. Nereistoxin precursors.

932

INSECTICIDE RESISTANCE ACTION (IRAC) COMMITTEE

at almost all stages of development, on many crops. Its structure is based on that of the naturally occurring neurotoxin, nereistoxin. Cartap is hydrolyzed in base to the dihydronereistoxin, which is oxidized to the insecticide, nereistoxin (32). The conversion occurs within plants, and the monoxide (33) was identified as a minor metabolite. In rats, cartap was rapidly excreted in urine. It was hydrolyzed, converted to the sulfoxide, and N-demethylated (9). Nereistoxin does not inhibit cholinesterase. Instead, it acts as an antagonist at the nicotinic acetylcholine receptor and blocks neural transmission (10).

CH2SCONH2

BIBLIOGRAPHY 1. E. Stedman and G. Barger, J. Chem. Soc. 127: 247 (1925). 2. E. Engelhart and O. Loewi, Arch. Exptl. Pathol. Pharmakol. Naunwyn-Schmiederberg’s 150: 1 (1930). 3. J. M. Harkin et al., in W. Y. Garner, R. C. Honeycutt, and H. N. Nigg, eds., Evaluation of Pesticides in Ground Water, Vol. 315, ACS Symposium Series, Am. Chem. Soc., Washington, D.C., 1986, pp. 219–254. 4. V. E. Clay et al., J. Agric Food Chem. 28: 1122–1129 (1980). 5. T. R. Roberts and D. H. Hutson, eds., Metabolic Pathways of Agrochemicals, Vol. 2, Royal Society of Chemistry, Cambridge, U.K., 1999, pp. 17–24.

CHN(CH3)2

6. C. D. S. Tomlin, ed., Pesticide Manual, 11th ed., British Crop Prot. Council, Farnham, Surrey, U.K., 1997, p. 813.

CH2SCONH2

7. J. L. Hansen and M. H. . Spiegel, Environ. Toxicol. Chem. 2: 147–153 (1983).

cartap (31) Thiocyclam Thiocyclam (34) [31895-21-3], N,N -dimethyl-1,2,3-trithian-5-ylamine (IUPAC), C5 H11 NS3 , MW 181.3, mp 125– 128 ◦ C [decomp.]. Thiocyclam is a pro-insecticide of the natural toxin nereistoxin and is rapidly converted into the latter in biological media. It has limited systemic activity with stomach and contact action. It causes paralysis by ganglionic blocking action on the insect central nervous system. It is used as the hydrogen oxalate salt. This is converted to the active nereistoxin, and its oxide in soils and plants and these are ultimately broken down into smaller molecules. DT50 in soil 1 day (pH 6.8, 22 ◦ C, organic content 2.8%) (11).

8. T. R. Roberts and D. H. Hutson, eds., Metabolic Pathways of Agrochemicals, Vol. 2, Royal Society of Chemistry, Cambridge, U.K., 1999, p. 544. 9. C. D. S. Tomlin, ed., Pesticide Manual, 11th ed., British Crop Prot. Council, Farnham, Surrey, U.K., 1997, p. 195. 10. V. E. Clay et al., J. Agric Food Chem. 28: 131–132 (1980). 11. T. R. Roberts and D. H. Hutson, eds., Metabolic Pathways of Agrochemicals, Vol. 2, Royal Society of Chemistry, Cambridge, U.K., 1999, p. 1195.

FURTHER READING

CH3

Kuhr, R. J. and Dorough, H. W., Carbamate Insecticides: Chemistry, Biochemistry and Toxicology, CRC Press, Cleveland, Ohio, 1976. ¨ Kulic, M. and Braunling, H., Meded. Fac. Landbouwwet. Rijksuniv. Gent 39: 847 (1974). Roberts, T. R. and Hutson, D. H., eds., Metabolic Pathways of Agrochemicals, Vol. 2, Royal Society of Chemistry, Cambridge, U.K., 1999, pp. 3–78, 127–137, 535–572.

thiocyclam (34)

Tomlin, C. D. S., ed., Pesticide Manual, 11th ed., British Crop Prot. Council, Farnham, Surrey, U.K., 1997, p. 164.

S H3C N

S S

Bensultap Bensultap (35) [17606-31-4], S,S -2-dimethylaminotrimethylene di(benzenethiosulfonate) (IUPAC), M.W. 431.6, C17 H21 NO4 S4 , pale yellow crystalline powder mp 83–84 ◦ C. Solubility in water 0.7–0.8 mg/kg (30 ◦ C). Solubility in methanol 25 g/kg (25 ◦ C), in acetone, acetonitrile, and N,Ndimethyl formamide >1000 g/kg. Hydrolyzed in neutral or alkaline solution. It is an insecticide with contact and stomach action for control of major insect pests. Acts as a pro-insecticide or analog of nereistoxin. It inhibits the action of acetylcholine by blocking the receptor site at the post-synaptic membrane.

CH2SSO2 CHN(CH3)2 CH2SSO2 bensultap (35)

INSECTICIDE RESISTANCE ACTION (IRAC) COMMITTEE This committee was formed in 1984 to provide a coordinated crop protection industry response to the development of resistance in insect and mite pests. It obtains information by surveys, reports, and other means to determine the extent of resistance. Its mission is to develop resistance management strategies to enable growers to use crop protection products in a way to maintain the efficacy. Its web site is http://www.plantprotection.org/IRAC/. At the same web site (http://www.plantprotection.org/HRAC/ MOA/) information is provided on the Herbicide Resistance Action Committee, which has developed a system, in part in cooperation with the Weed Science Society of America (WSSA), to create a uniform classification of herbicide modes of action in as many countries as possible. There are many cases in which such a classification system may be useful, but in some cases weeds may exhibit multiple

INSECTICIDES, IMIDACLOPRID

resistance across many of the groups. In such cases the key may be of limited value. The system itself is not based on resistance risk assessment, but can be used by the farmer or advisor as a tool to choose herbicides in different mode of action groups. This may be useful in selecting mixtures or rotations of active ingredients.

top selling insecticides in the world in 2000. Following imidacloprid, insecticidal molecules of analogous structure have been developed as listed in Figure 1. Nitenpyram (2), acetamiprid (3), and thiacloprid (4), which also carry the ‘‘activator’’ 6-choloro-3-pyridinylmethyl (trivial name: 6-chloronicotinyl) group in the structure, are already on the market. Then followed thiamethoxam (5) of oxadiazine skeleton carrying the 2-chloro-5-thiazolylmethyl group. Further, clothianidin (6) and dinotefuran (7) are likely going to be marketed soon. These new insecticides have been named chloronicotinyls or neonicotinoids, which show a similar mechanism of action (4). The chloronicotinyl insecticides are growing in importance compared with conventional organophosphates, carbamates, and pyrethroids (4). Imidacloprid, besides its agricultural use, is also used for the control of subterranean pests and pet ectoparasites.

INSECTICIDES Substances that kill insects and other arthropods (USEPA).

INSECTICIDES, IMIDACLOPRID SHINZO KAGABU Gifu University Gifu, Japan

NOMENCLATURE AND PHYSICOCHEMICAL PROPERTIES

The insecticide world market has long been dominated by well-established products belonging to the organophosphate and carbamate classes, which act by inhibition of acetylcholin-esterase, and pyrethroids, which act on voltage-gated sodium ion channels. In the late 1980s, these three classes accounted for more than 87% of sales, and other insecticide classes with different modes of action were of limited economic importance. There was an increasing demand for new broad-spectrum insecticides with new modes of action, especially since the insects’ resistance to the major insecticides became a serious problem. In 1986, imidacloprid (1) was discovered as a new insecticide with a unique structure and with a hitherto unrecognized insecticidal performance (1–4). Since being launched on the Japanese market in 1992, its sales potential has increased yearly, and it ranked as one of the

N

The nomenclature and physicochemical properties are listed in Table 1.

AGRICULTURAL USES Formulations The product is available as dustable powder, granules (including nursery box granules), seed dressing (flowable suspension, water-dispersible powder for slurry), emulsion concentrate, soluble liquid, suspension concentrate, wettable powder, water-dispersible granule, and tablet.

N N

CH2

Cl

NH

N

CH2

Cl

S

O

CH2

H

H

N

N Me

NNO2

NCN

Me N CH2

Cl

N

N

CH2

Cl

N

Me NNO2

2 (nitenpyram)

5 (thiamethoxam*)

Me CH2

N

N

S

CHNO2

Cl

7 (dinotefuran*) O

N

H Me

N

N Me

CH2

Cl

H

H

N

N Me

S NCN 3 (acetamiprid)

NNO2

4 (thiacloprid*)

1 (imidacloprid)

933

NNO2 6 (clothianidin*) * proposed common name

Figure 1. Imidacloprid and developed chloronicotinyl/neonicotinoid insectides.

934


Table 1. Nomenclature and Physicochemical Properties of Imidacloprid (35,36) Common Name Developing name IUPAC name Chemical abstract name CAS Registration number Commercial names

Imidacloprid (ISO) NTN 33893 1-(6-chloro-3-pyridylmethyl)-Nnitroimidazolidin-2-ylideneamine 1-(6-chloro-3-pyridinylmethyl)-Nnitro-2-imidazolidinimine [138261-41-3] For crop protection: Admire, Confidor, Gaucho, Provado; for termite control: Premise, Hachikusan; for animal health applications: Advantage

Structural formula

CH2

Cl

N

NH

N N Molecular formula (Mol. Wt) Physical state (20 ◦ C) Melting point Density (20 ◦ C) Vapor pressure (20 ◦ C) Hendry’s constant (calcd, 20 ◦ C) Water solubility (g/L, 20 ◦ C) Solubility in organic solvents (g/L, 20 ◦ C)

Partition coefficient n-octanol/water (21 ◦ C)

NO2

C9 H10 ClN5 O2 (255.7) colorless solid with a faint odor 144 ◦ C 1.41 g cm−3 4 × 10−10 Pa 2 × 10−10 Pa m3 mol−1 0.61 Hexane: 200, Acetonitrile: 50, Dichloromethane: 67, Methanol: 10 log POW = 0.57

Efficacy on Target Pests Imidacloprid is highly effective for the control of hemipteran pests, i.e., bugs, aphids, leafhoppers, planthoppers, and whiteflies. The compound is also active against some species of the orders Isoptera, Thysanoptera, Coleoptera, Diptera, and Lepidoptera. No activity against nematodes and spider mites has been found. Imidacloprid has a systemic activity that makes it especially useful for seed treatment and soil application, but it is equally effective after foliar application. Foliar Application The spray application targets especially pests attacking crops such as cereals, maize, rice, potatoes, vegetables, sugar beet, cotton, citrus, tea, and deciduous fruits. Table 2 shows the acute activity (estimated LC95 in ppm a.i.) of imidacloprid against a variety of pests following foliar application (dip and spray treatment) of host plants under laboratory conditions (3). Imidacloprid is very active on a wide range of aphids. Most susceptible is the damson hop aphid Phorodon humuli (LC95 = 0.32 ppm), which is often highly resistant against other classes of insecticides.

Imidacloprid is highly effective against some of the most important sucking rice pests, leafhoppers and planthoppers. The product is generally less effective against biting insects with the LC95 of 8 and 200 ppm against eggs, larvae, and pupae of Chilo suppressalis, Heliothis virescens, Plutella xylostella, Spodoptera frugiperda, Lema oryzae, Leptinotarsa decemlineata, and Lissorhoptrus oryzophilus. The translaminar transport, where the ingredient moves from the treated upper side of a leaf to the lower surface, is very effective for controlling pests with a furtive lifestyle. For imidacloprid, the reinforcement of efficacy by translaminar action is observed in the tests using cabbage leaves (3). Soil and Seed Treatment. A major strength of imidacloprid is based on efficacy in soil application and seed dressing due to its considerable mobility from the roots to the upper parts of plants through the xylem and the adequate residual activity. These applications have the advantage over conventional methods not only in agronomic value such as uniform distribution of the active ingredient, accurate dosing, reduced dose rates, and longer application intervals, but also in their environmental benefit in that only a small fraction of the land is exposed to the insecticide. As shown in Tables 2 and 3 (3,5), various soil and foliar insects are controlled for long periods by incorporation of ingredient into the soil or by seed dressing or seed coating. It is highly effective against early season sucking pests such as Myzus persicae at a soil concentration of 0.31 ppm for more than 8 weeks and Aphis fabae at a concentration of 1.25 ppm for more than 5 weeks. The likely long period of efficacy is shown by seed treatments. The seedling-box treatment, a method of placing granules in a nursery box before machinery transplanting to the field, made possible more than 11-week control of noxious insect pests for rice (6). The relation of pest control efficacy to the systemic property and the residual activity was investigated. Studies on seeds of winter wheat treated with radioactive imidacloprid revealed a continuous uptake of the applied radioactivity into the sprouting wheat from 1% at the first leaf stage to approximately 19% at full maturity. Concentrations as low as 0.12 mg/kg of younger leaves at the end of the shooting phase 195 days after sowing showed an insecticidal efficacy of 98% against Rhopalosiphum padi (7). In a seedling-box treatment, a continuous movement of the ingredient from the roots to the leaves and sheaths occurs and 0.01 ppm exist in the aerial parts 80 days after application. As a result, a one-shot application of imidacloprid to the nursery box (1 g a.i. per box) can control the brown hopper for the entire season (8). It can be calculated that an 85% less amount of pesticides is needed to achieve equivalent control of pests by imidacloprid-coated seeds for sugar beet production in the United Kingdom and less than 5% of the land exposed to granules or sprays of conventional products (9). Antifeedant Activity Against Homopteran and Coleopteran Species The antifeeding effect of imidacloprid at lower (sublethal) doses is different from the fast-acting insecticidal efficacy


935

Table 2. Spectrum of Activity (LC95 in ppm a.i.) of Imidacloprid After Foliar and Soil Application under Laboratory Conditions [after Elbert et al., 3] Foliar Application Pest Species Homoptera Aphis fabae Aphis gossypii Aphis craccivora Aphis pomi Brevicoryne brassicae Myzus nicotianae Myzus persicae Phorodon humuli Laodelphax striatellus Nephotettix cincticeps Nilaparvata lugens Sogatella furcifera Pseudococcus comstocki Bemisia tabaci Hercinothrips femoralis Lepidoptera Chilo suppressalis Helicoverpa armigera Plutella xylostella Spodoptera frugiperda Heliothis virescens Coleoptera Leptinotarsa decemlineata Lema oryzae Lissorhoptrus oryzophilus Phaedon cochleariae Diabrotica balteata

Developmental Stage

LC95 (≥ppm)

Mixed Mixed Mixed Mixed Mixed Mixed Mixed Mixed Larvae third instar Larvae third instar Larvae third instar Larvae third instar Larvae Larvae second instar Mixed

8 1.6 1.6 8 40 8 1.6 0.32 1.6 0.32 1.6 1.6 1.6 8 1.6

Larvae first instar Larvae second instar Egg, Larvae second instar Egg, Larvae second instar Egg

8 200 200,200 200,40 40

Larvae second instar, Adult Adult Adult Larvae second instar Larvae third instar

40,40 8 40 40 1.6

Soil Application Pest Species Soil insects Hylemyia antiqua Diabrotica balteata Agriotes spp. Agriotis segetum Reticulitermes flavipes Foliar insects Phaedon cochleariae Myzus persicae Aphis fabae Spodoptera frugiperda

Developmental Stage

LC95 (≥ppm)

Larvae Larvae Larvae Larvae Imago

5 2.5 5 20 10

Larvae Mixed population Mixed population Larvae

5 0.16 0.16 10

at recommended field rates. The sublethal effects may contribute particularly to the biological activity of imidacloprid against plant-feeding homopteran pest species. Low concentrations of imidacloprid have been shown to elicit behavioral changes in aphids, whiteflies, and planthoppers (10–15). Such behavioral changes include the depression of honeydew excretion and wandering, resulting in death due to starvation. Similar effects were also described for coleopteran pests such as black maize beetles and wireworms when feeding on stems of seed-treated maize plants (16,17). Furthermore, sublethal concentrations have been shown to reduce the fertility of aphids, thus, demonstrating its potential to prevent the buildup

or spread of populations later in the season when the concentration of imidacloprid in systemically treated plants declined. Control of Virus Vectors The transmission of plant-pathogenic viruses by plantsucking homopteran pest species is one of the major threats in many cropping systems, e.g., sugar beet, cereals, and vegetables. It has been shown that imidacloprid—foliarly and systemically applied—particularly prevents the persistent transmission of circulative, phloemrestricted viruses, due to the rapid action on homopteran virus vectors. Some successful examples include the

936

INSECTICIDES, IMIDACLOPRID Table 3. Residual Activity (Weeks) Against Pest Species (>95% Mortality) [after Elbert et al., 5] Method/Dose Rate (a.i.) Soil treatment 2.5 1.25 0.63 0.31 0.15

(ppm)a

Seed dressing (g/kg seed)b 1.0 0.25 0.06

Pest Species

Residual Activity (Weeks)

Myzus persicae (green peach aphid) >8 >8 >8 >8 4

Aphis fabae (black bean aphid) >5 5 4 3 2

Aphis gossypii (cotton aphid) >5 2 5 5 3

Seed coating (g/unit)c 30 70 110

Aphis fabae (black bean aphid) 7.7 8.3 9.3

Seedling-box treatment (g/box)d 3 1

Nephotettix cincticeps (green rice leafhopper) >11 >11

Nilaparvata lugens (brown planthopper) >11 >11

Sogatella furcifera (whitebacked planthopper) >11 >11

a

Tested in broad bean. Tested in cotton (A. gossypii) and in broad bean (A. fabae). c Tested for sugar beet seed. d Tested in rice. b

prevention of the secondary spread of barley yellow dwarf virus transmitted by R. padi and S. avenae (18–20), the control of virus yellows and potato leafroll virus transmitted by M. persicae (21,22), the leafstripe virus transmitted by L. striatellus (6), and the tomato-spotted virus transmitted by B. tabaci (23,24). Activity on Resistant Insect Species The chloronicotinyl/neonicotinoid insecticide imidacloprid acts agonistically on nicotinic acetylcholine receptors (nAChR). Because this is a new biochemical target addressed by modern insecticides, in principle, existing resistance mechanisms are not expected to confer cross resistance to imidacloprid. Table 4 shows that imidacloprid is equally effective against insecticide-susceptible and M. persicae resistant to organophosphates, carbamates, and pyrethroids (5,25). Many examples were reported in which the resistant strains were as susceptible to imidacloprid as were the sensitive ones: aphids (25–27), whiteflies (28), planthoppers (6), and the Colorado potato beetles (25). Table 4. Efficacy of Imidacloprid and Conventional Insecticides (LC95 , ppm) Against a Susceptible (S) and Resistant (R) Greenhouse Strain of Myzus persicae (Leaf Dip Bioassay) (5,25) Insecticide Imidacloprid Ethylparathion Pirimicarb Cypermethrin a

Resistance factor.

LC95 (ppm) Against S/R Strains 8/8 8/>1000 40/1000 8/1000

RFa 1 >125 25 125

Although no practical loss of field efficacy seems to be observed at present, selection studies in laboratories revealed that certain pest insects can be selected for lesser susceptibility. A population of small brown hopper reared under malathion and propoxur pressure showed 18-fold tolerance to imidacloprid (29,30). In another example, the continuous hydroponic laboratory selection of a population of Bemisia argentifolii resulted in more than 80-fold resistance after 24 generations (31,32). A clear indication of cross-resistance was reported between imidacloprid and other chloronicotinyls in B. tabaci from southern Spain (33). Resistance management strategies have been proposed to prevent or delay the development of resistance to imidacloprid in pest insects (25,34). They include deploying several active ingredients of different insecticidal mechanisms in an annual or regional rotation, applying the preparations at the recommended doses and spray intervals, alternating practice with transgenic crops, combining biological pesticides and physical pest control methods, performing crop rotation to delay colonization, reducing immigrant density, and decreasing the number of generations or manipulating crop cutout to force insect survivors into refuge areas prior mating. For applying such strategies, collecting baseline data and continuous monitoring of susceptibility of pests to imidacloprid is indispensable (28–30). It is also argued that systemic applications of persistent insecticides such as imidacloprid should be limited to minimize the dangers of continuing to select when control is no longer needed. Agricultural Significance Due to the novel biochemical target, broad-spectrum, and high intrinsic activity as well as good plant compatibility, the control target of imidacloprid encompasses a large


937

Table 5. Use Rates for Foliar and Soil Application for Representative Crops [Extracted from 35,36] Crops

Target Pest

Vegetables Vegetables Cotton Cotton Potatoes Tobacco Pome fruits Pome fruits Citrus Rice Rice

Recommended Rates of Active Ingredient Foliar Treatment (g/ha) Soil Treatment (g/unit)

Aphids, thrips whiteflies aphids whiteflies aphids, Colorado potato beetle aphids, thrips leafminer aphids aphids hoppers rice water weevil

50–200 100–200 25–100 100–350 50–100

100–300 100–300 0.05–0.075 (g/m) 0.05–0.075 (g/m) 180–200 (in furrow)

50–100 50–100 50–100 100–200 25–50

100–250 (drip/soil drench) 0.5–1.0/tree (drip/soil drench) 0.25–0.5/tree (drip/soil drench) 0.2–2.0 (drip/soil g/tree) 100–200 (0.5–1.0 g/box) 100–200 (0.75–1.0 g/box)

Table 6. Target Insects of Imidacloprid After Seed Treatment [Extracted from 35,36] Crop Corn

Soil-dwelling Insects

Early Leaf-feeding and Sucking Insects

Sorghum Cotton Canola (oilseed rape) Cereals

Wireworms, black maize beetle, ground beetle, rootworms, seed corn maggot wireworms, false wireworms, fire ants cotton root weevil, wireworms, termites Flea beetle Wireworms, ground beetle

Rice

Termites

Sugar beet

Sunflower Beans

Pygmy mangold beetle, wireworms, millipedes, springtails, beet root weevil, flea beetle Wireworms, ground beetle Rootworms, grubs

Potato

Wireworms

variety of insect species for various plant crops. Further, the appropriate physicochemical properties such as a good systemic activity, adequate stability in field, and the amphipathy allow imidacloprid to be applied in unusually diverse ways, e.g., as a foliar spray, or systemically as a drench or in irrigation water, as a granular soil application, as seed dressing, or as a paint-on formulation. The representative use rates and target pests are summarized in Tables 5 and 6. Further technical information is available from the product brochures (35,36).

CHEMISTRY Spectral Data and Dissociation Constant Electronic absorption λmax (nm)/log ε(water): 269 (4.17) (37); 1 H-NMR (δ, CDCl3 ): 3.54 (m, 2H), 3.83 (m, 2H), 4.55 (s, 2H), 7.35 (d, J = 8.4, 1H), 7.70 (dd, J = 7.0/2.6, 1H), 8.32 (d, J = 2.6, 1H), 8.20 (bs, 1H) (38); IR (KBr, ν, cm−1 ): 3155, 1580, 1565, 1300, 1280; EIMS (70eV, m/e, rel int): 209 (55%), 173(80%), 126 (100%) (38). The crystal

Argentine stem weevil, fruit fly, aphids, jassids, Armyworm Aphids, green bug, chinch bug Cotton aphid, thrips, jassids, white fly Aphids, thrips, cabbage root fly Bird cherry aphid, grain aphid, Russian wheat aphid, Hessian fly Green leafhopper, smaller brown planthopper, brown planthopper, rice leaf beetle, rice water weevil, Stechaenothrips spp. Green peach aphid, black bean aphid, beet leafminer, flea beetle, lygus (bugs) Aphids, Zygogramma exclamationis Black bean aphid, pea and bean aphid, whitefly, leafhopper Colorado potato beetle, green peach aphid, jassids

structure with the structure parameters is reproduced in Figure 2 and Table 7 (39). Synthesis The first laboratory synthesis of imidacloprid is outlined in Figure 3 (1,2). Reduction of 2-chloro-5-pyridinecarbonyl chloride (8) to 2-chloro-5-hydroxymethylpyridine (9) was carried out by excess NaBH4 in water, which was converted to the chloride (10) by SOCl2 . Imidacloprid was obtained by the coupling reaction of 10 with 2-nitroiminoimidazolidine (11) in acetonitrile with potassium carbonate as base. This method was successfully applied to the synthesis of [3 H]imidacloprid (12) using NaB[3 H]4 as the tritium source (40). Technical production starts with the Tschitschibabin reaction of 3-methylpyridine giving 2-amino-5-methylpyridine (14), which is transformed to the chloride (15) by the Sandmeyer reaction in the presence of hydrogen chloride. A successive operation of chlorination of the methyl group to 10 and the subsequent substitution of the active chloride with ethylene diamine to 16 are carried out without isolation of the intermediates. The final product is produced

938


CL

C1

N1

H9

H6 H1

C5

C9

C8

H3

H8

C2

H7

N3

C3 H2

C6

C4

H10 C7

N2 H4

N5

H5

O3

N4

O2

Figure 2. Crystal structure of imidacloprid.

by ring formation with nitroguanidine. This multistep process affords the product at a purity of >95% (41). Biological Activity and Mode of Action In vivo symptomatology in American cockroach, Periplaneta americana, after injecting an LD50 dose of 1 mg a.i./insect is characterized by the following sequence: walking up and down with a loss of strength in legs within 15 min; leg tremor followed by whole body shaking and prostration with a curled abdomen 30 min later; and death (42). The symptoms are similar to poisoning with nicotine, suggesting that both molecules act on a similar set of receptors in the central nervous system. Imidacloprid displaces radiolabeled α-bungarotoxin (α-BTX), a competitive antagonist of acetylcholine, from its binding site on the insect nicotinic acetylcholine receptors (nAChR) like nicotine. For imidacloprid and

nicotine, the IC50 values in 10−6 M, the concentrations to displace 50% specific binding of the probe [3 H] or [125 I]α-BTX on nAChR, are respectively, 1.95 and 1.25 in honeybee head membrane (43), 2.9 and 0.086 in stable fly head membrane (44), and 0.20 and 9.8 in American cockroach cholinergic motor neuron (45). Direct evidence of the molecular target of imidacloprid is that [3 H]imidacloprid (12) binds with 95% specificity to membranes from housefly, Musca domestica, and other insect nerve tissue, and that the binding is displaceable by acetylcholine, nicotine, and other nicotinic ligands (46). The high affinity binding to nAChR is also described for the aphid (47). Electrophysiological results support its primary site of action at the nAChR in insects. At concentrations below 10−6 M, imidacloprid induces a rapid depolarization of postsynaptic neurons accompanied by the induction of action potentials and subsequently by a complete block of the nerve impulse propagation. The induction responses are very similar to acetylcholine responses and are blocked by nicotinic receptor antagonists. These biphasic effects are evident in patch-clamp and two-electrode voltage-clamp studies on insects (45,48,49), and the insecticidal potency of imidacloprid-related compounds correlates with their capacity to cause excitation in cockroach nerve cords (50). In contrast to insects, the affinity to the vertebrate nAChRs is very weak. It failed to recognize the specific binding sites in brain membranes from human, dog, mouse, and chicken or electric organ of the electric eel (46). The low mammalian activity was demonstrated further by the low potency as an inhibitor of [3 H]nicotine binding in rat brain and [3 H]α-BTX binding to the muscle-type nAChR from Torpedo spp. (51), and the weak agonistic action in mouse NIE-115 neuroplasma and BC3H1 muscle cells (48), low activity in ion channel activation compared with acetylcholine with rat α 4 β 2 and α 7 subtypes expressed in Xenopus oocytes (52), weak or partial agonistic nature with recombinant chick α 4 β 2 receptor (53), and very low

Table 7. Selected Interatomic Atom Distances and Angles of Imidacloprid (39) Bond Lengtha C4-C6 C6-N2 N2-C7 N2-C8 C7-N3 N3-C9 C8-C9 C7-N4 N4-N5 N5-O1 N1-N2 N2-O1

1.50 1.45 1.34 1.45 1.32 1.45 1.53 1.34 1.35 1.23 4.35(5.45)b 4.47(5.80)b

Bond Angle (degree) C3-C4-C6 C4-C6-N2 C6-N2-C7 C6-N2-C8 C7-N2-C8 N2-C8-C9 C8-C9-N3 C7-N3-C9 N2-C7-N3 N2-C7-N4 N3-C7-N4 C7-N4-N5 N4-N5-O1 N4-N5-O2

122.2 113.1 125.7 122.2 112.0 102.7 102.8 112.5 109.6 117.1 133.3 116.7 115.4 122.9

10−10 m. Including the van der Waals surface (in parenthesis). c Between pyridine and imidazolidine rings. a b

Tortional Angle (degree) C4-C5-C6-N2 C7-N2-C6-C4 C6-N2-C7-N4 N2-C7-N4-N5 C7-N4-N5-O1 C7-N4-N5-O2 N4-C7-N2-C8 N3-C7-N4-N5 C9-N3-C7-N4

117.8 106.9 −0.5 176.3 0.0 179.4 176.5 −3.0 178.8

Interplane angle (degree)c 75.7

Laboratory synthesis of imidacloprid

CO2H

Cl

Cl

COCl

N

Cl

Cl

Water

N 7

NaBH4

+

HN

N 10

K2CO3

NH

Cl acetonitrile

N

N

CH2

NH

C[3H]2

Cl

N

N

NNO2

NNO2 1 imidacloprid

11

NH

N

NNO2 10

CH2Cl

Cl

N 9

8

CH2Cl

SOCl2

CH2OH

12 [3H]imidacloprid

Technical production of imidacloprid

CH3

NaNH2

N

CH3ONO / HCl H2N

CH3

Cl CH3OH

N 13

Cl N 10

H2NCH2CH2NH2

N 15

Cl

CH2NHCH2CH2NH2

10 (H2N)2C

NNO2

N 16 Figure 3. Laboratory and technical preparation of imidacloprid (1).

939

CH2Cl

Cl

N

14

CH2Cl

Cl2

CH3

imidacloprid (1)

940

INSECTICIDES, IMIDACLOPRID Table 8. Pharmacological Characterization of Imidacloprid and Nicotine in Radioligand Binding Sites in Insects, Torpedo, and Rodents IC50 (10−6 M) Ligand/Probe

Houseflya [3 H]IMI

Aphidb [3 H]IMI

Torpedoc [3 H]α-BTX

Ratd [3 H]NIC

Moused [3 H]α-BTX

Imidacloprid (−)-Nicotine

0.002 0.6

0.00076 2.1

1060 23

0.98 0.0096

42 1.9

a

Head membrane preparation 41. M. persicae: whole aphid homogenate 47. c Electric organ 51. d Brain membrane 54. b

affinity to immuno-isolated nAChRs of varying subunit composition (54). Table 8 compares the pharmacological characterizations with nicotine in radioligand binding sites in insects, Torpedo spp., and rodents. The safety factor, the ratio of the lethal doses for mammals to insects, can be estimated as 7300 from the LD50 of 0.062 mg/kg for M. persicae by topical application and an LD50 for rats of 450 mg/kg after oral administration, which ranks imidacloprid as one of the insecticides of highest selectivity known (13). The binding models of chloronicotinyl/neonicotinoid insecticides with the recognition site on the nAChR were proposed (4,39,51), and a comparative molecular field analysis (CoMFA) for the binding mode was performed (56). ENVIRONMENTAL FATE

Sample Rice straw: 10 g Crops: 20 g Soil: 20 g Extraction Add. water / acetonitrile (80:20), 200 −400 mL. Crops: homogenizer Soil: ultra sonic bath Washing with cyclohexane

Partition with dichloromethane

Washing with alkaline (0.05M K 2CO3)

Stability in Storage Formulated imidacloprid is stable at room temperature in the dark under usual storage conditions; the active ingredient is labile in alkaline media (35–37). The calculated half-life is 57 min based on the direct photolysis in water. The photostability of nitroimine with nitromethylene or cyanoimine chromophore, which functions as an electron-attracting group in chloronicotinyl molecules in Figure 1, was compared on the quantum chemical basis (57). Residue Analysis The strong absorption at 270 nm attributable to the nitroimino chromophore is a reliable indication for the analysis of imidacloprid. The HPLC-UV method is standardized for the detection and quantification of the parent molecule. The imidacloprid concentration can be measured by this method at levels >10 ng/g in different fruits and vegetables (58), and at 5–20 ng/g in rice plants and soil (8). Other detectors are also used for HPLC analysis of the parent molecule such as diodearray (59,60) and pulsed reductive amperometry (61). The metabolites or derivatives may be subject to an alternative determination; photochemically induced fluorimetry of 2-hydroxyimino derivative (62), GC-MS of 3N-perfluoroalkanoyl derivatives (63,64), or the urea (23) after hydrolysis (65,66). Total residue analysis based on 6chloronicotinic acid is a well-established method (58). The

Column chromatography 15 mm id, 300 mm long 10 g of silicagel containing 10% water Fr.1 n-hexane:ethylacetate (1:1), 60 mL, discard Fr.2 ethylacetate, 20 mL, discard Fr.3 (imidacloprid Fr.) ethylacetate, 60 mL, collect HPLC analysis column: ODS mobile phase: water / acetonitrile (80:20) UV: 270 nm

Figure 4. Analytical procedure to separate imidacloprid from crops and soil (8).

recovery procedures from the test plant tissues and the HPLC measurement conditions including the total residue analysis are described in detail (58). Figure 4 shows a representative residue analysis scheme for crops and soil. Fate in Soil The aerobic decomposition rates in soil are variable depending on type of soil, vegetation, and conditioning of the soil, e.g., by treatment with manure. The mean of the half-lives reported in the literature (63,67–69) is 80 days.


Fate in Plants

This is in line with a laboratory experiment simulating groundcover when a half-life of 48 days was found (70). According to laboratory trials, degradation is more rapid under anaerobic than under aerobic conditions. The halflives are 27 days in a water/sediment system after it had attained anaerobic conditions, and 53 and 69 days in two Japanese paddy soil types, volcanic and alluvial, respectively. More than half of the amount is decomposed in less than 1 week in the presence of light. The sunlight and microbial activity of a water/sediment system are important factors for the degradation of imidacloprid (71). The study in soil using [14 C]imidacloprid over 1 year shows that the metabolism proceeds via loss of the nitro group to form guanidine (20) and simultaneously via cleavage and oxidation of the imidazolidine ring to yield 6-chloronicotinic acid (7) and finally carbon dioxide (cf. Fig. 5). The main product in recovery was imidacloprid in aerobic soils or guanidine in anaerobic and paddy soils, and none of the identified metabolites was found in concentrations above 10 ppb after 100 days (71). According to a 5-year lysimeter study, ca. 40% of the total radioactivity is lost as CO2 in 2 years and the radioactive residues of leachate are adsorbed within 20 cm of the soil surface (72).

Imidacloprid is well translocated through xylem from the roots to the upper parts of plants due to its adequate water solubility and lack of an acidic or basic moiety. The active ingredient is accumulated in younger leaves after seed treatment. The degree of metabolism is significantly influenced by the application method. In the case of sprays or surface treatments, an unchanged parent compound is the main constituent of the residue because only a part of the applied component penetrates into the plant and is metabolized after application. Following granular application or box treatment, the active ingredient is readily taken up via the roots and the degree of metabolism is higher. The metabolic route in plants is presented in Figure 5 (71). The main degradation routes are 1) hydroxylation of the imidazolidine ring leading to the mono- and dihydroxylated compounds (17,19) with subsequent removal of water to form the olefin metabolite (18), 2) transformation of the nitro group to the guanidine metabolite (20) possibly partly the nitrosoimine intermediate (21), and 3) oxidative cleavage of the methylene bridge to form 6-chloropicolyl alcohol (9) (and the conjugates 24,25) and further oxidation to 6chloronicotinic acid (7). These metabolites are detected in

CO2H

Cl N

CH2O-(1-α-gentiobiosyl)

Cl 7

Cl

CH2

N

N

25 CH2OH

NH O

N N

Cl

NO2

Cl

CH2OH

CH2 N

N

18

5 CH2

N

NH

Cl

CH2

N

NH

Cl

N N

CH2

N

NO2

NO2

NO

OH

N

NH

Cl

CH2

N

NH

CH2NH

N

NO2

NH

22 20

N

NH2

N

N

N 19

NH N

21

Cl CH2

N

N 1 (imidacloprid)

HO Cl

OH

24

OH 4

N 17

HO

O OH

9

Cl

941

H Cl

CH2

N

NH

N 23 Figure 5. Metabolic degradation of imidacloprid (1) in plants (71).

O

942


variable amount in treated cultures. The following values in parentheses describe in order the total residue in ppm and the main metabolites with the amounts at the harvesting times for representative plants after the recommended application dose (71): spray tomato (0.85, 1: 88.0%), spray potato (Leaves; 1.35, 1: 37.9%, 20: 12.6%, 17: 7.0%/Tubers; 0.009, 7: ca. 30%, 1: ca. 5%), granular potato (Leaves; 5.76, 1: 26.7%, 7: 8.3%, 20: 8.2%, 17: 4.6%/Tubers; 0.091, 1: 48.3%, 20: 11.3%, 7: 9.4%, 17: 8.0%), granular corn (Straw; 3.08, 1: 22.2%, 20: 10.9%, 17: 6.0%/Grain; 0.039, 1: 25.2%, 18: 13.1%, 17: 9.3%), granular cotton (Leaves; 0.11, 9: 13.2%, 20: 9.8%, 24: 6.3%), granular rice (Straw; 1.47, 20: 25.6%, 1: 11.5%/Grain; 0.036, 1: 6.3%), nurserybox rice (Straw; 1.31, 20: 36.2%, 1: 8.1%/Grain; 0.014, 1: 13.6%). The residues in the storage organs of the plants in the crops and fruits are very low. TOXICITY AND SAFETY ASPECT Metabolism in Animals After oral administration of [14 C]imidacloprid to rats, the radioactivity was readily absorbed and distributed to all organs within 1 hour and eliminated by 96% after 48 hours, about 75% thereof with the urine and about 21% with the feces (73). At the time, the radioactive residues in the body amounted to ca. 0.5% of the administered radioactivity. Therefore, bioaccumulation of imidacloprid is low in rats. Maximum plasma concentration was reached at 1.1 and 2.5 hours. Two major routes of biotransformation are proposed. The first route includes an oxidative cleavage of the parent compound rendering 6-chloronicotinic acid (7) and its glycine conjugate. Dechlorination of this metabolite formed 6-hydroxynicotinic acid and its mercapturic acid derivative. The second route includes the hydroxylation to 17 followed by elimination of water of the parent compound rendering olefin 18. There were no sex, dose, or label differences in toxicokinetics. The metabolism in hens and goats is similar to that in rats (73). Acute Toxicity The following data for acute toxicity are reported: acute oral LD50 for male and female rats 424-475, for mice 131168 mg/kg; dermal LD50 for rats >5000 mg/kg; inhalation

LC50 (4h) for rats 69 mg/m3 aerosol, >5323 mg/m3 dust; nonirritating to eyes and skin (rabbits) and not a skin sensitizer (guinea pig). The toxicity class is II on World Health Organization (WHO) (a.i.) and II on U.S. Environmental Protection Agency (EPA) (formulation). Chronic Toxicity The following no-observed-effect levels (NOELs) are established: rat, 5.7 mg/kg body weight per day; and dog, 15 mg/kg body weight per day. The ADI is 0.057 mg/kg body weight. The compound has no mutagenic or carcinogenic potential. In the developmental toxicity study, the maternal and fetal NOELs for rats were 10 mg/kg and 30 mg/kg body weight per day, respectively. The corresponding NOELs for rabbits were 8 mg/kg and 24 mg/kg body weight per day. No fetal malformations were observed in rats and rabbits at any dose level. From the reproductive toxicity study in two-generation and two-litter rats, a pup reproductive NOEL of 12.5 mg/kg body weight per day was estimated. No effect on mating behavior, fertility, gestation, conception, litter size, or mortality was observed. Malformation did not occur. There was no primary neurotoxicity, and the NOELs for neurotoxicity were 307 mg/kg body weight per day after acute exposure and 196 mg/kg body weight per day in the 3month study (73). The tolerances by EPA are listed in Table 9. Ecotoxicology The median tolerance limits (TLms) in milligrams/liter are 190 (48 h) for carp, 237 (96 h) for golden orfe, 211 (96 h) for rainbow trout, and 85 (48 h) for water fleas. The EC50 value for algae (Scenedesmus subspicatus) is also larger than 10 mg/L. The hydrophilic properties are responsible for the low fish toxicity and low bioaccumulation. The acute bird toxicity is high; oral LD50 is 150 mg/kg for hen, 31 mg/kg for Japanese quail, 152 mg/kg bobwhite quail, and 33 mg/kg for canary and pigeon (74). In practice, however, considering the very low residues in seeds and the reported repellent effect on birds (75,76), the avian hazard may be negligible. The activity of soil microorganisms is not impaired even at very high dose rates of 2000 g a.i./ha (74).

Table 9. Tolerances in ppm for Residuesa of Imidacloprid (EPA) [Extracted from 35,36] Crop

Tolerance

Apple

0.6

Pear Tomato Peas (legume vegetables) Sugar beet (roots) Potato Legume vegetables (seed) Leafy green vegetables

0.6 1.0 0.1b 0.05 0.5 0.3b 3.5

a

Crop/Product Sweet corn (kernel plus cob with husk removed) Soybean (meal) Wheat (grain) Rice (grain) Meat Milk Egg

Tolerance 0.05 0.5 0.05 0.2c 0.3 0.1 0.02

Residue definition: the total amount of imidacloprid and its metabolites containing 6chloronicotinic acid. b Regional tolerance and time limited tolerance, expires June 30, 2002. c In Japan.


For earthworm (Eisenia foetida), the LC50 is 10.7 mg/kg dry soil. Spray applications, four times overdosed, had a transient effect on the population, which had been recovered by autumn of the year of application. The coated sugar beet seeds had no effect on this worm (77). The effects of imidacloprid on beneficials have been tested. It was harmless to the predator insects Deraeocoris nebulosus (Uhler), Olla v-nigrum (Say), Chrysoperla rufilabris (Burmeister), and a few predatory mites after foliar application at the near-recommended field rate of 127 mg a.i./L (78). No significant mortality was observed for the predator Perillus bioculatus (F.) after 24 h contact with potato foliage after foliar spray (79). On the other hand, harmful effects on nymphs and adults of predators Podisus maculiventris and Orius laevigatus were reported (80,81). The product is harmful to honeybees by direct contact and should not be applied during the flowering period (35,36,82). Also, it is suspected to be harmful to silkworms. Mulberry leaves should not be fed to silkworms until 40 days after spray (35,36). Because virtually no apparent impact on most beneficial insects has been observed after seed dressing, whenever possible, all types of systemic applications such as seed dressing, soil treatment, or stem painting/injection may be recommended.

943

7. U. Stein-Dönecke et al., Pflanzenschutz Nachr. Bayer 45: 327–368 (1992). 8. Y. Ishii et al., J. Agric. Food Chem. 42: 2917–2921 (1994). 9. A. M. Dewar and M. J. C. Asher, Pesticide Outlook 5: 11–17 (1994). 10. R. Nauen, Pestic. Sci. 44: 145–153 (1995). 11. G. L. Devine Z. K. Harling, A. W. Scarr, and A. W. Devonshire, Pestic. Sci. 48: 57–62 (1996). 12. R. Nauen and A. Elbert, Pestic. Sci. 49: 252–258 (1997). 13. R. Nauen et al., Pestic. Sci. 53: 133–140 (1998). 14. H. J. Knaust and H. M. Poehling, Pflanzenschtz Nachr. Bayer 45: 381–408 (1992). 15. R. Nauen, B. Koob, and A. Elbert, Entomol. Exp. Appl. 88: 287–293 (1998). 16. T. W. Drinkwater, Crop. Prot. 13: 341–345 (1994). 17. T. W. Drinkwater and L. H. Groenewald, Crop. Prot. 13: 421–423 (1994). 18. G. M. Tatchell, Pflanzenschutz Nachr. Bayer 45: 409–422 (1992). 19. D. J. Bluett and P. A. Birch, Pflanzenschutz Nachr. Bayer 45: 455–490 (1992). 20. S. J. McKirby and R. A. C. Jones, Plant Dis. 80: 895–901 (1996).

APPLICATION IN NONAGRICULTURAL FIELDS

21. A. M. Dewar, L. A. Read, J. Prince, and P. Ecclestone, Brit. Sugar Beet Rev. 61: 5–8 (1993).

High insecticidal potencies together with nonvolatility and stability under storage conditions, especially under the shelter of sunlight, impart imidacloprid, a good candidate for protecting wooden structures from subterranean pests. Imidacloprid has been successfully applied as a termiticide (83,84). Suitable toxicological properties such as high insecticidal activity, low mammalian toxicity, and absence of eye/skin irritation and skin sensitization potential allows imidacloprid to be used to control parasites on pets (cats and dogs) and in homes. Monthly topical application of 10 mg/kg kills rapidly existing and reinfesting flea infestations on pets and breaks the flea life cycle by killing adult fleas before egg production begins. Owing to accurate, quick, and simple dosing through dose unit pipettes, which helps to ensure compliance by pet owners, the flea spot-on solution is finding a substantial veterinary market (83,84).

22. J. A. T. Woodford, Pflanzenschutz Nachr. Bayer 45: 527–546 (1992).

BIBLIOGRAPHY

32. L. Williams, III., T. J. Dennehy, and J. C. Palumbo, Proc. Beltwide Cotton Conf., Vol. 2, 1998, pp. 1250–1255.

1. European Patent EP 0,192,060 A1 (1986), K. Shiokawa, S. Tsuboi, S. Kagabu, and K. Moriya. 2. K. Moriya et al., Biosci. Biotech. Biochem. 56: 364–365 (1992). 3. A. Elbert, B. Becker, J. Hartwig, and C. Erdelen, Pflanzenschutz Nachr. Bayer 44: 113–136 (1991). 4. S. Kagabu, Review. Toxicol. 1: 75–129 (1997). 5. A. Elbert, R. Nauen, and W. Leicht, in I. Ishaaya and D. Degheele, eds., Insecticides with Novel Modes of Action, Mechanism and Application, Springer, Berlin, 1998, pp. 50–73. 6. K. Iwaya and S. Tsuboi, Pflanzenschutz Nachr. Bayer 45: 197–230 (1992).

23. N. E. Ahmed et al., Plant Dis. 85: 84–87 (2001). 24. G. Rubinstein, S. Morin, and H. Czosnek, J. Econ. Entomol. 92: 658–662 (1999). 25. A. Elbert et al., Pflanzenschutz Nachr. Bayer 49: 5–54 (1996). 26. R. Nauen et al., Bull. Entomol. Res. 86: 165–171 (1996). 27. M. D. Barber et al., Bull. Entomol. Res. 89: 17–23 (1999). 28. M. Cahill et al., Bull. Entomol. Res. 86: 343–349 (1996). 29. S. Sone, Y. Hattori, S. Tsuboi, and Y. Otsu, J. Pesticide Sci. 20: 541–543 (1995). 30. S. Endo and M. Tsurumachi, J. Pesticide Sci. 26: 82–86 (2001). 31. N. Prabhaker, N. C. Toscano, S. J. Castle, and T. J. Henneberry, Pestic. Sci. 51: 419–428 (1997).

33. A. Elbert and R. Nauen, Pest Manag. Sci. 56: 60–64 (2000). 34. M. Cahill and I. Denholm, in I. Yamamoto and J. E. Casida, ed., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, Tokyo, 1999, pp. 253–270. 35. A. G. Bayer, Technical Information Confidor, Leverkusen, Sept. 2000. 36. Technical Information 2001, Nihon Bayer Agrochem Co., Tokyo, Sept. 2000. 37. S. Kagabu and S. Medej, Biosci. Biotech. Biochem. 59: 980–985 (1995). 38. S. Kagabu, K. Yokoyama, K. Iwaya, and M. Tanaka, Biosci. Biotechnol. Biochem. 62: 1216–1224 (1998).

944

INSECTICIDES, MODE OF ACTION

39. S. Kagabu and H. Matsuno, J. Agric. Food Chem. 45: 276–281 (1997).

71. O. Klein, 8th Int. Cong. Pestic. Chem., Washington, D.C., Poster 2A, No.157 (1994).

40. B. Latli et al., J. Labeled Compd. Radiopharm. 38: 971–978 (1996).

72. E. Hellpointer, ACS Symp. Ser. 699: 40–51 (1998).

41. H.-J. Diehr et al., Pflanzenschutz Nachr. Bayer 44: 107–112 (1992).

73. J. Thyssen and L. Machemer, in I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, Tokyo, 1999, pp. 213–222.

42. S. Sone, K. Nagata, S. Tsuboi, and T. Shono, J. Pesticide Sci. 19: 69–72 (1994).

¨ 74. W. Pfluger and R. Schmuck, Pflanzenschutz Nachr. Bayer 44: 145–158 (1991).

43. M. Tomizawa et al., J. Pesticide Sci. 20: 57–64 (1995).

75. M. L. Avery, D. G. Decker, and D. L. Fischer, Crop Protect. 13: 535–540 (1994).

44. J. Abbink, Pflanzenschutz Nachr. Bayer 44: 183–194 (1991). 45. D. Bai et al., Pestic. Sci. 33: 197–204 (1991). 46. M.-Y. Liu and J. E. Casida, Pestic. Biochem. Physiol. 46: 40–46 (1993).

76. M. L. Avery, D. L. Fischer, and T. M. Primus, Pestic. Sci. 49: 362–366 (1997). 77. F. Heimbach, Soil Biochem. 24: 1749–1753 (1992).

47. R. J. Lind, M. S. Clough, S. E. Reynolds, and F. G. P. Earley, Pestic. Biochem. Physiol. 62: 3–14 (1998).

78. R. F. Mizell III and M. C. Sconyears, Fla. Entomol. 75: 277–280 (1992).

48. R. Zwart, M. Oortgiesen, and H. P. M. Vijverberg, Pestic. Biochem. Physiol. 48: 202–213 (1994).

79. J. Hough-Goldstein and J. Whalen, Biol. Control. 3: 343–347 (1993).

49. S. D. Buckingham et al., J. Exp. Biol. 200: 2685–2692 (1997).

80. A. De Cock, P. De Clercq, L. Tirry, and D. Degheele, Environ. Entomol. 25: 476–480 (1996).

50. K. Nishimura, Y. Kanda, A. Okazawa, and T. Ueno, Pestic. Biochem. Physiol. 50: 51–59 (1994). 51. I. Yamamoto et al., J. Pesticide Sci. 20: 33–40 (1995). 52. I. Yamamoto et al., Arch. Insect Biochem. Physiol. 37: 24–32 (1998). 53. K. Matsuda et al., Brit. J. Pharmacol. 123: 518–524 (1998). 54. M. Tomizawa and J. E. Casida, Brit. J. Pharmacol. 127: 115–122 (1999). 55. M. Tomizawa, B. Latli, and J. E. Casida, in I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer, Tokyo, 1999, pp. 271–292.

81. F. Delbeke et al., Entomophaga 42: 349–358 (1997). 82. D. G. Boucias, C. Stokes, G. Storey, and J. C. Pendland, Pflanzenschutz Nachr. Bayer 49: 103–144 (1996). 83. D. E. Jacobs, M. J. Hutchinson, M. T. Fox, and K. J. Krieger, Am. J. Vet. Res. 58: 1260–1262 (1997). 84. M. W. Dryden, H. R. Hector, and D. M. Daniel, J. Am. Vet. Med. Assoc. 215: 36–39 (1999).

INSECTICIDES, MODE OF ACTION

56. A. Okazawa et al., Pestic. Sci. 54: 134–144 (1998).

Insecticide Resistance Action Committee

57. S. Kagabu and T. Akagi, J. Pesticide Sci. 22: 84–89 (1997). 58. F. J. Placke and E. Weber, Pflanzenschutz Nachr. Bayer 46: 109–182 (1993). 59. A. R. Fernandez-Alba et al., J. Chromatogr. A721: 97–105 (1996). 60. M. Martinez, A. G. Frenich, J. L. Martinez, and P. P. Vazquez, J. Chromatogr. A799: 149–154 (1998). 61. N. R. de Erenchun, Z. G. de Balugera, M. A. Goicolea, and R. J. Barrio, Anal. Chim. Acta 349: 199–206 (1997). 62. J. L. Vilchez, R. El-Khattabi, R. Blanc, and A. Navalón, Anal. Chim. Acta 371: 247–253 (1998). 63. J. Rouchaud, F. Gustin, and A. Wauters, Bull. Environ. Toxicol. 53: 344–350 (1994). 64. L. M. MacDonald and T. R. Meyer, J. Agric. Food Chem. 46: 3133–3138 (1998).

The following document was prepared by the Insecticide Resistance Action Committee. The Insecticide Resistance Action Committee was formed in 1984 to provide a coordinated crop protection industry response to the development of resistance in insect and mite pests. ‘‘The mission of IRAC is to develop resistance management strategies to enable growers to use crop protection products in a way to maintain the efficacy. The organization is implementing comprehensive strategies to confront resistance.’’ The Mode of Action Classification document is available from the Internet at http://www.plantprotection.org/ IRAC. IRAC-US is working with the U.S. Environmental Protection Agency (EPA) to match the document published by EPA in the Federal Register.

65. J. L. Vilchez et al., J. Chromatogr. A746: 289–294 (1996). 66. A. Navalón et al., Analyst 122: 579–581 (1997). 67. J. Rouchaud, F. Gustin, and A. Wauters, Toxic. Environ. Chem. 45: 149–155 (1994).

IRAC MODE OF ACTION CLASSIFICATION

68. J. Rouchaud, F. Gustin, and A. Wauters, Bull. Environ. Contam. Toxicol. 56: 29–36 (1996).

Issue January 2002. Label will contain a box marking the group and type of material:

69. J. Rouchaud et al., Arch. Environ. Contam. Toxicol. 31: 98–106 (1996). 70. K. Scholz and M. Spiteller, Brighton Crop Protect. Conf.— Pest. Dis. 1: 883–888 (1992).

CHEMICAL GROUP

IA

INSECTICIDE

Table. Primary Target Site

Chemical Subgroup

1. Acetyl choline esterase inhibitors

Aa . carbamates Ba . organophosphates

2. GABA-gated chloride channel antagonists

Aa . cyclodienes Ba . fiproles

3. Sodium channel modulators

Pyrethroids and pyrethrins

4. Acetyl choline receptor agonists/antagonists

Aa . chloronicotinyls Ba . nicotine, Ca . cartap, bensultap

5. Acetyl choline receptor modulators

Spinosyns

6. Chloride channel activators

Avermectin, emamectin benzoate Milbemycin

7. Juvenile hormone mimics

Aa . methoprene, hydroprene Ba . fenoxycarb Ca. pyriproxifen

8. Compounds of unknown or nonspecific mode of action (fumigants)

Aa . methyl bromide Ba . aluminum phosphide Ca . sulfuryl fluoride

9. Compounds of unknown or nonspecific mode of action (selective feeding blockers)

Aa . cryolite Ba . pymetrozine

10. Compounds of unknown or nonspecific mode of action (mite growth inhibitors)

Aa . clofentezine, hexythiazox Ba . etoxazole

11. Microbial disrupters of insect midgut membranes (includes Transgenic B.t. crops)

A1a,b . B.t. israelensis A2a,b . B.t. sphaericus B1a,b . B.t aizawai B2a,b . B.t. kurstaki Ca,b . B.t. tenebrionis

12. Inhibition of oxidative phosphorylation, disrupters of ATP formation 13. Uncoupler of oxidative phosphorylation via disruption of H proton gradient 14. Inhibition of magnesium-stimulated ATPase 15. Inhibit chitin biosynthesis 16. Inhibit chitin biosynthesis type 1-Homopteran 17. Inhibit chitin biosynthesis type 2-Dipteran 18. Ecdysone agonist/disruptor 19. Octopaminergic agonist 20. Site II electron transport inhibitors 21. Site I electron transport inhibitors 22. Voltage-dependent sodium channel blocker

Aa . diafenthiuron Ba . organotin miticides Chlorfenapyr Propargite Benzoylureas Buprofezin Cyromazine Tebufenozide Amitraz Hydramethylnon, dicofol Rotenone, METI acaricides Indoxacarb

a Not all members of this class have been shown to be cross-resistant. Different resistance mechanisms that are not linked to target site of action, such as enhanced metabolism, are common for this group of chemicals. Alternation of compounds from different subgroups within this class may be an acceptable part of an integrated pest management program. b Products containing multiple toxins would be differentiated from those containing single toxins only. This would be done by adding a suffix of ‘‘m’’ for multiple toxin products and ‘‘s’’ for single toxin products. Products containing spores would be differentiated from those without spores by adding ‘‘+’’ for spore-containing products and ‘‘−’’ for those products that do not contain spores. For example, B. thuringiensis subsp. kurstaki product containing multiple toxins and spores could be designated as Group 11Dm+, whereas the same product without spores and expressing only one toxin would be designated as Group 11Ds−.

945

946

INSECTICIDES, ORGANOCHLORINES

Label will have the statement: For resistance management, X (name of product) is a group 0X insecticide. Any insect population may contain individuals naturally resistant to X and other 0X insecticides. The resistant individuals dominate the insect population if these insecticides are used repeatedly. These resistant insects may not be controlled by X or other 0X group insecticides, although local experts should be consulted for local resistance recommendations. The following classification scheme is based on mode of action. It is recognized that resistance of insects and mites to insecticides and acaricides can also result from enhanced metabolism, reduced penetration or behavioral changes that are not linked to any site of action classification but are specific for individual chemicals or chemical groupings. Despite this, alternation of compounds from different chemical classes remains a viable management technique. To delay insecticide resistance: • Avoid exclusive repeated use insecticides from the same chemical subgroup. • Integrate other control methods (chemical, cultural, biological) into insect control programs. For further information contact your local distributor.

[This is the minimum statement. More can be added by the individual company.] Acknowledgments We are grateful to the Insecticide Resistance Action Committee for permission to include this document.

INSECTICIDES, ORGANOCHLORINES JACK R. PLIMMER Tampa, Florida

DEREK W. GAMMON California EPA Sacramento, California

Few chlorinated organic insecticides remain in use in north America and Europe. Several have been classified as Persistent Organic Pollutants (q.v.) and proscribed globally. However, their considerable benefits to humanity in the past should not be overlooked nor should the lessons that were learned during the period when they were applied to control disease vectors and agricultural pests throughout the world. Unfortunately, their injudicious use and, at the time of their introduction, ignorance of processes affecting fate and transport of pesticides and their residues led to the widespread occurrence of pollutants. The problems ranged from gross contamination at manufacturing sites to low level contamination in the water of lakes, rivers, and estuaries. Organochlorine compounds were not only used as pesticides, but they also had many industrial uses. Large quantities were used in heat-exchange systems and insulators. Chlorinated organic compounds were also byproducts of a number of chemical manufacturing processes, and as a result of the careless disposal and handling of wastes, quantities were

spilled into the environment. Pollution of the environment was not the only problem. Associated with the increasing use of organochlorine insecticides were questions of longterm toxicity, other effects on wildlife, and the issue of increasing insect resistance. The following sections describe the properties of the different classes of organochlorine compounds used as insecticides, miticides, or acaricides. Table 1 summarizes the nomenclature and properties of the principal organochlorine compounds that have been used in pest control. A stimulus for the investigations that led to the initial discoveries of the insecticidal activity of organochlorine compounds was the spread of World War II. In Europe, supplies of traditional botanical insecticides used in crop production, such as pyrethrum extract and nicotine, were limited by wartime blockades and shortages. The critical need to protect crops from insect pests and to protect personnel in tropical areas from malaria and other insect-borne diseases accelerated the search for synthetic replacements. The insecticidal properties of hexachlorocyclohexane were discovered almost simultaneously in France and England in 1940 (1). The discovery of the insecticidal activity of lindane (the gamma isomer of hexachlorocyclohexane), followed by the well-known successes of DDT for controlling vector-borne diseases, stimulated the evaluation of synthetic organic compounds as new insecticides and their commercialization. During the 1950s, the application of the Diels–Alder reaction to chemical synthesis gave rise to a new group of organochlorine insecticides, the cyclodienes. The insecticidal and acaricidal properties of halogen derivatives of benzene depend on the number and type of halogen atoms and their positions of substitution in the benzene molecule. The insecticidal activity of the fluorobenzenes is relatively weak but greater than that of benzene. Insecticidal activity of the chlorobenzenes increases with the number of chlorine substituents up to three; trichlorobenzenes are the most active. 1,4-Dichlorobenzene has been used as a mothproofing agent. Bromobenzene and dibromobenzenes are somewhat more active than are the corresponding chlorobenzenes. Toluenes containing halogen in the aromatic nucleus are similar in activity to the corresponding benzene derivatives. Activity is considerably higher when halogen is present in the methyl substituent, but the resulting derivatives are unsuitable for practical use because they have a strong irritating effect on mucous membranes. The fungicidal activity of chlorobenzenes increases from monochlorobenzene to hexachlorobenzene. There are several classes of organochlorine insecticides, but the use of many of them was discontinued or restricted because they were found to persist in the environment and accumulated or bioconcentrated through the food chain. Directly or indirectly they showed potential for adverse effects in humans and the environment. Their effectiveness diminished as many insect species showed resistance after repeated applications. Initially, organochlorine insecticides were heavily used, but their disadvantages soon became apparent. An advantage was that, generally, they were not expensive to manufacture. For example, toxaphene and related

Table 1. Insecticides: Organochlorine Compounds CAS RN CYCLODIENES Aldrin 41

IUPAC Name

Mol. Formula

Mol. Wt.

Mp

[309-00-2]

Technical aldrin contains not less than 90% HHDN (1R,4S,4aS,5S,8R,8aR)1,2,3,4,10,10-hexachloro1,4,4a,5,8,8a-hexahydro-1,4 : 5,8dimethanonaphthalene (IUPAC)

C12 H8 Cl6

364.9

HHDN mp 104-104.5 ◦ C.

Chlordane; 46 cis,49 trans

[57-74-9] [12789-03-6] Tech.Grade [5103-71-9] cisisomer(formerly [22212-52-8]). [5103-74-2] trans-isomer

IUPAC NAME: 1,2,4,5,6,7,8,8Octachloro-2,3,3a,4,7,7a-hexahydro4,7-methano-1H-indene Chlordane contains 60–75% of chlordane isomers. Major components are two stereoisomers: alpha or cis-isomer α,2α,3α, 4β,7β7aα, and the trans-isomer 1α,2b,3aα,4β,7β,7α, usually known as gamma, but occasionally as beta-chlordane. Nomenclature at C(1) and C(2)has been confused in the literature. The remainder of the technical grade comprises other stereoisomers (each not more than 7%) and heptachlor.

C10 H6 Cl8

409.8

Trans mp 106.5 ◦ C and cis mp 104.5 ◦ C.

Dieldrin (material containing > 85% HEOD) 42

[60-57-1]

Technical dieldrin contains not less than 85% of HEOD (1R,4S,4aS,5R,6R,8S,8aR)-, 1,2,3,4,10,10-hexachloro1,4,4a,5,6,7,8,8a-octahydro-6,7epoxy-1,4:5,8-dimethanonaphthalene (IUPAC)

C12 H8 Cl6 O

380.9

HEOD mp 176–177 ◦ C.

Dienochlor 37

[2227-17-0].

Perchloro-1,1 -bicyclopenta-2,4-diene

C10 Cl10

474.6

mp 122-123 ◦ C

Endosulfan alpha 70a, beta 70b

[115-29-7] [959-98-8]; formerly [33213-66-0], alpha- = endosulfan; [33213-65-9] formerly [891-86-1] and [19670-15-6], beta- = endosulfan

6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9ahexahydro-6,9-methano-2,4,3benzodioxathiepin-3-oxide [The technical product is a mixture of two isomers: αendosulfan:3α5aβ,6α,9α,9αβ(64–67%) and β-endosulfan 3α5aα,6β,9β,9aα(29–32%)]

C9 H6 Cl6 O3 S

406.9

mp 108-110 ◦ C (alpha isomer) 208-210 ◦ C (beta)

Endrin 40

[72-20-8]

(1R,4S,4aS,5S,6S,7R,8R,8aR)1,2,3,4,10,10-hexachloro1,4,4a,5,6,7,8,8a-octahydro-6,7epoxy-1,4 : 5,8-dimethanonaphthalene (IUPAC) Product contains not less than 85% 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8ahexahydro-6,7-epoxy-1,4-endo,endo5,8-dimethanonaphthalene

C12 H8 Cl6 O

380.9

Dec. 245 ◦ C

Heptachlor 45

[76-44-8]

1,4,5,6,7,8,8-heptachloro-3a,4,7,7atetrahydro-4,7-methano-1Hindene (IUPAC)

C10 H5 Cl7

373.3

mp 95–96 ◦ C

Isodrin 39

[465-73-6]

(1R,4S,5R,8S)-1,2,3,4,10,10hexachloro-1,4,4a,5,8,8a-hexahydro1,4 : 5,8-dimethanonaphthalene (IUPAC)

C12 H8 Cl6

365.5

mp 240–242 ◦ C


947

948


Table 1. (Continued) CAS RN

IUPAC Name

Mol. Formula

Mol. Wt.

Mp

Kepone 76 (Chlordecone)

[143-50-0]

decachloro-5-oxo-pentacyclo[5.3.0.02,6, 03,9 ,04,8 ]-decane; 1,1a,3,3a,4,5,5a,6decachlorooctahydro-1,3,4-metheno2H-cyclobuta(cd)pentalen-2-one (CA);

C10 Cl10 O

490.7

mp 350 ◦ C dec

Mirex 75

[2385-85-5]

1,1a,2,2,3,3a,4,5,5,5a,5b,,6dodecachloro-octahydro-1,3,4metheno-1H-cyclobuta-[c, d]pentalene (CA)

C10 Cl12

545.5

dec 485 ◦ C

Organochlorines DDD (TDE) 3

[72-54-8]

1,1-dichloro-2,2-bis-(4chlorophenyl)ethane

C14 H10 Cl4

320.1

mp 109–110 ◦ C

DDE 2

[72-55-9]

1,1-dichloro-2,2-bis-(4chlorophenyl)ethylene

C14 H8 Cl4

318.0

mp 85 ◦ C

DDT 1

[50-29-3]

1,1,1-trichloro-2,2-bis(4chlorophenyl)ethane (IUPAC); 1,1,1-trichloro-2,2-di-(4chlorophenyl)ethane; Tech. product: 1,1,1-trichloro-2,2bis(chlorophenyl)ethane - contains 30% o,p’-DDT (1,1,1-trichloro-2-(2-chlorophenyl)-2(4-chlorophenyl)ethane)

C14 H9 Cl5

354.5

mp 108.5–109 ◦ C (p,p )

Dicofol (kelthane) 13

[115-32-2].

2,2,2-trichloro-1,1-bis(4chlorophenyl)ethanol

C14 H9 Cl5 O

370.5

mp 77–78 ◦ C

hexachlorocyclohexane 32

HCH [608-73-1] (formerly [39284-22-5]) mixed isomers; [319-84-6] alpha HCH, [319-85-7] beta HCH; [58-89-9] gamma HCH; [319-86-8] delta HCH; [6108-10-7] epsilon HCH

1,2,3,4,5,6-hexachlorocyclohexane (mixed isomers) (IUPAC); gamma isomer, lindane [58-89-9] (contains not less than 99% gamma isomer (ααβ4α5α6β) when sold for pharmaceutical or medical purposes. (it is used as a pediculicide and scabicide, medically and as an ectoparasiticide for veterinary purposes)

C6 H6 Cl6

290.8

(gamma isomer) mp 112.5–113.5 ◦ C

Methoxychlor 22

[72-43-5]

1,1,1-trichloro-2,2-bis-(4methoxyphenyl)ethane (IUPAC)

C16 H15 Cl3 O2

345.7

mp 77 ◦ C tech.) 86–88 ◦ C

Perthane

[72-56-0]

1,1-dichloro-2,2-bis-(4ethylphenyl)ethane

C18 H20 Cl2

307.3

mp 60–61 ◦ C

Toxaphene (camphechlor BSI approved name)

[8001-35-2]

Chlorinated camphenes

C10 H10 Cl8

414

mp 65–90 ◦ C

Hexachlorobenzene 77

[118-74-1]

hexachlorobenzene

C6 Cl6

284.8

mp 231 ◦ C

Pentachlorophenol 85

[87-86-5]

pentachlorophenol

C6 HCl5 O

266.3

mp 191 ◦ C

insecticides were produced by chlorination of camphene to give an insecticidal product, which, although consistent in properties and composition, was a mixture of more than 175 individual products. The technical product was an effective insecticide and was used in large quantities in the United States (some cotton received each year up to 22 pounds per acre cumulatively).

After withdrawal of DDT and the cyclodienes in the 1970s, toxaphene sales in the United States in 1976 continued and were higher than those of any other insecticide, but the problems of resistance and the association of toxaphene spraying with the occurrence of spine injury in fish were among factors that led to its discontinuance.


Implementation of national environmental policies became, from 1970, a major driving force in the choice of molecules that were appropriate for development as pesticides. Research on mode of action was a guide to the design of molecules that might be more effective or selective. Understanding of the mode of action also served to indicate potential resistance problems that might occur when compounds were used injudiciously. When an insect species had developed resistance to a class of insecticides, there was the likelihood that resistance to compounds of other classes possessing the same mode of action might develop. In a particular strain of insects, such resistance due to the same mechanism is termed ‘‘cross resistance’’ in contrast to ‘‘multiple resistance,’’ which is the resistance of a strain to different compounds but resulting from different mechanisms. The organochlorine compounds affect neural transmission. The elucidation of their modes of action and the structure-toxicity relationships have demanded many years of investigation (2). They were succeeded by carbamate and organophosphate insecticides, which showed more acceptable environmental behavior because they were much more readily degraded than were the majority of

organochlorine pesticides, but both classes were inhibitors of acetylcholinesterase. Consequently, the potential for resistance was a major concern, as was high acute toxicity to mammals, and it was important to exploit other modes of action in designing new insecticides. This was addressed by introduction of the synthetic pyrethroids and many new structural types, such as imidacloprid, that acted at different target sites. SECTION 1 DDT DDT: 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (IUPAC); 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane; the technical product: 1,1,1-trichloro-2,2-bis(chlorophenyl)ethane contains 30% o,p -DDT (1,1,1-trichloro-2-(2chlorophenyl)-2-(4-chlorophenylethane)ethane); CAS RN [50-29-3]; C14 H9 Cl5 ; m.wt. 354.5 practically insoluble in water; vapor pressure 0.025 mPa (20 ◦ C, p,p -DDT). DDT is produced by condensation of chlorobenzene with chloral (obtained by oxidation of ethanol with bleaching powder) in the presence of strong sulfuric acid (Fig. 1). The product is p,p -DDT (1) with o,p -DDT (4) as a significant

Cl

Cl

Cl

Cl Cl Cl +

CCl3CHO H2SO4

Cl Cl Cl Cl

Cl p,p′-DDT

o,p′-DDT (4)

(1) Cl

Cl

Cl

Cl

Cl

Cl

H

Cl

Cl

(1) p,p′-DDT CAS [50-29-3] Cl

Cl (2) DDE CAS [72-55-9] Cl

Cl

Cl

Cl Cl

Cl

Cl (3) DDD CAS [72-54-8]

Cl (4) o,p′-DDT CAS [789-02-06] Cl

CO2H

Cl (5)

H

O

Cl

949

Cl

Cl (6)

Cl

Cl (7) DDMU

Figure 1. Manufacture of DDT: DDT structures 1–4; DDT structures 5–7.

950


impurity (20–30% depending on conditions) and a trace of o,o -DDT. DDT was relatively inexpensive to produce on a large scale, and as it had a wide spectrum of insecticidal activity with low mammalian toxicity, it became rapidly the insecticide of choice for control of many insectborne diseases, such as malaria. It was also used on a widespread scale for control of some forest pests, such as the gypsy moth. Initially, its environmental stability appeared to present a considerable advantage, but following its widespread application, residues of DDT and other organochlorine insecticides were shown to be ubiquitous in environmental samples and in wildlife. DDT was widely used in many agricultural crops. The primary use of DDT currently is as a vector control for eradication of malaria-bearing mosquitoes. Less-persistent insecticides have replaced DDT for control of insects on crops and in forests. DDT is a nerve poison that affects the sodium channel of nerve membranes. It is a nonsystemic insecticide with contact and stomach action. The most important reactions of DDT (1) are dehydrochlorination to DDE (2) and reductive dechlorination to DDD (3). These reactions occur abiotically, in vivo and in soils. The products resemble DDT in their recalcitrance toward environmental degradation. The stability of DDT and its principal metabolites DDD and DDE, in combination with their lipid solubility and resistance to biological degradation, resulted in their bioconcentration in fish and other organisms exposed to extremely low levels of these compounds in water. Although metabolism of DDT in mammals may proceed via DDD to give 4,4 dichlorodiphenylacetic acid (5), DDE is also formed and stored in fat. It may be slowly depleted by oxidative reactions, and ring hydroxylated derivatives have been detected in mammals and wildlife samples. Consumption of DDT residues in wildlife and fish by predators resulted in adverse effects. Although its mammalian toxicity is low, DDT is highly toxic to fish. It is only moderately toxic to birds, but DDE, which occurs as a significant environmental residue, is associated with thinning of eggshells in raptorial birds. Decreased eggshell thickness resulted in considerable breakage of eggs with a concomitant decline in population. It has been shown by Lundholm (3) that eggshell thinning caused by p,p -DDE in susceptible species, such as the duck, is due to the inhibition of prostaglandin synthetase. This leads to reduced levels of prostaglandin E2 and lower uptake of calcium by the eggshell gland mucosa. Analogs of p,p -DDE, such as o,p -DDE, p,p -DDT, o,p -DDT and p,p -DDD, were inactive, in both the enzyme assays and in causing eggshell thinning. Reductive dechlorination to DDD occurs readily in flooded soils. A variety of microorganisms convert DDT to DDE or DDD. Both DDE and DDD resemble DDT in their recalcitrance toward environmental degradation, and in mammals, residues may be stored in lipids. Further oxidation gives 4,4 -dichlorodiphenylacetic acid (5), the predominant excretory metabolite. DDT, DDD, and DDE occur widely as residues of DDT, but subsequent metabolism is generally slow in most organisms.

In alkaline solution and at temperatures above its melting point (the technical product has m.p. 108.5–109 ◦ C), DDT decomposes thermally with elimination of hydrogen chloride to form DDE. In solution in a proton donor solvent, such as methanol, DDT decomposes thermally (e.g., in a heated metal gas chromatographic inlet) to give products that include DDD. Environmental Fate. DDT decomposed very slowly in sunlight, and 93% was recovered unchanged from the surface of an apple after 3 months. DDE decomposed more rapidly than DDT in sunlight. Other reports indicate that DDT was photolyzed under field conditions to give products, including DDE, 4,4 dichlorobenzophenone (6), 4-chlorobenzoyl chloride, 4chlorobenzoic acid, and 4-chlorophenyl 4-chlorobenzoate (Fig. 2). Irradiation of DDT at shorter wavelengths under laboratory conditions gave a variety of products that arose from reactions of photolytically generated radicals. The nature of the products and the composition of the product mixture depended on the solvent and the presence or absence of oxygen. Some of the many compounds isolated or detected after irradiation of DDT in solvents by energetic ultraviolet irradiation (less than 260 nm) are shown (Fig. 2). Irradiation of a methanolic solution of DDT by wavelengths around 260 nm gave a complex mixture of products. In methanol under nitrogen, major products were DDD and 1,1-bis(4-chlorophenyl)-2-chloroethylene (DDMU, 7) (Fig. 2). More than 30 components of the mixture obtained by irradiation of DDT in oxygenated methanolic solution were characterized by gas chromatography–mass spectrometry. Many of these probably arose by the interaction of photolytically generated free radicals with oxygen or solvent. Products included DDD, DDE, 4,4 dichlorobenzophenone, and methyl 4-chlorophenyl acetate. Reduced products were formed by hydrogen abstraction from the solvent. Bond rearrangements also generated a variety of structures. For example, expulsion of a molecule of carbon monoxide from 4,4 -dichlorobenzophenone (6) gave 4,4 -dichlorobiphenyl (8). Chlorobenzoic acid and chlorophenol may have been formed by an alternative pathway from 4,4 -dichlorobenzophenone (4). Under similar irradiation conditions, DDE was photooxidized to 3,6dichlorofluorenone (9) in approximately 10% yield (5). Subsequent photooxidation reactions may result in a chlorinated biphenylcarboxylic acid. When DDT was exposed to light (253.7 nm) on quartz for 2 days, 80% of the original DDT degraded to 4,4 dichlorobenzophenone, DDE, and DDD. Irradiation of solid DDT moistened with benzene at 235.7 nm gave 4,4 -dichlorobenzophenone and a number of unidentified ketonic compounds. An intermediate DDT hydroperoxide was later identified. The irradiation of DDT in water (5 mg in 100 ml) with a 1.2-kW high pressure mercury lamp with a quartz filter gave 17 photoproducts identified by gas chromatography–mass spectrometry (with electron impact, chemical ionization, and negative ion chemical ionization). The major products included DDD, DDE, and DDMU (7). DDT was also converted into

INSECTICIDES, ORGANOCHLORINES Cl

951

Cl Cl

Cl Cl

hn

•

Cl

Cl

Cl R=

Cl

Cl + Cl •

(1) hn

+H

−H

•

Cl

R

Cl

Cl Cl

R

Cl

R

R

Cl

R

R

Cl

O

H Cl (3)

(2)

(6)

Cl

Cl (8)

Cl

Cl

Cl R

H

R

Cl

Cl

Cl Cl

(7)

(10)

O (9)

Cl

H

Cl Cl

Cl

Cl

Cl

Cl Cl (11)

Cl

(12)

Cl

Figure 2. Products of DDT irradiation: DDT structures 10–12.

rearrangement products, including 1-(2-chlorophenyl)-1(4-chlorophenyl)-2,2-dichloroethylene (o,p -DDE, (10) and 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethylene (o,p -DDE, 11). Subsequently, DDD, DDE, and DDMU were irradiated separately (6). The mixture of products formed is consistent with a free radical mechanism. Homolytic fission of C−Cl bonds gave dechlorinated radicals that may abstract hydrogen, lose hydrogen chloride, or undergo bond rearrangement. The addition of 5% acetone to the aqueous solution increased the rate of photolysis 1.5 to 2 times. A dimer, 2,3-dichloro-1,1,4,4-tetrakis (p-chlorophenyl)2-butene (12), was isolated in 10% yield after 26 hours of irradiation (360-watt mercury lamp) of DDT dissolved in ethanol with a lamp in the absence of air. In the presence of air, 4,4 -dichlorobenzophenone (6) was a photoproduct (7). The photolysis of DDT at long wavelengths was induced by the presence of an aromatic amine. DDT in cyclohexane in the presence of diethylaniline (or other aromatic amine) decomposed by irradiation at 310 nm to give the same products as those obtained by direct photolysis, including DDD, DDE, and 4,4 -dichlorobenzophenone (8).

Transformations in Soils and Plants. Many microorganisms are capable of degrading DDT, but degradation in soils is very slow, DT50 equalling 3800 days. The primary metabolites of DDT in soils are DDD and DDE. Conversion of DDT to DDD occurred rapidly under anaerobic conditions and slowly to DDE under aerobic conditions. Minor amounts of dicofol (13) were also detected. Soil metabolites of DDT under aerobic conditions (Fig. 3) also reported were 4,4 dichlorobenzophenone, 4,4 -dichlorodiphenylacetic acid 14, 15, 16, and 4-chlorophenylacetic acid (17) (Fig. 4). The fate of DDT in soils is influenced by water content. Under flooded conditions, there was virtually no release of carbon dioxide and DDT was rapidly converted to DDD. Flooding significantly reduced formation of DDE from DDT, and DDD comprised almost half the extractable products after flooding. Data support the hypothesis that loss of DDT associated with increasing soil water content is partly due to the creation of anaerobic microenvironments for microbial degradation of DDT via DDE (9). A proposed pathway of microbial degradation of DDT by Aerobacter

Cl

R H R

R H R

Cl Cl DDT

Cl H Cl

DDD (TDE)

(1)

(3)

Cl =R

Cl

R R

R

Cl

R

Cl

H DDMU (7)

DDE (2) R

H

R

H

R H R

DDNU R R

H Cl H DDMS

R H R

H CHO

H

R OH R

H DDOH

probable intermed

COOH H DDA (5)

Figure 3. Metabolism of DDT.

Cl

R

R

Cl

R

R

Cl

R

R

H

Cl

O

CO2H

Cl

Cl

R

Cl

HO

R

R

R

Cl

DDT

DDD

DDA

4,4′-dichlorobenzophenone

dicofol

(1)

(3)

(5)

(6)

(13)

R

H

R

H

R

OH

R

H

R R (14)

R R.CH2CO2H

CN

R

p,p′-dichlorobenzhydrol

4,4′-dichlorophenylmethane

p-chlorophenyl acetic acid

bis(p-chlorophenyl)acetonitrile

(15)

(16)

(17)

(18)

Cl =R

Figure 4. Products of DDT microbial and/or soil transformations.

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

MeSO2

HO

Cl

Cl

Cl

OH (19)

(20) DDT structures 19–21

952

(21)


aerogenes, based on studies with whole cells or cell free extracts, is supported by the presence of 4,4 dichlorobenzophenone in crude extract of flooded soil (10). In microcosms, mineralization of DDT and DDE was less than 1% of the added radiolabel (14 C-labeled DDE), consistent with half-lives observed in the field. However, in tropical soils, DDE mineralized at a considerably greater rate possibly due to the higher temperatures at which experiments were conducted. Activated sludge degraded DDT with a half-life of 7 hours. DDD, 4,4 -dichlorobenzophenone 4, and a compound identified as bis(4-chlorophenyl)acetonitrile (18) (Fig. 4) were formed. This compound was also found in the sediment layer of a Swedish lake and isolated from sewage sludge of a water treatment plant in Uppsala (11,12). DDT slowly degraded after application to spinach and cabbage. Metabolites identified were DDD, DDE, 4,4 -dichlorodiphenylacetic acid 4, and conjugates of 4, 4 -dichlorodiphenylacetic acid and 4,4 -dichlorobenzhydrol (13). In ensiled pasture herbiage, DDT was extensively converted to DDD and DDE (14). Mammalian Metabolism. Humans excreted 4,4 -dichloro diphenylacetic acid in urine after oral ingestion of DDT. DDD is metabolized and degrades and is rarely found as a stored metabolite, whereas DDE resists breakdown to 4,4 -dichlorodiphenylacetic acid. The higher levels of DDE than DDT found in the general human population reflect its stability (15,16). Human embryonic lung cells incubated with 14 C-labeled DDT produced DDD by reductive dechlorination and gave 4,4 -dichlorodiphenylacetic acid as the only other metabolite (17). The primary metabolites of DDT in the rat are DDE and DDD. The latter is converted to 4,4 -dichlorodiphenylacetic acid, which is excreted in feces or urine conjugated with glucuronic acid or amino acids. DDE was largely excreted unchanged after oral ingestion, but about 5% of the dose was excreted in feces as metabolites in which the aromatic rings were hydroxylated. The dichloroethylene moiety remained intact, and the shift in the position of the aromatic chlorine substituent suggested that an arene oxide intermediate is involved (18). When 14 C-labeled DDT was administered orally to mice, urine samples contained DDT, DDD, DDE, 4,4 dichlorobenzophenone, 4,4 -dichlorodiphenylacetic acid, and dicofol. Five unidentified metabolites and some conjugates were also obtained (19). Metabolism of DDT in mammals may proceed by oxidation of DDE to the acid, 4,4 -dichlorodiphenylacetic acid, followed by excretion as conjugated products or oxidative attack on the aromatic rings of DDE (Fig. 3). Tissue samples from wildlife specimens collected in the field contained many metabolites of DDT. Many of these were hydroxylated in the phenyl rings. Pooled tissue extracts from guillemots and gray seals contained two hydroxylated derivatives of DDE, identified as 1,1-dichloro-2-(4-chloro-3-hydroxyphenyl)-2-(4chlorophenyl)ethylene (19) and 1,1-dichloro-2-(3-chloro4-hydroxyphenyl)-2-(4-chlorophenyl)ethylene (20), and

953

some samples also contained 4,4 -dichlorobenzophenone and 4,4 -dichlorodiphenylacetic acid. These two compounds and a third, identified as 1,1-dichloro-2-(4chloro-2-hydroxyphenyl)-2-(4-chlorophenyl)ethylene, were obtained from rats fed DDD (20). These structures suggest that the metabolic pathway involves an arene oxide intermediate with subsequent ring opening accompanied by a shift of the chlorine substituent. A methanesulfonyl derivative of DDE (21) found in seal blubber and human milk may arise in a mechanistically similar manner involving the addition of the sulfhydryl group of glutathione to an arene oxide. A principal pathway of resistance to DDT in houseflies may be enzymic dehydrochlorination by the enzyme DDTase to the relatively less toxic DDE. There is evidence that the enzyme exists in insects in a number of forms. However, other mechanisms of resistance are important. Mode of Action in Invertebrates. DDT has been shown to interfere with nerve axon ion channels, resulting in a prolongation of the sodium inactivation mechanism and suppression of the potassium conductance increase (21,22). The combined effect is to slow down the repolarization of the nerve membrane after an action potential, resulting in sustained depolarization and repetitive action potentials. DDT has little or no capacity to cause nerve conduction blockage, and this is therefore considered to arise through secondary mechanism(s) that are initiated by the intense nerve activity in poisoned insects. Some of the proposed secondary effects may be related to one or more of the biochemical effects listed below. However, the likely relevance of such secondary effects playing a role in poisoning in vivo must be addressed by the consideration of inactive isomers of DDT (e.g., o,p -DDT) that are often significant impurities in samples of p,p -DDT. Metabolites of DDT and less active analogs also need to be considered in the structure-activity relationship before a putative effect can be ascribed a role in the poisoning process that results in insect mortality. Another interesting feature of DDT action in vivo is its negative temperature coefficient of toxicity in insects (similar to Type I pyrethroids). This is not simply a result of more rapid metabolism/excretion of DDT at higher temperatures because doses can be chosen that allow insects to go into and out of tremors, just by changing the temperature. Thus, any effect of DDT that becomes more pronounced as temperature is raised probably does not play a key role in the development of the signs leading to insect death. In an attempt to correlate the nerve effects with the in vivo poisoning signs, electrode-implanted, free-walking cockroaches (23) (Fig. 5 shows the electrode positions) were dosed with LD95 doses at three temperatures (24,25). Effects on the peripheral and central nervous systems were measured along with the stage of poisoning. Abnormal nerve activity commenced prior to the development of poisoning signs at all three temperatures. Repetitive firing following stimulation began within 2 hours of dosing, and an example is shown in Figure 6a, recorded 6 hours after dosing at 16 ◦ C (5.25 µg/insect). These discharges became more pronounced as poisoning progressed to the tremoring

954

INSECTICIDES, ORGANOCHLORINES Al

R2 ls ↑ ↓ AIR

Ri

S2

A6

Rl

Figure 7. Nerve responses of a DDT-dosed cockroach at 16.5 ◦ C: Top trace R2; second trace, R1 (nerve chord); third trace, Rc (cercus); bottom trace air-puff marker. Recorded 5.5 h after treatment with DDT (5.25 µg) about 1 h before tremors developed. The cockroach was ataxic with an occasional kick of a leg. (After D. W. Gammon, Pesticide Science, 9, 95–104 (1978)).

Cercus

Epoxy resin

Copper wire to P.C.B.

Sl

0

Rc

1.5

Tungsten needle

Figure 5. The ventral surface of a cockroach abdomen. The first (A1) and sixth ganglia (A6) are marked and the cercal nerves are shown leaving the posterior of A6. Recording electrodes are Rc (circus), R1 and R2 (abdominal connectives) and Ri (common indifferent). Nerves were stimulated electrically by applying rectangular pulses at S1, to stimulate a cercal nerve and S2, the abdominal nerve chord. Dashed line on left cercal nerve indicates the site of severance in some experiments. Inset: the electrode used for stimulating and recording from the cercus. P.C.B. indicates printed circuit board used for electrode attachments. (After D. W. Gammon, Pesticide Science, 9, 79–81 (1978)).

(a)

8

30

37

(b)

47 500 µV

500 µV

1 mV 10 ms

100 ms

Figure 6. Nerve responses of a DDT-dosed cockroach at 16.5 ◦ C: a) 6 h after treatment with DDT (5.25 µg), single electrical stimulation of a cercal nerve (S1) resulted in after-discharge of abdominal neurons, recorded by R2 (upper trace) and R1 (lower trace). Stimulation of the nerve chord at S2, gave a normal response. The cockroach exhibited no symptoms of poisoning; b) 19 h after treatment the same individual gave a more pronounced after-discharge following cervical nerve stimulation. Stimulation of the nerve chord, at S2, also yielded an after-discharge. The cockroach was undergoing periodic tremors. (After D. W. Gammon, Pesticide Science, 9, 95–104 (1978)).

stage, shown at 19 hours (Fig. 6(b)). Sensory axons (in the cercus) also fired repetitively following a brief air-puff stimulus, from an early stage, shown in Figure 7 at 5.5 hours, about an hour before tremors developed, at 16 ◦ C. At 25 ◦ C, an estimated LD95 (20 µg/insect) caused effects that

4 ms

500 µV

20 ms

500 µV

40 ms

Figure 8. Nerve responses of a DDT-dosed cockroach at 25 ◦ C. Evoked responses to a single electrical stimulation of a cercal nerve (at S1) recorded in the nerve chord at R2 (upper) and R1 (lower) at different sweep speeds. The records were taken from the same experiment and the numbers on the left indicate time (h) since treatment with DDT (20 µg). (After D. W. Gammon, Pesticide Science, 9, 95–104 (1978)).

were qualitatively similar. Examples of repetitive firing in the central nervous system (CNS) following electrical stimulation, presynaptically (Fig. 8) and postsynaptically (Fig. 9), were recorded from the same experiment at 25 ◦ C. In the experiments at 25 ◦ C, tremoring developed at about 6.5 hours after dosing and prostration followed at 24–30 hours. The insects at 25 ◦ C became paralyzed around 37 hours after dosing. At both 16 ◦ C and 25 ◦ C, as well as at 32 ◦ C, these after-discharges reached a peak in terms of both duration and intensity during the paralyzed stage, which was often many days after dosing. This was also true for the discharges in both sensory and motor neurones (25). The duration of the repetitive


955

Table 2. Approximate Maximum Duration of Abdominal After-Discharges Following Single Electrical Stimulation of a Cercal Nerve (S1) and the Nerve Cord (S2) of DDT-Dosed Periplaneta

0

1.5

Temperature (◦ C)

8

Dose of DDT µg

S1 Maximum Duration (ms)

S2 Maximum Duration (ms)

5.25 20.0 27.6

420 180 90

140 120 6000 mg/kg. It is useful in the home garden, for the control of insect pests of vegetable and fruits, for veterinary hygiene, and for the ∗

FOB = Functional Observational Battery.

959

control of bark beetles that are the vectors of Dutch elm disease. Methoxychlor is a contact insecticide and also has stomach action. It is much less readily dehydrochlorinated in alkaline solution or in biological systems than is DDT. However, the p,p -methoxy groups are rapidly attacked by microsomal oxidase systems in higher animals to form phenols that are conjugated and eliminated. Pathways of degradation of methoxychlor in microorganisms, mammals, mosquito larvae, algae, fish, and snails, are primarily dechlorination and O-dealkylation. The principal route of degradation in mammals is by O-dealkylation to the corresponding phenol and bisphenol and by dehydrochlorination to 4,4 -dihydroxybenzophenone. Thus, methoxychlor does not bioaccumulate as does DDT and is favored for general environmental use. However, it is more expensive than DDT and has little effectiveness toward a number of insects. It is more readily degraded by biota than is the fully chlorinated analog and is less likely to store in the body fat of animals or be excreted in milk. Methoxychlor is stable to oxidizing agents and to ultraviolet light, but it becomes pink- or tan-colored on irradiation. It reacts with alkalies, especially in the presence of catalytic metals, with the loss of hydrogen chloride. The major product of photolysis of methoxychlor in air-saturated water, irradiated at wavelengths >280 nm, was 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethylene (23), whereas 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane (24) was formed along with 23 in degassed water-acetonitrile solutions (Fig. 10). Subsequently, 23 was photolyzed to benzaldehyde (59). Products of photolysis in aqueous alcoholic solutions were 4,4 -dimethoxybenzophenone (25), 4-methoxybenzoic acid, and 4-methoxyphenol (26) (60). When 22 was irradiated by ultraviolet light (carbon arc, 220–330 nm) in milk, 4-methylanisole (27), 25, 26, 1,1,4,4-tetrakis(4-methoxy)-2,3-dichloro-2-butene (28) and 1,1,4,4-tetrakis(4-methoxy)-1,2,3-butatriene (29) were identified as products (61). Chemical decomposition was slow in water, and at 27 ◦ C, DT50 at pH 5 to 9 was 100 days. Major products of hydrolysis are anisoin, anisil, and 23 (62). DT50 of methoxychlor in water is about 46 days. Dechlorinated and dehydrochlorinated methoxychlor are major products observed in studies with bacteria. Degradation by Aerobacter aerogenes gave 23 and 24 (63). Methoxychlor degraded more rapidly in flooded soils than under aerobic conditions. In sediments, DT50 was 5000 mg/kg. Heptachlor may be obtained by treatment of chlordane with N-bromosuccinimide, followed by chlorination with hydrogen chloride in nitromethane in the presence of aluminum trichloride or with monochloro iodide in carbon tetrachloride.


Cl

ClHO

Cl

Cl

Cl Cl

Cl Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

(59)

cis chlordane (46)

Cl Cl

(45) Cl

Cl Cl Cl

Cl Cl

OH

Cl

O

Cl Cl

Cl

OH Cl

Cl (54)

Cl

(55)

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl Cl

OH

OH

Cl

Cl

Cl

Cl

Cl

Cl

Cl

(57)

Cl

Cl

Cl Cl

O

Cl

Cl

(53)

Cl

Cl Cl

Cl Cl

Cl

Cl

Cl

967

(58)

(56)

Figure 17. Metabolism of chlordane.

Cl

Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl

Cl

Cl

in acetone solution

Cl

Cl (60)

Cl Cl

Cl Cl

Cl

(62)

heptachlor (45)

Cl

Cl

Cl

Cl Cl

Cl Cl

Cl

Cl (61)

Cl

Cl O

Cl (63)

Figure 18. Heptachlor photochemistry.

Irradiation of heptachlor at 253.7 nm in hexane or cyclohexane solution afforded two isomeric monodechlorination products (60) and (61) (Fig. 18). In acetone at 300 nm, a cage compound (62) was the sole product. Cage

formation by heptachlor to give (62) may be postulated on the basis of analogous photochemical reactions of endocyclopentadiene derivatives. Photodechlorination in cyclohexane occurred via an excited singlet state (98). In the

968


presence of a photosensitizer, such as acetone, cage formation occurs via a triplet state (99). Under these conditions, heptachlor epoxide (64) gave a ketonic product (63) in an isomerization reaction in which the epoxide ring was opened.

Metabolism. Soil microorganisms transform heptachlor by epoxidation, hydrolysis, and reduction. Heptachlor incubated with a mixed culture of organisms gave chlordene (43), which was further metabolized to chlordene epoxide (65). Also detected were 1-hydroxychlordene (66), 1hydroxy-2,3-epoxychlordene (67), and heptachlor epoxide (64). The hydrolysis product of heptachlor (45) was metabolized by soil organisms to a product, which may be a ketochlordene (Fig. 19). Metabolic products identified when rats were fed a diet containing 100 ppm of heptachlor during a 4-week period were the epoxide (64) and 1-exo-1-hydroxyheptachlor epoxide (13) and 1,2-dihydroxydihydrochlordene (68) (100,101). Additional metabolites formed from heptachlor epoxide after incubation with microsomal preparations from the liver of pigs and from houseflies were the diol (69) and 67. Toxicology. Heptachlor was toxic to the rodent liver, resulting in hepatic hyperplasia, with a NOEL of 5 ppm (0.25 mg/kg/day) in the rat. Hepatocellular carcinoma in mice was observed, but this may have been secondary to liver damage because the genotoxicity assays were considered negative. IARC has designated heptachlor as a Class 2B or ‘‘possible human carcinogen.’’ No adverse effects were noted in developmental or reproductive toxicity tests in the rabbit and rat, respectively. Aldrin. Aldrin, (41) [309-00-2], 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4-endo,exo-5,8-dimethanonaphthalene. It is a colorless solid, mp 104–104.5 ◦ C, vapor pressure 5.2 mPa at 20 ◦ C. It is chemically stable and is not degraded by water or caustic alkalies at room temperature. This compound is very slightly soluble in water (to 0.027 µg/L) but soluble in petroleum hydrocarbons. Aldrin has rat LD50 values of 39, 60 mg/kg (oral) and 98 mg/kg (dermal). It has been widely used as a seed treatment and soil insecticide, where it is gradually oxidized to its epoxide dieldrin.

Toxicology. According to the U.S. Environmental Protection Agency, there is good evidence that aldrin/dieldrin is oncogenic in the mouse, causing a dose-related increase in benign and malignant liver tumors, along with some evidence of lung tumors. There is some evidence of oncogenicity in the rat. Liver toxicity was noted in both rodents and the dog, at doses as low as 0.5 ppm (0.025 mg/kg/day) in the rat. DNA damage and UDS recorded in transformed human cells following dieldrin administration indicated possible genetic toxicity. Nonetheless, IARC has designated aldrin and dieldrin as Class 3 or ‘‘not concluded to be a human carcinogen.’’ Signs of neurotoxicity were reported in rodents in dietary studies, in the form of hyperactivity and tremors, at doses as low as 2.5 ppm (0.4mg/kg/day) in the mouse.

Dieldrin. Dieldrin (42) [60-57-1] or 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-6,7-epoxy-1,4-endo, exo-5, 8-dimethanonaphthalene (mp 176 ◦ C, vp 0.4 mPa at 20 ◦ C) is formed from aldrin by epoxidation with peracetic or perbenzoic acids. It is soluble in water to 27 µg/L. Aldrin and dieldrin have had extensive use as soil insecticides and for seed treatments. Dieldrin, which is very persistent, has had wide use to control migratory locusts, as a residual spray to control the Anopheles vectors of malaria, and to control tsetse flies. Because of its environmental persistence and propensity for bioaccumulation, registrations in the United States were canceled in 1974. Endrin. Endrin (40) [72-20-8] is 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-6,7-epoxy-1,4-endo,endo5,8-dimethanonaphthalene (mp 245 dec, vp 0.022 mPa at 25 ◦ C) and is soluble in water to 23 µg/L. This compound is the endo,endo isomer of dieldrin, which is less stable and more toxic than dieldrin with rat LD50 values of 17.8 and 7.5 (oral) and 15 (dermal) mg/kg. It was used as a cotton insecticide, but because of its high toxicity to fish, its use was restricted.

Toxicology. Endrin did not appear to be carcinogenic in rat and mouse chronic dietary studies. It has been designated by IARC as Class 3 or ‘‘not considered to be a human carcinogen.’’ Enlarged liver and kidneys were reported in both rodents and the dog. The NOEL was 1 ppm (0.05 mg/kg/day) in the rat. Tissue degeneration was observed in both organs in the dog. All studies were compromised by high mortality, resulting from neurotoxic effects causing convulsions. Endosulfan. Endosulfan (70) [115-29-7], 6,7,8,9,10,10hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3,benzo-dioxathiepine 3-oxide (IUPAC) [The technical product is a mixture of two isomers: α-endosulfan: 3α,5αβ,6α, 9α,9αβ (64–67%) (70a) and β-endosulfan 3α,5aα,6β,9β, 9aα, (29–32%) (70b)]; [959-98-8] (formerly [33213-660]) (β-endosulfan);[33213-65-9] (formerly [891-86-1] and [19670-15-6]) (β-endosulfan) is the adduct of hexachlorocyclopentadiene and 1,4-dihydroxy-2-butene reacted further with SOCl2 to produce 6,7,8,9,10,10-hexachloro1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxa thiepin-3-oxide. The technical product is a brownish solid, mp 70–100 ◦ C, vapor pressure 1.3 mPa at 25 ◦ C, soluble in petroleum solvents but having low solubility in water. It consists of about four parts of α-isomer (mp 108 ◦ C, cis with regard to the sulfite group) and one part of the β-isomer (mp 206 ◦ C, trans with regard to the sulfite group). The α-isomer, which is somewhat more insecticidal, is slowly converted to the more stable β-isomer at high temperature, and both isomers are oxidized slowly to endosulfan sulfate [1031-07-8] (mp 181 ◦ C). In acid media, both isomers form endosulfan diol [2157-19-9] (mp 203 ◦ C). The rat LD50 values are 43, 18 mg/kg (oral) and 130, 74 mg/kg (dermal). The α-isomer has somewhat greater insecticidal activity and is slowly converted to the more stable β-isomer at a high temperature. Both isomers oxidize slowly in air and in biological systems to endosulfan sulfate [1031-07-8], mp 181–182 ◦ C. In acid media, both isomers form endosulfan diol [2157-19-9], mp 203–205 ◦ C.


Cl

Cl

Cl

Cl Cl

Cl

Cl

OH

Cl Cl

Cl

969

Cl

Cl

(66)

(43) chlordene

Cl

Cl

Cl

Cl

Cl

O

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl OH

Cl Cl

Cl

OH Cl

Cl

Cl Cl

O

Cl OH

Cl

(64) heptachlor epoxide

Cl

Cl

Cl

OH

Cl

Cl

Cl

Cl

(68)

Cl

Cl

Cl

heptachlor (45)

(65)

Cl

O

Cl

OH

Cl (69)

(67) Figure 19. Heptachlor metabolism.

Cl

Cl

Cl

Cl

Cl

O Cl

Cl

Cl

O

SO

Cl Cl

Cl

Cl alpha endosulfan (70a)

aldrin (41)

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl Cl

O O

Cl

O

dieldrin (42)

SO

Cl beta endosulfan (70b)

Structures 41 and 42; structures 70a and 70b

Endosulfan is a broad-spectrum insecticide used to control pests of vegetables, fruit, field crops, and ornamentals. Unlike other cyclodiene insecticides, it is biodegradable by hydrolysis at the sulfite ester bonds and is more readily metabolized. It is also less persistent on plant surfaces,

and 50% of the residues are lost in 3–7 days. Volatilization may be the major route of loss. Endosulfan is readily hydrolyzed in water to the diol (74), but it is moderately persistent in soil. Endosulfan (αand β-endosulfan) is degraded in soil with DT50 30 to 70

970


days. The major metabolite is usually endosulfan sulfate (71), which is degraded more slowly. In the field DT50 for total endosulfan (α- and β-endosulfan and endosulfan sulfate) is 5 to 8 months.

Metabolism. Endosulfan is metabolized rapidly in mammalian organisms to less toxic metabolites and to polar conjugates. The sulfate is also a major metabolite in plants and occurs as a metabolite in some mammals. Endosulfan is quite toxic to water organisms, and residues were found in runoff water, sediment, infiltration water, and soil following a single application. Rats dosed orally or intraperitoneally with endosulfan (4–8 mg/Kg) excreted unchanged endosulfan, the hydroxy ether (72), the lactone (73), and unidentified metabolites in urine in the ratio 3 : 1 : 1 : 2. The diol, the hydroxy ether, and the lactone were identified in most samples of urine and feces. The metabolite most frequently recovered from tissues, organs, and feces was endosulfan sulfate (Fig. 20). Transient amounts of endosulfan and endosulfan sulfate were detected in the body fat and liver of mice after they were dosed with 14 C labeled endosulfan. The mice excreted endosulfan metabolites. Cows fed 2.5–5 ppm endosulfan for 30 days excreted 0.1–0.2 ppm endosulfan sulfate in milk. After a single dose of 14 mg/kg of 14 C endosulfan, sheep excreted 0.25 ppm in milk in the 6–24-h period following ingestion (102). Residues fell to 0.04 ppm and 0.01 ppm after 3 and 11 days, respectively. The main metabolites in urine were the diol and the hydroxy ether. Endosulfan sulfate, the ether, the hydroxy ether, the lactone, and one unidentified metabolite were detected on the surface when male migratory locusts (Pachytilus migratoides) were exposed to endosulfan by oral, cutaneous, or subcutaneous administration. Similar metabolic pathways were observed in the housefly and the cockroach. Six to

Cl

Cl

Cl

Cl

OH Cl

Cl

OH

seven days after the last dose, neither endosulfan nor its metabolites could be detected in locusts (103). Endosulfan is very toxic to fish and caused many fish deaths when the Rhine River became contaminated in June 1969 (concentration was 0.1 ppm). The only residues detected in fish exposed to acute and multiple subchronic concentrations of endosulfan were endosulfan and the diol and the glucuronic acid conjugate of the diol.

Toxicology. Endosulfan was not carcinogenic in the rat or mouse in chronic dietary studies. It was not genotoxic in a variety of tests. Kidney toxicity was observed in the rat at dietary levels of 100 ppm (5 mg/kg/day) and above. Clinical signs of hyperactivity and tremors were reported in many studies. No evidence was found of developmental or reproductive toxicity. Mirex. Mirex (75) [2385-85-5] is 1,2,3,4,5,5,6,7,8,9, 10,10-dodecachloro-octahydro-1,3,4-metheno-2H-cyclobuta-[c,d]-pentalene. The rat LD50 s are 306, 600 (oral) and >2000 (dermal) mg/kg. Mirex was patented in 1954. It is extremely resistant to biodegradation and was once considered the perfect stomach poison insecticide for use in baits to control imported fire ants. However, even at doses of a few milligrams per 10 m2 , it was found to bioaccumulate in birds and fish and its registrations were canceled in the United States in 1976. Kepone. Kepone (76) or chlordecone [143-50-0] decamp chloro-5-oxo-pentacyclo-[5.3.0.02,6, 03,9 ,04,8 ]-decane, 350 ◦ C (dec.) is the 2-keto analog of mirex and is soluble in water to 4 g/L by hydration. The rat LD50 s are 95, 140 (oral) and > 2000 (dermal) mg/kg. Chlordecone is a stomach poison used in baits for the control of cockroaches and ants and for the control of banana thrips. Because

Cl

Cl

Cl

Cl

O Cl

Cl

Cl

O

Cl

SO

O Cl

Cl

Cl

O

SO2

Cl

diol

alpha endosulfan

endosulfan sulfate

(74)

(70)

(71)

Cl

Cl

Cl OH

Cl

Cl NoH

Cl

conjugates Cl

Cl

Cl

Cl

O

Cl

Cl (72)

Figure 20. Endosulfan transformations.

(73)

O


of bioaccumulation, its registrations were canceled in the United States in 1978. Cl Cl

Cl Cl

Cl

Cl

Cl Cl

Cl Cl Cl

Cl

Cl

Cl Cl

O Cl Cl

Cl Cl Cl

Cl

mirex

kepone

(75)

(76)

Cl

Structures (75) and (76) mirex and kepone

SECTION 4 Chlorinated Terpenes A group of incompletely characterized insecticidal compounds has been manufactured by the chlorination of the naturally occurring terpenes. Toxaphene [8001-35-2] is prepared by chlorination of the bicyclic terpene, camphene [79-92-5] to yield a product containing 67–69% chlorine and has the empirical formula C10 H10 Cl8 . The technical product is a yellowish, semicrystalline gum (mp 65–90 ◦ C, d 1.64) and is a mixture of 175 polychlorinated derivatives. Toxaphene is unstable in the presence of alkali, upon prolonged exposure to sunlight, and at temperatures above 155 ◦ C, liberating hydrogen chloride and losing some of its insecticidal potency. It is very soluble in organic solvents, but only soluble to 0.4 mg/L in water. The oral LD50 to the rat is 69 mg/kg. The most active ingredients in technical toxaphene are 2,2,5-endo-6-exo-8,9,10-heptachlorobornane [51775-36-1] (mouse intraperitoneal LD50 6.6 mg/kg) and 2,2,5-endo6-exo-8,9,9,10-octachlorobornane [58002-18-9] (mouse ip LD50 3.1 mg/kg). Each constitutes ca 2–6% of the technical mixture. Environmental. Toxaphene is extremely toxic to fish LC50 values to trout and bluegill of 0.003–0.006 ppm. At water concentrations as low as 0.00005 ppm, toxaphenetreated fish suffer broken-back syndrome, a crippling collagen deformity. Bioaccumulation occurs from water to fish at levels up to 100,000-fold. Toxaphene also is highly toxic to birds (oral LD50 to pheasant 40 and 71 mg/kg). The soil persistence of toxaphene is difficult to assess because of the complex mixture, but published estimates for half-life range from 2 months to 10 years. Toxaphene is a broad-spectrum, persistent pesticide that was widely used on cotton and other field crops. Its registration was revoked by the U.S. Environmental Protection Agency in 1983. Toxicology. Toxaphene has been found to be carcinogenic in rats and mice. It was found to cause an increased incidence of thyroid and pituitary adenomas in the rat and hepatocellular carcinomas in the mouse. Developmental

971

toxicity in the rat was reported as a significant reduction in the number of fetal ossification centers with increasing dose. SECTION 5 Hexachlorobenzene Hexachlorobenzene (77) [118-74-1], HCB, a fungicide, is used as a seed protectant. Hexachlorobenzene acts as a selective fungicide and exerts a fumigant action on fungal spores. It is a white crystalline compound mp 226 ◦ C and is almost insoluble in water. Hexachlorobenzene is very stable, unreactive toward acids and bases, and persistent in the environment. Photolysis is very slow, and in artificial sunlight, solid hexachlorobenzene photodecomposed after 5 months. In sunlight, 20 g of hexachlorobenzene contained in a borosilicate flask gave 64 ppm of pentachlorobiphenyl after 56 days (104). Sensitized photolysis of HCB at wavelengths greater than 285 nm in acetonitrile/water containing acetone gave dechlorinated products: pentachlorobenzene (78) (71%), 1,2,3,4-tetrachlorobenzene (79) (0.6%), 1,2,3,5-tetrachlorobenzene (80) (2.2%), and 1,2,4,5- tetrachlorobenzene (81) (3.7%). Without acetone, products included pentachlorobenzene (78) (76.8%), 1,2,3,5-tetrachlorobenzene (80) (1.2%), 1,2,4,5- tetrachlorobenzene (81) (1.7%), and 1,2,4-trichlorobenzene (82) (0.2%) (105). Irradiation of hexachlorobenzene in methanol solution at wavelengths greater than 260 nm gave a mixture of reductively dechlorinated products (pentachlorobenzene and a tetrachlorobenzene, probably 80) and pentachlorobenzyl alcohol 83, and also a tetrachlorodi(hydroxymethyl)benzene (106). A similar product mixture was obtained by exposing a methanolic solution of hexachlorobenzene in methanol to sunlight outdoors. After 15 days, only 30% of hexachlorobenzene was recovered. Photolysis rates were enhanced by the addition of sensitizers (diphenylamine, tryptophane, and naturally occurring organic substances), but no products were identified (Fig. 21). In an anaerobic sewage sludge, hexachlorobenzene was reductively dechlorinated and the principal product was 1,3,5-trichlorobenzene (84). Pentachlorobenzene, 1,2,3,5tetrachlorobenzene, and dichlorobenzenes were also identified (107). In activated sludge, 1.5% of hexachlorobenzene was mineralized as carbon dioxide after 5 days. Metabolism. Metabolism in mammals is slow, and metabolites include polychlorinated phenols and benzenes, and many sulfur derivatives. Enzyme preparations from liver, lung, kidney, and small intestine dechlorinate HCB, and hepatic mixed function oxidases are responsible for the formation of pentachlorophenol and other phenols. The principal metabolites in mammals are pentachlorophenol (85), tetrachlorohydroquinone, and pentachlorothiophenol. Lesser amounts of pentachlorobenzene, tetrachlorobenzene, 2,3,4,6- and 2,3,5,6- tetrachlorophenols, and 2,4,5and 2,4,6-trichlorophenols are also produced. Adult male rats excreted less than 1% of a single oral dose of 14 C-labeled HCB in urine within 7 days.

972

INSECTICIDES, ORGANOCHLORINES Cl Cl Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

(80)

Cl

Cl

Cl

Cl

Cl

(78)

Cl

Cl

Cl

(77) Cl irradiation in methanol

Cl

Cl (81)

Cl

Cl

Cl

Cl

Cl

CH2OH

Cl

Cl

Cl (79) Cl

Cl

Cl

Cl

(83)

(82)

Cl (84)

Figure 21. Hexachlorobenzene photolysis.

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

SCyst Cl

Cl

Cl

Cl

Cl

(78)

Cl

Cl Cl

(77)

SMe Cl

Cl

SCH2CH(NHCOMe)CO2H

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

OMe

Cl4 Cl

OH

Cl

Cl

Cl

(85) SOMe

Cl4

SO2Me OMe

Cl4

OMe

Figure 22. HCB metabolism—initial steps in pathways showing formation of sulfur- and oxygen-containing compounds.

Pentachlorophenol, 2,4,5-trichlorophenol, tetrachlorobenzene, and pentachlorobenzene were detected. A total of 16% of the dose was excreted in feces, but no metabolites were detected. A total of 70% of the dose remained in the body, mainly in fat, and this was mostly HCB with

traces of dechlorinated metabolites (108). Enzyme studies in vitro indicated that HCB was dechlorinated by enzyme preparations from liver, lung, kidney, and small intestine and that pentachlorophenol was formed by hepatic mixed function oxidases.


The identification of sulfur-containing metabolites, including N-acetyl-S-pentachlorophenyl cysteine (pentachlorophenylmercapturic acid) (86) and pentachloromethylthiobenzene (109) indicated that the reaction of glutathione with HCB may be an intermediate step in metabolism (Fig. 22). A key step in the metabolic pathway is the formation of pentachlorophenylmercapturic acid as a polar intermediate. The distribution pattern of metabolites was similar in rat urine and feces, except that tetrachloro-1,4hydroquinone was detected only in urine. When rats were treated orally with 50 mg Kg−1 HCB every other day for 2 weeks, 21 urinary metabolites could be separated by capillary gas chromatography. There may have been more than 21 metabolites in the excreta because conjugates were hydrolyzed during the procedure. Of the known HCB metabolites in humans, pentachlorophenol occurred in urine in larger amounts than in feces, and urinary concentrations were dependent on the concentrations of HCB in human adipose tissue. Metabolites were formed by reductive dechlorination, ring hydroxylation, or replacement of chlorine by sulfurcontaining moieties (104). Pentachlorophenol, 2,4,5-trichlorophenol, and pentachlorobenzene were detected in vivo (103). Enzyme studies in vitro indicated that HCB was dechlorinated by enzyme preparations from liver, lung, kidney, and small intestine and that pentachlorophenol was formed by hepatic mixed function oxidases. A variety of sulfur-containing metabolites, including N-acetyl-S-pentachlorophenyl cysteine and pentachloromethylthiobenzene, were identified (110). In higher animals, HCB metabolites are excreted primarily in feces, which contain unchanged HCB and polar metabolites. In lactating animals, HCB may be excreted in milk, but an important route may be passive elimination across the intestinal wall into the contents of the gut. Toxicology. According to WHO (111), HCB has been found to be carcinogenic in several animal studies as well as having adverse non-neoplastic effects on a number of organ systems. An accidental human poisoning took place in Turkey in 1955–1959, during which HCB-contaminated wheat flour was used to make bread. There were over 600 cases of porphyria cutanea tarda, related to disturbances in porphyrin metabolism, with high mortality. Severe developmental toxicity was also reported: Nursing infants of dosed mothers developed pembe yara or pink sore, and most died within a year. Although follow-up studies on survivors were conducted over 20 to 30 years, no consistent epidemiological evidence was developed for an increased cancer incidence, but other abnormalities persisted. Pentachlorophenol Pentachlorophenol [87-86-5] mp. 178 ◦ C (technical), 191 ◦ C (anhydrous) is an insecticide, fungicide, and nonselective contact herbicide. It is used to control termites, and it has been used extensively as a wood preservative to protect timber against rot and marine borers, and in 1972, 38 million pounds of an estimated U.S. production of more than 50 million pounds was used for this purpose. The

973

sodium salt has been used as a general disinfectant. It was widely used as a wood preservative, but its use in proximity to water led to leaching of pentachlorophenol and its associated impurities. It has now been displaced because of its potential for contamination of many ecosystems. Its metabolism, toxicology, and environmental effects have been investigated intensively, and pentachlorophenol and its associated impurities are the subject of extensive literature (112,113). Impurities of manufacture included dioxins, such as TCDD, dibenzofurans, and hexachlorobenzene. Pentachlorophenol was manufactured in the United States by direct chlorination of phenol or chlorophenols. Chlorination is performed at atmospheric pressure. The temperature in the primary reactor is in the range 65–130 ◦ C (preferably 105 ◦ C) and is held in this range until the melting point of the product reaches 95 ◦ C. The temperature is increased to maintain a temperature of about 10 ◦ C above the product melting point until the reaction is complete in 5–15 hours. The process gave rise to a number of related impurities. The commercial product contained about 10% tetrachlorophenol. Dow commercialized a product containing 88% chlorophenol, 2,3,4,6-tetrachlorophenol, less than 30 ppm octachlorodibenzo-p-dioxin, and less than 1 ppm hexachlorodibenzo-p-dioxin. Some commercial products contained up to 2500 ppm octachlorodibenzo-p-dioxin and up to 27 ppm of the hexachlorodibenzo-p-dioxins. Such compounds are extremely toxic to a variety of organisms, including mammals. Pentachlorophenol (PCP) (85) decomposed on exposure to sunlight. There have been a number of studies. Ultraviolet irradiation in hexane or methanol gave 2,3,5,6-tetrachlorophenol (86) by reductive loss of chlorine, whereas irradiation of a suspension of the free phenol in water afforded polymeric substances as the major products with a little tetrachlorophenol, chloranil (90), and chloranilic acid (88). When an aqueous solution was exposed to sunlight for 10 days, the violet-colored solution contained a number of reaction products (114). The major products were chloranilic acid (88) and 3,4,5-trichloro-6-(2 -hydroxy3 ,4 ,5 ,6 -tetrachlorophenoxy)-o-benzoquinone (92). Minor products also identified were tetrachlororesorcinol (0.10%) (87), 2,5-dichloro-3-hydroxy-6-pentachlorophenoxy-p-benzoquinone (91) (0.16%), and 3,5-dichloro-2-hydroxy5- (2 ,4 , 5 ,6 -tetrachloro-3-hydroxyphenoxy-p-benzoquinone (93) (0.08%). In hexane, 2,3,5,6-tetrachlorophenol (86) was the major product (30% after 32-h irradiation) and a 10% of a compound tentatively identified as a tetrachlorophenol (115). Trace amounts of octachlorodibenzo-pdioxin (94) were obtained when sodium pentachlorophenate was irradiated by natural or artificial sunlight (116) (Fig. 23). In soils, PCP may undergo reductive dechlorination under anaerobic conditions to give tetra-, tri-, and dichlorophenols and m-chlorophenol. In aerobic and anaerobic soils, the major metabolite was pentachloroanisole and lesser chlorinated phenols were also formed (117,118). Microbial conversion in aquatic situations or in activated sludge also gives rise to lesser chlorinated phenols, and pentachloroanisole was also identified among the products

974


OH Cl

Cl

O Cl

Cl

Cl

Cl

Cl

Cl

(86)

O (90)

OH

Cl

OH

Cl

OH

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl O

Cl

(87)

(85)

O

HO

Cl

O

O

Cl

Cl

Cl

OH O (88)

OH

OH

Cl

Cl

Cl

Cl OH

(91)

Cl

Cl

Cl

OH

Cl

Cl

Cl

Cl

Cl

OH

(89)

Cl

O

O

Cl

O

Cl

Cl

Cl

O

Cl

O

Cl

Cl

O

OH

Cl

O

Cl Figure 23. Photolysis of pentachlorophenol.

when the disappearance of pentachlorophenol was investigated under aerobic or anaerobic conditions in aquaria. Metabolism. Pentachlorophenol was metabolized in rats by conjugation with glucuronic acid and eliminated as the glucuronide. P450 catalyzed oxidative dechlorination also occurred to form tetrachlorohydroquinone, and this was conjugated to form a monoglucuronide representing 27% of the dose administered. Other metabolites have been reported, including isomeric tetrachlorophenols, tetrachlorocatechol and tetrachlororesorcinol. Trace amounts of benzoquinones were also noted. Metabolites in female rats were tetrachloromonophenols, diphenols, and hydroquinones. Mode of Action. PCP acts as a biocide through its ability to uncouple mitochondrial oxidative phosphorylation. Toxicology. The toxicology has been addressed in a recent risk assessment (119). Acutely, pentachlorophenol was reported to have LD50 values in the rat of 12 mg/kg

O (94)

Cl

OH (93)

Cl (92)

(inhalation) and 146 mg/kg (M)–175 mg/kg (F) by oral gavage. More detailed studies of the toxicology of pentachlorophenol have been compromised by the toxicity of impurities present in most of the earlier samples used in the evaluation process. These include dioxins, such as TCDD, dibenzofurans, and hexachlorobenzene, each causing a range of toxicological effects, some of which may have overlapped those caused by PCP itself. In addition, the principal rodent metabolite of PCP is genotoxic, tetrachloro1,4-hydroquinone (TCHQ) (89). Although it is apparently not formed in humans in vivo, its formation has nevertheless been demonstrated in vitro using human microsomal mixed function oxygenases. Although a number of toxicity studies have been conducted with both known impurities and TCHQ, it is often difficult to know whether animal experiments are valid for human health risk assessment. Nevertheless, it appears that the main target organ of purified TCP in animals is the liver. This toxicity was manifested as liver inflammation, increased relative weight, and increased serum alkaline phosphatase. The estimated chronic NOEL in the dog for these effects was 0.15 mg/kg/day, from a 1-year study, based on a LOEL


of 1.5 mg/kg/day. In the rat, a significantly increased incidence of mesotheliomas (p1 cm) compared to homogenized soil. Experiments and model simulations of biodegradation in artificial aggregates showed that biodegradation was slower in soil with larger aggregates, and that the effects of diffusion and sorption were required to model this process (11,16). Diffusion of Gases Through Soil Unlike the positive relationship between soil moisture content and the diffusion of solutes through soil solution discussed in the preceding section, soil moisture content strongly limits the rate of gas diffusion in soil because gas diffuses much more slowly through water-filled pore spaces than through air-filled pore spaces (17). This fact is illustrated by the one-dimensional equation for diffusion of gas through soil derived from Fick’s law: δC (5) F = τ θA D δz where F is the flux rate (mol m−2 s−1 ), τ is the tortuosity of the air-filled pore space (unitless), θA is the fraction of pore space filled with air (unitless), D is the diffusion constant for the gas in free air (m2 s−1 ), and δC/δz is the concentration gradient (mol m−4 ). This tortuosity factor (τ ) (not to be confused with f mentioned earlier in terms of water films) has been modeled (18) as: (1/3)

τ = θA

(6)

τ = 1 in free air. Both, the tortuosity and the pore space decrease with increasing water content, in turn slowing the diffusion of the gas through soil.

982

KINETICS OF MICROBIAL PROCESSES AND POPULATION GROWTH

The availability of oxygen (O2 ) can be the limiting factor for aerobic microbial activity, and can determine the extent of anaerobic activities such as denitrification or methane production. Therefore, CH4 and N2 O production are frequently related to soil water content (19–23). However, Sierra and Renault (23) found that immediately after heavy rainfall events, O2 can be trapped in soil pore water and that an equilibrium between soil water and atmosphere is not reached for 12 to 24 hours in these cases. Sorption The kinetics of microbial processes in soil is greatly complicated by the mineral and organic matter fractions of soil. These solid phases bind substrate both reversibly and irreversibly. Reversible sorption leads to slowed rates of movement and lower substrate concentrations in the soil solution, whereas irreversible sorption leads to overall decreases in the quantity of substrate available to microbes. Soils with high levels of organic matter or high clay contents generally retain compounds more than sandy soils (4,24). The types of clay and aluminum and iron hydroxides in soil can influence the ability of soil to adsorb molecules (25). Because many phenolic acids have a relatively low pKa ( Ks

S0 Ks

(7)

Nongrowth (S0 X0 )

Logistic

First order

−dS/dt = dS(S0 + X0 − S)

dS/dt = µmax (X0 /Ks )S

Monod

Michaelis-Menten

−dS/dt = µmax S(S0 + X0 − S)/(Ks + S)

−dS/dt = µmax X0 S/(Ks + S)

Logarithmic

Zero order

−dS/dt = µmax (S0 + X0 − S)

−dS/dt = µmax X0

984


curves (62,63). Nutrient limitation can also enhance degradation; some nitrogen- or phosphorous-containing organic compounds will only be utilized if the microbial community is nutrient limited (64). Another important characteristic of a given functional group is the range of substrates it can utilize. In one study (65), workers induced growth kinetics in the degradation of low levels of para-nitrophenol by adding larger levels of phenol to the soil. Thus, the rate of transformation of a compound in soil is affected by the presence of similar compounds that the microbial population can utilize.

RESPONSES OF MICROBIAL ACTIVITY TO ENVIRONMENTAL FACTORS From what has been stated in the preceding text, it is clear that the rate of a microbially mediated process in soil at any given moment is affected by a large number of variables. The picture is further complicated by the everchanging soil environment. Microbial processes are subject to nonlinear effects from the dynamics of temperature, soil moisture, and predation. These are discussed in the following section. Temperature

Effects of Microbial Community Composition Soil microbial communities are complex, containing thousands of bacterial types (66). A study of the kinetics of DNA reassociation showed soil microbial communities to be orders of magnitude more complex than aquatic microbial communities (67). The composition of the microbial community can cause deviations from standard growth models and has a profound influence on kinetics in soil. Filamentous microbes, such as fungi and actinomycetes, grow linearly over time on agar, and in liquid culture the cube root of the mass increases linearly over time (68). The predominance of filamentous microbes in a community could lead to nonexponential growth rates in soil. There is evidence that first-order kinetics does not always apply to fungal cultures in soil under conditions in which first-order kinetics would be expected with bacterial populations (63). The fungal-to-bacterial ratio in soil can also affect the apparent growth yield of functional groups in soil (69). Changes in growth yield by microbial biomass have been observed in response to fertilization (70,71). In both studies, microbial biomass was not affected by inputs of mineral fertilizer, but rates of respiration were affected. Long-term inputs of nitrogen can also cause shifts in the relative sizes of functional groups. Nitrogen limitation during decomposition of wheat straw caused a reduction in fungi but bacteria were unaffected (72). Nitrogen-fertilized alpine soils showed significantly reduced phenol degradation but degradation of glutamate and glucose were relatively unchanged (Schmidt and Lipson, unpublished data). Community shifts can also occur in response to seasonal changes, causing shifts in substrate utilization (73), and temperature response (74). Competition experiments using bacteria with different temperature optima and substrate affinities show that different organisms would dominate in different seasons (75), and so kinetic properties of the community could change as well. Microbial community composition can also affect the kinetics of microbial processes in cases in which multiple populations are utilizing the same substrate. For example, it has been observed (9,76) that the kinetics of 2,4-dinitrophenol mineralization in soil indicated that two physiologically distinct populations were responsible for the process. One population functioned at high dinitrophenol concentrations (high Ks population) whereas the other was dominantly active at much lower dinitrophenol concentrations (low Ks population). Such ‘‘multiphasic’’ kinetics has been observed in several other aquatic and soil studies (77,78).

The rate of most chemical reactions increase exponentially with higher temperature according to the Arrhenius equation: −Ea (8) k = A exp RT where k is the first-order rate constant (s−1 ), A is a constant called the frequency factor (s−1 ), Ea is the activation energy (J mol−1 ), R is the ideal gas constant (8.3145 J mol−1 K−1 ), and T is the absolute temperature (K). However, the effect of temperature on biological processes is harder to predict. Biologically mediated reactions are usually the result of several processes working in concert, each with its own response to temperature. Furthermore, enzymes and membranes can only function within a certain temperature range and so biological processes peak at some optimum temperature close to normal environmental conditions. Near the freezing point of water, physiological and diffusional effects further complicate rates of biological processes. Despite these factors, a narrow range usually exists where process rates obey the expected exponential relationship with temperature. This is especially true for enzymatic activity measured in isolation from other biological activities. One study (79) found that several soil exoenzymes obeyed Arrhenius kinetics within the range of 2 to 30 ◦ C. They reported Q10 s (defined as the proportional increase in rate with a 10 ◦ C increase in temperature) ranging from 1.3 to 4.1, with most falling close to 2.0. Workers (80) reported Q10 s for proteolysis and microbial uptake of amino acids as 1.98 and 2.57, respectively. As an example of how temperature responses of complex biological reactions can lead to subtle results, it was noted in the previous study that the higher temperature sensitivity of amino acid uptake compared to production could lead to higher availability of amino acids at lower temperatures. In an experiment with laboratoryincubated forest soils, researchers (81) reported that net nitrogen mineralization increased with temperature over the range of 5 to 25 ◦ C, but that respiration showed a relatively flat response to temperature and peaked at 10 ◦ C. The authors reasoned that microbial respiration became substrate limited at higher temperatures. In one study (82) it was observed that more labeled carbon and nitrogen from added substrate was retained in microbial biomass at lower temperatures, indicating that microbial turnover increased with temperature. This illustrates that mineralization is a balance between uptake and microbial turnover and that these processes have different temperature responses. A wide variety of positive, neutral,


or negative relationships between microbial biomass and temperature have been reported (83). Temperature affects not only the growth rate of microorganisms, but their growth yield and substrate affinity, as well. Lower growth yields are associated with higher growth rates (84), and hence, with higher temperatures. On the other hand, very slow growth rates can lead to low growth yields as well, as the energy required for maintenance of cells becomes significant relative to the carbon being assimilated into biomass (60,85). Substrate affinity sometimes decreases near 0 ◦ C (75), although it has also been reported to increase with decreasing temperature (86). Microbial and enzymatic activity can occur below the freezing point of water, and biological activities in soils at subzero temperatures have been reported (51,87–89). Significant mass loss of litter during winter has been observed in several ecosystems, although physical effects could also be involved (90,91). The limit to biological activity in cold soils is not temperature, itself, but the availability of liquid water. There is always some liquid water present in frozen soils, but the width of the liquid film decreases, more or less sharply depending on mineral type, as temperature drops from 0 to −5 ◦ C; ˚ or wider exist at 0 ◦ C, and of about liquid films of 50 A ◦ ˚ at −5 C or below (92). This creates a tortuous and 6A discontinuous distribution of water in frozen soils, just as in very dry soils, that limits diffusion of molecules to microorganisms. Thus, diffusion coefficients of ions decrease with temperature in the same way the liquid water film width does (93). Another important effect of freezing on the kinetics of soil processes is the disruptive effect of freeze-thaw events on soil aggregates and microbial cells. Freezethaw events tend to stimulate microbial activity in the short term (94–96) by releasing nutrients occluded in soil aggregates (97) or from lysis of cells (98,99). Researchers (95) have argued that repeated freeze-thaw cycles can have a long-term inhibitory effect on microbial activity by reducing the microbial population size. The rate of freeze determines the severity of effect on the soil microbial biomass. In an alpine soil, microbial biomass was not affected by freeze-thaw events designed to simulate spring and fall conditions (74,100). Soil Water Content In addition to the direct effects of soil water content on diffusion mentioned above, kinetics in soils are also affected indirectly by physical effects of fluctuating water content and by biological effects of osmotic stress on microbes. In dry soils, microbial activity can be limited both by osmotic stress and by diffusional limitation (101). Dry-rewet cycles can physically disrupt soil aggregates and microbial biomass much like freeze-thaw cycles do, as mentioned in the preceding text. This effect can enhance microbial activity by freeing substrate occluded in aggregates and by releasing nutrients from microbial biomass (102,103), but can reduce activity by lowering population sizes (104). As discussed for temperature in the preceding section, the effect of soil water content on a given process depends

985

on a complex set of factors. For most processes there is an optimum moisture content that balances both O2 and water availability. In a field study of several alpine communities, CH4 oxidation rates were stimulated by rainfall in drier soils, whereas the oxidation rates were negatively correlated with soil moisture in more moist soils (105). The stimulation of CH4 oxidation by rainfall in dry soil may not have been purely because of release from water limitation of the methanotroph population, but may have been caused indirectly by creating anaerobic microsites in which methanogenic activity provided substrate for the growth of the methanotroph population (22,106). This again illustrates the complex interdependency among processes and environmental factors in soil. Soil Texture and Carrying Capacity of Soil Microbial growth in soil frequently follows a logistic pattern in which growth approaches a finite limit because of some environmental factors, such as substrate limitation (63). The carrying capacity can be also determined by some extrinsic factor such as the number of ideal sites for an organism. The abundance of colonizable surfaces and pores depends on soil texture; finer textured soils have smaller pores and more surface area. Bacteria living in small pores would be protected from predation by protozoa, whereas those in larger pores could be readily eaten. Various modeling studies (e.g., 107,108) predicted the turnover of microbial biomass according to the clay and silt content of soil. MODELS OF MICROBIAL GROWTH AND SUBSTRATE USE Many approaches have been used to model kinetics in soils. These include empirical models and models based on biological and/or physical theory. The shape of substrate disappearance curves depends on all the factors mentioned herein: the soil physical factors that affect substrate movement and availability, the initial substrate and biomass levels, the kinetic properties of the microbial community, and the many potential environmental factors, such as temperature and the availability of nutrients and water. In the following section we briefly review models of microbial activity, and discuss the practical matter of how to analyze respiration data from soils. For thorough reviews of mathematical models of microbial growth and biodegradation, see references (63,109). The Use of Respiration Data in Studies of Kinetics in Soils Carbon dioxide evolution from soil is commonly used to monitor degradation of added substrates (e.g., 48,49,110). Special caution should be exercised when analyzing carbon dioxide evolution data from soil incubations. Several research groups (e.g., 49,110,111) have stressed that analyzing accumulated CO2 data leads to a number of statistical problems including nonrandom residuals, autocorrelated parameters and residuals, and underestimation of experimental error (Fig. 2). It is therefore preferable to use nonaccumulated rate data and

986


inhibitory substrates using the variations of the Haldane equation: S (9) µ = µmax S (Ks + S) 1 + Ki

60

40

0.4

Residual

% DNP mineralized

100 µg DNP g−1 soil

20

0.0

−0.4 0

dS/dt (ng g−1 h−1)

Residual

0

200

100 Hours

200

40 0

−40 0

400

100 Hours

where Ki is the inhibition constant, defined as the highest concentration of S at which µ = µmax /2. The Monod equation has also been modified to include the effect of metabolic maintenance requirements when microbial populations are subsisting on very low concentrations of substrate. The modified equation takes the form: S (10) µ = µmax (Ks + S) − a where a is the specific maintenance rate associated with nongrowth metabolic processes. This modification improved the modeling of degradation of low levels of pentachlorophenol and dinitrophenol in soils (85).

200 4

0 0

50

100 Hours

150

Accumulated carbon dioxide

100 µg DNP g−1 soil 200

Figure 2. Respiration of 14 C-labeled dinitrophenol in soil graphed as cumulative CO2 produced vs. time (A) and as CO2 evolution rate vs. time (B). The residuals are shown in the inset of each panel, and show that fitting of accumulated data leads to nonrandom residuals. (Modified from Hess and Schmidt (110)).

Non-growth kinetics (michaelis-menten)

3

Growth kinetics (logistic)

2

1

the differential form of kinetic models to analyze soil respiration data. Examples of this approach to analyzing soil respiration data can be found in the literature (49,78). Most commercially available statistics packages contain algorithms for performing nonlinear regression and several reviews of this approach are available (109,112,113).

0 0

10

20

30 Time

40

50

60

0.14 0.12

Variations on the Monod Equation

Growth kinetics (logistic)

0.1

Rate

In liquid culture growth of bacteria occurs when initial substrate concentration (S0 ) is high compared to initial biomass (X0 ). Exponential growth occurs if S0 is much higher than the half-saturation constant for the microbial population (S0 Ks ), Monod kinetics occurs when S0 is slightly higher than Ks (S0 > Ks ) and logistic growth occurs when S0 Ks . If S0 is low relative to X0 , nongrowth kinetics occur, and conditions when S0 Ks , S0 > Ks , and S0 Ks , result in zero-order, Michaelis-Menten, and first-order kinetics, respectively. The forms of these equations are all derived from the Monod equation, and are shown in Table 1. Examples of some of these types of curves are shown in Figure 3. The Monod equation has been modified to include various biological effects (109). For example, several studies (e.g., 114) have modeled the biodegradation of

0.08 0.06 0.04 Non-growth kinetics (michaelis-menten)

0.02 0 0

10

20

30 Time

40

50

60

Figure 3. Shapes of typical growth (logistic) and nongrowth (Michaelis-Menten) kinetic curves: (A) graphed as accumulated product vs. time, and (B) as rate of product formation or substrate disappearance vs. time.


The maintenance term, and other zero-order terms added to other models discussed in the following text, seem to be most important when very slow growth is occurring. This is when theory predicts growth yields would be low and maintenance requirements would be high (60).

987

effects of the abiotic soil fractions present. In short, when observing microbial processes in soil, anything can happen. Fortunately, a variety of models have been developed, and existing models can be modified for most any given system. CONCLUSION

Models of Microbial Activity in Soil In soil, the effects of diffusion and sorption cause further deviations from the Monod model. Theoretically, observed kinetics in soils are best dealt with using two-compartment models that take into account the slow and fast release of substrate from soil aggregates and surfaces (e.g., 13–16). These models can be quite complex but represent a fairly realistic view of kinetics in soil. Data can also be modeled effectively with an empirical approach, most commonly using 3/2 power kinetics (e.g., 29,56,62). The linear and exponential growth versions of the 3/2-order model are represented, respectively, as: (k2 t2 ) + k0 t (11) P = S0 1 − exp −k1 t − 2 E0 P = S0 1 − exp −k1 t − (exp(µt) − 1) + k0 t µ

(12) where P is product concentration, S0 is the initial substrate concentration, and the kn are rate constants. In Equation (11), k2 is the linear growth rate, and in Equation (12), µ is the exponential growth rate. E0 is related to the initial biomass by the relation, initial biomass = E0 /a, in which the constant, a, describes the dependence of the first-order substrate disappearance rate on biomass. It was found (62) that the linear model generally fit the data better except when microbial populations were first greatly reduced by gamma irradiation. The authors attributed this effect to the slow diffusion of substrates through thick layers of cells coating soil surfaces. The 3/2-order model is generally most effective under conditions in which little or no growth occurs, and has the advantage of fitting an initial acclimation phase and a late slow mineralization phase (62,63,115). Empirical models have the disadvantage of providing little heuristic information about the mechanisms affecting the observed kinetics. The parameters in the above 3/2 models are hard to interpret in a biologically or physically meaningful way. Some workers have interpreted the zero-order rate constant, k0 , to represent the ‘‘indigenous’’ slow, steady turnover of carbon in soil (62), others as the slow release of substrate from the soil matrix (29), and yet others as the metabolic energy expended by the microbes to maintain their biomass during long periods of slow growth (85). Nutrient limitation or microbial community effects can cause growth to be linear or biphasic under conditions where exponential growth would otherwise be expected. The presence of stable soil colloid-extracellular enzyme complexes can further decouple kinetics from existing microbial populations. One study (29) found no correlation between the mineralization of contaminants and total microbial biomass or activity, but rather found major

Soil is a labyrinth of mineral and organic fractions with ever-changing microclimatic conditions and a diverse community of poorly understood microorganisms. To realistically include all potentially important factors in predicting a process rate would result in an excessively complicated and unworkable model. But despite the seemingly intractable complexity of the soil ecosystem, researchers have made great progress in understanding and predicting the kinetics of microbially mediated processes in soils. If one is flexible in one’s model selection and allows for the myriad of possibilities that could arise, the situation is not hopeless. However, gaps in our knowledge still exist. More research is particularly needed to understand microbial community-level effects on processes in soil. Although we know almost nothing about the characteristics of most of the thousands of microbial species in soils, how they interact with each other, or their spatial and temporal distributions in soil microenvironments, this type of information is becoming increasingly available by way of powerful molecular techniques (116). It would be fruitful to integrate the current rapid advances in microbial ecology with the existing knowledge of microbial kinetics in soils. BIBLIOGRAPHY 1. S. Simkins and M. Alexander, Appl. Environ. Microbiol. 47: 1299–1306 (1984). 2. S. K. Schmidt, S. Simkins, and M. Alexander, Appl. Environ. Microbiol. 50: 323–331 (1985). 3. M. Alexander, Biodegradation and Bioremediation, 2nd ed., Academic Press, San Diego, CA, 1999. 4. B. R. Dalton, U. Blum, and S. B. Weed, Soil Sci. Soc. Am. J. 53: 757–762 (1989). 5. B. DeScisciolo, D. J. Leopold, and D. C. Walton, J. Chem. Ecol. 16: 1111–1130 (1990). 6. P. B. Barraclough and P. B. Tinker, J. Soil Sci. 32: 225–236 (1981). 7. D. D. Warncke and S. A. Barber, Soil Sci. Soc. Am. Proc. 36: 42–46 (1972). 8. D. D. Focht and D. Shelton, Appl. Environ. Microbiol. 53: 1846–1849 (1987). 9. S. K. Schmidt and M. J. Gier, Appl. Environ. Microbiol. 56: 2692–2697 (1990). 10. G. Sposito, The Chemistry of Soils, Oxford University Press, New York, 1989. 11. K. M. Scow and M. Alexander, Soil Sci. Soc. Am. J. 56: 128–134 (1992). 12. P. R. Darrah, Plant Soil 138: 147–158 (1991). 13. D. R. Shelton and M. A. Doherty, Soil Sci. Soc. Am. J. 61: 1078–1084 (1997).

988


14. W. Schlebaum, G. Schraa, and W. H. Van Riemsdijk, Environ. Sci. Technol. 33: 1413–1417 (1999).

43. K. Tatsumi, A. Freyer, R. D. Minard, and J.-M. Bollag, Environ. Sci. Technol. 28: 210–215 (1994).

15. S. Dhawan, L. E. Erickson, and L. T. Fan, Ground Water 31: 271–284 (1993).

44. M. Rahouti, F. Seiglemurandi, R. Steiman, and K. E. Eriksson, Appl. Environ. Microbiol. 55: 2391–2398 (1989).

16. K. M. Scow and J. Hutson, Soil Sci. Soc. Am. J. 56: 119–127 (1992).

45. N. Lahav, D. White, and S. Cheng, Science 201: 67–69 (1978).

17. R. G. Striegl, Chemosphere 26: 715–720 (1993).

46. M. C. Wang and C. H. Lin, Soil Sci. Soc. Am. J. 57: 88–93 (1993).

18. R. W. Healey et al., Soil Sci. Am. J. 60: 740–749 (1996). 19. M. K. Firestone and E. A. Davidson, Microbiological Basis of NO and N2 O Production and Consumption in Soil, in M. O. Andrae and D. S. Schimel, eds., Exchange of Trace Gases between Terrestrial Ecosystems and the Atmosphere, John Wiley and Sons, Chichester, 1989, pp. 7–21.

47. M. J. Morra and L. L. Freeborn, Soil Biol. Biochem. 21: 645–650 (1989). 48. J. P. E. Anderson and K. H. Domsch, Soil Biol. Biochem. 10: 215–221 (1978).

20. E. A. Davidson et al., Ecology 74: 130–139 (1993).

49. G. M. Colores, S. K. Schmidt, and M. C. Fisk, Soil Biol. Biochem. 28: 1569–1577 (1996).

21. J. C. Neff et al., Biogeochemistry 27: 23–33 (1994).

50. S. K. Schmidt, FEMS Microbiol. Ecol. 101: 197–206 (1992).

22. A. E. West and S. K. Schmidt, FEMSMicrobiol. Ecol. 25: 349–353 (1998).

51. D. A. Lipson, S. K. Schmidt, and R. K. Monson, Ecology 80: 1623–1631 (1999).

23. J. Sierra and P. Renault, Soil Sci. Soc. Am. J. 62: 1398–1405 (1998). 24. P. M. Huang et al., Soil Sci. 123: 213–219 (1977). 25. T. S. C. Wang, S. W. Li, and Y. L. Ferng, Soil Sci. 126: 15–21 (1978). 26. T. H. Dao, Sorption and Mineralization of Plant Phenolic Acids in Soil, in G. R. Waller, ed., Allelochemicals: Role in Agriculture and Forestry, American Chemical Society Symposium Series 330, American Chemical Society, Washington, D.C., 1987, pp. 358–370. 27. H. Nommik, Plant Soil 33: 581–595 (1970). 28. M. Schnitzer and H. Kodama, Soil Sci. Soc. Am. J. 56: 1099–1105 (1992). 29. D. B. Knaebel, T. W. Federle, D. C. McAvoy, and J. R. Vestal, Appl. Environ. Microbiol. 60: 4500–4508 (1994). 30. N. A. Marshman and K. C. Marshall, Soil Biol. Biochem. 13: 127–134 (1981). 31. F. Asmar, F. Eiland, and N. E. Nielsen, Biol. Fertil. Soils 14: 288–292 (1992). 32. P. Nannipieri et al., Soil Biol. Biochem. 28: 107–112 (1996). 33. S. Klose and M. A. Tabatabai, Soil Biol. Biochem. 31: 205–211 (1999).

52. K. Alef and D. Kleiner, Soil Biol. Biochem. 18: 233–235 (1986). 53. D. A. Lipson, T. K. Raab, S. K. Schmidt, and R. K. Monson, Biol. Fertil. Soils 29: 257–261 (1999). 54. G. M. Colores, P. M. Radehaus, and S. K. Schmidt, Appl. Biochem. Biotechnol. 54: 271–275 (1995). 55. I. S. Fomsgaard, Ecol. Modell. 102: 175–208 (1997). 56. T. K. Reffstrup, H. Sorensen, and A. Helweg, Pesticide Sci. 52: 126–132 (1998). 57. B. Hoyle, K. M. Scow, G. Fogg, and J. Darby, Biodegradation 6: 283–293 (1995). 58. R. M. Gersberg et al., World J. Microbiol. Biotechnol. 11: 549–558 (1995). 59. E. M. D’Angelo and K. R. Reddy, Soil Biol. Biochem. 31: 815–830 (1999). 60. D. W. Tempest and O. M. Neijssel, Adv. Microb. Ecol. 2: 105–153 (1978). 61. D. K. Button, Antonie van Leeuwenhoek 63: 225–235 (1993). 62. W. Brunner and D. D. Focht, Appl. Environ. Microbiol. 47: 167–172 (1984).

35. O. Dilly and P. Nannipieri, Z. Pflanzenernaehr. Bodenkunde 161: 243–248 (1998).

63. M. Alexander and K. M. Scow, Kinetics of Biodegradation in Soil, in B. L. Sawney and K. Brown, eds., Reactions and Movement of Organic Chemicals in Soils, SSSA Special Publication no. 22, ASA and SSSA, Madison, Wis, 1989, pp. 243–269.

36. C. Marzadori, S. Miletti, C. Gessa, and S. Ciurli, Soil Biol. Biochem. 30: 1485–1490 (1998).

64. G. K. Sims, and A. M. Cupples, Pesticide Sci. 55: 598–601 (1999).

37. F. Leprince and H. Quiquampoix, Eur. J. Soil Sci. 47: 511–522 (1996).

65. K. M. Scow, S. K. Schmidt, and M. Alexander, Soil Biol. Biochem. 21: 703–708 (1989).

¨ ¨ 38. P. Lahdesm aki and R. Piispanen, Soil Biol. Biochem. 24: 1173–1177 (1992).

66. A. Chatzinotas et al., Syst. Appl. Microbiol. 21: 579–587 (1998).

39. M. S. Carrasco, J. C. Rad, and C. S. Gonzalez, Bioresour. Technol. 51: 175–181 (1995).

67. K. Ritz, B. S. Griffiths, V. L. Torsvik, and N. B. Hendriksen, FEMS Microbiol. Lett. 149: 151–156 (1997).

40. M. A. Rao, L. Gianfreda, F. Palmiero, and A. Violante, Soil Sci. 161: 751–760 (1996).

68. K. C. Marshall and M. Alexander, Agronomy paper no. 473, Department of Agronomy, New York State College of Agriculture, Cornell University, Ithaca, N.Y., 1960.

34. M. J. Rowell, J. N. Ladd, and E. A. Paul, Soil Biol. Biochem. 5: 699–703 (1973).

41. H. Shindo et al., Soil Sci. Plant Nutr. 42: 141–146 (1996). 42. K. Haider and J. P. Martin, Soil Sci. Soc. Am. J. 39: 657–662 (1975).

69. K. Sakamoto and Y. Oba, Biol. Fertil. Soils 17: 39–44 (1994). 70. S. Jonasson et al., Oecologia 106: 507–515 (1996).

KOW (OCTANOL WATER PARTITION COEFFICIENT)

989

71. M. C. Fisk, PhD Dissertation, University of Colorado, Boulder, CO, 1995.

98. C. R. Morley, J. A. Trofymow, D. C. Coleman, and C. Cambardella, Microb. Ecol. 9: 329–340 (1983).

72. T. M. Henriksen and T. A. Breland, Soil Biol. Biochem. 31: 1121–1134 (1999).

99. D. A. Soulides and F. E. Allison, Soil Sci. 91: 291–298 (1961).

73. G. P. Zogg et al., Soil Sci. Soc. Am. J. 61: 475–481 (1997). 74. D. A. Lipson, S. K. Schmidt, and R. K. Monson, Soil Biol. Biochem. 32: 441–448 (2000). 75. D. B. Nedwell and M. Rutter, Appl. Environ. Microbiol. 60: 1984–1992 (1994). 76. S. K. Schmidt and M. J. Gier, Microb. Ecol. 18: 285–296 (1989). 77. D. L. Lewis, R. E. Hodson, and L. F. Freeman, Appl. Environ. Microbiol. 50: 553–557 (1985). 78. G. Soulas, Soil Biol. Biochem. 25: 443–449 (1993). 79. C. A. McClaugherty and A. E. Linkins, Soil Biol. Biochem. 22: 29–33 (1990). 80. D. A. Lipson, T. K. Raab, S. K. Schmidt, and R. K. Monson, Soil Biol. Biochem. 33: 189–198 (2001). 81. D. R. Zak, W. E. Holmes, N. W. MacDonald, and K. S. Pregitzer, Soil Sci. Soc. Am. J. 63: 575–584 (1999). 82. B. Nicolardot, G. Fauvet, and D. Cheneby, Soil Biol. Biochem. 26: 253–261 (1994). 83. D. A. Wardle, Biol. Rev. 67: 321–358 (1992). 84. G. Kuenen, Arch. Microbiol. 122: 183–188 (1979). 85. T. F. Hess, S. K. Schmidt, and G. C. Colores, Soil Biol. Biochem. 28: 907–915 (1996). 86. R. B. Mullen, PhD Dissertation, University of Colorado, Boulder, CO, 1995. 87. P. W. Flanagan and F. L. Bunnell, Microflora Activities and Decomposition, in J. Brown, P. C. Miller, L. L. Tieszen, and F. L. Bunnell, eds., An Arctic Ecosystem. The Coastal Tundra at Barrow, Alaska, Dowden, Hutchinson and Ross, Stroudsburg, 1980, pp. 291–334. 88. J. S. Clein and J. P. Schimel, Soil Biol. Biochem. 27: 1231–1234 (1995). 89. P. D. Brooks, M. W. Williams, S. K. Schmidt, Biogeochemistry 32: 93–113 (1996).

100. D. A. Lipson and R. K. Monson, Oecologia 113: 406–414 (1998). 101. J. M. Stark and M. K. Firestone, Appl. Environ. Microbiol. 61: 218–221 (1995). 102. L. T. Kieft, E. Soroker, and M. K. Firestone, Soil Biol. Biochem. 19: 119–126 (1987). 103. M. Van Gestel, R. Merckx, and K. Vlassak, Geoderma 56: 617–626 (1993). 104. J. S. Clein and J. P. Schimel, Soil Biol. Biochem. 26: 403–406 (1994). 105. A. E. West et al., Biogeochemistry 45: 243–264 (1999). 106. A. E. West and S. K. Schmidt, Soil Biol. Biochem. 31: 1649–1655 (1999). 107. J. A. Van Veen, J. H. Ladd, and M. J. Frissel, Plant Soil 76: 257–274 (1984). 108. W. J. Parton, D. S. Schimel, C. V. Cole, and D. S. Ojima, Soil Sci. Soc. Am. J. 51: 1173–1179 (1987). 109. S. K. Schmidt, Models for studying the population ecology of microorganisms in natural systems, in C. J. Hurst, ed., Modeling the Metabolic and Physiologic Activities of Microorganisms, John Wiley & Sons, New York, 1992, pp. 31–59. 110. T. F. Hess and S. K. Schmidt, Soil Biol. Biochem. 27: 1–7 (1995). 111. B. R. Taylor and D. Parkinson, 1948–1959 (1988).

Can.

J.

Botany

66:

112. J. A. Robinson, Adv. Microb. Ecol. 8: 61–114 (1985). 113. D. M. Bates and D. G. Watts, Nonlinear Regression Analysis and its Applications, John Wiley & Sons, New York, 1988. 114. V. H. Edwards, Biotechnol. Bioeng. 12: 679–712 (1970). 115. M. P. Lindequist et al., Environ. Sci. Toxicol. 33: 2601– 2606 (1999). 116. R. I. Amann, W. Ludwig, and K.-L. Schleifer, Microbiol. Rev. 59: 343–369 (1995).

90. J. F. McBrayer and K. Cromack Jr., Pedobiologia 20: 47–54 (1980). 91. S. E. Hobbie and F. S. Chapin III, Biogeochemistry 35: 327–338 (1996).

KOC (SOIL ORGANIC PARTITION COEFFICIENT). See SOIL ORGANIC PARTITION

92. D. M. Anderson, Cold Regions Research and Engineering Laboratory Report 274, U.S. Army Corps of Engineers, 1970.

COEFFICIENT (KOC)

93. R. P. Murrman and P. Hoekstra, Research Report no. 284, U.S. Army Cold Regions Research and Engineering Laboratory, Hanover, NH, 1970. 94. T. H. DeLuca, D. R. Keeney, and G. W. McCarty, Biol. Fertil. Soils 14: 116–120 (1992). 95. J. P. Schimel and J. S. Clein, Soil Biol. Biochem. 28: 1061–1066 (1996). 96. T. Skogland, S. Lomeland, and J. Goksoyr, Soil Biol. Biochem. 20: 851–856 (1988). 97. A. C. Edwards, M. S. Cresser, Freezing and its effect on chemical and biological properties of soil, in B. A. Stewart, ed., Advances in Soil Science, vol. 18, Springer, New York, 1992, pp. 59–79.

KOW (OCTANOL WATER PARTITION COEFFICIENT) Experimental ratio of a pesticide’s concentration in the octanol phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The Kow is a partition coefficient reflecting the relative hydrophobicity of a pesticide and its potential for bioconcentration. For convenience the term is often expressed in logarithmic form (log Pow ). A high value may be regarded as an indicator that the substance may bioaccumulate (IUPAC).

L LABEL

Current methods typically use gas chromatography (GC) with confirmation by GC-mass spectrometry (MS) or with high performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection or fluorescence detection to determine thermally labile compounds. Confirmatory analyses are very important for HPLC methods because these methods, especially UV detection, are often less specific than are GC methods. For example, confirmation by UV detection is problematic because the UV spectra often are very similar, and many thermally labile compounds lack a strong UV chromophore. On-line Liquid chromatography/Mass Spectrometry (LC/MS) provides the necessary specificity for confirmation, especially when two compounds are co-eluted. Under these conditions, LC-MS allows the identification and quantitation of both compounds, provided that each has different characteristic ions. Coupling HPLC to a mass spectrometer is far more complicated than to a GC system because of the large amount of mobile phase solvent. Typical mobile phase flow rates for HPLC are 0.5–2 mL/min, which translates into gas flow rates of 100–3000 mL/min. During the past 20 years, researchers have used several approaches to remove the solvent during ionization. Among the different interfaces, thermospray has been used because of its compatibility with HPLC system mobile phases. Thermospray, however, has its limitations, such as imprecision, compound dependent responses, and limited ruggedness (1). The particle-beam interface (2,3) has been commonly used to generate electron impact (EI) spectra in LC/MS. However, the importance of particle beam is declining, mainly because of insufficient sensitivity. Table 1 shows how LC/MS technology has evolved in the past 25 years. This chapter will focus on some applications, along with helpful references in getting started with LC/MS. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the two popular atmospheric pressure ionization (API) techniques that will be discussed in this chapter. The practical applications to the analysis of pesticides that will be discussed are sulfonylurea herbicides, imidazolinone herbicides, phenoxyacid herbicides, and N-methyl carbamate insecticides. Matrix effects with respect to quantitation also will be discussed. For the discussions below about the ESI and APCI techniques, the following references were used (4–13). These references are highly recommended, particularly

In the USA, all agricultural chemicals must be approved by the United States Environmental Protection Agency and carry a label as an indication that they have been approved by the appropriate regulatory authority (the USEPA) for the purpose for which they were intended. The label describes the ingredients of the material and gives recommendations for the control of a pest and indicates the crops for which its use is approved. It prescribes the manner in which it must be used, providing application rates and safety precautions. The use of any pesticide in a manner inconsistent with the instructions provided on the label is prohibited and the label may be regarded as a legal document.

LABORATORY SAMPLE Sample or subsample received by a laboratory (IUPAC).

LC Liquid chromatography

LC/MS Liquid chromatography/mass spectrometry

LC/MS, PESTICIDE RESIDUE ANALYSIS ALEXANDER J. KRYNITSKY U. S. Environmental Protection Agency Fort Meade, Maryland

DAYAN B. GOODENOWE Yol Bolsum Inc. Rycroft, Alberta, Canada

JAMES STRY Dupont Crop Protection Newark, Delaware

STEVEN J. STOUT Table 1. LC/MS Evolution

BASF Corporation Princeton, New Jersey

The very low concentration limits prescribed by regulatory agencies such as the U.S. Environmental Protection Agency (EPA) and the U.S. Food and Drug Administration (FDA) for pesticides in food, water, and soils have prompted analysts to develop analytical techniques that can determine these compounds at trace levels. 990

Year

Interface

1974 1978 1980 1984 1985 1985

Moving Wire/Belt Direct Liquid Introduction Thermospray Particle Beam Continuous FAB Electrospray/APCI

LC/MS, PESTICIDE RESIDUE ANALYSIS

for the individuals just getting started in API mass spectrometry.

solute ions formed are transferred from the atmosphericpressure region into the mass analyzer through a low pressure transport region (∼1 torr). The transport region is equipped with a heated capillary, a skimmer, and lens arrangements. The electrospray interface uses an additional octopole lens for ion focusing and ‘‘in-source’’ collision-induced dissociation experiments (CID) (in the case of a single quadrupole instrument). Figures 1–3 give a complete graphical description of the ESI process. The ability to form analyte ions from the electrospray process depends on pH and mobile-phase composition.

ELECTROSPRAY IONIZATION (ESI) In an electrospray ion source, the mobile phase is forced through a narrow metal capillary needle at a high electrical potential relative to the walls of the atmosphericpressure region. The potential causes the mobile phase to explode into a fine spray of charged droplets; a drying gas helps the solvent evaporate rapidly. The dry

Nebulizer gas (N2)

Electrostatic lenses Capillary

Liquid in Electrospray needle assembly

Quadruple mass spectometer

Skimmer

Electrodes Analyzer chamber

CID region

APCI Heated pneumatic nebulizer LC/MS interface Makeup gas

Discharge needle

Drying gas 1st 2nd 3rd Pumping Pumping Pumping stage stage stage

Nebulizer

−2 Torr

760 Torr

4th Pumping stage

−0.1 Torr −10−4 Torr

−10−6 Torr

Discharge needle

Figure 1. API interface.

5000 V

Atmosphere

N2 nebulization ++

+ ++ +

LC +

++

++

+ ++

++ +

+ −

Charged residues

+

+

++ +

+

+

+ +

+

+ + + ++ + + +

+

+ + + ++ + + +

Ion evaporation

+

++ + + + + +

Drying gas N2 300 °C

Charged residue (noise)

Sampling capillary Vacuum

Mechanisms of ionization • Ion evaporation: Electric field charge liquid → charged droplets → desolvation to reach high field strengths (108 V/cm2) → ionization

991

Figure 2. API-electrospray mechanism.

992

LC/MS, PESTICIDE RESIDUE ANALYSIS Table 2. Solvents and Additives Compatible for API-MS

Ion formation • Pneumatic nebulization forms µm size droplets

Suitable for ESI and APCI

• High voltage electrodes charges liquid • Drying gas desolvates charged droplets • Droplets continue to undergo successive cycles of evaporation and “explosion” • When droplets get to a certain size, the required field for ion desorption (approx. 108 V/cm3) can be achieved without exceeding the Rayleigh stability limit • At this point, solute ions can desorb into the ambient gas and be transported through the capillary • Even solute species that are not ionic can attach solute cations or anions to their polar groups and be desorbed Field necessary for ionization Droplet size/ charge

Jet

Charge

Methanol Ethanol Propanol Isopropanol Butanol Acetonitrile Water DMF (10% or less) DMSO (10% or less) Acetic acid Formic acid Ammonium acetate Ammonium hydroxide Trifluoroacetic acid (TFA) Triethylamine (TEA)

Suitable for only APCI Toluene Benzene Hydrocarbons (e.g., Hexane) Styrene CCl4 CS2

Explosions

10−9 M + + +++ ++ +

Droplet radius

10−6 M

++ ++ + +

++ + + + + + +++

Rayleigh jets

+++

Figure 3. ESI mechanisms.

micro columns, which lower the mobile phase flow rates. Electrospray ionization performs well for the more polar compounds such as imidazolinone herbicides, sulfonylurea herbicides, triazine herbicides, phenoxyacid herbicides, and carbamate pesticides (to name a few). Also, ESI performs well with proteins and peptides. ATMOSPHERIC-PRESSURE CHEMICAL IONIZATION (APCI)

To generate analyte ions from the mobile phase, a volatile organic acid such as formic acid or acetic acid is added to the mobile phase. Adding acids is suitable to generate positive ions, [M + H]+ , but not the first choice for negative ions, [M + H]+ . The electrospray ionization process is ‘‘soft,’’ generating [M + H]+ or [M − H]− ions even for very thermally unstable and nonvolatile molecules. The choice of mobile phase is very important in that it must be volatile and not having strong ion pairing properties. For example, HPLC mobile phases containing acetic acid, formic acid, and ammonium acetate are acceptable. Similarly HPLC mobile phases containing non-volatile buffers such as phosphate buffers are not acceptable, because their strong ion paring properties will favor the formation of neutral products. Stronger acids such as trifluoroacetic acid (TFA), although popular with HPLC, are less desirable because strong acid anions pair with analyte cations, reducing analyte ion abundance. Ion pairing strengths are as followed: sulfates, borates, phosphate > TFA > formate > acetate. Table 2 shows what solvents are acceptable for both ESI and APCI. The applicable HPLC flow rate, in ESI, is lower than that of thermospray or atmospheric pressure chemical ionization, usually below the 0.5-mL/min range. The typical flow rate is 0.10 mL to 0.20 mL/min for ESI. The effluent flow, introduced into the electrospray, is reduced by splitting, when using a conventional HPLC column (4.6 mm ID × 250 mm). It also can be reduced by decreasing the inner diameter of the columns, such as using narrow bore columns (2.1 mm ID) or packed

The atmospheric-pressure chemical ionization interface uses pneumatic nebulization in an atmospheric pressure region. Typically, these systems use a heated nebulizer (300–650 ◦ C) for spray formation at atmospheric pressure, and then chemical ionization is achieved by a corona discharge in the same region. The probe is connected to the mass analyzer through a transport region that is identical to that used in an electrospray system. In fact, modern atmospheric-pressure ionization systems allow a rapid change between the electrospray and atmosphericpressure chemical ionization modes. Figures 1 and 4 describe the mechanism for APCI. With atmospheric-pressure chemical ionization, just as with thermospray techniques, analyte response usually depends on the proton affinities of the analytes. Compounds with high proton affinities usually show high analyte response. Therefore, atmospheric-pressure chemical ionization is often more selective than is electrospray. Atmospheric-pressure chemical ionization is generally good for the less polar compounds such as polynuclear aromatic hydrocarbons, alcohols, aldehydes, ketones, and esters. Because the vaporization temperatures, using APCI, are between 300 and 600 ◦ C, thermally labile compounds will not perform well with APCI. The HPLC flow rates are typically equal to or greater than 1.0 mL/min. Table 2 shows the appropriate solvents and additives that can be used in APCI. There are several advantages and disadvantages in using each API technique. For example, the U.S. Geological Survey has done extensive comparisons between APCI


Makeup gas

+ • Analyte

Nebulizer

LC

Heater

993

+ • Analyte + + + •+ Analyte + + + Charge transfer

Gas molecules Discharge

Mechanisms of ionization • Vaporation → Solvent ionized → charge transfer to analyte N2 + e−

N2+• + 2e−

N2+• + 2N2

N4+• + N2

N4+• + H2O

H2O+• + 2N2

H2O+• + H2O

H3O+ + OH•

H3O+ + M

[M + H]+ + H2O Figure 4. APCI mechanisms.

and ESI, for 30–75 pesticides (14–15). Neutral pesticides in solution often are more sensitive in APCI (especially positive ion), and ionic pesticides in solution are more sensitive in ESI. Also in ESI, the formation of sodium adducts are common, but not in APCI. Matrix effects (signal suppression or enhancement) is common with ESI, but to a less of an extent with APCI. Examples later on in this chapter will demonstrate how APCI is less susceptible to matrix effects than is ESI. Certain compounds in the mid-polarity range, such as carbamate pesticides, perform well with both APCI and ESI. In this situation, ESI will usually give the better sensitivity. OPTIMIZATION OF API-MS RESPONSE If the API signal is not satisfactory, performance can be greatly improved with post-column modification of LC solvent. This is achieved by adding a post-column mixing tee and a pump (capable of delivering 4–400 uL/min of modifier). For example, isopropanol can be added at ∼0.10 mL/min to aid in the desolvation of aqueous solvents and dilute ionic buffers, in order to achieve acceptable API-MS performance. Also, sodium acetate (50 uM) can be added post-column to aid in cationization of samples. This is used for samples that have weak sites for protonation. If TFA must be used in the mobile phase, ESI sensitivity can be improved by adding: 20% propionic acid/80% isopropanol (also known as the ‘‘TFA Fix’’). In this case, the propionic acid displaces the TFA based on volatility. This favors the formation of [M + H]+ . Triethylamine (TEA) is sometimes used as an additive for signal enhancement. However, in the positive ESI mode, TEA readily ionizes to give an intense [M + H]+ ion at m/z 102. This then suppresses the ionization of the less

basic compounds in the positive ESI mode. In the negative mode, TEA can enhance ionization for certain compounds because of its basic properties. Other examples, using post-column modification, will be shown in the phenoxyacid herbicide portion of this chapter. Figure 5 shows a flow chart, which illustrates the recommended steps needed to be optimizing the API signal.

SULFONYLUREA HERBICIDES Several papers discuss the applications of LC/MS (electrospray) to the analysis of trace levels of sulfonylurea herbicides in complex matrices (16–18). Goodenowe et al. (19) have used LC/MS/MS (electrospray) to determine 19 sulfonylurea herbicides in 11 European soils at the 0.05-ppb and 0.5-ppb levels. The extraction procedure, C-18 solid-phase extraction (SPE) trapping of analytes, strong anion exchange (SAX) SPE cleanup, silica gel SPE cleanup, and LC/MS/MS determination of the sulfonylurea herbicides are briefly described below. The soil samples were extracted with 2 × 100-mL 10% acetone in 0.1 M ammonium acetate, using a wrist action shaker (for 15 minutes per each extraction). The soil solution was then centrifuged and filtered through a 0.22-um acetate filter, and the pH adjusted to 3.0–3.5, with 20% phosphoric acid, prior to C-18 solid-phase extraction. It was important to keep all aqueous solutions containing sulfonylurea herbicides on ice during the procedure to minimize degradation. A 2-g C-18 SPE was conditioned with acetone followed by water. The acidified soil extracts were loaded onto the C-18 SPE, and after loading the C-18, SPE is washed with 2 × 5-mL water. The analytes were then eluted with 10-mL 0.1% acetic acid (contained in ethyl acetate).

Tune and calibrate API-MS

Analyze test sample (1–10 ng/mL) Decrease sample pH (1% acetic acid)

Increase sample pH (0.1% NH4 OH or TEA)

ES-positive ion detection

ES-negative ion detection

APCI positive ion detection

APCI negative ion detection Evaluate different probe temperatures

Evaluate data: choose optimal mode of operation

Poor APCI sensitivity

Optimize capillary exit voltage to obtain CID structural information

LC separation developed? Yes

Are conditions compatible with API-MS

Yes

No

No

Post column addition to achieve compatibility

No

Develop API-MS compatible LC separation (use post-column addition if necessary)

Yes Evaluate LC/API-MS method for sensitivity, specificity, accuracy, percision, Linearity

Method meets analysis goals

No

Evaluate all aspects of method to improve results

Yes Analyze sample Figure 5. Optimization scheme for API-MS.

994


The samples were then passed through a 1-g strong anion exchange (SAX) SPE prior to cleanup on silica gel SPE. The purpose of the SAX SPE was to serve as a chemical filter by trapping the nonpolar acids, thus, removing most of the color from the extract. Sample was then taken to dryness under nitrogen at 37 ◦ C and then reconstituted in 80/20 ethyl acetate/hexane. A 1-g Silica SPE was conditioned with 80/20 (ethyl acetate/hexane) prior to loading on the sample extract. The samples were then loaded and finally eluted with 15-mL 0.1% acetic acid in acetone. Samples were then evaporated to dryness via nitrogen at 37 ◦ C and reconstituted in the mobile phase, prior to reverse phase LC/MS/MS analysis. Also, 10-mM ammonium acetate buffer was added to the sample solution to prevent degradation of some of the sulfonylurea herbicides. The acidic HPLC mobile phase consisted of methanol/ water and 0.05% acetic acid. Methanol was preferred over acetonitrile as an organic modifier because higher sensitivity with methanol vs. acetonitrile was observed. Because the mobile phase required a higher percentage of organic modifier (to elute the analytes), the desolvation in the electrospray source was better and the ion generation in the electrospray source was more efficient. The reverse phase analytical column was a Zorbax RX-C8 (2.1 × 150 mm). Positive ion LC/MS provides greater sensitivity, and for the majority of the sulfonylurea analytes, the greatest amount of structural information for the sulfonylurea herbicides. The positive [M + H]+ parent ions typically generate 2–5 good fragments under low collision energy conditions. Table 3 lists the ion transitions, primary and secondary for 19 sulfonylurea herbicides. Two parentproduct ion transitions were monitored for each of the 19 sulfonylurea herbicides. The peak area resulting from the most sensitive (primary) transition was used to quantitate the amount of analyte observed. The peak area, of the Table 3. Ion Transition Data for 19 Sulfonylurea Herbicides (LC/ESI/MS/MS) Sulfonylurea Herbicide Amidosulfuron Azimsulfuron Bensulfuron Chlorimuron Chlorsulfuron Cinosulfuron Ethametsulfuron Flupyrsulfuron Halosulfuron Metsulfuron Nicosulfuron Primisulfuron Prosulfuron Rimisulfuron Sulfometuron Thifensulfuron Triasulfuron Tribenuron Triflusulfuron

Primary Ion Transition

Secondary Ion Transition

370 ⇒ 261 425 ⇒ 182 411 ⇒ 149 415 ⇒ 186 358 ⇒ 141 414 ⇒ 183 411 ⇒ 196 466 ⇒ 182 435 ⇒ 182 382 ⇒ 167 411 ⇒ 182 469 ⇒ 254 420 ⇒ 141 432 ⇒ 182 365 ⇒ 150 388 ⇒ 167 402 ⇒ 261 396 ⇒ 155 493 ⇒ 264

370 ⇒ 218 425 ⇒ 270 411 ⇒ 182 415 ⇒ 213 358 ⇒ 167 414 ⇒ 215 411 ⇒ 168 466 ⇒ 156 435 ⇒ 252 382 ⇒ 199 411 ⇒ 213 469 ⇒ 199 420 ⇒ 167 432 ⇒ 325 365 ⇒ 199 388 ⇒ 205 402 ⇒ 141 396 ⇒ 181 493 ⇒ 236

995

secondary ion transition, was divided by the peak area of the primary ion transition, in order to obtain the ion ratios. The ion ratios generated from the individual standards and fortified samples were compared with the mean ion ratio of the six standards comprising the daily standard curve to obtain a percent difference value. The confirmation criteria used was as follows: The appropriate retention time (±2%); two parent-product ion transitions at S/N > 5; and the appropriate ion ratio (±30%).

IMIDAZOLINONE HERBICIDES The utility of LC/ESI/MS and LC/ESI/MS/MS for method simplification in pesticide residue analysis has been well demonstrated recently for the analysis of imidazolinone herbicides (imis) and their metabolites (Fig. 6) in a variety of matrices (20). With the sensitivity and specificity of LC/ESI/MS, the parent compounds (Structures A through F in Fig. 6) were directly determined in water at 1 ppb (21). In comparison with hundreds of milliliters of water, several cartridges, and organic solvents used in the conventional procedures (22,23), only a simple filtration was required prior to LC/ESI/MS analysis. Compared with a sample throughput of about six samples/day by the conventional route, sample preparation time for LC/ESI/MS was as fast as water could be forced through a 0.22-um filter from a 10-mL disposable syringe. For the determination of imazethapyr and its metabolites (Structures E, G, and H in Fig. 6) in a variety of plant commodities (24), the amount of initial extract requiring processing was reduced at least 20-fold to 100-mg equivalents from 2- to 4-g equivalents typically processed by conventional procedures (22,25). With the reduced sample requirements and the specificity of LC/ESI/MS, the initial extract could be directly loaded onto a 500-mg strong cation exchange (SCX) cartridge for cleanup, eliminating two evaporation steps, a precipitation step, and a C-18 cartridge from the conventional route. Economically speaking, sample throughput with LC/MS increased about four times. More importantly, over a range of 11 different commodities, this same generic approach was successfully demonstrated on each commodity in 1–2 days. A multiresidue method (26), using LC/ESI/MS, was developed to analyze six imidazolinone herbicides in five different soil types. Good recoveries (80%–120%) and adequate sensitivity at the 2.0-ppb level (LOQ) were obtained, for the compounds investigated. A 50-g aliquot of the of soil was extracted for 1 hour in 0.5-N sodium hydroxide. A portion of the extract was acidified, to precipitate the humic acids, and the supernatant was then loaded on to a preconditioned C-18 solid-phase extraction (SPE) cartridge and eluted with ethyl acetate. Further cleanup was achieved using a tandem strong anion exchange (SAX) SPE/strong cation exchange (SCX) SPE. Analytes were eluted off the SCX SPE with saturated KCl/methanol. After cleanup, the sample was then desalted using an RP-102 SPE cartridge. The sample was diluted to the appropriate volume with water, prior to LC/MS analysis.

996


O

O

N

OH

OH

N

N

N

N O

O H

H

B (imazamethabenz)

A (imazapyr) O

O

N

OH

OCH3

N

N

N

N O

O H

H

D (imazamethabenz-methyl)

C (imazameth) O

O OH

OH

N

N

N

N

N

N O

O H

H

F (imazaquin)

E (imazethapyr) OH

CH2OH

O

O O

O

OH N

N

OH

OH

N O H Figure 6. Structures of the imidazolinone herbicides.

G (Hydroxy-E)

The HPLC mobile-phase gradient started at 15% acetonitrile/85% 0.15% acetic acid in water and ended at 90% acetonitrile and 10% 0.15% acetic acid in water (0–32 min.). The HPLC column was a Zorbax RX-C8 2.1 mm ID × 150 mm, 5-um column with a flow rate of 0.2 mL/min and 100-uL injection. Quantitation was achieved by using a time-scheduled selective ion monitoring program (positive mode), monitoring the [M + H]+ ions for each compound. Also, the characteristic fragment ions were monitored using ‘‘insource’’ collision–induced dissociation (CID). One caveat that cannot be overstated, about using ‘‘in-source’’ CID, unlike MS/MS, is that the extract from the sample must be clean enough so that the relative abundances from the fragment ions can be matched to a standard (i.e., fragment ions from the matrix could interfere with ions of the same m/z from the analytes, which could affect the ion ratio in comparison with a reference standard). In this experiment, the extracts were

N

N

OH OH

N O H

H (glucoside of G)

clean enough to successfully confirm the presence of the residues found in a 2.0-ppb fortification of all six imidazolinones investigated. Table 4 lists the ions monitored for confirmation using ‘‘in-source’’ CID. The confirmation criteria used was as follows: The appropriate retention time (±2%); Fragment ions are S/N > 5, the appropriate ion ratio (±20%) when compared with a standard. The utility of LC/ESI/MS/MS for method simplification was illustrated in analysis of imazethapyr in soil at 1 ppb (27). Compared with the laborious conventional approach of processing 20- to 25-g equivalents of a 0.5 N NaOH extract through an extensive cleanup, as shown above (26), with a sample throughput of 2 hours/sample (28), the LC/ESI/MS/MS approach required processing only 200-mg equivalents of soil extract through a single 200-mg C-18 cartridge. As shown in Figure 7, LC/ESI/MS monitoring of the [M + H]+ ion of imazethapyr was almost adequate by itself for a

LC/MS, PESTICIDE RESIDUE ANALYSIS Table 4. List of Ions Monitored for the Imidazolinone Herbicides, Using ‘‘in-source’’ CID (LC/ESI/MS)

997

5:34 39555 M

Ions: [M + H]+ and Fragments Ions (Relative Intensities)

Analyte Imazapyr

262 (100), 217 (28), 243 (30)

Imazamethabenz acid (m,p)

275 (100), 229 (28), 257 (15)

Imazamox

306 (100), 278 (18), 193 (11)

Imazapic

276 (100), 248 (26), 163 (18)

Imazethapyr

290 (100), 177 (34), 230 (19)

Imazaquin

312 (100), 252 (23), 199 (48)

1:40

3:20

5:00

Figure 7. Determination of 1 ppb of imazethapyr in soil: HPLC/ESI/MS with selected ion monitoring of the [M + H]+ ion 290.

method. For the imis, CID of [M + H]+ ion in the MS/MS generated principal product ions at m/z 86 and 69, as shown in Figure 8. The additional specificity of monitoring the ion transition of m/z 290 to m/z 86 in MS/MS gave a chromatogram free of co-extractive peaks (Fig. 9). Using LC/ESI/MS/MS for detection reduced sample preparation time to about 10 min/sample.

(2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), 2-(4-chloro-2-methylphenoxy) propionic acid (MCPP), 4(4-chloro-2-methylphenoxy) butyric acid (MCPB), and 6- hydroxybentazone and 8-hydroxybentazone. This method was combined with a prior automated online liquid–solid-phase extraction step using an OSP-2 autosampler containing C-18 cartridges, and it was applied to the trace determination of acidic herbicides in environmental waters. The proposed method required only 50 mL of water with a limit of detection between 0.01 and 0.03 ppb, employing selected ion monitoring of the [M-H]− ion. Gradient elution was accomplished using an eluent

DETERMINATION OF ACIDIC HERBICIDES Chiron et al. (29) have used LC/ESI/MS in the negative mode for the determination of acidic herbicides in environmental waters. The acidic herbicides investigated were benazolin, bentazone, 2,4-dichlorophenoxyacetic acid

E + 04 8.20

69.3

100

−NH3 86.3 80 +C

CH3

NH2 M+H 290.3

60

COOH

40 N

+

C

NH

−H2O 230.2 248.6

20

−HCN

177.0 159.6 58.5

50

96.9 100

133.1 150

−

203.3 202.4 226.7 184.1 200

262.2

250

Figure 8. Product ion spectrum from collisionally activated dissociation of [M +

H]+

300 ion of imazethapyr.

998


5:34 4237 M

1:40

3:20

5:00

Figure 9. Determination of 1 ppb of imazethapyr in soil: HPLC/ESI/MS/MS with selected reaction monitoring of the ion transition: 290 = >86.

Table 5. List of Ions Monitored for the Acidic Herbicides (LC/ESI/MS) Analyte

Ions: [M − H]− and Fragment Ions

8-hydroxybentazone

255, 192

6-hydroxybentazone

255

Benazolin

198, 170

Bentazone

239

2,4-D

219, 161

MCPA

199, 141

MCPP

213, 141

MCPB

227, 141

containing 20% solvent A (methanol) and 80% solvent B (water, pH 2.9 with formic acid) to 80%A–20%B in 30 min at a flow rate of 0.25 mL/min. The analysis involved a Lichrospher cartridge column (125 × 3 mm i.d.) packed with Lichrospher 60RP select B material of 5-um particle size. Post-column addition of 0.1 mL/min of tripropylamine (4 g/L methanol) was needed for better sensitivity. Table 5 lists the ions monitored for the above study. Similar work (unpublished work at U.S. EPA) was performed for the direct determination of 2,4-D in runoff water at the 1.0-ppb level. Sample preparation involved filtering the water followed by the direct injection of water sample into the LC/MS system (without sample preconcentration). The mobile phase was 50/50 acetonitrile/0.15% acetic acid in water. The HPLC column was a Zorbax RX-C8 2.1 × 150 mm; 5-um column with a flow rate of 0.2 mL/min and 150-uL injection. The lower limit of quantitation for 2,4-D was 1.0 ppb. Also, sensitivity at the 1.0-ppb level was sufficient enough that post-column addition of 0.1 mL/min of tripropylamine was not necessary for that level.

N -METHYL CARBAMATE INSECTICIDES The increased use of N-methyl carbamate insecticides in agriculture demands the development of selective and sensitive analytical procedures to determine trace level residues of these compounds in crops and other food

products. HPLC is the technique most widely used to circumvent heat sensitivity for pesticide analysis. However, HPLC/UV lacks sensitivity. HPLC, using postcolumn hydrolysis and derivatization, was developed (30) and refined (31). This technique is currently the most widely used HPLC method for the determination of carbamates in water (32) and in fruit and vegetables (33,34). The technique relies on the post-column hydrolysis of the carbamate moiety to methylamine with subsequent derivatization to a fluorescent isoindole product. In addition to HPLC/fluorescence, there are several references that use both APCI or ESI HPLC/MS for the determination of N-methyl carbamate insecticides in a variety of matrices (35–37). Described below is a description of ongoing work at the U.S. EPA for the determination of N-methyl carbamate insecticides in nine fruit and vegetables at the 1.0-ppb level (manuscript in preparation). The fruits and vegetables investigated were cranberries, peaches, blueberries, kiwi, carrots, tomatoes, potatoes, lettuce, and grapefruit juice. The purpose of showing this work is to illustrate why LC/MS/MS is the method of choice for residue work at the 1.0-ppb level, especially for difficult matrices. In this study, HPLC/fluorescence was compared with HPLC/ESI/MS and HPLC/ESI/MS/MS. Summary of the procedure goes as follows. Sample is prepared using the method of Luke et al. (38). The sample is then cleaned up using a 1000-mg aminopropyl SPE. The sample is loaded onto the SPE in a solution of 5% dichloromethane in hexane. The SPE is then washed with 5 mL of 5% dichloromethane/hexane and then eluted with 10 mL of 1% methanol/dichloromethane. The sample is then evaporated to dryness and then diluted to the appropriate volume with 1 : 1 methanol/pH 3 buffer. Samples are ready to be analyzed by HPLC/fluorescence (post-column derivatization) or by HPLC/ESI/MS or HPLC/ESI/MS/MS. For ESI, the HPLC gradient started at 15% acetonitrile/85% 10-mm ammonium acetate for the first 3 minutes and then went to 90% acetonitrile/10% 10-mm ammonium acetate in 31 minutes (held for 4 minutes). The HPLC column was a Zorbax RX-C8 2.1 ID × 150 mm, 5-um column with a flow rate of 0.15 mL/min and 20-uL injection. For APCI (if matrix effects become a problem with ESI), the mobile phase consisted of A: 90% methanol/10% water/ 50-mM ammonium acetate; B: 90% water/50-mM ammonium acetate/10% methanol. The gradient started at 50% A/50% B for 10 minutes and then went to 90% A/10% B in 22 minutes (held for 3 minutes). The HPLC column was a Zorbax RX-C8 4.6 mm ID × 250 mm, 5-um column with a flow rate of 1.0 mL/min and 50-uL injection. Table 6 shows the ion transitions (parent to product ions) that were monitored for LC/ESI/MS/MS. For singlestage LC/ESI/MS, Table 7 shows the ions that were monitored. In this study, the preliminary findings show that the HPLC/fluorescence data agreed favorably, for all 12 carbamates, with HPLC/ESI/MS/MS, for most of the nine fruits and vegetables at the 1.0-ppb fortification level. The recoveries were generally within 70%–120%. However, at the 1.0-ppb level, in each commodity, HPLC/ESI/MS (single-stage MS) had difficulty with interferences for 3

N-Methyl Carbamate

Primary Ion Transition

Secondary Ion Transition

Aldicarb sulfoxide Aldicarb sulfone Oxamyl Methomyl 3-hydroxycarbofuran Aldicarb Propoxur Carbofuran Carbaryl Thiodicarb Isoprocarb Methiocarb

207 ⇒ 132 223 ⇒ 148 237 ⇒ 71.6 163 ⇒ 88 238 ⇒ 163 208 ⇒ 116 210 ⇒ 168 222 ⇒ 165 202 ⇒ 145 355 ⇒ 87.6 194 ⇒ 95 226 ⇒ 121

207 ⇒ 89 223 ⇒ 166 237 ⇒ 90 163 ⇒ 106 238 ⇒ 181 208 ⇒ 89 210 ⇒ 153 222 ⇒ 123 202 ⇒ 116.8 355 ⇒ 163 194 ⇒ 137 226 ⇒ 169.1

999

11.242 11.634 - 3-hydroxycarbofuran 12.210

Table 6. Ion Transition Data for 12 N-Methyl Carbamate Insecticides (LC/ESI/MS/MS)

9.412 - methomyl


Note: In the case of Aldicarb, Oxamyl, and 3-hydroxycarbofuran, the ion transitions go from [M + NH4 ]+ ⇒ product ions.

Table 7. Parent Ions, [M + H]+ , Monitored for 12 N-Methyl Carbamate Insecticides (LC/ESI/MS) N-Methyl Carbamate Aldicarb sulfoxide

10 Figure 10. HPLC/fluorescence of grapefruit juice control.

Parent Ions 207

Aldicarb sulfone

223

Oxamyl1

237

Methomyl

163

3-Hydroxycarbofuran1

238

Aldicarb2

116

Propoxur

210

Carbofuran

222

Carbaryl

202

Thiodicarb

355

Isoprocarb

194

Methiocarb

226

Notes: 1 In the case of Oxamyl and 3-hydroxycarbofuran, the ion monitored is [M + NH4 ]+ . 2 In the case of Aldicarb, the ion monitored is [MH-75]+ .

out of the 12 carbamate pesticides: aldicarb sulfoxide, aldicarb sulfone, and 3-hydroxycarbofuran. Quantitation was not possible for the above three compounds. In addition, there were problems with interferences using HPLC/fluorescence with carrots and grapefruit juice for most of the carbamates at the 1-ppb level. For example, Figure 10 shows a control grapefruit juice sample that is showing reportable levels of 3-hydroxycarbofuran using HPLC/fluorescence. (Other false positives were also reported for most of the other carbamate insecticides, in the control grapefruit juice, using HPLC/fluorescence.) Figure 11 shows a control grapefruit juice sample (bottom) and a 1.0-ppb fortification of 3-hydroxycarbofuran in grapefruit juice (top), using LC/ESI/MS (single-stage mass spectrometry). The control, with LC/ESI/MS, also shows reportable levels of 3-hydroxycarbofuran. Figure 12 is similar to Figure 11, except LC/ESI/MS/MS was used.

From this example, it is obvious that LC/ESI/MS/MS is the preferred technique for analyzing residues at the 1.0-ppb level and was therefore the primary tool for quantitating the carbamate residues in the difficult matrices (carrots and grapefruit juice). In the case of carbamate insecticides, both ESI and APCI can be used. However, in this study, we have found that the sensitivity of APCI is reduced by a factor of three- to five-fold, when compared with ESI. This can vary, depending on the configuration of the API source (orthogonal, off axis, on axis, or Z-spray). In this case, we used the Z-spray configuration. APCI can help with matrix effects, when analyzing for carbamate insecticides. For example, when analyzing for methiocarb in citrus products, the apparent recoveries were in the 50% range with ESI. However, when changing to APCI, the apparent recoveries were increased to 110%. This is an example where APCI can be an alternate API method if matrix effects are a problem with ESI. The only caveat, of course, is that the analyte must have sensitivity with both API techniques. MATRIX EFFECTS WITH ELECTROSPRAY LC/MS Despite the success of electrospray for quantitative analysis, the technique does have certain limitations. One such fundamental problem is limited dynamic range (39). Kerbarle and coworkers (4,40) have reported linear response from 10−8 to 10−5 M for various organic bases. At about 10−5 M, the response no longer increases with concentration but levels off and eventually begins to decrease. The cause of the nonlinear response is under investigation by various research groups. Experiments by Bruins (41) indicate that the limited dynamic range is caused by an inability of droplet charge to be converted to gas-phase ions that can be mass analyzed. As already mentioned in the carbamate section, a challenge with electrospray, at least in the area

1000


2: SIR of 1 channel ES+ 238 9.65e3 Area Time Height Area Area% 12.48 5002 3561.69 100.00

12.48 3562

3-hydroxycarbofuran; 1ppb in grapefruit juice; LC/MS

2: SIR of 1 channel ES+ 238 8.07e3 Area Area Area% Time Height 12.52 3010 1583.21 100.00

12.52 1583

Grapefruit juice control; LC/MS

Time 10.00

15.00

20.00

25.00

30.00

35.00

40.00

Figure 11. HPLC/ESI/MS of grapefruit juice fortified with 1.0 ppb 3-hydroxycarbofuran (top) and grapefruit juice control (bottom). Ion monitored: [M + NH4 ]+ ion 238.

of quantitation, is ion suppression. Another example with matrix effects is the discussion of the quantitative analysis of pyrithiobac sodium (sodium 2-chloro-6[4,6-dimethoxypyrimidin-2-yl)thio]benzoate, in cotton gin trash, by ESI LC/MS/MS. When using external standards, the recoveries ranging from 50%–55%. Recoveries for control extracts fortified just prior to injection ranged from 60%–70%. Low recoveries for extracts fortified just prior to analysis indicate that matrix suppression is occurring in the electrospray source (42,43). Although MS/MS removes co-eluting compounds from the baseline, it does not reduce matrix effects. Matrix effects result from changes in ionization efficiency due to competition for charge at the droplet level (4,44). In an attempt to minimize matrix effects, the APCI interface was used to analyze samples using the same purification procedure (accelerated solvent extraction followed by liquid/liquid partitioning). The method generated acceptable recoveries (70%–120%) at the 0.02- and 0.04-ppm levels.

The APCI interface is less susceptible to matrix suppression than is the electrospray interface for pyrithiobac sodium in cotton gin trash (45). The corona discharge in the APCI source appears to produce enough charge to ionize all compounds present at any given time, regardless of the presence of co-eluting compounds. The thermal stability of pyrithiobac sodium enabled it to withstand the increased temperature of the APCI interface without significant thermal degradation. Compounds, which are thermally labile, often do not give sufficient response for low level quantitation by APCI. In contrast to the signal suppression, observed in ESI, signal enhancement is occasionally observed in APCI. Other ways to minimize on matrix effects include improve sample cleanup, more dilution of sample, labeled internal standards, standard addition, and standard diluted in matrix. The latter solution (standard diluted in matrix) is not permitted for enforcement methods with the U.S. EPA or the U.S. FDA.


1001

2: MRM of 2 channels ES+ 238 > 163 1.11e4

12.87

3-hydroxycarbofuran; 1ppb in grapefruit juice; LC/MS/MS

2: MRM of 2 channels ES+ 238 > 163 3.03e3

12.76 14.37 15.91

Grapefruit juice control; LC/MS/MS

Time 10.00

12.50

15.00

17.50

20.00

22.50

25.00

27.50

30.00

32.50

35.00

37.50

Figure 12. HPLC/ESI/MS/MS of grapefruit juice fortified with 1.0 ppb 3-hydroxycarbofuran (top) and grapefruit juice control (bottom). Selected reaction monitoring of the ion transition: 238 => 163.

CONCLUSIONS Finally, Table 8 gives a summary of references along with the ionization modes used for the various classes of pesticides. These classes include the ones that were discussed earlier in this chapter plus other classes that were not discussed. Hopefully this information will provide some basic tools in performing successful API HPLC/MS, for the compounds of interest. HPLC/MS and HPLC/MS/MS has become more commercially available and is more affordable for the enforcement laboratories than several years ago. Although a mass spectrometer is still initially a more

expensive and a complex device than are most other LC detectors, once a mass spectrometer is up and running, it can be very dependable and reliable. Mass spectrometry can then eliminate many of the other variables that consume time in sample analysis and method development. For residue work at or below the 10-ppb level, it is strongly recommended that LC/MS/MS be used. Acknowledgments The authors wish to thank Robert D. Voyksner, LCMS Limited, for providing Figures 1–5. The authors also wish to thank Lynda Podhorniak, U.S. EPA, for providing the grapefruit juice

Table 8. Summary of Literature References for HPLC/MS Applications of Various Classes of Pesticides Compound Class Sulfonylurea herbicides Imidazolinone herbicides N-methyl carbamate insecticides Triazine herbicides

API Mode ESI ESI APCI and ESI APCI and ESI

Literature References 16–19, 46–49 20, 21, 23–27, 50 35–37 14, 15, 51–54

1002

LC50

extracts and the HPLC/fluorescence chromatogram of the grapefruit juice control.

28. K. N. Reddy and M. A. Locke, Weed Science 42: 249–253 (1994).

BIBLIOGRAPHY

29. S. Chiron, S. Pipilloud, W. Haerdi, and D. Barcelo, Anal. Chem. 67: 1637–1643 (1995).

1. C. M. Whitehouse, R. N. Dreyer, M. Yamashita, and J. B. Fenn, Anal. Chem. 57: 675–679 (1985). 2. R. D. Smith et al., Anal. Chem. 62: 882–899 (1990). 3. J. B. Fenn et al., Mass Spectrom. Rev. 9: 37–70 (1990). 4. P. Kebarle and L. Tang, Anal. Chem. 65: 972A–986A (1993). 5. J. B. Fenn, J. Rosell, and C. K. Meng, J. Am. Soc. Mass Spectrom. 8: 1147 (1997). 6. S. J. Gaskell, J. Mass Spectrom. 32: 677–688 (1997). 7. C. Enke, Anal. Chem. 69: 4885–4893 (1997). 8. A. P. Bruins, J. Chromatography A. 794: 345–357 (1998). 9. D. Barcelo, ed., Journal of Chromatography Library, Vol. 59, Elsevier, Amsterdam, 1996. 10. R. B. Cole, ed., Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications, John Wiley and Sons, New York, 1997, pp. 3–65. 11. D. A. Volmer and D. L. Volmer, LC-GC 14: 236–242 (1996).

30. H. A. Moye, S. Scherer, and P. A. St. John, Anal. Lett. 10: 1049–1073 (1997). 31. R. T. Krause, J. Chromatogr. Sci. 16: 281–288 (1978). 32. K. M. Hill, R. H. Hollowell, and L. A. Dal Cortivo, Anal. Chem. 56: 2465–2468 (1984). 33. R. T. Krause and E. M. August, J. AOAC Int. 66: 234–240 (1983). 34. R. T. Krause, J. AOAC Int. 63: 1114–1124 (1980). 35. S. Pleasance, J. F. Anacleta, M. R. Bailey, and D. H. North, J. Am. Soc. Mass Spectrom. 3: 378–397 (1992). 36. A. Di Corcia et al., J. Agric. Food Chem. 44: 1930–1938 (1996). 37. D. A. Volmer, D. L. Volmer, and J. G. Wilkes, LC-GC 14: 216–224 (1996). 38. M. L. Luke, J. E. Froberg, and H. T. Masumoto, J. AOAC Int. 58: 1020–1026 (1975).

12. P. J. Arpino, Mass Spectrom. Rev. 8: 35 (1989).

39. D. L. Buhrman, P. I. Price, and P. J. Rudewicz, J. Am. Soc. Mass Spectrom. 7: 1099–1105 (1996).

13. R. C. Willoughby and R. F. Browner, Anal. Chem. 56: 2626 (1984).

40. M. G. Ikonomou, A. T. Blades, and A. T. Kerbarle, Anal. Chem. 62: 957–967 (1990).

14. E. M. Thurman, I. Ferrer, and D. Barcelo, Anal. Chem. 73: 5441–5449, 2001.

41. A. P. Bruins, Proceedings of the 7th Sanibel Conference on Mass Spectrometry, Sanibel Island, FL, January 21–24, 1995.

15. E. M. Thurman, I. Ferrer, and D. Barcelo, The IonizationContinuum Diagram: A Concept for Selection of APCI and ESI Conditions for HPLC/MS of Pesticides: 17th Montreux Symposium on Liquid Chromatography/Mass Spectrometry, Montreux, Switzerland, November 8–10, 2000, p. 31. 16. A. J. Krynitsky, J. AOAC Int. 80: 392–400 (1997). 17. L. J. Marek and W. C. Koskinen, J. Agric. And Food Chem. 44: 3978–3881 (1996). 18. D. Volmer, J. G. Wlikes, and K. Levsen, Rapid Commun. Mass Spectrom. 9: 767–771 (1995). 19. D. B. Goodenowe and M. J. Duffy, Trace Level Confirmation and Quantitation of Sulfonylurea Herbicides in Soil by Electrospray LC/MS/MS, Presented at IUPAC, London, August 2–7, 1998. 20. S. J. Stout, A. R. daCunha, G. L. Picard, and M. M. Safarpour, J. AOAC Int. 81: 685–690 (1998).

42. C. G. Enke, Anal. Chem. 69: 4885 (1997). 43. B. K. Matuszewski, M. L. Constanzer, and C. M. ChavezEng, Anal. Chem. 70: 882 (1998). 44. A. T. Blades, M. G. Ikonomou, and P. Kerbarle, Anal. Chem. 63: 2109 (1991). 45. W. J. Blanchflower, P. J. Hughes, and D. G. Kennedy, J. AOAC Int. 80: 1177 (1997). 46. M. Rodriguez and D. B. Orescan, Anal. Chem. 70: 2710 (1998). 47. R. Bossi, B. Koppen, N. H. Spliid, and J. C. Streibeg, J. AOAC Int. 81: 775 (1998). 48. A. Di Corcia, C. Crescenzi, R. Samperi, and L. Scappaticcio, Anal. Chem. 69: 2819–2826 (1997). 49. H. Nejad et al., J. Capillary Electrophoresis 005: 81–87 (1998).

21. S. J. Stout, A. R. daCunha, G. L. Picard, and M. M. Safarpour, J. Agric. Food Chem. 44: 2182–2186 (1996).

50. C. Crescenzi, in R. A. Meyers, ed., Encyclopedia of Analytical Chemistry Instrumentation and Applications, John Wiley and Sons, Chichester, West Sussex, England, 2000.

22. J. M. Devine, in D. L. Shaner and S. L. O’Connor, eds., The Imidazolinone Herbicides, CRC Press, Boca Raton, FL, 1991, ch. 13.

51. A. Di Corcia, C. Crescenzi, E. Guerriero, and R. Samperi, Environ. Sci. Tech. 31: 1658–1663 (1997).

23. M. J. M. Wellos and J. L. Michael, J. Chromatogr. Science 25: 345–350 (1987). 24. S. J. Stout, A. R. daCunha, G. L. Picard, and M. M. Safarpour, J. Agric. Food Chem. 44: 3548–3553 (1996). 25. R. D. Mortimer and D. F. Weber, J. AOAC Int. 80: 426–432 (1993). 26. A. J. Krynitsky, S. J. Stout, H. Nejad, and T. C. Cavalier, J. AOAC Int. 82(4): 956–962 (1999). 27. S. J. Stout, A. R. daCunha, and M. M. Safarpour, J. AOAC Int. 80: 426–432 (1997).

52. E. M. Thurman and C. Batian, Determination of Atrazine and Atrazine Mercapture in Drinking Water Samples and in Urine Using Immunoaffinity SPE with Positive Ion Spray HPLC/MS: 15th Symposium on Liquid Chromatography/Mass Spectrometry, Montreux, Switzerland, November 9–10, 1998. 53. I. Ferrer, E. M. Thurman, and D. Barcelo, Environ. Sci. Tech. 34: 714–718 (2000).

LC50 .

See MEDIAN LETHAL CONCENTRATION (LC50 )

LD50

LD50 . See MEDIAN LETHAL DOSE (LD50 )

LD50 ALLAN FELSOT Washington State University Tri-Cities Richland, Washington

LD50 (or median lethal dose) is a statistically derived estimate of a single dose of a substance that can be expected to cause death in 50% of animals when administered by the oral route or when applied directly to the skin (dermal route) (1,2). The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (milligrams per kilogram or mg/kg). When a substance is administered via the inhalational route, the dosage per unit of body weight is not known. Therefore the concentration in air is measured and the resulting toxicity is expressed as LC50 or concentration in air (as milligrams per liter or mg/L) expected to cause death in 50% of exposed animals (3). LD50 is useful for determining hazards of pesticides resulting from short-term (24 hours or less) exposure by either oral, dermal, or inhalational routes. Its value serves as a basis for hazard categorization, product labeling, imposition of child-resistant packaging, or designation of application by only certified applicators (4). It is also an initial step in establishing a dosage regimen in long-term studies and may provide information on absorption of these pesticides and the mode of action of a pesticide. In isolation the value of LD50 has little meaning. However, it is useful for comparing the hazards of different compounds. The Environmental Protection Agency (EPA) has somewhat arbitrarily ranked the oral, dermal, and inhalational LD50 values into four toxicity categories that define labeling of pesticide products (Table 1). For example, all compounds with oral LD50 less than 50 mg/kg would have on the product label the signal word ‘‘Danger’’ accompanied by a skull and crossbones. In addition to

LD50 , qualitative results from dermal and eye sensitization studies are also used to categorize pesticide hazards. LD50 values can be obtained from the material safety data sheets that should be available with the purchase of a pesticide product. The greatest utility for these values is informing applicators of relative hazards between specific chemicals. In general, for any given pesticide the oral LD50 is greater than the dermal LD50 . Absorption through skin is nearly always less efficient than absorption through the intestine. However, pesticides having oral and dermal LD50 values less than 50 mg/kg would be extremely hazardous because skin absorption is very efficient and very rapid. The vast majority of fungicides have dermal and oral LD50 s that would place them in toxicity category III or IV. However, formulation ingredients may cause eye or skin irritations sufficient to label the fungicide product in a more hazardous toxicity category. In addition to defining the human health hazard of a substance, LD50 and LC50 values have had very important roles in determining the pest control potential of candidate pesticides. The endpoint of pesticide effectiveness need not be lethal. Growth inhibition or suppression especially relevant for fungicide efficacy can also be expressed as a median lethal dose. In this latter case, the term ED50 , signifying effective dose would be used to describe any physiological effect in 50% of exposed organisms. In addition to the utility of the LD50 or ED50 for comparing potency of different fungicides, the slope of the curve from which the LD50 is estimated can provide information about the variability of susceptibility in any pest population, and thus serve as an early warning sign for resistance development. In any population of organisms, some will be affected by comparatively low doses and some by increasingly higher doses. If the numbers of organisms (or in the case of fungi, isolates) were plotted relative to the dose causing an effect, the resulting dose-response curve would tend toward normality (Fig. 1a). If the distribution of responses at different doses were then expressed as the cumulative proportion or percentage of the population response, the curve would approximate a sigmoidal shape that could be described by a logistic

Table 1. LD50 and Sensitization Descriptors for Categorizing Hazards of Pesticides Study

Category I

Category II

Category III

Category IV

Product label Signal word Acute oral Acute dermal Acute inhalation Eye irritation

Danger

Warning

Caution

Caution

≤50 mg/kg ≤200 mg/kg ≤0.05 mg/L Corrosivea

Skin irritation

Corrosiveb

>50–≤500 mg/kg >200–≤2000 mg/kg >0.05–≤0.5 mg/L Corneal injury or irritation, clearing in 8–21 days Severe irritation at 72 hoursc

>500–≤5000 mg/kg >2000–≤5000 mg/kg >0.5–≤2 mg/L Corneal injury or irritation, clearing in 7 days or less Moderate irritation at 72 hoursc

>5000 mg/kg >5000 mg/kg >2 mg/L Minimal effects, clearing in less than 24 hours Mild or slight irritation

a

Irreversible destruction of ocular tissues, injury to cornea, or irritation persisting for more than 21 days. Tissue destruction into the dermis and scarring. c Severe irritation includes severe skin reddening (erythema) or swelling (edema); moderate irritation refers to moderate erythema only; mild irritation includes no irritation or only slight erythema. b

1003

1004

LIMIT OF DETECTION (LOD) (a)

(b)

(c)

LD50

Cumulative percent responding

Frequency of response

Dose or concentration

LD50

Probit

Dose or concentration

LD50

log Dose or concentration

Figure 1. Theoretical dose-response relationship. (a) Distribution of response of two populations to increasing doses or concentrations of a toxicant. The dotted line represents a population exhibiting greater variability in response than the population represented by the solid line. The dose associated with the median response (50th percentile) is the LD50 . (b) Expression of response as a cumulative percent of the population responding to increasing doses. Note the LD50 s for the two populations are the same, but the populations represented by the dotted line exhibit responses over a greater range of doses. (c) Transformation of cumulative percent responding to probit units and concentration to log units. Note the flatter slope corresponding to the population exhibiting greater variability in response to the test substance (dotted line).

function (5) (Fig. 1b). When the dose or concentration is expressed as a logarithm and the percentage response is changed to probability units called probits, the doseresponse curve becomes linear (Fig. 1c). The wider the distribution in doses necessary to cause a response in the entire population, the flatter is the slope of the transformed curve (Fig. 1c). Populations that are susceptible to a pesticide and have had little exposure to it normally respond to a very narrow dose range. As selection occurs for resistant individuals, however, the distribution of doses widens and the transformed dose-response curve begins to flatten out. As a pesticide resistance trait becomes more prevalent in the population, the dose-response curves become steeper, shifting the estimated LD50 or ED50 to higher values. BIBLIOGRAPHY 1. U.S. Environmental Protection Agency, 1996a, Health Effects Test Guidelines, OPPTS 870.1100, Acute Oral Toxicity, EPA 712-C96-190. 2. U.S. Environmental Protection Agency, 1996b, Health Effects Test Guidelines, OPPTS 870.1200, Acute Dermal Toxicity, EPA 712-C96-192. 3. U.S. Environmental Protection Agency, 1996c, Health Effects Test Guidelines, OPPTS 870.1300, Acute Inhalation Toxicity, EPA 712-C96-193. 4. U.S. Environmental Protection Agency, 1996c, Health Effects Test Guidelines, OPPTS 870.1000, Acute Toxicity Testing—Background, EPA 712-C96-189. 5. W. J. Hayes, Jr., in W. J. Hayes, Jr. and E. R. Laws, Jr., eds., Dosage and Other Factors Influencing Toxicity, in Handbook

of Pesticide Toxicology: General Principles, vol. 1, Academic Press, New York, 1991, pp. 39–105.

LIMIT OF DETECTION (LOD) Lowest concentration of a pesticide residue in a defined matrix, where positive identification can be achieved using a specified method (IUPAC).

LIMIT OF QUANTITATION (LOQ) Lowest concentration of a pesticide residue in a defined matrix where positive identification and quantitative measurement can be achieved using a specified method. The term limit of quantitation (LOQ) is preferred to limit of determination to distinguish it from LOD. LOQ has been defined as three times the LOD (1) or as 50% above the fortification level used to validate the method (USEPA) (IUPAC). See also Analysis. BIBLIOGRAPHY 1. L. H. Keith, Environmental sampling and analysis—a practical guide, Lewis Publishers, Boca Raton, Florida, 1991.

LOEL Lowest observed effect level. The lowest dosage (in units of mg/kg (body wt.)/day) causing a particular toxicological effect.

ENCYCLOPEDIA OF


ENCYCLOPEDIA OF AGROCHEMICALS Editor-in-Chief Jack R. Plimmer

Editorial Staff

Associate Editor Derek W. Gammon California EPA

Executive Editor: Jacqueline I. Kroschwitz

Associate Editor Nancy R. Ragsdale Agricultural Research Service, USDA

Executive Publisher: Janet Bailey Managing Editor: Shirley Thomas Publishing Technology Associate Manager, Books: David Blount Illustration Manager: Dean Gonzalez Editorial Assistant: Audrey Roker

ENCYCLOPEDIA OF

AGROCHEMICALS VOLUME 3 Jack R. Plimmer Derek W. Gammon Nancy N. Ragsdale

The Encyclopedia of Agrochemicals is available Online at www.mrw.interscience.wiley.com/eoa

A John Wiley & Sons, Inc., Publication

Copyright  2003 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, e-mail: [email protected]. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services please contact our Customer Care Department within the U.S. at 877-762-2974, outside the U.S. at 317-572-3993 or fax 317-572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print, however, may not be available in electronic format. Library of Congress Cataloging in Publication Data: ISBN 0-471-19363-1 Encyclopedia of agrochemicals / Jack R. Plimmer, editor-in-chief. p. cm. ISBN 0-471-19363-1 (cloth) 1. Agricultural chemicals—Encyclopedias. I. Plimmer, Jack R., 1927S584.4 .E53 2002 631.8 03—dc21 2002027418 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

ENCYCLOPEDIA OF


M MACROPORE AND PREFERENTIAL FLOW

and the principles underlying some existing models describing preferential flow.

NICHOLAS JARVIS Swedish University of Agricultural Sciences Uppsala, Sweden

HISTORY OF PREFERENTIAL FLOW RESEARCH The significance of macropores was recognized as long ago as the mid-19th century by Schumacher (6), who stated that ‘‘the permeability of a soil during infiltration is mainly controlled by large pores, in which the water is not held under the influence of capillary forces’’ and by Lawes and colleagues (7), who noted that ‘‘in a heavy soil, channel drainage will in most cases precede general drainage, a portion of the water escaping by the open channels before the body of the soil has become saturated; this will especially be the case if the rain fell rapidly, and water accumulates on the surface.’’ Such qualitatively accurate descriptions of flow phenomena occurring in natural structured soils were largely ignored, despite early experimental confirmation of the importance of large noncapillary pores for saturated water flow (8). Instead, it was the empirical but quantitative work of a contemporary, an engineer named Darcy, working on flow through artificial packed sand beds that laid the foundation for soil water physics in the twentieth century. Darcy’s law was later extended by L. A. Richards, who combined it with an equation of mass conservation to enable prediction of transient unsaturated flow in soils (9). The underlying assumption of homogeneity, that single values of water potential, water content, and hydraulic conductivity could adequately characterize a representative elementary soil volume at a given soil depth, remained virtually unchallenged for nearly half a century. Solute transport theory developed along conceptually similar lines, with the convective–dispersive (CDE) theory of transport gaining overwhelming popularity in the last half of the twentieth century. This theory assumes that lateral mixing processes are fast in relation to vertical convective transport (10), so that a single value of solute concentration is assumed to characterize the soil at any given depth. Starting in the 1960s and early 1970s, new experimental observations of rapid nonequilibrium flow of water in a variety of different soils, and the resulting effects on patterns of solute displacement, began to challenge the established paradigm (11–22). These observations led in the early 1980s to the first attempts to develop new theoretical frameworks that could account for nonequilibrium flow of water in macropores (23,24). In the 1970s, soil scientists had already extended the CDE concept to account for nonequilibrium of solute concentrations under steady-state saturated water flow in laboratory columns (25), but it was not until the late 1980s and early 1990s that models were developed that could account for both nonequilibrium flow of water and solute transport in unsaturated macroporous soils under transient conditions in the field (26,27). Research on unstable finger flow dates

Until recently, the prevailing conceptual model of water infiltration into soils was based on the idea that ‘‘new’’ incoming water displaced existing ‘‘old’’ water uniformly, with water moving downward in the soil profile as a broad and well-defined ‘‘wetting front.’’ Similarly, the prevailing view of agrochemical transport was that leaching took place as a chromatographic process, with the chemical as fully exposed to adsorption sites in undisturbed field soils as it would be both in laboratory batch experiments on water–slurry mixtures, and in column leaching experiments on repacked soils. The idea that water flow and chemical transport normally takes place as a uniform displacement process in soils has now been abandoned, and it has been replaced by an understanding that the heterogeneity of undisturbed soils in the field often leads to markedly nonuniform patterns of water flow and agrochemical displacement. The term preferential flow is used to describe this irregular wetting. It is a generic term, covering several processes with different physical causes, but with the common feature that nonuniform wetting leads to an increase in the effective velocity of the water flow through a small portion of the soil unsaturated zone. For example, in structured soils, macropores (shrinkage cracks, worm channels, root holes) may dominate the soil hydrology, particularly in fine-textured soils, where they operate as high conductivity flow pathways bypassing the denser impermeable soil matrix (1). Preferential flow also occurs in unstructured sandy soils in the form of unstable flow or fingering (2,3) caused by profile heterogeneities such as horizon interfaces or water repellency (4). Thus, preferential flow can occur in virtually all types of soils (5) caused by heterogeneities at scales ranging from the single pore to the soil horizon or profile. Preferential flow greatly increases the risk of leaching of surface-applied agrochemicals to groundwater and surface water bodies, because infiltrating water is channeled through only a small fraction of the total pore space, at rates that are too fast to allow sufficient time for equilibration with slowly moving ‘‘old’’ water stored in the bulk of the soil matrix. Thus, much of the adsorption and degradation capacity of the chemically and biologically reactive topsoil is ‘‘bypassed,’’ and a significant fraction of the applied agrochemical quickly reaches subsoil layers where these attenuation processes are generally less effective. This article discusses the history of preferential flow research, some physical principles of preferential flow, the range of experimental approaches available to investigate and quantify the processes, the main factors affecting the impact of preferential flow on leaching of agrochemicals, 1005

1006

MACROPORE AND PREFERENTIAL FLOW

Preferential flow in soil (+ synonyms) 600 AGRICOLA database 500

Publications

400

300

200

100

0 1970

1980

1990

2000

Year Figure 1. Publications on preferential flow and macropore flow, as revealed by a computer literature search.

back to the 1970s (28), but there are still today relatively few examples of applications to agrochemical leaching in the field. Research into all aspects of preferential flow and macropore flow has intensified during the last ten years, and it has now become a mainstream research topic in the geosciences. Based on a computer literature search using a large number of combinations of relevant keywords, Figure 1 shows that the number of publications in this research field has increased exponentially in the last 30 years, and that more than a hundred papers per year are now published on preferential flow and macropore flow in soils. These numbers are likely to be gross underestimates, because this kind of search cannot hope to discover all the relevant articles, but Figure 1 does at least illustrate the general trend. It remains to be seen whether the yearly output of publications continues at its present rate, or if the curve reaches a plateau as the subject ‘‘matures.’’ PHYSICAL PRINCIPLES Classic theory of water flow (Richards’s equation) and solute transport (convection–dispersion equation, CDE) is based on continuum physics, with the underlying assumption that unique values of soil water pressure and solute concentration can be defined for a representative elementary volume of soil. Physical nonequilibrium occurs when soil heterogeneities result in the generation of lateral differences (nonuniformity) either in water pressures or solute concentrations, or both. More specifically, preferential flow or transport results when rates of lateral equilibration of water pressures or solute concentrations, respectively, are slow in relation to the vertical flow rates (10,29). From these definitions, it can be noted that preferential transport can occur without preferential flow,

for example, in water-saturated soil characterized by a broad pore size distribution and thus a large range of pore water velocities. Thus, preferential flow is a generic term encompassing a range of different processes with similar characteristics and consequences for solute leaching in soils, although the underlying physical mechanisms may be different. The defining feature in all cases is a nonuniform lateral moisture distribution during vertical flow. Broadly, three different types of preferential flow mechanism are recognized: finger flow, heterogeneous flow, and macropore flow. Unstable ‘‘finger’’ flows can be initiated by small- and large-scale heterogeneities within the soil (2) or by flow concentration at the surface either due to interception by vegetation and stem flow, or by water repellency. When dry, many soils possess water-repellent properties caused by the presence of hydrophobic organic materials and coatings on soil particles (30). Rain falling on nearly airdry water-repellent soil tends to accumulate in shallow depressions, where aided by the hydrostatic pressure, it eventually infiltrates as finger flows (31–34). It is also well known that layer and horizon interfaces can generate fingers, particularly where a coarse-textured sand layer underlies a finer material (28). The downward movement of a wetting front is temporarily interrupted at the interface, because the water pressure must increase to the ‘‘water-entry’’ pressure of the coarse sand (i.e., near saturation). Due to local heterogeneities, the waterentry pressure may be exceeded at one or several points, rather than uniformly along the interface. This leads to the development of fingers moving rapidly into the subsoil layer at a rate slightly less than the saturated conductivity of the sand, whereas the remainder of the soil remains dry. Once formed, the fingers can only persist, and the flow field remains ‘‘unstable,’’ if lateral dispersion due to capillary forces is relatively weak. It has been shown theoretically that hysteresis in the soil water characteristic curve of narrow-graded sands can sustain finger flow if the water-entry pressure on the wetting curve is larger (i.e., closer to zero pressure potential) than the air-entry pressure on the draining curve (35). This is likely to be the case in hydrophobic soils. Theoretical and experimental studies on somewhat idealized porous media indicate that the finger width and hence the effective transport volume largely depends on the hydraulic properties of the porous media, especially the saturated hydraulic conductivity in relation to the applied flux, and the soil pore size distribution (3,36). Preferential solute transport may be further enhanced by the slow equilibration of concentrations between wet fingers and surrounding dry soil, due to the small water content in the bulk soil, which implies a negligible effective diffusion coefficient. Preferential flow may also occur in heterogeneous soils characterized by nonrandom arrangements of lenses and admixtures of various particle size fractions (37–39). With ‘‘heterogeneous’’ flow, the pathways taken by the infiltrating water should depend on the relative values of unsaturated hydraulic conductivity of the component materials at the applied water flux. For example, at saturation, a coarse sand fraction might comprise a


preferred flow region, whereas at small fluxes under unsaturated conditions, interconnected regions of a finer textured material may conduct all the water because soil water pressures may not increase sufficiently to saturate the coarse sand. Macropores are large, continuous, structural pores that constitute preferred flow pathways for infiltrating water in most soils (1). At the macroscopic scale of measurement, this is reflected in large increases in unsaturated hydraulic conductivity across a small soil water pressure head range close to saturation (40,41). At the pore scale, macropore flow is generated when the water pressure locally increases to near saturation at some point on the interface with the surrounding soil matrix, such that the water-entry pressure of the pore is exceeded. Macropore flow can be sustained if the vertical flux rates in the macropore are large in relation to the lateral infiltration losses into the matrix due to the prevailing capillary pressure gradient. This is most likely to be the case in clay soils with a slowly permeable matrix. These lateral losses can be further restricted by relatively impermeable interfaces between macropores and the bulk soil, including clay linings on aggregate surfaces (cutans), and organic linings in biotic pores (42). Thus, macropore flow can sometimes be significant, even in lighter textured soils of large matrix hydraulic conductivity. Nothing is known about the relative significance of finger flow, heterogeneous flow, and macropore flow for leaching of agrochemicals. However, intuitively, macropore flow ought to be the most important process, for two main reasons: macropores are ubiquitous, and the transport volume (often fractions of 1% of the soil volume) is appreciably smaller, which should give shorter transit times and minimal adsorption interaction with the matrix. Thus, in a numerical simulation study based on field experiments, the transit time for a nonadsorbed chemical through a sandy vadose zone was predicted to be four times faster in the presence of heterogeneous flow (43). From observed flow velocities (1), we may expect macropore flow to decrease the transit time through the unsaturated zone by up to two orders of magnitude (i.e., hours instead of years). On the other hand, macropore flows are clearly highly intermittent, whereas heterogeneous flows in the matrix are more or less continuous. EXPERIMENTAL APPROACHES A wide range of experimental techniques have been employed to characterize and quantify preferential flow processes in soils (see ‘‘Suggestions for Further Reading’’). Broadly, these techniques can be divided into the use of dyes to stain flow pathways, morphometric and related methods that quantify soil structure at the pore scale, and tracer and leaching experiments in soil columns (monoliths, lysimeters) or in the field. Dye tracing experiments produce visible qualitative evidence of preferential flow, and they have been widely used to identify flow patterns and their relation to soil profile and horizon characteristics, water application methods, tillage treatments, and other management practices (5,21,22,44,45). In recent years, progress has

1007

been made in quantifying the observable dye patterns in terms of two-dimensional concentration profiles using image analysis of photographs taken with near-infrared film (46). This kind of methodology should expand the range of applications of dye tracing studies to include quantitative testing of models, thereby enabling the elucidation of processes (47). Micromorphological techniques have been used since the 1970s to characterize macropores and soil structure in soil thin sections (48,49). Application of tracers to the soil prior to sectioning is necessary to distinguish functioning or conducting macroporosity from ‘‘dead-end’’ or nonconnected macropores (50). One major disadvantage with these methods is that the samples represent small two-dimensional sections, so that it may be difficult to draw conclusions concerning the connectivity of larger pores and the significance of the observed pore structures for preferential flow at larger scales. However, methods have been recently developed that allow quantitative estimates of pore connectivity to be made from serial sections (51). Macromorphometric imaging techniques (52,53) have been developed to study macropore structures at the soil horizon scale. Plaster of paris casting has occasionally been used as a direct field method to obtain information on the sizes, shapes, and connectivity of larger soil macropores (54). Computed tomography (CT) scanning, originally developed in the medical sciences, has in recent years been increasingly used for the nondestructive analysis of macropores in undisturbed soil columns (55,56). The advantage of such nondestructive techniques lies in the ability to generate two- and three-dimensional quantitative information on the shape, continuity, and size distribution of soil macropores in relatively large undisturbed soil samples. To distinguish total macroporosity and conducting macroporosity, X-ray absorbing tracers such as iodide can be infiltrated into the soil column. Recently, images obtained from CT scanning combined with solute breakthrough experiments have been used to develop and test pore scale models of water flow and solute transport (57). Leaching experiments are widely used to identify the extent of preferential flow and transport, and to determine model parameters by calibration or inverse modeling. Broadly, two types of experiment have been carried out: 1) long-term transient tests under field conditions, either by monitoring tile drainage flows (58,59) or by measurements in field lysimeters (60), and 2) short-term steady-state ‘‘breakthrough’’ experiments (often under saturated conditions) in the laboratory (61). With respect to column and lysimeter experiments, it is essential that undisturbed soil monoliths are used, because the natural soil structure is destroyed by sieving and repacking soil, and this effectively eliminates macropore flow (11). Saturated flow experiments on undisturbed laboratory columns may yield detailed information on solute transport characteristics, but they cannot be used to investigate preferential water flow because it is a transient unsaturated flow phenomenon. Furthermore, simple saturated steady-state flow experiments may not always yield sufficient information to properly characterize preferential solute transport processes. In

1008


this situation, flow interruption methods have been successfully used to identify the extent of nonequilibrium in solute concentrations (61,62). In these experiments, the steady water flow is interrupted for a short period (usually a few days), which allows diffusion equilibration to occur. On restarting the flow, the concentrations in the outflow display a sharp discontinuity, the size of which reflects the rate of solute diffusion between pore regions. FACTORS AFFECTING THE IMPACT OF PREFERENTIAL FLOW ON LEACHING Soil and Vadose Zone Properties Macropore flow strongly depends on both soil texture and structure. The smaller hydraulic conductivity in the matrix of clay soils compared with loamy soils and sands means that soil water pressures exceeding the water entry pressure of larger macropores are attained much more frequently, even for low intensity rainfalls. A variety of factors related to structural development, such as macropore network continuity, the size of structural units, wall roughness, and the strength of structure development (including the presence of cutans on aggregate surfaces), have all been shown to influence the strength of macropore flow. For example, early work on aggregated finetextured soils showed that strongly developed structures resulted in more pronounced macropore flow than did weaker structures (63–65). More recently, in bromide leaching experiments, preferential flow was found to be more significant in well-structured clay-enriched subsoil horizons, compared with weakly structured ploughed and bleached (eluvial) horizons (66). In field dye tracing studies, marked preferential flow behavior was found in 13 out of 14 Swiss agricultural soils and the observed flow patterns were shown to be strongly correlated with the texture and visible structural development in individual horizons and profiles (5). In outdoor lysimeter experiments, leaching of isoproturon was greater from both a structured clay loam and a medium loam soil than from a structureless sandy soil and a weakly structured light loam soil (67). In contrast, nonstructured sands may show larger leaching for very mobile compounds that move easily through the bulk soil matrix with little retardation (68). These interactions between soil and agrochemical properties are discussed in more detail in a later section. Most work on preferential flow and transport has either been performed by soil scientists in surface horizons, or by hydrogeologists investigating contaminant transport in saturated fissured rock formations. Comparatively few studies have investigated the extent of preferential solute transport occurring in the deeper unsaturated vadose zone below the rooting depth of crops. In many cases, the extent of preferential flow may diminish with depth in the vadose zone, because structural development generally becomes weaker in the absence of biotic macropores and physical processes that generate structure such as wetting and drying and freezing/thawing. Li et al. (69) investigated the movement of water, bromide, and adsorbing red and

blue dyes through the Bt (illuvial or clay enriched) horizon of a clay loam soil and the underlying weathered rock (saprolite). They found pronounced macropore flow and transport in the structured Bt horizons, whereas transport in the saprolite occurred largely through the matrix pores, with little preferential movement, even though some structural features (infilled fissures) inherited from the parent rock were visible. However, in other widespread hydrogeological formations, such as glacial clayey tills, fracture flow has been demonstrated to be continuous to great depth during periods of seasonal saturation and is the dominant mechanism of contaminant transport toward underlying aquifers (70–72). In the deep unsaturated fissured chalk vadose zone that overlies important drinking water aquifers in southern England, isotope profiling combined with investigations of rock hydraulic properties indicate that at some locations, recharge processes are dominated by flow in the highly porous chalk matrix with steady downward percolation rates of c. 0.5 to 1 m/year, whereas at other locations, fissure flows during periods of heavy rain seem to contribute significantly to the recharge (73–75). The key factors determining whether recharge is either matrix dominated or influenced by fissure flow were shown to be the saturated hydraulic conductivity of the chalk matrix (of the order of 0.5 to 5 mm/day) and the depth of soil overlying the chalk (75), because a deeper soil cover results in the dissipation of macropore flows before they reach the fissured chalk. However, the significance of preferential flow in the root zone should not be underestimated even if matrix processes dominate transport from the base of the root zone to the groundwater. This is because the attenuation of agrochemical transport by sorption and degradation is generally much weaker in deep vadose zones and groundwater (76,77). The properties of macropore linings and aggregate surfaces (cutans) are very different than those of the bulk soil. Macropores represent microsites in the soil with larger clay and organic carbon contents, better nutrient supply and oxygen status, and larger microbiological activity. All of these factors generally contribute to a larger sorption and degradation capacity per unit mass of soil (78–81). The extent to which these differences may also be important for the leaching of agrochemicals in the presence of macropore flow is not well understood, and this is likely to be an important research topic for the future. Much depends on the characteristic time scales of different processes. In breakthrough experiments for individual earthworm channels, concentrations of atrazine and metolachlor in the effluent were reduced by 20% and 50%, respectively, even at macropore flow velocities of c. 1.5 m/hour (79). Nevertheless, despite higher adsorption capacities per mass of soil, agrochemical transport in macropores usually shows smaller sorption retardation than does the bulk soil (47), perhaps because the surface area per volume of flow pathway is small, or due to kinetic sorption effects, or both. Because degradation is relatively slow in comparison to both macropore flow and sorption, enhanced microbial degradation in macropore microsites may not be important, although some preliminary studies seem to suggest otherwise (80,82).


We should expect finger flow to occur in homogeneous very narrowly graded sands. This is because such soils are characterized by a very steep portion of the soil water characteristic on the primary wetting curve close to saturation. This means that small differences in water pressure close to saturation can result in very large differences in water saturation, hydraulic conductivity, and flux rates. Steep wetting curve water retention functions close to saturation are also characteristic of soils exhibiting water-repellent behavior (33). Finger flow should be much less common in heterogeneous, wettable, poorly sorted materials with a broad pore-size distribution, because, in such soils, lateral dispersion cannot easily be prevented. In these kinds of soils, heterogeneous flow can be expected instead. Climate Differences in long-term average recharge to groundwater in contrasting climates are important for leaching of agrochemicals, both with and without preferential flow. For example, in lysimeter experiments where the amount of ‘‘rainfall’’ was adjusted by artificial irrigation, leaching from a sandy loam soil consistently increased with total rainfall, whereas the pattern of leaching from a structured clay loam soil was more complex, being greater in both very wet and dry rainfall climates (83). In wet climates, macropore flow clearly will be generated more frequently, whereas presumably in very dry conditions, the macropore system in the soil is better developed with shrinkage cracks contributing proportionally more to the flow. A similar effect was noted for dichlorprop leaching in lysimeters containing clay and peat soils (84). For preferential flow mechanisms occurring in the matrix of sandy soils, such as heterogeneous flow and finger flow, leaching may be less affected by the rainfall climate, and it may even actually increase at smaller groundwater recharge rates, because as input rates increase, more of the soil matrix becomes wetted, and the uniformity of the flow pattern increases (43). In a field experiment on a loamy sand soil (85), no significant differences were found in the extent of preferential flow caused by four different irrigation methods (flood irrigation, sprinkler, continuous, or intermittent). The extent of leaching in the presence of macropore flow depends not only on total rainfall, but also perhaps more importantly on rainfall distribution and intensity. It is almost self-evident that increases in rainfall intensity will enhance macropore flow, because the soil water pressures attained during rainfall will be closer to saturation (and may even reach saturation if the intensity is greater than the saturated conductivity of the soil). This means that larger macropores, which tend to be less tortuous and fewer in number, will conduct water, which in turn will lead to a faster effective pore water velocity. Many experiments have demonstrated that higher rainfall intensities usually lead to greater bypass flow in macropores and enhanced leaching of tracers and agrochemicals (64,86–90). The timing of rainfall events in relation to pesticide application is especially critical for soils exhibiting preferential flow. For surfaceapplied pesticides, the resident concentrations will be

1009

very large in the first few millimeters of soil immediately after application. Heavy rain soon after application may therefore lead to large leaching losses (91), because before flowing into macropores at the surface, incoming water interacts by diffusion and physical mixing (‘‘rainsplash’’) with the resident soil water at and close to the soil surface (92). Conversely, leaching can be considerably reduced, either if dry weather follows application, so that sufficient time is allowed for the pesticide to diffuse away from the soil surface, or if the chemical is ‘‘washed’’ into the soil matrix by one or more light rain showers that do not generate macropore flow (89,93,94). Once the bulk of the compound has penetrated into the matrix away from the soil surface, it is no longer so readily exposed to macropore flow (91), because the micropore volume is much larger than the volume of macropores, and very slow diffusion toward macropores becomes rate-limiting. Agrochemical Properties The impact of preferential flow on solute transport depends strongly on the nature of the chemical under consideration, particularly its sorption characteristics, and the nature of any source/sink terms, including biological transformations, which affect the transport process (i.e., whether the solute is surface-applied and consumed in the soil, or whether it is produced within the soil). Because agrochemicals have a very wide range of sorption and degradation characteristics, we can expect the impact of preferential flow on leaching to vary widely from compound to compound. In principle, the occurrence of preferential flow should significantly increase the leaching of otherwise ‘‘nonleachable’’ (i.e., strongly sorbed or fast degrading) compounds, whereas it will have little effect on highly mobile and persistent chemicals (95). Indeed, in some cases, preferential flow may actually decrease agrochemical leaching. For example, leaching of the highly mobile compound bentazone to tile drains in a structured clay soil was reduced by approximately 50% due to macropore flow (59). This is because, after the first few weeks following application, the bulk of the compound was stored in the soil matrix and, therefore, not exposed to water flowing in macropores, instead moving downward through the matrix at a reduced effective transport velocity. However, for most compounds registered for use, preferential flow will certainly increase leaching, because most highly leachable compounds will be denied registration (95). Exceptions to this rule are those compounds for which high leaching fractions due to weak sorption or long half-lives may be acceptable simply because the dose is very low (e.g., sulfonylurea herbicides). One consequence of the differential effects of preferential flow on inherently ‘‘leachable’’ and ‘‘nonleachable’’ compounds is that the differences in leaching losses between agrochemicals of widely differing properties should be significantly reduced in the presence of preferential flow. Thus, in one simulation case study, two compounds that showed a 100-fold difference in leaching in the absence of macropore flow were predicted to have only a four-fold difference in the presence of macropore flow (95). These model predictions are also supported by the results of

1010


field experiments where leaching in the presence of preferential flow has been monitored for several compounds of different chemical characteristics applied simultaneously (58,96,97). These studies often show an equally fast transport of agrochemicals irrespective of sorption characteristics, but that concentrations (and therefore loads) are clearly dependent on sorption. Thus, although it is not true to say that preferential flow completely overshadows the effects of compound properties on leaching, the significance of compound properties is certainly reduced. Management Soil tillage affects the total macroporosity, size distribution of large pores, and their continuity. Different tillage implements and systems affect soil structure differently, and this affords possibilities to ‘‘manage’’ macropore flow to limit leaching losses. For example, a considerable body of experimental data confirms that macropore flow is usually more pronounced under reduced tillage systems compared with conventional tillage management, and that this can significantly increase agrochemical leaching (91,93,98–101). In one study, significantly greater preferential water flow and bromide transport under notill compared with four other tillage systems was found at high irrigation rates, but not at medium and low rates (30%, higher tier tests can be conducted under more realistic conditions of exposure in the field. Semi-field tests are conducted with single species released into cages or enclosures, while field tests use naturally occurring beneficial arthropod populations under normal agronomic conditions. At each stage, the product may be classified as ‘‘harmless’’ (in which case, no further testing is necessary) or assigned to one of the ‘‘harmful’’ categories according to the level of effects seen. For IPM purposes, this testing scheme has been effective as it produces a ranking of effects at the different levels of testing for a range of beneficial arthropods, thus allowing a comparison to be made among different products. This sequential or tiered testing approach has also formed the basis of the nontarget arthropod regulatory schemes that have been developed. REGULATORY TESTING PROCEDURES The assessment of the effects of agrochemicals on nontarget arthropods as part of the regulatory process has been largely confined to European countries. Prior to 1990, only a few countries required any information to be generated in this area as part of their national regulatory requirements for pesticides. In Germany, the Biologische Bundesanstalt ¨ Land und Forstwirtschaft (BBA) established a workfur ing group to test the effects of plant protection products against beneficial arthropods in 1970 (27). Such testing became an obligatory part of the approval procedure in Germany in 1989. This testing was based on the sequential approach, starting in the laboratory and moving where necessary through to semi-field and field testing (28). Testing was conducted according to BBA guidelines (Series VI) as well as a number of the IOBC test methods. In the United Kingdom, more specific concerns were raised by the Pesticides Safety Directorate, e.g., assessing the acceptability of the use of dimethoate and synthetic pyrethroids on summer cereals (29). More recently, requirements about the effects of agrochemicals on nontarget arthropods have become a requirement for the evaluation and authorization of agrochemicals according to the Uniform Principles of the European Union (EU) (30,31). These are now implemented in all member countries of the EU. RECENT DEVELOPMENTS IN REGULATORY TESTING ESCORT 1 The Uniform Principles of Council Directive 91/414/EEC refers for specific guidance on risk assessment and testing for nontarget arthropods to the scheme of EPPO/Council of Europe (CoE) (32) and on the guidance document of the first European Standard Characteristics of Beneficials Regulatory Testing (ESCORT) workshop, Wageningen (33). This workshop was organized by BART, EPPO/CoE, and IOBC, in conjunction with the Society of Environmental Toxicology and Chemistry

NATURAL ENEMIES

(SETAC)-Europe and with the support of the European Commission. The regulatory procedure laid down in Council Directive 91/414/EC is divided into two stages: 1) evaluation of the active substance under Annex II for inclusion into Annex I of the Directive and 2) additional testing of a lead formulation for authorization of products containing the active substance in EU Member States in accordance with Annex III. The EPPO/CoE nontarget arthropod risk assessment scheme can be subdivided to meet these requirements. Initially, under Annex II, two sensitive species and two crop relevant species are tested in IOBC Tier 1 tests. Due to the technical requirements of the available methodology, this testing is conducted using a lead formulation as representative of the active substance. The ESCORT guidance document provides a list of 13 possible test species. These are categorized according to crop relevance (orchard/greenhouse/forest/vineyard and arable crops) and functional grouping (parasitoids, predatory mites, ground-dwelling predators, and foliagedwelling predators). Where significant effects are seen in the Annex II tests, i.e., exceeding the 30% threshold (taken from the IOBC ‘‘harmless’’ categorization), additional testing on a further two crop relevant species is initially required under Annex III. In addition, further testing is required as part of a sequential testing scheme as proposed in the EPPO/CoE scheme. This follows a tiered path through extended laboratory, semi-field, or field tests, as appropriate. Test methodology and the principles of testing are outlined in the ESCORT guidance document. The data generated in these various levels of testing are classified in the EPPO/CoE scheme according to four categories: 1. negligible risk (no exposure) 2. low risk (effects 25% in higher tier tests and dependent on the results of field trials) In order to facilitate the risk assessment, the ESCORT guidance document identifies three different situations that should be taken into account when assessing the acceptability of effects: 1. Within-crop nontarget arthropods, where IPM is not practiced. These species are normally subjected to perturbation through agricultural practices, but it is recognized that there needs to be some limit to the impact of pesticides. 2. Within-crop nontarget arthropods, where IPM is practiced, and so it is necessary to maintain the natural control capacity. 3. Off-crop nontarget arthropods, which increase species diversity, provide food to other nontarget species, and provide a reservoir to aid the recovery of affected in-crop populations. At each level of testing, the risk assessment assesses the acceptability of any significant effects identified, taking

1087

into account these different situations. This may conclude that further testing is necessary or that the risk can be managed through appropriate label statements. Guidance is given on these label statements, with suggested references (e.g., to the EPPO risk categories, major taxonomic categories, crop groupings, applicability to IPM) and for appropriate use restrictions (e.g., buffer zones). ESCORT 2 After about 5 years of implementation, a number of limitations were identified with ESCORT 1. 1) The scheme does not precisely discriminate among in-field, off-field, and IPM situations. 2) The trigger value for Tier 1 data is inappropriate as it produces too many false positives. 3) The available guidance does not clearly define the data requirements (in terms of methodology and evaluation), especially for higher tier studies. 4) The data generated under ESCORT 1 do not allow a satisfactory risk assessment for in-field and off-field habitats. 5) The risk assessment scheme requires excessive testing compared with other nontarget groups under Council Directive 91/414/EEC. 6) New proposals on risk management have been developed since the ESCORT 1 workshop. Accordingly, a second ESCORT workshop was held on the same basis as the first one (jointly organized by BART, EPPO/CoE, OECD, and IOBC and in conjunction with SETAC Europe and the EC). Thus, 53 invited scientists representing government, industry, and academia, mainly from the EU but with some North American representation, met in Wageningen, The Netherlands, in March 2000 (34). The aim was to develop updated regulatory guidance for terrestrial nontarget arthropod testing and risk assessment for pesticides, consistent with a revised draft EPPO nontarget arthropod scheme and addressing the problems identified with ESCORT 1. It is clearly recognized that the ESCORT 2 guidance document is concerned with regulatory testing and risk assessment and not with the assessment of the suitability of pesticides for IPM (although some of the data used may be the same in both cases). At the Tier 1 level, two standard sensitive species are tested, the predatory mite, Typhlodromus pyri, and the aphid parasitoid, Aphidius rhopalosiphi. Sensitivity analyses of available test species have shown this to be sufficient (35,36). As before, testing is carried out with an inert substrate but now generates dose response data based on a lethality endpoint (generating LR50 values). An option is included for a limit test, where no insecticidal activity is expected, and specific exceptions are identified for products with special modes of action, e.g., seed treatments, solid formulations, and insect growth regulators. For the Tier 1 risk assessment, the 30% effect threshold is replaced by a Hazard Quotient (HQ), which is derived from the maximum application rates or appropriate drift rates for in-field and off-field assessments, respectively, and the LR50 values. For products that have two or more applications in a season, a multiple application factor (MAF) is applied. For the off-field assessment, additional factors are included to take into account the uncertainty in extrapolating to off-field species diversity and to produce appropriate drift rates for the terrestrial

1088

NATURAL ENEMIES

environment. Trigger values were initially set on the basis of an empirical comparison of hazard quotients and field effects for known pesticides: 12 for T. pyri and 8 for A. rhopalosiphi (37). However, these were subsequently reduced to 2 for both species to take into account uncertainties in this validation exercise (due to the limited information currently available), although it was recognized that they could be revised when more data become available. The Tier 1 risk assessment thus considers HQ values for both standard species, and taking into account both the in-field and off-field habitats, it determines the extent of any risk and the nature of any higher tier testing necessary. Higher tier testing follows the typical sequential pattern of extended laboratory, semi-field, and field testing applied as appropriate to the specific concerns of each assessment (i.e., in-field or off-field risk). At the extended laboratory and semi-field level, additional species are required, one if only the in-field HQ values is exceeded and two if the off-field HQ value is also exceeded. For this purpose, the following species are recommended: Orius laevigatus, Chrysoperla carnea, Coccinella septempunctata, and Aleochara bilineata. A trigger value for lethal or sublethal effects of 50% after exposure of the test organisms to fresh or aged residues is applied to both in-field and off-field assessments. Validated laboratory test methods for these and other species have been developed by the Joint Initiative of IOBC, BART, and EPPO (38). For semi-field and field testing, the recommendations of the Joint Initiative document on ‘‘Principles for regulatory testing and interpretation of semi-field and field studies with nontarget arthropods’’ (39) was adopted. In particular, it was noted that the objective of field trials is to assess any effects on one or more taxa, as appropriate, using naturally occurring populations. The acceptability of effects is not based on fixed trigger values for acceptability of effects; rather it takes into account a range of factors, e.g., persistence of effect, range of taxa involved, and the recovery of populations affected. RISK MANAGEMENT STRATEGIES In the ESCORT 2 guidance document, it is recognized that the question of appropriate risk management strategies for nontarget arthropods, where identified as necessary by the risk assessment, should be left to individual Member States. Factors such as the local habitat types, regional environmental conditions, and specific agricultural practices will influence the approaches to risk management considered appropriate. Some general guidance is given with regard to possible risk management options for in-field and off-field areas, which need to be addressed separately. In addition, specific proposals for national risk management policies have been presented, e.g., for the United Kingdom (41) and Germany (42). In the U.K. scheme (implemented into the working procedures of the Pesticides Safety Directorate), pesticides

that pass the appropriate Tier 1 trigger values are not classified or labeled with respect to nontarget arthropods. If the Tier 1 trigger values are not met, further data are usually submitted or requested and on the basis of this, appropriate labeling is applied. This may be accepted or additional higher tier (semi-field or field) data can be used to present a case for reducing or removing the risk management and labeling requirements. Factors that can be used in assessing the acceptability of effects seen in field studies are identified, e.g., application details; specificity, intensity, and duration of effects; comparative effects with soft and toxic reference substances; and information about the crop; and pest/disease to be controlled. The risk label proposal identifies two categories: ‘‘high risk to non-target insects or other arthropods’’ and ‘‘risk to non-target insects or other arthropods’’. The latter is applied to all application situations, whereas the high-risk label applies only to ‘‘high-risk’’ arable and tractor-mounted spray boom applications, identified by consideration of a number of factors, such as level of risk, crop, acreage, application type and timing, economic viability, and agronomic implications. For arable and tractor-mounted spray boom applications as well as for solid-based products (e.g., pellets and granules), where a pesticide is classified as a risk to nontarget insects, an advisory restriction is applied (‘‘Avoid application within 6 m of the field boundary’’). For pesticides with specific effects on certain nontarget arthropods, there may be additional management options, including timing or method of application as well as, or instead of, a buffer zone. A high-risk label attracts a statutory restriction (‘‘Do not spray within 6 m of the field boundary’’), the only example given being for summer cereals. In the case of broadcast air-assisted spray applications (e.g., orchards, hops, and grapes), it is recognized that a buffer zone is not a practical restriction. In this case, a risk classification would result in the advisory phrase ‘‘The best available application technique, which minimizes of-target drift should be used to reduce effects on nontarget insects or other arthropods’’. For handheld spray applications identified as a risk to nontarget arthropods, it is considered that the use is localized, and so does not represent a major risk. Here the appropriate advisory phrase is given as ‘‘Avoid application within 6 m of field boundary to reduce effects on non-target insects or other arthropods’’. In Germany, new risk management measures for nontarget terrestrial organisms have been implemented into the national authorization of plant protection products. Restrictions on use may be imposed as a result of the assessment of risk likely to occur. As a first step, risk mitigation is based on the use of the most appropriate application technique available to reduce spray drift to a level that is safe for nontarget arthropods and plants. In these cases, the use of spray drift–reducing equipment (with 50% to 90% reduction) must be used in a strip of at least 20 m to adjacent areas except for specific exceptions (e.g., agricultural or public areas).

NATURAL ENEMIES

Where drift-reducing equipment is not available, a second risk management option is the use of a 5 m buffer zone where this does not interfere with the principles of good agricultural practice (e.g., the requirements for plant health or protection). A third risk management group is used where drift-reducing equipment or a buffer zone of 5 m alone do not provide the level of protection required. In these cases, both the drift-reducing techniques and a buffer zone must be used, again provided they do not interfere with the principles of good agricultural practice. However, factors affecting the magnitude of the risk at a local landscape level also may be taken into account. Thus, restrictions are not required if specific agricultural or environmental conditions apply in the proposed area of use: (1) If application is conducted using handheld sprayers (because they produce less spray drift). (2) If adjacent areas (field margins hedges etc) are less than 3 m wide (due to lower biodiversity and especially to avoid eradication of these margins by farmers). (3) Application in areas where there is sufficient abundance of natural and seminatural habitats. Overall, it is considered that this approach is based on current technical progress and is both economically acceptable and ecologically effective.

BIBLIOGRAPHY 1. W. E. Ripper, Annual Review of Entomology 1: 403–438 (1956). 2. B. A. Croft and A. W. A. Brown, Annual Review of Entomology 20: 285–335 (1975). 3. B. A. Croft, Arthropod Biological Control Agents and Pesticides, John Wiley and Sons, New York, 1990. 4. P. C. Jepson, Pesticides and Non-Target invertebrates, Intercept, Wimborne, UK, 1989. 5. P. C. Jepson, in P. Calow, ed., Handbook of Ecotoxicology, Vol. 1, Blackwell Science, Oxford, UK, 1994, pp. 299–325. 6. M. W. Johnson and B. E. Tabashnik, in T. S. Bellows and T. W. Fisher, eds., Handbook of Biological Control, Academic Press, San Diego, CA, 1999, pp. 297–317. 7. K. M. Thieling and B. A. Croft, Agriculture Ecosystems and the Environment 21: 191–218 (1988). 8. T. S. Bellows and T. W. Fisher, Handbook of Biological Control, Academic Press, San Diego, CA, 1999. 9. J. A. Pickett, Philosophical Trans. Roy. Soc. Lond. B 318: 203–211 (1988). 10. B. A. Croft, and M. E. Whalon, Entomophaga 27: 3–21 (1982). 11. L. D. Newsome, et al., in C. B. Huffaker and P. S. Messenger, eds., Theory and Practice of Biological Control, Academic Press, New York, 1976, pp. 565–591. 12. W. E. Ripper, Publications of the Entomological Society of America 2: 153–156 (1959). 13. J. E. Cranham, et al., in D. A. Griffiths and C. E. Bowman, eds., Acarology VI, Vol. 2, Ellis Horwood, Chichester, 1984, pp. 690–685.

1089

14. R. L. Metcalf, in M. Kogan, ed., Ecological Theory and IPM in Practice, Wiley Interscience, New York, 1986, pp. 251–292. 15. J. K. Waage, in P. C. Jepson, ed., in Pesticides and Non-target Invertebrates, Intercept, Andover, 1989, pp. 81–93. 16. N. J. Aebischer, Functional Ecology 4: 369–373 (1990). 17. N. J. Aebischer and G. R. Potts Proceedings of the BCPC Conference, BCPC Publications, Thornton Heath, Surrey, UK, 1990, pp. 163–173. 18. A. J. Burn, in P. Greig-Smith, et al., eds., Pesticides and the Environment: The Boxworth Study, HMSO, London, UK, 1992, pp. 110–131. 19. T. N. Sherratt and P. C. Jepson, J. Appl. Ecol. 30: 696–705 (1993). 20. N. M. Van Straalen and J. P. Van Rijn, Reviews of Pesticide Contamination and Toxicology 154: 83–141 (1998). 21. J. E. Cohen et al., J. Appl. Ecol. 31: 747–763 (1994). 22. W. H. Settle et al., Ecology 77: 1975–1988 (1996). 23. IOBC/WPRS Bulletin, XI/4, Working Group, Pesticides and Beneficial Organisms, Guidelines for testing the effects of pesticides on beneficials: short description of test methods, 1988. 24. IOBC/WPRS Bulletin, XV/3, Working Group, Pesticides and Beneficial Organisms, Guidelines for testing the effects of pesticides on beneficial organisms: description of test methods, 1992. 25. L. Samsoe-Petersen et al., Z. Pflanzenkrankh. Pflanzensch. 96: 289–316 (1989). 26. G. Sterk et al., BioControl (formerly Entomophaga) 44: 99–117 (1999). 27. D. Von Brasse, Nachrichtenbl. Deut. Pflanzenschutzd. (Braunschweig) 42: 81–86 (1990). 28. D. Von Brasse and H. Rothert, Abteilung fur ¨ Pflanzenschutzmittel und Anwengdungstechnik, Mitt. Biol. Bunde¨ Land- und Forstwirtsch, Berlin-Dahlem, Heft sanstalt. fur 285, 1993. 29. Anon., Data Requirements for Approval under the Control of Pesticides Regulations 1986. Working Document 7/7: Guideline to Study the Within-Season Effects of Insecticides on Beneficial Arthropods in Cereals in Summer, 1986. 30. Council Directive 91/414/EEC (15 July 1991): Concerning the placing of plant protection products on the market. Official Journal of the European Communities, L 230, 19 August 1991, pp. 1–31. 31. Council Directive 94/43/EC (27 July 1994): Establishing Annex VI to Directive 91/414/EEC concerning the placing of plant protection products on the market. Official Journal of the European Communities, L 227, 1 September 1994, pp. 1–55. 32. OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 24: 17–35 (1994). 33. K. L. Barrett et al., eds., Guidance Document on Regulatory Testing Procedures for Pesticides with Non-Target Arthropods. From the ESCORT Workshop (European Standard Characteristics of Beneficial Regulatory Testing), Wageningen, The Netherlands, March 1994, 28–30. 34. M. P. Candolfi et al., eds., Guidance Document on Regulatory Testing and Risk Assessment Procedures for Plant Protection

1090

NATURAL PRODUCT PESTICIDES

Products with Non-Target Arthropods. From the ESCORT II Workshop (European Standard Characteristics of nontarget arthropod Regulatory Testing), Wageningen, The Netherlands, 21–23 March 2000. SETAC Office. 35. M. Candolfi et al., Chemosphere 39: 1357–1370 (1999). 36. H. Vogt, IOBC Bulletin 23: 3–15 (2000). 37. P. J. Campbell et al., J. Pest Science 73: 117–124 (2000). 38. M. P. Candolfi et al., eds., IOBC/WPRS Guidelines to evaluate side-effects of plant protection products to non-target arthropods; IOBC, BART and EPPO Joint Initiative. IOBC/WPRS, Germany, 2000. 39. M. Candolfi et al., J. Pest Science 73: 141–147, 2000. 40. P. J. Campbell, in P. T. Haskell and P. McEwen, eds., Ecotoxicology, Pesticides and Beneficial Organisms, Conference Proceedings, Cardiff, UK, 1996, Kluwer Academic Publishers, 1998, pp. 232–240. 41. R. Forster et al., IOBC Working Group, ‘‘Pesticide and Beneficial Organisms,’’ Meeting, San Michele, Istituta Agroria, Italy, October 2001.

NATURAL PLANT REGULATORS Natural plant regulators are chemicals produced by plants that have toxic, inhibitory, stimulatory, or other modifying effects on the same or other species of plants. Some are termed plant hormones or phytohormones (1). BIBLIOGRAPHY 1. EPA Pesticide Assessment Guidelines Subdivision M: Part A Microbial. EPA No. 540/09-89-056, March 1989; USEPA; Washington, DC; 1989.

NATURAL PRODUCT PESTICIDES ´ UJVARY ´ ISTVAN

Hungarian Academy of Sciences Budapest, Hungary

During their evolution, living organisms have developed a broad array of defense strategies often involving complex and dynamic chemistries that assure survival, coexistence, or in some cases, territorial dominance of the species and the individual. These chemicals, also labeled as ‘‘secondary metabolites,’’ indicating their yet largely unknown functions in the producing organism, offer extraordinary diversity both in their chemical structure and biological activity. Since prehistoric times, people in all parts of the world have exploited natural products in one form or another in curing diseases and fighting pests that endanger their health or compete for their food. The importance of natural products faded with the maturity of the synthetic chemical industry by the middle of the 20th century, but there has been a resurgence of interest in these materials that is due as much to ecological as to economic reasons. Environmental concerns, the appearance of resistance to many widely used crop protection agents, and the

emergence of new pests and diseases continue to fuel the search for new chemical entities, preferable with new modes of action. Improvements in separation and analytical techniques, complemented by biochemical and often receptor-based assay methods and high throughput screening techniques developed in the late 1980s now allow ready isolation and structure identification of bioactive constituents of plants and other terrestrial or marine organisms. The sources of natural products are usually renewable, and breeding or genetic engineering often yields strains producing substances originally obtained in economically unacceptable low yield. Moreover, directed biosynthesis using non-natural precursors (e.g., amino acid analogs) that are incorporated into the final molecule can give rise to novel and complex structures that otherwise would be unattainable by chemical synthesis. In addition, these biologically produced complex substances can be utilized by industry as feedstocks for novel semisynthetic products with special biological properties. Serendipitous discovery by random screening of extracts from field or botanical garden collections, soil samples, microbial fermentation broths, and other natural sources is complemented by more rational tests of organisms based on available ethnobotanical or (chemo)taxonomic information. The biologically active fractions obtained from natural sources are usually blends of metabolically related compounds with differing activity profile. Often, both the mixture and its components display an array of activities against various organisms and can affect multiple targets (e.g., membranes, various enzymes, or DNA) that, from a practical point of view, could be advantageous in pesticide resistance management. Nevertheless, the joint effect of minor or even trace constituents of a crude preparation can complicate the biological evaluation. Natural products, even those having no evident agricultural relevance, can often contribute to the understanding of essential life processes and the mode of action and selectivity of agrochemicals. A legendary example is the elucidation of the physiological mode of action of the neurotoxic alkaloid physostigmine (1, Fig. 1) and the clarification of the neurophysiology and biochemistry of acetylcholine (ACh). Natural products can be used either directly in pest control or can serve as models (lead compounds) for the development of new synthetic analogs with favorable biological and physicochemical properties. For direct use in agriculture, a natural product should be 1) sufficiently efficacious against target species, 2) safe and selective, 3) environmentally stable, 4) standardized for composition and formulation, and 5) readily available. If these criteria are not met, appropriate structural modifications guided by structure-biological activity relationship studies, often using computer-aided molecular modeling methods, can afford a marketable pest control agent. The inspection of the ‘‘chemical evolution’’ from a ‘‘failed’’ natural product to a commercial pest control agent is instructive in our understanding of how various structural changes affect

NATURAL PRODUCT PESTICIDES O O

N H

N H

N

(1)

O O

O N

O

N H O

N+ (2) O O

(3) O

O P

O

O

O P

O

H N H (4)

O

O (5)

Figure 1. Representative natural and synthetic acetylcholinesterase inhibitors: physostigmine (1), neostigmine (2), carbofuran (3), monocrotophos (4), and CGA 134 736 (5).

biological activity, metabolism, environmental behavior, and, ultimately, selectivity. Although, as mentioned, natural products can affect several different organisms, they can be categorized according to their main use as follows: 1) insect control agents; 2) weed control agents, including plant growth regulators; 3) disease control agents, including fungicides and bactericides; 4) nematicides; and 5) rodenticides. INSECT CONTROL AGENTS There is a large number of insecticides, either commercial or structural prototypes, obtained from plants, microorganisms, or other natural sources, including specific insect control agents making use of the insects’ unique hormonal regulatory system (Table 1). Botanical Insecticides Because of their ready accessibility and elaborate biochemistry, terrestrial plants were the earliest natural pest control agents. In practice, flowers, leaves, twigs, bark, and roots of the often home-grown plants or their extractions of various purity are used. Some of the substances are still utilized, and some of them are of historical importance.

1091

Physostigmine or eserine (1, Fig. 1) is obtained from the Calabar bean, the seed of the plant Physostigma venenosum Balf., once used by native tribes of West Africa as an ‘‘ordeal poison’’ (1). The carbamate-type alkaloid was introduced into medicine for glaucoma treatment in 1877. The clarification of its physiological mode of action as an inhibitor of acetylcholinesterase (AChE) prompted the preparation of a series of aryl N,N-dialkylcarbamates, including the synthesis of the water-soluble prototype drug neostigmine (2) in 1931. The natural alkaloid is weakly insecticidal, but subsequent studies with uncharged and lipid-soluble compounds culminated in the discovery of N-methylcarbamate insecticides exemplified by carbofuran (3). The development of organophosphorus neurotoxicants by Schrader prior to and during World War II as potential warfare agents and later as selective insecticides is also associated with this botanical alkaloid. Both insecticide groups act by inhibiting AChE. Interestingly, the synthetic organophosphate insecticides, e.g., monocrotophos (4), also have their natural counterparts, such as the cyclic phosphate 5 isolated from a Streptomyces antibioticus strain (2). Pyrethrum, the most widely used botanical insecticide (3), is a mixture of cyclopropanecarboxylic acid esters, including those of chrysanthemic acid as in pyrethrin I (6, Fig. 2). The insecticidal extract is obtained from the flowers of the Tanacetum cinerariifolium (Trev.) SchultzBip., also known as Chrysanthemum cinerariaefolium Vis. or Dalmatian pyrethrum, cultivated mainly in Kenya. Pyrethrum is a contact neurotoxic insecticide with a rapid action (knockdown). It is remarkably selective, but the photochemical lability of the active ingredients greatly limits its use. Commercial pyrethrum formulations usually contain a synergist (vide infra) to prevent enzymatic detoxification in insects. Despite the lack of field stability and limited availability, the unique potency of the pyrethrins stimulated the development of synthetic analogs, the pyrethroids. Systematic structural modifications led to various photostable synthetic products with a wide range of agricultural applications. The ‘‘evolution’’ of pyrethroids is demonstrated by selected examples, including deltamethrin (7), esfenvalerate (8) lacking the cyclopropane ring, and the nonester-type etofenprox (9) having little structural resemblance to the original natural product (Fig. 2). Rotenone is the bioactive principal of insecticidal preparations from the roots of the plants Derris and Lonchocarpus genera. Pure rotenone is also used in fishery management as a piscicide. It is a classic inhibitor of NADH:ubiquinone oxidoreductase in the mitochondrial respiratory chain (4). Rotenone and its structural relatives have antiproliferative properties in human cancer cells in vitro (5). Preparations obtained from the plants of the Simaroubaceae family are known for their multifaceted biological activities. The major active principles are the bitter, triterpenoid quassinoids having insecticidal, antifeedant, anthelmintic, as well as antimalarial and antitumor properties (6,7). Herbicidal activity for several quassinoids has also been reported (8).

Table 1. Insect Control Agents Common Name

Common Source

Biological Activity Type

Use1

Reference

From Plants Pyrethrum or pyrethrins, e.g., pyrethrin I (6)

Tanacetum cinerariifolium (Trev.) Schultz-Bip.

Na+ channel activator

+++

3

Rotenone

Derris elliptica (Wallich) Benth., Lonchocarpus utilis A. C. Smith

Mitochondrial respiration inhibitor2

+++

4,49

Quassia

Quassia amara L., Picrasma excelsa Planch, Ailanthus altissima (Miller) Swingle

Neurotoxicant, growth regulator, antifeedant2

+++

6,7

Nicotine (10)

Nicotiana tabacum L. and N. rustica L.

Nicotinic ACh receptor activator

#+++

9,10

Ryania

Ryania speciosa Vahl.

Specific Ca2+ channel opener2

+++

11,12

Sabadilla

Schoenocaulon officinale A. Gray


+++

9

Unsaturated isobutylamides

Several Piper species


−#

9,13

Annonaceous acetogenins

Annona and Asimina species


−

15

Juvabione (16)

Abies balsamea (L.) Miller

Growth regulator

−#

regulator2

−

18,19,20

−

21

+++

22

Phytoecdysones, e.g., ecdysone (22)

Various plants and fungi

Insect growth

Precocenes

Ageratum houstonianum Miller

Insect growth regulator

Azadirachtin

Azadirachta indica A. Juss

Growth regulator, antifeedant2

α-Terthienyl

Tagetes erecta L.

Photodynamic2

−#

25,49

1,4-Benzoxazin-3-ones

Gramineae

Allelochemical2

−

26,27,28

Monoterpene essential oils

Various plants

Attractant, repellent, neurotoxic, etc.

+++

23,24

Phytooils and fatty acids

Various plants

Diverse

++

51

Various plants Cucurbita species

Attractant Phagostimulant2

+++ +++

44

Sesamum indicum L.

Synergist by inhibiting oxidative metabolism

#+++

30

Plant kairomones, e.g., Methyl eugenol Cucurbitacins Sesamin (20)

From Microorganisms Avermectins (24–27)

Streptomyces avermitilis

Glutamate-gated Cl− channel activator2

#+++

38,52

Milbemycins

Streptomyces hygroscopicus

Glutamate-gated Cl− channel activator2

+++

52

Polynactins

Streptomyces aureus

Mitochondrial membrane disruptor

Bacillus thuringiensis endotoxins

Bacillus thuringiensis strains

Poration/disruption of midgut membrane

+++

33,34

Bacillus thuringiensis β-exotoxin (thuringiensin)

Bacillus thuringiensis strains

Nucleic acid synthesis inhibitor

+

36,37

Dioxapyrrolomycin

Streptomyces species


#

40

Spinosyns

Saccharopolyspora spinosa Mertz et Yao

Nicotinic ACh receptor activator2

+++

41

53


1092


1093

Table 1. (Continued) Common Name

Common Source


Use1

Reference

−#

9

From Marine Organisms Nereistoxin (13)

Lumbriconereis heteropoda Marenz

Nicotinic ACh receptor activator

Diatomaceous earth

Marine or fresh water algae

Physicochemical (desiccant)

+++

48

Sex pheromones

Common in insects

Attractant

+++

43,45,46

Host kairomones: e.g., phenols, 1-octen-3-ol

Host mammals of flies or mosquitoes

Attractants

+++

44,54

Photorhabdus toxin

Photorhabdus luminescens

Unknown

−

55

From Animals

− = laboratory use only; # = served as model for (semi)synthetic analogues; + = limited or historical use; ++ = under development; +++ = currently used in pest control. Other types of biological activities are also known.

1

2

O

H

H O trans H

O

(6)

Br

H

H

H

O

cis Br

O

O

CN (7)

H

O H Cl

O

O

CN (8)

O

O

O (9) Figure 2. Structural evolution of pyrethroids from pyrethrum: pyrethrin I (6) and its synthetic analogs deltamethrin (7), esfenvalerate (8), and etofenprox (9).

A major chemical group of botanical insecticides are the alkaloids (9). Historically, the most important is the tobacco alkaloid nicotine (10, Fig. 3). This highly toxic compound is usually obtained from waste of the tobacco industry, and its use is mainly confined to small-scale or glasshouse application. A new group of highly selective nicotinoid insecticides are 6-chloropyridinyl derivatives such as imidacloprid (11) and acetamiprid (12), which, like the natural alkaloid, are cholinergic acting on the insect nicotinic ACh receptors (10). Ryania insecticide is the ground stem wood of the tropical tree Ryania speciosa Vahl., and it contains over a dozen of several structurally related alkaloids. Nowadays, this botanical preparation is of minor importance, but one of its major ingredients, ryanodine, is a widely used pharmacological tool in studies of the calcium release channels in sarcoplasmic reticulum membranes of skeletal and cardiac muscle of both insects and mammals (11,12). Veratrum alkaloids, a distinctive group of steroid alkaloids, are the major biological principles of liliaceous plants and have notable hypotensive as well as insecticidal properties. Insecticidal preparations from dried sabadilla seeds are used against thrips in citrus and some minor crops. Veratridine and cevadine, the major components of sabadilla, activate, that is, prevent closure, of the sodium ion channels of excitable membranes. This action is similar to that of pyrethrins, but the alkaloids bind at a separate site. The fruits of the pepper (Piper nigrum L.) and other plants of the Piperaceae as well as the Compositae and Rutaceae families contain pungent, sialagogue, anesthetic, and insecticidal compounds (13). The bioactive principles are unstable N-isobutylamides of polyunsaturated C10 –C18 acids. The naturally co-occurring amides show synergistic properties; i.e., the insecticidal activity of their mixture is higher than the additive effect of the pure ingredients. These lipophilic amides show symptoms characteristic of DDT and pyrethroids, indicating the involvement of voltage-sensitive sodium ion channels. These amides are active against pyrethroid-resistant insects initiating efforts to find synthetic analogs with improved properties (14).

1094


N

H N

S S (13)

N (10)

N N Cl

N H

N NO2

S

O

NH2

(11)

N

O NH2

(14)

N

N Cl

S

N CN

(12)

S

S

S

(15)

factor,’’ the structure of which was identified and named juvabione in 1966 (16; Fig. 4). Interestingly, a Malaysian plant, as part of its defense mechanism against insects, produces a sesquiterpenoid identical with the most abundant natural insect JH, JH III (17) (17). Another group of phytochemicals with IGR activity are the phytoecdysones, steroids having structural resemblance and biological activity similar to that of the ecdysteroids (18). Producing plants, presumably for deterrence, are able to accumulate these steroids in concentrations much higher than are those found in insects (19,20). Certain plant-derived chromenes from Ageratum species, called precocenes, target the JH-producing corpora allata gland of developing insect larvae (21). The resulting precocious—and ultimately fatal—metamorphosis is preventable by the administration of natural JHs or JH mimics. These compounds are also termed anti-JHs or antiallatotropins. Seeds and other parts of the Indian neem tree contain triterpenoid limonoids with multiple biological effects, including insecticidal, growth inhibitory, antifeedant, and oviposition-deterring and fecundity-reducing activity against many insect pests. Neem preparations from the bark, roots, flowers, and seeds of the tree have also been used for centuries for medical, agricultural, hygienic, and cosmetic purposes. The major component responsible for most of the effects on insects is azadirachtin, which was isolated in a pure form in 1968 (22). This

Figure 3. Natural and synthetic insecticides acting on the nicotinic acetylcholine receptor: (S)-nicotine (10), imidacloprid (11), acetamiprid (12), nereistoxin (13), cartap (14), and thiocyclam (15).

H

O

O O

Ethnobotanical observations and wide-scale screening have led to the characterization of other insecticidal alkaloids from Aconitum, Aglaia, Cocculus, Celastrus, Delphinium, Erythrina, Haplophyton, Stemona, and Tripterygium genera, but their current use is confined to the laboratory (9). Preparations from the seeds, leaves, and bark of the tropical Annonaceous plants, such as the custard apple (Annona reticulata L.) or the paw paw (Asimina triloba Dunal), are known for several types of biological activity, including insecticidal, antifeedant, antimicrobial, antiparasitic, anticancer, and cytotoxic activities (15). The principal bioactive ingredients are oxidized long-chain fatty acid derivatives, the acetogenins. The compounds inhibit the mitochondrial electron transport system, a mode of action shared by rotenone.

(16) O

O

O

H (17)

O O

O (18)

Insect Growth Regulators from Plants The molting and metamorphosis of insects, regulated in a concerted manner by ecdysteroids and the sesquiterpenoid juvenile hormone (JH) (16), offer a unique target for selective insect control agents. Studies to find practically useful insect growth regulators (IGRs) relied either on screening natural products for IGR activity or, as in the case of JH, on the structure of the insect hormone as a lead compound (vide infra). The search for insect JHs led to the unexpected discovery of a botanical IGR in 1965 ´ when Slama and Williams discovered the so-called ‘‘paper

O O

N H

O

O (19) Figure 4. A natural insect juvenile hormone (JH III (17)), and its natural and synthetic mimics: juvabione (16), methoprene (18), and fenoxycarb (19).


complex compound has low environmental persistence, but hydrogenated derivatives are more stable.

O

Others

O

Many plants produce volatile, odorous substances that are commercially obtained by distillation. These compounds are collectively called essential oils and have a broad spectrum of agriculturally important biological activity, including attractant, repellent, and insecticidal effects (23,24). They are also used as general antiseptics as well as fragrances and flavoring agents. Due to their biocidal properties against various organisms, these essential oils are promising nonpersistent crop protection agents. Among the important monoterpenoid components, there are simple hydrocarbons (e.g., limonene), alcohols (e.g., geraniol, menthol, terpineol), cyclic ethers (e.g., 1,8-cineole), aldehydes (e.g., citronellal), ketones (e.g., pulegone), phenols (e.g., carvacrol, thymol), as well as various esters (e.g., linalyl acetate). Of the phenylpropanoids, some phenol derivatives (e.g., asarone, eugenol) and aldehydes (e.g., cinnamaldehyde) have significance. α-Terthienyl was first recognized as a nematicidal constituent of marigolds, but in the presence of light, it is also highly toxic to larvae of several insect species, including mosquitoes. This electron donor thiophenederivative phototoxin is biosynthesized from polyacetylene precursors and appears to function as a photosensitizer catalyzing the formation of reactive oxygen species at the target site (25). 1,4-Benzoxazin-3-ones are major chemical defenses involved in the resistance of maize and rye and other members of the Gramineae family to insects, bacteria, and fungi (26,27). These reactive cyclic hydroxamic acids inhibit insect gut proteases, but their precise mode of action has not been fully elucidated (28). Certain plant chemicals possess male or female sterilant action in insect, and an extensive list of various chemosterilants of natural origin is available (29). Sesamin (also known as fagarol) (20; Fig. 5), a component of sesame oil, has insignificant insecticidal properties, but it potentiates the toxicity of pyrethrins. This synergism is due to the methylenedioxyphenyl (1,3-benzodioxole) moiety inhibiting the oxidative metabolism of a variety of insecticides (30). Based on the structure of 20, several synergistic chemicals were developed, of which piperonyl butoxide (21), manufactured from the essential oil of Sassafras trees, found general use. Insect Hormones as Models for Insect Control Agents The discovery in the late 1960s that insect JHs, such as JH III (17, Fig. 4), are sesquiterpenoid esters, initiated an extensive search for their synthetic analogs. The efforts were quickly rewarded in the early 1970s with the development of the first biorational insect control agents, including methoprene (18, Fig. 4) (31). A departure from the terpenoid structure is illustrated by the non-neurotoxic carbamate juvenoid, fenoxycarb (19). Another important group of insect hormones are the ecdysteroids. Both ecdysone (22; Fig. 6), first detected by Butenandt in 1954, and its 20-hydroxy derivative regulate molting by controlling gene activity and

1095

O H

H O

O

O (20)

O O

O

O O (21) Figure 5. Structural relation of insecticide synergists: sesamin (20) and its synthetic analog, piperonyl butoxide (21).

OH 20

H

HO H HO

OH

H

OH

O (22)

O N

H N O

(23) Figure 6. Steroidal and nonsteroidal inducers of insect molting: the ecdysteroid hormone ecdysone (22) and the insecticide tebufenozide (23).

subsequent protein synthesis (16). These hydroxylated steroids are metabolites of dietary cholesterol and have been found either in free form or as conjugates not only in insects, but also in other invertebrates as well as in plants (phytoecdysones; vide supra) and certain fungi. The serendipitous discovery in the late 1980s of diacylhydrazines, such as tebufenozide (23),

1096


lacking the steroid skeleton but binding to the same receptor and inducing similar morphogenetic disturbances to 20-hydroxy ecdysone in developing larvae, resulted in the commercialization of a novel group of IGRs (32). Microbial Insecticides Several strains of the common gram-positive bacterium Bacillus thuringiensis Berliner (Bt) produce crystalline proteinaceous endotoxins (δ-endotoxin) that are the major commercial bioinsecticides (33). The 130–135-kDa crystalline proteins are inactive protoxins and must be solubilized and activated in the insect gut to 55–65-kDa toxins that, by incorporation into the midgut membrane at specific sites, kill the insect in a few days after ingestion (34). Expression of the genes responsible for endotoxin production in other organisms, including plants such as cotton and maize, confers resistance to insect pests (35). B. thuringiensis also produces an exotoxin (β-exotoxin or thuringiensin), an insecticidal nucleic acid analog obtainable as a water-soluble fermentation by-product of the manufacture of Bt endotoxin (36,37). Avermectins, isolated in the mid-1970s from a soil actinomycete originating from Japan, represent a novel class of sugar-containing macrocyclic lactones with anthelmintic, acaricidal, and insecticidal activity (38,39) (Fig. 7). Abamectin, containing over 80% of avermectin B1a (24), is used against agricultural and household arthropod pests. Among the semisynthetic derivatives is doramectin (25), obtained by directed biosynthesis using unnatural amino acids in the fermentation broth. Ivermectin (26), obtained by selective hydrogenation of abamectin, is also an effective antifilarial agent and used widely to control and treat onchocerciasis that causes river

R2 4"

O

O

O O 23 22

O

O

X

O

25

13

O

O

R1

O

OH

O H

OH

(24) R1 = sec-butyl, R 2 = b-OH, X = CH

CH

(25) R1 = cyclohexyl, R 2 = b-OH, X = CH

CH

(26) R1 = sec-butyl, R 2 = b-OH, X = CH2

CH2

(27) R1 = sec-butyl, R 2 = a-NHCH3, X = CH

blindness in humans. Emamectin (27) is an amino-group containing avermectin analog. These exceptionally potent compounds selectively stimulate glutamate–gated chloride ion channels of endo- and ectoparasites. A related group of macrocyclic lactones lacking the disaccharide moiety at C-13 are the milbemycins possessing biological activities similar to that of the avermectins. Dioxapyrrolomycin, a tricyclic pyrrole-containing antibiotic isolated first from Streptomyces fumanus Sveshnikova in the mid-1980s, displays moderate insecticidal and acaricidal activity, but systematic structural changes led to novel synthetic pyrrole derivatives with increased insecticidal activity and mammalian safety (40). The natural product and its synthetic congeners are uncouplers of mitochondrial oxidative phosphorylation. Spinosyns are structurally novel carbohydrate macrocyclic lactones produced by an actinomycete discovered in the mid-1980s (41). The commercial preparation, called spinosad, contains two structurally related fermentation products. Their insecticidal activity is mainly due to the persistent activation of the nicotinic ACh receptors in a manner distinct from the botanical alkaloid nicotine and the synthetic nicotinoids but γ -aminobutyric acid receptors are also affected (42). Semiochemicals The importance of chemical signals in food location, reproduction, and defense of animals and primitive organisms is well recognized and utilized in crop protection. Of the practically significant semiochemicals utilized by insects, the volatile, fatty acid–derived sex pheromones are emitted for intraspecific communication (43), whereas kairomones (methyl eugenol, cucurbitacins, phenols, and 1-octen-3-ol, etc.) are used for interspecific communication (44). The first sex pheromone was identified from females of the silk moth, Bombyx mori L., in 1959. There are now hundreds of synthetically produced insect sex pheromone compositions employed in species-selective traps to detect and monitor insects (45,46). Some of them attained importance in direct insect control methods that rely on either mass trapping or on the disruption of communication between sexually mature females and mate-seeking males of the target pest. Another group of semiochemicals with practical potential are the less volatile oviposition-deterring pheromones added by females to their eggs to make the marked area deterrent to other egg-laying females. Some kairomones, utilized by insects to find their food source, are also employed in traps or baits. The phagostimulant steroidlike triterpenoid cucurbitacins, for example, when coformulated with neurotoxic carbamates, compel chrysomelid cucumber beetles to persistent feeding on the poisoned bait until death. Among kairomones, we can find structural analogies between trimedlure (28, Fig. 8), a synthetic attractant discovered by routine screening of hundreds of compounds and now used worldwide in traps for the Mediterranean fruit fly, and α-copaene (29), an attractant terpenoid component of the seed oil of Angelica archangelica L., the assumed ancestral host plant of the fly (44).

CH

Figure 7. The natural macrocyclic lactone avermectin B1a (24) and its semisynthetic derivatives doramectin (25), ivermectin (26), and emamectin (27).

Insecticides of Marine Origin Marine organisms are rich in structurally diverse bioactive metabolites, but very few pest control products have


WEED CONTROL AGENTS AND PLANT GROWTH REGULATORS

Cl

O

2 4 1

(29)

1097

When compared with insecticides, relatively few herbicides and plant growth regulators of natural origin attained commercial importance (Table 2).

O

(28)

Figure 8. Kairomones of the Mediterranean fruit fly: (1S,2S,4R)trimedlure (28) and α-copaene (29).

Plant Hormones

emerged from this abundant source (47). A notable exception is nereistoxin (13, Fig. 3) isolated in 1934 from a marine annelid used as a fish bait in Japan and found to be insecticidal in 1962. This cyclic disulfide served as the lead compound for the development of a structurally related family of insecticides exemplified by cartap (14) and thiocyclam (15). These simple amines act on the nicotinic acetylcholine receptor, also the target of nicotine (10) and its relatives. Diatoms are a type of microscopic algae, abundant in marine and fresh water ecosystems that have hard cell walls consisting of pectin and silica. The cell walls of dead diatom shells sink and accumulate in large fossilized deposits, which are mined for various industrial, agricultural, and household uses. Chemically, diatomaceous earth is mostly amorphous silicon dioxide, accompanied by mineral salts, with large surface area. Its use as an insecticide dust is based on its capability to absorb moisture, oils, and waxes (48).

The identification of the growth-promoting plant hormone auxin or, chemically, indole-3-acetic acid (30, Fig. 9) paved the way for the development of a large number of the structurally analogous aryloxyacetic acid weed control agents, such as 2,4-dichlorophenoxyacetic acid (2,4-D, 31) disclosed in the 1940s. These herbicides, being synthetic analogs of a natural plant hormone, can be considered the first biorational pest control agents (56). Another group of structurally simple plant hormones is the cytokinins, of which 6-benzylaminopurine (or 6-benzyladenine) is used alone or in combination with other plant growth regulators in orchards to increase fruit set and delay senescence (56,57). Brassinosteroids are a family of widely distributed plant growth-promoting and stress-resistance enhancing steroidal lactones, the first member of which (brassinolide) was characterized from Brassica napus L. (rape) pollen in the 1970s (58,59). The polyhydroxylated steroidal structure of brassinosteroids resembles that of ecdysteroids, which is also reflected by the IGR activity of brassinolide and relatives.

Table 2. Weed Control Agents and Plant Growth Regulators Common Name

Common Source


Use1

Reference

From Plants Indoleacetic acid (30)

Common hormone

Plant growth regulator

#+

56

Cytokinins

Common hormones


#+++

56,57

Brassinosteroids

Common hormones

Plant growth regulator2

+++

58,59

1-Triacontanol

Medicago sativa L.

Plant growth regulator2

+

71

1,8-Cineole (eucalyptol) (32)

Eucalyptus species

Growth and germination inhibitor2

#

60,72

α-Terthienyl

Tagetes erecta L.

Photodynamic2

−

72

Artemisinin (34)

Artemisia annua L.

Growth and germination inhibitor2

−

61,62

Gibberellins, e.g., gibberellic acid (37)

Gibberella fujikuroi Wr.; common plant hormone!


#+++

56,57

Anisomycin (35)


Photosynthetic pigment inhibitor

#

65

Bilanafos3

Streptomyces hygroscopicus and S. viridochromogenes

Inhibitor of ammonia assimilation

#+++

53,63

L-Phosphinothricin (glufosinate4 )


Inhibitor of ammonia assimilation

+++

63,64

From Microorganisms

− = laboratory use only; # = served as model for (semi)synthetic analogues; + = limited or historical use; +++ = current use. Other types of biological activities are also known. 3 Proherbicide of L-phosphinothricin. 4 The natural product is the L-isomer; commercial glufosinate is a synthetic isomeric mixture. 1 2

1098


OH

OH O

O O

N H

Cl

Cl

(30)

(31)

O O

O

(32)

(33) H O O H

O H

O O (34)

O

O

O

O OH

O

N H (35)

(36) OH H O

O

OH

H HO

O

a higher plant species limits the growth of other plants, and practical utilization appears straightforward. The structural similarity between the terpene 1,8-cineole (32), one of the first allelopathic substances characterized, and the commercial herbicide cinmethylin (33) is apparent. Other essential oils are also known for their allelopathic properties (60). Another group of allelopathic substances are cyclic hydroxamic acids (1,4-benzoxazin-3-ones), commonly found in many cereals and also involved in herbicide detoxification (26,28). One of the few botanicals products that crossed the boundaries of medicinal use is the antimalarial artemisinin (quinghaosu; 34). This sesquiterpenoid lactone endoperoxide from the annual wormwood displays herbicidal activity against several plant species (61,62). Weed Control Agents and Plant Growth Regulators of Microbial Origin Of the large number of phytotoxic microbial natural products (53,63), only two, the organophosphorus amino acid derivatives bilanafos (bialaphos) and phosphinothricin (glufosinate), are of commercial importance. Bilanafos is a tripeptide derivative (phosphinothricyl-Ala-Ala) originally isolated from a soil-borne Streptomyces strain in the early 1970s. In the plants, it is metabolically hydrolyzed to the actual nonselective herbicide, the glutamic acid analog phosphinothricin, an inhibitor of the ammonia-fixing enzyme glutamine synthetase. The synthetic variant of phosphinothricin is a stereoisomeric mixture and sold as glufosinate (64). The hydroxypyrrolidine anisomycin (35), a protein biosynthesis inhibitor antibiotic, served as a lead compound for the development of the now superseded rice herbicide methoxyphenone (36) (65). Gibberellins were first isolated in Japan prior to World War II from abnormally tall rice infected with the ‘‘bakanae’’ fungus, Gibberella fujikuroi Wr. Subsequent studies identified additional tetracyclic diterpenoid gibberellins from culture filtrates of the plant pathogen, including gibberellic acid (37). Later, the gibberellins were found in many plants as another group of growth hormones (56). Adaptation of plants to environmental stress also involves the regulation of gibberellin biosynthesis (66). In addition, several plant growth retardants are known to act by blocking gibberellin biosynthesis. For commercial purpose, 37 is produced by fermentation of G. fujikuroi.

(37) Figure 9. Natural and synthetic plant growth regulators and herbicides: indole-3-acetic acid (30), 2,4-D (31), 1,8-cineole (32), cinmethylin (33), artemisinin (34), anisomycin (35), methoxyphenone (36), and gibberellic acid (37).

Allelopathic Agents and Other Botanicals Allelopathy, defined as chemical interaction between plants, was first demonstrated for black walnut, Juglans nigra L., in 1925, which involves the phytotoxic juglone (5hydroxyanthraquinone) suppressing the growth of nearby plants. When this interaction is of a defensive nature, e.g.,

Others Certain cyclohexene-1,3-diones, existing in equilibrium with their enolized form, are interesting examples of the structural relation of synthetic herbicides and a group of natural products (Fig. 10). The development of the 2acylcyclohexene-1,3-dione sethoxydim (39), an inhibitor of plant acetyl-CoA carboxylase (67), predates the discovery of the structurally similar acetogenins such as 38 that are produced by insects as kairomones (68) and also occur in plants (69). Variations on the side-chain of 39 provided another herbicide with a different mode of action: the carotenoid biosynthesis inhibitor sulcotrione (40) (70).


O O

10

OH (38)

O

O

S

N

O

O Cl OH O

O OH

S (39)

(40)

Figure 10. Structural similarity of an insect kairomone (38) and the cyclohexanedione herbicides sethoxydim (39) and sulcotrione (40).

DISEASE CONTROL AGENTS Although a number of antifungal compounds have been isolated from plants, including the important and structurally diverse group of phytoalexins, the plant’s induced defense system against infecting pathogens has not attained practical importance (73). A recently recognized endogenous signal system of plants utilizes salicylic acid (SA) (41, Fig. 11) and jasmonic acid that are not antimicrobial per se but trigger the natural defense mechanism (systemic acquired resistance) against certain diseases. These hormone-like substances regulate genes involved in defensive processes or in the biosynthesis of secondary metabolites that ultimately ward off pathogens (74,75). Although inexpensive, SA cannot be used as antimicrobial agent because it is readily metabolized in plants, but synthetic surrogates, such as acibenzolar-S-methyl (42; Fig. 11), are suitable for such a protective treatment (76). Cyclooctasulfur, the stable form of elemental sulfur supplied by the chemical industry for many decades as an indispensable fungicide and acaricide, was recently

1099

found to be an essential phytoalexin component of certain pathogen-resistant cocoa genotypes (77). Virtually all natural products used to control plant diseases caused by pathogens originate from microorganisms (Table 3). Among the structurally complex microbial fungicides of current importance are blasticidin-S, an aminohexose nucleoside analog identified in 1966; kasugamycin, an aminoglycoside antibiotic identified in 1965; mildiomycin, an amino acid modified nucleoside derivative characterized in 1978; validamycin A, an aminosugar isolated in 1972; and the polyoxins, which are peptidic nucleoside antibiotics identified in the 1960s (53,78). In the early 1970s, novel antibiotics, the β-methoxyacrylates such as strobilurin A (also called mucidin; 43, Fig. 12) were characterized from tree-inhabiting basidiomycetes fungi. Their unusual structure and remarkable antifungal activity inspired the development of a new class of fungicides, the strobilurins (79,80). Systematic modifications of the light-sensitive polyenic structure led to the synthetic fungicides kresoxim-methyl (44) and azoxystrobin (45). These compounds selectively inhibit fungal mitochondrial respiration, establishing it as a novel fungicidal target. Similarly, structural optimizations of the photolabile chlorinated antibiotic pyrrolnitrin, isolated in 1964 from various Pseudomonas bacteria and used in human medicine, led to a new family of agricultural fungicides such as fenpiclonil (81). Pyrrolnitrin is one of the bioactive metabolites of Burkholderia cepacia and Pseudomonas fluorescens bacteria used in agriculture as biological fungicides. Among the few agricultural antibacterial antibiotics, the aminoglycoside streptomycin, and, to some extent, oxytetracycline and their mixture are of importance (53). The former has had a longer and much wider use record in human and veterinary medicine (82). NEMATICIDES Although several natural, mainly plant-derived compounds have been shown to be nematicidal in the laboratory and, presumably, in the natural environment of the originating organisms, no such product is marketed as a nematicide. Among the compounds shown to be active against nematodes are several phytoalexins, lectins, quassinoids, neem preparations, the isothiocyanate precursor glucosinolates and other simple sulfur derivatives, phenols, and essential oils (85). α-Terthienyl and related polyacetylenes, already mentioned as photodynamic insecticides (vide supra), are also nematicidal. RODENTICIDES

O

OH

O

S

OH

S N N

(41)

(42)

Figure 11. Inducers of the antimicrobial defense system of plants: salicylic acid (41) and its synthetic analog, acibenzolar-Smethyl (42).

Rodenticides are used to eliminate unwanted rodents such as mice, rats, moles, and voles feeding on crops and stored products. They are also extensively used to control rats that spread human diseases as well as to protect underground electric cables, building structures, and dams from being damaged. Rodenticides are highly toxic to most vertebrates and thus are exceptional among crop protection agents. Rotation of various rodenticides with different modes of action, e.g., synthetic anticoagulants for

1100

NATURAL PRODUCT PESTICIDES Table 3. Antimicrobial Agents Common Name

Common Source


Use1

Reference

Diverse3 Diverse3

+++ −

77 26,28

Protein biosynthesis inhibitor3 Protein biosynthesis inhibitor Protein biosynthesis inhibitor Chitin biosynthesis inhibitor3 Protein biosynthesis inhibitor Cell division inhibitor Disruption of fungal cell membrane Disruption of fungal cell membrane Trehalase inhibitor

+++

78

+++

78

+++

78

+++

78

+++

78

+ +

78

+

83

+++

78

Mitochondrial respiration and protein kinase inhibitor3 Mitochondrial respiration inhibitor

#+++

81,84

#

79,80

−#

84

#+++

53

+

53

From Plants Sulfur (S8 ) 1,4-Benzoxazin-3-ones

Theobroma cacao L.2 Gramineae species

Blasticidin-S

Streptomyces griseochromogenes Streptomyces kasugaensis

From Microorganisms

Kasugamycin Mildiomycin Polyoxins Cycloheximide Griseofulvin Natamycin (pimaricin) Nystatin Validamycin Pyrrolnitrin

Strobilurins, e.g., strobilurin A (43)

Streptoverticillium rimofaciens Streptomyces cacaoi var. asoensis Streptomyces griseus Penicillium griseofulvum Streptomyces natalensis and S. chattanoogensis Streptomyces species Streptomyces hygroscopicus spp. limoneus Pseudomonas pyrrocinia and P. fluorescence

Soraphens

Oudemansiella mucida (Schr. ex Fr.) Höhnel and Strobilurus tenacellus (Pers. ex Fries) Singer Sorangium cellulosum

Streptomycin4

Streptomyces griseus

Oxytetracycline4

Streptomyces rimosus

Acetyl-CoA carboxylase inhibitor Protein biosynthesis inhibitor Protein biosynthesis inhibitor

1 − = laboratory use only; # = served as model for (semi)synthetic analogues; + = limited or historical use; +++ = currently used in pest control. 2 As phytoalexin in pathogen-resistant genotypes of this and other plants. 3 Other types of biological activities are also known. 4 Antibacterial agents.

botanical neurotoxicants, has been essential for prudent resistance management (86). Traditionally, parts of a toxic plant or, more recently, their purified active ingredients were used in rodent control (Table 4). The seeds of Strychnos nux-vomica L. were introduced for rodent control first in Germany in the late 17th century. Its poisonous principle is a very bitter alkaloid, strychnine, that was isolated from the beans of the related St. Ignatius plant, Strychnos ignatii Berg, in 1818 but is now obtained from S. nux-vomica. Strychnine formulations are used to kill vertebrate pests, including moles, skunks, gophers, mice, rabbits, coyotes, as well as unwanted birds. The alkaloid, however, is ineffective against rats because its bitterness causes bait shyness. Strychnine has also been used as a tonic and stimulant in veterinary and human medicine. Strychnine excites the central

nervous system by specifically antagonizing the inhibitory neurotransmitter amino acid glycine at receptors involved in regulation of motor functions. In humans, strychninepoisoning symptoms are tetanic convulsions characterized by an arched-back body (opisthotonos) and fixed jaws (risus sardonicus) (87). Red squill or the sea onion is a large onionlike plant growing wild around the Mediterranean Sea and cultivated elsewhere. Its major bioactive principle, the bitter and emetic steroid glycoside, scilliroside, is concentrated in the bulbs. Red squill preparations have been used since the 13th century as a rodenticide. It is emetic and affects the cardiovascular and the central nervous systems. Due to its quick and potent emetic action, red squill is considered to be a safe rodenticide because most nontarget animals, including humans, can regurgitate any ingested material. Rodents, however, are unable to vomit, and they are


OH

1101

OH

O O

N

O

O

OO (46)

O (44)

O

O OH O

O O

O (43)

O (47)

Figure 13. Anticoagulant rodenticides: dicumarol (46) and its synthetic analog, warfarin (47).

N

N

O

O

CN

O

O O

(45) Figure 12. The fungal metabolite strobilurin A (43) as a lead for the synthetic fungicides kresoxim-methyl (44) and azoxystrobin (45).

Warfarin (47, Fig. 13) was the first of the synthetic anticoagulant rodenticides with structural features inspired by a natural product (88). This prototype coumarin derivative was developed in the 1940s by systematically altering the structure of dicumarol (46), recognized earlier as the causative agent of the sweet clover disease causing severe bleeding in grazing cattle (89). These rodenticides act by inhibiting the oxidoreductive recycling of vitamin K, a cofactor necessary for prothrombin synthesis involved in blood coagulation. BIBLIOGRAPHY

thus slowly killed. Another steroidal rodenticide is cholecalciferol, which is in fact the naturally occurring vitamin D3 . This compound is an essential factor for vertebrates but in large doses causes hypercalcemia, resulting in calcification and degeneration of various soft tissues, ultimately leading to death. In baits, cholecalciferol may be combined with other, usually anticoagulant, rodenticides. The main natural source of cholecalciferol is fish liver oil, but it is manufactured from ergosterol.

1. B. Holmstedt, in T. Swain, ed., Plants in the Development of Modern Medicine, Harvard University Press, Cambridge, MA, 1972, pp. 303–360. 2. R. Neumann and H. H. Peter, Experientia 43: 1235–1237 (1987). 3. J. E. Casida and G. B. Quistad, eds., Pyrethrum Flowers: Production, Chemistry, Toxicology, and Uses, Oxford University Press, Oxford, 1995, pp. 1–356. 4. R. M. Hollingworth and K. I. Ahammadsahib, in R. M. Roe and R. J. Kuhr, eds., Reviews of Pesticide Toxicology,

Table 4. Rodenticides Substance

Common Source


Use1

Reference

From Plants Strychnine

Strychnos nux-vomica L.

Glycine receptor antagonist2

+++

86

Red squill (scilliroside)

Urginea maritima (L.) Baker

Neuro- and cardiotoxicant

+++

86

Dicumarol (46)

Melilotus officinalis (L.) Medikus

Anticoagulant2

#

86,88

Reserpine

Rauvolfia serpentina (L.) Kurz. and R. vomitoria Afzel.

Hypotensive and sedative2

+#

90

Ricin

Ricinus communis L.

Protein synthesis inhibitor

+

91,92

1 2

# = served as model for synthetic analogues; + = limited or historical use; +++ = currently used in pest control. Other types of biological activities are also known.

1102


Vol. 3, Toxicology Communications Inc., Raleigh, NC, 1995, pp. 277–302.

32. T. S. Dhadialla, G. R. Carlson, and D. P. Lee, Annu. Rev. Entomol. 43: 545–569 (1998).

5. N. Fang and J. E. Casida, Proc. Natl. Acad. Sci. USA 95: 3380–3384 (1998).

33. J. A. Baum, T. B. Johnson, and B. C. Carlton, in F. R. Hall and J. J. Menn, eds., Biopesticides: Use and Delivery, Humana Press, Totowa, NJ, 1999, pp. 189–209.

6. J. Polonsky, Fortschr. Chem. Org. Naturst. 47: 221–264 (1985). 7. Z. Lidert et al., J. Nat. Prod. 50: 442–448 (1987). 8. F. E. Dayan et al., Pestic. Biochem. Physiol. 65: 15–24 (1999).

34. F. Rajamohan, M. K. Lee, and D. H. Dean, Progr. Nucleic Acid Res. Mol. Biol. 60: 1–27 (1998). 35. V. A. Hilder and D. Boulter, Crop Prot. 18: 177–191 (1999).

´ 9. I. Ujvary, in I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer-Verlag, Tokyo, 1999, pp. 29–69.

ˇ ´ and J. Vankov ˇ ´ in 36. K. Sebesta, J. Farkaˇs, K. Horska, a, H. D. Burges, ed., Microbial Control of Pests and Plant Diseases, 1970–1980, Academic Press, London, 1981, pp. 249–281.

10. I. Yamamoto and J. E. Casida, eds., Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, Springer-Verlag, Tokyo, 1999, pp. 1–300.

37. T.-H. Hsu, Y.-M. Tzeng, and M.-M. Wu, Pestic. Sci. 50: 35–41 (1997).

11. P. R. Jefferies, P. Yu, and J. E. Casida, Pestic. Sci. 51: 33–38 (1997). 12. J. L. Sutko, J. A. Airey, W. Welch, and L. Ruest, Pharmacol. Rev. 49: 53–98 (1997). 13. V. S. Parmar et al., Phytochemistry 46: 597–673 (1997). 14. M. L. Elliott et al., Pestic. Sci. 26: 199–208 (1989). 15. F. Q. Alali, X. X. Liu, and J. L. McLaughlin, J. Nat. Prod. 62: 504–540 (1999). 16. L. M. Riddiford, K. Hiruma, Q. Lan, and B. Zhou, Amer. Zool. 39: 736–746 (1999). 17. Y. C. Toong, D. A. Schooley, and F. C. Baker, Nature 333: 170–171 (1988). 18. R. Lafont and I. D. Wilson, The Ecdysone Handbook, 2nd ed., The Chromatographic Society, Nottingham, U.K., 1996, pp. 1–524. 19. L. Dinan, T. Savchenko, P. Whiting, and S. D. Sarker, Pestic. Sci. 55: 331–335 (1999). 20. E. A. Schmelz, R. J. Greenok, D. W. Galbraith, and W. S. Bowers, J. Chem. Ecol. 25: 1739–1757 (1999). 21. W. S. Bowers, in J. R. Coats, ed., Insecticide Mode of Action, Academic Press, New York, 1982, pp. 403–427. 22. S. V. Ley, A. A. Denholm, and A. Wood, Nat. Prod. Rep. 109–157 (1993). 23. S. Lee, R. Tsao, C. Peterson, and J. R. Coats, J. Econ. Entomol. 90: 883–892 (1997). 24. M. Isman, Pesticide Outlook 10(April): 68–72 (1999). 25. G. K. Cooper and C. I. Nitsche, Bioorg. Chem. 13: 362–374 (1985). 26. H. M. Niemeyer, Phytochemistry 27: 3349–3358 (1988). 27. R. A. Assabgui, J. T. Arnason, and R. I. Hamilton, J. Econ. Entomol. 88: 1482–1493 (1995). 28. Y. Hashimoto and K. Shudo, Phytochemistry 43: 551–559 (1996). 29. A. B. Borkovec, in E. D. Morgan and N. B. Mandava, eds., CRC Handbook of Natural Pesticides, Vol. III, Insect Growth Regulators, Part B, CRC Press, Inc., Boca Raton, FL, 1987, pp. 1–19.

38. W. C. Campbell, Ivermectin and Abamectin, Springer-Verlag, New York, 1989, pp. 1–363. 39. M. H. Fisher, in P. A. Hedin et al., eds., Phytochemicals for Pest Control, ACS Symp. Ser. Vol. 658, American Chemical Society, Washington, DC, 1997, pp. 220–238. 40. D. A. Hunt and M. F. Treacy, in I. Ishaaya and D. Degheele, eds., Insecticides with Novel Modes of Action: Mechanisms and Application, Springer, Berlin, 1998, pp. 138–151. 41. T. C. Sparks et al., in F. R. Hall and J. J. Menn, eds., Biopesticides: Use and Delivery, Humana Press, Totowa, NJ, 1999, pp. 171–188. 42. V. L. Salgado, Pestic. Biochem. Physiol. 60: 91–102 (1998). 43. P. E. Howse, I. D. R. Stevens, and O. T. Jones, Insect Pheromones and Their Use in Pest Management, Chapman and Hall, London, 1998, pp. 1–369. 44. R. L. Metcalf and E. R. Metcalf, eds., Plant Kairomones in Insect Ecology and Control, Chapman and Hall, New York, 1992, pp. 1–178. 45. M. S. Mayer and J. R. McLaughlin, Handbook of Insect Pheromones and Sex Attractants, CRC Press, Inc., Boca Raton, FL, 1991, pp. 1–1083. 46. H. Arn, M. Tóth, and E. Priesner, The Pherolist, Available online: http://www.nysaes.cornell.edu/pheronet and http://www.quasimodo.Versailles.inra.fr/pherolist. 47. K. A. El Sayed et al., J. Agric. Food Chem. 45: 2735–2739 (1997). 48. Z. Korunic, J. Stored Prod. Res. 34: 87–97 (1998). 49. J. Fukami, in C. F. Wilkinson, ed., Insecticide Biochemistry and Physiology, Plenum Press, New York, 1976, pp. 353–396. 50. R. Marles et al., Pharmacol. Toxicol. 77: 164–168 (1995). 51. E. Shaaya and M. Kostjukovsky, in I. Ishaaya and D. Degheele, eds., Insecticides with Novel Modes of Action: Mechanisms and Application, Springer, Berlin, 1998, pp. 171–187. 52. G. I. Kornis, in C. R. A. Godfrey, ed., Agrochemicals from Natural Products, Marcel Dekker, Inc., New York, 1995, pp. 215–255. 53. I. Yamaguchi, in L. G. Copping, ed., Crop Protection Agents from Nature: Natural Products and Analogues, The Royal Society of Chemistry, Cambridge, U.K., 1996, pp. 27–49.

30. J. E. Casida, J. Agric. Food. Chem. 18: 753–772 (1970).

54. C. H. Green, Advances in Parasitology 34: 229–291 (1994).

31. C. A. Henrick, in C. R. A. Godfrey, ed., Agrochemicals from Natural Products, Marcel Dekker, Inc., New York, 1995, pp. 147–213.

55. D. Bowen et al., Science 280: 2129–2132 (1998). 56. N. Takahashi, ed., Chemistry of Plant Hormones, CRC Press, Inc., Boca Raton, FL, 1986, pp. 1–277.

NATURAL PRODUCT PESTICIDES 57. L. G. Nickell, Plant Growth Regulators, Springer-Verlag, Berlin, 1982, pp. 1–173. 58. H. G. Cutler, T. Yokota, and G. Adam, eds., Brassinosteroids: Chemistry, Bioactivity, and Applications, ACS Symp. Ser. No. 474, American Chemical Society, Washington, DC, 1991, pp. 1–358. 59. S. D. Clouse and J. M. Sasse, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 427–451 (1998). 60. N. Dudai et al., J. Chem. Ecol. 25: 1079–1089 (1999). 61. S. O. Duke, K. C. Vaughn, E. M. Croom, Jr., and H. N. Elsohly, Weed Sci. 35: 499–505 (1987).

1103

83. H. vanden Bossche, in H. Lyr, ed., Modern Selective Fungicides: Properties, Application, Gustav Fischer Verlag, Jena, 1995, pp. 431–484. 84. J. P. Pachlatko, Chimia 52: 29–47 (1998). 85. D. J. Chitwood, in H. N. Nigg and D. Seigler, eds., Phytochemical Resources for Medicine and Agriculture, Plenum Press, New York, 1992, pp. 185–204. 86. A. C. Elliott, in L. G. Copping, ed., Crop Protection Agents from Nature: Natural Products and Analogues, The Royal Society of Chemistry, Cambridge, U.K., 1996, pp. 341–368. 87. J. A. Perper, J. Forensic Sci. 30: 1248–1255 (1985).

62. F. E. Dayan et al., Phytochemistry 50: 607–614 (1999).

88. R. S. Overman et al., J. Biol. Chem. 153: 5–24 (1944).

63. S. O. Duke, H. K. Abbas, T. Amagasa, and T. Tanaka, in L. G. Copping, ed., Crop Protection Agents from Nature: Natural Products and Analogues, The Royal Society of Chemistry, Cambridge, U.K., 1996, pp. 82–113.

89. C. F. Huebner and K. P. Link, J. Biol. Chem. 138: 529–534 (1941).

64. G. Hoerlein, Rev. Environ. Contam. Toxicol. 138: 73–145 (1994). 65. O. Yamada et al., Agric. Biol. Chem. 38: 2017–2020 (1974).

90. A. P. Meehan, Pestic. Sci. 11: 555–561 (1980). 91. F. Stirpe et al., Biotechnology (New York) 10: 405–412 (1992). ´ 92. I. Ujvary, in R. I. Krieger, ed., Handbook of Pesticide Toxicology, 2nd ed., Academic Press, San Diego, 2001, pp. 109–179.

66. P. C. Bethke and R. I. Jones, Curr. Opin. Plant Biol. 1: 440–446 (1998).

FURTHER READING

67. N. Sato et al., Chemtech 18: 430–433 (1988).

Beier, R. C., Natural pesticides and bioactive components in foods, Rev. Environ. Contam. Toxicol. 113: 47–137 (1990) (a thorough review on the chemistry and toxicology of the subject matter). Coats, J. R., Risks from natural versus synthetic insecticides, Annu. Rev. Entomol. 39: 489–515 (1994) (a valuable review on the subject). Copping, L. G., ed., Crop Protection Agents from Nature: Natural Products and Analogues, The Royal Society of Chemistry, Cambridge, U.K., 1996, pp. 1–501 (a collection of thorough reviews on various topics). Copping, L. G., ed., The BioPesticide Manual, The British Crop Protection Council, Farnham, Surrey, U.K., 2001, pp. 1–528 (a fine collection of important data of natural product and biological pest control agents). Crombie, L., Natural product chemistry and its part in the defence against insects and fungi in agriculture, Pestic. Sci. 55: 761–774 (1999) (a personal account on an amazingly broad range of compounds). Godfrey, C. R. A., Agrochemicals from Natural Products, Marcel Dekker, Inc., New York, 1995, pp. 1–424 (a fine collection of thorough reviews).

68. A. Mudd, J. Chem. Soc. Perkin Trans. 1 2161–2164 (1983). 69. N. R. Azevedo, S. C. Santos, E. G. De Miranda, and P. H. Ferri, Phytochemistry 46: 1375–1377 (1997). 70. D. L. Lee et al., Pestic. Sci. 54: 377–384 (1998). 71. S. Ries, Plant Physiol. 95: 986–989 (1991). 72. S. O. Duke, Rev. Weed Sci. 2: 15–44 (1986). 73. R. J. Grayer and J. B. Harborne, Phytochemistry 37: 19–42 (1994). 74. A. J. Parr and M. J. C. Rhodes, in L. G. Copping, ed., Crop Protection Agents from Nature: Natural Products and Analogues, The Royal Society of Chemistry, Cambridge, U.K., 1996, pp. 301–328. 75. R. A. Creelman and J. E. Mullet, Plant Cell 9: 1211–1223 (1997). 76. W. Kunz, R. Schurter, and T. Maetzke, Pestic. Sci. 50: 275–282 (1997). 77. R. M. Cooper et al., Nature 379: 159–162 (1996). 78. I. Yamaguchi, in H. Lyr, ed., Modern Selective Fungicides: Properties, Application, Gustav Fischer Verlag, Jena, 1995, pp. 415–429. 79. H. Sauter, E. Ammermann, and F. Roehl, in L. G. Copping, ed., Crop Protection Agents from Nature: Natural Products and Analogues, The Royal Society of Chemistry, Cambridge, U.K., 1996, pp. 50–81. 80. J. M. Clough, Nat. Prod. Rep. 565–574 (1993). 81. R. Nyfeler and P. Ackermann, in D. R. Baker, J. G. Fenyes, and J. J. Steffens, eds., Synthesis and Chemistry of Agrochemicals III, ACS Symp. Ser., Vol. 504, American Chemical Society, Washington, DC, 1992, pp. 395–404. 82. H. F. Chambers and M. A. Sande, in J. G. Hardman and L. E. Limbird, eds., Goodman & Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, 1996, pp. 1103–1121.

Harborne, J. B., Introduction to Ecological Biochemistry, Academic Press, London, 1993, pp. 1–318 (an outstanding reference work on the subject). Hedin, P. A. et al., eds., Phytochemicals for Pest Control, ACS Symp. Ser., Vol. 658, American Chemical Society, Washington, DC, 1997, pp. 1–356 (a collection of articles on the subject). ¨ Henkel, T., Brunne, R. M., Muller, H., and Reichel, F., Statistical investigation into the structural complementarity of natural products and synthetic compounds, Angew. Chem. Int. Ed. Engl. 38: 643–647 (1999). Isman, M. B., Leads and prospects for the development of new botanical insecticides, in R. M. Roe and R. J. Kuhr, eds., Reviews in Pesticide Toxicology, Vol. 3, Toxicology Communications Inc., Raleigh, NC, 1995, pp. 1–20. Jacobson, M. and Crosby, D. G., Naturally Occurring Insecticides, Marcel Dekker, Inc., New York, 1971, pp. 1–585. Pachlatko, J. P., Natural products in crop protection, Chimia 52: 29–47 (1998) (a review with emphasis on newer results).

1104

NEMATICIDES

Smith, A. E. and Secoy, D. M., Plants used for agricultural pest control in western Europe before 1850, Chem. Ind. 12–17 (1981). Stoll, G., Natural Crop Protection Based on Local Farm Resources in the Tropics and Subtropics, Verlag Josef Margraf, Aichtal, 1986, pp. 1–186. ´ Ujvary, I., Pest control agents from natural products, in R. I. Krieger, ed., Handbook of Pesticide Toxicology, 2nd ed., Academic Press, San Diego, 2001, pp. 109–179 (a thorough work on the chemistry, biochemistry and toxicology of natural products used in pest control). ¨ Wink, M., Schmeller, T., and Latz-Bruning, B., Modes of action of allelochemical alkaloids: Interaction with neuroreceptors, DNA, and other molecular targets, J. Chem. Ecol. 24: 1881–1937 (1998) (a comprehensive paper on the subject). Pest Management Science 56: 649–723 (2000) (this Natural Product/Biocontrol special issue of the journal contains a series of articles on the chemistry, regulation, use, and environmental fate of a broad range of biopesticides).

NEMATICIDES DAVID J. CHITWOOD USDA-ARS Beltsville, Maryland

Nematodes are nonsegmented, bilaterally symmetric worm-like invertebrates that possess a body cavity and a complete digestive system but lack respiratory and circulatory systems. The body wall is composed of a multilayered cuticle, a hypodermis with four longitudinal cords, and internal musculature. The most conspicuous feature of the nervous system is the nerve ring near the nematode pharynx. The so-called excretory system has never been associated with removal of metabolic wastes; instead, it functions in osmoregulation or in the secretion of compounds essential to the life history of the nematode, depending on the species and the developmental stage. The digestive and reproductive systems constitute much of the body contents. Most nematode species are ‘‘free-living’’; i.e., they feed on microorganisms in water and soil. A smaller number of species are ubiquitous parasites of animals or plants. Indeed, Nathan A. Cobb (1), the father of American nematology, stated in 1914: If all the matter in the universe except nematodes were swept away, our world would still be recognizable, and if, as disembodied spirits, we could then investigate it, we should find its mountains, hills, vales, rivers, lakes, and oceans represented by a film of nematodes. The location of towns would be decipherable, since for every massing of human beings there would be a corresponding massing of certain nematodes. Trees would still stand in ghostly rows representing our streets and highways. The location of the various plants and animals would still be decipherable, and had we sufficient knowledge, in many cases even their species could be determined by an examination of their erstwhile nematode parasites.

soil or within plant roots, delivery of a chemical to the immediate surroundings of a nematode is difficult. The outer surface of nematodes is a poor biochemical target and is impermeable to many organic molecules. Delivery of a toxic compound by an oral route is nearly impossible because most phytoparasitic species ingest material only when feeding on plant roots. Therefore, nematicides have tended to be broad-spectrum toxicants possessing high volatility or other properties promoting migration through the soil. The resulting record of less-than-perfect environmental or human health safety has resulted in the widespread deregistration of several agronomically important nematicides (e.g., ethylene dibromide and dibromochloropropane). The most important remaining fumigant nematicide, methyl bromide, faces immediate severe restrictions and future prohibition because of concerns about atmospheric ozone depletion (2). This review focuses on the chemical compounds presently used against plant-parasitic nematodes and the compounds with the greatest likelihood to replace some of the current problematic compounds. Chemical control of nematodes of veterinary or medical importance is achieved through use of several compounds useful in management of several types of vermiform parasites besides nematodes. In general, mammalian anthelmintics are poorly suited as agronomic nematicides because of lack of mobility in soil, expense, or other undesirable properties. Readers curious about mammalian anthelmintics should refer to several excellent reviews (3–5). The mode of action of some mammalian nematicides is briefly discussed in this review. AGRICULTURAL IMPACT OF NEMATODES As with damage caused by other crop pests and pathogens, the extent of crop losses caused by nematodes is a topic of debate. The most comprehensive estimate was obtained in a 1986 survey incorporating the responses of 371 nematologists in 75 countries (6). Estimates of nematode damage to specific crops ranged from 3.3% to 20.6%, with a mean of 12.3%. Annual production losses at the farm gate (in year 2000 dollars) were $121 billion globally and $9.1 billion in the United States. Developing nations reported greater yield loss percentages than did developed countries. Figures for mean crop losses can be deceptive; yield reduction in specific crops can exceed 75% in some locations (7). More typically, growers are forced to select less profitable crops. In addition to directly causing crop losses, nematodes can vector many plant viruses or create wounds that allow the entry of other root pathogens. Several nematodes are major pests of quarantine importance and interfere with free trade of several agricultural commodities. SPECIFIC NEMATICIDES: AN INTRODUCTION

The development of chemical controls for plant-parasitic nematodes is a formidable challenge. Because most phytoparasitic nematodes spend their lives confined to the

Although the discovery of nematicidal activity in a synthetic chemical dates from the use of carbon disulfide

NEMATICIDES

as a soil fumigant in the second half of the nineteenth century, research on the use of nematicides languished until surplus nerve gas (chloropicrin) became readily available following World War I (8). In the 1940s, the discovery that D-D (a mixture of 1,3-dichloropropene and 1,2-dichloropropane) controlled soil populations of phytoparasitic nematodes and led to substantial increases in crop yield provided a great impetus to the development of other nematicides, as well as the growth of the science of nematology. Subsequently, other halogenated hydrocarbons and other volatile compounds were developed as nematicidal soil fumigants. In the 1960s, a new generation of nematicides was introduced—carbamates and organophosphates that served as contact nematicides, devoid of fumigant activity. Many of the carbamates and organophosphates are systemic within plants, but only one contact nematicide has registered systemic nematicidal activity. For most systemics, the high concentrations needed to retard nematode development within plant roots is not likely to occur under field conditions (9). Most soil nematicides are also registered as insecticides or fungicides and are discussed in greater detail elsewhere in this volume. This broad-spectrum activity is a result of the difficulty in discovering or designing compounds capable of movement through the soil. In addition, the small size of the commercial market for nematicides in comparison to other pesticides dictates that nematicide discovery is often an appendage to research programs pursuing controls for other organisms. Compounds included in the following compilation of chemical nematicides are not necessarily registered for usage in the United States or elsewhere, particularly when viewed through their ever-changing regulatory context. FUMIGANTS D-D This mixture of 1,2-dichloropropane and 1,3-dichloropropene had widespread use as an effective nematicide until problems with groundwater contamination resulted in its withdrawal from use in 1984. The 1,2-dichloropropane component was relatively inactive as a nematicide at concentrations used in agricultural fields. 1,3-Dichloropropene Because of the relative lack of nematicidal activity in 1,2-dichloropropane and the desire to eliminate groundwater contamination by a compound not useful for nematode control, 1,3-D became a highly successful nematicide. Although it also has fungicidal activity and insecticidal activity against wireworms in particular, the primary use of the compound is as a nematicide. On a weight basis, 1,3-D is the sixth most abundantly used pesticide in the United States (11); 1,3-D is classified as a possible or probable human carcinogen. Commercial formulations are liquids and contain two isomers. In one series of experiments, aqueous trans-1,3-D was 60% as toxic as the cis isomer, whereas in the vapor phase, trans-1,3-D was 90% as toxic as cis-1,3-D (12). In laboratory experiments simulating field situations, the

1105

trans isomer was completely ineffective against the potato cyst nematode Globodera rostochiensis (13). Ethylene Dibromide Once the most abundantly used nematicides in the world, use of EDB was prohibited in the United States in 1983 because of groundwater contamination (8,10). It was available in liquid formulations and is regarded as a probable human carcinogen. 1,2-Dibromo-3-Chloropropane Liquid formulations of this fumigant with substantial nematode-specific activity were once popular. The compound was notable because of its usefulness in postplant applications. The discovery that over one-third of the male workers at a DBCP manufacturing plant in California were sterile led to the immediate 1977 prohibition of its use in the United States, except for usage in pineapple production (14). Sterility problems were also reported among some DBCP applicators (14). All uses were prohibited in the late 1980s. DBCP is classified as a possible or probable human carcinogen. Methyl Bromide Methyl bromide is a broad-spectrum fumigant toxic to nematodes. In 1997, methyl bromide was the fourth most commonly used pesticide in the United States (11). It is agronomically useful against soil fungi, nematodes, insects, and weeds. The Montreal Protocol, an international treaty regulating the use of ozone-depleting substances, mandates the elimination of methyl bromide use in developed countries by 2005. Under a 1999 amendment to the Clean Air Act, the United States phaseout of usage will not be more restrictive than that mandated by the Montreal Protocol. Research pursuing the development of nematicidal methyl bromide alternatives has been intensive, but no single compound appears likely to substitute for it. Methyl bromide is used as a gas; because of its lack of odor, small amounts of chloropicrin are often added as an indicator of exposure to applicators and are often required by specific governmental agencies, such as the state of Florida. Methyl bromide is the fastest moving fumigant in soils, followed by chloropicrin, 1,3-D, EDB, methyl isothiocyanate, and DBCP (15). Chloropicrin One of the oldest soil fumigants, chloropicrin’s primary agricultural use in soils is as a fungicide, although it does have herbicidal and nematicidal activity. It is often added to 1,3-D formulations in order to increase their fungicidal activity. The compound is acutely toxic and is used in liquid formulations. In 1997, it was the 25th most abundantly used U.S. pesticide (11). Metam Sodium, Dazomet, and Methyl Isothiocyanate (MITC) Metam sodium is a soil fumigant used to control nematodes, fungi, insects, and weeds; it is the third most commonly used U.S. pesticide (11). When applied to soils, metam sodium is converted to MITC, which is the active biocidal agent. MITC is no longer registered for

1106

NEMATICIDES

use as a soil pesticide in the United States, except as a wood preservative. Metam sodium and related compounds have provided excellent control of nematodes in some circumstances but not in others (8,16,17). Dazomet is one of the few compounds with activity as a fumigant that is supplied as a granular formulation. Research on the use of isothiocyanates as nematicides began in the 1930s (18). Several brassicaceous plants contain nematicidal isothiocyanates or glucosinolates that release isothiocyanates when incorporated into soils (19). Sodium Tetrathiocarbonate Sodium tetrathiocarbonate is more recently registered preplant soil fumigant active against fungi, insects, and nematodes. It is supplied as a liquid formulation and may be applied via drip or surface irrigation. Sodium tetrathiocarbonate rapidly degrades in soil into carbon disulfide, sodium hydroxide, hydrogen sulfide, and sulfur. Carbon disulfide is the active principle. Although carbon disulfide has a long history as a fumigant, its flammability is legendary. Carbonates and sulfates are the terminal degradation products. Unlike other commonly used fumigants, sodium tetrathiocarbonate does not readily move through soil air and requires a high level of soil moisture when applied in order to be distributed throughout the soil.

lily (20). Oxamyl is widely used throughout the world and is less persistent in soil than is aldicarb (8). ORGANOPHOSPHATES While this review is being written, the U.S. EPA is actively reviewing the uses of all organophosphates. It is possible that several of the following compounds will face mandatory or voluntary withdrawals from use in the United States. Ethoprop Introduced in the 1960s, ethoprop is a nonsystemic insecticide/nematicide. The mobility of ethoprop in soil and its half-life are strongly dependent on soil organic matter (21). It is not known to be carcinogenic and is available as granules or emulsifiable concentrates. Fenamiphos Also introduced in the 1960s, fenamiphos does have some systemic insecticidal activity. It is widely used as a nematicide. Like ethoprop, it is strongly adsorbed onto organic matter. It is acutely toxic but not shown to be a carcinogen. Cadusafos

CARBAMATES Aldicarb Like most other carbamate nematicides, aldicarb was introduced in the 1960s. It is active against a wide variety of nematodes (as well as insects and mites) and is useful in a variety of soil types throughout the world (8). Aldicarb is available in granular formulations and possesses systemic activity. Aldicarb, carbofuran, and oxamyl are highly toxic but have not been shown to be carcinogens.

This nonsystemic organophosphate not registered for U.S. usage is used to control nematodes and soil insects on bananas and other crops in several countries. The U.S. EPA has granted tolerances for cadusafos in imported bananas, where it provides excellent control of the burrowing nematode, Radopholus similis (22). Cadusafos reportedly possesses reduced risk for contaminating groundwater and provided good control of the citrus nematode, Tylenchulus semipenetrans (23). Cadusafos is commercially available in granular and microencapsulated formulations.

Aldoxycarb Aldicarb is oxidized in soils to aldicarb sulfone, which is available in some parts of the world as the insecticide/nematicide aldoxycarb. A flowable formulation is available. Carbofuran Carbofuran is another systemic insecticidal/nematicidal carbamate available in granular and liquid formulations. Because use of carbofuran granules was associated with bird kills, the U.S. Environmental Protection Agency (EPA) prohibited the use of carbofuran granules in 1994. Oxamyl Like carbofuran, oxamyl is a carbamate that is manufactured in liquid and granular form, but the latter is no longer registered in the United States because of concerns about its consumption by birds. Oxamyl is the only nematicide with downward-moving systemic activity and thus has registered foliar nematicidal applications; foliar applications did reduce Pratylenchus penetrans on

Fosthiazate Fosthiazate is a somewhat recently developed (1992) systemic organophosphorus nematicide with broad-spectrum activity (24). A clay-based microgranule formulation is available. Fosthiazate provided control of the lesion nematode Pratylenchus penetrans on potato (25) and root knot nematodes (Meloidogyne spp.) on tobacco (26) and M. arenaria on peanut (27), but it failed to control M. javanica on tobacco and Rotylenchulus reniformis on pineapple as well as fumigation with 1,3-D (28,29). It is not registered for U.S. usage. Other Organophosphates Terbufos is a less widely used organophosphate with insecticidal and a few nematicidal uses. It is available in granular formulations. Fensulfothion is a systemic previously but not currently registered for insecticidal and nematicidal activity in the United States. Granular and emulsifiable concentrate formulations were available. Phorate is primarily used as a soil insecticide but has nematicidal uses. Its current U.S. reregistration process

NEMATICIDES

involves the use of several risk mitigation measures. Organophosphate nematicides with limited worldwide use but not registered in the United States include thionazin, fosthietan, and isazofos. BIOCHEMICALS DiTera The nematode-parasitic fungus Myrothecium verrucaria produces a mixture of compounds registered in 1996 as a biologically based nematicide named DiTera. Toxicity apparently results from the synergistic action of lowmolecular-weight, water-soluble compounds. DiTera is active against many plant-parasitic nematodes but not the free-living and mammalian-parasitic nematodes studied thus far (30). Toxic effects observed with G. rostochiensis include disruption of hatching, movement, and response to potato root diffusate; toxicity to M. incognita did not involve inhibition of hatching (31,32). DiTera is available as granules, a powder, and an emulsifiable suspension. ClandoSan ClandoSan is a granular product made from processed crab and crawfish exoskeletons. The material contains large amounts of chitin and urea and was registered in the United States in 1998 as a nematicide. Its nematicidal activity (33) is believed to result from the stimulation of populations of nematode-antagonistic microorganisms, particularly those that produce chitinase, a major component of nematode eggshells. Proper application is necessary to avoid phytotoxicity (33). Sincocin Sincocin is the trade name of the mixture registered in 1997 as ‘‘Plant Extract 620’’ with the U.S. EPA. It consists of a blend of extracts from the prickly pear Opuntia lindheimeri, the oak Quercus falcata, the sumac Rhus aromatica, and the mangrove Rhizophora mangle. Sincocin has provided control of the citrus nematode on orange roots (34), the reniform nematode on sunflower (35), and the sugarbeet cyst nematode (36); but control of M. incognita on cassava and R. similis on anthurium was less successful than that provided by other methods (37,38). Its mode of action has not been fully elucidated. MODE OF ACTION In general, nematode developmental stages that are active are more susceptible to nematicides than are resting stages (12,39). The detailed 20-year-old review by Wright (40) on nematicidal mode of action remains relevant because few new nematicides have been introduced since its publication. Moreover, the broad-spectrum activity of most nematicides has resulted in much of their basic biochemical effects being documented in insects or mammals instead of nematodes.

1107

Fumigants A primary effect of halogenated hydrocarbons is to serve as alkylating agents. The sulfhydryl groups of proteins, in particular, are labile to methyl bromide–induced methylation (41). With respect to research performed with nematodes, EDB alkylated proteins and oxidized Fe+2 centers in the cytochrome-mediated electron transport chain, thereby blocking respiration (40). The mode of action of methyl isothiocyanate generators in nematodes is even more poorly understood (42); amino and hydroxyl groups have been speculated as sites of attack (40). Beyond a minimal threshold lethal concentration of a fumigant, the susceptibility of a nematode to a fumigant has long been known to be proportional to the product of the concentration of the fumigant and the duration of exposure, i.e., the concentration-time product. Nonfumigants Carbamates and organophosphates are well-known reversible inhibitors of acetylcholinesterase activity in insects. Several nonfumigant nematicides have been demonstrated to inhibit cholinesterase in nematodes, e.g., aldicarb, carbofuran, fenamiphos, and oxamyl in M. incognita and M. javanica (43) and Aphelenchus avenae (44). Interestingly, although carbofuran inhibits Meloidogyne cholinesterase approximately 10,000 times higher than fenamiphos (43), the latter has greater nematicidal activity against Meloidogyne; this discrepancy is correlated with a much quicker metabolism of fenamiphos than carbofuran by root-knot nematodes (45). Chang and Opperman (46) discovered five molecular forms of acetylcholinesterase in M. arenaria and M. incognita; the forms could be divided into three classes, one of which was highly resistant to aldicarb and fenamiphos. Given that nonfumigant nematicides inhibit nematode acetylcholinesterase, it is not surprising that many of the symptoms induced in nematodes reflect nervous system dysfunction. These symptoms include stylet thrusting, twitching, trembling, convulsions, soiling and uncoiling, other uncoordinated movements, inhibited penetration, and eventual paralysis if the concentration is sufficiently high (39,47,48). Nematode recovery from acetylcholinesterase inhibitor treatment can occur within a short time, even for the case of the stem and bulb nematode, Ditylenchus dipsaci, exposed to 10-mg/ml oxamyl for a day (48). In some cases, however, recovery may not occur, as with A. avenae exposed to fenamiphos, but not carbofuran (49). The speed of recovery from acetylcholinesterase inhibition varies among inhibitors, and nematodes that grossly appear fully recovered still can exhibit pronounced acetylcholinesterase inhibition in enzyme assays. Because contact nematicide concentration in agricultural soils following application is usually not sufficiently high to kill nematodes, the primary organismal mode of action may be temporary paralysis, interference with host finding, inhibition of hatching, or disruption of some other process (10). For example, the three carbamates aldicarb, carbofuran, and cloethocarb inhibited H. schachtii juvenile mobility at concentrations of nematicide that occur in

1108

NEMATICIDES

field situations, whereas inhibition of hatching occurred at concentrations not likely to occur in the field (50). Because soil is a heterogeneous mixture, complete eradication of a nematode population with a chemical nematicide, even a fumigant, is an unlikely achievement. Moreover, contact nematicides are used at levels insufficient to induce immediate death. Nonetheless, the inhibition of movement and penetration is usually substantial enough to result in lack of economic damage. Sometimes the reduction in nematode populations is not sufficiently long to eliminate the need for postplant reapplication of nematicides, however, especially for perennials or crops with long growing seasons. Nonetheless, higher initial nematicide application rates are often not cost-effective and may be associated with increased environmental or other risks. The metabolism of nematicides by nematodes has not been extensively studied. In one interesting investigation of the metabolism of carbofuran and fenamiphos by root-knot nematodes, detected metabolites included 3-hydroxycarbofuran, 3-ketocarbofuran, fenamiphos sulfoxide, and various unidentified watersoluble products (45). Mammalian Anthelmintics Although the purpose of this review is not to focus on nematicides of veterinary or human medical importance, the modes of action of these compounds have been reviewed (4) and are relevant. Representatives of the most popular classes of compounds include the following: 1) nicotinic agonists such as the imidazothiazole levamisole, the tetrahydropyrimidines pyrantel and morantel, and the pyrimidine methyridine, which act as agonists on muscle acetylcholine receptors and induce paralysis; 2) the GABA agonist piperazine, which induces muscular paralysis, particularly in large nematodes in oxygen-poor environments; 3) macrocyclic lactones such as avermectins and milbemycins, with mode of action as discussed in this review; 4) benzimidazoles such as thiabendazole and mebendazole, which bind to β-tubulin and interfere with nematode microtubule formation; and 5) diethylcarbamazine, which appears to interfere with host and possibly nematode arachidonic acid metabolism. RESISTANCE TO NEMATICIDES Resistance of field populations to nematicides has not been well characterized and is remarkably insignificant in comparison to the levels of resistance observed with mammalian parasites. Indeed, a recent National Academy of Sciences monograph stated, ‘‘Resistance of nematodes to soil fumigants has yet to be observed but systemic nematocides are relatively new and it is probably only a matter of time until resistance does appear’’ (51). In one interesting study, Moens and Hendrickx (52) evaluated populations of Meloidogyne naasi, G. rostochiensis, and Pratylenchus crenatus exposed to aldicarb for 15 years. Although some developmental differences were noticed between treated and control populations when challenged with aldicarb, the differences were species specific and were concluded to be not significant.

In another investigation, the free-living nematode Rhabditis oxycerca was bred for 400 generations in order to obtain strains adapted to reproducing on concentrations of 600- and 480-µg/ml aldicarb and oxamyl, respectively. Compared with wild type, the two mutant strains were characterized by decreased size (particularly in the tail region), tolerance of warm temperature, production of offspring, and migration in electric fields, among other characteristics. In nematicide solutions, the wild type exhibited decreased motility, electric field migration, and reproduction (53). In a third study, genetically selected strains of the insect pathogen Heterorhabditis bacteriophora possessed 8–70-fold increased resistance to fenamiphos, avermectin, and oxamyl (54). The enhanced resistance was generally stable in the absence of further nematicide pressure; the strains have obvious potential utility in integrated pest management systems.

APPLICATION METHODS The methods for treating agricultural soils with nematicides are similar to those used for other pesticides examined in this volume. Nematicide application research is being driven by the need to maximize efficacy while minimizing groundwater and atmospheric contamination. Fumigation Soil fumigation requires prior preparation to be effective (55). Prior to fumigant or nonfumigant application, soil is often turned or tilled to increase porosity and uniformity and promote decomposition of residual plant roots, which can serve as hiding places for nematodes or interfere with fumigant movement. Adequate but not excessive soil moisture is critically important to the success of some fumigants. Fumigants are typically injected with chisels or shanks into the upper 15–40 cm of soil, with the actual depth a function of compound, soil structure, and crop. Although deep injection is often required to minimize the escape of fumigant into the surrounding air, inadequate levels of nematicide in the upper soil layers may result in some situations. Following fumigation, the soil surface is often compacted in order to retard fumigant loss from the soil surface. The design of injection equipment modified for minimization of fumigant escape into the surrounding air is an active research area (56). Because the shallow chisel traces left in treated soils provide a means for fumigant to escape into the atmosphere, some nematicide labels mandate that the traces be covered with soil. Experimental chisels angled to the side 45◦ in order to eliminate chisel trace formation have provided control of root-knot nematodes on tomato equivalent to conventional chisels (57). Another example of minimizing atmospheric loss is through use of single chisel injections for crops traditionally fumigated with dual chisels (58). Fumigation usually involves the use of plastic tarpaulins to minimize atmospheric losses and deliver nematicide to the target organism. Sometimes, tarpaulins

NEMATICIDES

must be in place for 10 days. Even when plastic sheeting is employed, fumigant losses can exceed 50% and approach 80% under extreme conditions (55,59). A variety of injection temperatures and plastic sheeting compositions have been employed to maximize nematicidal activity and reduce atmospheric losses of methyl bromide and other fumigants. Impervious sheeting, warm temperatures, and deep injection often enhance nematicidal activity and permit the use of much smaller quantities of fumigant (41,59). A recovery system involving a double layer of polyethylene sheeting through which air is blown to a methyl bromide collection unit has reduced methyl bromide emissions in a laboratory setting (60). Buffer zones around fumigated areas are often required to reduce the exposure of the general population to airborne fumigants. Irrigation Liquid and emulsifiable formulations of nematicides can often be applied through surface or drip irrigation systems. The goal of delivering sufficient nematotoxic materials without excessive leaching is researchable but sometimes difficult to achieve (61). Drip irrigation in particular offers a means of precisely controlling the amount of active ingredient delivered to a field, as well as regulating the amount of water, so that leaching of active ingredient beyond the root zone and into groundwater can be eliminated. Drip irrigation also is useful for postplant applications, and it avoids the use of granular materials that may pose risks to birds. Use of drip irrigation also reduces the amount of personal protective equipment required for field workers. A substantial percentage of pineapple production in Hawaii is drip irrigated, and drip irrigation with ethoprop, fenamiphos, or soluble liquid formulations of 1,3-D have been used to provide control of nematodes in pineapple production in Hawaii (61). In order to minimize leaching of nematicides below the root zone and maximize effectiveness, fields are not irrigated for 2 weeks following application. Successful control of P. penetrans on lilies was provided with drip-irrigated ethoprop, fenamiphos, sodium tetrathiocarbonate, 1,3-D, and oxamyl (20); similarly, drip-irrigated emulsifiable 1,3-D provided control of the citrus nematode, Tylenchulus semipenetrans (62). Although less precise than drip irrigation in delivering nematicide to targeted areas, overhead spray irrigation can also effectively convey nematicides (63). However, injection of metam sodium into a center pivot irrigation system was associated with higher airborne concentrations of MITC than that which occurred in fields receiving metam sodium at depths of 5, 15, and 25 cm (64). Granules and Broadcast Sprays The most widely practiced method of applying nonfumigant nematicides is with granular formulations. Methods for application of nonfumigants to soil have been thoroughly reviewed (65). In some cases, adequate control can be achieved by band application of nematicides at or before sowing. In band application, plant roots may eventually grow beyond the treated area at a time when the root system will be sufficiently vigorous to not suffer serious

1109

damage. In-furrow application sometimes is practiced but may result in lack of delivery to the root zone; in other cases, in-furrow application may be preferable. In some cases, sidedress applications of nematicides are useful replacements or additions to at-plant applications. In other cases, broadcast application of granules or sprays followed by a thorough mixing of the soil may be effective. Tillage is necessary to distribute nematicide to a broad enough area to provide control, and a thorough mixing is particularly important for nematicides with poor soil mobility characteristics. Use of broadcast sprays instead of granules often promotes greater uniformity in distribution. For many annual crops, incorporating nematicides into the upper 10–15 cm of soil provides the best balance of efficacy, expense, ease, and safety to wildlife. Research on the distribution of granules to soils by various types of tillage equipment can be facilitated via the use of sepiolite granules containing a fluorescent dye (66). Nematodes are usually distributed unevenly in a given field; nematicide treatment deposits expensive chemical throughout a grower’s field, even in areas where it may not be needed. In one interesting study, Baird et al. (67) quantified the numbers of root-knot nematode juveniles at specific locations in experimental cotton fields treated with variable rates of aldicarb or 1,3-D applied with prototype equipment designed to apply nematicide at rates dependent on initial nematode population levels. Although final nematode population levels did not vary among treatments, the variable rate applications of 1,3-D (but not aldicarb) resulted in yield increases and lowered nematicide costs that justified the additional costs of nematode sampling and enumeration. Seed Dressing and Bare Root Dip The reasons why few nematicides have been registered as seed coatings include the difficulty in applying a sufficient quantity of nematicide needed to provide control beyond the seedling stage, the expense of registration relative to market size, and the attraction of such products to wildlife (65). Nonetheless, experimental formulations have provided some successes, as with control of P. penetrans on corn by seed treatment with oxamyl (68). In addition, seed-transmitted nematodes can be successfully treated with nematicidal treatment of seeds (69). Much experimental research with biocontrol organisms or nematicidal natural products is performed with seed formulations. The principle behind bare root dips is similar to that for seed dressings; i.e., sufficient nematicide is applied to transplants to protect them at a highly vulnerable time. Root dips have provided nematode control in several situations (8). NEMATICIDE ECOLOGY Effects of Temperature on Activity The effects of temperature on nematicide efficacy are complex and not well studied. Increases in temperature may stimulate the metabolic activity of the target nematode, alter the solubility of the chemical in the aqueous or vapor phases, and alter the rate of microbial

1110

NEMATICIDES

or chemical destruction of the nematicide. Because nematicides are often applied at the beginning of a growing season, low soil temperature may be of concern with respect to efficacy in some cases (70). The activity of EDB and 1,3-D against the motility and infectivity of M. javanica in fumigation chambers was much less at 5 ◦ C than at 15 ◦ C (12). Similarly, methyl bromide exhibited greater activity against the dagger nematode Xiphinema index and M. incognita at 30 ◦ C than at 15 ◦ C in soils in sealed cans (71). The enhancement of methyl bromide and 1,3-D activity against Tylenchulus semipenetrans by high temperature in controlled-temperature experiments indicated that nematicide efficacy could possibly be improved by soil solarization (72). Effects of Soil Structure on Activity The physicochemical composition of soil is a critical factor influencing nematicidal efficacy. Nematicides diffuse more slowly through soils with small pore spaces, fine particle size, and low moisture content (73). A high clay content can result in increased adsorption and poorer movement of nematicide (47,61,74). Nematicide adsorption onto organic matter is strongly correlated with lipophilicity (10); organic matter can reduce efficacy, either by increasing moisture content, by acting as an adsorbent, by providing receptors for alkylating agents, or by increasing microbial populations that are capable of degrading the applied nematicide (75). The movement of contact nematicides away from their application zone is similarly a function of adsorption onto organic matter. Fumigants, ethoprop, and fenamiphos are less effective in soils with large amounts of organic matter, but aldicarb and oxamyl are effective in soils with a wide range of organic matter concentrations (65). Riegel et al. (76) noted that 1,3-D applied to microplots supplemented with yard waste compost was less effective in suppressing M. incognita reproduction on tomato than in control microplots. Adsorption onto soil organic matter, although undesirable from the perspective of nematicide efficacy, may be negatively correlated with tendency to contaminate groundwater. Degradation of Nematicides Once applied to soils, any pesticide is subject to biological and physicochemical transformations. Transformation products may have less or greater toxicity than the parent compound. An analysis of various values reported in the literature indicated half-lives of parent compounds of 2–190 days, depending on the parent compound and the physicochemical properties of the soil (75). Nordmeyer (10) regarded a 14-day half-life as ideal for a balance between efficacy and environmental safety. In soils, 1,3-D is first biologically or chemically hydrolyzed to 3-chloroallyl alcohol, which is then oxidized to chloroacrylic acid, which in turn is converted to simple short-chain organic acids (77). Chloroallyl alcohol and chloroacrylic acid also are toxic to humans and are of regulatory concern (78). The primary route of chemical degradation of methyl bromide in soil is through hydrolysis to yield methanol and bromide ions and through methylation. Some bacteria, particularly nitrifying bacteria, are

capable of oxidizing methyl bromide to form formaldehyde and inorganic bromide (77). Aldicarb and fenamiphos are initially degraded in soils into sulfone and sulfoxide derivatives with target and nontarget toxicity and with enhanced mobility correlated with increased solubility in water (73,79). Transformation of fenamiphos sulfoxide into sulfone progresses much more rapidly in subsurface soils than in surface soils (80). Aldicarb and fenamiphos sulfoxides may be the major active materials (73,81). Aldicarb is further degraded into oximes and nitriles. The sulfoxide and sulfone derivatives of fenamiphos and aldicarb are more mobile in soils than are the parent nematicides and have the potential to more readily contaminate groundwater (82). Unlike aldicarb, the carbamate group is hydrolyzed in oxamyl. The degradation of oxamyl into nontoxic oximes at 10 different sites was generally associated with increased pH, temperature, and moisture (83). Microbial transformation of nematicides is an important factor affecting efficacy. As with other types of pesticides, repeated application of nematicides to agricultural soils can result in enhanced microbial degradation and decreased efficacy (77). For example, decreased efficacies of aldicarb, ethoprop, and oxamyl against potato cyst nematodes following multiple applications were associated with increased transformation of the nematicides (75). When previously treated soils were autoclaved, these effects did not occur. Similar phenomena have been observed in fenamiphos-treated soils; the amount of time required for enhanced degradation to disappear has been reported as being from 1 to 5 or more years, depending on the study (79,84,85). Enhanced biological degradation of 1,3-D or methyl isothiocyanate has been described in a number of soils, and various bacteria capable of mineralizing 1,3-D have been isolated (77,86,87). In at least some of these bacteria, a haloalkane dehalogenase gene carried on a plasmid is involved in enhanced degradation (86,87). One such organism (Pseudomonas cichorii) can grow on low concentrations of 1,3-D as its sole carbon and energy source (88). Enhanced microbial degradation of nematicides is a somewhat unpredictable phenomenon, has not been reported with some nematicides, and is generally unpredictable in occurrence (75,77,89). When accelerated transformation exists, the responsible microorganisms generally transform compounds chemically related to the original nematicide (75). Exceptions occur when the enhanced biodegradation occurs as a result of metabolism of a specific part of the nematicide, such as occurred in a situation when enhanced ethoprop degradation resulted from increased hydrolysis of the P−S bond in the S-propyl moiety of ethoprop (90). In this case, two strains of Pseudomonas putida capable of rapidly degrading ethoprop were isolated from the soil (91). Effects on Nontarget Organisms The nontarget effects of nematicide applications are reviewed in this volume and elsewhere; a detailed evaluation is beyond the scope of this review. Because of their broad-spectrum activities, most nematicides radically alter soil flora and fauna. Fumigant usage

NEMATICIDES

may result in the absence of nematode competitors, predators, and parasites in soils (92). The elimination of mycorrhizae by methyl bromide can result in poorer plant growth (55). Long-term aldicarb treatment of potato fields decreased the number of bacterial genera and species, decreased the population levels of plant growthpromoting rhizobacteria, and increased total bacterial biomass compared to untreated soils (93). Nematicides can greatly alter the subsequent structure of nematode communities in soils; for example, Pratylenchus recolonized methyl bromide–treated pasture soil, replacing Helicotylenchus as the dominant phytoparasitic nematode (94). Nematodes and other organisms play a complex role in agroecosystems (7); use of broad-spectrum biocides makes it difficult to exploit some of these roles. Environmental Contamination One of the greater environmental problems sometimes associated with nematicide usage is groundwater contamination. Indeed, the initial detection of the nematicides DBCP and aldicarb in groundwater in the United States over 20 years ago led to the stimulation of scientific and regulatory interest in pesticide contamination of groundwater that continues to this day (95). Even though DBCP usage was prohibited in 1977, groundwater contamination persists (96). In 1990, the manufacturer of Temik (aldicarb) announced a voluntary halt on its sale for use on potatoes because of concerns about groundwater contamination. The following year, a train wreck released 72,000 L of metam sodium into the Upper Sacramento River and resulted in soil microbial changes that persisted for at least a year (97). When the special review of 1,3-D by the U.S. EPA was terminated, several measures for reducing potential groundwater contamination were instituted, such as prohibition of usage within 100 feet of drinking-water wells, in areas overlying karst geology, and in several states with certain soil types and where groundwater is 50 feet from the soil surface (78). As previously indicated, 1,3-D use was suspended in California in 1990 for several years because of its detection in air distant from application sites, specifically in a school. This has resulted in the creation of 300-foot–wide buffer zones around residences for fumigation (100 feet wide if fields are drip irrigated). In addition, ‘‘township caps’’ limit the total amount of 1,3-D that can be used in a given area in California (98). THE FUTURE Presently, only a few chemical nematicides remain, and some of these will undoubtedly be withdrawn before the end of the decade, if not before the end of this year. The economic cost of research and registration of new chemicals is an enormous hurdle for a new chemical nematicide to overcome. Of the 497 new active ingredients registered for use as pesticides from 1967 to 1997, only seven were registered as nematicides (11). Nonetheless, the decreasing number of compounds and the enormous economic damage caused by phytoparasitic nematodes continues to maintain the interest of private and public

1111

sector researchers in pursuing the development of new chemical nematicides. In some countries, demand for nematicides is high. Although the nematicide market in the United States represents a small fraction of total pesticide usage, in The Netherlands, nematicides represent more than 60% of the total pesticides used in agriculture (13). Future control of nematodes will increasingly rely on site-specific, sustainable management practices, as well as on integrated pest management involving the judicious use of nematicides. Nonchemical strategies available to growers for some nematode-host combinations include crop rotation, altered planting time, resistant germplasm, solarization, fallow, and nematodesuppressive soil amendments. Many of these strategies are less expensive and sometimes less effective than is traditional chemical control. The development of new nematicides has been reviewed (8,10,99). Prospective compounds can originate from empirical screening or by rational design of compounds that can exploit biological or biochemical weaknesses of nematodes. The underlying biochemistry of plants and nematodes is similar in many respects; successful transfer of a rationally designed compound from laboratory to the field has not yet been achieved, in no small part because of the previously described difficulties in nematicide design. It is beyond the scope of this review to list every compound described as possessing nematotoxicity. However, the following compounds are worthy of discussion. Biorationals are listed at the conclusion. Methyl Iodide and Propargyl Bromide The immediate demand for methyl bromide replacements makes it likely that the next nematicides to be registered could be compounds similar to methyl bromide; for example, methyl iodide and propargyl bromide. The latter has provided experimental control of M. incognita on tomato, although the explosiveness of the compound requires that innovative formulations be developed (100). Methyl iodide exhibits greater toxicity to phytoparasitic nematodes than does methyl bromide, perhaps because of greater reactivity or lower volatility than methyl bromide (101), and it is degraded in the atmosphere before it has the opportunity to react with ozone (102). Because it is a liquid at ambient temperature, methyl iodide is easier than methyl bromide to apply safely. Methyl iodide has provided control of M. incognita on carrot (102), but it also eliminated Rhizobium nodules (101). DMDP One compound moving closer to agricultural utilization is 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP), a naturally occurring sugar analog from the tropical legume Lonchocarpus felipei, which inhibited hatching of G. pallida and movement of G. rostochiensis (103). The compound is downwardly mobile in plant phloem; foliar applications on tomato decreased galling induced by M. incognita. Use as a nematicide has been patented, and plans are underway to produce this compound from natural sources in tropical America.

1112

NEMATICIDES

Avermectins The avermectins are often drugs of choice for treatment of human and veterinary nematode infections. These macrocyclic lactones have experimentally provided successful control of nematodes in the field (104,105) but are not registered for use against phytoparasitic nematodes. Meloidogyne javanica and R. similis on banana were controlled by injections of abamectin into the pseudostem as well as preplant applications of fenamiphos (106). The effects of avermectin have been best documented in the mammalian intestinal parasite Ascaris and the free-living nematode Caenorhabditis elegans. Avermectin paralyzes somatic musculature in Ascaris and pharyngeal musculature in C. elegans by irreversibly opening glutamine-gated chloride channels (5,107). Sodium Azide Sodium azide is a potent inhibitor of cytochrome oxidase and disrupts the respiratory electron transport chain. It was registered as a nematicide in the United States in 1974, but its nematicidal use was withdrawn. Preplant applications provided successful control of M. incognita and Helicotylenchus dihystera on potato (108). Interest in this compound is intensifying because of the urgent need for methyl bromide replacements. Furfural Like sodium azide, furfural is being investigated as a replacement for methyl bromide. Furfural has provided control of nematodes on pineapple and cotton (109,110). Phytochemicals Several researchers are attempting to develop phytochemical-based strategies for nematode control (19). To some extent, this research has its roots in the complex chemical interactions between plants and nematodes. In addition, there has been a vast body of work involving the application of green manures to or within soils. Moreover, because members of the plant kingdom produce a variety of secondary metabolites, many investigators have ventured beyond allelopathic interactions and looked for nematode-antagonistic substances in plant parts unlikely to be involved in nematode-plant interactions, such as leaves, or in algae or fungi. A rich assortment of over 100 different secondary metabolites has been identified as being responsible for plant- or fungalmediated nematotoxicity (19). In recent years, various plant-based products have appeared with putative antinematodal activity. Most of these have not been available long enough to permit satisfactory evaluation by agricultural researchers. A few of these products may curtail nematode damage by stimulating plant growth. Systemic Acquired Resistance Inducers Systemic acquired resistance (SAR) is a phenomenon in which exposure of plants to one pathogen or elicitor can result in resistance to several diverse kinds of pathogens. A few laboratories are currently investigating the use

of SAR inducers such as salicylic acid and benzo-(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester as nematode control agents (111–113). Hatching Stimulants and Inhibitors Although not nematicidal, hatching stimulants could be used to induce hatch in the absence of host plants, resulting in the death of host-deprived nematodes. Stimulation of potato cyst nematode hatching by potato root diffusate has been investigated for decades and results from a complex mixture of at least 20 distinct compounds (114). A hatching stimulant for the soybean cyst nematode was isolated from 1058 kg of dried kidney bean roots and identified as a complex triterpenoid derivative named glycinoeclepin A (115). Two simpler analogs stimulated hatch, although at higher concentrations than required than for glycinoeclepin A (116). Two other simpler analogs were also synthesized (117); one inhibited hatch but the other stimulated it. Transgenic Proteins As with most other classes of plant pests and pathogens, transgenically based plant resistance is expected by many to provide the basis for future management of phytoparasitic nematodes. Although no transgenic system has resulted in commercial success equivalent to that of insect-resistant plants expressing Bacillus thuringiensis toxins, substantial progress is being made. For example, transgenic plants expressing a proteinase inhibitor resulted in a 50% decrease in the reproduction of M. incognita, compared to control rice plants (118). Strains of B. thuringiensis are known that produce toxins to the free-living nematode Caenorhabditis elegans (119). Behavior-Modifying Compounds A variety of behaviors are involved in host- and matefinding by nematodes. The only nematode compound with sex attractant activity is vanillic acid, which is produced by soybean cyst nematode females. Several synthetic analogs did lower cyst production in field and microplot experiments (120). The possibility of using specific compounds to attract nematodes to toxic baits was shown in laboratory experiments with T. semipenetrans, and three different nematicides whose activity was increased by the attractant sodium acetate (121). When precise molecular interactions between nematodes and their hosts important to parasitism are discovered, these could be exploited. Steroids and Hormones Nematodes possess a nutritional requirement for sterols; dietary sterols are converted to sterols typical of nematodes. Several compounds interfere with the conversion of plant sterols to nematode sterols and disrupt the nematode life cycle (122). The identification of a nematode hormone has not been achieved, a necessary first step to permit their exploitation in a manner similar to that of insect juvenile and molting hormones.

NEMATICIDES

Acknowledgments Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

BIBLIOGRAPHY 1. N. A. Cobb, U.S. Department of Agriculture Yearbook, 1914, pp. 457–490. 2. W. B. Thomas, Suppl. J. Nematol. 28: 586–590 (1996). 3. W. C. Campbell, J. Parasitol. 72: 45–61 (1986). 4. R. J. Martin, Parasitology 114: S111–S124 (1997). 5. P. Köhler, Int. J. Parasitol. 31: 336–345 (2001). 6. J. N. Sasser and D. W. Freckman, in J. A. Veech and D. W. Dickson, eds., Vistas in Nematology, Society of Nematologists, Hyattsville, MD, 1987, pp. 7–14. 7. K. R. Barker and S. R. Koenning, Annu. Rev. Phytopathol. 36: 165–205 (1998). 8. N. G. M. Hague and S. R. Gowen, in R. H. Brown and B. R. Kerry, eds., Principles and Practice of Nematode Control in Crops, Academic Press, Sydney, Australia, 1987, pp. 131–178. 9. R. A. Sikora and J. Hartwig, Rev. Nématol. 24: 531–536 (1991). 10. D. Nordmeyer, in F. J. Gommers and P. W. T. Maas, eds., Nematology: From Molecule to Ecosystem, European Society of Nematologists, Invergowrie, Scotland, 1992, pp. 281–293.

1113

27. N. A. Minton, T. B. Brenneman, K. Bondart, and G. W. Harrison, Peanut Sci. 20: 66–70 (1993). 28. J. R. Rich et al., Suppl. J. Nematol. 26: 701–704 (1994). 29. B. S. Sipes, Fruits 51: 173–177 (1996). 30. P. Warrior et al., Pestic. Sci. 55: 376–379 (1999). 31. U. Twomey, P. Warrior, B. R. Kerry, and R. N. Perry, Nematology 2: 355–362 (2000). 32. R. N. Perry, U. Twomey, R. N. Rolfe, Aspects Appl. Biol. 59: 53–58 (2000).

and

P. Warrior,

33. T. D’Addabbo, Nematol. Medit. 23: 299–305 (1995). 34. G. Y. Osman and F. M. Salem, Anz. Schadlingskde. ¨ Pflanzenschutz Unweltschutz 68: 179–181 (1995). 35. A. A. Farahat, A. A. Osman, H. I. El-Nagar, and H. H. Hendy, Bull. Fac. Agric. Univ. Cairo 44: 191–204 (1993). 36. H. Banaszak and M. Jagusz, Prog. Plant Prot. 39: 154–159 (1999). 37. Medina, R. Crozzoli, and D. Rivas, Fitopatol. Venez. 5: 26–29 (1992). 38. B. S. Sipes and K. M. Delate, Nematropica 26: 171–175 (1996). 39. A. A. F. Evans, Ann. Appl. Biol. 75: 469–473 (1973). 40. D. J. Wright, in B. M. Zuckerman and R. A. Rohde, eds., Plant Parasitic Nematodes, Vol. III, Academic Press, New York, 1981, pp. 421–449. 41. O. C. Macdonald and C. Reichmuth, in C. H. Bell, N. Price, and B. Chakrabarti, eds., The Methyl Bromide Issue, Wiley, Chichester, U.K., 1996, pp. 150–189.

11. A. L. Aspelin and A. H. Grube, Pesticides Industry Sales and Usage, 1996 and 1997, U.S. Environmental Protection Agency, Publication 733–R-99-001, 1999.

42. B. S. Sipes and D. P. Schmitt, in K. R. Barker, G. A. Pederson, and G. L. Windham, eds., Plant and Nematode Interactions, American Society of Agronomy, Madison, WI, 1998, pp. 173–185.

12. M. V. McKenry and I. J. Thomason, Hilgardia 42: 422–438 (1974).

43. D. Nordmeyer and D. W. Dickson, Rev. Nématol. 23: 311– 316 (1990).

13. C. H. Schomaker and T. H. Been, Nematology 1: 19–29 (1999).

44. D. J. Pree, J. L. Townshend, and D. E. Archibald, J. Nematol. 19: 188–193 (1987).

14. L. A. Thrupp, Int. J. Health Serv. 21: 731–757 (1991).

45. D. Nordmeyer, D. W. Dickson, L. T. Ou, and H. L. Cromroy, Pestic. Biochem. Physiol. 34: 179–184 (1989).

15. D. E. Munnecke and S. D. Van Gundy, Annu. Rev. Phytopathol. 17: 405–429 (1979). 16. A. S. Csinos et al., Crop Prot. 19: 39–49 (2000).

46. S. Chang and C. H. Opperman, Mol. Biochem. Parasitol. 49: 205–214 (1991).

17. S. J. Locascio et al., HortScience 32: 1208–1211 (1997).

47. A. G. Whitehead, Ann. Appl. Biol. 75: 439–453 (1973).

18. E. M. Smedley, J. Helminthol. 17: 31–38 (1939). 19. D. J. Chitwood, Annu. Rev. Phytopathol. 40: 221–249 (2002).

48. C. H. Opperman, in F. J. Gommers and P. W. T. Maas, eds., Nematology: From Molecule to Ecosystem, European Society of Nematologists, Invergowrie, Scotland, 1992, pp. 60–72.

20. B. B. Westerdahl et al., Suppl. J. Nematol. 25: 758–767 (1993).

49. D. J. Pree, J. L. Townshend, and K. J. Cole, J. Nematol. 22: 182–186 (1990).

21. F. A. Norris, E. G. Jordan, and A. Guardigli, Anal. Meth. Pestic. Plant Growth Regulators 16: 3–20 (1988).

50. J. Hartwig and R. A. Sikora, Rev. Nématol. 14: 525–530 (1991).

22. P. Quénéherve, T. Mateille, and P. Topart, Rev. Nématol. 14: 221–229 (1991). 23. M. A. McClure and M. E. Schmitt, Suppl. J. Nematol. 28: 624–628 (1996).

51. M. Berenbaum, Committee on the Future Role of Pesticides, National Academy of Sciences, The Future Role of Pesticides in U.S. Agriculture, National Academy Press, Washington, D.C., 2000, p. 48.

24. T. Koyanagi, O. Imai, and K. Yoshida, J. Pestic. Sci. 23: 174–183 (1998).

52. M. Moens and G. Hendrickx, Fund. Appl. Nematol. 21: 199–204 (1998).

25. J. Kimpinski, Suppl. J. Nematol. 29: 685–689 (1997).

¨ ¨ Nematologica 41: 449–467 (1995). 53. L. Kampfe and H. Schutz,

26. M. P. Pullen and B. A. Fortnum, Suppl. J. Nematol. 31: 694–699 (1999).

54. I. Glazer, L. Salame, and D. Segal, Biocontrol Sci. Technol. 7: 499–512 (1997).

1114

NEMATICIDES

55. L. Klein, in C. H. Bell, N. Price, and B. Chakrabarti, eds., The Methyl Bromide Issue, Wiley, Chichester, U.K., 1996, pp. 191–235.

85. J. H. Smelt, A. E. van de Peppel-Groen, L. J. T. van der Pas, and A. Dijksterhuis, Soil Biol. Biochem. 28: 1757–1765 (1996).

56. Anonymous, Methyl Bromide Alternatives, Vol. 7, USDA, Washington, D.C., 2000, pp. 5–6.

86. C. Verhagen, E. Smit, D. B. Janssen, and J. D. van Elsas, Soil Biol. Biochem. 27: 1547–1557 (1995)

57. C. Riegel et al., Nematropica 31: 291–295 (2001).

87. C. Verhagen, G. Lebbink, and J. Bloem, Soil. Biol. Biochem. 28: 1753–1756 (1996).

58. R. C. Schneider et al., Pestic. Sci. 30: 243–257 (1990). 59. D. Wang et al., Environ. Sci. Technol. 31: 3017–3022 (1997).

88. G. J. Poelarends et al., Appl. Environ. Microbiol. 64: 2931– 2936 (1998).

60. D. E. Chitwood and M. A. Deshusses, Environ. Sci. Technol. 35: 636–642 (2001).

89. A. W. Johnson, Suppl. J. Nematol. 30: 40–44 (1998).

61. R. C. Schneider, R. E. Green, and W. J. Apt, Acta Hort. 334: 351–360 (1993). 62. D. Wang and S. R. Yates, Pestic. Sci. 55: 154–160 (1999). 63. A. W. Johnson, Suppl. J. Nematol. 26: 697–700 (1994). 64. I. A. M. Saeed, D. I. Rouse, and J. M. Harkin, Pest Manag. Sci. 56: 813–817 (2000). 65. A. G. Whitehead, Brit. Crop Prot. Counc. Monograph 39: 309–317 (1988). 66. S. Woods et al., Soil Tillage Res. 51: 17–23 (1999). 67. R. E. Baird, J. R. Rich, and D. Waters, Nematol. Medit. 29: 247–254 (2001). 68. M. Chiba, G. J. Fulop, B. D. McGarvey, and J. W. Potter, J. Agric. Food Chem. 41: 2160–2163 (1993). 69. R. Tacconi, R. Santi, and R. Gironi, Nematol. Medit. 27: 291–293 (1999). 70. I. G. Grove and P. P. J. Haydock, Aspects Appl. Biol. 59: 103–108 (2000).

90. D. G. Karpouzas and A. Walker, Pest Management Sci. 56: 540–548 (2000). 91. D. G. Karpouzas and A. Walker, J. Appl. Microbiol. 89: 40–48 (2000). 92. B. S. Sipes and D. P. Schmitt, Suppl. J. Nematol. 27: 639–644 (1995). 93. A. V. Sturz and J. Kimpinski, Plant Pathol. 48: 26–32 (1999). 94. G. W. Yeates and H. van der Meulen, Biol. Fert. Soils 21: 1–6 (1996). 95. S. Z. Cohen, J. Environ. Sci. Health B31: 345–352 (1996). 96. L. A. Soutter and K. Loague, J. Environ. Qual. 29: 1794– 1805 (2000). 97. G. E. Taylor et al., Environ. Toxicol. Chem. 15: 1694–1701 (1996). 98. J. Carpenter, L. Lynch, and T. Trout, Calif. Agric. 55(3): 12–18 (2001).

71. N. Abdalla and B. Lear, Plant Dis. Reptr. 59: 224–228 (1975).

99. J. Feldmessr, J. Kochansky, H. Jaffe, and D. Chitwood, in J. L. Hilton, ed., Agrochemicals of the Future, Rowman and Allanheld, Totowa, NJ, 1985, pp. 327–344.

72. S. Xue, J. Gan, S. R. Yates, and J. O. Becker, Pest Management Sci. 56: 737–742 (2000).

100. J. W. Noling and J. P. Gilreath, USDA Methyl Bromide Alternatives Vol. 6: 9–10 (2000).

73. R. H. Bromilow, Ann. Appl. Biol. 75: 473–479 (1973).

101. J. O. Becker et al., Pestic. Sci. 52: 58–62 (1998).

74. S. Q. Zheng et al., Sci. Total. Environ. 156: 1–9 (1994).

102. C. M. Hutchison et al., Nematology 14: 407–414 (1999).

75. J. H. Smelt and M. Leistra, in F. J. Gommers and P. W. T. Maas, eds., Nematology: From Molecule to Ecosystem, European Society of Nematologists, Invergowrie, Scotland, 1992, pp. 266–280.

103. A. N. E. Birch et al., Nematologica 39: 521–535 (1993).

76. C. Riegel et al., J. Nematol. 33: 289–293 (2001). 77. L.-T. Ou, J. Nematol. 30: 56–64 (1998). 78. U.S. Environmental Protection Agency, Fed. Reg. 66: 58468–58472 (2001). 79. L.-T. Ou, Soil. Sci. Soc. Am. J. 55: 716–722 (1991). 80. R. S. Kookana, C. Phang, and L. A. G. Aylmore, Aust. J. Soil Res. 35: 753–761 (1997). 81. R. F. Davis, R. D. Wauchope, and A. W. Johnson, J. Nematol. 26: 511–517 (1994). 82. E. Loffredo, N. Senesi, V. A. Melilli, and J. Environ. Sci. Health B26: 99–113 (1991).

F. Lamberti,

104. J. N. Sasser, T. L. Kirkpatrick, and R. A. Dybas, Plant Dis. 66: 691–693 (1982). 105. K. Blackburn, S. R. Alm, and T. S. Yeh, Suppl. J. Nematol. 28: 687–694 (1996). 106. R. K. Jansson and S. Rabatin, Suppl. J. Nematol. 29: 695– 702 (1997). 107. L. Avery and J. H. Thomas, in D. L. Riddle, T. Blumenthal, B. J. Meyer, and J. R. Preiss, eds., C. elegans II, Cold Spring Harbor Laboratory Press, Plainview, NY, 1997, pp. 679–716. ´ 108. R. Rodr´ıguez-Kabana and P. A. Backman, Plant Dis. Reptr. 59: 528–532 (1975). 109. B. S. Sipes, Acta Hort. 425: 457–464 (1997). 110. E. M. Bauske et al., Nematropica 24: 143–150 (1995).

83. E. Ambrose, P. P. J. Haydock, and A. Wilcox, Aspects Appl. Biol. 59: 41–51 (2000).

111. B. Chinnasri, B. S. Sipes, and D. P. Schmitt, J. Nematol. 33: 253 (2001).

84. J. P. E. Anderson and A. Lafuerza, in J. P. E. Anderson, D. J. Arnold, F. Lewis, and L. Torstensson, eds., Proc. Int. Symp. Environ. Aspects Pestic. Microbiol, Sigtuna, Sweden, 1992, pp. 184–192.

112. V. N. Kempster, K. A. Davies, and E. S. Scott, Nematology 3: 35–43 (2001). 113. B. Nandi, N. C. Sukul, and S. P. Sinha Babu, Allelopathy J. 7: 285–288 (2000).

NITRATE IN GROUNDWATER 114. K. J. Devine, J. Byrne, N. Maher, and P. W. Jones, Ann. Appl. Biol. 129: 323–334 (1996).

1115

120. S. L. F. Meyer et al., J. Nematol. 29: 282–288 (1997).

this period. Environmental concern has centered mainly on the formation of algal blooms and excessive growth of water plants in surface fresh waters and in the coastal areas of the sea. Worries about our health spring from fears that nitrate in potable water might cause stomach cancer in adults or methemoglobinemia (‘‘blue-baby’’ syndrome) in infants. Recent medical research, however, suggests not only that nitrate is beneficial to our health but also that we produce it within our bodies. Water supplies are drawn from both ground and surface waters according to their availability. This article is concerned with nitrate in groundwater, which has health, rather than environmental, implications, but environmental issues are not ignored.

121. L. W. Duncan and M. M. Abou-Setta, Nematropica 25: 173– 175 (1995).

NOMENCLATURE

115. A. Fukuzawa, A. Furusaki, M. Ikura, and T. Masamune, J. Chem. Soc. Chem. Commun. 1985: 222–224,748 (1985). 116. A. Miwa, Y. Nii, H. Okawara, and M. Sakakibara, Agric. Biol. Chem. 51: 3459–3461 (1987). 117. G. A. Kraus, B. Johnston, A. Kongsjahju, and G. L. Tylka, J. Agric. Food Chem. 42: 1839–1840 (1994). 118. P. Vain et al., Theor. Appl. Genet. 96: 266–271 (1998). 119. J. S. Griffitts, J. L. Whitacre, D. E. Stevens, and R. V. Aroian, Science 293: 860–864 (2001).

122. D. J. Chitwood, Crit. Rev. Biochem. Mol. Biol. 34: 273–284 (1999).

FURTHER READING Barker, K. A., Pederson, G. A., and Windham, G. L., eds., Plant and Nematode Interactions, American Society of Agronomy, Madison, WI, 1998. Brown, R. H. and Kerry, B. R., eds., Principles and Practice of Nematode Control in Crops, Academic Press, Sydney, Australia, 1987. Perry, R. N. and Wright, D. J., eds., The Physiology and Biochemistry of Plant-Parasitic and Free-Living Nematodes, CAB International, Wallingford, U.K., 1998. Sharma, S. B., ed., The Cyst Nematodes, Kluwer Academic Publishers, Dordrecht, 1998. Whitehead, A. G., Plant Nematode Control, CAB International, Wallingford, UK, 1998.

NITRATE IN GROUNDWATER THOMAS ADDISCOTT Rothamsted Experimental Station Harpenden, Herts, United Kingdom

INTRODUCTION The nitrate ion is one of the more ubiquitous chemical substances on the planet and is nearly always found in water. Most of the water around us contains nitrate, but the water with which we are concerned here is groundwater, which is water accumulated in the saturated zones of certain rock formations, usually at depth. Most of this water has passed through the soil before it accumulates, so that activities at the soil surface, particularly agriculture, can have a strong influence on the concentrations of nitrate and other agrochemicals in groundwater. Despite its commonplace nature, nitrate has for at least two decades been a source of widespread concern because of its perceived effects on our environment and our health. As a result, the ‘‘nitrate problem’’ has been a major influence on agroecological research in the developed world during

‘‘Nitrate’’ is the chemical name for the NO3 − ion, and it is not known by any other. The practice of referring to ‘‘nitrates’’ in natural waters and water supplies is incorrect because, as in all dilute electrolyte solutions, the anions and cations are dissociated from each other. The species with which we are concerned is, therefore, the free nitrate ion, which is unique rather than plural. Structural Formula The nitrate ion, NO3 − , has a symmetrical planar trigonal structure in which the nitrogen atom has a formal positive charge. Two negative charges are shared between the three oxygen atoms in a resonance structure comprising three electronic conformations in which each of the oxygen atoms, in turn, is without charge. The uncharged atom has two electron pairs and is attached to the nitrogen atom by a π -bond, and the charged atoms have three electron pairs. PHYSICAL PROPERTIES Solubility The salts formed by the nitrate ion are generally soluble, and calcium nitrate has such a high affinity for water that it is deliquescent, which means that it will pick up moisture from the air and dissolve in it. The main cations in groundwater are likely to be calcium, magnesium, potassium, sodium, iron, and aluminium, and the salts they form with nitrate are all very soluble (Table 1). Ammonium nitrate is also highly soluble. Calcium is usually the dominant cation in groundwater, and the nitrate concentration at the limit of solubility for calcium nitrate is 32,000 times greater than the U.S. limit for nitrate concentration in potable water and 28,000 times greater than the E.C. limit. Solubility cannot, therefore, limit nitrate concentrations in groundwater. Sorption Nitrate, being an anion, is attracted to positively charged surfaces. Nearly all agricultural soils in the developed world are usually maintained at pH values that are not acid enough to permit the development of the positive charges that will retain nitrate. However, there are some soils, particularly highly weathered soils in the Tropics,

1116

NITRATE IN GROUNDWATER

Table 1. Solubilities in Cold Water1 of the Salts of the Nitrate Ion (26) Solubility (g m−3 ) Salt

Nitrate2

Ca(NO3 )2 · 4H2 O

2.66 × 106

1.40 × 106

Mg(NO3 )2 · 6H2 O

1.25 × 106

0.61 × 106

KNO3

0.32 × 106

0.18 × 106

NaNO3

0.92 × 106

0.67 × 106

NH4 NO3

1.18 × 106

0.91 × 106 0.36 × 106

Cation Ca++ Mg

++

+

K

Na

+

NH4 + ++

Fe(NO3 )2 · 6H2 O

0.84 × 106

+++

Fe(NO3 )3 · 6H2 O

1.50 × 106

0.80 × 106

+++

Al(NO3 )3 · 9H2 O

0.64 × 106

0.32 × 106

Fe Fe Al

Salt

1 The temperatures at which the solubilities in the table (26) had been determined were not all the same and ranged from 0 K to 25 K. 2 The ‘‘nitrate’’ concentration is that corresponding to the solubility of the salt.

which are sufficiently acid for nitrate retention to occur. In the absence of clear evidence that the soil is positivelycharged, it will be advisable to assume that sorption, like solubility, does nothing to limit nitrate concentrations in groundwater. Other Properties Nitrate and its salts do not exert a vapor pressure. The melting point of each salt depends on the cation in the salt. AGRICULTURAL USES Nitrate is used in agriculture solely as a constituent of fertilizers. Formulations Guano, the oldest form of nitrate fertilizer, is the accumulated excrement of sea-birds and is found most notably on the cliffs of the Peruvian coast and nearby islands. Because of the marine origin of this material, the main cation associated with the nitrate is sodium. Pure sodium nitrate would contain 16.5% of N, but guano contains a smaller and somewhat variable percentage of N. Ammonium nitrate is widely used as a nitrogen fertilizer, particularly in Europe, because both the cation and the anion contain nitrogen, so the pure salt has 36% of N. The solid fertilizer contains fillers and stabilizers for quality and safety reasons (see also the discussion of Reactivity in the Chemistry section) and usually has a stated N content of 33% to 35.5%. Ammonium nitrate is also used in liquid fertilizer formulations, often in combination with urea (see below). Calcium ammonium nitrate is also used as a fertilizer. It contains 26% to 28% of N, depending on the manufacturer. We also need to note three other fertilizers, urea, ammonium sulphate, and directly injected ammonia, which do not contain nitrate but are transformed to nitrate by soil microbes.

Urea is a very useful fertilizer where transport is a problem because it contains 46% of N, more than any other solid N source, so that the least possible noneffective weight has to be carried. Nearly half the world’s fertilizer production is as urea. Urea is converted to ammonium by the Urease enzyme, which is very widespread. Ammonium sulfate used to be a popular fertilizer because it was a cheap by-product of gas production from coal. Because of the increased use of natural gas in many countries, its use has declined during the last 20 years. Injected ammonia. Ammonia liquified under pressure can be taken to the field and injected directly into the soil, usually to a depth of about 100 mm. The machinery used cuts a slot in the soil with a disc. The nozzle feeding the liquified ammonia is directly behind the disc and is followed by a flat wheel, which closes the slot. This is a very efficient source in that ammonia is 82% N, but the specialized equipment needed for storing and injecting the ammonia tends to localize its use. Compound Fertilizers All the above sources of nitrogen are described as ‘‘straight’’ because they supply nitrogen alone and do not provide phosphate or potassium, the other two major nutrients. In compound fertilizers, nitrogen is mixed, usually in granules but sometimes in liquid form, with either or both of these nutrients and occasionally others. The nutrient composition of a compound depends on the crop for which it is manufactured. Cereal crops, for example, need a large proportion of nitrogen, whereas potatoes need more phosphate and potassium than other crops (see Biological role discussion in the Chemistry section). Ammonium phosphate is often a constituent of compound fertilizers. The diammonium phosphate seems to be the more widely used. Saltpeter, natural potassium nitrate, could have been an early compound fertilizer because it contains two of the three main nutrients needed by plants. However, it was not greatly used as a fertilizer in the past, almost certainly because it was more valuable as a preserving agent for meat and as a constituent of gunpowder. It is still not used on a wide scale, although it may be used in foliar applications. Table 2 shows world fertilizer nitrogen consumption in 1994–1995 for the various sources of nitrogen. More recent figures were not found, but overall consumption of

Table 2. World Consumption in 1994/1995 of Nitrogen in Various Types of Fertilizer (27) Type of Fertilizer Ammonium nitrate Calcium ammonium nitrate Urea Ammonium sulphate Injected ammonia N solutions Other straights Compound fertilizers Total

N Consumed (Tonnes) 6.58 × 106

3.69 × 106 31.57 × 106 2.37 × 106 4.20 × 106 3.79 × 106 9.05 × 106 11.68 × 106 72.93 × 106

% of Total 9.0 5.1 43.3 3.2 5.8 5.2 12.4 16.0 100.0


nitrogen fertilizer has increased by about 9% since then, probably without much change in the ratios between the sources. CHEMISTRY Reactivity Nitrate is the most fully oxidized compound of nitrogen and is, therefore, stable to oxidation but potentially a strong oxidizing agent. Saltpeter (potassium nitrate) has long been the oxidizing constituent of constituent of gunpowder. Solid ammonium nitrate can explode because the nitrate moiety can oxidize the ammonium moiety. Mixed with aluminium powder it formed Ammonal, one of the most widely used explosives in the Second World War. There is, however, an important difference between the salts of nitrate in the solid and dissolved states. Because of the stability conferred by the resonance structure of the ion, nitrate in a near neutral dilute solution of its dissociated salts (as found in groundwater) is unreactive chemically. Its biological reactivity is discussed below. Synthesis and Manufacture Synthetic nitrate is manufactured (1) in two main stages, ammonia (NH3 ) being produced and then oxidized to nitrate. Ammonia has long been synthesized from nitrogen and hydrogen in the Haber process in which the two elements are reacted over a catalyst at high temperature and pressure. Modern methods of production often involve the steam reforming of natural gas, in which the methane (CH4 ) from the natural gas and the steam (H2 O) react with the air to give carbon dioxide (CO2 ) and hydrogen (H2 ). Production often involves a secondary reforming process. Nitrogen (N2 ) left from the air reacts with the hydrogen over a nickel catalyst to give ammonia (NH3 ). Sulpfur and oxygen compounds (particularly the CO2 ) have to be removed before the reaction over the catalyst, because they inhibit its activity. Other sources of carbon and hydrogen, such as naphtha, oil, and coal can be used but give poorer energy efficiency than natural gas. The ammonia is oxidized to nitric acid over a platinum catalyst, which is alloyed with rhodium for strength, and the nitric acid is reacted either with ammonia to give ammonium nitrate or with the appropriate oxide, hydroxide, or carbonate to provide the nitrate salt required. The ammonia may also be reacted with phosphoric acid to give ammonium phosphates, which are also fertilizer materials. Biological Nitrate Production Because nitrate is chemically stable and cannot be oxidized further, it is the end product of a key biological nitrogen chain in the soil (2,3). The topsoil (first 250 mm of the soil) contains large quantities of nitrogen, often of the order of 5,000 kg ha−1 , in organic forms (‘‘organic’’ is used here in its original chemical sense of ‘‘pertaining to the special chemistry of carbon’’ rather than in that of recent farming philosophy). The organic carbon and

1117

nitrogen come from the debris of green plants, including dead roots, and dead tops where they are not harvested, exudates from roots, and animal excreta. This organic matter is colonized by soil organisms of various sizes, ranging from earthworms, through springtails and mites, to bacteria and fungi. They form a chain in which the largest organisms make the organic matter more available to the smallest but also predate on them. The process is described as mineralization, because the end products are the most highly oxidized forms of carbon and nitrogen, carbon dioxide and nitrate, which are in the realm of inorganic—or mineral—chemistry. The final stage of the process for nitrogen, the oxidation of ammonium to nitrate, is important where ammonium fertilizers are used. It proceeds in two stages (2,3), each of which is effected by a chemoautotrophic bacterium. First, ammonium is oxidized to nitrite by Nitrosomonas species: 2NH4 + + 3O2 −−−→ 2NO2 − + 4H+ + 2H2 O + energy, (1) and then the nitrite is oxidized to nitrate by Nitrobacter species: 2NO2 − + O2 ←−−→ 2NO3 − + energy.

(2)

Some nitrous oxide (N2 O) is formed during the second stage. The mineralization of organic nitrogen is an entirely natural process, and it cannot be controlled to more than a limited extent because the nitrate is produced in the soil without any human intervention. Measurements made at Rothamsted from 1877 to 1915 and summarized more recently (4) show that, even back in the 1870s, nitrate concentrations in water draining from an uncropped, unploughed soil that had received no nitrogen fertilizer for at least 10 years exceeded the present day U.S. and E.C. limits for potable water of 44 g and 50 g m−3 . During the 38 years of the study, the soil lost more than 1,000 kg ha−1 of nitrogen from its organic matter, all of which emerged as nitrate in the drainage from the soil. Losses of nitrate produced by mineralization need to be considered in any discussion of groundwater issues because there is evidence (5) that they are usually greater than direct losses from fertilizer. Adventitious Nitrate Production Many industrial processes emit substantial quantities of oxides of nitrogen to the atmosphere. Further emissions of this kind come from vehicles with internal combustion engines, which cause a reaction between the nitrogen and oxygen of the atmosphere by compressing them and subjecting them to high temperature and a spark. Industry and motor traffic each generates about half of these man-made nitrogen oxides. Nitrogen oxides are also produced naturally by lightning (6). Other activities, notably farming, emit ammonia to the atmosphere, and this is readily converted to nitrate, particularly when it reaches the soil. Between 1877 and 1915, during the experiments mentioned in the previous section, about 6 kg ha−1 of mineral nitrogen, as ammonium and nitrate, was deposited in

1118


rainfall at Rothamsted each year. By 1990, measurements at four sites in southeast England showed an annual deposition of 35–40 kg ha−1 (7), but these measurements included deposition of nitrate on particulate matter and dry deposition of nitrogen oxides, nitric acid, and ammonia in addition to ammonium and nitrate in rain. A more recent estimate (8) suggests that about 37 kg ha−1 of nitrogen is deposited annually on bare soil and 48 kg ha−1 on soil carrying the extra deposition area supplied by a winter wheat crop. The latter amount is one-quarter of the average application of nitrogen fertilizer in England and Wales. This deposited nitrogen probably contributes 10–15 kg ha−1 of nitrate-N to annual losses from the soil. Biological Role Nitrogen is vital to the growth of plants. It is part of all the essential constituents of cells, including the chlorophyll needed for photosynthesis; the DNA and RNA, which encode the plant’s program for growth and development; the proteins—including the enzymes, which catalyze all biochemical processes; and the cell walls, which do more than just hold the cell together. All plant nutrients increase the growth and yield of crop plants but nitrogen has the largest effect, except in plants that form large storage organs such as potato tubers. These plants store large amounts of phosphorus and potassium in their storage organs and, therefore, have a larger demand for these nutrients. The nitrate ion is usually the main form of nitrogen taken up by the plant’s roots from nonacid aerobic soils, although ammonium ions can also contribute to its uptake. The ammonium ion predominates in anaerobic soils such as those found in rice paddies and may be the main form taken up there. The form of nitrogen supplied has a considerable effect on the cation-anion balance of the plant and, hence, its growth (9). As might be expected, supplying nitrogen as the NH4 cation rather than the NO3 anion lessens the uptake of other cations, particularly potassium. The apparent preference of many plants for nitrate rather than ammonium in aerobic soils probably reflects not only the effects of charge but also the ubiquity of nitrate in soils and the sorption by nonacid soils of ammonium but not nitrate. ENVIRONMENTAL FATE The fate of nitrate and the type of environmental problem it causes depend on two main processes, leaching and denitrification. Leaching Nitrate is leached from the soil because neither solubility nor sorption withholds it from the water passing through the soil, so, whenever water moves, nitrate tends to move with it. The only restriction on such movement is afforded by the structure of the soil, which holds some water in aggregates and larger structural units (10). This water does not move appreciably, so any nitrate within it is temporarily safe from leaching. Nitrate may be in the immobile water because it was produced within

the structural unit or because it diffused into it. Inward diffusion would result from a greater nitrate concentration on the outside than on the inside and could be the result of fertilizer application. Once the concentration outside the structural unit has fallen below that on the inside, nitrate diffuses out into water that may move downwards, but the ‘‘hold-back’’ provided by the soil structure can help usefully to restrict nitrate concentrations in water draining from the soil. The effect is greatest in the soils with the bestdefined soil structure, which are usually clay soils. The environmental problems caused by nitrate leaching spring from the fact that it is not only land plants that use nitrogen for extra growth. Plants growing in water respond too, and their extra growth is usually not welcome. Increased nitrate concentrations caused by leached nitrate in rivers and lakes encourage reeds to grow to excess, narrowing waterways and potentially overloading and damaging banks. The proliferation of underwater plants fouls the propellers of boats, entangles the tackle of fishermen, and blocks water supply conduits, thereby damaging machinery. The large plants are usually not as great a problem as algal blooms. The blue-green algae are very small singlecelled plants of the Cyanobacteria species that grow on the surface of practically anything, including water (11). Some of them are toxic, and others are a problem because of buoyancy conferred by the gas vesicles they contain, which enable them to rise to the surface of the water during calm conditions. The resulting ‘‘bloom’’ or ‘‘scum’’ is often blown by even gentle breezes to the edge of the lake or river where it is particularly unpleasant—and a hazard if it is toxic. A further problem is that when algae die, the bacteria that consume them use oxygen to do so, and this lessens the supply to fish and other desirable organisms, which may die as a result. Algal blooms and other problems can also occur in the sea, particularly in partially enclosed water bodies such as the Gulf of Mexico and the Baltic Sea, which are not flushed by strong currents. Algal blooms are usually triggered in fresh water by the phosphate concentration to which they are sensitive over five orders of magnitude of concentration (11,12), but they also depend on the presence of nitrate. They are probably more sensitive to nitrate in the sea. Denitrification Nitrate is, as noted above, the most fully oxidized compound of nitrogen, and, when certain microbes in the soil need oxygen and are unable to get enough, they take it from nitrate ions. The reduction is effected by facultative anaerobic bacteria, mainly of the Pseudomonas and Bacillus species once the partial pressure of oxygen has become low (8), there may be measurable concentrations of NO2 − , an intermediate in several nitrogen cycle processes. Nitrogen associated with the solid matrix of the soil is largely insoluble organic matter, although soluble forms can be held on cation and anion exchange sites of the solid matrix and NH4 + can be sequestered, or fixed, within the interlayers of certain 2 : 1 clay minerals, such as the illites. Soil organic nitrogen typically comprises most (>90%) of the total soil nitrogen and exists in numerous forms, many of which defy clear chemical characterization. Those forms of organic nitrogen that can be identified are termed nonhumic substances, and include fragments of nucleic acids, peptides, and oligomers of amino sugars found in microbial cell walls. The bulk of soil organic nitrogen is made up of humic substances, nitrogenous compounds of varying size and complexity, which make up the amorphous organic material in soil. Historically, several chemical-fractionation schemes, on the basis of solubility in organic solvents or inorganic acids and bases, have been used to characterize soil organic nitrogen and assess its availability to plants. These methods, which have been greatly enhanced by modern spectroscopic methods, have provided some insight into the chemical nature of soil organic nitrogen but have not been particularly useful in explaining the biological availability of soil organic nitrogen. It should be noted that generally 2 to 5% of soil organic nitrogen is found in the living microbial biomass, which represents a source of organic nitrogen that is biologically active and turns over relatively quickly as will be discussed later. THE NITROGEN CYCLE AND NITROGEN BALANCES The conversions of nitrogen from one form to another are generally depicted in a diagram consisting of boxes, which represent the several pools of nitrogen, connected by arrows, which represent the various biological processes that transform nitrogen into its different forms. Such abstractions of the nitrogen cycle can be quite simple, showing only the major pools and transformations, or can be exceedingly intricate, showing all pools and known reactions. A relatively simple nitrogen cycle model often suffices to represent the nitrogen balance for a given system, whether

Table 1. Amounts of Important Forms of Soil Nitrogen (1). For Comparison, a Typical Agronomic Crop Might Contain About 25 g N m−2 Nitrogen Form N2 N2 O NH4 + NO3 − Organic N

Concentration

Contenta (g N m−2 )

Relative Fraction (%)

78 kPa ≥31 mPa 560 mg/m3 air. ADI is 0.025 mg/kg. Azamethiphos is metabolized via the cleavage of phosphorothiolate C−S bond, followed by N-demethylation and opening of the oxazolinone ring and conjugation of the resultant aminochloropyridinol as the glucuronide and sulfate ester. AZINPHOS-ETHYL

S (C2H5O)2P

N S

CH2

N

N O

IUPAC name. S-(3,4-Dihydro-4-oxobenzo[d]-[1,2,3]-triazin-3-ylmethyl) O,O-diethyl phosphorodithioate CASRN. 2642-71-9 Selected trade names. Ethyl guthion, Gusathion A, Crysthion Azinphos-ethyl is synthesized by condensation of O,O-diethyl phosphorodithioic acid with N-chloromethylbenzazimide or directly from benzazimide, formaldehyde, and the phosphorodithioic acid [refer to (10)]. It forms colorless needles, mp 50 ◦ C, bp 147 ◦ C/1.3 Pa, vp 0.32 mPa (20 ◦ C). It is nearly insoluble in water 4.5 mg/L but readily soluble in common organic solvents except aliphatic hydrocarbons. Log Kow = 3.18. It is rapidly hydrolyzed in alkaline and acid media; DT50 (22 ◦ C) at pH 4, 7, and 9 are about 3 h, 270 d, and 11 d, respectively. The half-life in soils is several weeks. Azinphos-ethyl is a nonsystemic insecticide and acaricide with contact and stomach action. It is used to control both chewing and sucking insects and spider mites on a wide range of crops. Major formulation types are dustable powder, emulsifiable concentrate, wettable powder, and ultra low volume liquid. The acute oral LD50 for rats is about 12 mg/kg. Inhalation LC50 (4 h) for rats is ca. 0.15 mg/L air. NOEL (2 yr) for rats is 2 mg/kg diet (0.1 mg/kg/d). In mammals, more than 90% of orally administered azinphosethyl is eliminated in the urine and feces within 48 h.

1157

The major metabolites are desethylazinphos-ethyl and benzazimide. AZINPHOS-METHYL

S (CH3O)2P

N S

CH2

N

N O

IUPAC name. S-(3,4-Dihydro-4-oxobenzo[d]-[1,2,3]-triazin-3-ylmethyl) O,O-dimethyl phosphorodithioate CASRN. 86-50-0 Selected trade names. Gusathion-M, Cotnion-methyl, Guthion For synthesis of azinphos-methyl, see (10). Azinphosmethyl is a colorless crystalline substance, mp 73 ◦ C, vp 5 × 10−4 mPa (20 ◦ C), practically insoluble in water (28 mg/L at 20 ◦ C) but soluble in most organic solvents. Log Kow = 2.96. It is rapidly hydrolyzed in alkaline media; DT50 (22 ◦ C) at pH 4, 7, and 9 are 87, 150, and 4 d, respectively. Azinphos-methyl is a nonsystemic insecticide with contact and stomach action for use to control both chewing and sucking insects of the orders Coleoptera, Diptera, Homoptera, Hemiptera, and Lepidoptera on a wide range of crops. Major formulation types are emulsifiable concentrate, wettable powder, and liquid flowable. The acute oral LD50 for rats is about 9 mg/kg. Inhalation LC50 (4 h) for rats is ca. 0.15 mg/L air. NOEL (2 yr) for rats and mice is 5 mg/kg diet (0.25 mg/kg/d), and ADI is 0.5 µg/kg body weight. In mammals, more than 95% of orally administered azinphos-methyl is eliminated in the urine and feces within 48 h. The major metabolic routes are demethylation and the P−S bond cleavage to give desmethylazinphos-methyl and O,O-dimethyl phosphorothioate. Another route is oxidative desulfuration to the oxon, followed by hydrolysis to dimethyl phosphate. Benzazimide is one of the major metabolites. In the environment, oxidation, demethylation, and hydrolysis are the main degradation routes. The half-life in soils is several weeks. CADUSAFOS

O CH3 C2H5O

P(SCHC2H5)2

IUPAC name. S,S-Di-sec-butyl O-ethyl phosphorodithioate CASRN. 95465-99-9 Selected trade name. Rugby, Apache Cadusafos is a colorless to yellow liquid, bp 112–114 ◦ C/0.8 mm Hg, vp 0.12 Pa (25 ◦ C). Solubility in water is 248 mg/L. It is completely miscible with most organic solvents. Log Kow = 3.9. It is stable up to 50 ◦ C.

1158

ORGANOPHOSPHORUS INSECTICIDES

Cadusafos is a nematicide with contact and stomach action for use to control nematodes, cutworms, and other soil insects in a variety of crops at 3–10 kg/ha. Granules and emulsions are the usual formulation types. The acute oral LD50 values for rats and mice are 37.1 and 71.4 mg/kg, respectively. Inhalation LC50 (4 h) for rats is 0.026 mg/L air. NOEL (2 yr) for rats is 1 mg/kg diet (0.05 mg/kg/d) and ADI is 0.3 µg/kg. In mammals, it is readily metabolized and is eliminated in the urine and feces. The major metabolic route is hydrolysis to give 1-methyl-1-propanothiol, followed by oxidation. The halflife in soils is 11–55 d. CHLORETHOXYFOS

S (C2H5O)2P

Cl OCHCCl3

IUPAC name. O,O-Diethyl O-(1,2,2,2-tetrachloroethyl) phosphorothioate CASRN. 54593-83-8 Selected trade name. Fortress Chlorethoxyfos is a liquid, bp 110–115 ◦ C/0.8 mm Hg, vp ca. 0.11 Pa (20 ◦ C). It is almost insoluble in water (700 h (pH 1.1, 38 ◦ C), >400 h (pH 9.1, 38 ◦ C), and 1.28 h (pH 13, 20 ◦ C). Chlorfenvinphos was first reported for use in the control of animal ectoparasites as a sheep and cattle dip. Because it persists a rather long time in soils, it is useful to control soil insects such as rootworms on vegetables and bulb flies in wheat and maize. By foliar application, it is used to control Colorado beetles on potatoes, scale insects and mite eggs on citrus fruit, and stemborers and leafhoppers on rice. Formulation types are granules and emulsifiable concentrates. Mixtures with cypermethrin are often formulated. The toxicity is particularly high to rats (acute oral LD50 10–40 mg/kg) but is much lower to other mammals (acute oral LD50 for mice 117–200, rabbits 300–1,000, dogs >12, 000 mg/kg). Inhalation LC50 (4 h) for rats is about 0.05 mg/L air. NOEL (2 yr) for rats and dogs is 1 mg/kg diet (0.05 mg/kg/d). ADI is 0.5 µg/kg b.w. The selective toxicity of chlorfenvinphos to rats is attributed to the poor degradative activity of rat liver microsomal enzymes. In other mammals, liver microsomal mfo catalyzes the detoxification of the insecticide through oxidative deethylation to give 2chloro-1-(2,4-dichlorophenyl)vinyl hydrogen phosphate. Ultimate metabolites include the glucuronides of 2,4dichlorophenylethanediol and 1-(2,4-dichlorophenyl)ethanol and N-(2,4-dichlorobenzoyl)glycine. In soils, chlorfenvinphos is gradually decomposed to form α-dichlorophenylethanol, dichloroacetophenone, diethyl hydrogen phosphate, and desethyl chlorfenvinphos. CHLORMEPHOS

S CHLORFENVINPHOS

(C2H5O)2P O (C2H5O)2P

O Cl

O C

(C2H5O)2P

C

H

O C

C

H

Cl

Cl (Z)

Cl (E)

Cl

Cl

IUPAC name. 2-Chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate CASRN. 470-90-6

SCH2Cl

IUPAC name. S-Chloromethyl O,O-diethyl phosphorodithioate CASRN. 24934-91-6 Selected trade name. Dotan Chlormephos is a colorless liquid, bp 81–85 ◦ C/0.1 mm Hg, vp 7.6 Pa (30 ◦ C). Solubility in water is 60 mg/L (20 ◦ C). It is miscible with most organic solvents. Log Kow = 3.0. It is stable in neutral and weakly acidic media at room temperature but rapidly hydrolyzed in alkaline media. Chlormephos is a nonsystemic insecticide with contact and vapor phase action for control of wireworms and


other soil-dwelling insects in a number of crops by soil application of granules. The acute oral LD50 for rats is 7 mg/kg. NOEL (90 d) for rats is 0.39 mg/kg diet (0.002 mg/kg/d). In rats, orally administered chlormephos is almost completely eliminated within 24 h in the urine as diethyl hydrogen phosphate and O,O-diethyl hydrogen phosphorothioate. In soils, chlormephos is converted to ethion by reaction with the hydrolytic product, O,O-diethyl hydrogen phosphorothioate. CHLORPYRIFOS

Cl

S (C2H5O)2P

N O

Cl Cl

IUPAC name. O,O-Diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate CASRN. 2921-88-2 Selected trade names. Dursban, Lorsban

1159

Selected trade names. Reldan, Pyriban-M Chlorpyrifos-methyl is a white crystalline substance with a slight mercaptan odor, mp 45.5–46.5 ◦ C, vp 3 mPa (25 ◦ C), nearly insoluble in water (2.6 mg/L at 25 ◦ C) but highly soluble in most organic solvents. Log Kow = 4.24. The hydrolytic DT50 values are 27, 21, and 13 d at pH 4, 7, and 9, respectively. It is a nonsystemic insecticide with contact, stomach, and respiratory action to be used for control of many types of insects in cereals and on foliage of various crops. It is also used for public health. Formulation types are emulsifiable concentrate, ultra low volume liquid, and fogging concentrate. Chlorpyrifos-methyl is much less toxic to mammals in comparison with the ethyl homologue, chlorpyrifos. The acute oral LD50 for rats is >3000 mg/kg. NOEL (2 yr) for rats and dogs is 0.1 mg/kg daily. Chlorpyrifos-methyl is rapidly metabolized in mammals, the principal metabolite being 3,5,6-trichloro-2pyridinol that is then conjugated and excreted in the urine. Chlorpyrifos-methyl in soil undergoes microbial degradation to 3,5,6-trichloro-2-pyridinol. DT50 in soil is 1.5–33 d, depending upon soil type and microbial activity. COUMAPHOS

Chlorpyrifos is colorless crystalline substance, mp 42–43.5 ◦ C, vp 2.7 mPa (25 ◦ C), nearly insoluble in water (ca. 1.4 mg/L at 25 ◦ C) but highly soluble in most organic solvents. Log Kow = 4.7. The hydrolytic rate increases with pH and in the presence of copper or other metal ions that form chelates; DT50 values are 1.5 d in water, pH 8, at 25 ◦ C and 100 d in phosphate buffer, pH 7, at 15 ◦ C. Chlorpyrifos is a nonsystemic insecticide with contact, stomach, and respiratory action to be used for control of many types of insects in soils or on foliage in a wide range of crops and ornamentals. It is also used for control of household pests, including termites. Formulation types are wettable powder, emulsifiable concentrate, granules, smoke, and microcapsule. The acute oral LD50 for rats is 135–163 mg/kg. NOEL (2 yr) for rats is 0.03 mg/kg daily. Chlorpyrifos is rapidly metabolized in mammals, the main initial metabolite being 3,5,6-trichloro-2-pyridinol that is then conjugated and excreted principally in the urine. The metabolic fate of chlorpyrifos in soil and plants is similar to that in animals. Dechlorination of the chloropyridine occurs by photolysis in the environment. DT50 in soil is 60–120 d. CHLORPYRIFOS-METHYL

Cl

S (CH3O)2P

N O

Cl Cl

IUPAC name. O,O-Dimethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate CASRN. 5598-13-0

S (C2H5O)2P

O

O

O Cl

CH3 IUPAC name. O-3-Chloro-4-methyl-2-oxo-2H-chromen7-yl O,O-diethyl phosphorothioate CASRN. 56-72-4 Selected trade name. Asuntol Coumaphos is a colorless crystalline substance; mp 95 ◦ C, vp 0.013 mPa (20 ◦ C), nearly insoluble in water (1.5 mg/L at 20 ◦ C) but more soluble in organic solvents. Log Kow = 4.13. It is relatively stable in aqueous media, though the pyrone ring is opened in dilute alkali, reclosing on acidification. It is used for control of Diptera and ectoparasites on cattle. It also controls parasitic mites, Varroa jacobsoni, on bees. It is formulated to powders, sprays, and liquids. The acute oral LD50 values for male and female rats are 41 and 16 mg/kg, respectively. Inhalation LC50 values (1 h) for male and female rats are >1081 and 341 mg/m3 air. In 2-yr trials, rats tolerated 100 mg/kg diet. Degradation occurs rapidly in the liver of the cow and rat. The principal metabolite excreted in urine is diethyl hydrogen phosphorothioate. Deethylation products are also found in lesser amounts. Photolytic DT50 on soil surface is 23.8 d. CYANOPHOS

S (CH3O)2P

O

CN

1160


IUPAC name. O-4-Cyanophenyl O,O-dimethyl phosphorothioate CASRN. 2636-26-2 Selected trade name. Cyanox Cyanophos is an amber liquid, mp 14 ◦ C, decomposes at 119–120 ◦ C, vp 105 mPa (20 ◦ C). Solubility in water is 46 mg/L (30 ◦ C). It is soluble in most organic solvents. Log Kow = 2.65. Cyanophos is effective in controlling a variety of insect pests including rice stem borers. Major formulation types are dustable powder, emulsifiable concentrate, wettable powder, and ultra low volume liquid. It has a low mammalian toxicity: acute oral LD50 for rats is 710–730 mg/kg. Inhalation LC50 (4 h) for rats is >1500 mg/m3 air. The main biodegradation pathways in mammals are demethylation and aryl ester bond cleavage; the liberated cyanophenol is excreted in the form of the sulfate ester, formed by conjugation. The metabolism in plants is similar to that in mammals except the cyanophenol conjugation. DEMETON-S-METHYL

O (CH3O)2P

SCH2CH2SC2H5

IUPAC name. S-2-Ethylthioethyl O,O-dimethyl phosphorothioate CASRN. 867-27-6 Selected trade names. Metaphor, Mifatox Preparations manufactured from dimethyl phosphorochloridothionate and 2-ethylethanol are the 3 : 7 mixtures of demeton-S-methyl and the isomeric demetonO-methyl (O-2-ethylthioethyl O,O-dimethyl phosphorothioate) and known as demeton-methyl or methyldemeton. Demeton-S-methyl is produced by an improved manufacturing process [refer to (4) and (5)]. It is a pale yellow oil, bp 89 ◦ C/0.15 mm Hg, vp 40 mPa (20 ◦ C). Solubility in water is 22 g/L (20 ◦ C). It is readily soluble in common polar organic solvents. Log Kow = 1.32(20 ◦ C). Demeton-Smethyl is rapidly hydrolyzed in alkaline media and more slowly in acidic and neutral aqueous media; DT50 at pH 4, 7, and 9 are 63, 56, and 8 d at 22 ◦ C, respectively. Demeton-S-methyl is a systemic insecticide and acaricide. The emulsifiable concentrates are used for the control of aphids, other sucking insects, and spider mites in fruit, vegetables, cereals, potatoes, and ornamentals. The acute oral LD50 for rats is about 30 mg/kg. Inhalation LC50 (4 h) for rats is about 0.13 mg/L air. NOEL (2 yr) for rats is 1 mg/kg diet (0.05 mg/kg/d). ADI is 0.3 µg/kg b.w. for the sum of demeton-S-methyl, its sulfoxide, i.e., oxydemeton-methyl, and the sulfone. The metabolic routes of demeton-S-methyl are essentially the same in plants, insects, and mammals, involving the oxidation of the sulfide group into the sulfoxide and then sulfone, and hydrolysis to dimethyl phosphate. In mammals, following oral administration, excretion occurs rapidly in the urine (97–99% within 24 h). Degradation in soil is also rapid.

DIAZINON

CH(CH3)2

S (C2H5O)2P

N O

N CH3

IUPAC name. O,O-Diethyl O-(2-isopropyl-6-methylpyrimidin-4-yl) phosphorothioate CASRN. 333-41-5 Selected trade names. Basudin, Dianon Diazinon is a colorless liquid, bp 125 ◦ C/1 mm Hg, vp 12 mPa (25 ◦ C). Solubility in water is 60 mg/L (20 ◦ C). It is miscible with common organic solvents. Log Kow = 3.30. Diazinon is stable in neutral aqueous media, slowly hydrolyzed in alkaline media, and much more rapidly in acidic media; DT50 (20 ◦ C) at pH 3.1, 7.4, and 10.4 are 11.8 h, 185 d, and 6.0 d. Diazinon is a nonsystemic insecticide and acaricide with contact, stomach, and respiratory action and is used for the control of sucking and chewing insects and mites on a very wide range of crops. It is also used as a veterinary ectoparasiticide. Major formulation types are granules, wettable powder, emulsifiable concentrate, dustable powder, aerosol, and coating agent. It is incompatible with copper-containing compounds. The acute oral LD50 for rats and mice are 1250 and 80–135 mg/kg. Inhalation LC50 (4 h) for rats is >2330 mg/m3 air. NOEL (2 yr) for rats is 0.06 mg/kg/d. The main biodegradation pathway in mammals, plants, and soils is pyrimidinyl ester bond cleavage; the principal metabolites are diethyl phosphorothioate and diethyl phosphate. Degradation in the environment involves oxidation to diazoxon and hydrolysis. DICHLORVOS

O (CH3O)2P

OCH

CCl2

IUPAC name. 2,2-Dichlorovinyl dimethyl phosphate CASRN. 62-73-7 Selected trade names. Vapona, Nuvan, Phosvit Dichlorvos is manufactured by the Perkow reaction from trimethyl phosphite and chloral, though it was synthesized first by the alkali-catalyzed conversion of trichlorfon (2). It is a colorless liquid, bp 74 ◦ C/1 mm Hg, vp 2.1 Pa (25 ◦ C). Solubility in water is ca. 18 g/L (25 ◦ C). It is completely miscible with aromatic hydrocarbons, chlorinated hydrocarbons, and alcohols and moderately soluble in kerosene. Log Kow = 1.9. Dichlorvos is stable to heat and slowly hydrolyzed in water and acidic media and rapidly in alkaline media to dimethyl phosphate and dichloroacetaldehyde; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 31.9, 2.9, and 2.0 d, respectively.


Dichlorvos is a contact and stomach insecticide and acaricide with fumigant action and low residual activity, usable for control of household and public health insect pests. It is also used for the control of sucking and chewing pests in a wide range of crops. It has an anti-AChE activity without activation. Formulation types are emulsifiable concentrate, aerosol, impregnated strip, and smoke. The acute oral LD50 for rats is about 50 mg/kg. Inhalation LC50 (4 h) for rats is 340 mg/m3 air. NOEL (2 yr) for rats is 10 mg/kg/d. In mammals, orally administered dichlorvos is rapidly degraded in the liver by hydrolysis and O-demethylation, with a half-life of ca. 25 min. The hydrolytic product, dichloroacetaldehyde, is further metabolized to dichlorethanol and glycolic acid. It is nonpersistent in the environment and rapidly decomposes in the atmosphere. The half-lives in soils and water systems are less than 1 d.

DICROTOPHOS

O (CH3O)2P

H

O C

C CON(CH3)2

H3C (E)

IUPAC name. Dimethyl (E)-1-methyl-2-(dimethylcarbamoyl)vinyl phosphate CASRN. 141-66-2 Selected trade names. Bidrin, Carbicron Dicrotophos is synthesized by the Perkow reaction from trimethyl phosphite and N,N-dimethyl-αchloroacetoacetamide, consisting mainly of the (E)-form. It is a yellowish liquid, bp 130 ◦ C/0.1 mm Hg, vp 9.3 mPa (20 ◦ C). It is miscible with water and most organic solvents except kerosene. Log Kow = −0.5. Dicrotophos is rather stable to heat and slowly hydrolyzed in acidic media and more rapidly in alkaline media; DT50 (20 ◦ C) at pH 5, 7, and 9 are 88, 72, and 28 d, respectively. Dicrotophos is a moderately persistent systemic insecticide and acaricide with contact and stomach action and is effective for the control of household and public health insect pests. It is used for the control of sucking, chewing, and boring insects and mites in a wide range of crops. It is also used as an animal ectoparasiticide. Formulation types are emulsifiable concentrate and water-soluble concentrate. The acute oral LD50 for rats is 17–22 mg/kg. Inhalation LC50 (4 h) for rats is about 0.09 mg/L air. NOEL in a three-generation reproduction study with rats is 2 mg/kg daily. In mammals, orally administered dicrotophos is rapidly metabolized, and 63–71% was excreted in the urine within 48 h. The main degradation routes are O-demethylation to des-O-methyldicrotophos and hydrolysis to dimethyl phosphate and N,N-dimethylacetoacetamide. Oxidative Ndemethylation also occurs.

1161

DIMETHOATE

S (CH3O)2P

SCH2CONHCH3

IUPAC name. O,O-Dimethyl S-methylcarbamoylmethyl phosphorodithioate CASRN. 60-51-5 Selected trade names. Cygon, Rogor For the synthesis of dimethoate, see Equation 8. Dimethoate is colorless crystalline substance, mp 49 ◦ C, bp 117 ◦ C/0.1 mm Hg, vp 0.25 mPa (25 ◦ C). The solubility in water is 23.8 g/L at 20 ◦ C and pH 7. It is readily soluble in polar organic solvents. Log Kow = 0.7. Dimethoate is relatively stable in aqueous media at pH 2–7 and hydrolyzed in alkaline media; DT50 (20 ◦ C) at pH 9 is 12 d. Dimethoate is a systemic insecticide and acaricide with contact and stomach action and is used to control a wide range of insects and mites in many crops. It is also used to control flies in animal houses. Formulation types are emulsifiable concentrate, wettable powder, ultra low volume liquid, granules, and aerosol. The acute oral LD50 for rats is 387 mg/kg. Inhalation LC50 (4 h) for rats is >1.6 mg/L air. NOEL (2 y) for rats is 5 mg/kg diet (0.25 mg/kg/d). ADI is 2 µg/kg (sum of dimethoate and its oxon, i.e., omethoate). Oxidative desulfuration to form omethoate, the active AChE inhibitor, occurs both in mammals and plants. The main degradation routes are O-demethylation and amide hydrolysis that is important particularly for the selective species toxicity in animals. The cleavage of the P−S and S−C linkages also occurs to a considerable degree. Aerobic DT50 in soil is 2–4 d, whereas it was 22 d under anaerobic conditions. DIMETHYLVINPHOS

O (CH3O)2P

O

Cl C

C Cl

H

Cl IUPAC name. (Z)-2-Chloro-1-(2,4-dichlorophenyl)vinyl dimethyl phosphate CASRN. 2274-67-1 Trade name. Rangado Dimethylvinphos consists of >95% of the (Z)-isomer in contrast to the diethyl homologue, chlorfenvinphos, which is a mixture of (Z)- and (E)-isomers. It is a pale white crystalline solid, mp 69–70 ◦ C, vp 1.3 mPa (25 ◦ C). The solubility in water is 130 mg/L at 20 ◦ C. It is readily soluble in polar organic solvents. Log Kow = 3.12. It is hydrolyzed in water with DT50 (25 ◦ C, pH 7) of 40 d and unstable in sunlight.

1162


Dimethylvinphos is a moderately persistent insecticide with contact and stomach action and is used to control stem borers and leaf rollers in rice with a dust formulation. The acute oral LD50 for rats is 155–210 mg/kg. Inhalation LC50 (4 h) for male and female rats is 970–1189 and >4900 mg/m3 air. The main degradation route in mammals is O-demethylation to desmethyl dimethylvinphos, which is hydrolyzed to 2,2 ,4 trichloroacetophenone. DISULFOTON

S (C2H5O)2P

SCH2CH2SC2H5

IUPAC name. O,O-Diethyl S-2-(ethylthioethyl) phosphorodithioate CASRN. 298-04-4 Selected trade names. Disyston, Frumin AL, Solvirex, Prosper Disulfoton is a colorless oil, bp 128 ◦ C/1 mm Hg, vp 7.2 mPa (20 ◦ C). It is practically insoluble in water (25 mg/L at 20 ◦ C) but miscible with common organic solvents. Log Kow = 3.95. Disulfoton is relatively stable in aqueous media; DT50 (22 ◦ C) at pH 4, 7, and 9 are 133, 169, and 131 d, respectively. Disulfoton is a systemic insecticide and acaricide, absorbed by the roots, translocated to the whole plant, giving long-lasting control of aphids, other sucking insects, and spider mites in a wide range of crops. Main formulation types are granules, emulsifiable concentrate, and seed treatment powder. The acute oral LD50 for rats is 2–12 mg/kg. Inhalation LC50 (4 h) for rats is 0.06–0.015 mg/L air. NOEL (2 yr) for rats is 1 mg/kg diet (0.05 mg/kg/d). ADI is 0.3 µg/kg b.w. The metabolic routes of disulfoton are essentially the same in plants, insects, and mammals, involving the oxidation of the sulfide group into the sulfoxide and then sulfone, oxidative desulfuration to the corresponding oxons, and hydrolysis to diethyl phosphorothioate. In mammals, orally administered disulfoton is rapidly metabolized and excreted in the urine. Disulfoton is rapidly degraded in soil; DT50 (20 ◦ C) was 1.3–2 d. EPN

S P

O

NO2

OC2H5 IUPAC name. O-Ethyl O-4-nitrophenyl phenylphosphonothioate CASRN. 2104-64-5 Trade name. EPN EPN is a light yellow crystalline solid, mp 34.5 ◦ C, vp 5.02. EPN is relatively stable in neutral and acidic media but hydrolyzed by alkali to liberate p-nitrophenol; DT50 at pH 4, 7, and alkaline are 70, 22, and 3.5 d, respectively. EPN is a nonsystemic insecticide and acaricide effective in controlling Lepidoptera larvae, especially including rice stem borer, bollworms, and other leaf-eating larvae on a number of crops. Formulation types are dust, emulsifiable powder, and granules. The acute oral LD50 for rats is 24–36 mg/kg. NOEL (104 w) for rats is 0.67 mg/kg b.w. daily. The principal biodegradation pathway in mammals is oxidative dearylation to afford nitrophenol and O-ethyl hydrogen phenylphosphonothioate. The oxidative desulfuration followed by hydrolysis occurs to a lesser extent. The reduction of the nitro group to an amino group is observed in soils, microorganisms, and animals. The major metabolite in plants is ethyl hydrogen phenylphosphonate. The DT50 in paddy soil was less than 15 d. ETHION

S

S

(C2H5O)2P

SCH2S

P(OC2H5)2

IUPAC name. O,O,O ,O -Tetraethyl S,S -methylene bis(phosphorodithioate) CASRN. 563-12-2 Selected trade names. Cethion, Nialate Ethion is synthesized by the reaction of diethyl hydrogen phosphorodithioate (2 mole) with bromochloromethane or dibromomethane (1 mole) in the presence of alkali. The product is a yellow liquid, bp 164–165 ◦ C/0.3 mm Hg, vp 0.2 mPa (25 ◦ C). It is practically insoluble in water (2 mg/L at 25 ◦ C) but miscible with most organic solvents. Log Kow = 5.07. Ethion is slowly hydrolyzed in aqueous acids and alkalis; DT50 at pH 9 is 390 d. Ethion is a nonsystemic acaricide and insecticide with contact action and is used for the control of spider mites, aphids, sucking insects, lepidopterous larvae, and soil-dwelling insects in a wide range of crops. Main formulation types are granules, emulsifiable concentrate, wettable powder, and seed treatment powder. The acute oral LD50 for rats is 208 mg/kg. Inhalation LC50 (4 h) for rats is 0.45 mg/L air. NOEL (2 yr) for rats is 6 mg/kg diet (0.3 mg/kg/d). ADI is 2 µg/kg b.w. The main biodegradation routes of ethion in animals are cleavage of the P−S and C−S linkages to give O,O-diethyl hydrogen phosphorothioate and O,O-diethyl hydrogen phosphorodithioate, respectively. Oxidative desulfuration to its mono- and dioxons also occurs.

ETHOPROPHOS

O C2H5OP(SCH2CH2CH3)2


IUPAC name. O-Ethyl S,S-dipropyl phosphorodithioate CASRN. 13194-48-4 Trade name. Mocap Ethoprophos is a pale yellow liquid, bp 86–91 ◦ C/ 0.2 mm Hg, vp 46.5 mPa (26 ◦ C). The solubility in water is 700 mg/L at 25 ◦ C, and that in common polar organic solvents is more than 300 g/kg at 20 ◦ C. Log Kow = 3.59. Ethoprophos is stable in neutral and weakly acidic media but rapidly hydrolyzed in alkaline media. Ethoprophos is used for the control of plant parasitic nematodes and soil insects in ornamentals and many crops by incorporating into soil in the form of granule or emulsifiable concentrate formulation at 1.6–6.6 kg a.i./ha. The acute oral LD50 for rats is 62 mg/kg. Inhalation LC50 (4 h) for rats is 123 mg/m3 air. ADI is 0.3 µg/kg b.w. The main degradation route of ethoprophos in both plants and animals is hydrolytic cleavage of the P−S linkage to give O-ethyl S-propyl hydrogen phosphorothioate and propanethiol. The DT50 in sandy loam at pH 7.2–7.3 was ca. 14–28 d. ETRIMFOS

FAMPHUR

S (CH3O)2P

O

SO2N(CH3)2

IUPAC name. O-(4-Dimethylsulfamoylphenyl) O,O-dimethyl phosphorothioate CASRN. 52-85-7 Selected trade names. Bo-Ana, Warbexol Famphur is a colorless crystalline powder, mp 52.5–53.5 ◦ C, vp 0.03 mPa (20 ◦ C). Solubility in water is about 100 mg/L. It is soluble in acetone, chlorinated hydrocarbons, and aromatic hydrocarbons. Log Kow = 1.5. It is stable at ambient temperatures. Famphur is a veterinary ectoparasiticide used to control grubs, hornfly, and lice in cattle by pour-on or feeding. The acute oral LD50 for rats is 35–62 mg/kg. The principal degradation routes in mammals are P−Ophenyl bond cleavage and O-demethylation. Oxidative desulfuration and N-demethylation also take place to form toxic metabolites.

C2H5

S (CH3O)2P

1163

FENAMIPHOS

N O

N

CH3

O OC2H5 IUPAC name. O-(6-Ethoxy-2-ethylpyrimidin-4-yl) O,Odimethyl phosphorothioate CASRN. 38260-54-7 Selected trade names. Ekamet, Satisfar Etrimfos is a colorless liquid, mp −3.35 ◦ C, vp 6.5 mPa (20 ◦ C). Solubility in water is 40 mg/L (23–24 ◦ C). It is miscible with most organic solvents. Log Kow > 3.3. Neat etrimfos is unstable and degrades ca. 40% in 28 d at 50 ◦ C, but the dilute solutions in nonpolar solvents and its formulations are stable (about 5% loss in 1 y at ca. 20 ◦ C). It is hydrolyzed in aqueous media; DT50 (25 ◦ C) at pH 3, 6, and 9 are 0.4, 16, and 14 d, respectively. Etrimfos is a nonsystemic insecticide and acaricide with contact and stomach action and it is used for the control of chewing insects on fruit trees and a number of other crops. It is also used to control Lepidoptera, Coleoptera, and mites in stored products. Major formulation types are dustable powder, granules, emulsifiable concentrate, ultra low volume liquid, etc. The acute oral LD50 for rats is 1,600–1,800 mg/kg. Inhalation LC50 (1 h) for rats is >200 mg/L air. NOEL (2 yr) for rats is 6 mg/kg diet (0.3 mg/kg/d). ADI is 3 µg/kg b.w. The principal degradation pathway in mammals, plants, and soils is pyrimidinyl ester bond cleavage: the major metabolites are 6-ethoxy-4-hydroxy2-ethylpyrimidine and its further transformed products. Degradation in mammals involves demethylation to desmethyl etrimfos. The DT50 in soils at pH 6.8 was 3–10 d.

C2H5O

P

O

SCH3

NHCH(CH3)2 IUPAC name. Ethyl 3-methyl-4-(methylthio)phenyl isopropylphosphoramidate CASRN. 22224-92-6 Trade name. Nemacur Fenamiphos is a colorless crystalline substance, mp 49.2 ◦ C, vp 0.12 mPa (20 ◦ C). Solubility in water is 400 mg/L (20 ◦ C). It is soluble in polar organic solvents. Log Kow = 3.3(20 ◦ C). It is stable in aqueous media; DT50 (22 ◦ C) at pH 4, 7, and 9 are 1, 8, and 3 yr. Fenamiphos is a systemic nematicide with contact action, used primarily for the control of nematodes in a wide range of crops. It also has activity against sucking insects and mites. It is absorbed by the roots and the leaves and translocated in the whole plant, displaying not only protective but also curative activity for a long time. Major formulation types are granules and emulsifiable concentrate. The acute oral LD50 for rats is about 6 mg/kg. Inhalation LC50 (4 h) for rats is about 0.12 mg/L air. NOEL (2 yr) for rats is 1 mg/kg diet (0.05 mg/kg/d). ADI is 0.5 µg/kg b.w. The oxidation of the thiomethyl group to the sulfoxide and sulfone is required for both translocation and nematicidal activity in plants. The oxidation products are more susceptible to hydrolysis than fenamiphos itself. In mammals, orally administered fenamiphos is rapidly metabolized to the sulfoxide and sulfone, followed by

1164


subsequent hydrolysis, conjugation, and excretion in the urine. N-Dealkylation also occurs. The DT50 in soils is several weeks; the major degradation products are fenamiphos sulfoxide and sulfone and their phenols. FENITROTHION

CH3

S (CH3O)2P

O

NO2

IUPAC name. O,O-Dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate CASRN. 122-14-5 Selected trade names. Sumithion, Folithion Fenitrothion is a yellow liquid, bp 95 ◦ C/0.01 mm Hg, vp 15 mPa (20 ◦ C). The solubility in water is 21 mg/L at 20 ◦ C. It is readily soluble in common polar organic solvents. Log Kow = 3.5. Fenitrothion is relatively stable in aqueous media under usual conditions: DT50 (22 ◦ C) at pH 4, 7, and 9 are 108.8, 84.3, and 75 d. Fenitrothion is a nonsystemic insecticide and is used for controlling chewing, sucking, and boring insects in cereals, fruit, sugarcane, vegetables, turf, and forestry. It is also used for the control of flies, mosquitoes, and cockroaches in public health programs. It is formulated as granules, emulsifiable concentrates, ultra low volume liquids, dusts, oil-based sprays, and in combination with other pesticides. The acute oral LD50 values in mammals range from 330 mg/kg in rats to 1850 mg/kg in the guineapig. Inhalation LC50 (4 h) for rats is >1.2 mg/L air. NOEL (2 y) for rats and mice is 10 mg/kg diet (0.5 mg/kg/d). ADI is 5 µg/kg b.w. The main biotransformation routes involve oxidative desulfuration to the oxon and dearylation to give dimethyl hydrogen phosphate, O,O-dimethyl hydrogen phosphorothioate and 3-methyl-4-nitrophenol. Demethylation dependent on glutathion-S-alkyl transferase is particularly important in mammals. Oxidation of the 3-methyl group to hydroxymethyl and then carboxyl group is also a degradative route. Reduction of the nitro group to an amino group occurs in anaerobic soils and ruminants. The DT50 in soils under upland and submerged conditions were 12–28 and 4–20 d, respectively. FENTHION

CH3

S (CH3O)2P

O

SCH3

Fenthion is a colorless liquid, mp 7.5 ◦ C, bp 87 ◦ C/ 0.01 mm Hg, vp 0.37 mPa (20 ◦ C). The solubility in water is 0.42 mg/L at 20 ◦ C. It is readily soluble in most organic solvents except for aliphatic hydrocarbons. Log Kow = 4.84. Fenthion is relatively stable in aqueous media and heat: DT50 (22 ◦ C) at pH 4, 7, and 9 are 223, 200, and 151 d, respectively. Fenthion is a nonsystemic insecticide and is used for controlling chewing, sucking, and boring insects in numerous crops. It is also used for the control of insects in human and animal health situations. It is formulated as granules, emulsifiable concentrate, ultra low volume liquid, dustable powder, and wettable powder. The acute oral LD50 for rats is about 250 mg/kg. Inhalation LC50 (4 h) for rats is about 0.5 mg/L air. NOEL (2 yr) for rats is 1, and >1 yr, respectively. Isofenphos is a systemic insecticide with contact and stomach action. It is used to control cabbage root flies, onion flies, corn rootworms, wireworms, and other soil insects on a wide range of crops. Main formulations are emulsifiable concentrates, wettable powders, and granules. The acute oral LD50 for rats is about 20 mg/kg. Inhalation LC50 (4 h) for rats is 0.3–0.5 mg/L air. NOEL (2 yr) for rats 1 mg/kg diet (0.05 mg/kg/d). ADI is 1 µg/kg. In mammals, administered isofenphos is rapidly metabolized and eliminated; almost 95% is excreted within 24 h in the urine and feces. The active metabolite is des-N-isopropylisofenphos oxon. Main degradation route is cleavage of the P−O-aryl ester linkage through oxidative desulfuration to isofenphos oxon followed by hydrolysis and oxidative dearylation from isofenphos. In plant, the major metabolites are salicylic acid and dihydroxybenzoic acid.

ISOXATHION

S

N O

(C2H5O)2P

O

IUPAC name. O,O-Diethyl phosphorothioate CASRN. 18854-01-8 Trade name. Karphos

O-(5-phenylisoxazol-3-yl)

Isoxathion is a pale yellow liquid, bp 160 ◦ C/0.15 mm Hg, vp 200 g/L at 20 ◦ C) but slightly in hexane. Log Kow = −0.8(20 ◦ C). It is stable in aqueous media at pH 3–8; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 1.8 y, 120 h, and 70 h, respectively. Methamidophos is a systemic insecticide-acaricide absorbed through the roots and leaves. It is used for the control of chewing and sucking insects and spider mites on a variety of crops. It is formulated as emulsifiable concentrates or liquids. Acute oral LD50 for rats is about 20 mg/kg. Inhalation LC50 (4 h) for rats is 0.2 mg/L air. NOEL (2 yr) for rats is 2 mg/kg diet (0.1 mg/kg/d). ADI is 4 µg/kg b.w. Methamidophos itself shows only a poor anti-AChE activity, oxidative activation being suggested. It appears to cause delayed neuropathy. The major part of administered methamidophos in animals is rapidly eliminated from the body through urine and respiration. The major metabolic routes are O-demethylation, S-demethylation, and deamination. In plants, deaminated methamidophos is a major metabolite. Methamidophos is degraded rapidly in soil by deamination and demethylation, to eventually form carbon dioxide and phosphoric acid. METHIDATHION

O

S (CH3O)2P

SCH2

N

S

N OCH3 IUPAC name. S-(2,3-Dihydro-5-methoxy-2-oxo-1,3,4-thiadiazol-3-ylmethyl) O,O-dimethyl phosphorodithioate CASRN. 950-37-8 Selected trade names. Supracide, Ultracide Methidathion is colorless crystals, mp 39–40 ◦ C, vp 0.25 mPa (20 ◦ C). Solubility in water is 200 mg/L (25 ◦ C).

1168


It is soluble in common organic solvents. Log Kow = 2.2. It is relatively stable in neutral and slightly acidic media but readily hydrolyzed in alkali; DT50 (25 ◦ C) at pH 13 is 30 min. Methidathion is a nonsystemic insecticide-acaricide with a wide spectrum, particularly effective to control scale insects, lepidopterous larvae, aphids, and spider mites on citrus and other fruit trees and a number of crops. Major formulations are emulsifiable concentrates, wettable powders, and ultra low volume liquids. The acute oral LD50 for rats is 25–54 mg/kg. Inhalation LC50 (4 h) for rats is 3.6 mg/L air. NOEL (2 yr) for rats is 4 mg/kg diet (0.2 mg/kg/d). ADI is 1 µg/kg b.w. Methidathion administered to animals is rapidly metabolized and excreted. The principal degradation route is similar both in animals and plants, that is, cleavage of the P−S bond via oxidative desulfuration (activation) to the oxon followed by hydrolysis to O,O-dimethyl hydrogen phosphorothioate and the 3-thiomethyl-5-methoxythiadiazole derivative, which is further degraded or conjugated. Methidathion is rapidly degraded in soil; DT50 in soil is 3–18 d. MEVINPHOS

Inhalation LC50 (1 h) for rats is 0.125 mg/L air. No ill effects were observed in rats receiving 4 mg/kg diet (0.2 mg/kg/d) for 2 yr. ADI is 1.5 µg/kg b.w. In comparison with the Z-isomer, E-mevinphos is a more effective insecticide and is detoxified more quickly in plants but more slowly in animals. The hydrolysis of the P−O vinyl ester bond is the major route of detoxication in plants. Demethylation by glutathion-S-alkyl transferase is an important degradation route of the E-mevinphos in mammals. Mevinphos is rapidly broken down in soils with a DT50 of 3–12 d. MONOCROTOPHOS

O (CH3O)2P

H

O C

C CONHCH3

CH3

IUPAC name. Dimethyl (E)-1-methyl-2-(methycarbamoyl) vinyl phosphate CASRN. 6923-22-4 Selected trade names. Azodrin, Nuvacron

O (CH3O)2P

CO2CH3

O C

C H

CH3 (Z) O (CH3O)2P

H

O C CH3

C CO2CH3

(E) IUPAC name. 2-Methoxycarbonyl-1-methylvinyl dimethyl phosphate CASRN. 26718-65-0 Selected trade names. Phosdrin, Duraphos For the synthesis of mevinphos, see (1). Technical products consist of the E and Z isomers in about 2 : 1 ratio. It is a colorless liquid, bp 99–103 ◦ C/0.3 mm Hg, vp 17 mPa (20 ◦ C). Mp of the E and Z isomer is 21 and 6.9 ◦ C, respectively. It is miscible with water and most organic solvents, except alkanes and carbon disulfide. Log Kow = 0.127. It is relatively stable in aqueous media but hydrolyzed in alkaline solution; DT50 values at pH 7, 9, and 11 are 35 d, 3 d, and 1.4 h, respectively. Mevinphos is a systemic insecticide-acaricide with short residual activity. It is used to control chewing and sucking insects and spider mites on a wide range of crops including fruit trees and vegetables. Major formulation types are emulsifiable concentrate and soluble concentrate. Acute oral LD50 for rats is 3–12 mg/kg.

Monocrotophos is a colorless crystalline substance, mp 54–55 ◦ C, bp 125 ◦ C/0.0005 mm Hg, vp 0.29 mPa (20 ◦ C). It is readily soluble in water and polar organic solvents but slightly in alkanes. Log Kow is −0.22. It is relatively stable in aqueous media but hydrolyzed in alkaline solutions; DT50 values (20 ◦ C) at pH 3, 7, and 9 are 131, 66, and 17 d, respectively. Monocrotophos is a systemic insecticide-acaricide effective against a broad spectrum of pests, including chewing, sucking, and boring insects and spider mites on a wide range of crops, especially against bollworms on cotton. Major formulations are granules, soluble concentrates, and ultra low volume liquids. The acute oral LD50 for rats is 18–20 mg/kg. Inhalation LC50 (4 h) for rats is 0.09 mg/L air. NOEL (2 yr) for rats was estimated as 0.5 mg/kg diet (0.025 mg/kg/d). ADI is 0.6 µg/kg b.w. In mammals, 60–65% of administered monocrotophos is excreted within 24 h, predominantly in the urine. The main metabolic degradation routes are demethylation to O-desmethylmonocrotophos and hydrolysis of the P−O vinyl ester bond to give dimethyl hydrogen phosphate and N-methylacetoacetamide. Monocrotophos is rapidly degraded in soils; DT50 is 1–5 d. NALED

O (CH3O)2P

Br OCHCCl2Br

IUPAC name. 1,2-Dibromo-2,2-dichloroethyl dimethyl phosphate CASRN. 300-76-5 Selected trade names. Dibrom, Bromex


Naled is produced by bromination of dichlorvos and is a colorless liquid, mp 26–27.5 ◦ C, bp 110 ◦ C/0.5 mm Hg, vp 266 mPa (20 ◦ C). It is slightly soluble in water and is readily soluble in aromatic and chlorinated organic solvents. Log Kow = 1.38. It is hydrolyzed in aqueous media (over 90% in 48 h at room temperature) and more rapidly in alkaline and acidic media. Naled is a short-lived insecticide-acaricide for use on vegetable crops. It is also used for the control of flies and mosquitoes in public and animal health. Major formulations are emulsifiable concentrates and ultra low volume liquids. The acute oral LD50 for rats is 430 mg/kg. No harm was observed in mice exposed to 1.5 mg/L air for 6 h. No ill effect was observed in rats that were fed 100 mg/kg diet (5 mg/kg/d) for 2 yr. The main degradation routes of naled are nonenzymatic debromination and hydrolytic cleavage of the phosphate ester bonds. OMETHOATE

O (CH3O)2P

1169

Oxydemeton-methyl, i.e., demeton-S-methyl sulfoxide, is a colorless oil, bp 106 ◦ C/0.01 mm Hg, vp 3.8 mPa (20 ◦ C). It is miscible with water and soluble in most organic solvents, except petroleum ether. Log Kow = −0.74(21 ◦ C). Oxydemeton-methyl is relatively stable in acidic media but hydrolyzed in alkaline media; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 107, 46, and 2 d, respectively. Oxydemeton-methyl is a systemic insecticide with a quick knockdown effect and is used to control sucking insects on fruit, vegetables, ornamentals, etc. It is usually formulated as emulsifiable or soluble concentrates. The acute oral LD50 for rats is about 50 mg/kg. Inhalation LC50 (4 h) for rats is 0.47 mg/L air. NOEL (2 yr) for rats is 1 mg/kg diet (0.05 mg/kg/d). ADI is 0.3 µg/kg b.w. for the sum of oxydemeton-methyl, demeton-S-methylsulfone, and demeton-S-methyl. Almost 99% of orally administered oxydemeton-methyl to animals is excreted within 48 h in the urine. It is oxidized to the sulfone, followed by hydrolytic cleavage of the P−S bond. The thiol metabolites are conjugated or methylated. O-Demethylation is also an important degradation route both in mammals and plants. Oxydemeton-methyl is rapidly degraded in soils.

SCH2CONHCH3 PARATHION

IUPAC name. O,O-Dimethyl S-methylcarbamoylmethyl phosphorothioate CASRN. 1113-02-6 Trade name. Folimat Omethoate, the oxon of dimethoate, is an undistillable colorless oil, vp 3.3 mPa (20 ◦ C). It is readily soluble in water, alcohols, and dichloromethane but almost insoluble in petroleum ether. Log Kow = −0.74(20 ◦ C). Omethoate is relatively stable in acidic media but hydrolyzed in alkaline media; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 102 d, 17 d, and 28 h, respectively. Omethoate is a systemic insecticide–acaricide and is used to control sucking insects and mites on a variety of crops. Major formulation types are emulsifiable concentrate, ultra low volume liquid, and soluble concentrate. The acute oral LD50 for rats is about 25 mg/kg. Inhalation LC50 (4 h) for rats is 0.3 mg/L air. ADI is 0.3 µg/kg b.w. Orally administered omethoate to rats is rapidly metabolized and excreted in the urine; the main metabolites are O-demethylomethoate and N-methyl-2methylsulfinylacetamide. O-Demethylation and hydrolysis of the P−S bond are main degradation routes both in mammals and plants. Omethoate is rapidly degraded in soils with DT50 of a few days. OXYDEMETON-METHYL

O (CH3O)2P

O

S (C2H5O)2P

O

NO2

IUPAC name. O,O-Diethyl O-4-nitrophenyl phosphorothioate CASRN. 56-38-2 Selected trade names. Folidol, E605, Chimac Par H Parathion is a pale yellow liquid, bp 113 ◦ C/0.05 mm Hg, vp 0.89 mPa (20 ◦ C). Solubility in water is 11 mg/L (20 ◦ C). It is miscible with most organic solvents. Log Kow = 3.83. It is very slowly hydrolyzed in aqueous media at pH 7, or below, and more rapidly at higher pHs; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 272, 260, and 130 d, respectively. Parathion is a nonsystemic insecticide effective for the control of sucking and chewing insects and mites on a wide range of crops. Main formulations are emulsifiable concentrates, wettable powders, dusts, and granules. The acute oral LD50 for rats is about 2 mg/kg. Inhalation LC50 (4 h) for rats is 0.03 mg/L air. NOEL (2 yr) for rats is 2 mg/kg diet (0.1 mg/kg/d). ADI is 4 µg/kg b.w. The principal degradation routes of parathion in animals, plants, and soil are dearylation and dealkylation to give O,O-diethyl hydrogen phosphorothioate, p-nitrophenol, and desethylparathion. Oxidative desulfuration also occurs to form the active methabolite paraoxon, which is quickly detoxified by hydrolysis. DT50 in soil was 65 d.

SCH2CH2SC2H5 PARATHION-METHYL

IUPAC name. S-(2-Ethylsulfinylethyl) O,O-dimethylphosphorothioate CASRN. 301-12-2 Trade name. Metasystox-R

S (CH3O)2P

O

NO2

1170


IUPAC name. O,O-Dimethyl O-4-nitrophenyl phosphorothioate CASRN. 298-00-0 Selected trade names. Metacide, Folidol-M Parathion-methyl is a colorless crystalline powder, mp 35–36 ◦ C, vp 0.2 mPa (20 ◦ C). Solubility in water is 55 mg/L (20 ◦ C). It is readily soluble in most organic solvents except petroleum ether. Log Kow = 3.0. It is hydrolyzed about five times faster than the ethyl homologue parathion in acidic and alkaline media; DT50 values (25 ◦ C) at pH 5, 7, and 9 are 68, 40, and 33 d, respectively. Parathion-methyl is a nonsystemic insecticide-acaricide effective for the control of sucking and chewing insects on a wide range of crops. Major formulation types are emulsifiable concentrate, wettable powder, dustable powder, and ultra low volume liquid. The acute oral LD50 for rats is about 3 mg/kg. Inhalation LC50 (4 h) for rats is about 0.17 mg/L air. NOEL (2 yr) for rats is 2 mg/kg diet (0.1 mg/kg/d). ADI is 3 µg/kg b.w. Orally administered parathion-methyl in animals is almost completely excreted in the urine within 24 h. The principal degradation routes in animals are dearylation and demethylation to give O,O-dimethyl hydrogen phosphorothioate, p-nitrophenol and desmethylparathionmethyl. Oxidative desulfuration also occurs to form the active metabolite paraoxon-methyl, which is quickly detoxified by hydrolysis to dimethyl hydrogen phosphate and p-nitrophenol. PHENTHOATE

S (CH3O)2P

SCH CO2C2H5

IUPAC name. S-Ethoxycarbonyl(phenyl)methyl dimethyl phosphorodithioate CASRN. 2597-03-7 Selected trade names. Elsan, Cidial, Papthion

O,O-

Phenthoate is a colorless crystalline substance, mp 17–18 ◦ C, bp 70–80 ◦ C/2 × 10−5 mm Hg, vp 5.3 mPa (40 ◦ C). Solubility in water is 10 mg/L (25 ◦ C). It is readily soluble in most organic solvents. Log Kow = 3.69. It is relatively stable in neutral and acidic aqueous media but decomposed under alkaline conditions. Phenthoate has a broad spectrum of nonsystemic insecticidal and acaricidal activities on a wide range of crops, being particularly effective against codling moth and scale insects. It is also used for mosquito control. It is formulated as emulsifiable concentrates or dusts. Acute oral LD50 for rats is 410 mg/kg. Inhalation LC50 (4 h) for rats is 3.17 mg/L air. NOEL (104 w) for dogs is 0.29 mg/kg daily. ADI is 3 µg/kg b.w. Phenthoate is degraded by hydrolysis of the carboethoxy moiety. Demethylation and the cleavage of P−S−C linkages are

also important degradation routes. Oxidative desulfuration to the oxon followed by hydrolysis occurs in animals and plants. The major metabolites excreted in the urine and feces are demethyl phenthoate, demethyl phenthoate acid, demethyl phenthoate oxon, and O,O-dimethyl hydrogen phosphorodithioate and phosphorothioate. It is rapidly degraded in soils; DT50 was less than 1 d in both upland and submerged soil. PHORATE

S (C2H5O)2P

SCH2SC2H5

IUPAC name. O,O-Diethyl S-ethylthiomethyl phosphorodithioate CASRN. 298-02-2 Trade name. Thimet For the synthesis of phorate, see (9). It is a colorless oil, bp 118–120 ◦ C/0.8 mm Hg, vp 85 mPa (25 ◦ C). The water solubility is 50 mg/L (25 ◦ C). It is miscible with common organic solvents. Log Kow = 3.92. Phorate is relatively unstable to hydrolysis in aqueous media; DT50 values at pH 7 and 9 are 3.2 and 3.9 d, respectively. Phorate is effective against sucking plant pests as a systemic insecticide-acaricide and also has good contact and vapor actions. It is usually formulated as granules. The acute oral LD50 for rats is 1.6–3.7 mg/kg. Inhalation LC50 (1 h) for rats is 0.06–0.011 mg/L air. ADI is 0.5 µg/kg b.w. The metabolic routes of phorate are essentially the same in plants, animals, and soils, involving the oxidation of the sulfide group into the sulfoxide then sulfone, and oxidative desulfuration to the corresponding oxons, followed by hydrolysis to diethyl hydrogen phosphorodithioate, phosphorothioate, and phosphate. Phorate protects plants for a relatively long time because of the persistency of the sulfoxide metabolite in plants and in soils. DT50 in soil is 2–14 d. PHOSALONE

O

S (C2H5O)2P

SCH2

N

O

Cl IUPAC name. S-(6-Chloro-2,3-dihydro-2-oxobenzoxazol3-ylmethyl) O,O-diethyl phosphorodithioate CASRN. 2310-17-0 Selected trade names. Zolone, Rubitox Phosalone is a colorless crystalline solid, mp 47–48 ◦ C, vp < 0.06 mPa (25 ◦ C). The water solubility is 3.05 mg/L


(25 ◦ C). It is readily soluble in common organic solvents. Log Kow = 4.01(20 ◦ C). Phosalone is hydrolyzed in acid and alkaline media; DT50 at pH 9 is 9 d. Phosalone is a nonsystemic insecticide-acaricide useful for the control of caterpillars, aphids, and the active stages of mites on fruit trees and vegetables. It is used in integrated pest management, being selective of most beneficial insects. The major formulation types are emulsifiable concentrate and wettable powder. The acute oral LD50 for rats is 120 mg/kg. Inhalation LC50 (4 h) for rats is 0.7 mg/L air. NOEL (2 y) for rats is 2.5 mg/kg/d. ADI is 1 µg/kg b.w. Orally administered phosalone in mammals is rapidly degraded by oxidation and hydrolysis to give O,O-diethyl hydrogen phosphorothioate and phosphorodithioate and 6-chloro-2,3-dihydro-2-oxobenzoxazole, which is further metabolized and excreted in the urine. It is strongly adsorbed to soil and rapidly degraded with DT50 values of 1–4 d.

PHOSMET

O

S (CH3O)2P

SCH2

N O

IUPAC name. O,O-Dimethyl S-phthalimidomethyl phosphorodithioate CASRN. 732-11-6 Selected trade names. Imidan, Prolate Phosmet is an off-white crystalline solid, mp 72 ◦ C, vp 0.065 mPa (25 ◦ C). The water solubility is 25 mg/L (25 ◦ C). It is readily soluble in most organic solvents except aliphatic hydrocarbons. Log Kow = 2.95. Phosmet is relatively stable in acid conditions but rapidly hydrolyzed in alkaline media; DT50 values (20 ◦ C) at pH 4.5, 7, and 8.3 are 13 d, 4.0 mg/L air. NOEL (2 yr) for rats is 15 mg/kg diet (0.75 mg/kg/d). ADI is 1 µg/kg b.w. The high selective toxicity of phoxim may be due to the specificity of the oxon against insect AChE and to the rapid degradation in mammals. Phoxim is oxidatively desulfurated to the oxon, which inhibits housefly AChE 270 times as quickly as bovine AChE. In mammals, the oxon is immediately hydrolyzed into diethyl hydrogen phosphate. The direct cleavage of the oxime ester bond of phoxim, the hydrolytic transformation of the nitrile group into carboxyl, and deethylation also contribute to the low mammalian toxicity. Elimination is very quick, 97% of the dose being excreted in the urine and feces in 24 h. Degradation in soils is also very rapid. By photochemical reactions, the thiooxime phosphate isomer, tetraethyl pyrophosphate, and its monothio analog are produced in small amounts on foliage.

PIRIMIPHOS-ETHYL

CH3 S (C2H5O)2P

PIRIMIPHOS-METHYL

CH3 S (CH3O)2P

N

O

N

N(C2H5)2

IUPAC name. O-(2-Diethylamino-6-methylpyrimidin4-yl) O,O-dimethyl phosphorothioate CASRN. 29232-93-7 Trade name. Actellic Pirimiphos-methyl is a straw-colored liquid, mp 15–18 ◦ C, decomposes on distillation, vp 2 mPa (20 ◦ C). Solubility in water is 8.6 mg/L (pH 7.3). It is miscible with most organic solvents. Log Kow = 4.2. It is hydrolyzed in acidic and alkaline media; DT50 values (25 ◦ C) in pH range 5.8 and 8.5 are 7.5–35 d. Pirimiphos-methyl is a broad spectrum insecticideacaricide with contact and respiratory action. Owing to the low mammalian toxicity, it is useful for animal and public health as well as crop protection. The major formulation types are emulsifiable concentrate, dustable powder, smoke, ultra low volume liquid, and aerosol. The acute oral LD50 for rats is 2050 mg/kg. Inhalation LC50 (4 h) for rats is >5 mg/L. NOEL (2 y) for rats is 10 mg/kg diet (0.5 mg/kg/d). ADI is 0.03 mg/kg b.w. In mammals, pirimiphos-methyl is degraded by extensive cleavage of the P−O pyrimdine bond and N-dealkylation followed by conjugation of the pyrimidine moiety. The half-life in soils is 5 ppm. NOEL (90 d) for rats is 1.6 mg/kg diet (0.08 mg/kg/d). The half-life in soils is 21–70 d.

C2H5O

P

O

Br

CH3CH2CH2S IUPAC name. O-(4-Bromo-2-chlorophenyl) O-ethyl Spropyl phosphorothioate CASRN. 41198-08-7 Trade name. Curacron Profenofos is a pale yellow liquid, bp 100 ◦ C/1.80 Pa, vp 0.124 mPa (25 ◦ C). Solubility in water is 28 mg/L (25 ◦ C). It is miscible with most organic solvents. Log Kow = 4.44. It is relatively stable in neutral and mild acid media but hydrolyzed in alkaline media; DT50 values (20 ◦ C) at pH 5, 7, and 9 are 93 d, 14.6 d, and 5.7 h, respectively. Profenofos is a nonsystemic insecticide–acaricide effective for the control of Lepidoptera and mites on a wide range of crops. It has ovicidal properties. The (R)-(−) isomer is biologically more active than the other isomer. Profenofos is mainly formulated as emulsifiable concentrates, ultra low volume liquids, and granules. The acute oral LD50 for rats is 358 mg/kg. Inhalation LC50 (4 h) for rats is about 3 mg/L air. NOEL (2 yr) for rats is 0.3 mg/kg


diet (0.015 mg/kg/d). ADI is 0.01 mg/kg b.w. Profenofos orally administered to rats is rapidly excreted, mainly in the urine. The principal degradation route is hydrolysis to 4-bromo-2-chlorophenol followed by conjugation. DT50 in soil is about 1 week. PROPAPHOS

O (CH3CH2CH2O)2P

O

SCH3

IUPAC name. 4-(Methylthio)phenyl dipropyl phosphate CASRN. 7292-16-2 Trade name. Kayaphos Propaphos is a colorless liquid, bp 175–177 ◦ C/0.85 mm Hg, vp 0.12 mPa (25 ◦ C). Solubility in water is 125 mg/L (25 ◦ C). It is soluble in most organic solvents. Log Kow = 3.67. It is stable in neutral and mild acid media, but is hydrolyzed in alkaline media. Propaphos is a systemic insecticide with contact and stomach action and is used for the control of both rice hoppers and stem borers in paddy rice. It is effective against strains resistant to other OP and carbamate insecticides. Propaphos is formulated as emulsifiable concentrates, dusts, and granules. The acute oral LD50 for rats is 70 mg/kg. Inhalation LC50 (4 h) for rats is 39.2 mg/m3 . NOEL (2 yr) for rats is 0.08 mg/kg/d. Propaphos orally administered to rats is rapidly excreted, mainly in the urine. The principal metabolic routes of propaphos are oxidation of the sulfide group to the sulfoxide and sulfone, and hydrolysis of phenyl phosphate ester bond in both animals and plants. PROPETAMPHOS

CH3O

S P

C2H5NH

H

O C CH3

C CO2CH(CH3)2

IUPAC name. (E)-O-2-(Isopropoxycarbonyl-1-methylvinyl) O-methyl ethylphosphoramidothioate CASRN. 31218-83-4 Trade name. Safrotin Propetamphos is a yellowish liquid, bp 87–89 ◦ C/0.005 mm Hg, vp 1.9 mPa (20 ◦ C). Solubility in water is 110 mg/L (24 ◦ C). It is miscible with most organic solvents. Log Kow = 3.82. It is relatively stable in aqueous solutions; DT50 values (20 ◦ C) at pH 3, 6, and 9 are 11 d, 1 yr, and 41 d, respectively. Propetamphos is an insecticide with contact and stomach action, having long residual activity. It is mainly used for control of household and public health pests. It is also used for the control of animal ectoparasites. Main

1173

formulation types are emulsifiable concentrate, wettable powder, aerosol, and dustable powder. The acute oral LD50 for rats is 59.5–119 mg/kg. Inhalation LC50 (4 h) for rats is 0.69 (female) and >1.5 (male) mg/L air. NOEL (2 yr) for rats is 6 mg/kg diet (0.3 mg/kg/d). Propetamphos administered in animals is rapidly metabolized and excreted mainly via urine and exhaled air. The major pathways of detoxication in mammals are O-demethylation and cleavage of the P−O-vinyl linkage to give isopropyl acetoacetate, which is finally metabolized to carbon dioxide. Hydrolysis of the carboxylic ester bond is also involved. Activation by oxidative desulfuration also occurs. PROTHIOFOS

Cl

S C2H5O

P

O

Cl

CH3CH2CH2S IUPAC name. O-(2,4-Dichlorophenyl) O-ethyl S-propyl phosphorodithioate CASRN. 34643-46-4 Trade name. Tokuthion Prothiofos is a colorless liquid, bp 125–128 ◦ C/13 Pa, vp 0.3 mPa (20 ◦ C). It is nearly insoluble in water (1.7 mg/L at 20 ◦ C) but readily soluble in most organic solvents. Log Kow = 5.67. It is relatively stable in aqueous media; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 120, 280, and 12 d, respectively. Prothiofos is a nonsystemic insecticide with contact and stomach action and is used to control chewing insects in a range of crops including vegetables, maize, sugarcane, and ornamentals. It is mainly formulated as emulsifiable concentrates and wettable powder. The acute oral LD50 for rats is 1390–1569 mg/kg. Inhalation LC50 (4 h) for rats is >2.7 mg/L air. NOEL (2 yr) for rats is 5 mg/kg diet (0.25 mg/kg/d). ADI is 0.1 µg/kg b.w. Prothiofos administered to rats is rapidly metabolized, and 98% of the dose is excreted in 72 h. The principal metabolic routes are activation by oxidative desulfuraton and detoxification by dearylation and cleavage of the P−S bond in both animals and plants. Prothiofos is strongly adsorbed in soil; the half-life under field conditions is 1–2 months. PYRACLOFOS

O N C2H5O

P

O

N

Cl

CH3CH2CH2S IUPAC name. (RS)-[O-1-(4-Chlorophenyl)pyrazol-4-yl O-ethyl S-propyl phosphorothioate] CASRN. 77458-01-6

1174


Trade name. Boltage Pyraclofos is a pale yellow oil, bp 164 ◦ C/0.01 mm Hg, vp 0.0016 mPa (20 ◦ C). Solubility in water is 33 mg/L (20 ◦ C). It is miscible with most organic solvents. Log Kow = 3.77. It is slowly hydrolyzed in aqueous media; DT50 (25 ◦ C) at pH 7 is 29 d. Pyraclofos is a nonsystemic insecticide with respiratory, contact, and stomach action and is used to control Lepidoptera, Coleoptera, Acarina, and nematodes in vegetables, fruit, ornamentals, and forestry. Pyraclofos is formulated as emulsifiable concentrates, wettable concentrates, and granules. The acute oral LD50 for rats is 237 mg/kg. The inhalation LC50 for rats is 1.69 mg/L air. NOEL (2 yr) for rats is 0.10–0.12 mg/kg diet (0.005–0.006 mg/kg/d). Pyraclofos administered to rats is rapidly degraded, and more than 90% of the dose is excreted principally in the urine within 24 h. The degradation routes are cleavages of the P−S, P−O-alkyl, and P−O-aryl bonds in both animals and plants. Pyraclofos is metabolized to an active AChE inhibitor, probably by the oxidation of the sulfur atom in the phosphorothiolate linkage. The half-life in soil is 3–38 d, depending on soil type.

IUPAC name. O,O-Diethyl O-quinoxalin-2-yl phosphorothioate CASRN. 13593-03-8 Selected trade names. Bayrusil, Ekalux Quinalphos is a white crystalline powder, mp 35–36 ◦ C, vp 0.346 mPa (20 ◦ C). Solubility in water is 17.8 mg/L (22 ◦ C). It is readily soluble in most organic solvents. Log Kow = 4.44. Quinalphos is rather unstable, not only in alkaline solution but also under acid conditions. It is a nonsystemic insecticide-acaricide and is used to control sucking and chewing insects and mites on a large number of crops. Quinalphos is mainly formulated as emulsifiable concentrates and granules. The acute oral LD50 for rats is 66 mg/kg. The major metabolic route of quinalphos is dearylation to quinoxolinol, which is conjugated in plants and animals. Oxidative desulfuration to the oxon occurs photochemically. The half-life in soil is about 3 weeks. SULFOTEP

S (C2H5O)2P

PYRIDAPHENTHION

O

S (C2H5O)2P

O

N

Pyridaphenthion is a pale yellow solid, mp 54.5–56 ◦ C, vp 0.00147 mPa (20 ◦ C). Solubility in water is 100 mg/L (20 ◦ C). It is very soluble in acetone, methanol, and diethyl ether. Log Kow = 3.2. Pyridaphenthion is a nonsystemic insecticide-acaricide with contact and stomach action and is used to control sucking and chewing insects and spider mites on rice, vegetables, fruit, and ornamentals. Pyridaphenthion is formulated as emulsifiable concentrates, wettable concentrates, and dustable powder. The acute oral LD50 for rats is 769–850 mg/kg. Inhalation LC50 (4 h) for rats is >1.13 mg/L air. More than 70% of the dose administered to rats and mice was excreted within 24 h in the urine. The major metabolites excreted are phenylmaleic hydrazide and desethyl pyridafenthion-oxon. The half-life in soil is 11–24 d.

N O

P(OC2H5)2

IUPAC name. O,O,O ,O -Tetraethyl dithiopyrophosphate CASRN. 3689-24-5 Trade name. Bladafum Sulfotep is synthesized by the action of water on diethyl phosphorochloridothionate in the presence of pyridine and is a pale yellow liquid, bp 92 ◦ C/0.1 mm Hg, vp 14 mPa (20 ◦ C). Solubility in water is 10 mg/L (20 ◦ C). It is miscible with most organic solvents except petroleum ether. Log Kow = 3.99. Sulfotep is slowly hydrolyzed in aqueous media; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 10.7, 8.2, and 9.1 d, respectively. It is a nonsystemic insecticide-acaricide and is used to control aphids, thrips, whiteflies, and mites on glasshouse crops by fumigation. Sulfotep is formulated as fumigant. The acute oral LD50 for rats is about 10 mg/kg. Inhalation LC50 (4 h) for rats is about 0.05 mg/L air. NOEL (2 yr) for rats is 10 mg/kg diet (0.5 mg/kg/d). ADI is 1 µg/kg b.w. Sulfotep is activated by oxidative desulfuration to tetraethyl monothiopyrophosphate and pyrophosphate in the environment. Sulfotep administered to rats is quickly eliminated. The major elimination product is diethyl hydrogen phosphorothioate. SULPROFOS

S C2H5O

QUINALPHOS

(C2H5O)2P

O

N

IUPAC name. O-(1,6-Dihydro-6-oxo-1-phenylpyridazin3-yl) O,O-diethyl phosphorothioate CASRN. 119-12-0 Trade names. Ofunack, Oreste

S

S

N

P

O

SCH3

CH3CH2CH2S IUPAC name. O-Ethyl O-4-(methylthio)phenyl S-propyl phosphorodithioate


CASRN. 35400-43-2 Trade name. Bolstar Sulprofos is a colorless liquid, bp 125 ◦ C/1 Pa, vp 0.084 mPa (20 ◦ C). It is nearly insoluble in water (0.31 mg/L at 20 ◦ C) but readily soluble in common organic solvents. Log Kow = 5.48. It is slowly hydrolyzed in aqueous media; DT50 values (22 ◦ C) at pH 4, 7, and 9 are 26, 151, and 26 d, respectively. Sulprofos is a nonsystemic insecticide with contact and stomach action and is used to control Lepidoptera, thrips, and other insects in cotton, soya beans, vegetables, tobacco, and tomatoes. Sulprofos is formulated as emulsifiable concentrates and ultra low volume liquids. The acute oral LD50 for rats is 176–304 mg/kg. Inhalation LC50 (4 h) for rats is >4.1 mg/L air. NOEL (2 yr) for rats is 6 mg/kg diet (0.3 mg/kg/d). ADI is 3 µg/kg b.w. Sulprofos administered to rats is rapidly metabolized, and 92% of the dose is excreted within 24 h. Major metabolic routes are by oxidation to the sulfoxide and sulfone and oxidative desulfuration to the oxons. Detoxification by dearylation to the phenols occurs rapidly. Sulprofos is degraded in soil with a half-life ranging from a few days to several weeks, depending on the soil type. TEBUPIRIMFOS

S C2H5O

P

N O

C(CH3)3 N

(CH3)2CHO

IUPAC name. O-(2-tert-Butylpyrimidin-5-yl) O-ethyl Oisopropyl phosphorothioate CASRN. 96182-53-5 Trade name. Aztec Tebupirimfos is an amber liquid, bp 152 ◦ C, vp 5 mPa (20 ◦ C). Solubility in water is 5.5 mg/L (20 ◦ C). It is soluble in ketones, alcohols, and toluene. Tebupirimfos is hydrolyzed under alkaline conditions. It is a nonsystemic insecticide with contact action and good residual activity. Tebupirimfos controls soil-dwelling insects by treating soil with granules. The acute oral LD50 for rats is 1.3–3.6 mg/kg. Inhalation LC50 (4 h) for rats is 36–82 mg/m3 air. NOEL (2 yr) for rats is 1 mg/kg diet (0.05 mg/kg/d). ADI is 0.2 µg/kg b.w. TEMEPHOS

(CH3O)2P

Temephos is a colorless crystalline solid, mp 30 ◦ C, vp 0.0095 mPa (20 ◦ C). Solubility in water is 0.03 mg/L (25 ◦ C). It is soluble in common organic solvents. Log Kow = 4.91. Temephos is hydrolyzed by strong acids and alkalis. It is a nonsystemic insecticide used for the control of mosquito and black fly larvae in public and animal health. Temephos is formulated as emulsifiable concentrates, granules, fumigants, etc. Temephos has a very low mammalian toxicity; acute oral LD50 for male and female rats is 4204 and >10,000 mg/kg, respectively. NOEL (2 yr) for rats is 300 mg/kg diet (15 mg/kg/d). Temephos administered orally to rats is eliminated in the feces and urine. The major elimination compound is unchanged temephos. Other urinary metabolites are sulfate ester conjugates of 4,4 -thiodiphenol, its sulfoxide, and sulfone. TERBUFOS

S (C2H5O)2P

SCH2SC(CH3)3

IUPAC name. S-tert-Butylthiomethyl O,O-diethyl phosphorodithioate CASRN. 13071-79-9 Trade name. Counter Terbufos is the S-tert-butyl homologue of phorate. It is a slightly yellow liquid, bp 69 ◦ C/0.01 mm Hg, vp 34.6 mPa (25 ◦ C). The water solubility is 4.5 mg/L (27 ◦ C). It is readily soluble in most organic solvents. Log Kow = 4.5. It is hydrolyzed by strong alkalis (pH > 9) and acids (pH < 2). Terbufos is used as a soil insecticide and nematicide by applying granules in soil. It is also effective against various above-ground pests on plants grown in the treated soil. The acute oral LD50 for rats is 1.6 mg/kg. Inhalation LC50 (4 h) for rats is 1.2–6.1 µg/L air. ADI is 0.2 µg/kg b.w. The metabolic routes of terbufos are essentially the same in plants, animals, and soils, involving the oxidation of the sulfide group into the sulfoxide, then sulfone, and oxidative desulfuration to the corresponding oxons, followed by hydrolysis to diethyl hydrogen phosphorodithioate, phosphorothioate, and phosphate. DT50 in soil is 9–27 d. TETRACHLORVINPHOS

O (CH3O)2P

S S

IUPAC name. O,O,O ,O -Tetramethyl phenylene) bis(phosphorothioate) CASRN. 3383-96-8 Trade name. Abate

O

P(OCH3)2

O,O -(thiodi-p-

Cl

O C

S O

1175

Cl

C Cl

H

Cl (Z) IUPAC name. (Z)-2-Chloro-1-(2,4,5-trichlorophenyl) vinyl dimethyl phosphate

1176


CASRN. 22248-79-9 Selected trade names. Gardona, Rabond, Debantic The raw products of tetrachlorvinphos synthesized by the Perkow reaction are the E/Z mixtures in 1 : 9 ratio. The technical grade contains 98% Z-isomer, the E-isomer being removed by crystallization. It is a white crystalline solid, mp 97–98 ◦ C, vp 0.0056 mPa (20 ◦ C), practically insoluble in water (11 mg/L at 20 ◦ C), and readily soluble in most organic solvents. Log Kow = 3.53. It is hydrolyzed slowly in acid media and more rapidly in alkaline media; DT50 (50 ◦ C) values at pH 3, 7, and 10.5 are 54 d, 44 d, and 80 h, respectively. Tetrachlorvinphos is a nonsystemic insecticide effective in controlling the larvae of Lepidoptera, Diptera, and Coleoptera in a variety of crops. It is also used against flies in livestock barns, animal ectoparasites, and stored product pests. Major formulation types are wettable powder, dustable powder, and emulsifiable concentrate. The mammalian toxicity is very low; acute oral LD50 for rats is 4000–5000 mg/kg. NOEL (2 yr) for rats is 125 mg/kg diet (6.25 mg/kg/d). In mammals, orally administered tetrachlorvinphos is rapidly metabolized through demethylation and hydrolysis of the vinyl phosphate linkage, followed by excretion within a few days. Metabolites found in the urine include the glucuronides of 2,4,5-trichlorophenylethanediol and 1-(2,4,5-trichlorophenyl)ethanol, 2,4,5-trichloromandelic acid, and desmethyl tetrachlorvinphos.

IUPAC name. O,O-Diethyl O-(1-phenyl-1H-1,2,4-triazol3-yl) phosphorothioate CASRN. 24017-47-8 Trade name. Hostathion Triazophos is a pale yellow oil, mp 2–5 ◦ C, exothermally decomposes above 140 ◦ C, vp 0.39 mPa (30 ◦ C). Solubility in water is 39 mg/L (20 ◦ C). It is readily soluble in common organic solvents. Log Kow = 3.34. It is hydrolyzed in aqueous acids and alkalis. Triazophos is a broad-spectrum insecticide–acaricide with contact and stomach action. It is used to control a wide range of insects and mites in many crops and forestry. It is also effective against some free-living nematodes. It is formulated as emulsifiable concentrates and ultra low volume liquids. The acute oral LD50 for rats is 57–59 mg/kg. Inhalation LC50 (4 h) for rats is 0.531 mg/L air. Rats receiving 1 mg/kg diet (0.05 mg/kg/d) for 2 yr were unaffected, except for serum cholinesterase inhibition. ADI is 1 µg/kg b.w. In mammals, administered triazophos is rapidly metabolized and excreted, mainly in the urine. The main metabolic route is disruption of P−O (triazolyl) bond either by hydrolysis of triazophos oxon or through oxidative cleavage of triazophos itself. The half-life in soils is 6–12 d. TRICHLORFON

O THIOMETON

(CH3O)2P S

CCl3

OH

(CH3O)2P

SCH2CH2SC2H5

IUPAC name. S-(2-Ethylthioethyl) O,O-dimethyl phosphorodithioate CASRN. 640-15-3 Trade name. Ekatin Thiometon is the thion (P=S) analog of demeton-Smethyl. It is a colorless oil, bp 104 ◦ C/0.3 mm Hg, vp 39.9 mPa (20 ◦ C). The water solubility is 200 mg/L (27 ◦ C). It is highly soluble in most organic solvents except alkanes. Log Kow = 3.15. It is hydrolyzed in alkaline and acidic media; DT50 (25 ◦ C) values at pH 3, 6, and 9 are 25, 27, and 17 d, respectively. Thiometon is a systemic insecticide–acaricide with contact activity and is effective against aphids, sawflies, thrips, and mites. The acute oral LD50 for rats is 70–120 mg/kg. Thiometon is metabolized oxidatively in plants, forming demeton-S-methyl sulfoxide and sulfone, which are the active principles. TRIAZOPHOS

S N (C2H5O)2P

CH

N

O N

IUPAC name. O,O-Dimethyl 2,2,2-trichloro-1-hydroxyethylphosphonate CASRN. 52-68-6 Selected trade names. Dipterex, Tugon For the synthesis of trichlorfon, see (16). The product is a racemate, i.e., the 1 : 1 mixture of (1R)- and (1S)enantiomers. Trichlorfon is a colorless crystalline powder; mp 83–84 ◦ C, bp 100 ◦ C/0.1 mm Hg, vp 0.21 mPa (20 ◦ C). It is soluble in water (120 g/L) and most organic solvents, except aliphatic hydrocarbons. Log Kow = 0.43. Trichlorfon is rapidly converted to dichlorvos by alkalis (2) and then hydrolyzed; DT50 (22 ◦ C) values at pH 4, 7, and 9 are 510 d, 46 h, and 0.5 mg/L air. NOEL (2 yr) for rats is 100 mg/kg diet (5 mg/kg/d). ADI is 0.01 mg/kg b.w. Trichlorfon administered to mammals is

OVICIDES

rapidly metabolized and excreted almost completely in the urine within 6 h. Major metabolites are dimethyl hydrogen phosphate, methyl dihydrogen phosphate, and conjugates of dichloroacetic acid and trichloroethanol. Trichlorfon is rapidly broken down in soil. VAMIDOTHION

O (CH3O)2P

CH3 SCH2CH2SCHCONHCH3

IUPAC name. O,O-Dimethyl S-2-(1-methylcarbamoylethylthio)ethyl phosphorothioate CASRN. 2275-23-2 Trade name. Kilval Vamidothion is a colorless crystalline substance, mp 46–48 ◦ C, vp 0.009 mPa (20 ◦ C). It is readily soluble in water (4 kg/L) and most organic solvents except aliphatic hydrocarbons. Log Kow = 0.12. It decomposes in strong alkaline and acidic media. Vamidothion is a systemic insecticide-acaricide with long persistence. It is formulated as emulsifiable concentrates and used for control of Homoptera in cotton, hops, fruit, and rice. The acute oral LD50 for rats is 64–105 mg/kg. Inhalation LC50 (4 h) for rats is 1.73 mg/L air. ADI is 8 µg/kg b.w. In mammals, the sulfide group of vamidothion is oxidized into the sulfoxide, then sulfone, followed by cleavage of the P−S and PS−C bonds to give watersoluble metabolites. O-Demethylation and hydrolytic P−S bond cleavage of vamidothion itself are also important degradation routes. Vamidothion sulfoxide is more persistent in plants but is less toxic to mammals than

1177

the parent compound. Vamidothion is rapidly degraded in soil with DT50 (22 ◦ C) of 1–1.5 d under aerobic conditions. FURTHER READING Chambers, J. E. and Levi, P. E., eds., Organophosphates Chemistry, Fate, and Effects, Academic Press, San Diego, 1992, p. 443. Eto, M., Organophosphorus Pesticides: Organic and Biological Chemistry, CRC Press, Cleveland, 1974, p. 387, contains 1,097 references. Eto, M. and Casida, J. E., eds., Progress and Prospects of Organophosphorus Agrochemicals, Kyushu Univ. Press, Fukuoka, 1995, p. 190. Roberts, T. R. and Hutson, D. H., eds., Metabolic Pathways of Agrochemicals, Part 2: Insecticides and Fungicides, The Royal Society of Chemistry, Cambridge, 1999, pp. 187–522. Schrader, G., Die Entwicklung neuer insektizider Phosphorsaure¨ Ester, Verlag Chemie, Weinheim, 1963, p. 444. Shibuya, S. and Shimazaki, I., Shibuya Index, 8th edn., Zen-Noh, Tokyo, 1999, p. 873, a complete index of pesticides. Tomlin, C. D. S., ed., The Pesticide Manual, 11th edn., British Crop Protection Council, Farnham, 1997. WHO, Environmental Health Criteria 63, Organophosphorus Insecticides: A General Introduction, WHO, Geneva, 1986, p. 181. WHO, Environmental Health Criteria 132, Trichlorfon, WHO, Geneva, 1992, p. 162. WHO, Environmental Health Criteria 133, Fenitrothion, WHO, Geneva, 1992, p. 184.

OVICIDES Substances that kill eggs of insects and mites (USEPA).

P PAN

transport within the body, to be reviewed later in this chapter, provide appropriate rationales for size-selective aerosol sampling approaches and/or usage of biomarkers of exposure. Finally, this chapter discusses the choices of sampling times, intervals, rates, durations, and schedules most appropriate for exposure measurements and/or modeling that are most relevant to risk assessment strategies that reflect data needs for 1) documenting compliance with exposure standards; 2) performing epidemiological studies of exposure–response relationships; 3) developing improved exposure models; and 4) facilitating secondary uses of exposure data for epidemiological research, studies of the efficacy of exposure controls, and analyses of trends.

Pesticide Action Network (http://www.panna.org)

PATHWAYS AND MEASURING EXPOSURE TO TOXIC SUBSTANCES MORTON LIPPMANN

INTRODUCTION For toxic substances in the environment to exert adverse effects on humans, they must deposit on and/or penetrate through a body surface and reach target sites where they can alter normal functions and/or structures. The critical pathways and target sites can vary greatly from substance to substance and, for a given substance, can vary with its chemical and physical form. A further complication arises from the fact that chemical and/or metabolic transformations can take place between deposition on a body surface and the eventual arrival of a toxic substance or metabolite of that substance at a critical target site. A critical target site is where the toxic effect of first or greatest concern takes place. This chapter reviews and summarizes current knowledge concerning the generic aspects of the environmental pathways and processes leading to 1) deposition of toxicants on body surfaces (skin, respiratory tract, gastrointestinal tract); 2) uptake of toxicants by epithelial cells from environmental media (air, waste, food); 3) translocation and clearance pathways within the body for toxicants that penetrate a surface epithelium; and 4) the influence of chemical and physical form of the toxicant on the metabolism and pathways of the chemical of concern. Where the physical attributes of the toxicant such as the length and biopersistence of airborne fibers are of generic concern, these are also discussed in this chapter. Other aspects of the pathways and the fates of toxicants that are specific to the chemical species that are the subject of the following chapters of this volume are discussed, as appropriate, in those chapters. This chapter also summarizes and discusses techniques for measuring personal and population exposures to environmental toxicants and their temporal and spatial distributions. Quantitative exposure assessment, as a component of risk assessment, involves consideration of 1) the nature and properties of chemicals in environmental media, 2) the presence in environmental media of the specific chemicals that are expected to exert toxic effects, 3) the temporal and spatial distributions of the exposures of interest, and 4) the ways that ambient or workplace exposure measurements or models can be used to draw exposure inferences. In this context, the knowledge of deposition, fate, pathways, and rates of metabolism and

NATURE OF TOXIC SUBSTANCES Physical Properties of Toxic Air Contaminants Chemicals can be dispersed in air at normal ambient temperatures and pressures in gaseous, liquid, and solid forms. The latter two represent suspensions of particles in air and were given the generic term ‘‘aerosols’’ by Gibbs (1) by analogy with the term ‘‘hydrosol,’’ used to describe dispersed systems in water. Although hydrosols generally have uniformly sized particles, aerosols do not. Gases and vapors, which are present as discrete molecules, form true solutions in air. Particles composed of moderate- to high-vapor-pressure materials evaporate rapidly because those small enough to remain suspended in air for more than a few minutes (i.e., those smaller than about 10 µm) have large surface to volume ratios. Some materials with relatively low vapor pressures can have appreciable fractions in both vapor and aerosol forms simultaneously. Once dispersed in air, contaminant gases and vapors generally form mixtures so dilute that their physical properties, such as density, viscosity, and enthalpy, are indistinguishable from those of clean air. Such mixtures follow ideal gas law relationships. There is no practical difference between a gas and a vapor except that the latter is generally the gaseous phase of a substance that can exist as a solid or liquid at room temperature. While dispersed in the air, all molecules of a given compound are essentially equivalent in their size and capture probabilities by ambient surfaces, respiratory tract surfaces, and contaminant collectors or samplers. Aerosols are dispersions of solid or liquid particles in air and have the very significant additional variable of particle size. Size affects particle motion and, hence, the probabilities of physical phenomena such as coagulation, dispersion, sedimentation, impaction onto surfaces, interfacial phenomena, and light-scattering. It is not possible to characterize a given particle by a single size parameter. For example, a particle’s aerodynamic properties depend on density and shape, as well as linear dimensions, and the effective size for light scattering depends on refractive index and shape. 1178

PATHWAYS AND MEASURING EXPOSURE TO TOXIC SUBSTANCES

In some special cases, all of the particles are essentially the same size. Such aerosols are considered monodisperse. Examples are natural pollens and some laboratorygenerated aerosols. More typically, aerosols are composed of particles of many different sizes and hence are called heterodisperse or polydisperse. Different aerosols have different degrees of size dispersion. Therefore, it is necessary to specify at least two parameters in characterizing aerosol size: a measure of central tendency, such as a mean or median, and a measure of dispersion, such as an arithmetic or geometric standard deviation. Particles generated by a single source or process generally have diameters that follow a log-normal distribution, i.e., the logarithms of their individual diameters have a Gaussian distribution. In this case, the measure of dispersion is the geometric standard deviation, which is the ratio of the 84.16th percentile size to the 50th percentile size. When more than one source of particles is significant, the resulting mixed aerosol will usually not follow a single log-normal distribution, and it may be necessary to describe it by the sum of several distributions. Particle and Aerosol Properties Many properties of particles, other than their linear size, can greatly influence their airborne behavior and their effects on the environment and health. These include Surface: For spherical particles, the surface varies as the square of the diameter. However, for an aerosol of given mass concentration, the total aerosol surface increases with decreasing particle size. For nonspherical or aggregate particles, the particles may have internal cracks or pores, and the ratio of surface to volume can be much greater than for spheres. Volume: Particle volume varies as the cube of diameter; therefore, the few largest particles in an aerosol dominate its volume (or mass) concentration. Shape: A particle’s shape affects its aerodynamic drag, as well as its surface area, and therefore its motion and deposition probabilities. Density: A particle’s velocity in response to gravitational or inertial forces increases as the square root of its density. Aerodynamic diameter: The diameter of a unit-density sphere that has the same terminal settling velocity as the particle under consideration is equal to its aerodynamic diameter. Terminal settling velocity is the equilibrium velocity of a particle that is falling under the influence of gravity and fluid resistance. Aerodynamic diameter is determined by the actual particle size, the particle density, and an aerodynamic shape factor. Types of Aerosols Aerosols are generally classified in terms of their processes of formation. Although the following classification is neither precise nor comprehensive, it is commonly used and accepted in the industrial hygiene and air pollution fields.

1179

Dust: An aerosol formed by mechanical subdivision of bulk material into airborne fines that have the same chemical composition. Dust particles are generally solid and irregular in shape and have diameters greater than 1 µm. Fume: An aerosol of solid particles formed by condensation of vapors formed at elevated temperatures by combustion or sublimation. The primary particles are generally very small (less than 0.1 µm) and have spherical or characteristic crystalline shapes. They may be chemically identical to the parent material, or they may be composed of an oxidation product such as a metal oxide. Because they may be formed in high concentrations, they often coagulate rapidly and form aggregate clusters of low overall density. Smoke: An aerosol formed by condensation of combustion products, generally of organic materials. The particles are generally liquid droplets whose diameters are less than 0.5 µm. Mist: A droplet aerosol formed by mechanical shearing of a bulk liquid, for example, by atomization, nebulization, bubbling, or spraying. The droplet size can cover a very large range, usually from about 2 to greater than 50 µm. Fog: An aqueous aerosol formed by condensation of water vapor on atmospheric nuclei at high relative humidities. The droplet sizes are generally larger than 1 µm. Smog: A popular term for a pollution aerosol derived from a combination of smoke and fog. The term is commonly used now for any atmospheric pollution mixture. Haze: A submicrometer-sized aerosol of hydroscopic particles that take up water vapor at relatively low relative humidities. Aitken or condensation nuclei (CN): Very small atmospheric particles (mostly smaller than 0.05 µm) formed by combustion processes and by chemical conversion from gaseous precursors. Accumulation mode: A term given to the particles in the ambient atmosphere ranging in diameter from 0.1 to about 1.0 µm. These particles generally are spherical, have liquid surfaces, and form by coagulation and condensation of smaller particles that derive from gaseous precursors. Too large for rapid coagulation and too small for effective sedimentation, they accumulate in the ambient air. Coarse particle mode: Ambient air particles larger than about 2.5 µm in aerodynamic diameter and generally formed by mechanical processes and surface dust resuspension. Physical Properties of Toxic Liquid and Solid Components For liquids and solids deposited on human skin or taken into the gastrointestinal (GI) tract by ingestion, penetration to and through the surface epithelium depends upon their physical form, their solubility in the fluids on the surface, and the structure and nature of the epithelial barrier. Dissolved chemicals can penetrate by diffusion,

1180


whereas chemicals present as particles or droplets must find access via pores or defects in the barrier associated with injury caused by trauma or corrosive chemicals or by dissolution in solvents that alter the barrier function.

Exposure is a key and complex step in this continuum. The concept of total human exposure developed in recent years is essential to the appreciation of the nature and extent of environmental health hazards associated with ubiquitous chemicals at low levels. It provides a framework for considering and evaluating the contribution to the total insult from dermal uptake, ingestion of food and drinking water, and inhaled doses from potentially important microenvironments such as workplace, home, transportation, recreational sites, etc. More thorough discussions of this key concept have been prepared by Sexton and Ryan (3), Lioy (4), and the National Research Council (5). Guidelines for Exposure Assessment have been formalized by the U.S. Environmental Protection Agency (6). Figure 2 outlines possible approaches for estimating contaminant exposures of populations, as well as individuals, in a conceptual sense, and Figure 3 indicates terminologies used by EPA to describe exposures and their distributions within a population. Toxic chemicals in the environment that reach sensitive tissues in the human body can cause discomfort, loss of function, and changes in structure leading to disease. This section addresses the pathways and transport rates of chemicals from environmental media to critical tissue sites, as well as retention times at those sites. It is designed to provide a conceptual framework as well as

HUMAN EXPOSURE PATHWAYS AND DOSIMETRY People can be exposed to chemicals in the environment in numerous ways. The chemicals can be inhaled, ingested, or taken up by and through the skin. Effects of concern can take place at the initial epithelial barrier, i.e., the respiratory tract, the gastrointestinal (GI) tract, or the skin, or can occur in other organ systems after penetration and translocation by diffusion or transport by blood, lymph, etc. As illustrated in Figure 1, exposure and dose factors are intermediate steps in a larger continuum ranging from the release of chemicals into an environmental medium to an ultimate health effect in an exposed individual. There are, of course, uncertainties of varying magnitude at each stage. The diagram could also be applied to populations as well as to individuals. In that case, each stage of the figure would include additional variance for the interindividual variability within a population associated with age, sex, ethnicity, size, activity patterns, dietary influences, use of tobacco, drugs, alcohol, etc.

Outdoor emission sources

Indoor emission sources

Dispersion, conversion, and removal factors (including weather) Building penetration

Outdoor concentrations

air exchange, conversion, and removal factors

Time-activity patterns

Total personal exposures

Dispersion, conversion, and removal factors (including ventilation) Indoor concentrations

Time-activity patterns

Host factors (Body and airway sizes, activity modified intake rates) Applied doses (to skin, airways, GI tract) Host factors (Internal translocation, metabolism) Biologically effective doses (to critical target tissues) Host factors (Generic determinants, prior injury or disease) Figure 1. Framework for personal exposure assessment and exposure-response (modified from Ref. 2).

Health effects (Mortality, morbidity, function decrements)


1181

Exposure analysis approaches

Environmental models and source inventories

Methods related to individuals

Personal monitoring

Biological monitoring

Mitigation factors

Questionnaires and diaries

Methods related to populations

Environmental concentrations

Demographics and lifestyle data

Population exposure models

Individual exposure models Exposure assessments

Figure 2. Possible approaches for analyzing contaminant exposures.

Prior EPA terminology

EPA guidance (EPA, 1992)

Estimators of levels of exposure within a known* (or default)** Distribution of exposure to a chemical agent

Typical %ile of exposure 50%

90%

95% 98% 99%

99.9%

High end of exposure

Semi-quantitative terminology

Reasonable worst case Maximum exposure

brief discussions of 1) the mechanisms for—and some quantitative data on—uptake from the environment; 2) translocation within the body, retention at target sites, and the influence of the physicochemical properties of the chemicals on these factors; 3) the patterns and pathways for exposure of humans to chemicals in environmental media; and 4) the influence of age, sex, size, habits, health status, etc. Terminology An agreed on terminology is critically important when discussing the relationships among toxic chemicals in the environment, exposures to individuals and populations,

Bounding estimate

Worst case MEI

Figure 3. EPA guidance on terminology for exposures in the general population.

and human health. Key terms used in this chapter are defined as follows: Exposure: Contact with external environmental media containing the chemical of interest. For fluid media in contact with the skin or respiratory tract, both concentration and contact time are critical. For ingested material, concentration and amount consumed are important. Microenvironments: Well-defined locations that can be treated as homogeneous (or well characterized) in the concentrations of a chemical or other stressor. Deposition: Capture of the chemical at a body surface site on the skin, the respiratory tract, or the GI tract.

1182


Clearance: Translocation from a deposition site to a storage site or depot within the body or elimination from the body. Retention: Presence of residual material at a deposition site or along a clearance pathway. Dose: The amount of chemical deposited on (applied dose) or translocated to a site on or within the body where toxic effects can take place (delivered dose). Target tissue: A site within the body where toxic effects lead to damage or disease. Depending on the toxic effects of concern, a target tissue can extend from whole organs to specific cells and to subcellular constituents within cells. Exposure surrogates or indices: Indirect measures of exposure, such as: 1) concentrations in environmental media at times or places other than those directly encountered; 2) concentrations of the chemical of interest, a metabolite of the chemical, or an enzyme induced by the chemical in circulating or excreted body fluids, generally referred to as a biomarker of exposure; and 3) elevations in body burden measured by external probes. PATHWAYS Respiratory Tract The respiratory system extends from the breathing zone just outside of the nose and mouth through the conductive airways in the head and thorax to the alveoli, where respiratory gas exchange takes place between alveoli and the capillary blood flowing around them. The prime function of the respiratory system is to deliver oxygen (O2 ) to the gas-exchange region of the lung, where it can diffuse to and through the walls of the alveoli to oxygenate the blood passing through the alveolar capillaries, as needed over a wide range of work or activity levels. In addition, the system must also: 1) remove an equal volume of carbon dioxide (CO2 ) that enters the lungs from the alveolar capillaries; 2) maintain body temperature and water vapor saturation within the lung airways (to maintain the viability and functional capacities of the surface fluids and cells); 3) maintain sterility (to prevent infections and their adverse consequences); and 4) eliminate excess surface fluids and debris, such as inhaled particles and senescent phagocytic and epithelial cells. It must accomplish all of these demanding tasks continuously during a lifetime and do so with highly efficient performance and energy utilization. The system can be abused and overwhelmed by severe insults, such as high concentrations of cigarette smoke and industrial dust, or by low concentrations of specific pathogens that attack or destroy its defense mechanisms or cause them to malfunction. Its ability to overcome and/or compensate for such insults as competently as it usually does is a testament to its elegant combination of structure and function. Mass Transfer The complex structure and numerous functions of the human respiratory tract have been summarized concisely

by a Task Group of the International Commission on Radiological Protection (7), as shown in Figure 4. The conductive airways, also known as the respiratory dead space, occupy about 0.2 liter (L). They condition the inhaled air and distribute it by convective (bulk) flow to approximately 65,000 respiratory acini that lead off the terminal bronchioles. As tidal volumes increase, convective flow dominates gas exchange deeper into the respiratory bronchioles. In any case, within the respiratory acinus, the distance from the convective tidal front to alveolar surfaces is short enough so that efficient CO2 –O2 exchange takes place by molecular diffusion. By contrast, submicrometer sized airborne particles whose diffusion coefficients are smaller by orders of magnitude than those for gases, remain suspended in the tidal air and can be exhaled without deposition. A significant fraction of the inhaled particles do deposit within the respiratory tract. The mechanisms that account for particle deposition in the lung airways during the inspiratory phase of a tidal breath are summarized in Figure 5. Particles larger than about 2 µm in aerodynamic diameter (the diameter of a unit density sphere that has the same terminal settling (Stokes) velocity) can have significant momentum and deposit by impaction at the relatively high velocities present in the larger conductive airways. Particles larger than about 1 µm can deposit by sedimentation in the smaller conductive airways and gas-exchange airways where flow velocities are very low. Particles smaller than 0.1 µm are in Brownian motion, and their random walk while in small airways causes them to diffuse to and deposit on small airway walls at a rate that increases with decreasing size. Finally, particles whose diameters are between 0.1 and 1 µm, which have a very low probability of depositing during a single tidal breath, can be retained within the approximately 15% of the inspired tidal air that is exchanged with residual lung air during each tidal cycle. This volumetric exchange occurs because of the variable time constants for airflow in the different segments of the lungs. Because of the much longer residence times of residual air in the lungs, the low intrinsic particle displacements of 0.1 to 1 µm particles within such trapped volumes of inhaled tidal air become sufficient to cause their deposition by sedimentation and/or diffusion over the course of successive breaths. The essentially particle-free residual lung air that accounts for about 15% of the expiratory tidal flow acts like a clean-air sheath around the axial core of distally moving tidal air, so that particle deposition in the respiratory acinus is concentrated on interior surfaces such as airway bifurcations, whereas interbranch airway walls have relatively little particle deposition. The number of particles deposited and their distribution along the respiratory tract surfaces, along with the toxic properties of the material deposited, are the critical determinants of pathogenic potential. The deposited particles can damage the epithelial and/or the mobile phagocytic cells at or near the deposition site or can stimulate the secretion of fluids and cell-derived mediators that have secondary effects on the system. Soluble materials deposited as, on, or within particles can diffuse into and

Mucous membrane, respiratory epithelium, no cartilage, no glands, smooth muscle layer

Nose mouth

Mucous membrane, respiratory or stratified epithelium, glands

Larynx

1

Trachea main bronchi

2− 8

Bronchi

0

Mucous membrane, respiratory epithelium, cartilage rings, glands

ET1

Pharynx ET2 LNET (N-P) posterior Esophagus

BB

Mucous membrane, respiratory epithelium, cartilage plates, smooth muscle layer, glands

(T-B)

Gas exchange; very slow particle clearance

Conduction

bb Mucous membrane, single-layer respiratory epithelium, less ciliated, smooth muscle layer Mucous membrane, single-layer respiratory epithelium of cubodial cells, smooth muscle layers

15

16−18

Squamous alveolar epithelium cells Wall consists of alveolar entrance (type I), covering 93% of alveolar rings, squamous epithelium layer, surface areas surfactant

**

Cuboidal alveolar epithelial cells (type II. Surfactant-producing), covering 7% of alveolar surface area

**

Interalveolar septa covered by squamous epithelium, containing capillaries, surfactant

Terminal bronchioles

LNTH

Respiratory bronchioles

Alveolar ducts A]

P

Alveolar ducts

Alveolar macrophages Lymphatics * Previous ICRP model ** Unnumbered because of imprecise information † Lymph nodes are located only in BB region but drain the bronchial and alveolar interstitial regions as well as the bronchial region.

Figure 4. Structure and function of the human respiratory tract.

1183

L

Gas-exchange transitory

Air conduction; gas Respiratory epithelium consisting exchange; slow mainly of clara cells (secretory) particle clearance and few ciliated cells

9−14

Bronchioles

Extrathoracic

Respiratory epithelium with clara cells (no goblet cells) cell types: – Ciliated cells – Nonciliated cells • Clara (secretory) cells

Anterior nasal passages

Extrapulmonary

Mucous membrane, respiratory epithelium (pseudostratified, ciliated, mucous), glands

Number of airways

2 × 10–3m2

—

4.5 × 10–3m2

—

3 × 10–2m2

511

2.6 × 10–1m2 6.5 × 104 Thoracic

Respiratory epithelium with goblet cells: cell types: – Ciliated cells – Nonciliated cells: • Goblet cells • Mucous (secretoey) cells • Serous cells • Brush cells • Endocrine cells • Basal cells • Intermediate cells

Airway surface

Pulmonary

Air conditioning; temperature and humidity, and cleaning; fast particle clearance; air conduction

Anatomy

Conditioning

Histology (walls)

0.175 × 10–3m3 (anatomical dead space)

Cylology (eplthillum)

0.2 × 10–3m3

Functions

Regions used in model Zones New Old* (air) Location

4.5 × 10–3m3

Generation number

7.5m2

4.6 × 105

140m2

4.5 × 107

1184


Interception Diffusion

Sedimentation Impaction – + Electrostatic deposition Flow streamline Particle trajectory Figure 5. Mechanism for particle deposition in lung airways.

through surface fluids and cells and be rapidly transported throughout the body by the bloodstream. The aqueous solubility of bulk materials is a poor guide to particle solubility in the respiratory tract. Generally solubility is greatly enhanced by the very large surface to volume ratio of particles small enough to enter the lungs. Furthermore, the ionic and lipid contents of surface fluids within the airways are complex and highly variable and can lead to enhanced solubility or to rapid precipitation of aqueous solutes. In addition the clearance pathways and residence times for particles on airway surfaces are very different in the different functional parts of the respiratory tract. The ICRP (7) Task Group’s clearance model identifies the principal clearance pathways within the respiratory tract that are important in determining the retention of various radioactive materials and thus the radiation doses received by respiratory tissues and/or other organs after translocation. The ICRP deposition model is used to estimate the amount of inhaled material that enters each clearance pathway. These discrete pathways are represented by the compartment model shown in Figure 6. They correspond to the anatomic compartments illustrated

Anterior nasal

in Figure 4 and are summarized in Table 1, along with those of other groups that provide guidance on the dosimetry of inhaled particles. Extrathoracic Airways As shown in Figure 4, the extrathoracic airways were partitioned by ICRP (7) into two distinct clearance and dosimetric regions: the anterior nasal passages (ET1 ) and all other extrathoracic airways (ET2 ), i.e., the posterior nasal passages, the naso- and oropharynx, and the larynx. Particles deposited on the surface of the skin that lines the anterior nasal passages (ET1 ) are assumed to be subject only to removal by extrinsic means (nose blowing, wiping, etc.). The bulk of material deposited in the nasooropharynx or larynx (ET2 ) is subject to fast clearance in the layer of fluid that covers these airways. The 1994 ICRP model recognizes that diffusional deposition of ultrafine particles in the extrathoracic airways can be substantial, whereas earlier ICRP models did not (8–10). Thoracic Airways Radioactive material deposited in the thorax is generally divided between the tracheobronchial (TB) region, where

ET1 14

Extrathoracic:

1

0.001 Naso-oropharynx/ larynx

LNET 13

Sequestered in tissue 0.01 Bronchi

100

ETseq 12

ET2

0.03

BBseq 9

BB2 8

BB1

0.01 LNTH

bbseq

bb2 6

Alveolarinterstitium Figure 6. Compartment model.

7 2

bb1

5

0.0001 0.001 0.02 0.00001 AI 3 AI 2 AI 10 Thoracic:

3

2

GI tract 15

Surface transport 10

0.03 Bronchioles

11

Environment 16

4 1

1


1185

Table 1. Respiratory Tract Regions as Defined in Particle Deposition Models Anatomic Structures Included

ISO and CEN Regions

ACGIH Region

Nose, nasopharynx

1966 ICRP Task Group Region

1994 ICRP Task Group Region

Head airways (HAR)

Extrathoracic (E)

Nasopharynx (NP)

Anterior nasal passages (ET1 )

Mouth, oropharynx, laryngopharynx Trachea, bronchi, and conductive bronchioles (to terminal bronchioles)

Tracheobronchial (TBR)

Tracheobronchial (B)

Tracheobronchial (TB)

All other extrathoracic (ET2 ) Trachea and large bronchi (BB) Bronchioles (bb)

Respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli

Gas exchange (GER)

Alveolar (A)

Pulmonary (P)

Alveolar-interstitial (Al)

deposited particles are subject to relatively fast mucociliary clearance (duration in hours to 1 or 2 days), and the alveolar-interstitial (AI) region, where macrophagemediated particle clearance is much slower (duration up to several weeks), and dissolution rates for insoluble particles not cleared by macrophages can have half-times measured in months or years. For purposes of dosimetry, the ICRP (7) divided the deposition of inhaled material in the TB region between the trachea and bronchi (BB) and in the more distal, small conductive airways, known as bronchioles (bb). However, the subsequent efficiency with which mucociliary transport in either type of airway can clear deposited particles is controversial. To be certain that doses to bronchial and bronchiolar epithelia would not be underestimated, the ICRP Task Group assumed that as much as half the number of particles deposited in these airways is subject to relatively ‘‘slow’’ mucociliary clearance that lasts up to about 1 week. The likelihood that an insoluble particle is cleared relatively slowly by the mucociliary system depends on its size. Gas-Exchange Airways and Alveoli The ICRP (7) model also assumed that material deposited in the AI region is subdivided among three compartments (AI1 , AI2 , and AI3 ) each of which is cleared more slowly than TB deposition, and the subregions clear at different characteristic rates.

Regional Deposition Estimates Figure 7 depicts the predictions of the ICRP (7) Task Group Model in terms of the fractional deposition in each region as a function of the size of the inhaled particles. It reflects the minimal lung deposition between 0.1 and 1 µm, where deposition is determined largely by the exchange in the deep lung between tidal and residual lung air. Deposition increases below 0.1 µm as diffusion becomes more efficient with decreasing particle size. Deposition increases with increasing particle size above 1 µm as sedimentation and impaction become increasingly effective. Although aerodynamic diameter is an excellent index of particle behavior for relatively compact particles that differ greatly in shape and density, it is inadequate for fibers that deposit by interception, as well as by inertia, gravitational displacement, or diffusion. The aerodynamic diameter of mineral or vitreous fibers whose aspect ratio (length/width) is greater than 10 is about three times their physical diameter. Fibers whose diameters are less than 3 µm can penetrate into bronchioles whose diameters are less than 500 µm. For thin fibers longer than 10 or 20 µm, interception, whereby an end of the fiber touches a surface and is collected, accounts for a significant enhancement of deposition (11). Less complex models for size-selective regional particle deposition have been adopted by occupational health and community air pollution professionals and agencies, and these have been used to develop inhalation exposure limits

60

Regional deposition, %

AMAD

AMAD AI ET2

40

ET1 20

bb

BB 0 0.0001

0.001

1 0.01 0.1 Particle diameter, µ m

10

100

Figure 7. Fractional deposition in each region of the respiratory tract for a reference light worker (normal nose breather) in the 1994 ICRP model.

1186


within specific particle size ranges. Distinctions are made between: 1) those particles that are not aspirated into the nose or mouth and therefore represent no inhalation hazard; 2) the inhalable (aka inspirable) particulate mass (IPM), i.e., those that are inhaled and are hazardous when deposited anywhere within the respiratory tract; 3) the thoracic particulate mass (TPM), i.e., those that penetrate the larynx and are hazardous when deposited anywhere within the thorax; and 4) the respirable particulate mass (RPM), i.e., those particles that penetrate through the terminal bronchioles and are hazardous when deposited within the gas-exchange region of the lungs. These criteria are described in more detail later in this chapter in the sections devoted to exposure assessment. Translocation and Retention Particles that do not dissolve at deposition sites can be translocated to remote retention sites by passive and active clearance processes. Passive transport depends on movement on or in surface fluids that line the airways. There is a continual proximal flow of surfactant to and onto the mucociliary escalator, which begins at the terminal bronchioles, where it mixes with secretions from Clara and goblet cells. Within midsized and larger airways are additional secretions from goblet cells and mucus glands that produce a thicker mucous layer that has a serous subphase and an overlying more viscous gel layer. The gel layer that lies above the tips of the synchronously beating cilia is found in discrete plaques in smaller airways and becomes more of a continuous layer in the larger airways. The mucus that reaches the larynx and the particles carried by it are swallowed and enter the GI tract. The total transit time for particles cleared during the relatively rapid mucociliary clearance phase varies from ∼2 to 24 hours in healthy humans (12). Macrophagemediated particle clearance via the bronchial tree takes place during a period of several weeks. Compact particles that deposit in alveolar zone airways are ingested by alveolar macrophages within about 6 hours, but the movement of the particle-laden macrophages depends on the several weeks that it takes for the normal turnover of the resident macrophage population. At the end of several weeks, the particles not cleared to the bronchial tree via macrophages have been incorporated into epithelial and interstitial cells, from which they are slowly cleared by dissolution and/or as particles via lymphatic drainage pathways, passing through pleural and eventually hilar and tracheal lymph nodes. Clearance times for these later phases depend strongly on the chemical nature of the particles and their sizes, and half-times range from about 30 to 1,000 days or more. All of the characteristic clearance times cited refer to inert, nontoxic particles in healthy lungs. Toxicants can drastically alter clearance times. Inhaled materials that affect mucociliary clearance rates include cigarette smoke (13,14), sulfuric acid (15,16), ozone (17,18), sulfur dioxide (19), and formaldehyde (20). Macrophagemediated alveolar clearance is affected by sulfur dioxide (21), nitrogen dioxide and sulfuric acid (22), ozone (17,22), silica dust (23), and long mineral and vitreous fibers (24,25). Cigarette smoke affects the later

phases of alveolar zone clearance in a dose-dependent manner (26). Clearance pathways and rates that affect the distribution of retained particles and their dosimetry can be altered by these toxicants. Long mineral and manufactured vitreous fibers cannot be fully ingested by macrophages or epithelial cells and can clear only by dissolution. Most glass and slag wool fibers dissolve relatively rapidly within the lung and/or break up into shorter length segments. Chrysotile asbestos is more biopersistent than most vitreous fibers and can subdivide longitudinally, creating a larger number of long fibers. The amphibole asbestos varieties (e.g., amosite, crocidolite, and tremolite) dissolve much more slowly than chrysotile. The close association between the biopersistence of inhaled long fibers and their carcinogenicity and fibrogenicity has been described by Eastes and Hadly (27), and additional data on the influence of fiber length on the biopersistence of vitreous fibers following inhalation was described by Bernstein et al. (28). Ingestion Exposures and Gastrointestinal (GI) Tract Exposures Chemical contaminants in drinking water or food reach human tissues via the GI tract. Ingestion may also contribute to the uptake of chemicals that were initially inhaled, because material deposited on or dissolved in the bronchial mucous blanket is eventually swallowed. The GI tract may be considered a tube running through the body, whose contents are actually external to the body. Unless the ingested material affects the tract itself, any systemic response depends on absorption through the mucosal cells that line the lumen. Although absorption may occur anywhere along the length of the GI tract, the main region for effective translocation is the small intestine. The enormous absorptive capacity of this organ results from the presence in the intestinal mucosa of projections, termed villi, each of which contains a network of capillaries; the villi have a large effective total surface area for absorption. Although passive diffusion is the main absorptive process, active transport systems also allow essential lipid-insoluble nutrients and inorganic ions to cross the intestinal epithelium and are responsible for the uptake of some contaminants. For example, lead may be absorbed via the system that normally transports calcium ions (29). Small quantities of particulate material and certain large macromolecules such as intact proteins may be absorbed directly by the intestinal epithelium. Materials absorbed from the GI tract enter either the lymphatic system or the portal blood circulation; the latter carries material to the liver, from which it may be actively excreted into the bile or diffuse into the bile from the blood. The bile is subsequently secreted into the intestines. Thus, a cycle of translocation of a chemical from the intestine to the liver to bile and back to the intestines, known as the enterohepatic circulation, may be established. Enterohepatic circulation usually involves contaminants that undergo metabolic degradation in the liver. For example, DDT undergoes enterohepatic circulation; a product of its metabolism in the liver is excreted into the bile, at least in experimental animals (30).


Various factors modify absorption from the GI tract and enhance or depress its barrier function. A decrease in gastrointestinal mobility generally favors increased absorption. Specific stomach contents and secretions may react with the contaminant and possibly change it to a form with different physicochemical properties (e.g., solubility), or they may absorb it, alter the available chemical, and change the translocation rates. The size of ingested particulates also affects absorption. Because the rate of dissolution is inversely proportional to particle size, large particles are absorbed to a lesser degree, especially if they are fairly insoluble in the first place. Certain chemicals, e.g., chelating agents such as EDTA, also cause a nonspecific increase in the absorption of many materials. As a defense, spastic contractions in the stomach and intestine may eliminate noxious agents via vomiting or by accelerating the transit of feces through the GI tract. Skin Exposure and Dermal Absorption The skin is generally an effective barrier against the entry of environmental chemicals. To be absorbed via this route (percutaneous absorption), an agent must traverse a number of cellular layers before gaining access to the general circulation (Fig. 8) (31). The skin consists of two structural regions, the epidermis and the dermis, which rest on connective tissue. The epidermis consists of a number of layers of cells and varies in thickness depending on the region of the body; the outermost layer is composed of keratinized cells. The dermis contains blood vessels, hair follicles, sebaceous and sweat glands, and nerve endings. The epidermis represents the primary barrier to percutaneous absorption, the dermis is freely permeable to many materials. Passage through the epidermis occurs by passive diffusion. The main factors that affect percutaneous absorption are the degree of lipid solubility of the chemicals, the site

1187

on the body, the local blood flow, and the skin temperature. Some environmental chemicals that are readily absorbed through the skin are phenol, carbon tetrachloride, tetraethyl lead, and organophosphate pesticides. Certain chemicals, e.g., dimethyl sulfoxide (DMSO) and formic acid, alter the integrity of skin and facilitate penetration of other materials by increasing the permeability of the stratum corneum. Moderate changes in permeability may also result following topical applications of acetone, methyl alcohol, and ethyl alcohol. In addition, cutaneous injury may enhance percutaneous absorption. Interspecies differences in percutaneous absorption are responsible for the selective toxicity of many insecticides. For example, DDT is about equally hazardous to insects and mammals if ingested but is much less hazardous to mammals when applied to the skin. This results from its poor absorption through mammalian skin compared to its ready passage through the insect exoskeleton. Although the main route of percutaneous absorption is through the epidermal cells, some chemicals may follow an appendageal route, i.e., entering through hair follicles, sweat glands, or sebaceous glands. Cuts and abrasions of the skin can provide additional pathways for penetration. Absorption Through Membranes and Systemic Circulation Depending upon its specific nature, a chemical contaminant may exert its toxic action at various sites in the body. At a portal of entry—the respiratory tract, GI tract, or skin—the chemical may have a topical effect. However, for actions at sites other than the portal, the agent must be absorbed through one or more body membranes and enter the general circulation, from which it may become available to affect internal tissues (including the blood itself). Therefore, the ultimate distribution of any chemical contaminant in the body is highly dependent on its ability to traverse biological membranes. There are two main types

Idealized section of skin Surface layer (S)

Duct Keratin layer (K)

Pigment cells (P)

S

S

Epidermal cells Basal cells Sabaceous (oil) gland Hair follicle

DEFENDS AGAINST: INJURY BY: rapid entrance of water, Soap, solvents, alkalis water soluble chemicals and warm water and changes in pH

Sweat gland

K

K Mild acids, water loss from skin, Alkalis, detergents, solvents, water soluble chemicals, keratolytic chemicals, trauma and micro-organisms, ultraviolet certain internal diseases and physical injury

P P Ultraviolet Trauma (mechanical, physical or chemical) and internal diseases

Figure 8. Idealized section of skin. The horny layer is also known as the stratum corneum. From Birmingham (31).

1188


of processes by which this occurs: passive transport and active transport. Passive transport is absorption according to purely physical processes, such as osmosis; the cell has no active role in transfer across the membrane. Because biological membranes contain lipids, they are highly permeable to lipid-soluble, nonpolar, or nonionized agents and less so to lipid-insoluble, polar, or ionized materials. Many chemicals may exist in both lipid-soluble and lipidinsoluble forms; the former is the prime determinant of the passive permeability properties of the specific agent. Active transport involves specialized mechanisms, and cells actively participate in transfer across membranes. These mechanisms include carrier systems within the membrane and active processes of cellular ingestion, phagocytosis and pinocytosis. Phagocytosis is the ingestion of solid particles, whereas pinocytosis refers to the ingestion of fluid containing no visible solid material. Lipid-insoluble materials are often taken up by activetransport processes. Although some of these mechanisms are highly specific, if the chemical structure of a contaminant is similar to that of an endogenous substrate, the former may also be transported. In addition to its lipid-solubility, the distribution of a chemical contaminant also depends on its affinity for specific tissues or tissue components. Internal distribution may vary with time after exposure. For example, immediately following absorption into the blood, inorganic lead localizes in the liver, the kidney, and in red blood cells. Two hours later, about 50% is in the liver. A month later, approximately 90% of the remaining lead is localized in bone (32). Once in the general circulation, a contaminant may be translocated throughout the body. In this process it may 1) become bound to macromolecules, 2) undergo metabolic transformation (biotransformation), 3) be deposited for storage in depots that may or may not be the sites of its toxic action, or 4) be excreted. Toxic effects may occur at any of several sites. The biological action of a contaminant may be terminated by storage, metabolic transformation, or excretion; the latter is the most permanent form of removal. Accumulation in Target Tissues and Dosimetric Models Some chemicals concentrate in specific tissues because of physicochemial properties such as selective solubility or selective absorption on or combined with macromolecules such as proteins. Storage of a chemical often occurs when the rate of exposure is greater than the rate of metabolism and/or excretion. Storage or binding sites may not be the sites of toxic action. For example, carbon monoxide produces its effects by binding with hemoglobin in red blood cells; on the other hand, inorganic lead is stored primarily in bone but exerts its toxic effects mainly on the soft tissues of the body. If the storage site is not the site of toxic action, selective sequestration may be a protective mechanism because only the freely circulating form of the contaminant produces harmful effects. Until the storage sites are saturated, a buildup of free chemical may be prevented. On the other hand, selective storage limits the amount of contaminant

that is excreted. Because bound or stored toxicants are in equilibrium with their free form, as the contaminant is excreted or metabolized, it is released from the storage site. Contaminants that are stored (e.g., DDT in lipids and lead in bone) may remain in the body for years without effect. However, upon weight loss and mobilization of body reserves, the stored chemicals can enter the circulation and produce toxic effects. For example, pregnant women who had prior excessive exposure to lead can increase their own blood lead levels and also create high and possibly damaging levels of lead exposures to their fetus. Accumulating chemicals may also produce illnesses that develop slowly, as occurs in chronic cadmium poisoning. A number of descriptive and mathematical models have been developed to permit estimation of toxic effects from knowledge of exposure and one or more of the following factors: translocation, metabolism, and effects at the site of toxic action. More complex models that require data on translocation and metabolism have been developed for inhaled and ingested radionuclides by the International Commission on Radiological Protection (7–10). MEASURING AND MODELING HUMAN EXPOSURES Direct measurement data on personal exposures to environmental toxicants would be ideal for risk assessments for individuals, and personal exposure data on large numbers of representative individuals would be ideal for performing population-based risk assessments. However, considerations of technical feasibility, willingness and ability to participate in extensive measurement studies among individuals of interest, and cost almost invariably preclude this option. Instead, more indirect measures of exposure and/or exposure models are relied on that combine a limited number of direct measurements with general background knowledge, historic measurement data believed to be relevant to the particular situation, and some reasonable assumptions based on first principles and/or expert judgements. When monitoring exposures, it is highly desirable to have benchmarks (exposure limits) as references. There are well-established occupational exposure limits for hundreds of air contaminants, including legal limits such as the Permissible Exposure Limits (PELs) established by the U.S. Occupational Safety and Health Administration (OSHA), as well as a larger number of Threshold Limit Values (TLVs) recommended by the American Conference of Governmental Industrial Hygienists (ACGIH) as professional practice guidelines. For ingested chemicals, there are acceptable daily intake values (ADIs), such as those adopted by the Food and Drug Administration (FDA) and the U.S. Department of Agriculture. Until now, comparable exposure limits have not been available for dermal exposure. However, Bos et al. (33) recently proposed a procedure for deriving such limits, and Brouwer et al. (34) performed a feasibility study following the Bos et al. proposal. Table 2 from Bos et al. (33) summarizes the nature and applications of such dermal exposure limits.

Table 2. Some Characteristics of Available Exposure Limitsa Route of Entry Respiratory Tract Name

Qualitative or quantitative Target population Dimensions

Monitoring methods

Miscellaneous or Combined

Maximum accepted concentration (MAC) Threshold limit value (TLV) Quantitative

Acceptable daily intake (ADI)

Skin denotation

Biological limit value; (BEI, BAT-Werte, biological monitoring guidance value)

Quantitative

Qualitative

Quantitative

Working population mg/m3

General population mk/kg/food

parts per million (ppm)

mg/kg body weight

Working population Not applicable; however likely to be assessed as mg (mg/cm2 ) For example, environmental surface wipe-off; patches, gloves, coveralls; tracer methods; skin washings; or skin stripping

Working population or general population (a) mg/L blood, mg/L urine, mg/m3 exhaled air (b) cholinesterase inhibition, zinc protoporphyrin, DNA adducts, mutations, etc. Biological media: blood, urine, exhaled air, feces, hair

fibres n/m3 Environmental monitoring (EM)

Personal air sampling (PAS) a

Skin

Gastrointestinal Tract

Food residues or contaminants in combination with food intake data No specific worker monitoring method

From Bos et al. (33).

1189

1190


In routine monitoring of occupational exposures, it is quite common to collect shift-long (∼8 hour) integrated breathing zone samples using passive diffusion samplers (for gases and vapors) or battery-powered personal samplers that draw a continuous low flow rate stream of air from the breathing zone through a filter or cartridge located in the breathing zone that captures essentially all of the air contaminants of interest for subsequent laboratory analyses. Such sampling is typically performed on only a single worker or at most on a small fraction of the workforce on the basis that the exposures of the sentinel worker(s) represent the exposures of other, unmonitored workers in the same works environment. In this case, the modeling of the other worker’s exposures is relatively simple. Shift-long sampling can provide essential information for cumulative toxicants, but that information may be inadequate when peak exposure levels are important (as for upper respiratory irritants or asphyxiants). Continuous readout monitors would be ideal for evaluating such exposures, but may be impractical because of their size and/or cost. Spot or grab samples can be informative for evaluating of such exposures but require prior knowledge of the timing and locations of peak exposures. In such situations, peak exposures can be estimated using fixedsite continuous monitors in the general vicinity and supplementary information or experience-based models that relate breathing zone levels to general air levels in the room. Time-activity pattern data on each worker can be combined with measured or estimated concentrations at each work site or with specific work activities to construct a time-weighted average exposure (TWAE) for that worker to supplement estimates of peak exposures. The characteristics of equipment used for air sampling in industry are described in detail in Air Sampling Instruments (35). In constructing exposure estimates or models for community air or indoor air exposures for the general population, this time-weighted averaging approach is generally known as microenvironmental exposure assessment. For community air pollutants of outdoor origin, data are often available on the concentrations measured at central monitoring sites, and population exposures to these pollutants are based on models incorporating time-activity patterns (indoors and outdoors), as well as factors representing the infiltration and persistence of the pollutants indoors. Such models should recognize the substantial variability of time-activity patterns among and between subsegments of the population (children, working adults, elderly and/or disabled adults, etc.). Biomonitoring An alternate approach to measuring exposures directly is the use of biomarkers of exposures, determined from analyses of samples of blood, urine, feces, hair, nails, or exhaled air. The levels of the contaminant, its metabolites, changes in induced enzyme or protein levels, or characteristic alterations in DNA may be indicative of recent peak or past cumulative exposures. Exposure biomarkers may be complementary to and, in some cases, preferable to direct measures of environmental exposures.

In any case, they are more biologically informative than indirect measures based on models and knowledge of sources or qualitative measures of exposure such as questionnaires about work and/or residential histories. There are diverse types of biomarkers that range from simple to complex in measurement requirements, and they are diverse in their relationships to either remote or recent exposures. There is also a range of biological relevance among exposure biomarkers: some provide indices that are directly biologically relevant, e.g., the level of carbon monoxide in end-tidal air samples and the risk of myocardial ischemia, whereas others, although broadly related, may not cover the temporally appropriate exposure window, e.g., nicotine levels in biological fluids and lung cancer risk from smoke exposure. For the near term, extensive development of new molecular level biomarkers relevant to malignant and nonmalignant diseases can be anticipated. However, most of these new exposure biomarkers remain to be validated, and few will be ready for translation to the population in the short term. Anticipated applications include epidemiological studies of responses to low-level exposures to environmental agents. Biomarkers will also be used to validate other exposure assessment methods and to provide more proximate estimates of dose. Exposure biomarkers may be applied to groups that have unique exposure or susceptibility patterns, to monitor the population in general, and to document the consequences of exposure assessment strategies designed to reduce population exposures. Exposure biomarkers validated against the end point of disease risk and used in conjunction with other measurements and metrics of exposure should prove particularly effective in risk assessment. However, biomarkers of exposure may pose new and unanticipated ethical dilemmas. Information gained from biomolecular markers of exposure may provide an early warning of high risk or preclinical disease; capability for early warning will require a high level of, and an accepted social-regulatory framework for follow-up actions. They may also cause false alarms and needless stress for individuals warned about the presence of uncertain signals. In summary, exposure represents contact between a concentration of an agent in air, water, food, or other material and the person or population of interest. The agent is the source of an internal dose to a critical organ or tissue. The magnitude of the dose depends on a number of factors: 1) the volumes inhaled or ingested; 2) the fractions of the inhaled or ingested material transferred across epithelial membranes of the skin, the respiratory tract, and the GI tract; 3) the fractions transported via circulating fluids to target tissues; and 4) the fractional uptake by the target tissues. Each of these factors can have considerable intersubject variability. Sources of variability include activity level, age, sex, and health status, as well as such inherent variabilities as race and size. With chronic or repetitive exposures, other factors affect the dose of interest. When the retention at, or effects on, the target tissues are cumulative and clearance or recovery is slow, the dose of interest can be represented by cumulative uptake. However, when the agent is rapidly


eliminated or when its effects are rapidly and completely reversible on removal from exposure, the rate of delivery may be the dose parameter of primary interest. Determining Concentrations of Toxic Chemicals in Human Microenvironments The technology for sampling air, water, and food is relatively well developed, as are the technologies for sample separation from copollutants, media, and interferences and for quantitative analyses of the components of interest. However, knowing when, where, how long, and at which rate and frequency to sample to collect data relevant to the exposures of interest is difficult and requires knowledge of the temporal and spatial variability of exposure concentrations. Unfortunately, we seldom have enough information of these kinds to guide our sample collections. Many of these factors that affect occupational exposures are discussed in detail in the chapters of Patty’s Industrial Hygiene, 5th ed. (35) The following represents a very brief summary of some general considerations. Water and Foods Concentrations of environmental chemicals in food and drinking water are extremely variable, and there are further variations in the amounts consumed because of the extreme variability in dietary preferences and food sources. The number of foods for which up-to-date concentration data for specific chemicals are available is extremely limited. Relevant human dietary exposure data are sometimes available in terms of market basket survey analyses. In this approach, food for a mixed diet is purchased, cleaned, processed, and prepared as for consumption, and one set of specific chemical analyses is done for the composite mixture. The concentrations of chemicals in potable piped water supplies depend greatly on the source of the water, its treatment history, and its pathway from the treatment facility to the tap. Surface waters from protected watersheds generally have low concentrations of dissolved minerals and environmental chemicals. Well waters usually have low concentrations of bacteria and environmental chemicals but often have high mineral concentrations. Poor waste disposal practices may contribute to groundwater contamination, especially in areas of high population density and/or industrial sources of wastes. Treated surface waters from lakes and rivers in densely populated and/or industrialized areas usually contain a wide variety of dissolved organics and trace metals, whose concentrations vary greatly with the season (because of variable surface runoff), with proximity to pollutant sources, with upstream usage, and with treatment efficacy. The uptake of environmental chemicals in bathing waters across intact skin is usually minimal compared to uptake via inhalation or ingestion. It depends on both the concentration in the fluid surrounding the skin surface and the polarity of the chemical; more polar chemicals have less ability to penetrate intact skin. Uptake via skin can be significant for occupational exposures to concentrated liquids or solids.

1191

Air Although chemical uptake through ingestion and the skin surface is generally intermittent, inhalation provides a continuous means of exposure. The important variables that affect the uptake of inhaled chemicals are the depth and frequency of inhalation and the concentration and physicochemical properties of the chemicals in the air. Exposure to airborne chemicals varies widely among inhalation microenvironments, whose categories include workplace, residence, outdoor ambient air, transportation, recreation, and public spaces. There are also wide variations in exposure within each category, depending on the number and strength of the sources of the airborne chemicals, the volume and mixing characteristics of the air within the defined microenvironment, the rate of air exchange with the outdoor air, and the rate of loss to surfaces within the microenvironment. For community air pollutants that have national ambient air quality standards, particulate matter (PM), sulfur dioxide (SO2 ), carbon monoxide (CO), nitrogen dioxide (NO2 ), ozone (O3 ), and lead (Pb), there is an extensive network of fixed-site monitors, generally on rooftops. Although the use of these monitors generates large volumes of data, the concentrations at these sites may differ substantially from the concentrations that people breathe, especially for tailpipe pollutants such as CO. Data for other toxic pollutants in the outdoor ambient air are not generally collected routinely. Workplace Exposures to airborne chemicals at work are extremely variable in composition and concentration and depend on the materials being handled, the process design and operation, the kinds and degree of engineering controls applied to minimize release to the air, the work practices followed, and the personal protection provided. Residential Airborne chemicals in residential microenvironments are attributable to air infiltrating from out of doors and to the release from indoor sources. The latter include unvented cooking stoves and space heaters, cigarettes, consumer products, and volatile emissions from wallboard, textiles, carpets, etc. Indoor sources can release enough nitrogen dioxide (NO2 ), fine particle mass (FPM), and formaldehyde (HCHO) that indoor concentrations for these chemicals can be much higher than those in ambient outdoor air. Furthermore, their contributions to the total human exposure are usually even greater because people usually spend much more time at home than outdoors. Conventions for Size-Selective Inhalation Hazard Sampling for Particles In recent years, quantitative definitions of Inhalable particulate matter (IPM), Thoracic particulate matter (TPM), and Respirable particulate matter (RPM) have been internationally harmonized. The size-selective inlet specifications for air samplers that meet the criteria of ACGIH (36), ISO (37), and CEN (38) are enumerated in Table 3 and illustrated in Figure 9. They differ from

1192


Table 3. Inhalable, Thoracic and Respirable Dust Criteria of ACGIH, ISO and CEN, and PM10 Criteria of U.S. EPA Inhalable

Thoracic

Respirable

PM10

Particle Inhalable Particle Thoracic Particle Respirable Particle Thoracic Aerodynamic Particulate Aerodynamic Particulate Aerodynamic Particulate Aerodynamic Particulate Diameter (µm) Mass (IPM) (%) Diameter (µm) Mass (TPM) (%) Diameter (µm) Mass (RPM) (%) Diameter (µm) Mass (TPS) (%) 0 1 2 5 10 20 30 40 50 100

100 97 94 87 77 65 58 54.5 52.5 50

Fine mode mass (condensation aggregation)

0 2 4 6 8 10 12 14 16 18 20 25

100 94 89 80.5 67 50 35 23 15 9.5 6 2

Coarse mode mass (mechanically generated) TPM (PM10)

Mass concentration

IPM

RPM TSP PM2.5

0.02

0.05 0.1

0.2 1 2 5 10 Aerodynamic diameter dae (m)

20

100

Figure 9. Effect of size-selective inlet characteristic on the aerosol mass collected by a downstream filter. IPM = inhalable particulate matter; TSP = total suspended particulate; TPM = thoracic particulate matter; (aka PM10 ); RPM = respirable particulate matter; and PM2.5 = fine particulate matter in ambient air.

the deposition fractions of ICRP (7), especially for larger particles, because they take the conservative position that protection should be provided for those engaged in oral inhalation and thereby bypass the more efficient filtration efficiency of the nasal passages. The U.S. Environmental Protection Agency (39) set a standard for ambient air particle concentration known as PM10 , i.e., for particulate matter less than 10 µm in aerodynamic diameter. It replaced a poorly defined sizeselective criterion known as total suspended particulate matter (TSP), whose actual inlet cut varied with wind speed and direction. PM10 has a sampler inlet criterion that is similar (functionally equivalent) to TPM but,

0 1 2 3 4 5 6 7 8 10

100 97 91 74 50 30 17 9 5 1

0 2 4 6 8 10 12 14 16

100 94 89 81.2 69.7 55.1 37.1 15.9 0

as shown in Table 3, has somewhat different numerical specifications. In 1997, following its most recent thorough review of the literature on the health effects of ambient PM, the EPA concluded that most of the health effects attributable to PM in ambient air were more closely associated with the fine particles in the fine particle accumulation mode (extending from about 0.1 to 2.5 µm) than with the coarse mode particles within PM10 and promulgated new National Ambient Air Quality Standard (NAAQS) based on fine particles, defined as particles whose aerodynamic diameters (dae ) are less than 2.5 µm (PM2.5 ), to supplement the PM10 NAAQS that was retained (40). The selection of dae = 2.5 µm as the criterion for defining the upper bound of fine particles in a regulatory sense was, inevitably, an arbitrary selection made from a range of possible options. It was arrived at using the following rationales: • Fine particles produce adverse health effects more because of their chemical composition than their size (see Table 4) and need to be regulated using an index that is responsive to control measures applied to direct and indirect sources of such particles. • Any separation by aerodynamic particle size that attempts to separate fine mode from coarse mode particles cannot include all fine mode particles and exclude all coarse mode particles because the modes overlap (see Fig. 9). • The position of the ‘‘saddle point’’ between the fine mode and coarse mode peaks varies with aerosol composition and climate. Data from Michigan indicates a volumetric saddle point at dae ∼2 µm. If the data were corrected for particle density, it might be somewhat higher. Data from Arizona have a lower saddle point at dae ∼1.5 µm. • Evidence of a need for a fine particle NAAQS came from studies based on PM2.5 or PM2.1 . If PM2.5 errs, it also does so on the conservative side with respect to health protection. Further, it was deemed to be impractical to have different cut sizes in different parts of the United States.


1193

Table 4. Comparisons of Ambient Fine and Coarse Mode Particlesa Fine Mode

Coarse Mode

Formed from Formed by

Gases Chemical reaction; nucleation; condensation; coagulation; evaporation of fog and cloud droplets in which gases have dissolved and reacted

Large solids/droplets Mechanical disruption (e.g., crushing, grinding, abrasion of surfaces); evaporation of sprays; suspension of dusts

Composed of

Sulfate, SO4 2− ; nitrate, NO3 − ; ammonium, NH4 + ; hydrogen ion, H+ ; elemental carbon; organic compounds (e.g., PAHs, PNAs); metals (e.g., Pb, Cd, V, Ni, Cu, Zn, Mn, Fe); particle-bound water

Resuspended dusts (e.g., soil dust, street dust); coal and oil fly ash; metal oxides of crustal elements (Si, Al, Ti, Fe); CaCO3 , NaCl, sea salt; pollen, mold spores; plant/animal fragments; tire wear debris

Solubility Sources

Largely soluble, hygroscopic, and deliquescent Combustion of coal, oil, gasoline, diesel, wood; atmospheric transformation products of NOx , SO2 , and organic compounds including biogenic species (e.g., terpenes); high temperature processes, smelters, steel mills, etc.

Largely insoluble and non hygroscopic. Resuspension of industrial dust and soil tracked onto roads; suspension from disturbed soil (e.g., farming, mining, unpaved roads); biological sources; construction and demolition; coal and oil combustion; ocean spray

Lifetimes

Days to weeks

Minutes to hours

Travel distance

100s to 1000s of kilometers

95% of the tertiary phosphine are obtained. Tributylphosphine is readily converted to tetraalkylphophonium salts by reaction with an alkyl halide. These compounds are used commercially as biocides and phase-transfer catalysts. In contrast, if the olefin is more sterically hindered (branched) and/or the reaction is operated at a higher pressure (4 MPa), formation of the primary and secondary phosphines is favored as in the reaction with 2,4,4trimethyl-1-pentene [107-39-1]. PH3 + C8 H16 −−−→ (C8 H17 )PH2 + (C8 H17 )2 PH + (C8 H17 )3 P 43%

51%

6% (7)

The mixture can be separated by distillation. The primary phosphine is recycled for use in the subsequent autoclave batch, the secondary phosphine is further derivatized to the corresponding phosphinic acid which is widely employed in the industry for the separation of cobalt from nickel by solvent extraction. With even more hindered olefins, such as cyclohexene [110-83-8], the formation of tertiary phosphines is almost nondetectable. Other typical alkylphosphines that can be prepared through phosphine chemistry are monoisobutylphosphine [4023-52-3], trioctylphosphine [4731-53-7], monocyclohexylphosphine [822-68-4], dicyclohexylphosphine [82984-5], and triethylphosphine [554-70-1]. Textile Flame Retardants The first known commercial application for phosphine derivatives was as a durable textile flame retardant for cotton and cotton–polyester blends. The compounds are tetrakis(hydroxymethyl)phosphonium salts (10) which are prepared by the acid-catalyzed addition of phosphine to formaldehyde. The reaction proceeds in two stages.

PHOSPHINE AND ITS DERIVATIVES

Initially, the intermediate tris(hydroxymethyl)phosphine [2767-80-8] is formed. H+

PH3 + 3 CH2 O −−−→ (HOCH2 )3 P

(8)

This compound is unstable, particularly at alkaline pH, and decomposes to release hydrogen. It is not isolated but reacts in situ with an additional mole of formaldehyde and a mineral acid, for example hydrogen chloride [7647-01-1], to form the phosphonium salt. (HOCH2 )3 P + HCl + CH2 O −−−→ (HOCH2 )4 P+ Cl−

(9)

The salt in this case is tetrakis(hydroxymethyl)phosphonium chloride [124-64-1]. The corresponding sulfate salt [55566-30-8] is also produced commercially as are urea-containing formulations of both salts. The latter formulations are actually used to flame retard the textiles. After application to the fabric, the compounds are polymerized by reaction with gaseous ammonia (11,12), then oxidized to phosphine oxides by reaction with hydrogen peroxide. The structure of the polymer is shown (13). O C H2

P

H C H2

CH2

N

H C

N n

O

This provides a durable finish which, unlike many other flame retardants, can withstand repeated (50–100) launderings without a loss of efficiency. An added advantage is that the feel of the cloth (hand) is little effected. Principal markets are in the treatment of industrial protective clothing, military uniforms, and, in Europe, for furnishings. These products are available from Albright & Wilson Ltd. and Cytec Industries Inc. Flotation Reagents Only one sulfide mineral flotation collector is manufactured from phosphine, i.e., the sodium salt of bis(2methylpropyl)phosphinodithioic acid [13360-78-6]. It is available commercially from Cytec Industries Inc. as a 50% aqueous solution and is sold as AEROPHINE 3418A promoter. The compound is synthesized by reaction of 2-methyl-1-propene [115-11-7] with phosphine to form an intermediate dialkylphosphine which is subsequently treated with elemental sulfur [7704-34-9] and sodium hydroxide [1310-73-2] to form the final product (14). The reactions described in equations 10 and 11 3.3 MPa

→ (C4 H9 )2 PH 2 C4 H8 + PH3 −−− ◦

(10)

80 C 60◦ C

(C4 H9 )2 PH + 2 S + NaOH −−−→ (C4 H9 )2 P(S)SNa + H2 O (11) are carried out in an autoclave and a glass-lined kettle, respectively. The primary phosphine formed during the autoclave reaction is removed from the autoclave liquor

1219

by distillation and is recycled for use in the next autoclave batch. The tertiary phosphine reacts with sulfur in equation 12 to produce tris(2-methylpropyl)phosphine sulfide [3982-87-4], a solid which is separated from the product using a centrifuge. (C4 H9 )3 P + S −−−→ (C4 H9 )3 P(S)

(12)

AEROPHINE 3418A promoter is widely used in North and South America, Australia, Europe, and Asia for the recovery of copper, lead, and zinc sulfide minerals. Advantages in comparison to other collectors (15) are said to be improved selectivity and recoveries in the treatment of complex ores, higher recoveries of associated precious metals, and a stable grade–recovery relationship which is particularly important to the efficient operation of automated circuits. Additionally, AEROPHINE 3418A is stable and, unlike xanthates (qv), does not form hazardous decomposition products such as carbon disulfide. It is also available blended with other collectors to enhance performance characteristics. Phase-Transfer Catalysts The use of phase-transfer catalysts to improve kinetics and yields in heterogeneous reactions has been growing rapidly since the 1960s. The five to ten commercial processes in use in 1970 had risen to 550 (16) by 1989. The principal areas of application are in the preparation of polymers, accounting for 50% of catalyst consumption, followed by pharmaceuticals (20%) and agricultural chemicals (10%). Details of the chemistry and applications have been given elsewhere (17). The most common phase-transfer catalysts are quaternary ammonium salts containing either alkyl or mixed alkaryl groups. However, these compounds are being displaced in some applications by the corresponding phosphonium salts mainly because of the enhanced thermal stability of the phosphorus compounds (17). Additionally, the phosphonium salts tend to be more efficient than the nitrogen-based analogues and can promote more rapid reaction kinetics (18). Phosphonium salts are readily prepared by the reaction of tertiary phosphines with alkyl or benzylic halides, eg, the reaction of tributylphosphine [998-40-3] with 1-chlorobutane [109-69-3] to produce tetrabutylphosphonium chloride [2304-30-5]. 60◦ C

(C4 H9 )3 P + C4 H9 Cl −−−→ (C4 H9 )4 P+ Cl−

(13)

Kinetics are slow and many hours are required for a 95% conversion of the reactants. In the case of the subject compound, there is evidence that the reaction is autocatalytic but only when approximately 30% conversion to the product has occurred (19). Reaction kinetics are heavily dependent on the species of halogen in the alkyl halide and decrease in the order I > Br > Cl. Tetrabutylphosphonium chloride exhibits a high solubility in a variety of solvents, for example, >80% in water, >70% in 2-propanol, and >50% in toluene at 25◦ C. Its analogues show similar properties. One of the latest

1220


applications for this phosphonium salt is the manufacture of readily dyeable polyester yarns (20,21). In addition to tetrabutylphosphonium chloride, typical phosphonium salts that can be produced include tetraoctylphosphonium bromide [23906-97-0], tetrabutylphosphonium acetate [17786-43-5] (monoacetic acid), and tetrabutylphosphonium bromide [3115-68-2]. In most cases, these compounds can be prepared with alternative counterions. Biocides Two phosphine derivatives are in commercial use as biocides. These are tetrakis(hydroxymethyl)phosphonium sulfate [55566-30-8] and tributyl(tetradecyl)phosphonium chloride [8741-28-8]. These compounds are sold by Albright and Wilson Ltd. and FMC, respectively. The preparation of the hydroxymethylphosphonium salt has been discussed. Synthesis of the tetraalkylphosphonium chloride follows the reaction described in equation 13 except that 1-chlorotetradecane [2425-54-9] is employed in place of 1-chlorobutane. Various patents (22–24) have been issued claiming the use of tetrakis(hydroxymethyl)phosphonium sulfate in, for example, water treating, pharmaceuticals (qv), and in the oil industry where this compound shows exceptional activity toward the sulfate-reducing bacteria that are a primary cause of hydrogen sulfide formation and consequent problems associated with souring and corrosion (25). Tributyl(tetradecyl)phosphonium chloride was developed specifically (26,27) as a broad-spectrum biocide for the control of biological fouling in cooling water systems where it is particularly effective. Ultraviolet Photoinitiators Photoinitiators are used in increasing volume for a multitude of applications. The most important of these are in the formulation of uv-curable inks and in the production of coatings on vinyl flooring, wood, and electronics components (28,29). The most common types of photoinitiators are phenone derivatives, for example, acetophenones and benzophenones (30). However, Ciba-Geigy has introduced (31,32) a type of phosphine-based photoinitiator. In general, the compound can be described as a bis(acyl)phosphine oxide and is prepared by the reaction of a monoalkylphosphine with a substituted benzoyl chloride (33). The composition of the first commercial product is proprietary. However, advantages in comparison with conventional photoinitiators, including monoacylphosphine oxides, lie in the ability to prepare thicker coatings that have improved scratch resistance and do not yellow with age. The compound is self-bleaching. Pigmented coatings can also be prepared. This enables formulators, for the first time, to prepare white, uv-cured coatings. Initial areas of application are for furniture coatings and white screen inks. Solvent Extraction Reagents Solvent extraction is a solution purification process that is used extensively in the metallurgical and chemical industries. Both inorganic (34,35) and organic (36) solutes

are recovered. The large commercial uses of phosphine derivatives in this area involve the separation of cobalt [7440-48-4] from nickel [7440-02-0] and the recovery of acetic acid [61-19-7] and uranium [7440-61-1]. Uranium Recovery from Wet-Process Phosphoric Acid. In the mid- to late 1960s, work at the Oak Ridge National Laboratory (37,38) led to the invention of a process to recover the low concentrations (100–200 mg/L) of uranium [7440-61-1], which occur naturally in the wet-process phosphoric acid [7664-38-2] used to make fertilizers (qv). Key to the development of this process was the discovery of the synergic interaction between the bis(2-ethylhexyl) ester of phosphoric acid [298-07-7] (D2EHPA) and trioctylphosphine oxide [78-50-2] (TOPO) in extracting U6+ (37). D2EHPA is prepared by conventional organophosphorus chemistry and TOPO is readily manufactured by the reaction of phosphine with octene [25377-83-7] to form intermediate trioctylphosphine [4731-53-7] which is subsequently oxidized to TOPO with hydrogen peroxide [772284-1] as outlined in equations 14 and 15. TOPO is a white, waxy solid with a melting point of approximately 50 ◦ C. 90◦ C

3 C8 H16 + PH3 −−−→ (C8 H17 )3 P

(14)

1.5 MPa 75◦ C

(C8 H17 )3 P + H2 O2 −−−→ (C8 H17 )3 P(O) + H2 O

(15)

Following further development (38), a two-cycle process has been adopted by industry. In the first concentration cycle, the clarified feed acid containing 100–200 mg/L U3 O8 [1334-59-8] is oxidized, for example, with hydrogen peroxide or sodium chlorate [7775-09-9] to ensure that uranium is in its 6+ valence state; U4+ is not extracted. Uranium is extracted with a solvent composed of 0.5 M D2EHPA and 0.125 M TOPO dissolved in an aliphatic hydrocarbon diluent. Uranium is subsequently stripped reductively from the loaded solvent using a bleed stream of the raffinate acid to which ferrous iron has been added to reduce uranium to its nonextractable, quadravalent state. Raffinate is acid from which uranium has already been extracted. By controlling the organic-to-aqueous volume phase ratios in the extraction and stripping circuits, uranium is concentrated by a factor of approximately 70. Raffinate acid from the first cycle, containing approximately 7 to 14 g/L U3 O8 is then reoxidized and re-extracted in the second, purification cycle using a solvent containing 0.3 M D2EHPA and 0.075 M TOPO. The loaded solvent is washed with iron-free acid to remove iron and then with water to remove extracted and entrained acid. The solvent is stripped with ammonium carbonate [506-876] to yield ammonium uranyl tricarbonate [18077-77-5] which is subsequently calcined to U3 O8 (yellow cake). The stripped solvent is regenerated with mineral acid before recycling (39). Beginning in approximately 1975, both IMC and Freeport Minerals operated large uranium recovery plants in the United States using this technology. Several plants continue to run but a number have been closed because of the depressed uranium prices that resulted when uranium from the former Soviet Union flooded Western


1221

Table 3. Organophosphorus Extractants for Co–Ni Separationa Extractanta type

Commercial homologue, R =

Structure

Phosphoric acid (D2EHPA)

RO

Co–Nic separation factor

2-ethylhexyl

14

2-ethylhexyl

280

P(O)OH RO Phosphonic acid (PC-88A)

RO P(O)OH R

Phosphinic acid (CYANEX 272)

2,4,4-trimethylpentyl

R

7000

P(O)OH R Conditions: temperature = 25 ◦ C; equilibrium pH = 4; A/O = 1. 0.1 M extractant in MSB 210. c Each metal ion concentration 2.5 × 10−2 M. a b

markets. A relatively small plant is operated by Prayon in Belgium (40). TOPO is available from Cytec Industries Inc. as CYANEX 921 extractant. D2EHPA is available from Albright & Wilson Ltd. and is also sold by Daihachi as DP-8R.

In a similar application, Cape Industries has announced its intention to commission a solvent extraction plant to recover acetic acid from an effluent generated at its dimethyl terephthalate [120-61-6] facility (Wilmington, North Carolina) (44,45). The plant was commissioned in February 1995. In this case, the solvent will be CYANEX 923 extractant [100786-00-3]. CYANEX 923 is also a phosphine oxide, but unlike TOPO is a liquid and can be used without a diluent (46,47). This has the benefit of reducing plant size, capital, and operating costs.

Acetic Acid Recovery. Sulfite wood pulping operations produce dilute, aqueous effluents containing 10–20 g/L acetic acid. In some cases, 2-furancarboxaldehyde [98-011], more commonly known as furfural, can also be present at lower concentrations (∼1 g/L) (41). Lenzing (Austria) recovers both of these by-products by solvent extraction with TOPO. Although few data concerning the plant have been published (41,42), it is known (43) that the solvent is 30% TOPO in undecane [1120-21-4]. The extraction column is operated at 50 ◦ C and the aqueous-to-organic volume phase ratio (A/O) is 1. The loaded solvent is distilled to strip the extracted species, first to remove most of the water for recycle, then to strip an azeotrope of water, acetic acid, and furural. The azeotrope is further distilled to yield pure acetic acid and furfural. Both compounds are sold. The plant has been operating successfully since 1983 and supplies approximately 50% of Austria’s demand for food-grade acetic acid.

Cobalt–Nickel Separation. The bis(2,4,4-trimethylpentyl)phosphinic acid [83411-71-6] became commercially available during the early 1980s (48,49). It is sold by Cytec Industries Inc. as CYANEX 272 extractant and was developed specifically to selectively extract cobalt from weakly acidic, nickeliferous solutions. It is a member of one of three groups of organophosphorus extractants that have been examined for cobalt–nickel separation. These are derivatives of phosphoric (50), phosphonic (51), and phosphinic (52) acids. CYANEX 272 has two significant advantages over its competing reagents. The first is superior cobalt–nickel selectivity, as illustrated by the

100 Ca (D2EHPA)

% Extraction

80

60

Co

40

Ca CYANEX 272 extractant

Ca (PC 88A)

20

0

0

1

2

3

4

Equilibrium, pH

5

6

7

Figure 1. Cobalt–calcium selectivity with organophosphorus extractants. Conditions: solvent = 0.6 M extractant in Kermac 470B; aqueous = 0.015 M metal ion as sulfate; temperature = 50C; and A/O = 1.

1222


results of some batch equilibrium tests shown (52) in Table 3. The benefits of high selectivity lie in the ability to produce high purity cobalt in a limited number of stages. This minimizes capital and operating costs. It is particularly important when the solution in question contains low concentrations of cobalt. For example, solutions derived from laterite deposits may only contain 0.5–2 g/L Co but 90–100 g/L Ni. The second principal advantage is that CYANEX 272 is the only one of the three above-mentioned compounds that extracts cobalt in preference to calcium (52). This property can minimize or eliminate the solvent losses that are associated with calcium extraction and the subsequent precipitation of gypsum cruds in the scrubbing or stripping circuits. This is illustrated in Figure 1 where calcium extraction is shown as a function of pH for the three subject reagents. The first commercial plant to use CYANEX 272 became operational in 1985. An additional three plants were constructed between 1985 and 1989. Of the four, one is in South America and three in Europe. An additional three plants have been built; two in Europe (1994) and one in North America (1995). Approximately 50% of the Western world’s cobalt is processed using CYANEX 272. Both high purity salts and electrolytic cobalt metal are recovered from solutions ranging in composition from 30 g/L each of cobalt and nickel to 0.2 g/L Co, 95 g/L Ni. Operating companies usually regard use of CYANEX 272 as confidential for competitive reasons and identities cannot be disclosed. CYANEX 272 is being evaluated on the pilot-plant scale in many additional projects involving the recovery of cobalt and other metals. BIBLIOGRAPHY

13. W. A. Reeves and R. M. Perkins, Colourage, (18): 1–7 (1971). 14. Brit. Pat. Appl. 2,068,381A (Aug. 12, 1981), A. J. Robertson and T. Ozog (to Cytec Technology Corp.). 15. P. A. Mingione, ‘‘Use of AEROPHINE 3418A Promoter for Sulphide Minerals Flotation,’’ Proceedings of the 22nd Annual Meeting of the Canadian Mineral Processors, Ottawa, 1990. 16. Phase Transfer Catalysis in Industry, PTC Interface, Inc., Marietta, Ga. 17. C. M. Starks and C. Liotta, Phase Transfer Catalysis, Academic Press, Inc., New York, 1978. 18. C. M. Starks, ‘‘Selecting Chemtech (Feb. 1980).

a

Phase

Transfer

Catalyst,’’

19. A. J. Robertson, private communication, Cytec Canada Inc., Phosphine Technical Centre, Jan. 1994. 20. Jpn. Kokai Tokkyo Koho JP 03241024 (Jan. 28, 1991), M. Yanagihara, K. Kawakami, and H. Nagai (to Taijin Ltd.). 21. Eur. Pat. Appl. EP 280028A2 (Aug. 31, 1998), T. Suzuki and co-workers (to Teijin Ltd.). 22. U.S. Pat. 4,673,509 (June 16, 1987), K. P. Davis and R. E. Talbot (to Albright and Wilson Ltd.). 23. Eur. Pat. Appl. EP 275207 A2 (July 20, 1988), R. E. Talbot and co-workers (to Albright and Wilson Ltd.). 24. U.S. Pat. 4,775,407 (Oct. 4, 1988), K. G. Cooper R. E. Talbot (to Albright and Wilson Ltd.).

and

25. Tolicide PS 72A Product Brochure, Albright and Wilson Biocides, Oldbury, Worley, West Midlands, U.K. 26. Eur. Pat. Appl. EP 0066544 (May 30, 1981), R. Grade and B. M. Thomas (to Ciba-Geigy AG). 27. Can. Pat. Appl. CA 2082994 AA (May 28, 1993), W. Wehner and R. Grade (to FMC Corp. (U.K.) Ltd.). 28. K. K. Dietliker and P. Oldring, ‘‘Chemistry and Technology of UV & EB Formulations for Coatings, Inks & Paints,’’ Vol. 3: Photoinitiators for Free Radical and Cationic Polymerization, Sholium International, 1991.

1. U.S. Pat. 2,977,122 (Mar. 28, 1961), R. W. Cummins (to Food Machinery and Chemical Corp.).

29. K. Lawson, ‘‘UV/EB Curing in North America—1994,’’ RadTech Rep. 21: (Mar./Apr. 1994).

2. Fr. 1,352,605 (Feb. 14, 1964), (to Albright & Wilson Ltd. and Hooker Chemical Corp.).

30. N. S. Allen, Photopolymerization and Photoimaging Science and Technology, Elsevier Applied Science, London, 1989.

3. N. Weferling, ‘‘Phosphine Based Organophosphorus Products and Their Applications’’, paper presented at Chemspec Europe ’88, Frankfurt, Germany, Mar. 22–23, 1988.

31. Eur. Pat. Appl. 0184095 A2 (Nov. 27, 1984), K. Ellrich and C. Herzig (to Espe Fabrik Pharmazeutischer Proparate GmbH).

4. Ger. Offen. DE 2,632,316 (Jan. 19, 1978), J. Stenzel, G. Heymer, and C. May (to Hoechst AG).

32. K. Dietliker and co-workers, ‘‘Novel High Performance Bisacylphosphine Oxide (BAPO) Photoinitiators,’’ paper presented at RadTech’94, Orlando, Florida, May 1–5, 1994.

5. N. Weferling, Phos. Sulf. 30: 641(1987). 6. S. Lian and co-workers, J. Vac. Sci. Technol. 11(6): 2914 (1993). 7. J. F. Carlin and co-workers, Mater. Sci. Eng. B21 (2–3): 293 (1993). 8. Chem. Week, 134(14): 14 (Apr. 1984). 9. Brit. Pat. Appl. 2,177,004A (Jan. 14, 1987), S. Latif and R. F. Ryan (to The BOC Group PLC). 10. U.S. Pat. 3,888,779 (June 10, 1975), C. T. Hsiang (to Cytec Technology Corp.). 11. Eur. Pat. Appl. EP 294234 A2 (July 12, 1988), G. W. Smith (to Albright and Wilson, Ltd.). 12. Can. Pat. Appl. Ca 2048402 AA (Feb. 11, 1992), R. Cole (to Albright and Wilson Ltd.).

33. Ger. Offen., DE 4,231,579 (Mar. 25, 1994) D. G. Leppard, M. Koehler, and L. Misev (to Ciba-Geigy AG). 34. G. M. Ritcey and A. W. Ashbrook, Solvent Extraction—Principles and Applications to Process Metallurgy, Part I and II, Elsevier, Amsterdam, the Netherlands, 1979. 35. T. C. Lo, M. H. I. Baird, and C. Hanson, Handbook of Solvent Extraction, John Wiley and Sons, Inc., New York, 1983. 36. F. J. Hurst, D. J. Crouse, and K. B. Brown, Solvent Extraction of Uranium from Wet Process Phosphoric Acid, ORNLTM-2522, U.S. Atomic Energy Commission, Washington, D.C., 1969. 37. F. J. Hurst, D. J. Crouse, and K. B. Brown, Ind. Eng. Chem., Process Des. Develop 13: 286–291 (1974). 38. P. D. Mollere, DECHMA 2: 49 (1986).

PHOTOSYSTEM I ENERGY DIVERTERS 39. U.S. Pat. 4,105,74 (Aug. 8, 1978), T. K. Wiewioroski and W. L. Thornsberry (to Freeport Minerals Co.). 40. W. W. Berry, Chem. Eng. Prog. 77(2): 76–82 (1981). 41. Eur. Pat. Appl. EP 36406 A1 (Sept. 23, 1981), W. Kanzler and J. Schedler (to Vereinigte Edelstahlwerke AG). 42. Eur. Pat. Appl. EP 38317 (Oct. 21, 1981), W. Kanzler and J. Schedler (to Vereinigte Edelstahlwerke AG).

1223

a sizeable literature exists on their use as herbicides, their biochemical effects on mammals and plants, and the development of herbicide-resistant weed biotypes. One whole book was even published on this group 20 years (20). Because of this voluminous literature, this review will cite mainly recent references, referring the reader to previous reviews (1–3) for further details of the earlier literature.

43. E. K. Watson and W. A. Rickelton, Solv. Extr. Ion Exch. 10(5): 879 (1992).

Nomenclature

44. Chem. Week, 152(10): 40 (Mar. 17, 1993).

The major group of PS I diverter herbicides (Fig. 1) is the bipyridilium (bipyridinium) herbicides, including the two most common herbicides in this classification, diquat ((6,7-dihydropyrido[1,2-a:2 ,1 -c] pyrazinediium ion)) and paraquat (1,1 -dimethyl-4,4 -bipyridinium ion). A third herbicide in this group, morfamquat ((1,1 -di-3,5-dimethylmorpholinocarbonylmethyl)-4,4 -bipyridilium)), selectively controls dicot weeds. However, the performance of this herbicide was erratic, leading to it being withdrawn as a commercial product (1). All other PS I diverter herbicides were produced only as experimental compounds that were not developed further for the weed control market. A most promising group of these diverters was developed by Shell UK (4,5), called the heteropentalenes [(the compound known as HEP II is 5H,7H-2,3 dioxa-2a,6-dithia(2e-S)-1,4 diazacyclopent[cd] indene and is shown structurally in Fig. 1)]. These are much less water-soluble than the bipyridilium herbicides; yet, they induce identical sorts of structural and biochemical effects as the bipyridiliums in plants (6). A most interesting approach to the PSI diverters was attempted by a group at Ciba Geigy (now Syngenta) in trying to produce molecules with characteristics of both the bipyridiliums and the triazine herbicides. The active members of this group of compounds induce responses identical to paraquat (6–8), and it may have been due to the lack of improvement over that well-established herbicide that they were not developed further.

45. Oil Gas J. 91(15): 35(Apr. 12, 1993). 46. Technical brochure, CYANEX 923 Extractant, SPT-032a, Cytec Industries, Inc., West Paterson, N.J., 1987. 47. U.S. Pat. 4,909,939 (Mar. 20, 1991), W. A. Rickelton and A. J. Robertson (to Cytec Technology Corp.). 48. U.S. Pat. 4,353,883 (Oct. 12, 1982), W. A. Rickelton, A. J. Robertson, and D. R. Burley (to Cytec Technology Corp.). 49. U.S. Pat. 4,374,780 (Feb. 22, 1983), A. J. Robertson (to Cytec Technology Corp.). 50. G. M. Ritcey, A. W. Ashbrook, and B. H. Lucas, CIM Bull. 68: 111–123 (1975). 51. J. S. Preston, J. S. Afr. Inst. Min. Metall. 83: 126–132 (1983). 52. W. A. Rickelton, D. S. Flett, and D. W. West, Solv. Extr. Ion Exch. 2(6): 815–838 (1984).

PHOTOLYSIS The fission of chemical bonds or other chemical reaction caused by light energy.

PHOTOOXIDATION Chemical reaction with oxygen or oxidizing species caused by light.

AGRICULTURAL USES

PHOTOSENSITIZED REACTIONS Molecules may undergo direct or indirect photochemical reactions. Indirect (sensitized) processes may occur by energy transfer from a second molecular species that has absorbed light to produce an excited molecule without itself undergoing chemical reaction.

PHOTOSYSTEM I ENERGY DIVERTERS KEVIN VAUGHN USDA-ARS-MSA Stoneville, Mississippi

The photosystem (PS) I diverter herbicides include several of the oldest in the herbicide arsenal, introduced in the mid-late 1950s by ICI (now Syngenta) and still commercially available. Because of this long-term use,

The PSI diverter herbicides are nonselective and thus are used for total vegetation control (1–3,9). These are contact herbicides that require actively photosynthetic tissue for maximum efficacy. Because these herbicides are watersoluble, formulated products contain nonionic surfactants or oily adjuvants to assist in penetration through the waxy cuticular layers of the plant surface. Both paraquat and diquat may be applied as preplanting treatments for many agronomic crops to eliminate weeds at or before planting. In addition, these herbicides may be used in established perennial crops such as alfalfa, mint, and rhubarb during the dormant cycle. Similarly, their use in vineyards, orchards, and coffee and rubber plantations, at times of crop dormancy or using directed sprays so that only the weed is contacted, are useful for total weed control measures. Diquat is also used extensively as an aquatic herbicide to control algae and submerged and floating aquatic weeds. Besides their use as herbicides, both diquat and paraquat have been used as ‘‘harvest aids’’ to remove

1224

PHOTOSYSTEM I ENERGY DIVERTERS

(b)

(a)

CH3 N

N

N

CO

CH3 (d)

O

+

CH3

CH3

CH3 O

Figure 1. Chemical structures of some of the photosystem I diverter herbicides. Paraquat (a), diaquat (b), and morfamquat (c) are bipyridilium herbicides, whereas HEP II (d) is a heteropentalene.

N

N+

+

(c)

+

S

CH2

+

N

+

N

CH2

CO

N

O

CH3

O N

N

S

unwanted leaf material from crop plants to assist in the mechanical removal of the plant parts of interest (1–3,9). Soybeans, sunflowers, and potatoes are treated in this manner. In addition to removing the leaves, the herbicide treatment results in desiccation of all of the aboveground plant parts, which improves the storage qualities of the crop. CHEMISTRY AND SYNTHESIS The structures of some of the PSI diverter herbicides are shown in Figure 1. Paraquat, diquat, and morfamquat are bipyridinium ions, whereas the heteropentalene herbicide known as HEP II is an uncharged molecule. Details of the reduction of these molecules by photosynthetic electron transport are discussed below. Both paraquat and diquat can be reduced chemically by dithionite, resulting in a strong blue dye, the other common name for paraquat being methyl viologen, for its blue dye color. Paraquat is synthesized by the direct quaternization of 4,4 -bipyridyl with chloromethane under pressure (1,9). Diquat is synthesized by reaction of 2,2 -bipridyl with di-n-propyl amine (1,9). MECHANISM OF ACTION/PHYSIOLOGICAL EFFECTS Chloroplasts, the chlorophyll-containing organelles of plants, have the ability to split water into H+ ions and molecular oxygen. The protons are used to generate a pH gradient that results in energy, whereas the electrons are passed through a chain of molecules that results in the production of NADPH. A schematic of this flow of electrons, called the Z scheme, is shown in Figure 2. Bipyridilium and other PS I diverter herbicides interrupt this chain by accepting electrons at the PSI primary acceptor site (X), reducing them from their normal state to a radical dication (Fig. 3). In the radical dication form, the molecule is able to react with molecular oxygen to generate superoxide, with the herbicide returned to its ground state. Further reaction of superoxide results in the production of the hydroxy

Figure 2. Diagrammatic representation of the light reactions of photosynthesis (so-called Z scheme) and the site of paraquat reduction in this pathway. Cyclic electron flow around PSI is not illustrated in order to simplify the model. Q = primary acceptor quinone, PQ = plastoquinone, cyt f = cytochrome f, PC = plastocyanin.

Figure 3. Steps in the reaction of paraquat with the photosynthetic electron transport chain (line 1) and its subsequent reduction of molecular oxygen to superoxide (line 2).

radical and hydrogen peroxide [(10); Fig. 4]. Hydrogen peroxide has been detected directly at the electron microscope level by the precipitation of the peroxide as cerium perhydroxide (11). All of these activated oxygen forms are highly reactive with the membranes of the photosynthetic apparatus, resulting in the production of lipid peroxides and leaky membranes of all types. One can detect this change in membranes by the formation of malondialdehyde-reactive lipid peroxides, as well as changes in conductivity in leaf disks in bathing solutions from the release of cellular components (6,12). At a structural level, these membrane perturbations can be


Figure 4. Reactions in the formation of other toxic oxygen species from superoxide. In the first reaction, two superoxide molecules combine with water to produce peroxide and oxygen. In the second reaction, peroxide reacts with iron to produce the highly reactive hydroxy radical. These further reactions of the superoxide molecules generated by paraquat action produce more toxic effects than superoxide alone and account for much of the membrane damage generated by paraquat.

detected first by a change in the stroma lamellae (the site of most of the PSI in the chloroplast) and by a curving of the grana membranes (6,12). At later stages of treatment, all photosynthetic membranes, the tonoplast, and plasma membrane are compromised, leading to a loss of any cellular integrity, and ensuring the death of the plant. PS I diverters would also cause a depletion of NADPH in the chloroplasts, although this would only cause a slow death of the plant, rather than the rapid changes brought about by the action of the activated oxygen species. By adding herbicides that disrupt electron flow at PS II, such as atrazine, the chloroplasts are protected from the effect of the PS I diverters. In order to be reduced effectively, the bipyridilium herbicides must have a potential of −0.35 to −0.45 eV (7) so that they may accept electrons from the primary electron acceptor of PSI. In a study of a number of different potential PSI diverters, only those compounds with this reduction potential proved to be herbicidal. One other experimental compound (‘‘B1000’’) was herbicidal even though it had a higher reduction potential. It is likely that the compound was transformed to a compound with greater reduction potential (something akin to paraquat) in the plant. Although animal cells do not contain chloroplasts, they contain mitochondria that do go through a similar sort of reduction mechanism in the electron transport chain and are thus also affected in a similar manner to plant cells. Particularly strong effects are noted in lung tissue. In plant cells treated in the dark or in tissue such as roots lacking chloroplasts, a similar sort of mitochondrial reduction occurs, leading to the death of these tissues too, albeit at a slower rate than in photosynthetic tissue (13). A number of chloroplast and extra-chloroplast enzymes have been promoted as having potential in ameliorating the effects of paraquat-induced damage, including the enzymes superoxide dismutase, ascorbate peroxidase, and glutathione reductase (Fig. 5). The results of these experiments have been variable, however. In some cases, an increase in these protective enzymes also offered some protection from paraquat damage, whereas in other

1225

cases, no such protection was obtained. Plants that have been genetically engineered to have as much as 100× the activity of superoxide dismutase or glutathione reductase were found to be actually more sensitive to paraquat (14). Moreover, these herbicides prevent the production of NADPH, which is required to keep this system of enzymatic protection in operation. Thus, in the long run, the effects of paraquat on the production of active oxygen species and the inhibition of NADPH would exhaust any potential enzymatic protection. Substantial information has recently been obtained on the mechanism of uptake of paraquat into the cell through the work of DiTomaso and colleagues (15,16). Bipyridilium herbicides structurally resemble some of the polyamine compounds such as putrescine and spermidine. Indeed, when paraquat is supplied simultaneously with some of these polyamines, the uptake of paraquat is severely limited. These data indicate that the polyamine transporters or uptake sites might be shared with paraquat. Although these experiments were performed on roots rather than on shoots because of the technical difficulties in measuring efflux in leaf pieces, it is likely that a similar mechanism exists in leaves as well as in roots. For example, leaf disks of Conyza could be protected from paraquat damage by higher concentrations of polyamines (see discussion below). Recently, Vaughn (17) has shown that the cell wall has paraquat-binding sites. These sites could be blocked by polyamines. These experiments were conducted with a probe with the same charge separation as paraquat coupled to a colloidal gold particle visible with the electron microscope. Relatively little binding of the paraquat charge analog was found in other cellular compartments. A correlation between the labeling of de-esterified pectins and the paraquat-binding probe indicated that these are sites of paraquat binding in the cell wall. A similar explanation has been put forth on the interactions between polyamines and de-esterified pectins (18). Thus, it is likely that a portion of the paraquat reaching plant leaves is bound in the apoplast in the de-esterified pectin fraction. Morfamquat is a selective herbicide in that dicots but not monocots are affected at the normal field rates (19). The mechanism of this difference is not clearly understood but probably relates to the ability of morfamquat to be enzymatically converted to paraquat by esterases that cleave the bulky side groups on the molecule. Once in the cell, morfamquat then behaves identically to paraquat. Less is known of the other herbicides that are photosystem I diverters, although the biochemical studies on isolated chloroplasts and structural studies on leaf material indicate that they have an identical mechanism to the bipyridilium herbicides (6–8). Because the heteropentalenes do not resemble the bipyridiliums in

Figure 5. Diagrammatic representation of the Halliwell–Asada pathway for protection of toxic oxygen species.

1226


structure, it is unlikely that they have similar uptake and translocation properties. HERBICIDE RESISTANCE When one considers that paraquat and other PSI diverters interact with a site critical to the growth of the plant and a site unlikely to be modified without lethality to the plant, the chances for resistance seem remote. However, resistance to PSI diverter herbicides (chiefly, paraquat) have been noted in virtually all countries where there has been extended use. To date, 25 weed species with resistance are known worldwide (20).

Conyza Herbicide resistance to the PSI diverters was first noted in populations of Conyza bonariensis in Egypt (21) after many years of continuous use of this herbicide solely for weed control. Other groups in other countries reported similar occurrences of paraquat resistance in this genus or in the related genus Erigeron (22). Despite 20 years of work by a number of laboratories, the molecular mechanism(s) for resistance has remained a contentious issue. A preliminary report of this work indicated a novel form of superoxide dismutase (21), although numerous other workers subsequently have been unable to detect any isozyme differences with respect to this enzyme (12). A subsequent report by Fuerst et al. (23) revealed that paraquat movement in the plant must be affected in the resistant biotype, using two lines of evidence. In one set of experiments, 14 C paraquat was used to directly monitor paraquat movement using excised leaves. These experiments revealed a restricted movement of the paraquat in the resistant (R) biotype but a general movement throughout the leaf in the susceptible (S) biotype. In a second set of experiments, chlorophyll fluorescence suppression was used to monitor the presence of the PSI diverter in the chloroplast. PSI diverters cause chlorophyll fluorescence suppression by accepting electrons very efficiently from PSI, quenching the active chlorophyll molecules. Thus, by monitoring the fluorescence, one can determine whether the herbicide has reached the chloroplast. These fluorescence experiments revealed that little or no paraquat reached the chloroplast in the R biotype. Despite the results that paraquat movement was restricted in the R biotype, some workers continued to argue in favor of increased protective enzymes as the cause of paraquat resistance. However, the results with the protective enzymes were variable, with some laboratories finding enhanced activity and other laboratories not. However, restricted movement was always found in the R biotypes, even in populations that were segregating both R and S biotype (24). Moreover, the resistance is controlled by a single gene, which makes it less likely that three or more protective enzymes could be induced by a single factor, although such regulatory genes are not without precedent. Moreover, as mentioned above, the protection pathway relies on a steady supply of NADPH in order to be functional. However, with paraquat-diverting electrons

at the primary acceptor of PSI, the supply of NADPH would be quickly exhausted. Thus, it is unlikely that the presence of small increases in protective enzymes would be effective over long time periods under field situations. Some of the most convincing data on potential paraquat resistance mechanisms comes from crossresistance studies. Although the R biotype of Conyza exhibits ∼100-fold resistance to paraquat, the resistance to diquat is only ∼10-fold, and no resistance is observed to morfamquat. Moreover, herbicides with the same mode of action, including the heteropentalenes, the triazinebipyridiliums, and the anti-protozoan drug metronidazole, were effective in causing necrosis, lipid peroxidation, and membrane damage at the same concentration in both R and S biotypes (6). In comparing the structures of the phytotoxins to which the R biotype exhibited resistance, a reasonable hypothesis from these data is that some facet of the charged N atoms on paraquat is responsible for the resistance. Moreover, because protoplasts (wall-less cells) of both biotypes displayed equal sensitivity to paraquat, it was likely that the resistance factor was somewhere in the cell wall. As a more direct proof of this, Vaughn (17) used a colloidal gold probe with the charge separation of paraquat to determine potential binding sites in the two biotypes. Both biotypes displayed binding in the cell walls, but there was eight-fold more labeling in the case of the R biotype. The probe could be displaced by paraquat but not by morfamquat. Polyamines do compete with the label, however, indicating that these are polyaminebinding sites as well. The increase in the probe binding in the R biotype correlated closely with changes in the quantity and distribution of de-esterified pectin. Thus, it is likely that paraquat resistance in Conyza is due to an alteration in the cell wall involving the de-esterified pectins.

Hordeum Glaucum and Other Cases Paraquat resistance in Hordeum glaucum was discovered in Australia not long after the appearance of resistance in Conyza species in Egypt and elsewhere (25). Although certain aspects of the resistance mechanisms appear to be similar, there are differences as well [(26,27) Vaughn, unpublished]. Similarities with the Conyza resistance include a pattern of restricted movement when applied to the plant, lack of resistance of protoplasts, lack of paraquat-induced chlorophyll fluorescence suppression, sensitivity to the heteropentalene herbicides, no increase in protective enzymes, and protection by certain polyamines. Differences include the following: The level of resistance is less than in Conyza, the resistant biotype is cross-resistant to morfamquat, and no substantial changes in de-esterified pectin. Inheritance is as a monogenic recessive. Thus, although the restriction of paraquat movement seems to be critical in both Hordeum and Conyza, the mechanisms may different. Hart et al. (28) compared the distribution of paraquat movement in the R and S Hordeum biotypes and came to the conclusion that resistance may be due to vacuolar translocation mechanisms related to polyamine transporters in that organelle rather than the cell wall.


Most of the other resistant biotypes have been investigated less intensively than are the two cases mentioned above. Basically two scenarios are obtained. A low level of paraquat resistance is associated with an increase in one or more of the protective enzymes (29), whereas a much higher level of resistance is associated with differences in the movement/uptake of paraquat (30). ENVIRONMENTAL FATE/DEGRADATION/TRANSPORT Because paraquat and diquat are positively charged ions, they are very quickly and tightly bound to negatively charged clay particles in the soil, rendering them totally inactive (1). Thus, these herbicides have no soil activity. Plants do not actively metabolize either paraquat or diquat (31); however, substantial photodegradation does occur on the leaf surface. Isonicotinic acid and methylamine hydrochloride are the decomposition products most often noted from paraquat (1). With diquat, the decomposition products included the pyrazinium salt, picolinamide, and picolinic acid (1). Photodegradation of diquat was greater than that of paraquat at equal irradiances (1–3). Neither paraquat nor diquat is generally transported great distances, as they tend to kill the tissues to which they come in contact, rendering little further movement. Under conditions of reduced light intensity or darkness, there is some movement of the herbicide that apparently occurs via the xylem [see (24) for an example of the translocation pattern of paraquat in leaf samples fed via the petiole]. Thus, most of the movement of paraquat is from the affected leaf area up the stem to higher positions in the plant. Autoradiography of 14 C paraquat spotted on leaf disks indicates that movement by mechanisms other than the xylem (24). Moreover, the accumulation of radioactivity in the trichomes may be associated with an increase in pectin content in these cells (17), wall sites associated with binding of the paraquat probe. ANTIBODIES TO PARAQUAT Although one normally does not think of small molecules such as herbicides as being antigenic, when coupled to suitable carrier proteins, immune response can be induced that specifically detect herbicide molecules. Antibodies to paraquat have been developed in a number of laboratories, both for the production of polyclonal serum or monoclonal antibodies (31,32). Specificity of these sera and monoclonals vary, although most seem to be fairly specific for paraquat and closely related molecules. With several of the immunoassay procedures, as little as 0.1-ppb paraquat may be detectable in a sample, comparable with the best analytical methods of detection. Because the immunoassays will work on crude samples and are faster and require less expensive equipment, they are a great alternative to standard laboratory protocols for herbicide detection and quantification. One of the more ingenious approaches to the use of paraquat antibodies was their production by plant cells. By introducing the gene for a paraquat antibody into tobacco

1227

cells, the genetically engineered plants were able to resist the effects of paraquat (33). This technology promises to add a potential selective use for these nonselective herbicides. TOXICITY AND SAFETY Both diquat and paraquat are relatively toxic compounds, with LD50 s (mg/kg) of 50 to 150 for many species of mammal, with man at the lower end of this spectrum (1–3,9). Paraquat poisoning in the 1960s and 1970s from inhalation of paraquat-treated cannabis was a serious safety issue of the time. Because paraquat is most active in rapidly respiring tissue and in the presence of oxygen, it is not surprising that most of the damage in mammalian systems is associated with lung tissue. Symptoms such as fibrosis and hemorrhage of the lungs are often detected after paraquat poisoning [reviewed in Summers (1)]. SUGGESTIONS FOR FURTHER READING Many of the reviews cited in the literature referring to the earlier literature on these herbicides should be consulted for material of more historical interest (1–3). An excellent review of the photosystem I reaction site is presented by Goldbeck (34). BIBLIOGRAPHY 1. L. A. Summers, The Bipyridinium Herbicides, Academic Press, London, U.K., 1980. 2. A. A. Akhavien and D. L. Linscott, Residue Rev. 23: 97–145 (1968). 3. P. Camilleri, J. R. Bowyer, and P. H. McNeil, Z. Naturforsch. 42C: 829–833 (1987). 4. P. Camilleri, M. T. Clark, I. J. Gillmore, and D. ColeHamilton, J. Chem. Soc. Perkin Trans. II 833–836 (1985). 5. K. C. Vaughn, M. A. Vaughan, and P. Camilleri, Weed Sci. 37: 5–11 (1989). 6. E. F. Elstner, H. P. Fischer, W. Osswald, and G. Kwiatkowski, Z. Nuturforsch. 35C: 770–775 (1980). 7. H. Fischer and L. A. Summers, Tetrahedron 35: 615–618 (1976). 8. W. H. Ahrens, Herbicide Handbook, 7th edn., Weed Science Society of America, Champaign, IL, 1994. 9. B. Halliwell, New Phytol. 73: 1075–1086 (1974). 10. E. P. Fuerst and M. A. Norman, Weed Sci. 39: 452–462 (1991). 11. K. C. Vaughn and S. O. Duke, Plant, Cell Environ. 6: 13–20 (1983). 12. K. C. Vaughn and E. P. Fuerst, Pestic. Biochem. Physiol. 24: 86–94 (1984). 13. B. M. R. Harvey and D. B. Harper, in H. M. LeBaron and J. Gressel, eds., Herbicide Resistance in Plants, Wiley, New York, pp. 215–233. 14. G. Creissen et al., Plant Cell 11: 1277–1291 (1999). 15. J. J. Hart, J. M. DiTomaso, and L. Kochian, Pestric Biochem. Physiol. 43: 212–222 (1992).

1228

PHYTOALEXIN

16. J. J. Hart, J. DiTomaso, and L. Kochian, Plant Physiol. 103: 963–969 (1993). 17. K. C. Vaughn, WSSA Ann. Meeting Absts. 40: 163–164 (2000). 18. J. Messiaen, P. Cambier, and P. Van Cutsem, Plant Physiol. 113: 387–395 (1997). 19. R. C. Brian, Pestic. Sci. 3: 409–414 (1972). 20. I. Heap, Weed Science.com, Available at http://www.weedscience.com, 2000. 21. R. J. Youngman and A. D. Dodge, in G. Akoyunoglou, ed., Photosynthesis and the Environment, Balaban Int. Sci., Philadelphia, 1981, pp. 537–544. 22. E. P. Fuerst and K. C. Vaughn, Weed Tech. 3: 150–156 (1990). 23. E. P. Fuerst et al., Plant Physiol. 77: 984–989 (1985). 24. M. A. Norman, R. J. Smeda, E. P. Fuerst, and K. C. Vaughn, Pestic. Biochem Physiol. 46: 236–249. 25. S. B. Powles and G. Cornic, Aust. J. Plant Physiol. 14: 81–89 (1987). 26. E. C. Purba, C. Preston, et al., Planta 196: 464–468 (1995). 27. C. Preston, in S. B. Powles and J. A. M. Holtum, eds., Herbicide Resistance in Plants: Biology and Biochemistry, Lewis Pub., Boca Raton, FL, 1994. 28. D. B. Harvey and B. M. R. Harvey, Plant Cell Environ. 1: 211–215 (1978). 29. E. W. Carroll, O. J. Schwarz, and L. G. Hickok, Plant Physiol. 87: 651–654 (1988). 30. P. Slade, Weed Res. 6: 158–167 (1966). 31. M. R. Bowles and S. M. Pond, Mol. Immunology 27: 847–852 (1990). 32. J. M. V. Emon, J. N. Seiber, et al., Am. Chem. Soc. Symposium Ser. 276: 307–316 (1985). 33. M. Longstaff, C. A. Newell, et al., Biochim. Biophys. Acta 1381: 147–160 (1998). 34. J. H. Goldbeck, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 293–324 (1992).

PHYTOALEXIN RALPH L. NICHOLSON Purdue University West Lafayette, Indiana

Antimicrobial compounds are found in all plants; some occur constitutively and function as preformed inhibitors of microorganisms. They are often thought to function in warding off attacks by nonpathogens or weak pathogens. These compounds have been referred to as phytoanticipins and are considered as part of the plant’s passive defense system (1). Other compounds are formed specifically in response to the attempted ingress of a pathogen and their synthesis is part of a plant’s active defense response. In this case the plant responds to the presence of a pathogen or, more specifically, to attempted infection by upregulation of synthesis of secondary products which act as specific toxicants of the potential pathogen. These compounds may be naturally present in the plant but when the plant is under stress they are synthesized at an accelerated rate and deposited at specific sites where their

toxicity is most effectively expressed. The phenylpropanoid phenols (2), which are found as glycosides and esters and ultimately are important constituents of the cell wall and of lignin, are excellent examples of these compounds. Such compounds act as toxicants if present as free acids, or they can be bound to carbohydrate polymers of the cell wall and prevent the growth of the pathogen; thus they can serve as either a chemical or mechanical barrier to the pathogen. If the compounds formed are new or different from those normally synthesized by the plant, they are considered as phytoalexins (1). The definition of phytoalexins has been revised many times and it is now generally accepted that they are low molecular weight antimicrobial compounds produced by plants in response to infection or stress (1,3). Since the time when secondary metabolites were suggested to have a role in the expression of resistance, a significant problem has been to prove that such metabolites are of primary importance as factors that account for resistance. This is associated with two concerns: first, the site of accumulation of the compounds within the host tissue, and second, the timing of the host response and eventual synthesis of compounds with respect to the stage of pathogen and disease development. Essentially the questions asked are when and where phytoalexins are synthesized, and whether synthesis occurs fast enough to limit the growth and development of the pathogen. A major problem is to demonstrate beyond doubt that a compound (or a family of compounds) actually is toxic to microorganisms that cause plant disease. Several reviews on the importance of phytoalexins to the disease response are available (2,4). Initial demonstrations that phytoalexins are significant components of plant defense required the isolation and identification of compounds from large amounts of tissue after inoculation (3). Although successful within the constraints of available technology, this approach had several problems, one of which was the demonstration that the phytoalexin actually accumulates within the infection site and not only in tissue surrounding it. The advent of high performance liquid chromatographic analysis coupled with new techniques of mass spectrometry now allow for the isolation and identification of small amounts of compounds from very small tissue samples (5), and this has changed our assumptions about the significance of metabolic intermediates to the expression of resistance. Pisatin, a pterocarpan isoflavonoid from peas, was one of the first phytoalexins to be chemically characterized (6). As a group, phytoalexins represent a variety of classes of compounds. Many have been characterized from a range of plant families, and plants that produce phytoalexins often synthesize several different compounds. Although phytoalexins are structurally diverse, any one plant family tends to produce similar compounds (7). This correlation of structures with plant species is intimate, and phytoalexins have even been used as chemotaxonomic markers. For example, isoflavonoid and pterocarpan phytoalexins are common in the Fabaceae, sesquiterpenoids are unique to members of the Solanaceae, and the sulfurcontaining indoles are characteristic of members of the

PLANT NUTRITION R1 OH O+

R3

R2

(I) Apigeninidin R1 = H, R2 = OH (II) Luteolinidin R1 = OH, R2 = OH (III) Caffeic acid ester of Arabinosyl 5-O-Apigeninidin O R1 = H R2 = HO CH C C O H HO

(IV) 5-Methoxy-Luteolinidin R1 = OH, R2 = OCH3 (V) 7-Methoxy-Apigeninidin R1 = H, R2 = OH, R3 = OCH3

Brassicaceae (4). Members of the Poaceae accumulate phytoalexins of a variety of chemical classes, including flavonoids (rice, sorghum), diterpenes (rice), stilbenes (sugarcane), and anthranilic acids (oat). Synthesis of phytoalexins occurs as a result of diversion of primary metabolic precursors into secondary metabolic pathways (2,6). The diversion often arises from the de novo induction of enzymes that control key branch points in the biosynthetic pathways. For phenylpropanoid-derived phytoalexins, phenylalanine ammonia-lyase and chalcone synthase are the major regulated enzymes in their biosynthesis (2). For mevalonatederived sesquiterpenoid phytoalexins, hydroxymethylglutaryl coenzyme A reductase, squalene synthase, and sesquiterpenoid synthase are the key regulatory enzymes (8). Because synthesis of phytoalexins requires a series of enzymatic activities, highly coordinated signaling events are believed to be involved in the challenged host cells regardless of the type of phytoalexin being synthesized. The antimicrobial properties of phytoalexins suggest that they are important components of plant defense. Phytoalexins are absent in healthy tissue and accumulate after infection by fungal or bacterial pathogens in monocot and in dicot plants (7). Surprisingly, few phytoalexins have been demonstrated to accumulate rapidly at the site of attempted infection and in sufficient quantities to inhibit the in vitro growth of pathogens. Considerable literature exists on phytoalexins in host–parasite interactions involving dicots, but there is little information on their role in diseases of monocots. A significant exception is the case of deoxyanthocyanidin flavonoid phytoalexins synthesized by sorghum (Fig. 1). As stated earlier, it is necessary to demonstrate the site and timing of phytoalexin accumulation and toxicity. The sorghum phytoalexins satisfy each of these criteria. They are synthesized in cellular inclusions within the cell that is under attack (9) and synthesis is localized at the site of attempted penetration. Synthesis occurs rapidly, indicating that the events that constitute recognition and signal transduction occur early in the plant–pathogen interaction. Microspectrophotometry showed that the phytoalexins can accumulate to as much as 0.15 M within individual inclusions, a

1229

MW = 255.0654 MW = 271.0603 MW = 549.3286 CH2 O OH H H OH

O

MW = 285

MW = 269

Figure 1. The sorghum 3-deoxyanthocyanins.

level of the compounds well above that required for fungitoxicity (10). BIBLIOGRAPHY 1. H. D. VanEtten, J. W. Mansfield, J. W. Bailey, and E. E. Farmer, Plant Cell 6: 1191,1192 (1994). 2. R. A. Dixon and N. L. Paiva, Plant Cell 7: 1085–1097 (1995). 3. R. L. Nicholson and R. Hammerschmidt, Annu. Rev. Phytopathol. 30: 369–389 (1992). 4. R. J. Grayer and J. J. Harborne, Phytochemistry 37: 19–42 (1994). 5. J. A. Sugui et al., Phytochemistry 48: 1063–1066 (1998). 6. D. R. Perrin and W. Bottomley, Nature 191: 76–78 (1961). 7. C. J. Smith, New Phytol. 132: 1–45 (1996). 8. D. Choi, R. Bostock, S. Avdiushko, and D. Hildebrand, Proc. Natl. Acad. Sci. USA 91: 2329–2333 (1994). 9. B. A. Snyder and R. L. Nicholson, Science 248: 1637–1639 (1990). 10. B. A. Snyder et al., Physiol. Mol. Plant Pathol. 39: 463–470 (1991).

PHYTOPHAGOUS Plant-eating (Greek: phyton plant and phago to eat).

PLANT GROWTH REGULATORS Substances (excluding fertilizers or other plant nutrients) that alter the expected growth, flowering, or reproduction rate of plants (USEPA).

PLANT NUTRITION HEINRICH W. SCHERER ¨ Bonn Universitat Agrikulturchemisches Institut Bonn, Germany

1230

PLANT NUTRITION Landbaues, Technische ¨ Munchen ¨ Universitat Freising, Germany

KONRAD MENGEL ¨ Justus-Liebig-Universitat Giessen Institute for Plant Nutrition Giessen, Germany

HEINRICH DITTMAR VILMOS CZIKKELY ¨ WALTER BRANDLEIN BASF Aktiengesellschaft Ludwigshafen, Germany

MANFRED DRACH RALF VOSSKAMP ¨ REINHARDT HAHNDEL KARL-HEINZ ULLRICH KARL-FRIEDRICH KUMMER BASF Aktiengesellschaft Limburgerhof, Germany

MARTIN E. TRENKEL Eusserthal, Germany

REINHOLD GUTSER ¨ Lehrstuhl fur ¨ Pflanzenernahrung, Technische ¨ Universitat ¨ Munchen-Weihenstephan Freising, Germany

INTRODUCTION Fertilizers in the broadest sense are products that improve the levels of available plant nutrients and/or the chemical and physical properties of soil, thereby directly or indirectly enhancing plant growth, yield, and quality. Fertilizers are classified as follows in terms of their chemical composition: 1. Mineral fertilizers consist of inorganic or synthetically produced organic compounds. 2. Organic fertilizers are waste products from animal husbandry (stable manure, slurry manure), plant decomposition products (compost, peat), or products from waste treatment (composted garbage, sewage sludge). 3. Synthetic soil conditioners are products whose main function is to improve the physical properties of soils, for example, friability and water and air transport. The following categories are distinguished with respect to nutrient content:

¨ GUNTER STEFFENS

Landwirtschaftliche Untersuchungs- und Forschungsanstalt Oldenburg, Germany

TITUS NIEDERMAIER formerly BASF Aktiengesellschaft Ludwigshafen, Germany

1. Straight fertilizers generally contain only one primary nutrient. 2. Compound (complex or multinutrient) fertilizers contain several primary nutrients and sometimes micronutrients as well. 3. Micronutrient fertilizers contain nutrients required in small quantities by plants, as opposed to macronutrients; quantities range from 1 to 500 g ha−1 a−1 .

¨ HANS PRUN

formerly BASF Aktiengesellschaft Limburgerhof, Germany ¨ HERMANN MUHLFELD

formerly Chemische Fabrik Kalk GmbH Köln, Germany

WILFRIED WERNER Agrikulturchemisches Institut ¨ Bonn der Universitat Bonn, Germany ¨ GUNTER KLUGE

¨ Bundesministerium fur ¨ Ernahrung, Landwirtschaft und Forsten Bonn, Germany

FRIEDRICH KUHLMANN ¨ Landwirtschaftliche Institut fur Betriebslehre Giessen, Germany

HUGO STEINHAUSER ¨ formerly Lehrstuhl fur Wirtschaftslehre des

Finally, fertilizers can be classified as solid or liquid fertilizers and as soil or foliar fertilizers, the latter being applied exclusively by spraying on an existing plant population.

History. Fertilizing substances were applied even in antiquity. Their use can be attributed to the observation in nature that plants developed especially well in locations where human or animal excreta, ash residues, river mud, or dying plants were left. For example, the Egyptians knew about the fertility of the Nile mud, and the Babylonians recognized the value of stable manure; for example, HOMER mentions manure in the Odyssey. PLINY reports that the Ubians north of Mainz used ‘‘white earth,’’ a calcareous marl, to fertilize their fields. The Romans acknowledged the advantages of green manuring, cultivating legumes and plowing them under. At the end of the first millenium, wood ash was much used as fertilizer in Central Europe. Not until the beginning of the 19th century did guano, at the suggestion of ALEXANDER VON HUMBOLDT (1800), and Chilean caliche, on the recommendation of HAENKES (1810), come into use as fertilizers. Up to that time, however, it was still believed that the organic matter of soil, humus, was the true source of plant nutrition.

PLANT NUTRITION

1231

Table 1. Physiological Classification of Plant Nutritive Elements, Nutrient Carriers, and Form in Which the Nutrient is Taken Up Nutritive Element

Nutrient Carrier

Uptake

First group C

CO2 , HCO3 −

CO2 by leaves, HCO3 − by roots

H

H2 O

H2 O by leaves, H2 O and HCO3 − by roots

O

CO2 , HCO3 − , O2

O2 and CO2 by leaves, HCO3 − and O2 by roots

N

NH4 + , NH3 , NO3 − , NOx

NH4 + and NO3 − by roots, NH3 and NOx by leaves

S

SO4 2− , SO2 , SO3 , H2 S

SO4 2− by roots, SO2 , SO3 , and H2 S by leaves

P

H2 PO4 − , HPO4 2−

H2 PO4 − and HPO4 2− by roots

B

H3 BO3 , borates

H3 BO3 and B(OH)4 − by roots

Si

Silicates

Si(OH)4 by roots

K

K+ , K salts

K+ by roots

Mg

Mg2+ ,

Mg salts

Mg2+ by roots

Ca

Ca2+ ,

Ca salts

Ca2+ by roots

Mn

Mn2+ ,

Mn salts

Mn2+ by roots

Second group

Third group

Fourth group Fe, Cu, Zn, Mo

Ionic form or metal chelates, minerals containing these elements

Around 1800, the nutrition problem entered a critical phase in Europe. In 1798, MALTHUS set forth his pessimistic theses, saying that the quantity of food could increase only in arithmetic progression while the population grew geometrically. Combining results obtained by others (SPRENGEL, BOUSSINGAULT) with his own pathbreaking studies, J. VON LIEBIG set forth the theoretical principles of plant nutrition and plant production in Chemistry in Its Application to Agriculture and Physiology (1840). He took the view, now considered obvious, that plants require nitrogen, phosphate, and potassium salts as essential nutrients and extract them from the soil. LIEBIG’s mineral theory was well supported by experimental data of J. B. BOUSSINGAULT (1802–1887) in France. He and also J. B. LAWES (1814–1900) and J. H. GILBERT (1827–1901) in England showed that plants benefit from inorganic N fertilizers. LIEBIG thus became the founder of the theory of mineral fertilizers, and his doctrines led to an increasing demand for them. A number of companies were subsequently founded in Europe to produce phosphate and potash fertilizers. Superphosphate was manufactured for the first time in 1846, in England. In Germany, this industrial development started in 1855. The importation of saltpeter on a large scale began in the area of the German Federation (56000 t in 1878). Peruvian guano soon came into heavy use (520000 t in 1870). Ammonium sulfate, a coke-oven byproduct, was

By roots in ionic form or in the form of soluble metal chelates, Mo in the form of the molybdate

later recognized as a valuable fertilizer, and the mining of water-soluble potassium minerals was undertaken in the 1860s (1). The demand for nitrogen that developed at the end of the 19th century soon outstripped the availability of natural fertilizers. A crucial breakthrough came about with the discovery and large-scale implementation of ammonia synthesis by HABER (1909) and its industrial realization by BOSCH (1913). Around the turn of the century, the technique of hydroponics led to the discovery of other essential plant nutrients. Research showed that plants in general require ten primary nutrients: carbon, hydrogen, oxygen. nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, and iron. JAVILLIER and MAZE (1908) pointed out for zinc and AGULHON (1910) pointed out for boron the nutritional effects on plants. WARINGTON (1923) first described the symptoms of boron deficiency, and BRANDENBURG (1931) clearly recognized dry rot in the sugar beet as boron deficiency. Generally micronutrients were made available to the plant as liquid foliar fertilizer, a method first suggested for iron by GRIS in 1844. By 1950, this list of micronutrients had been expanded to include manganese, copper, and molybdenum. Almost 70 years ago, serious research began on the best nutrient forms for individual plant species under various soil and climatic conditions. Besides

1232

PLANT NUTRITION

the classical fertilizers, for example, controlled-release fertilizers, improved foliar fertilizers, nutrient chelates, and nitrification inhibitors have been developed in recent decades. This development of new nutrient forms is still in full swing in the special fertilizers sector. In the developed market economies of Western Europe, the United States, and Japan, however, the level of mineral fertilizer use has not been increasing since the beginning of the 1980s. In some countries, genuine agricultural overproduction has occurred recently. Since better delivery of plant nutrients has led to increasing self-reliance even in the Third World economies (e.g., China, India, Brazil), these countries are not so important as purchasers of nutrients on the world market, so that surpluses cannot be exported without limit. The production of fertilizers is also on the increase in these countries. Thus overproduction plus regional environmental problems (nitrates entering the ground water) are actually leading to a decrease in mineral fertilizer use in some areas. This decline will be limited by diminishing soil fertility in localities where fertility has been enhanced by decades of proper fertilization. PLANT NUTRITION AND SOIL SCIENCE The science of plant nutrition is situated between soil science and plant physiology. It comprises the definition of the elements nutritive for plants; the uptake of plant nutrients and their distribution in the plant; the function of the nutritive elements in plant metabolism; their effect on plant growth; yield formation and quality parameters in crops; soil nutrient exploitation by plant roots; factors and processes that control the plant nutrient availability in soils; toxic elements in soils and their impact on plant growth; the application of plant nutrient carriers (fertilizers) and their turnover in soils; nutrient balance; and the maintenance of soil fertility. Plant nutrition is considered mainly from two aspects, an agronomic one and an ecological one. The former is focused on the question of fertilizing soil as an efficient means to increase crop yield and to maintain or even improve soil fertility. The latter, the ecological aspect of plant nutrition, is concerned with the nutritive condition of a soil and a location and with its effect on plant growth and plant communities. Since fertilizers are the topic of this article, the agronomic aspects of plant nutrition are treated with greater depth. The science of plant nutrition is closely associated with the science of soils. The latter comprises a broad field of scientific activity and thus cannot be considered here in all its facets. In this article only those problems of soil science relevant to understanding plant nutrition are treated.

1. A deficiency of the element makes it impossible for the plant to complete its life cycle. 2. The deficiency is specific for the element in question. 3. The element is directly involved in the nutrition of plants because of either its chemical or its physical properties. According to this definition, the following chemical elements are nutritive elements for plants: C, H, O, N, P, S, K, Ca, Mg, Fe, Mn, Cu, Zn, Mo, B. Further elements, such as Na, Cl, and Si, may affect plant growth positively, and there are particular plant species for which these elements are of great importance. Nevertheless, they are not essential nutritive elements for plants in the strict sense of the definition. Cobalt is required by some bacteria, e.g., by dinitrogen-fixing bacteria and thus may also benefit plant growth indirectly. Generally it is not the element itself that is provided to and taken up by the plant, but an ion or a molecule in which the nutritive element is present, e.g., C is present in CO2 , P in H2 PO4 − , N in NO3 − or NH4 + , and B in H3 BO3 . The particular molecule or ion in which the nutritive element is present is termed the nutrient carrier. In the case of metals, the corresponding ion or salts of ion species in question, e.g., K+ , Ca2+ , Zn2+ , can be considered the carrier. In this sense fertilizers are nutrient carriers. Plant nutrients may be grouped into macronutrients and micronutrients. Macronutrients are required in high amounts and thus are present in plant tissues in much higher concentrations than the micronutrients. Carbon, H, O, N, P, S, K, Ca, and Mg belong to the macronutrients. Their concentration in the dry plant matter is in the range 1–50 mg/g, except for C, H, and O, which have much higher concentrations (see Table 2). The concentration of the micronutrients in the dry plant matter is in the range 1–1000 µg/g. From the viewpoint of fertilization, those nutrients that are required by plants in high quantities and that must be regularly supplied by fertilization are of particular interest. These nutrients are N, K, P, and to a minor degree also Ca, Mg, and S. Calcium is a soil nutrient, which means that it is important for an optimum soil structure. Application of micronutrients is not a common practice, but they are applied at locations where soils are low in a particular micronutrient or where soils may bind this micronutrient very strongly. This is the case for heavy metals (Fe, Mn, Cu, Zn) and B in calcareous and alkaline

Table 2. Mean Content of Chemical Elements in the Dry Matter of Green Plant Material

Plant Nutrients

Element

Definition and Classification. From a scientific point of view, the term plant nutrient is not especially precise. More appropriate is to distinguish between nutritive elements of plants and nutritive carriers. Essential nutritive elements for plants are the chemical elements that are required for a normal life cycle and that satisfy the following criteria:

O C H N K P All other elements

Content, g/kg 440 420 60 30 20 4 26

PLANT NUTRITION

soils (soils with a high pH value), while Mo is strongly fixed in acid soils. Acid organic soils are known for their low available Cu content. According to the different quantitative requirements for macronutrients and micronutrients, the former are taken up in much higher quantities than the latter. Thus a wheat stand with a yield potential of 7 t of grain per hectare requires about 100 kg K but only 100 g Cu. From a physiological point of view, plant nutrients are grouped into four groups, as shown in Table 1. The first group, comprising C, H, O, N, and S, includes all major elementary constituents of organic plant matter. Their carriers are present mainly in the oxidized form, and they must be reduced during the process of incorporation. The energy required for this reduction originates directly or indirectly from photosynthetically trapped energy. Assimilation of H is basically an oxidation process, namely, the oxidation of water with the help of light energy (photolysis): hν

H2 O −−−→ 2H+ + 2e− + 0.5O2 The second group (P, B, Si) comprises elements that are taken up as oxo complexes in the partially deprotonated (P) and protonated (B, Si) form. The oxo complex is not reduced in the plant cell, but may form esters with hydroxyl groups of carbohydrates, thus producing phosphate, borate, and silicate esters. The third group comprises metals that are taken up from the soil solution in ionic form. They are only partially incorporated into the organic structure of the plant tissue: Mg in the chlorophyll molecule, Mn in the electron donor complex of photosystem II, and Ca2+ as countercation of indiffusible anions in cell walls and particularly in biological membranes. Potassium is virtually not incorporated into the organic plant matter. It is only weakly adsorbed by Coulombic forces. There exist, however, some organic molecules that may bind K+ very selectively (ionophores, see Section 3). These ionophores are likely to be involved in K+ uptake. The fourth group comprises heavy metals, of which Fe, Cu, and Zn are taken up as ions or in the form of soluble metal chelates, while Mo is taken up as molybdate. These molecules are easily incorporated into the organic structure, where they serve as essential elements of enzyme systems: Fe in the heme group and in ferredoxin, Mn in arginase (2), Cu in oxidases (polyphenol oxidase, cytochrome oxidase, ascorbate oxidase (3)), Zn in RNA polymerase (4), and Mo in nitrate reductase (5) and nitrogenase (6). All nutritive elements of plants, therefore, are taken up in the form of inorganic complexes, mostly in oxidized form or as metal ions, i.e., in forms characterized by a low energy level. This is a unique feature of plants, and a feature in which they contrast sharply with animals and most kinds of microorganisms (bacteria and fungi). Animals and most microorganisms must take up food that is rich in chemical energy in order to meet their energy requirements. Plants, at least green plants, meet their energy requirement by converting radiation energy into chemical energy. This energy conversion process

1233

is manifest in the reduction of plant nutrient carriers (NO3 − , SO4 2− , CO2 ) as already mentioned. Thus important processes of plant nutrition are closely linked with the unique function of plants in the great cycle of nature, i.e., the conversion of inorganic matter into organic form. Liebig (7) was correct in commenting on plant nutrition: ‘‘Die ersten Quellen der Nahrung liefert ausschließlich die anorganische Natur.’’ [The primary source of nutrition is provided exclusively by the inorganic materials in nature.] Function of Plant Nutrients Most plant organs and particularly plant parts that are metabolically very active, such as young leaves and roots, are rich in water (ca. 80–90 wt% of the total fresh matter), while their organic material is ca. 12–18 wt% and their mineral content is 2–6 wt%. As shown in Table 2, in the dry matter of plant material O and C are by far the most abundant elements, followed by H, N, and K. The elements C, O, H, and, to some extent, N are mainly structural elements in plant matter. They can, however, form chemical groups that are directly involved in metabolic processes, e.g., carboxyl groups, amino groups, hydroxyl groups. Since in many soils the available N is low, nitrogen [7727-37-9] is the most important fertilizer element, and for this reason its function in plant metabolism deserves particular interest. Nitrogen is an essential element for amino acids, proteins, nucleic acids, many coenzymes, and some phytohormones. Basic biochemical processes of meristematic growth, such as the synthesis of proteins and nucleic acids, require N. If this nutrient is not supplied in sufficient amounts, the growth rate is depressed and the synthesis of proteins affected. Nitrogen-deficient plants are characterized by low protein and high carbohydrate contents. This relationship is shown in Table 3(8). Nitrogen is also essential for the formation of chloroplasts, especially for the synthesis of chloroplast proteins. Hence N deficiency is characterized by low chlorophyll content; the leaves, especially the older ones, are pale and yellow; the stems thin and the plants small. Nitrogen-deficient plants senescence earlier, probably because of a deficiency of the phytohormone

Table 3. Effects of N Supply on Yield of Dry Matter and the Content of Organic N and Carbohydrates in the Dry Matter of Young Timothy Plants (Phleum Pratense) (8) N supply Yield and Content

Low

Yield, g/pot Content, mg/g Organic N, Sucrose Fructans∗ Starch Cellulose

15.7

20.2

20.5 46.9 22.2 32.8 169

31.5 22.6 9.2 11.7 184

∗

Polysaccharides of fructose.

Sufficient

1234

PLANT NUTRITION Table 4. Relationship Between N Fertilizer Rate and Nitrogenous Fractions in the Dry Matter of Rye Grass (9) Nitrogenous Fraction, g/kg Nitrogen Fertilizer rate, kg/ha 0 110 440

Total N

Protein N

Free Amino Acid N

NO3 − and NO2 − N

13.2 18.9 37.3

9.8 12.6 20.6

1.6 2.1 5.6

0.4 0.6 3.5

cytokinin. Abundant N supply increases the protein content, especially the content of free amino acids, and often also the content of NO3 − in plants. An example of this is shown in Table 4. Excess nitrogen nutrition results in luxurious plants that frequently are susceptible to fungi attack. The ratio of N to S in plant matter is ca. 10: 1. Hence sulfur [7704-34-9] is required in much lower quantities than N. Their functions are, however, similar. Sulfur is an elementary constituent of most proteins; the SH group in involved in various enzymatic processes and it is the reactive group of coenzyme A. Disulfide (S−S) bridges are essential structural elements in the tertiary structure of polypeptides and in many volatile S compounds, such as diallyl disulfide, which is the main component in garlic oil. Mustard oils occurring in many species of the Cruciferae contain a S-glycosidic bond and a sulfuryl group:

S R

glucose O

C NO

S

OH

O Insufficient S supply results in a decrease of growth rate with extremely low levels of SO4 2− and high concentrations of free amino compounds and NO3 − in the leaves, which is due to hampered protein synthesis. Sulfur plays an important role in the baking quality of wheat, since the concentration of S compounds in the gluten fraction is responsible for the linkages between the protein molecules (10). Sulfur deficiency may also affect N2 fixation of legumes by causing unfavorable conditions in the host plant or because of the relatively high S content of nitrogenase and ferredoxin (11). Deficiency symptoms of S appear at first in the youngest leaves, which turn light green to yellow. Abundant supply with S results in an accumulation of sulfate in plant tissues. Sulfur oxide can be taken up by the leaves and metabolized and thus can contribute to the S nutrition of plants. Too high SO2 concentrations in the atmosphere may be toxic. The toxicity symptoms are necrotic spots in the leaves. According to SAALBACH (12), the critical SO2 level in the atmosphere for annual plants is 120 µg/m3 . For trees and other perennials it is about half this level. The currently much discussed damage to trees in the forest of the Federal Republic of Germany (mainly spruce and silver fir) is not caused by toxic SO2 levels.

Phosphorus [7723-14-0] is an essential element in nucleic acids and various phospholipids (phosphoglyceride and phosphosphingolipids). In both cases. phosphate is esterified with sugars (nucleic acid) or with alcohol groups of glycerol or sphingosine. Phosphate is also present in various coenzymes; the most prominent is adenosine triphosphate (ATP), which carries a kind of universal energy that is used in a number of biochemical processes. Metabolites and enzymes can be activated by phosphorylation, a transfer of the phosphoryl group from ATP to the metabolite according to the following reactions: Activation of glucose Glucose + ATP −−−→ Glucose-6-phosphate + ADP Phosphorylation of an enzyme Enzyme − OH + ATP −−−→ Enzyme − O P + ADP Such reactions demonstrate the essential role of P not only in plant metabolism but also in all living organisms. Undersupply with P results in a reduced growth rate, and seed and fruit formation is affected. The leaves of P-deficient plants often show a gray dark green color; the stems may turn red. The P reserve in seeds is the Mg (Ca) salt of the inositol hexaphosphate (phytic acid):

P O

O

P O

O

O

P

P O

P

P

Myo-inositol hexaphosphate O P =

P

OH

OH Phosphoryl group The physiological role of boron has remained obscure until now, and therefore various hypotheses with numerous modifications exist concerning the physiological and biochemical role of boron in higher plants. Depending on

PLANT NUTRITION

the pH of the soil, boron seems to be taken up mainly as undissociated boric acid or as the borate anion. Plant species differ in their boron uptake capacity, reflecting differences in boron requirements for growth. However, there is still some controversy about boron translocation in plants. At least in higher plants, a substantial proportion of the total boron content is complexed in the cell walls in a cis-diol configuration (13). According to BIRNBAUM et al. (14), B is involved in the synthesis of uracil and thus affects UTP formation. (UTP is an essential coenzyme for the synthesis of sucrose and cell-wall components.) Also the synthesis of ribonucleic acid is hampered in the case of B deficiency. Since uracil is an integral part of ribonucleic acid (RNA), the formation of RNA may also be related to the synthesis of uracil. POLLARD et al. (15) suggest that B has a specific influence on plant membranes by the reaction of borate with polyhydroxy compounds. Boron deficiency appears as abnormal or retarded growth of the apical growing points. The youngest leaves are misshapen and wrinkled and show a darkish bluegreen color. The fact that B deficiency primarily affects the apex is in accord with the impaired synthesis of ribonucleic acids required for meristematic growth. High levels of available B in the soil may cause B toxicity in plants. This is mainly the case in arid areas; however, B toxicity can also be the consequence of industrial pollution (16). The toxicity is characterized by yellow leaf tips followed by progressive necrosis. The leaves take on a scorched appearance and drop prematurely. Silicon [7440-21-3] is not an essential element for plants; however, it has a beneficial effect on various plant species, mainly grasses (17). In plants well supplied with Si, cuticular water losses are diminished and resistance against fungal attack is improved (18). The favorable effect of Si on rice growth is well known. Silicon-containing fertilizer is frequently applied in rice production. Among the metal cation species, the potassium [744009-7] ion, K+ , is the nutrient plants take up from the nutrient medium at the highest rates. The K+ concentration in the cytoplasm is about 100 mM and thus much higher than the concentration of other ion species (19). Probably this high K+ concentration has a favorable influence on the conformation of various enzyme proteins (20). Potassium ions can easily penetrate plant membranes (see Section 3), which often leads to a depolarization of the membranes. Membrane depolarization, it is supposed, has a favorable effect on meristematic growth, photophosphorylation, aerobic phosphorylation, and phloem loading (21). These basic processes are important for the long-distance transport of photosynthates, the synthesis of various organic compounds, and CO2 assimilation. The data in Table 5 show that with an increase of K+ in alfalfa leaves (Medicago sativa), the CO2 assimilation rate increased, while the mitochondrial respiration rate decreased (22). In the case of low K, the respiration was about 2/3 of the CO2 assimilation, while with high K the C gained by assimilation was about 11 times higher than the C lost by respiration. This typical behavior indicates that under the conditions of K+ deficiency much of the stored carbohydrates must be respired in order to meet

1235

the ATP demand of the plant. Plants undersupplied with K+ have therefore a low energy status. Such plants are highly susceptible to fungal attack, water stress, and frost damage. Potassium is important in determining the osmotic pressure of plant fluids, and K+ -deficient plants are characterized by inefficient water use. Sodium ions may replace some K+ functions, e.g., the less specific osmotic functions. Important counterions of K+ in plant tissues are Cl− , NO3 − , and organic anions. The frequently observed favorable effect of Na+ and Cl− on plant growth is related to their osmotic functions. Plants suffering from K+ deficiency show a decrease in turgor, and under water stress they easily become flaccid. Plant growth is affected, and the older leaves show deficiency symptoms as necrosis beginning at the margins of tips and leaves. In K+ -deficient plant tissue, toxic amines such as putrescine and agmatine accumulate. The most spectacular function of magnesium [743995-4] is its integral part in the chlorophyll molecule. Besides this function, Mg2+ is required in various other processes and, the Mg fixed in the chlorophyll molecule amounts only to about 20% of the Mg present in green plant tissues. Magnesium is an essential ion in ribosomes and in the matrix of the cell nucleus. Here Mg2+ is bound by phosphate groups, since the Mg2+ is strongly electrophilic and thus attracts oxo complexes such as phosphate (23). The magnesium ion activates numerous enzymatic reactions in which phosphate groups are involved. The activation is assumed to be brought about by bridging the phosphate group with the enzyme or with the substrate. This is an universal function of Mg2+ not only relevant for plant metabolism but also for practically all kinds of organisms. Deficiency of Mg2+ affects chlorophyll synthesis: leaves turn yellow or red between the veins. The symptoms begin in the older leaves. Protein synthesis and CO2 assimilation are depressed under Mg2+ deficiency conditions. Recent results (24) have shown that the yellowing of spruce needles in the Black Forest is due to a Mg2+ deficiency and can be cured by Mg2+ fertilizer application. Calcium [7440-70-2] is the element of the apoplast (cell wall and ‘‘free space’’) and of biological membranes. Here it is adsorbed at the phosphate head groups of membrane lipids, thus stabilizing the membranes (25). Most of the Ca2+ present in plant tissues is located in the apoplast and in the vacuole, some in the mitochondria and in the chloroplasts, while the cytoplasm is extremely low in Ca2+ (10−7 to 10−6 M). The maintenance of this low cytoplasmic Table 5. Relationship Between K+ Concentration in the Dry Matter of Alfalfa Leaves, CO2 Assimilation, and Mitochondrial Respiration (22) Carbon gain and loss, mg dm−2 h−1 Concentration of K+ , mg/g 13 20 38

CO2 Assimilation 11.9 21.7 34.0

Mitochondrial Respiration 7.56 3.34 3.06

1236

PLANT NUTRITION

Ca2+ concentration is of vital importance for the plant cell (26). Higher cytoplasmic Ca2+ concentrations interfere with numerous enzymatic reactions and may even lead to a precipitation of inorganic phosphates. This low Ca2+ concentration suffices to form a complex with calmodulin, a polypeptide of 148 amino acids. The Ca–calmodulin complex is a universal enzyme activator. The activation is brought about by allosteric induction. Direct Ca2+ deficiency in plants is rare, since most soils are relatively rich in Ca2+ . Physiological disorders as a consequence of an insufficient Ca2+ supply of particular plant parts, however, occur frequently. Calcium is mainly translocated by the transpiration stream. Hence plant parts such as fruits, which mainly feed from the phloem and less from the xylem sap, may suffer from an insufficient Ca2+ supply. Shear (27) cites a list of 35 such Ca2+ -related disorders in fruits and vegetables. Two of the most important ones involve storage tissues and result in poor crop qualities (28): bitter pit in apples, characterized by small brown spots on the surface, and blossom-end rot in tomatoes, a cellular breakdown at the distal end of the fruit, which is then susceptible to fungal attack. Manganese [7439-96-5] is an integral part of the superoxide dismutase and of the electron donor complex of photosystem II. Manganese may activate enzymes in the same way as Mg2+ by bridging the phosphate group with the enzyme or the substrate. Deficiency of Mn2+ leads to the breakdown of chloroplasts. Characteristic deficiency symptoms are smaller yellow spots on the leaves and interveinal chlorosis. Manganese toxicity may occur, especially on flooded soils, because of the reduction and thus solubilization of manganese oxides. Toxicity symptoms are generally characterized by brown spots of MnO2 in the older leaves surrounded by chlorotic areas (29). Iron [7439-89-6] is an essential element for heme and ferredoxin groups. Iron deficiency leads to chloroplast disorders; the synthesis of thylakoid membranes is disturbed and the photochemical activity affected (30). Iron deficiency is characterized by yellow leaves. The symptoms are at first visible in the younger leaves. There is evidence that the deficiency, mainly occurring in plants growing on calcareous soils, is not induced by an insufficient Fe uptake from the soil but by a physiological disorder in leaves, affecting the Fe distribution in the leaf tissue (31). Iron toxicity can be a problem under reducing soil conditions, which prevail in flooded soils. Under such conditions iron(III) oxides are reduced and the iron is rendered soluble. This may increase the Fe concentration in the soil solution by a factor of 102 to 103 (32) so that plants may suffer from Fe toxicity, characterized by tiny brown spots on the leaves, which later may turn uniformly brown. Iron toxicity is known as ‘‘bronzing.’’ Copper [7440-50-8] is an essential element of various enzymes, such as superoxide dismutase, polyphenol oxidases, plastocyanin of the photosynthetic transport chain, and cytochrome c oxidase, the terminal oxidase in the mitochondrial electron transport chain. Deficiency in Cu leads to pollen sterility and thus affects the fruiting

of plants. Copper-deficient plants often are characterized by white twisted leaf tips and a tendency to become bushy. Zinc [7440-66-6] is an integral part of carbonic anhydrase, superoxide dismutase, RNA polymerase, and various dehydrogenases. It is closely involved in the N metabolism of plants. In Zn-deficient plants, protein synthesis is hampered and free amino acids accumulate. There is evidence that Zn is involved in the synthesis of tryptophan, which is a precursor of indole acetic acid, an important phytohormone. Zinc deficiency is characterized by short internodes, small leaves, and chlorotic areas in the older leaves. Frequently the shoots die off and the leaves fall prematurely. Molybdenum [7439-98-7] is present in the nitrate reductase and in the nitrogenase system that catalyzes the bacterial fixation (reduction) of dinitrogen. Deficiency of Mo frequently appears first in the middle and older leaves as a yellowish green coloration accompanied by a rolling of leaf margins. Cruciferae species are particularly susceptible to Mo deficiency. The most wellknown Mo deficiency is the ‘‘whiptail’’ of cauliflower. For further information on the physiology of plant nutrition, see (3,23,33). Soil Science Soil Classes, Soil Types, and Parent Material According to SCHROEDER (34), ‘‘soil is the transformation product of mineral and organic substances on the earth’s surface under the influence of environmental factors operating over a very long time and having defined organization and morphology. It is the growing medium for higher plants and basis of life for animals and mankind. As a space-time system, soil is four dimensional.’’ Soils are complex, quite heterogeneous, and may differ from each other considerably. Nevertheless, all soils have some common features. They possess a mineral, an organic, a liquid, and a gaseous component. In an ideal soil, the percentage proportions of these components are 45%, 7%, 23%, and 25%, respectively. The volumes of the liquid and gaseous components may change quickly. For example, in a water-saturated soil all pores are filled with water, and in a dry soil the soil pore volume is almost completely filled with air. The mineral and organic components contain plant nutrients and adsorb plant nutrients at their surfaces, and they are therefore of importance for the storage and retention of plant nutrients. The liquid phase of the soil is the soil solution. It contains dissolved plant nutrients and is the medium for the translocation of plant nutrients from various soil sites towards the plant roots. The gaseous soil component is essential for gas exchange, especially for the supply of plant roots with oxygen and for the release of CO2 from the soil medium into the atmosphere. For the description, comparison, and assessment of soils, a grouping according to general criteria is indispensable. There are two main grouping systems for soils: 1) soil classes or soil texture and 2) soil types. Textural classes are defined according to the particle size of soils. Soil types relate to the parent material of soils, to the pedological genesis, and to typical properties evident

PLANT NUTRITION

in the soil profile i.e., the horizontal layers of soils, called soil horizons.

Soil Classes. Soil particle sizes as a main characteristic of soil classes are grouped into four major groups as shown in Table 6. The major groups (sand, silt, and clay) are subdivided into coarse, medium, fine. Designation of the soil texture (soil class) depends on the percentage proportions of the sand, silt, and clay fraction in the total fine earth, which is sand + silt + clay. Soils in which the sand fraction dominates are termed sandy soils, soils consisting mainly of silt and clay are silty clays, and soils which contain all three fractions in more or less equal amounts are called loams. In the German terminology, abbreviations for the fractions are used (S = sand, U = silt, T = clay, L = loam). For example, if the major fraction is silt (U) and the next sand (S), the abbreviation is sU = sandy silt. Figure 1 shows the designations of the various soil classes according to the percentage proportion of the three main particle fractions. In the farmer’s practice, sandy soils are called light soils, soils rich in clay heavy soils. This distinction relates to the force required to work (plough, cultivate) a soil. Soils rich in clay, but also silty soils, tend to compaction when dried and hence are heavy to work. Although the grouping according to particle size is based on a physical factor, particle size is also associated with the chemical properties. This can be seen from

Table 6. Particle Size of Soil Fractions Relating to Soil Texture Diameter, mm

Designation

Abbreviation

>2

Pebbles, gravels

0.06–2

Sand

S

0.002–0.06

Silt

U

Mg2+ > K+ > Na+ At equilibrium, cation-exchange reactions are a helpful tool for predicting the distribution of ions between the adsorbed and solution phases of the soil as the amounts of cations present are changed. When a soil saturated with potassium is placed in a NaCl solution, the following equilibration occurs: − Ksoil + NaCl −

−− − − Nasoil + KCl The exchange equation for this reaction is [Na](K) = k1 [K](Na) Brackets refer to ions on the exchange site and parenthesis to the activity of ions in the solution. Since the proportionate strength of adsorption of the ions varies with the exchange site, values for k1 differ for different exchange materials. The divalent/monovalent system, which almost represents the situation in the soil, with K+ , Ca2+ , and Mg2+ as the dominant exchangeable cations, is more complex. The following equation, developed by GAPON (39), is widely used to describe monovalent/divalent exchange:

Anion Exchange. Soil particles may also adsorb anions. The adsorption occurs at the OH groups of aluminum and iron oxides as well as of some clay minerals. One may distinguish between a nonspecific adsorption and a specific anion adsorption. The nonspecific anion (A− ) adsorption originates from protonated hydroxylic groups. H

[K](Ca)1/2 = k1 [Ca](K)

M−O

Cation exchange capacity ∗ Specific weight, kg/L

cmol/kg

1.5 1.5 1.5 0.3

3 15 30 75

Sandy soil Loam Clay soil Organic soil

cmol/L 4.5 22.5 45.0 22.5

cmol = centimole.

Table 8. Cation Exchange Capacity and Inner and Outer Surfaces of Some Soil Colloids

Kaolinite Illite Smectite Humic acids

 A−

Protonation depends on soil pH and is particularly high under acid conditions. Hence nonspecific anion adsorption only plays a role in acid soils. The specific anion adsorption is a ligand exchange. This is, for example, the case for phosphate. In step 1 (shown

Table 7. Cation Exchange Capacity Based on Soil Weight and Soil Volume as Well as the Specific Weight of Some Soil Classes

∗

+

H

Cation Exchange Capacity (CEC) is defined as the quantity of cation equivalents adsorbed per unit soil or clay mineral. In Table 7 the exchange capacities of some soil classes are shown. The exchange capacity of the organic soil appears high if it is based on unit weight of soil. A more realistic picture is obtained, however, when the

Soil class

1239

Total surface, m2 /g

Inner surface, %

Cation exchange capacity, mol/kg

20 100 800 800

0 0 90 0

10 30 100 200

1240

PLANT NUTRITION

on the next page) H2 PO4 − replaces OH− , resulting in a mononuclear bond between the phosphate and the iron oxide. In step 2, a further deprotonation of the phosphate occurs, followed by a second ligand exchange (step 3) to form a binuclear bond between the surface of the iron oxide and the phosphate.

Fe

−

OH

O

+

O Fe

OH

OH−

OH P

HO

O

Fe

OH HO

O

1

O

Fe

P

H+

Fe

O

O

O P

Fe

O

OH

O

O

OH P

OH −O

Fe

OH−

O

2

Fe

3

OH

O

The final structure is supposed to be very stable, and the phosphate so bound is hardly available to plant roots. This reaction sequence explains why anion (phosphate) adsorption is promoted under low pH conditions. In mineral soils with pH arsenate > selenite = molybdate > sulfate = fluoride > chloride > nitrate Borate and silicate may also be adsorbed, but only at high pH. Under these conditions, boric acid and silicic acid may form anions according to the following equations: H3 BO3 + H2 O −−−→ B(OH)4 − + H+ −

+

H2 SiO3 + H2 O −−−→ H3 SiO4 + H

This is why in neutral to alkaline soils boron can be strongly adsorbed (fixed) by soil particles, which may lead to boron deficiency in plants. The formation of a silicate anion can improve phosphate availability since H3 SiO4 − and phosphates compete for the same ligands at anionadsorbing surfaces.

Soil pH, Buffer Power, and Liming Proton Concentration (pH) is of vital importance for all living organisms and also has an impact on soils and soil constituents. High H+ concentrations (pHxx mg/kg) will determine the toxicology classification; so it is to the benefit of the registrant to test the highest value feasible if the new material is believed to be nontoxic. A way to avoid substantial effort with this assay is to first perform a limit test. A limit test involves testing five males and five females at a dosing level of at least 5000 mg/kg and observing clinical signs/mortality for 14 days. If no toxicity is noted, the test is complete. The testing duration is typically 14 days, and a detailed FOB (functional observational battery) is required. Observations should include evaluation of skin and fur, eyes and mucous membrane, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, changes in activity, gait, reactivity to sensory

1503

stimuli, or bizarre behavior (i.e., self-mutilation). These observations should be carefully and accurately reported. They may be used in the future to determine a toxic endpoint and have a profound effect on the compound’s risk assessment and subsequent use pattern as well as lead to additional testing requirements. At the end of 14 days, surviving animals are sacrificed and a gross necropsy performed. Further analysis of visibly damaged organs is required. Additionally, any animals that expire prior to the 14th day are also autopsied. Acute Dermal Toxicity (OPPTS Guideline 870.1200, PAG 81-2). Required for Experimental-Use Permit Similar to the acute oral toxicity guidelines above, the dermal toxicity test involves testing rabbits and rats to determine an LD50 . However, as noted in the title of the study, the test substance is applied to the skin of the animal rather than orally introduced. Basically, the fur of the animal is clipped from the dorsal trunk of the test animal. The animal may be shaved, but this needs to be done 24 hours prior to test and care taken to avoid abrading the skin. The test substance is applied to 10% of the total animal surface area (from shoulders to hip). The material is kept in contact with the animal for 24 hours using a gauze pad. Afterward effects are observed over the next 14 days. Acute Eye Irritation (OPPTS Guideline 870.240, PAG 81-4). Required for Experimental-Use Permit Material is applied to one eye of one to three rabbits (the other eye serves as a control). Effects are observed for at least 72 hours but not more that 21 days. The 72-hour period is divided into 1-, 24-, 48-, and 72-hour periods to determine any severe acute effects. Evidence of irritation during this period allows for the test to terminate immediately. Otherwise, 7- and 21-day examination points are added. Effects to the eye are graded according to a scale provided in the OPPTS 870.240 guidance. Acute Dermal Irritation (OPPTS Guideline 870.2500, PAG 81-5). Required for Experimental-Use Permit This test, which also utilizes rabbits, involves application of the test substance to patches and subsequently to the animal’s skin. Patches are removed every hour over a 4-hour period. If irritation occurs within this period, then no further testing is required. Otherwise, two additional rabbits are patched for 4 hours; after which, the patch is removed and the animals observed for up to 14 days. Typically, this period would be 30 min, 60 min, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. Like ocular effects, skin reaction is graded and evaluated according to the table provided in the guideline. In addition to the acute test battery, a series of chronic and subchronic tests are required. Again, many are use specific, and below are highlighted the ones typically required for food use pesticides. 90-Day Feeding Toxicity in Rodent (OPPTS Guideline 870.3100, PAG 82-1). Required for Experimental-Use Permit, Although Interim Data May Be Acceptable As suggested in the title, this study tests the effects of a chemical when administered at a sublethal dose over

1504

STANDARD EVALUATION PROCEDURES

a period of 90 days. Rats are dosed by gavage or in feed at three different levels 5 days a week (if by gavage) or 7 days a week (if in feed) over a 90-day interval. The three dose levels should be, going from highest to lowest, a level that results in toxic effects but not fatalities, a dose that produces some symptomology of poisoning, and a dose that produces no evidence of toxicity. Depending on effects found in other toxicology tests at other doses, this latter concentration may become the No Observed Effect Level (NOEL). Consequently, one should always choose the lowest ‘‘safe’’ dose carefully. This study does have a limit test. If the registrant tests their product in rats at 1000 mg/kg and no effect is found, the other two dose levels are not required. Chronic Toxicity (Feeding) Study (OPPTS Guideline 870.4100, PAG Guideline 83-1). Required for Experimental-Use Permit, Although Interim Data May Be Acceptable Originally titled Chronic Feeding Study, it was renamed when the guidelines were harmonized with the European requirements. The route of exposure may be oral, inhalation, or dermal depending on the most likely route of human exposure, hence the reason for the name change in the harmonized guidelines. As with other toxicology studies, this study also has a limit test component. Basically if no effect is found at 1000 mg/kg body weight/day, then the test may be terminated. Otherwise, this is a very expensive and animal-intensive study. Forty rats (20 males and 20 females) for each of three dose levels are required. Also, this study is required for two different species of animals, preferably rat and dog. For dog, eight animals (four male and four female) are required at each dose level. Animals are fed by diet, capsule, or gavage for 1 year. Additionally, another group of animals fed at the highest dose level are monitored for an additional 28 days to examine any delayed effects. Following the 12-month period, the animals should be sacrificed and examined by autopsy. The purpose of the study is to determine the NOEL dosage for chronic exposure. The rat chronic toxicity study may be combined with the rat oncogenicity study described below for significant cost savings. Carcinogenicity (Oncogenicity) Study (OPPTS 870.4200, PAG 83-2) This study (also renamed during harmonization) is another long, animal-intensive chronic study. Application is oral, dermal, or inhalation, depending on the most likely route of human exposure. It also requires two animal species; however, both may be rodents; hence, rats and mice are the suggested species. The study requires a minimum of three dose levels and 100 animals (50 male and 50 female) for each dose level. The high dose must produce toxicity [defined as maximum tolerated dose (MTD)]. Animals are subjected daily to dosing for a period of 24 months and examined for the appearance of a myriad of organ carcinogenetic indicators. There is no limit test for this study. The results may be interpreted in conjunction with the mutagenicity studies.

Reproduction and Fertility Effects (Teratogenicity and Reproduction—Three Separate Studies) (OPPTS 870.3800, PAG 83-4). Required for Experimental-Use Permit, Although Interim Data Are Acceptable Also renamed when harmonized, the reproduction and developmental toxicity studies (rat and rabbit with dosing during gestation and subsequent fetal evaluation) measure the potential of a compound to disrupt reproduction or normal development. Since the passage of the Food Quality Protection Act in 1996 by the United States Congress, these tests have taken on additional importance as the EPA must consider the results and determine if a 10× safety factor is necessary for chemicals that pose a risk to childrens’ development. In a two-generation animal study, typically three doses of the test substance are given to parents and two generations of offspring daily by gavage or diet, two litter per generation. Determination of effects on time course and proper development of offspring, as well as disruption of sexual cycles and fertility, is determined. Metabolism and Pharmacokinetics (OPPTS 870.7485, PAG 85-1) This study provides the basic absorption, distribution, metabolism, and excretion (ADME) pattern for a new product in an animal test model (typically the rat). The test substance is radiolabeled technical material, and complete mass balance is required. This includes monitoring and collection of urine, feces, and expelled air as well as determination of tissue distribution for the material either as dosed substance or as a metabolite. Identification of all metabolites and conjugates comprising more than 5% of the total applied radioactivity is also required. Dosing is typically a single dose oral intubation. Mutagenicity Studies. Required for Experimental Use Permit, Although Interim Data Are Acceptable In addition to the above direct animal effects, a battery of genotoxicity determinations are required. These studies measure a chemicals’ ability to cause gene mutations or structural chromosomal aberrations. Two studies commonly used to evaluate these questions are OPPTS 870.5100 Bacterial Reverse Mutation Test and OPPTS 870.5375 In Vitro Mammalian Chromosome Aberration Test (4). These are not expensive or involved tests, and the reader is referred to the above guidelines for information. Depending on the results of these tests, other genetic studies may be required. This is a compound-specific issue that is best decided by direct communication with the EPA. Other studies that are likely to be required are Acute and Subchronic Neurotoxicology (OPPTS 870.6200), Developmental Neurotoxicology (OPPTS 870.6300) and Immunotoxicology (OPPTS 870.7800). Registrants need to stay aware of the status of these requirements as well as critically evaluate their own compound biochemistry to anticipate a need for the study and avoid a registration delay during its completion. Finally, with regard to toxicology studies, the EPA has specific flagging criteria. These criteria are listed in 40CFR158.34 and the reader is advised to consult


them with regard to effects determined in carcinogenicity (occurrence of neoplasms), subchronic feeding, teratogenicity (birth defects), neurotoxicity (apparently delayed neurotoxic effects), chronic and subchronic feeding, and reproduction studies. ENVIRONMENTAL FATE STUDIES The environmental fate battery of required studies determine the probable path and terminal points for a chemical in the external world. The results of these tests should not be taken lightly. They will dictate possible additional fate studies (including arduous, expensive, and time-consuming groundwater monitoring studies) as well as what parts of the country a chemical may not be used. As critical as these studies are, they are some of the most trying to conduct. The original guidelines have not been revised and EPA issues or changes have to be extracted through memos or precedents of other studies. The original Pesticide Assessment Guidelines do not provide any emphasis in the procedures. Consequently, the user is unable to determine if any procedural points are more important than others, and several are. The following sections provide a brief description of each key test and where the information of proper conduct can be found. Essential Documents Before attempting to conduct an environmental fate study, two pieces of information (in addition to the listed guideline) should be reviewed. 1. The FIFRA section 3 accelerated review guidelines (5). This checklist style document allows the Registrant to quickly determine of the proposed protocol contains all the key study elements: items such as temperature, radiolabel purity, light or dark conditions, and so on. These items are easily overlooked and can quickly result in study rejection. 2. The EFATE rejection rate guidance (6,7). This book allows the user to determine if study deviations or inadequacies are likely to lead to a study rejection. Unlike the other guidelines that have been revised to reflect the very important issues covered in the rejection rate guidance, no such revisions have occurred with these tests. Consequently, the environmental fate study results are a common cause of problems during the registration process. The required environmental fate tests are adsorption/desorption, aerobic soil metabolism, anaerobic soil metabolism, hydrolysis, and aquatic and soil photolysis. In some cases, data on relevant metabolites (those found at greater than 10% of the applied radioactivity) may be required. Aerobic Soil Metabolism (PAG 162-1) Required for Experimental-Use Permit The aerobic soil metabolism test is a difficult and expensive study. The registrant must utilize a soil type typical of that

1505

expected to be the most common the chemical is applied to. The test substance (as radiolabeled technical material) is applied to soil that has been maintained under conditions of aerobicity for at least 30 days. The soils are stored in the dark and samples taken at specific increments for the next 365 days. In this study, maintaining mass balance of 90% or greater is crucial and critical. The laboratory needs to collect all radiolabeled CO2 in KOH scrubbers and determine post-extracted solids (PES) at each interval. The laboratory needs to also characterize every metabolite with a concentration greater than 0.01 ppm or 10% of applied parent material. Levels of high PES (greater than 10% of applied radioactivity) should also be characterized. Failure to not well-characterize aerobic soil metabolites will likely lead to study rejection. Registrants should not cut corners with this study, especially in the characterization area. Adsorption/Desorption (Column Leaching, PAG 163-1). Required for Experimental-Use Permit This study is conducted on five soils, one of which was utilized in the aerobic soil metabolism study. The radiolabeled parent compound and its principal metabolites (as previously defined) are equilibrated in a soil/water slurry. After equilibration, the water is removed and the relative amounts of compound are determined in each matrix. Fresh water is then added to the soil and allowed to re-equilibrate. This water is removed, and the process is repeated once more. The data generated are then used to determine the Kd (partition characteristic between the soil and water) and a Koc (the Kd with respect to % organic carbon of test soil). This number is then compared with trigger values for determining a compound’s probability of leaching into groundwater. If such a probability is determined to be high, one or more prospective groundwater studies may be required. These studies are costly, time consuming (typically 1 or more years), and can kill or severely restrict a compound’s use by limiting the types of soils on which it can be used. Anaerobic Soil Metabolism (PAG 162-2). Required for Experimental-Use Permit Unlike the aerobic soil metabolism study that looks at the typical fate of a pesticide after application to soil, the anaerobic soil metabolism study is designed to measure the persistence of the pesticide and the formation of metabolites after flooding or an unusual event in which anaerobic (reduction is favored over oxidation) reactions can occur. Experimentally, the test involves adding radiolabeled material to the soil and allowing it to age aerobically for 30 days (or 1 half-life for parent material, whichever is shorter), then flooding the soil to create anaerobicity, and running the test for an additional 60 days, sampling after 30 and 60 days of anaerobicity. All residues exceeding 10% of applied parent material or 0.01 ppm are identified and quantified. The anaerobic metabolism study is frequently rejected for raising more questions than it answers. Because of its short duration, it produces incomplete metabolite profiles and the aerobic/anaerobic periods tend to lead to poor mass balance recovery. The perspective registrant is well

1506


advised to skip this study altogether and instead conduct an anaerobic aquatic study (PAG 162-3). This study begins with anaerobic conditions and is a 1-year study like the aerobic soil metabolism study. The EPA will accept this study to cover both the anaerobic soil and anaerobic aquatic study requirements. Even if an anaerobic aquatic study is not required at initial registration, typical label expansion usually finds applications near enough to bodies of water to find that the EPA will require this test. Hydrolysis (PAG 161-1). Required for Experimental-Use Permit This study utilizes radiolabeled active ingredient, is carried out in darkness at 25 ◦ C and utilizes buffered solutions of pH 5,7, and 9. The test interval is 30 days, and all metabolites greater than 10% of the initial test substance must be identified. Additionally, the degradation time curve for the parent compound must be plotted and a half-life determined. Photodegradation in Water (PAG 161-2) Although this is the only core photodegradation environmental fate test, typically photodegradation on soil is required for any chemical used in a terrestrial environment. This test (photodegradation in water) involves exposure of radiolabeled test material to environmentally typical radiation (wavelengths of 290 nm and greater) for up to 30 days or 1 half-life of parent, whichever is shorter. The material is dissolved in a buffered solution of a pH determined in the hydrolysis test to be its most stable. Like the hydrolysis study discussed above, the EPA requires determination of half-life, plotting of degradation curve, and identification and quantification of all metabolites that exceed 10% of the original parent concentration. ECOTOXICOLOGY Where the toxicology requirements are directed at measuring a pesticide’s level of hazard to humans, the ecotoxicology section determines the effects on the most susceptible organisms likely to be exposed in the environment. Although harmonized guidance documents are available, they are still very much in draft form and the reader needs to review the same material as listed above for environmental fate (except, of course, directed at ecotoxicology). Unlike environmental fate, however, ecotoxicology is covered under subdivision E (8) of the Pesticide Assessment Guidelines rather than Subdivision N (9). Four tests are required of all outdoor use pesticides, and other tests are likely to be required. These mostly longer term subchronic/chronic studies include avian reproduction (two species), invertebrate lifecycle (one, possibly two species), estuarine/marine tests (three species), nontarget insects (one, possibly two tests on bees), nontarget aquatic plants (one to five species), and nontarget terrestrial plants (ten species if the product is a herbicide; less testing may be needed for other types of products). Although most of the above are listed in 40CFR158 (1) as ‘‘CR,’’ they are in effect required because exceptions to the above battery are very rare. In addition,

testing of key degradates (degradates of concern) may also be required. The four certain core studies are summarized below, and the reader is directed to 40CFR 158.490 (1) for a description of the others. Avian Oral LD50 (OPPTS 850.2100, PAG 71-1). Required for Experimental-Use Permit This study, which utilizes either the mallard duck or bobwhite quail, determines the LD50 for the proposed new pesticide. Like the oral rat LD50 test, this study is first conducted by rangefinding with a finding of no toxicity at 2000 mg/kg (administered by oral gavage) sufficient to conclude the study. In other words, the registrant may conduct a study with only ten birds (five of each sex), and if no mortality is found after 14 days, then the test is concluded. Otherwise, a rangefinding study with doses typically at 2, 20, 200, and 2000 mg/kg should be utilized to determine the dose range. Once this range is determined, a series of doses within about 60% of each other should be utilized as described in the OPPTS guidelines. The test is run for 14 days using ten birds (five of each sex) for each dose level. Although pathology is not typically required, gross necropsies are required on all mortalities and at least 50% of the survivors. Avian Dietary LD50 (OPPTS 850.2200, PAG 71-2). Required for Experimental-Use Permit This study utilizes juvenile birds (10- to 14-day olds). Test material is administered in the feed for a 5-day period; two species (waterfowl and gamebird) are required with analytical support (dose verification, stability, and homogeneity). The guideline study consists of 5 days on treated diet followed by a 3-day (minimum) observation period. [Note: Test duration of 21 days discussed in OPPTS 850.2200 (4) is NOT acceptable.] For this study, a maximum dose of 5000 ppm (w/w to food) constitutes a noeffect level and the registrant may conclude this test after completion of rangefinding if this dose shows no toxicity. Freshwater Fish Acute LC50 (OPPTS 850.1075, PAG 72-1) Required for Experimental-Use Permit This study provides data on potential harm to freshwater fish. The fish acute study is actually two tests, requiring a warmwater (bluegill sunfish) and a coldwater (rainbow trout) species for testing. The test is conducted by adding test material in a static tank or a flow-through system. Seven to 10 fish per dose level are used for the test and monitored for mortality at 24, 48, 72, and 96 hours. The most common test design is the flow-through system (for all aquatics, not just acute fish testing). As in previous ecotoxicology studies, analytical support is required for acceptability. The limit test consists of 30 fish at >100 ppm (measured) or at/above the limit of solubility. If the new pesticide is nontoxic at an active ingredient concentration of 100 ppm, then the test and study can be terminated. If the definitive test must be run, product concentrations at five levels are utilized with results analyzed for determination of an LC50 level. Like the other animal studies, this one also contains a rangefinding portion. However, in general, and in contrast to avian testing,


aquatic testing usually involves two or more rangefinding tests before conducting a definitive test. Acute LC50 Freshwater Invertebrates (OPPTS 850-1010, PAG 72-2). Required for Experimental-Use Permit The final required ecotoxicology test determines the effects of a new product on freshwater invertebrates. Daphnids (Daphnia magna or Daphnia pulex) are typically used as the marker organism because they are a highly sensitive indicator of pond toxicity. As with the aforementioned fish acute studies, the limit test consists of 30 daphnids at >100 ppm (measured) of product or at/above the limit of solubility. If the new pesticide is nontoxic at an active ingredient concentration of 100 ppm, then the test and study can be terminated. For the definitive study, organisms are exposed to a rangefinding dilution series of test material (1, 10, 100 mg/L, etc.), after which a geometric series of dilutions is utilized. The results of the test are EC50 values for 24 and 48 hours. This test should be monitored very carefully to ensure no other effects (i.e., oxygen deprivation, solvent effects, etc.) are the cause of mortality. RESIDUE CHEMISTRY The last step in registration is the determination of what happens to a chemical when it is applied to a crop. The residue chemistry test guidelines are based solely on the proposed crops (or surfaces) on which application of the test material is planned. With regard to a crop, for example, cotton, one must determine the metabolism of the pesticide in cotton and then design the residue chemistry method to analyze unlabeled (‘‘cold’’) test material (active ingredient) and metabolites at levels of 0.01 ppm and higher. All residue chemistry studies utilize formulated material and application to test plots of crops. In addition, processing studies as well as rotational crop studies may also be required. The degree of variability in the residue trial programs make a general discussion of them beyond the scope of this work; however (and fortunately), the OPPTS 860 guidelines for residue chemistry are extremely well written and detailed (4). Unfortunately, two very large types of studies are not considered in these guidelines. They are the field dissipation studies (they are classified, unfortunately, as environmental fate studies). These studies determine the metabolism and movement of the pesticide when applied as a formulated product. One study (terrestrial field dissipation) deals with compounds applied to soilbased crops (i.e., corn) and the other to aquatic crops (i.e., rice) or applications near bodies of water (hence, aquatic field dissipation). These studies are covered by PAG 164-1 (field) and 164-2 (aquatic). The results of these studies are compared with soil and aquatic metabolism laboratory studies. Care must be taken to apply enough material to allow a metabolic profile to be determined. Failure to do so (i.e., loss of parent without detection of metabolites) may delay a registration. These are yearlong studies; so a considerable amount of time is at stake. Comparing a scaled-up field study with a small

1507

laboratory study is challenging in its own right, and it is not unusual to have difficulty even correlating zero time residues to application rate. This study should be monitored on an ongoing basis to be sure results such as parent loss, metabolite formation, and leaching are predictable. So difficult and perplexing are these studies that recent attempts by the EPA to revise the guidelines have not been successful. LABORATORY CONSIDERATIONS All field preparation and protocol work are worthless without proper laboratory analysis. According to the EPA, proper laboratory chemistry utilizes Good Laboratory Practices. Due to data fraud problems with some laboratories, the EPA promulgated under the Code of Federal Register a series of administrative and scientific requirements for generating data for required registration studies. These requirements are known as GLPs and are covered under 40CFR part160 (1). The reader is advised to review the guidelines specified in this section prior to conducting a required study. Although laboratories contracted to perform the aforementioned projects may well profess to being GLPcompliant, it is ultimately the registrant who may well suffer if the chosen facility is actually not in compliance. SUMMARY OF TEST PROCEDURES Health Effects (Toxicology) and Residue Chemistry test procedures have been revised and are available on the Internet at the following address: HTTP://www.epa.gov/OPPTS Harmonized/. (Note: This is case sensitive.) These guidelines are a compilation of the original Pesticide Assessment Guidelines, FIFRA88 checklist for acceptability guidelines and rejection rate guidelines and provide the registrant a complete guide for designing protocols for the required studies. The reader is still advised to read the various rejection rate guidance documents when evaluating the study data for report writing. Harmonized ecological effects guidelines are available. However, they are still in draft and the reader is advised to refer to the original Pesticide Assessment Guidelines for designing test protocols. Unfortunately the case is not as simple for environmental fate. The guidelines have not been revised, and the registrant must use the aforementioned documents (Pesticide Assessment Guidelines, FIFRA88 checklist for acceptability guidelines and rejection rate guidelines) to even design the protocol. These studies require radiolabel material and can be very expensive. A general rule of thumb for acceptability of environmental fate studies is mass balance. Failure to maintain mass balance will almost certainly result in study rejection. WAIVERS A final comment on study requirements is the submission of waivers. In general, a waiver for a study can be

1508

STANDARD SOLUTION, SECONDARY

requested if the product is not going to come into contact with the test system. Although waivers for the studies mentioned in this article are unlikely as they are core requirements, waivers for additional requirements (such as an aquatic field dissipation study) may be submitted if the registrant can prove the conditions that prompted the EPA to request the study are unlikely to occur. Some studies may not be required at all for certain use patterns, such as an aerobic soil metabolism study for a chemical proposed for indoor use only.

STANDARD SOLUTION, SECONDARY Standard prepared by dilution of an aliquot of a primary standard solution with a known volume of solvent, or by subsequent serial dilutions; or a standard solution measured by reference to a primary standard solution.

STARK-EINSTEIN LAW

CONCLUSION

This law states that only one molecule is activated to an excited state for each quantum of light absorbed. See Photolysis.

Registration of a new pesticide is a costly, time-consuming process. However, when one considers the importance of growing food, the cost and time are less of an issue. Help is always available, and prospective registrants should understand both the cost and timeframes involved in the process before undertaking the project.

STICKER

BIBLIOGRAPHY

A formulant that increases the adhesiveness of a formulation applied to a surface (1). See also Wetting Agent. A material that increases the retention of a product applied to a surface (CIPAC)

1. Code of Federal Regulations #40 Parts 150 to 189. Available at: http://www.epa.gov/pesticides/cfr.htm (Revised as of July 1, 1998).

BIBLIOGRAPHY

2. United States Environment Protection Agency—Pesticides and Toxic Substances. General Information on Applying for Registration of Pesticides in the United States—June 1989. Available at: http://www.epa.gov/pesticides.

1. G. S. Hartley and L. V. Graham Bryce, Physical Principles of Pesticide Behaviour, Vol. 2, Academic Press, New York, 1980, pp. 809–810.

3. Pesticide Assessment Guidelines Subdivision I: Experimental Use Permits, United States Environmental Protection Agency Office of Pesticides and Toxic Substances, 1982. 4. EPA-OPPTS Harmonized Testing Guidelines. Series 870 Health Effects, Series 850 Ecological Effects, Series 860 Residue Chemistry. Available at: http://www.epa.gov/OPPTS Harmonized/ (Note: This is case sensitive.) August 1998. 5. FIFRA Accelerated Reregistration Phase 3 Technical Guidance, United States Environmental Protection Agency Office of Pesticide and Toxic Substances, December 24, 1989. 6. Pesticide Reregistration Rejection Rate Analysis, Summary Report, United States Environmental Protection Agency Office of Pesticide and Toxic Substances, February 1995. 7. Pesticide Reregistration Rejection Rate Analysis, Environmental Fate, United States Environmental Protection Agency Office of Pesticide and Toxic Substances, September 1993. 8. Pesticide Assessment Guidelines, Subdivision E. Hazard Evaluation: Wildlife and Aquatic Organisms, United States Environmental Protection Agency Office of Pesticide and Toxic Substances, December 1986. 9. Pesticide Assessment Guidelines, Subdivision N. Chemistry: Environmental Fate, United States Environmental Protection Agency Office of Pesticide and Toxic Substances, October 1982.

STANDARD SOLUTION, PRIMARY Standard prepared by dissolving a weighed amount of an analytical standard pesticide in a known volume of solvent.

STORAGE STABILITY SUSHIL K. KHETAN New Delhi, India

Modern agrochemical formulations are usually complex mixtures of different organic and inorganic compounds. Over a period of time, these may undergo chemical or physical changes. The stability of an agrochemical product depends on the intrinsic stability of the active ingredient(s), the formulations, the packaging, and, in particular, the storage conditions (1). The storage stability or shelf life is defined as the length of time the product can be stored under normal local conditions, with changes in its properties and characteristics varying within predetermined, acceptable limits and remaining in a satisfactory and safe package and still giving the claimed biological activity (1). The user, at the end of distribution chain, has great interest in the product’s fitness for use. When users buy a package of pesticide, they expect that it will perform in the way they have been led to expect. This expectation comes from either the product labeling, advertising literature, or by the extension services of a local agricultural university. Also, registration authorities and other public organizations seek a manufacturer’s guarantee that a product will still comply with the specification during its specified time of storage. At the user’s end, the shelf life of an agrochemical product is measured by its correct

STORAGE STABILITY

biological performance. This performance should not be less than expected even at the end of declared shelf life, although some alteration in chemical and physical properties would be expected. How much change in physical and chemical properties should be accepted at which the product would reach the end of its shelf life? This is reflected in many of the FAO specifications, where the heat stability of the product is accepted satisfactory, provided the assay after the test is still 90% of its original value (2). These specifications are based on a GIFAP (Now GCPF, Global Crop Protection Federation) proposal recommending acceptance of a decline of active ingredient up to 10% during storage (3). In this case, there is more than one active ingredient the one degrading to 90% of its nominal value at the earliest determines the shelf life. It is acceptable if the manufacturer uses up to 10% of nominal content as an overage to compensate for the degradation of the active ingredient. On the other hand, if some of the physical properties are more critical than the active ingredient stability, e.g., thickening of a flowable, shelf life will be based on establishing appropriate limits for properties, which can be determined by laboratory test methods. A variety of factors impact stability of agrochemicals. Most substances in solid form that have been protected from extreme heat, humidity, and natural and artificial light are stable for long periods of time. The expiration date of powdered products is often several years after their production. Extensive studies have been carried out on stability of labile products in solution under varying conditions of ionic strength, pH, and temperature. Degradation of active ingredients in liquid formulations occurs through several pathways such as hydrolysis, oxidation, photolysis, and racemization. In many cases, degradation of dissolved agrochemicals is caused by the active role of solvent in the degradation reaction. Temperature can dramatically influence the rates of degradation and, thus, the shelf-lives of agrochemicals in solution. In liquid products, such as emulsifiable concentrates, which are usually true solutions and thermodynamically stable, the rate of degradation can increase many-fold at high temperatures. On the other hand, low temperatures may have a far more harmful effect than high temperatures on emulsions and suspension concentrates. When the outer aqueous phase begins to freeze, the physical properties in the remaining liquid between the water crystals change so enormously that very often irreversible alterations occur. Such alterations may be intensified by frequent changes of temperature.

STABILITY DURING TRANSPORTATION AND STORAGE Between production and application, the product has to undergo periods of storage and transportation, from formulator’s warehouse to area distributor and subsequently to dealers shops, under varied conditions. During transportation, alteration in relevant product properties, caused by environmental factors such as elevated temperature, humidity, vibration, and shock can be expected. The storage of the product would also bring down the acceptable

1509

quality due to deterioration. For desirable storage stability, only realistic general average conditions need be considered. There are typical storage conditions such as corrugated tin shed or similar building in tropical areas, whereas in more temperate climates, concrete buildings may be used. Products may sometimes be stored in the full heat of summer sun or above the snow line; these conditions have to be considered abnormal. PRODUCT–PACKAGE COMPATIBILITY The user first comes into contact with agrochemicals in their packed form. If the package is poor and in degraded form, then it is obviously a danger to the one handling it or to the contents. Shelf life must inevitably be associated with the packaging, and thus, any shelf life testing must be carried out on the relevant packaging. The agrochemical products belong to different chemical types and have varying properties. The essential prerequisite is that there should be no interaction between the agrochemical product and its container. The use of suitable packaging is also important for better storage stability of the products. For example, water-based formulations in mild steel container lead to rusting and contamination if not degradation of the product. Similarly, emulsifiable concentrates in lower density polyethylene bottles suffer from seepage through the polyethylene. Water-sensitive products in permeable bags can be degraded. On the other hand, use of drums instead of sacks for packing of powders will minimize pressure load resulting from stacking (1). Stability tests can lead to important conclusions on packaging configuration such as adequate protection of agrochemical products from climatic influences, catalytic reaction between packaging material and the formulation, and functioning of package normally, i.e., stress cracking of the container or difficult cap opening. ACCELERATED STABILITY TESTING Accelerated testing is widely used for the prediction of storage stability and quality, and for the estimation of shelf-lives and ‘‘safe’’ storage temperatures of labile products. Accelerated stability testing is used in many branches of industry. For example, the weathering of white paint films is established by exposing them to ultraviolet radiation of increasing intensity and measuring the degree of yellowing as a function of time. Similarly, the useful life of a plastic shower curtain can be estimated by monitoring its accelerated embrittlement resulting from exposure to high temperatures and humidities—conditions that promote the leaching and evaporation of the plasticizer (4). In industries such as food, dyestuffs, pharmaceuticals, and agrochemicals, products are stressed by testing at high temperatures. Temperature is the most important external factor that influences storage stability. Elevated temperatures enhance the rate of degradation of the active ingredient and can also lead to irreversible changes in physical properties. FAO guidelines specify a heat stability test for all products at 54 ◦ C for 14 days. The test provides registration authorities the possibility to verify shelf-life

1510

STORAGE STABILITY

statements of manufacturers in a short time. The active ingredient declines in the heat stability test should be less than 2% and 1%, respectively, for expecting 2 years shelf life at 20 ◦ C and 30 ◦ C. Similarly, if the physical properties of the product do not change significantly in heat stability test, it would be safe to assume that the product would be physically stable at ambient temperature. On the other hand, there are no laws to describe the temperature dependence of physical properties and it is not possible to predict the changes that may occur to the product for longer storage periods (5). The FAO specification on stability also raises some ambiguity, for example: 1. It is not clear that if the product passes the heat stability test, it would mean that it is stable for its declared shelf life, usually 2 years. 2. If a product is tested at the end of shelf life, the heat stability test would still be valid. The FAO guidelines have not found formal acceptance in the United States, and the active ingredient content is expected to remain within the certified limits throughout the shelf life of the product. There is no substitute for real-time stability studies. As the manufacturer cannot follow on a real-time basis all the potential transportation, storage, and environmental conditions that a product may be subjected to, tests need to be devised that will simulate such conditions as closely as possible. These tests are most valuable for the manufacturer, as they give information to make proper changes in product designs to prevent or, at best, defer failures. Accelerated tests, when possible, can be useful to predict the shelf life of the product in a relatively short time. The basic problem is the correlation of performance in the accelerated test with that in the real world. The most commonly used accelerated test is stability at elevated temperature. The most common approach is the accelerated degradation test at the temperature ranges based on realistic local average conditions. The ambient temperature should be the normal average encountered at average storage depots, warehouse, or dealer stores and accelerated temperatures adjusted accordingly. Another important point is to ensure that the appropriate packaging is used, as this is an integral part of the actual product being sold. It is often very useful to include tests in glass, as these will show whether any degradation taking place is inherent in the formulation or is a result of packaging. Several samples from a homogeneous batch of the agrochemical product are packed in final proposed packaging. If the packaging size is very large, then a reduced size simulation should be used. Most manufacturers use two or more temperatures in accelerated storage studies. The higher temperature is 54 ◦ C, as referred to in FAO guidelines, and the lower temperature is in the range of 30 ◦ C to 40 ◦ C. Typical time intervals for removing samples for testing are 4 weeks, 8 weeks, 3 months, 6 months, 12 months, and 24 months. Table 1 shows storage conditions and time intervals recommended for both accelerated and real-time studies (6). The test periods are arranged to avoid unnecessary time being wasted. If the sample degrades at one of the higher temperatures, then the decision regarding the lower temperature can be taken. However, every

Table 1. Storage Conditions and Test Intervals for Stability Studies Time Table Temperature ◦ C

Relative Humidity (%)

−10/0 Ambient 30 40 54

85 85 85

1

2

4

8

O

O O O O X

X O X O X

Weeks 16 24 X X X x

O O O O

52

104

O O O

O O O

X—Visual inspection. O—Analysis.

recommended test at every test point may not be necessary. Once the stability profile of the product has been established and stability parameters are known, one can be selective in the choice of tests to be carried out. The test method selected should be stability indicating and capable of providing accurate initial value. Also, all parameters that might possibly be affected by change in quality with time/storage conditions must be determined as an initial value at t = 0. Based on the decrease of the active ingredient content at elevated temperatures, a prediction of the shelf life at ambient temperature can be made. The relative degradation rates are used to fit the Arrhenius equation (relating degradation rate to temperature). The results are then extrapolated to predict stability under ‘‘normal’’ storage conditions. The results of stability studies can be used for fixing specification limits and for testing the influence of a changed formulation. INFLUENCE OF TEMPERATURE ON DEGRADATION The influence of temperature on the rate of chemical reactions is known, with Van’t Hoff’s law stating that a temperature rise of 10 ◦ C increases the rate of reaction by two- to four-fold (7). It thus follows that, with regard to active ingredient degradation, the shelf life of agrochemicals is shorter in warm climates than it is in the temperate zones. This empirical rule is useful for general prediction; it is necessary to conduct a planned schedule of accelerated tests to ascertain temperature dependency of the chemical changes in the product under evaluation. The influence of temperature on the rate of degradation obeys the Arrhenius equation (8): k = Ae−(E/RT) where k is the reaction rate constant, A is Arrhenius constant or frequency factor, E is the activation energy (cal mole−1 ), R is the gas constant (1.987 cal mole−1 0 K−1 ), and T is the absolute temperature (0 C + 273). This equation is often expressed logarithmically for linear presentation of data as follows: Log k = − E/2.303 RT + log A where log A is constant.

STORAGE STABILITY

Given the rates obtained at several elevated temperatures, it is possible to plot the logarithms of the rate constants against the reciprocal of absolute temperatures, giving a straight line with a slope of − E/2.303R. This line can be extrapolated to evaluate k25 o . Knowing the k25 o , one can calculate the time to reach 90% of the initial content (t90 ) or stability of the product at room temperature without conducting prolonged experiments. t90 can be obtained from the kinetic expression, −

dC = kC dt

or,

the range of 50 to 90 kJ/mole (12 to 22 kcal/mole). This range corresponds to reaction rate changes by a factor of 2 to 3.4, when the temperature rises from, say 20 ◦ C to 30 ◦ C. In case of simple degradation, accelerated test data can be used with a high degree of confidence, and the results can be extrapolated to provide an accurate stability estimate under ‘‘real’’ storage conditions. This procedure can be made more rigorous by measurements at two temperatures; E can then be calculated with a high degree of confidence, and back extrapolation can be performed. Given that shelf life or t90 = 0.105/k at any given temperature, Q10 can be defined in terms of t90 rather than rate constants. As follows:

dC = k · dt − C C = Co e(−kt) where k is rate constant, Co is initial concentration, and C is the remaining concentration after time t. The expression is valid for first-order reaction; i.e., rate of decomposition is dependent on the concentration of active ingredient only. Solution of an agrochemical, as in emulsifiable concentrate formulation, follows a first-order decomposition. Logarithmically, it can be expressed as Co 2.303 Co 1 = log t = ln k C k C When accepting a 10% decline in nominal content as a criterion for shelf life, the time (t90 ), during which active ingredient content decreases to 90% of the initial content, would be t90 =

0.104 2.303 100 = log o k25 90 k25 o

On the other hand, the same agrochemical in suspension would follow a zero-order reaction. Such accelerated stability testing enables the agrochemical formulation chemist to determine stability at elevated temperatures rapidly and to extrapolate this to normal storage temperatures with a great saving of time. However, the accelerated storage test at elevated temperatures cannot completely substitute for long-term real-time tests under practical conditions (8).

Q10 METHOD FOR ESTIMATION OF THERMAL STABILITY The Q10 method can be used for translation of accelerated storage data into real-time storage stability (9). This is obtained empirically, although the procedure is loosely based on the Arrhenius kinetic rate law. The Q10 factor is functionally related to the activation energy E as follows: Q10 =

(kT+10 ) kT = e−{( E/R){1/(T+10)−1/T}}

where kT is the rate constant at temperature T and kT+10 is the rate constant at a temperature 10 ◦ C higher. According to this method, a 10◦ rise in temperature enhances a given rate process by some known factor, frequently 2 to 3.4. It is known that in most cases, the activation energy lies in

1511

Q10 =

tT90 tT+10 90

where tT90 is shelf life at temperature T, and tT+10 is shelf 90 life at temperature 10 ◦ C higher than T. This approach has been further generalized for cases in which the prediction of shelf life is desirable for changes in temperature ( T) greater than 10 ◦ C: Q T = Q10 ( T/10) =

tT tT+ T

where Q T is an average value of Q10 over the temperature interval, T + T. An alternative practical expression would be: t90 at TUnknown = t90 at

TKnown Q10 ( T/10)

where TKnown and TUnknown are the temperatures in ◦ C of known and unknown t90 and T is the difference between TUnknown and TKnown . From this equation follows the general case: tU =

Q10

tK ( T/10)

where tU and tK are unknown and known exposure duration at temperature U and K, respectively. This equation enables estimation of the equivalent duration at two or more temperatures that have the same effect on product shelf life. REAL-TIME TESTING In accelerated storage tests, extrapolations from higher to lower temperatures, and from shorter to longer storage periods, may lead to some uncertainty. When accelerated tests result in marginal stability, i.e., approximately 2 years or less, reliability is normally not sufficient. In that case, only real-time testing at room temperature can provide the final answer for storage stability of the product. Real-time storage is carried out in simulated sales package and under thermostatic conditions. Samples are checked periodically for at least 2 years of storage. STORAGE STABILITY CONVERSION FOR DIFFERENT CLIMATIC ZONES A methodology to extend the predictions for chemical stability for drug products from the data obtained for

1512

STORAGE STABILITY TEST

Table 2. World Climatic Zones and Storage Conditions

7. H. J. Niessen, Pestic. Sci. 6: 181–188 (1975).

Climatic Zone

8. D. W. Newton and K. W. Miller, Amer. Jour. Hosp. Pharmacy 44: 1633–1640 (1987).

I

Definition Temperate climate

Storage Condition 21 ◦ C/45% RH ◦

II

Subtropical and mediterranean climates

25 C/60% RH

III

Hot, dry climate

30 ◦ C/35% RH

IV

Hot, humid climate

30 ◦ C/70% RH

9. K. A. Connors, G. L. Amidon, and V. J. Stella, Chemical Stability of Pharmaceuticals: A handbook of Pharmacists, 2nd edn., John Wiley & Sons, New York, 1986, pp. 819–823. 10. W. Grimm, Drug Dev. Ind. Pharm. 24: 313–325 (1998).

STORAGE STABILITY TEST one climatic zone to other climatic zones has been presented (10). Broadly, the world has been divided into four climatic zones, as given in Table 2. Although all the countries of Europe, North America, and several countries of Asia, Africa, and Australia fall in climatic zones I and II, a large part of the populated countries of the world are assigned in climatic zones III and IV. To make predictions for chemical stability and to cover the physicochemical test criteria, an accelerated storage condition is necessary. The International Conference on Harmonization (ICH) stability guideline for drug substances and products has been fixed for zones III and IV at 40 ◦ C/75% RH as an accelerated storage condition. The guideline for climatic zone II is 25 ◦ C/60% RH. A predictive factor of 5 has been found for a 15 ◦ C difference for the chemical stability between 25 ◦ C and 40 ◦ C. Therefore, the 6-month value at 40 ◦ C corresponds to the 30-month value at 25 ◦ C. Thus, the predictive factor for 15 ◦ C for the chemical stability is 5. On the other hand, for the difference of 10 ◦ C (40 − 30 ◦ C), the predictive factor is 3.3. CONCLUSION Accelerated storage stability tests are useful for getting an idea of the stability of the product. When the stability is marginal, real-time testing is necessary to provide the final answer. At the end of shelf life, a decline up to 10% in the active ingredient content should generally be acceptable. BIBLIOGRAPHY 1. J. Hartmann, S. K. Khetan, opments and UNIDO/New pp. 350–372.

in W. Van Valkenburg, B. Sugavanam, and eds., Pesticide Formulation: Recent DevelTheir Applications in Developing Countries, Age International, New Delhi, India, 1998,

2. Manual on the Development and Use of FAO Specifications for Plant Protection Products, 4th edn., FAO, Rome, Italy, 1995, p. 18. 3. Guidelines for Specifying the Shelf life of Plant Protection Products, GIFAP Technical Monograph No. 17, GIFAP (Now GCPF), Brussels, 1993. 4. F. Franks, TIBTECH 12: 114–117 (1994). 5. E. Neuenschwander, The shelf life of crop protection products, Association of Formulation Chemists Forum, USA, 1997, pp. 539–558. 6. A. R. Woodford, ‘‘Shelf life of pesticide formulations,’’ Workshop in Pesticide Formulation Technology, Pesticide Development Program India, Gurgaon, India, 1987.

For a pesticide formulation, a test that measures the chemical and physical stability of the product stored under defined, often worst-case, conditions. For pesticide residues, a test that measures stability of residues in stored analytical samples, usually held under frozen conditions at a specified temperature.

STREPTOMYCIN GEORGE W. SUNDIN Michigan State University East Lansing, Michigan

The antibiotic streptomycin, produced by Streptomyces griseus, was discovered by Waksman and Schatz in 1944 (1). Streptomycin was the second antibiotic (after penicillin) to be utilized in clinical medicine, and its class of antibiotics, the aminoglycosides, helped fuel the antibiotic age of modern clinical medicine. Streptomycin is also one of many antibiotics with importance as a feed amendment for growth promotion in agricultural animals. Since the early 1950s, streptomycin has been utilized for bacterial plant disease control. Initial field and greenhouse studies showed promising results in terms of disease management in a number of diverse pathosystems, including bacterial blight of celery (2), bacterial speck of tomato (3), bacterial wilt of chrysanthemum (4), blossom blast and bacterial canker of stone fruit (5,6), and fire blight of apple and pear (7). Conflicting reports have also appeared regarding the effectiveness of streptomycin as a seed treatment for controlling halo blight of bean caused by Pseudomonas syringae pv. phaseolicola (8,9). The most important use of streptomycin in plant disease control has probably been in the management of fire blight caused by Erwinia amylovora. Phytotoxicity problems limit the usefulness of copper-containing bactericides for this disease leaving few alternatives for chemical management. Streptomycin had been utilized effectively in most regions where fire blight is a problem for a number of decades. However, beginning in the 1970s, reports of the occurrence of streptomycin resistance in field populations of E. amylovora and associated control failures began to appear (10–12). Streptomycin resistance is now widespread in populations of E. amylovora, P. syringae, and Xanthomonas campestris in regions where the antibiotic is utilized (13). Although the usefulness of streptomycin is becoming more limited because of resistance problems, the success of alternative measures,

SUBSURFACE MICROBIAL COMMUNITIES: DIVERSITY OF CULTURABLE MICROORGANISMS

including the combined application of streptomycin along with a streptomycin-resistant biological control agent (14,15), may increase the longevity of this compound in agriculture today. BIBLIOGRAPHY 1. A. Schatz, E. Bugie, and S. A. Waksman, Proc. Soc. Exp. Biol. Med. 55: 66–69 (1944). 2. R. S. Cox, Plant Dis. Rep. 39: 484–486 (1955). 3. P. A. Ark, in S. A. Waksman, ed., Streptomycin, Williams & Wilkins, Baltimore, Md., 1953, pp. 607–612. 4. R. S. Robison, R. L. Starkey, and O. W. Davidson, Phytopathology 44: 646–650 (1954). 5. J. E. Crosse, Ann. Appl. Biol. 45: 226–228 (1957). 6. D. W. Dye, Ochardist N. Zeal. 29: 2,3 (1956). 7. W. J. Moller, M. N. Schroth, and S. V. Thomson, Plant Dis. 65: 563–568 (1981). 8. D. J. Hagedorn, Plant Dis. Rep. 51: 544–548 (1967). 9. J. D. Taylor and C. L. Dudley, Ann. Appl. Biol. 85: 223–232 (1977). 10. M. N. Schroth, S. V. Thomson, and W. J. Moller, Phytopathology 69: 565–568 (1979). 11. C.-S. Chiou and A. L. Jones, Phytopathology 81: 710–714 (1991). 12. J. E. Loper et al., Plant Dis. 75: 287–290 (1991). 13. G. W. Sundin and C. L. Bender, Mol. Ecol. 5: 133–143 (1996). 14. S. E. Lindow, G. McGourty, and R. Elkins, Phytopathology 86: 841–848 (1996). 15. V. O. Stockwell, K. B. Johnson, and J. E. Loper, Phytopathology 86: 834–840 (1996).

SUBSAMPLE 1) Portion of the sample obtained by selection or division; 2) individual unit of the lot taken as part of the sample; 3) final unit of multistage sampling (1). BIBLIOGRAPHY 1. W. Horwitz et al., Nomenclature for sampling in analytical chemistry, Pure Appl. Chem. 62: 1193–1208 (1990).

SUBSURFACE MICROBIAL COMMUNITIES: DIVERSITY OF CULTURABLE MICROORGANISMS DAVID L. BALKWILL Florida State University Tallahassee, Florida

Interest in the microbiology of the terrestrial subsurface has increased steadily since the early 1980s, when it was discovered that several comparatively shallow aquifers

1513

(99.5%). The subsurface Acinetobacter isolates, then, did not differ noticeably from species and strains that have been isolated from surface environments. Anaerobic and Thermophilic Metal-Reducing Isolates Many of the bacteria cultured from terrestrial subsurface environments before the mid-1990s were aerobic or facultatively anaerobic mesophiles (mostly heterotrophs). Strict anaerobes were detected or enumerated in most subsurface environments studied up to that point (using MPN assays and other methods), but there were few attempts to culture and isolate them. In recent years, though, anaerobic (both strict and facultative) and/or thermophilic forms have been cultured more frequently, as microbiologists have explored increasingly deeper and hotter environments. Several of the isolates obtained from such environments have been shown to reduce metals. This property is of interest to the U.S. Department of Energy and other agencies that are concerned about the fate of metal and radionuclide contaminants in the subsurface, especially in aquifers, in which the contaminants may migrate with the flow of groundwater. Microorganisms that can reduce metals could be significant in such environments, because many of the metals of interest (e.g., U, Tc, and Cr) are less

soluble—and, therefore, less mobile—in their reduced forms. Selected examples of metal-reducing bacteria from deep subsurface environments are described later, to illustrate the diversity and metabolic characteristics of these potentially useful organisms. Boone and coworkers (15) described a novel species of Bacillus—Bacillus infernus—that was isolated from a deep (2.7-km below land surface) soapstone within the Taylorsville Triassic Basin at a site in Virginia. B. infernus is a strict anaerobe (the only strict anaerobe in the genus Bacillus when it was first described) that can grow on formate or lactate with Fe(III), MnO2 , trimethylamine oxide, or nitrate (which is reduced to nitrite) as an electron acceptor. The organism also grows fermentatively on glucose. It is very slightly alkaliphilic (good growth at pH 7.8), halotolerant (growth up to 0.6 M Na+ ), and thermophilic (optimum growth at 61.4 ◦ C). Geologic evidence suggests that microbes inhabited the Taylorsville Triassic Basin between 200 and 140 million years ago, when penetration of meteoric water into the basin was probably greatest. Since then, most of the groundwater flow has been preferentially funneled through the overlying permeable sediments. It is unlikely that any subsequent introduction of microbes has taken place because they would have to be transported through, approximately, 2.5 km of sedimentary rock with low porosity and permeability (14). There is a good chance then that B. infernus has survived in the deep subsurface for a very long time. Kieft and coworkers (42) described a novel strain of Thermus, designated SA-01, that was isolated from groundwater in a South African gold mine. The groundwater was sampled from a horizontal borehole that was situated at a depth of 3.2 km and that penetrated 121 meters into the Witswatersrand Supergroup, a 2.9-billion-yearold formation composed of low-permeability shales and sandstones with minor volcanic units and conglomerates. The ambient temperature of the rock was approximately 60 ◦ C. Strain SA-01 grows over a temperature range of 35 ◦ C to 70 ◦ C, and has an optimum temperature of 65 ◦ C. It cannot grow fermentatively; an external electron acceptor is required for anaerobic growth. The organism can reduce soluble Fe(III), complexed with citrate or nitrilotriacetic acid (NTA). Only comparatively small quantities of hydrous ferric oxide are reduced unless the humic acid analog, 2,6-anthraquinone disulfonate, is added to the medium as an electron shuttle. Strain SA-01 is able to reduce Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI) in the presence of lactate. It can also mineralize NTA to carbon dioxide and couple its oxidation to growth and the reduction of Fe(III). Strain SA-01 is the first Thermus isolate known to couple oxidation of organic compounds to the reduction of Fe, Mn, or S. A novel strain of Shewanella putrefaciens (strain CN32), with relatively versatile metal-reducing capabilities, has been isolated from Cretaceous period sandstone at a depth of 250 m in the Morrison formation of northwestern New Mexico (43,44). Strain CN-32 grows over a temperature range of 2.7 ◦ C to 42 ◦ C. It utilizes several organic acids and other simple organic compounds as sources of carbon. With lactate as the electron donor, it is able to

SUBSURFACE MICROBIAL COMMUNITIES: DIVERSITY OF CULTURABLE MICROORGANISMS

reduce Fe(III), Co(III), Cr(IV), U(VI), and Tc(VII). Strain CN-32 can also reduce Fe(III) and Co(III) when complexed with chelating agents such as NTA or EDTA. This organism is of particular interest to researchers dealing with subsurface contamination at U.S. Department of Energy sites, where the movement of metals such as Cr(VI), U(VI), and especially Tc(VII) in the groundwater is a major concern. As noted earlier, microbially mediated reduction of these metals may limit their migration by reducing their solubility in the groundwater. The aforementioned isolates and studies in which organisms were not necessarily isolated (45,46), may indicate that diverse populations of metal-reducing bacteria are widely distributed in the deep subsurface. For more information on the possible significance of these organisms on subsurface mineralogy and geochemistry, see GEOCHEMICAL AND GEOLOGICAL SIGNIFICANCE OF SUBSURFACE MICROBIOLOGY and BIOMINERALIZATION BY BACTERIA, this Encyclopedia. CONCLUSION

1519

biological methods have detected a broad variety of asyet uncultured microbes in many natural environments including several in the subsurface. Hopefully, information from direct molecular techniques will eventually facilitate the culturing of a larger proportion of subsurface microorganisms and, thereby, enable scientists to study their potentially novel or unique characteristics. Acknowledgments Some of the research described herein was supported by the Subsurface Science Program, and (more recently) by the Natural and Accelerated Bioremediation Research (NABIR) Program, Office of Biological and Environmental Research, U.S. Department of Energy.

BIBLIOGRAPHY 1. W. C. Ghiorse and J. T. Wilson, Adv. Appl. Microbiol. 33: 107–172 (1984). 2. J. K. Fredrickson et al., Mol. Ecol. 4: 619–626 (1995). 3. T. L. Kieft et al., Microb. Ecol. 26: 59–78 (1993).

A wide variety of microorganisms (primarily eubacteria and archaea) have been cultured from terrestrial subsurface environments. These organisms are phylogenetically diverse, falling into dozens of different genera. Detailed studies on culturable microbial communities in selected subsurface environments indicate that most of the communities are quite diverse, although the numerically predominant forms often fall into a relatively small number of genera. Among the isolates that belong to a single genus, however, one usually sees a considerable amount of additional diversity at the species and strain levels. Given the diverse nature of many subsurface culturable communities, there is at least the potential for a wide range of microbially mediated chemical transformations (of organic and inorganic compounds) to take place in deep-earth environments. Many of the bacteria cultured from the subsurface appear to be new species or, in some cases, novel genera, although some of them are phylogenetically indistinguishable from previously described species that were isolated from surface environments. Several subsurface isolates that have been examined in detail have been shown to differ from the most closely related surface species in their physiological and/or genetic characteristics, most likely indicating that the subsurface organisms have a distinct evolutionary history. It seems likely, then that microorganisms cultured from deep subsurface environments represent a significant source of new genetic information. Some of these microbes also have potentially valuable metabolic capabilities, such as ability to degrade toxic organic compounds, or to immobilize metals and radionuclides in groundwater. Therefore, they may not only influence the fate of contaminants in the subsurface, but might also have applications in the field of bioremediation. Although much information has been derived from the study of microorganisms cultured from subsurface environments to date, it is recognized that the cultured strains probably represent only a small fraction of the total communities in these environments. Direct molecular

4. J. K. Fredrickson et al., Geomicrobiol. J. 11: 95–107 (1993). 5. F. J. Brockman et al., Microb. Ecol. 23: 279–301 (1992). 6. V. Boivin-Jahns et al., Appl. Environ. Microbiol. 61: 3400– 3406 (1995). 7. D. L. Balkwill, Geomicrobiol. J. 7: 33–52 (1989). 8. D. L. Balkwill et al., Appl. Environ. Microbiol. 55: 1058– 1065 (1989). 9. J. K. Fredrickson et al., Geomicrobiol. J. 7: 54–66 (1989). 10. J. K. Fredrickson et al., Appl. Environ. Microbiol. 402–411 (1991).

57:

11. R. E. Jones et al., Geomicrobiol. J. 7: 117–130 (1989). 12. T. J. Phelps et al., Geomicrobiol. J. 7: 79–91 (1989). 13. J. L. Sinclair and W. C. Ghiorse, Geomicrobiol. J. 7: 15–31 (1989). 14. D. L. Balkwill et al., Eos 75: 385,395–396 (1994). 15. D. R. Boone et al., Int. J. Syst. Bacteriol. 45: 441–448 (1995). 16. T. C. Onstott et al., Geomicrobiol. J. 15: 353–385 (1998). 17. P. S. Amy et al., Appl. Environ. Microbiol. 58: 3367–3373 (1992). 18. D. L. Haldeman and P. S. Amy, Microb. Ecol. 25: 183–194 (1993). 19. D. L. Haldeman et al., Microb. Ecol. 26: 145–159 (1993). 20. K. Pedersen and S. Ekendahl, Microb. Ecol. 20: 37–52 (1990). 21. K. Pedersen and S. Ekendahl, Microb. Ecol. 23: 1–14 (1992). 22. K. Pedersen et al., FEMS Microbiol. Ecol. 19: 249–262 (1996). 23. J. K. Fredrickson et al., Geomicrobiol. J. 14: 183–202 (1997). 24. L. R. Krumholz et al., Nature 386: 64–66 (1997). 25. D. L. Balkwill, ASM News 59: 504–506 (1993). 26. K. A. Sargent and C. B. Fliermans, Geomicrobiol. J. 7: 3–13 (1989). 27. D. L. Balkwill et al., FEMS Microbiol. Rev. 20: 201–206 (1997). 28. R. H. Reeves et al., J. Microbiol. Methods 1: 235–251 (1995).

1520

SURFACTANT

29. T. L. Kieft et al., Appl. Environ. Microbiol. 61: 749–575 (1995). 30. F. H. Crocker et al., Microbiology 146: 1295–1310 (2000).

ensure compliance with established MRLs. Surveillance may be directed at domestic, imported, or exported commodities.

31. S. Ekendahl et al., Microbiology 140: 1575–1583 (1994). 32. D. L. Balkwill et al., Microb. Ecol. 35: 156–171 (1998).

SUSPENSIBILITY

33. A. J. Francis et al., Geomicrobiol. J. 7: 103–116 (1989). 34. J. K. Fredrickson et al., Appl. Environ. Microbiol. 796–803 (1991).

57:

35. J. K. Fredrickson et al., Appl. Environ. Microbiol. 1917–1922 (1995).

61:

36. R. Imai et al., J. Bacteriol. 173: 6811–6819 (1991).

The amount of solid that remains suspended after a given time in a column of specified liquid, of stated height, under specific conditions (CIPAC). It is expressed as a percentage of the amount of active ingredient and/or carrier in the original suspension.

37. U. Karlson et al., Appl. Syst. Microbiol. 18: 539–548 (1996). 38. K. Taira et al., Biochemistry 27: 3990–3996 (1988). 39. R.-M. Wittich et al., Appl. Environ. Microbiol. 58: 1005– 1010 (1992). 40. D. L. Balkwill et al., Int. J. Syst. Bacteriol. 47: 191–201 (1997).

SUSPENSION CONCENTRATE (SC) Formulation in which the active ingredient is in the form of a stable dispersion of fine particles in water or organic liquid (1).

41. E. Kim et al., Appl. Environ. Microbiol. 62: 1467–1470 (1996). 42. T. L. Kieft et al., Appl. Environ. Microbiol. 65: 1214–1221 (1999).

BIBLIOGRAPHY

43. J. K. Fredrickson et al., Geochim. Cosmochim. Acta 62: 3239– 3257 (1999).

1. GIFAP Catalogue of Pesticide Formulation Types and International Coding System. GIFAP Technical Monograph No. 2, Brussels, 1989.

44. R. E. Wildung et al., Appl. Environ. Microbiol. 66: 2451–2460 (2000).

SURFACTANT A formulant for reducing interfacial tension of two boundary surfaces, thereby increasing the emulsifying, spreading, dispersability, or wetting properties of liquids or solids (IUPAC) (1). A material for reducing interfacial tension (CIPAC). BIBLIOGRAPHY 1. C. S. Hartley and L. J. Graham-Bryce, Physical Principles of Pesticide Behaviour, Vol. 2, Academic Press, New York, 1980, pp. 427–430.

SURVEILLANCE Systematic sampling and residue analysis of commodities, and collation and interpretation of data, in order to

SYNERGIST A substance that, although formally inactive or weakly active, can significantly enhance the activity of the active ingredient in a formulation (IUPAC).

SYNOMONES Chemicals emitted by one species that modify the behavior of a different species to the benefit of both the emitting and receptor species (EPA No. 540/09-89-056, March 1989; USEPA; Washington, DC; 1989).

SYSTEMIC A systemic pesticide is capable of being translocated internally to sites other than where it was absorbed in sufficient quantities to be biologically active (IUPAC).

T TARGET, BIOLOGICAL

TETRACYCLINE GEORGE W. SUNDIN

Any organism, organ, tissue, cell, enzyme, receptor or cell constituent that is subject to the action of a pesticide or its residue (IUPAC).

Michigan State University East Lansing, Michigan

The tetracyclines are a group of compounds with broadspectrum antimicrobial activity against a diverse range of gram-negative and gram-positive bacteria. Most of the important tetracyclines are bacteriostatic in vitro, inhibiting cell growth by reversibly binding to ribosomes and inhibiting protein synthesis (1). The first tetracycline, aureomycin (chlortetraclycine), was discovered by a plant pathologist, B.M. Duggar, in the late 1940s (2). The structure of chlortetracycline differs from tetracycline by the presence of a single Cl atom covalently bonded at the C7 position on the molecule (3). Oxytetracycline, which has seen significant usage in plant disease control, does not contain the Cl atom but has an additional -OH group at the C5 position on the tetracycline molecule (4). Tetracyclines are widely used therapeutic agents in clinical medicine [second to penicillins in total tons used each year (5)] for bacterial respiratory, periodontal, and urogenital tract diseases (6). Oxytetracycline is also used as a feed amendment for growth promotion and as a therapeutic agent for curing diseases of agricultural animals including fish. The use of oxytetracycline for plant disease control began in the early 1950s with the introduction of Agrimycin (15% streptomycin sulfate +1.5% oxytetracycline). Field studies documenting disease control using Agrimycin at 50–200 ppm against bacterial spot of tomato (Xanthomonas campestris pv. vesicatoria), fire blight (Erwinia amylovora), halo blight (Pseudomonas syringae pv. phaseolicola), and wildfire of tobacco (P. syringae pv. tabaci) were reported (7–10). As early as 1954, the inclusion of oxytetracycline in the Agri-mycin formulation was recognized as significant in delaying the emergence of streptomycin resistance in Erwinia and Xanthomonas strains (11). Annual injections of oxytetracycline into trunks or scaffold branches of fruit trees has also resulted in the reduction of symptoms of phytoplasma diseases including pear decline and X disease of peach (12,13). Currently oxytetracycline is mostly used in the control of fire blight and bacterial spot of nectarine and peach caused by Xanthomonas campestris pv. pruni. Oxytetracycline use is critically important for fire blight management in situations in which resistance to streptomycin (another important agent for fire blight management) already exists within the E. amylovora population. In one field study on apple, protective chemical sprays of oxytetracycline at 200 ppm were applied at 25–50% bloom, followed by inoculation 24 hours later with E. amylovora. This treatment reduced the percentage of blossom clusters infected compared to a water-sprayed control; however, oxytetracycline was significantly less effective than streptomycin in reducing infection for two

TECHNICAL MATERIAL The unformulated active ingredient (CIPAC). Commercial grade of the pesticide as it comes from the manufacturing plant comprising the active ingredient and associated impurities. It may also contain small quantities of additives necessary for stability (IUPAC).

TEST GUIDELINE Guideline published by an appropriate authority for the order or conduct of certain tests (IUPAC).

TEST PORTION (ANALYTICAL PORTION) Subsample, of proper size for a chemical analysis or other test, removed from the test sample [after (1)] (IUPAC). BIBLIOGRAPHY 1. W. Horwitz et al., Nomenclature for sampling in analytical chemistry, Pure Appl. Chem. 62: 1193–1208 (1990).

TEST SAMPLE (ANALYTICAL SAMPLE) Homogenous sample, prepared from the laboratory sample, grinding, blending, fine-chopping, etc., from which test portions are removed for analysis with minimal sampling error (1) (IUPAC). BIBLIOGRAPHY 1. W. Horwitz et al., Nomenclature for sampling in analytical chemistry, Pure Appl. Chem. 62: 1193–1208 (1990).

TEST SUBSTANCE The pesticide as a chemical substance or mixture that is under investigation in a GLP study (IUPAC).

TEST SYSTEM Each system (animal, plant, microbial, other cellular, subcellular; chemical, physical, or a combination thereof) used in a study. 1521

1522

THEORETICAL MAXIMUM DAILY INTAKE (TMDI)

consecutive years (14). Evaluations of oxytetracycline in reducing E. amylovora populations on detached apple blossoms also showed that it was less effective than streptomycin (14). However, only oxytetracycline was totally effective in reducing populations of a streptomycinresistant E. amylovora strain on blossoms (14). An important aspect in the use of any compound for bacterial control is the propensity of the target population to develop resistance. There are several genes known in clinical pathogens that confer tetracycline resistance; these genes typically confer resistance equally to chlortetracycline, oxytetracycline, and tetracycline (1). In contrast to the control problems stemming from the evolution of copper and streptomycin resistance in plantpathogenic bacteria (15), there is only one report to date of resistance to tetracycline although the antibiotic has been applied for over twenty years in some regions. R.A. Spotts and L.A. Cervantes (16) detected a small percentage of tetracycline-resistant strains of Pseudomonas syringae pv. syringae from pear orchards in Oregon. In contrast, a survey of 44 pear orchards in Washington did not detect any tetracycline-resistant isolates of E. amylovora (17). In an important recent study, Schnabel and Jones surveyed for tetracycline-resistant bacteria in two Michigan apple orchards, where oxytetracycline had been previously applied (18). In this study at least four different previously known tetracycline-resistance genes were detected in Pantoea agglomerans and Pseudomonas spp., but not E. amylovora (18). Schnabel and Jones also performed experiments showing that the likelihood of transfer of a tetracycline-resistance determinant(s) into E. amylovora by conjugation was currently not high (18). Thus oxytetracycline has survived for two decades in bacterial plant disease control with few resistance problems developing. However, the long-term outlook for this antibiotic must remain cautious. The large number and diversity of known tetracycline-resistance genes (1,6) coupled with the presence of some of these genes in organisms inhabiting orchard environments (18) increases the risk of future resistance problems following the continued intensive use of this antibiotic.

BIBLIOGRAPHY 1. I. Chopra, P. M. Hawkey, and M. Hinton, J. Antimicrob. Chemother. 29: 245–277 (1992).

10. H. E. Heggested and E. E. Clayton, Plant Dis. Rep. 38: 661–665 (1954). 11. A. R. English and G. van Helsema, Plant Dis. Rep. 38: 429–433 (1954). 12. A. L. Jones and H. S. Aldwinckle, eds., Compendium of Apple and Pear Diseases, APS Press, St. Paul, Minn., 1990. 13. J. M. Ogawa, ed., Compendium of Stone Fruit Diseases, APS Press, St. Paul, Minn., 1995. 14. P. S. Mcmanus and A. L. Jones, Phytopathology 84: 627–633 (1994). 15. D. A. Cooksey, Annu. Rev. Phytopathol. 28: 210–219 (1990). 16. R. A. Spotts and L. A. Cervantes, Plant Dis. 79: 1132–1135 (1995). 17. J. E. Loper et al., Plant Dis. 75: 287–290 (1990). 18. E. L. Schnabel and A. L. Jones, Appl. Environ. Microbiol. 65: 4898–4907 (1999).

THEORETICAL MAXIMUM DAILY INTAKE (TMDI) A prediction of the maximum daily intake of a pesticide residue, based on the assumption of levels of residues in food at maximum residue limits and average daily consumption of food per person. The TMDI is expressed in milligrams of residue per person calculated for a person of body weight 60 kg (IUPAC) (1). BIBLIOGRAPHY 1. WHO, Guidelines for Predicting Dietary Intake of Pesticide Residues. World Health Organization, Geneva, 1989. (See also Guidelines for Predicting Dietary Intake of Pesticide Residues (Revised) World Health Organization, Geneva, 1997 http://www.who.int/fof/!pest.pdf)

THRESHOLD Concentration of a pesticide in an organism or environmental compartment below which an adverse effect is not expected (IUPAC).

TID

2. B. M. Duggar, Ann. N.Y. Acad. Sci. 51: 177–181 (1948). 3. I. Chopra, Antimicrob. Agents Chemother. 38: 637–640 (1994). 4. L. G. Nickell and P. N. Gordon, in P. Gray, B. Tabenkin, and S. G. Bradley, eds., Antimicrobial Agents Annual 1960, Plenum Press, New York, 1961, pp. 588–593.

Thermionic detector (cf. NPD, AFID).

TILLAGE

5. N. F. Col and R. W. O’Connor, Rev. Infect. Dis. 9: 232–243 (1987).

RAVI G. BHAT KRISHNA V. SUBBARAO

6. M. C. Roberts, FEMS Microbiol. Rev. 19: 1–24 (1996).

University of California Davis, California

7. R. A. Conover, Plant Dis. Rep. 38: 405–409 (1954). 8. R. N. Goodman, Plant Dis. Rep. 38: 874–878 (1954). 9. W. J. Zaumeyer, Agric. Food Chem. 3: 112–116 (1955).

Tillage is a cultural practice of fragmenting and burying plant residues through plowing or in general, a process

TILLAGE

of soil inversion using practices such as disking, ripping, plowing, and chiseling to prepare land for new planting. Tillage enhances the degradation of plant materials left in the field after harvest by mixing them with soil and exposes subsoil to the top layer. Probably no cultural practice has a greater impact on the soil environment than tillage (1). Tillage is useful to farmers for smooth farm operations by keeping the field clean and reducing some of the weeds, insects, and plant diseases. However, conventional tillage in rain-fed agriculture has resulted in excessive soil erosion, high costs of labor, and energy. In recent years conservation or reduced tillage systems, including zero tillage, have been established to conserve soil moisture and reduce soil erosion. Generally conservation tillage is defined as a system that leaves 30% or more of the soil surface covered by crop residue after planting (2). Tillage practices can directly influence plant disease development. Some disease problems may not be easily controlled without some degree of tillage. Plant pathogens are a small part of the larger ecological web in the soil. Burying the crop debris creates an unfavorable environment for many soilborne plant pathogens. Decomposing crop residues are niches for saprophytic soil microorganisms that are more likely to compete with pathogens for nutrients and space. Physical displacements of pathogen propagules to the subsoil have adverse effects on survival and viability of pathogens. Even the resistant survival structures such as chlamydospores, oospores, and sclerotia are affected by tillage. When buried, pathogen propagules may die because of harsh conditions prevailing inside the soil or may not have the chance to be in contact with the succeeding crop. For example, sclerotia of Sclerotinia sclerotiorum do not survive for long periods when buried; thus tillage assists in reducing the level of initial inoculum in the field (3). Similarly, oospores of Peronosclerospora sorghi, the causal agent of downy mildew of grain sorghum cannot infect seedlings when buried deep in the subsoil. Deep plowing was developed as a disease management strategy for some soilborne diseases. The rationale for using this technique is to remove pathogen propagules concentrated in the upper soil profiles to depths at which they no longer are able to infect their hosts. While the utility of this strategy in disease management is intuitively known, detailed analyses of the pros and cons of this strategy were not available until recently. Subbarao et al. (4) studied the distribution patterns of S. minor sclerotia before and after deep plowing and followed the effects on the incidence of lettuce drop in successive lettuce crops. Deep plowing did not result in reduced lettuce drop incidence. While the desired effect of reducing the number of sclerotia was accomplished with deep plowing, the altered distribution of sclerotia increased the likelihood of infection of a greater number of lettuce plants. This was because the higher-than-normal tillage operations following deep plowing altered the distribution of sclerotia from highly aggregated patterns to less aggregated patterns approaching randomness. This provided the opportunity for infection of a greater number of lettuce plants in succeeding crops (4).

1523

Conservation tillage has posed a greater problem for disease control. It is generally believed that reduced tillage maximizes disease potential by allowing diseased crop residue to remain on the soil surface, whereas burial of crop residue reduces the potential for infection by residueborne pathogen that release airborne spores. From the considerable amount of literature on the subject, it is clear that conservation tillage practices can variously increase, decrease, or have no effect on plant diseases (5,6). Crop residues left on the soil surface degrade slowly over a period of time and create a new microclimate that changes the soil physical environment such as lower soil temperature, increased soil water, increased diffusion of plant exudates, increased soil compaction, decreased soil aeration, and soil porosity. Each one of these factors affects not only the succeeding crop but also the inoculum concentration and inoculum potential of plant pathogens. Also crop residues are a food source for longer pathogen survival and greater reproduction. In addition to these, soil microflora that affect both hosts and pathogens fluctuate depending upon the state of residue decomposition and microclimate. Conservation tillage is one component in the development of sustainable agriculture system, and plant diseases are only one variable considered in this process. Extensive studies on field crops such as corn, wheat, and soybean have been conducted for major disease problems individually, but it takes more time to develop an integrated pest management program to simultaneously control several pathogens on different crops (2). It is highly recommended that crop rotation and resistant varieties be included with conservation tillage for efficient crop production. This practice controls many diseases and yet allows as much of the crop residue as possible to be retained on the soil surface. Subbarao et al. (7) compared furrow and subsurface drip irrigation for lettuce drop development and inoculum augmentation in a field where the initial inoculum was identical and uniformly distributed under the two management systems. Implicit in this study was also the comparison of conventional and minimum tillage practices under furrow and subsurface drip irrigation systems. They found that although conventional tillage under furrow irrigation redistributed the inoculum added after each crop, the inoculum density was always higher under this system. In contrast, under subsurface drip irrigation and minimum tillage, the amount of inoculum was consistently lower and the distribution of sclerotia was altered little. The combination of lower inoculum added and its aggregated distribution resulted in lower lettuce drop incidence under this management system (7). Not all diseases react the same way to the conditions created by conservation tillage management. Since disease development is a function of host, pathogen, and environment, problems developing in each region may be unique. Therefore prediction of a disease problem in a particular field is difficult because observations from one area are not necessarily applicable to others. Cultivation practices impact upon diseases by changing their importance within the cropping system. The degree of impact depends on the level of host specificity the pathogen has in relation to the crops used in rotation,

1524

TLC

the pathogen’s biology and its mechanisms for dispersal and survival, and weather and microclimate effects. In the Pacific northwestern United States, conservation tillage has been practiced for more than 30 years (8). In the early years wheat yields were often reduced in continuous cultivation of cereals with minimum tillage but not with conventional tillage. At first, this effect was attributed to phytotoxins being released from the microbial breakdown of moist straw at the soil surface. Later on it was shown that three soilborne pathogens namely, Gaeumannomyces graminis var. tritici, Rhizoctonia solani AG 8, and Pythium spp. were favored by a lack of crop rotation in the farming systems and also by optimal moisture conditions in the top undisturbed soil (2).

but the samples are further processed as for domestic consumption, i.e., further trimming and cooking as appropriate to local practice (IUPAC).

TOTAL TERMINAL RESIDUE Summation of levels of all the compounds comprising residues of a pesticide in a food (IUPAC). See also Pesticide Residue.

TOXICITY TESTING IN SOILS

BIBLIOGRAPHY

MIKAEL PELL LENNART TORSTENSSON

1. C. S. Rothrock, Soil Sci. 154: 308–315 (1992).

Swedish University of Agricultural Sciences Uppsala, Sweden

2. W. W. Bockus and J. P. Shroyer, Annu. Rev. Phytopathol. 36: 485–500 (1998). 3. K. L. Bailey, Can. J. Plant Sci. 76: 635–639 (1996). 4. K. V. Subbarao, S. T. Koike, and J. C. Hubbard, Plant Dis. 80: 28–33 (1996). 5. K. L. Bailey and L. J. Duczek, Can. J. Plant Pathol. 18: 159–167 (1996). 6. A. V. Sturz, M. R. Carter, and H. W. Johnston, Soil and Tillage 41: 169–189 (1997). 7. K. V. Subbarao, J. C. Hubbard, and K. F. Schulbach, Phytopathology 87: 877–883 (1997). 8. R. J. Cook and W. A. Haglund, Soil Biol. Biochem. 23: 1125–1132 (1991).

TLC Thin-layer chromatography.

TOLERABLE DAILY INTAKE

The economic well-being of human society is dependent on the productivity and sustainability of arable soils. Soil is the ultimate receptor of, and the incubation chamber for, decomposing organic material and recycling nutrients back to plants, as well as detoxification of organic pollutants. Soil quality can improve or deteriorate depending on several factors. If mismanaged, the soil can work against us, for example, it can pollute the air by emissions of nitrous gases and pollute water by leaching of nitrogen and other plant nutrients or pesticides. We are becoming more aware of the frequent exposure of soils to anthropogenic substances and that there is a delicate balance between maintaining good soil quality and achieving production goals. Therefore, there is an urgent need for tools to measure toxicity in soils (Fig. 1), to interpret toxicity data, as well as to develop a strategy for evaluating soil quality (1). Our aim is to give a short overview of the soil microbial ecosystem and some important factors pertaining to soil formation, and against this background to discuss what

Term preferred by the European Commission for acceptable daily intake (ADI) of environmental contaminants. ADI is reserved for pesticides and food additives where extensive toxicological test data are available (IUPAC).

Toxicity testing • Chemicals • Heavy metals • Acidifying substances

TOLERANCE Permitted limits of variation from a given value (CIPAC). See also Maximum Residue Limit (MRL).

TOLERANCE, RESIDUE.

See MAXIMUM RESIDUE LIMIT

(MRL)

TOTAL DIET STUDY Pesticide residue monitoring to establish the pattern of residue intake by a person consuming a defined diet. Primary sampling is as for a market basket survey,

Need and use of soil tests

Grouping soils according to their quality Monitoring soils over long periods Follow restoration of polluted soils

Figure 1. The need of various soil tests to be able to assess soil quality.


kind and quality of information soil tests should deliver. Examples of useful microbial and enzymatic tests are given. Finally, we discuss strategies and problems in screening and testing the effects of chemicals and heavy metals, the assessment of soil quality, and the monitoring of long-term changes in both naturally managed and polluted soils. THE SOIL MICROBIAL ECOSYSTEM The soil ecosystem gains most of its energy from dead organic materials, for example, plant and animal residues. During the mineralization of these residues, carbon dioxide and inorganic nutrients such as nitrate, phosphate, and sulfate are released so that plants can utilize them again. The microbial biomass itself represents a major pool of readily available nutrients, which is continuously shunted into the growth cycles of macro- and microphytes. Consequently, soils that host a high level of microbial biomass are capable not only of storing more nutrients, but also have the potential of cycling more nutrients through the ecosystem. As microorganisms are adapted to survive under extreme conditions, they are present in soil both in large numbers and under almost all environmental conditions. Many catabolic and anabolic functions are widely distributed among soil micropopulations, and groups of taxonomically diverse organisms can therefore replace one another in the decomposition cycles. The microbial community carries out the majority of decomposition processes in soil and is stimulated by the activities of soil invertebrates, especially saprotrophs feeding on decaying organic matter and microbivores grazing the microflora. Furthermore, microorganisms are irreplaceable in the biological transformation and degradation of synthetic organic compounds and natural waste materials.

Soil stress situations: Temperature fluctuations Water potential extremes Soil pH extremes

1525

Soil microorganisms are critical in creating and maintaining good soil structure, which is important for proper soil aeration and the formation of humus and particle aggregates. Filamentous fungi and actinomycetes, in particular, entrap soil particles to form aggregates. In addition, bacteria produce extracellular metabolites, for example, polysaccharides, lipids, and proteins, which function as gums and cementing agents that stabilize the aggregates. Soil texture and structure, in combination with a variation in moisture levels, can drastically affect the aeration status, thus influencing the distribution of physiological groups in the microbial community. In all terrestrial ecosystems, microorganisms are more or less continuously exposed to changing environmental conditions. This means that the microbial community in soil is exposed to stress situations owing to both soil management and climatic conditions (Fig. 2). SOIL QUALITY In agriculture, soil quality is an integral part of agricultural sustainability, and is influenced by a number of degrading and conserving forces (2). Soil is a complex system created by a number of factors that cannot be easily influenced, such as geology, topography, and climate (Fig. 3). Soil quality is controlled by physical, chemical, and biological components. Soil quality factors that can be influenced are humus content; the number, composition, and activity of microorganisms; the degree of base saturation; nutrient status; and others. A number of short-term practices, such as soil cultivation or use of fertilizers and pesticides, are generally not included as soil quality factors. Neither are unintended deposition of chemicals, heavy metals, or acidifying substances considered soil quality factors. Many attempts have been made to define soil quality (3–5). All definitions have in common the capacity of a soil to function effectively both at present and in the future. As suggested by Doran and Parkin (3), soil quality can be defined as ‘‘the capacity of a soil to function within ecosystem boundaries to sustain biological productivity, maintain environmental quality, and promote plant and animal health.’’ Such a definition implies that good soil quality is relative, and must be

Decreased soil gas exchange Other factors

Environmental influence on the microbial community

Soil management: Organic and inorganic fertilisers Physical disturbances of soil The crop rotation Other factors Climate: Temperature Precipitation Evaporation Other factors

Figure 2. The environmental factors influencing soil microbial communities.

Topography

Climate

Physical factors

Chemical factors

Biological factors

Geology

Anthropogenic activities

Figure 3. Soil is a complex structure created by influences of geology, topography, and climate as well as anthropogenic activities.

1526


individually defined for each soil ecosystem (see SOIL QUALITY, THE ROLE OF MICROORGANISMS, this Encyclopedia). SOIL TEST DATA—A COMPREHENSIVE PICTURE With the development of new methods for assessing soil biomass and microbial or enzymatic activity, it is important to have a clear idea of what kind of information the test should deliver. It is important to consider whether to choose a test for the evaluation of a soil property or to test the effect of a specific substance. Furthermore, when interpreting data the basic test design, that is, the possibilities and limits of the test, must be considered. This work would be simplified if apparently different tests and test data could be treated in a common frame, that is, to use the same basic concept of kinetics. The kinetic discussion in the following text refers to tests of potential activities, that is, tests performed under optimized environmental conditions so that only the amounts of organisms and enzymes are rate limiting. In its simplest form, a quantitative soil test assay uses the assumption that no product is present at time zero. After a certain time of incubation the first and only sample is withdrawn for analysis of the product. A constant product formation rate must be assumed to allow calculation of a process rate from such limited data. A more accurate way is to also establish the initial concentration of the product. Even if in most cases the product formation rate of some tests is constant, the above test strategy has some uncertainty in the rate and, hence, should be viewed as semiquantitative information. The uncertainty will increase not only with time of incubation, but also when an unknown substance to be tested is added. A better experimental design is to take a reasonable number of samples during the test so that a straight-line relation can be established by linear regression. Moreover, many samples give more accurate data, that is, random errors in sampling and analysis cancel each other. The linear product formation can be described by the following general formula: (1) p = p0 + Et where p and p0 are the amounts of product at time t and t = 0, respectively, and E is the enzyme activity (Fig. 4a). When referring to the enzyme activity of a specific microbial process showing no growth during the test, E can be replaced by, for example, K, a rate constant for potential microbial activity (Fig. 4b). The complexity increases when nonlinear test data are generated because nonlinear processes, by necessity, demand a larger number of sampling points to establish the shape of the curve. Several means of linearization of such data have been proposed. Perhaps the most common way is to make only a very short incubation and to subjectively fit a straight line to the nonlinear data. A biased estimate will probably be achieved in this way. As a nonlinear product formation rate is most likely owing to de novo synthesis of enzymes, with or without cell growth, the use of antibiotics with inhibitory effect on protein synthesis has been proposed to maintain linear rates.

Another strategy is to analyze the phenomenon as a nonlinear process. Such a strategy generates more information of the process tested for. In most test situations enzyme activity, as well as growth of microorganisms, must be considered. These two properties are described in the formula p = p0 +

r µt e −1 µ

(2)

where r is the initial microbial activity and µ the specific growth rate (6). Thus, by assuming a constant amount of enzymes without cell growth (i.e., zero-order kinetics with respect to cell growth), a constant product formation rate (K), as discussed earlier, will explain the straight-line result of a test (Fig. 4b). Assuming a substrate saturated enzyme system with cell growth (i.e., first-order kinetics with respect to cell growth), both the initial rate of product formation (r) and specific rate of cell growth (µ) will explain the nonlinear curve of some tests (Fig. 4c). For general soil processes such as respiration, it is not likely that all microorganisms react to a substrate in the same way. It has been suggested that mineralization of glucose is performed by two main groups of microorganisms. The first group grows exponentially as a result of substrate addition, whereas the other group increases its respiration activity to a higher rate without multiplication. In a further modification of formula (2), the activity of the nongrowing group, denoted K, was introduced (7), resulting in the new formula: p = p0 +

r µt e − 1 + Kt µ

(3)

By applying this formula (Fig. 4d), a deeper understanding of underlying structures in the soil ecosystem can be achieved (8). From the preceding discussion it is evident that results from different tests permit different degrees of interpretation and understanding. Many enzymatic tests used today are simplifications chosen to get rapid and inexpensive tests. One reason for this is that authorities and the chemical industry have urged the development of these kinds of tests. Another reason is that the awareness of the enormous task ahead with so many untested chemicals necessitates the use of simple tests. On the other hand, the consequence of using such simple tests might be that important pieces of information are overlooked. Thus, a contradiction can be seen between using soil tests as research tools or as practical environmental indicators. The conclusion is, however, that both types are needed and their development must go hand in hand. MICROBIAL AND ENZYMATIC TESTS Commonly, microbial soil tests are grouped into biomass estimations, activity measurements, and assays of soil enzymes. The distinction between activity and enzymatic assays is not straightforward as several enzymes may simultaneously be located intracellularly, on cell surfaces, and also be actively exuded as free enzymes into the bulk


(a)

Phosphatase activity

(b)

1527

Ammonium oxidation

2500

2000

µg N2O-N kg−1 dry soil

µmol PNP kg−1 dry soil

1600

PAlk-P = P0 + EAlk-Pt

1500

1000

500

PPAO = P0 + KPAOt 1200

800

400

0

0 0

30

60

90

120

0

50

100

150

200

250

300

350

400

Minutes Minutes (c)

(d)

Denitrification

2000

Respiration 3000 2500

PPDA = P0 + rPDA/m(e(µt)−1)

1600

mg CO2 kg−1 dry soil

µg N2O-N kg−1 dry soil

PSIR = P0 + rSIR/m(e(µt)−1) + KSIRt

1200

800

400

2000 1500 1000

PB-res = P0 + KB-rest

500 SIR = rSIR + KSIR

0

0

30

60

90

120

150

180

210

Minutes

0 −3000 −2000 −1000

0

1000 2000 3000 4000

Minutes

Figure 4. Examples of microbiological test data and a kinetic approach to evaluating product formation rates: (a) rate of phosphatase activity (PAlk-P ), (b) potential NH4 + oxidation rate (PPAO ), (c) potential denitrification rate (PPDA ), and (d) substrate induced respiration rate (PSIR ) and basal respiration rate (PB-res ). Gray circles are data not included in the regression analysis.

soil. In addition, ‘‘dead’’ or dormant cells can be regarded as being in between these two states. Another common practice is to relate tests to the general biogeochemical cycles of carbon, nitrogen, phosphorus, and sulfur. To be more precise, we have chosen to classify the tests according to different functional levels within ecosystems: 1) basic microbial soil functions (biomass and processes performed by virtually all groups of microbes), 2) specific microbial soil functions (performed by a more or less well-defined group of microbes), and 3) general microbial growth. A review of some important tests that are commonly used for assessing the effects on the soil ecosystem is given later. Several manuals with detailed information on soil test performance (9–11) and soil toxicity testing (9,12) have been produced. Basic Microbial Soil Functions Biomass. (see BIOMASS: SOIL MICROBIAL BIOMASS, this Encyclopedia) The level of total soil biomass has often been regarded as a fertility indicator as a microbial biomass is correlated with nutrient turnover. Moreover, a high biomass contains a reserve of valuable nutrients.

Historically, the total biomass of soil microorganisms has been determined by viable plate counts or direct counts by microscopy. The former method seriously underestimates the biomass whereas the latter is tedious and time consuming. By combining the microscopic technique with immunological techniques or molecular genetic probing techniques, specific groups of soil bacteria can be enumerated. Also, various techniques for indirect estimation of the microbial biomass have been developed, of which the chloroform fumigation–extraction (CFE) techniques of biomass carbon, nitrogen, or phosphorus are the most widely used (13–16). Another method for indirect measurement of biomass is the substrate-induced respiration (SIR) technique (16,17). After a period of 7 to 10 days of preincubation the soil sample is amended with glucose in surplus. To ensure no nutrient limitation, ammonium and phosphate can also be added. Immediately after substrate addition, the respiration pattern is recorded for at least 8 hours. The maximum response in CO2 production or O2 consumption before start of cell growth is proportional to the amount of biomass. Several techniques exist for the determination of carbon dioxide produced, such as

1528


titration, gas chromatography, or infrared gas analysis. One convenient respirometer to use is a Respicond III ˚ Sweden). It is based on (Nordgren Innovations AB, Umea, the capture of carbon hydroxide in potassium hydroxide and measurement of the resulting decrease in conductivity of the KOH solution. The respirometer can measure the respiration of 96 soil samples two times every hour. A computer program allows the calculation of SIR. In addition, data can be exported to a computer program capable of nonlinear regression analysis. By fitting data to formula (3) above, the total biomass can be split into the amount of growing (r) and nongrowing (K) bacteria, respectively. Basal Respiration. Soil respiration is the degradation of organic forms of carbon. For organisms to access the carbon, several enzyme systems must be involved, resulting in the release of various nutrients bound in the organic matter. Thus, the ultimate products of respiration, or carbon mineralization, are carbon dioxide, water, and various nutrients. Owing to competition for energy among fast-growing microorganisms, the most available carbon fractions, such as simple sugars and amino acids, will be degraded first, leaving the more recalcitrant structures of hemicellulose and lignin to slow-growing organisms. In an undisturbed soil, respiration is dominated by the degradation of more complex forms of carbon and is often referred to as basal respiration. The simplest way to measure basal respiration is to preincubate a soil sample to allow the initial flush of carbon dioxide caused by sample manipulation to level off. Thereafter, carbon dioxide production is measured in a closed system by absorption of carbon dioxide in an alkaline KOH solution. By titration, the remaining potassium hydroxide can be determined and the amount of carbon dioxide produced can be calculated. By using the respirometer described earlier, basal respiration can conveniently be monitored during a 48hour period before the glucose addition for determination of SIR. The respirometer method gives very accurate estimates as many data points per assay are generated. Nitrogen Mineralization. Nitrogen mineralization is the process during which organic bound nitrogen is enzymatically degraded to the mineral form ammonium ion (18). In soil, the process is performed by many diverse bacteria, both aerobic and anaerobic. Thus, the mineralization process is more or less independent of the oxygen status. To degrade organic macromolecules, microorganisms have to excrete extracellular enzymes, many of which are unspecific. Owing to the importance of nitrogen in crop production, many methods have been proposed to estimate the nitrogen mineralization capacity. One technique is to make a standardized aerobic incubation of a soil sample over several weeks or months. During the incubation period, nitrogen that is soluble in water is leached, either at the end or repeatedly at specified intervals. Alternatively, many replicates are started and destructive extractions with a KCl solution are made. The leachates/extracts are analyzed for ammonium and nitrate, and eventually nitrite. This technique estimates net mineralization because virtually

all microbial nitrogen transformation processes might occur during the incubation. Moreover, if the effect of a test substance is to be evaluated, it is impossible to determine which group of organisms involved in nitrogen transformation are affected. Another commonly used technique to measure nitrogen mineralization capacity is the slurry assay (19) performed under anaerobic conditions with water as the only additive. The incubation period is 7 to 14 days. The advantage of an anaerobic slurry assay is that problems with determination and maintenance of an optimum soil water content is avoided. Substrate limitations because of restricted diffusion are also minimized. Moreover, nitrification is inhibited and assimilation is retarded by the anaerobiosis, as well as by uncontrolled losses of gaseous nitrogen. Thus, only ammonium has to be analyzed. Ammonium can effectively be analyzed by the indophenol blue method on a spectrophotometer provided with a flow cuvette system. The net mineralization capacity is calculated as the difference in ammonium content at the start and end of incubation. Specific Microbial Soil Functions Nitrification. Autotrophic nitrification is the two-step process by which ammonia is first oxidized to nitrite and then further to nitrate (20). In this aerobic process nitrifying bacteria gain energy for growth and reducing capacity to fix carbon dioxide. Nitrification ability is restricted to only a few bacterial species, all within the family Nitrobacteriaceae. Owing to their complicated metabolic machinery, nitrifying bacteria are sensitive to various environmental disturbances and thus can be used as organisms indicative of low levels of stress. Another reason for using nitrification in a test system is its important role in the biogeochemical cycle of nitrogen. Two basic nitrification assays are used: 1) assessing only the first step by analysis of nitrite after a short incubation period and 2) assessing the full nitrification pathway by analysis of ammonium and/or nitrate after a longer incubation period. The former assay has become increasingly popular because of its rapidity and simplicity (21). In the assay a soil slurry is generated by adding an optimum concentration of ammonium dissolved in a buffer (pH 7.2). The second step in the nitrification pathway is blocked with chlorate. The product, nitrite, can easily be analyzed colorimetrically. To increase the analysis capacity, the use of an automated spectrophotometer technique is recommended. As NH4 + oxidizing bacteria have long generation times (>10 hours), the rate of product formation will be constant when short incubation periods are used. This means that data can be evaluated by linear regression. The NH4 + oxidation test is now in its final revision in becoming an ISO standard (ISO DIS 15685). Denitrification. Biological denitrification is the process by which nitrogenous oxides, mainly nitrate and nitrite, are reduced to the nitrogen gases nitric oxide, nitrous oxide, and dinitrogen (20). Most denitrifiers prefer oxygen as the terminal electron acceptor and therefore reduce nitrogenous oxides only under anaerobic conditions. The


complex pathway of denitrification is not fully understood, but is thought to consist of more than 26 genes and to be regulated both at the enzyme and gene levels by a number of environmental factors. Moreover, the steps in the denitrifying pathway have different sensitivities to various kinds of disturbances. Denitrification is a functional trait found within many taxonomical and physiological groups of bacteria. The genera Pseudomonas, Alcaligenes, and Bacillus are thought to be the most frequently found denitrifiers in soil. However, the list of denitrifiers is increasing with the introduction of molecular biology techniques for identification and determination of taxonomic relationships. Thus, denitrifiers can be viewed as representatives of a broad range of soil microbial populations. Denitrification is not just a process where nitrogen is lost to the atmosphere but can also indicate easily available organic carbon, as most denitrifiers are organotrophic and mineralize organic matter both under aerobic and anaerobic conditions. A common way to characterize denitrification in soil is to determine the potential denitrifying activity (PDA). In this method a soil slurry is incubated anaerobically with additions of an optimum amount of nitrate and an easily available carbon and energy source, such as glucose (22). At the start of the incubation, acetylene is added to block the last step in the denitrification pathway, the reduction of nitrous oxide to nitrogen. The accumulated product, nitrous oxide, is then analyzed on a gas chromatograph (GC) equipped with an electron capture detector (ECD). In this assay, problems with substrate diffusion have been eliminated and thus only the amounts of denitrifying enzymes will be rate limiting. The use of chloramphenicol (CAP) has been suggested to lock the enzyme concentration at its initial test concentration. The result will be a prolonged initial phase of linear product formation. Unfortunately, CAP seems to affect not only the synthesis but also the activity of denitrification enzymes (22,23). Recently, it has been demonstrated that it might be possible to use low concentrations of CAP (

Encyclopedia of Agrochemicals, 3 Volume Set

Encyclopedia of Computational Mechanics (3 Volume Set)

Encyclopedia of Wars - 3 Volume Set

Encyclopedia of Revolutionary America, 3-Volume Set

Macmillan Encyclopedia of Energy (3 Volume Set)

The Internet Encyclopedia, 3 Volume Set

The Human Rights Encyclopedia, 3 Volume Set

Encyclopedia of Gastroenterology, Three-Volume Set, Volume 1-3

Encyclopedia of Archaeology, Three-Volume Set, Volume 1-3

International Encyclopedia of Human Geography, Twelve-Volume Set: Volume 3

The Internet Encyclopedia, 3 Volume Set (Vol 1-3)

Encyclopedia of Genocide and Crimes Against Humanity - 3 Volume Set

Encyclopedia of Toxicology, Four-Volume Set, Second Edition: 1 - 3

Encyclopedia of Genocide and Crimes Against Humanity - 3 Volume Set

Encyclopedia of Clothing and Fashion 3-Volume Set

Encyclopedia of Clothing and Fashion 3-Volume Set

Encyclopedia of Clothing and Fashion. 3-Volume Set

Encyclopedia of Food and Culture (3 Volume Set)

Encyclopedia of Grain Science, Three-Volume Set (Vol 1-3)

Encyclopedia of Clothing and Fashion 3-Volume Set

UXL Encyclopedia of Biomes, 2nd Edition 3 Volume Set

Encyclopedia of Genocide and Crimes Against Humanity - 3 Volume Set

Encyclopedia of Measurement and Statistics 3-Volume Set

Encyclopedia of Measurement and Statistics 3-Volume Set

Encyclopedia of Wars3 Volume of Set

Encyclopedia of Digital Government (3-Vol. Set)

Encyclopedia of Law Enforcement (3 Vol Set)

Encyclopedia of chemical compounds. 3-Vol. set

Encyclopedia of Aging (Two Volume Set)

Encyclopedia of Polymer Research (2 Volume Set)

Encyclopedia of Agrochemicals, 3 Volume Set

Encyclopedia of Computational Mechanics (3 Volume Set)

Encyclopedia of Wars - 3 Volume Set

Encyclopedia of Revolutionary America, 3-Volume Set

Macmillan Encyclopedia of Energy (3 Volume Set)

The Internet Encyclopedia, 3 Volume Set

The Human Rights Encyclopedia, 3 Volume Set

Encyclopedia of Gastroenterology, Three-Volume Set, Volume 1-3

Encyclopedia of Archaeology, Three-Volume Set, Volume 1-3

International Encyclopedia of Human Geography, Twelve-Volume Set: Volume 3

The Internet Encyclopedia, 3 Volume Set (Vol 1-3)

Encyclopedia of Genocide and Crimes Against Humanity - 3 Volume Set

Encyclopedia of Toxicology, Four-Volume Set, Second Edition: 1 - 3

Encyclopedia of Genocide and Crimes Against Humanity - 3 Volume Set

Encyclopedia of Clothing and Fashion 3-Volume Set

Encyclopedia of Clothing and Fashion 3-Volume Set

Encyclopedia of Clothing and Fashion. 3-Volume Set

Encyclopedia of Food and Culture (3 Volume Set)

Encyclopedia of Grain Science, Three-Volume Set (Vol 1-3)

Encyclopedia of Clothing and Fashion 3-Volume Set

UXL Encyclopedia of Biomes, 2nd Edition 3 Volume Set

Encyclopedia of Genocide and Crimes Against Humanity - 3 Volume Set

Encyclopedia of Measurement and Statistics 3-Volume Set

Encyclopedia of Measurement and Statistics 3-Volume Set

Encyclopedia of Wars3 Volume of Set

Encyclopedia of Digital Government (3-Vol. Set)

Encyclopedia of Law Enforcement (3 Vol Set)

Encyclopedia of chemical compounds. 3-Vol. set

Encyclopedia of Aging (Two Volume Set)

Encyclopedia of Polymer Research (2 Volume Set)

Recommend Documents