Advances in Environmental Research, Volume 13

ADVANCES IN ENVIRONMENTAL RESEARCH

ADVANCES IN ENVIRONMENTAL RESEARCH. VOLUME 13 No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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ADVANCES IN ENVIRONMENTAL RESEARCH

ADVANCES IN ENVIRONMENTAL RESEARCH. VOLUME 13

JUSTIN A. DANIELS EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

ISBN: 978-1-61209-049-8 (eBook)

ISSN: 2158-5717

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface

vii

Short Commentary The Global Extent of Black C in Soils: Is It Everywhere? Evelyn Krull, Johannes Lehmann, Jan Skjemstad, Jeff Baldock and Leonie Spouncer

3

Research and Review Studies Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Current and Emerging Microbiology Issues of Potable Water in Developed Countries William J. Snelling, Catherine D. Carrillo, Colm J. Lowery John E. Moore, John P. Pezacki, James S. G. Dooley and Roy D. Sleator Vermiculture Biotechnology: The Emerging Cost-Effective and Sustainable Technology of the 21st Century for Waste and Land Management to Safe and Sustainable Food Production Rajiv K. Sinha, Sunil Herat, Gokul Bharambe, Swapnil Patil, Uday Chaudhary, Priyadarshan Bapat, Ashish Brahambhatt, David Ryan, Dalsukh Valani, Krunal Chauhan, R. K. Suhane and P. K. Singh

11

41

Human Waste - A Potential Resource: Converting Trash into Treasure by Embracing the 5 R‘s Philosophy for Safe and Sustainable Waste Management Rajiv K. Sinha, Sunil Herat, Gokul Bharambe, Swapnil Patil, Pryadarshan Bapat, Krunal Chauhan and Dalsukh Valani

111

Effective Removal of Low Concentrations of Arsenic and Lead and the Monitoring of Molecular Removal Mechanism at Surface Yasuo Izumi

173

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) Loads in Mink Accompanying Parasitic Infection by the Giant Kidney Worm (Dioctophyme Renale) Glenn H. Parker and Liane Capodagli

187

vi

Contents

Chapter 6

Aerobically Biodegraded Fish-Meal Wastewater as a Fertilizer Joong Kyun Kim and Geon Lee

219

Chapter 7

Equity of Access to Public Parks in Birmingham, England Andrew P. Jones, Julii Brainard, Ian J. Bateman and Andrew A. Lovett

237

Chapter 8

An Idea for Phenomenological Theory of Living Systems Svetla E. Teodorova

257

Chapter 9

A New Trait of Gentoo Penguin: Possible Relation to Antarctica Environmental State? Roumiana Metcheva, Vladimir Bezrukov, Svetla E.Teodorova and Yordan Yankov

Chapter 10

Assessing Population Viability of Focal Species Targets in the Western Forest Complex, Thailand Yongyut Trisurat and Anak Pattanavibool

Chapter 11

Protection of Riparian Landscapes in Israel Tseira Maruani and Irit Amit-Cohen

Chapter 12

Hydraulic Characterization of Aquifer(s) and Pump Test Data Analysis of Deep Aquifer in the Arsenic Affected Meghna River Floodplain of Bangladesh Anwar Zahid, M. Qumrul Hassan, Jeff L. Imes and David W. Clark

Chapter 13

Application of DNA Microarrays to Microbial Ecology Research: History, Challenges, and Recent Developments John J. Kelly

271

285 305

325

357

Chapter 14

Food Safety in India: Challenges and Opportunities Wasim Aktar

Chapter 15

Impact of Pesticide Use in Indian Agriculture Their Benefits and Hazards Wasim Aktar

423

Ozone Decomposition by Catalysts and its Application in Water Treatment: An Overview J. Rivera-Utrilla, M. Sánchez-Polo and J. D. Méndez-Díaz

433

Chapter 16

Chapter 17

Index

Use of Microarrays to Study Environmentally Relevant Organisms: A UK Perspective Michael J. Allen, Andrew R. Cossins, Neil Hall, Mark Blaxter, Terry Burke and Dawn Field

385

465

481

PREFACE Short Communication - The latest projections of the Intergovernmental Panel on Climate Change (IPCC) estimate a 3°C increase in global temperatures within the next 100 years (IPCC 4th Assessment Report, 2007), and global warming is seen as a major driver in accelerating decomposition of soil organic matter, resulting in increased production of CO2 (e.g. Davidson and Janssens, 2006). The 2007 IPCC report recommended coupling models of terrestrial biogeochemical and atmospheric and oceanic processes in order to improve general circulation models and to recognize the quantitative value of soil organic carbon (SOC) in the global carbon cycle. Estimates of CO2 emissions from soil rely on predictions of the response of different SOC pools to global warming and correct estimation of the size of these pools. In comparison to the pool representing the most stable and biologically unreactive fraction, commonly referred to as passive or inert organic carbon (IOC), the decomposition of labile C is expected to be faster as a response to temperature increase. IOC is a fraction of the SOC pool that is not readily available for microbial decomposition and has turnover times exceeding 100 years (e.g. Krull et al., 2003). Black C (BC) is usually considered the most abundant form of IOC and is defined as the ‗carbonaceous residue of incomplete combustion of biomass and fossil fuels‘ (Schmidt and Noack, 2000). BC is important to several biogeochemical processes; for example, BC potentially modifies climate by acting as a potential carbon sink for greenhouse gases (Kuhlbusch, 1998) and leads to increasing solar reflectance of the Earth‘s atmosphere, but also to a heating of the atmosphere (Crutzen and Andreae, 1990). BC production from fossil fuel combustion contributes to aerosol C, decreasing surface albedo and solar radiation (IPCC 4th Assessment Report). Due to its condensed aromatic structure, BC has a low biochemical reactivity. 14C ages of BC in soils vary between 1160 and 5040 years (e.g. Schmidt et al. 2002). Chapter 1 - Water is vital for life; for commercial and industrial purposes and for leisure activities in the daily lives of the world‘s population. Diarrhoeal disease associated with consumption of poor quality water is one of the leading causes of morbidity and mortality in developing countries (especially in children 100,000), and in many countries, e.g. Denmark, Yersinia interest and concern have declined (Skovgaard, 2007).

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 21

PROTOZOAN PARASITES At least 325 water-associated outbreaks of parasitic protozoan disease have been reported (Karanis et al., 2007). North American and European outbreaks accounted for 93% of all reports and nearly two-thirds of outbreaks occurred in North America (Karanis et al., 2007). Over 30% of all outbreaks were documented from Europe, with the UK accounting for 24% of outbreaks, worldwide (Karanis et al., 2007). Giardia duodenalis and C. parvum account for the majority of outbreaks (132; 40.6% and 165; 50.8%, respectively), Entamoeba histolytica and Cyclospora cayetanensis have been the aetiological agents in nine (2.8%) and six (1.8%) outbreaks respectively, while Toxoplasma gondii and Isospora belli have been responsible for three outbreaks each (0.9%) and Blastocystis hominis for two outbreaks (0.6%) (Karanis et al., 2007). Balantidium coli, the microsporidia, Acanthamoeba and Naegleria fowleri were responsible for one outbreak each (0.3%) (Karanis et al., 2007). Waterborne parasites produce transmission stages which are highly resistant to external environmental conditions, and to many physical and chemical disinfection methods routinely used as bacteriocides in drinking water plants, swimming pools or irrigation systems (Gajadhar and Allen, 2004). Resistant stages include cysts of amoebae, Balantidium, and Giardia, spores of Blastocystis and microsporidia, oocysts of Toxoplasma gondii, Isospora, Cyclospora and Cryptosporidium and eggs of nematodes, trematodes, and cestode(Gajadhar and Allen, 2004). These exogenous transmission stages are microscopic in size and of low specific gravity, which facilitate their easy dissemination in fresh water, or seawater (Gajadhar and Allen, 2004). The exogenous stages of waterborne parasites possess outer surfaces capable of withstanding a variety of physical and chemical treatments (Gajadhar and Allen, 2004). The resistant surfaces are comprized of multiple polymeric layers of lipids, polysaccharide, proteins or chitin (Gajadhar and Allen, 2004). Examples of these are the two protein layers of coccidian oocysts derived from the coalescence of wall-forming bodies, the chitinous wall of microsporidian spores, the multi-layered (inner lipid/protein-, middle protein/chitin-, outer protein/mucopolysaccharide) shell of Ascaris eggs, and the impermeable embryophore of the Echinococcus egg which is constructed of polygonal blocks of keratinlike protein held together by a cement substance (Gajadhar and Allen, 2004). The intestinal protozoan parasites Cryptosporidium (Apicomplexan) and Giardia (G. duodenalis) are major global causes of diarrhoeal disease in humans (Smith et al., 2007). Significantly, normal concentrations of chlorine and ozone used in mass water treatment are not adequate to kill these microbes (Smith et al., 2007), which have life cycles suited to both waterborne and foodborne transmission (Smith et al., 2007). Giardia causes intestinal malabsorption and diarrhoea (giardiasis) in humans and other mammals worldwide (Smith et al., 2007). Giardia is one of the most prevalent pathogens that should be removed from drinking water (Smith et al., 2007). In developing countries, the prevalence of human giardiasis is on average 20% (4–43%), compared with 5% (3–7%) in developed countries, where it is associated mainly with travel and waterborne outbreaks (Smith et al., 2007). G. duodenalis assemblages A and B have been found in humans and most mammalian orders (Smith et al., 2007). Giardiasis is a disease with economic ramifications due to the large impact on domestic animals such as cattle and sheep (Smith et al., 2007). The role of animal transmission of human giardiasis is unclear, but the greatest risk of zoonotic transmission seems to be from companion animals such as dogs and cats. Interestingly, the capacity of

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Acanthamoeba to predate Cryptosporidium oocysts has recently been demonstrated (GómezCouso et al., 2007). Free-living-amoeba (FLA) may act as environmentally resistant carriers of Cryptosporidium oocysts and, thus, may play an important role in the transmission of cryptosporidiosis (Gómez-Couso et al., 2007). Zoonotic Cryptosporidium parvum and anthroponotic C. hominis are the major cause of human cryptosporidiosis, although other species including C. meleagridis, C. felis, C. canis, C. suis, C. muris and two corvine genotypes of Cryptosporidium have been associated with human gastroenteritis (Xiao and Ryan 2004; Caccio et al. 2005). Cryptosporidium can survive for months in a latent form outside hosts, as its oocysts retain their infectivity for several months in both salt and fresh water (Fayer et al. 1998; Sunnotel et al., 2006a). Cryptosporidium causes self-limited watery diarrhoea in immunocompetent subjects, but has far more devastating effects in the immunocompromized and in some cases can be lifethreatening due to dehydration caused by chronic diarrhoea (Caccio 2005, Chen et al. 2005). Cryptosporidiosis is responsible for significant neonatal morbidity in farmed livestock and causes weight loss and growth retardation, leading to significant economic losses (McDonald 2000). Over the last two decades, increasing numbers of cryptosporidiosis (in particular water related) outbreaks have been recorded in developed countries (Craun et al. 2005). In 1993, the largest Cryptosporidium outbreak was registered in Milwaukee, Wisconsin, USA where 403,000 people were infected through contaminated drinking water (MacKenzie et al. 1994). This outbreak was caused by C. hominis (Peng et al. 1997), and the total cost of outbreakassociated illness was estimated at >$96 million in both medical costs and productivity losses (Corso et al. 2003). Cryptosporidium has also been associated with treated water in swimming and wading pools (Craun et al. 2005). Shellfish have the ability to filter large amounts of water and concentrate oocysts within their gills (Gomez-Couso et al. 2006). Thus, despite prevention measures, e.g. standard UV depuration treatment, the consumption of raw or undercooked shellfish, may still be a potential health risk (Sunnotel et al., 2007). Also, the use of surface water for irrigation can indirectly cause human infection via the consumption of contaminated fresh produce, e.g. lettuce (Shigematsu et al., 2007). Outbreaks have been reported in healthcare facilities and day-care centres, within households, among bathers and water sports enthusiasts in lakes and swimming pools, and in municipalities with contaminated public water supplies or people served by private water supplies. In 2005 the European Basic Surveillance Network (BSN) recorded 7,960 human cases of cryptosporidiosis from 16 countries. Microsporidial gastroenteritis; a serious disease of immunocompromized people, can have a waterborne etiology (Graczyk et al., 2007). Microsporidia are obligate intracellular eukaryotes parasitizing a wide range of invertebrates and vertebrates with over 1,200 species, of which 14 are opportunistic human pathogens, with Encephalitozoon intestinalis, E. hellem, E. cuniculi, and Enterocytozoon bieneusi being the most common (Graczyk et al., 2007). Currently, Microsporidia are on the Contaminant Candidate List of the U.S. Environmental Protection Agency due to their unknown transmission routes, technologically challenging identification and the difficult treatment of human infections (Graczyk et al., 2007). Considerable evidence indicates involvement of water in the epidemiology of microsporidiosis, however, this link has not been conclusively substantiated (Graczyk et al., 2007). Risk factor analysis for encephalito zoonosis has previously suggested groundwater as a source of infection, and a massive outbreak of microsporidiosis was epidemiologically

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 23 linked to a drinking water distribution system (Cotte, et al., 1999). More accurate Microsporidial epidemiological data is urgently required to accurately assess the importance of this emerging pathogen.

EMERGING AND OPPORTUNISTIC PATHOGENS Opportunistic microbes are a subset of the emerging pathogens and include species of both fecal and environmental origin. Treated potable water contains a variety of microbes that are not well characterized (Stelma et al., 2004). Many of these organisms grow slowly and require nutrient-poor media for culturing (Stelma et al., 2004). Although there is evidence that these microbes are generally not hazardous to the general healthy population, there is a possibility that some of them may be opportunistic pathogens and may be capable of causing adverse health effects in individuals with impaired body defences (Stelma et al., 2004). Examples of opportunistic bacterial pathogens of potable water origin include Aeromonas hydrophila, L. pneumophila, M. avium, and Pseudomonas aeruginosa. There is no reason to assume that the currently known opportunistic pathogens are the only opportunistic pathogens indigenous to potable water (Stelma et al., 2004). Many cases of respiratory infections and digestive system infections are still of unknown etiology and it is possible that some of them could be due to pathogens that are currently unknown (Stelma et al., 2004). Opportunistic bacterial pathogens have the potential to reproduce in the natural environment, particularly in the presence of increased temperatures and nutrients, often when associated with free-living protozoa (Långmark et al., 2007). Free-living protozoa are in most cases non-parasitic, however, some species are known to cause human illness (Långmark et al., 2007). The potential significance of free-living protozoa, e.g. amoeba, as potential environmental reservoirs for aquatic pathogens has been recognized for more than twenty years (King et al., 1988), as has the often complex interspecies relationships between mixed biofilm populations, e.g. bacteria and protozoa (Snelling et al., 2006; Långmark et al., 2007). Yet despite this, with the exception of L. pneumophila, relatively little attention has been payed to studying and understanding interactions between free-living protozoa and other smaller human pathogens (Brown and Barker, 1999; Snelling et al., 2006). Some microorganisms have evolved to become resistant to protozoa digestion and these amoebaresistant microorganisms include established bacterial pathogens, such as Legionella spp., Chlamydophila pneumoniae, M. avium, P. aeruginosa, and C. jejuni, and emerging pathogens such Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens (Snelling et al., 2006). The fate of internalized bacteria can be divided into three main outcomes; i)

Bacteria which multiply and cause lysis of amoebal cells (FLA), e.g. Legionella and Listeria monocytogenes. ii) Bacteria which multiply without causing cell lysis, e.g. Vibrio cholera. iii) Bacteria which survive without multiplication, e.g. mycobacteria (Snelling et al., 2006).

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FLA feed on bacteria, fungi and algae and as such are ubiquitous predators that control microbial communities while at the same time acting as reservoirs for many human pathogens. FLAs are thus often regarded as the ―Trojan horses‖ of the microbial world (Chang and Jung, 2004; Greub and Raoult 2004; Molmeret et al., 2005). Dictyostelium discoideum is an established host model for several pathogens including, P. aeruginosa, Mycobacterium spp., and L. pneumophila (Snelling et al., 2006). Significantly, numerous bacterial species survive disinfection treatments when internalized within protozoa, compared to the exposed and thus more sensitive planktonic state which forms the basis of current disinfection policies (Snelling et al., 2005b; King et al., 1998; Whan et al., 2006). Interactions such as these are unaccounted for in current disinfection models (Berry, et al., 2006). FLA provide habitats for the environmental survival of L. pneumophila, which have been observed undergoing binary fusion within intracellular vacuoles of amoeba (Atlas, 1999; Colbourne et al., 1984). To date it has never been shown that Legionella can multiply in its own natural environment outside protozoa (Atlas, 1999; Colbourne et al., 1984). Legionella pneumophila are fastidious bacteria which develop mostly in water, causing legionnaires‘disease [(LD), legionellosis, or atypical pneumonia/flu-like illness] in humans (Stout et al. 1992; Stojek and Dutkiewicz, 2006). In environmental water bodies L. pneumophila proliferates intracellularly in more than 15 protozoan genera, e.g. Acanthamoeba, Hartmannella, Vahlkamphia and Ehinamoeba (Steinert et al., 1994). Infection of humans, generally the elderly and immunocompromized, occurs after inhalation of aerosolized bacteria from contaminated water sources (Steinert et al., 1994). The infectious particle is unknown, but may be excreted as legionellae-filled vesicles, intact legionellaefilled amoebae, or free legionellae that have lysed their host cell, resulting in L. pneuomphila, not amoeba, persisting within the respiratory tract (Steinert et al., 1994). The processing of L. pneumophila by A. castellanii shows many similarities to monocyte phagocytosis, e.g. the uptake of L. pneumophila by coiling phagocytosis (Steinert et al., 1994). Phagosome–lysosome fusion is prevented based upon L. pneumophila expressing dot/icm genes that code for a putative large membrane complex which forms a type IV secretion system that is used to alter the endocytic pathway (Steinert et al., 1994). Genes such as hfq appear to play a major role in exponential phase regulatory cascades of L. pneumophila (McNealy et al., 2005; Solomon and Isberg, 2000). Globally, potable water supplies which harbour L. pneumophila are important sources of community acquired LD (Stout et al. 1992). LD accounts for an estimated 8,000 to 18,000 cases of hospitalized community-acquired pneumonia in the United States annually (Phares et al., 2007). Individuals are most often infected with Legionella by inhaling bacteria-laden aerosol droplets, e.g. via bathing, but can also become infected by the oral route through drinking water or via traumatized skin or mucous membranes (Stojek and Dutkiewicz, 2006). Approximately 35% of all LD cases reported to the Centers for Disease Control and Prevention (CDC) are acquired in health care facilities (Phares et al., 2007). M. avium is a common cause of systemic bacterial infection in patients with AIDS (Miltner and Bermudez, 2000; Snelling et al., 2006). Infection with M. avium, commonly occurs through the gastrointestinal tract and has been linked to bacterial colonization of domestic water supplies, where A. castellanii may serve as an environmental host for M. avium AIDS (Miltner and Bermudez, 2000). M. avium can also survive within A. polyphaga cyst outer walls and bacterial growth occurs in cocultures (Steinert et al., 1998; Snelling et al., 2006). M. avium enters and replicates in A. castellanii, and similar to mycobacteria within

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 25 human macrophages, inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae (Cirillo et al., 1997). Growing M. avium in amoebae enhances invasion and intracellular replication of the bacterium in human macrophages, the intestinal epithelial cell line HT-29, as well as in mice (Miltner and Bermudez, 2000), and also increases resistance to rifabutin, azithromycin, and clarithromycin, which might have significant implications for prophylaxis of M. avium infection in AIDS patients (Miltner and Bermudez, 2000).

INDICATOR METHODS For more than thirty years the measurement of bacteria of the coliform group has been extensively relied on as an indicator of water quality (Yáñez et al., 2006). Among the coliforms, the specific determination of E. coli contamination can be performed as one of the best means of estimating the degree of recent fecal pollution (Edberg et al., 2000). The European Drinking Water Directive 98/83/EC (Anon, 1998) defines that the isolation of coliforms using Lactose TTC agar with Tergitol (Tergitol-7 agar) by membrane filtration (Anon, 2000) be the reference method for the enumeration of total coliforms, including E. coli in drinking water (Yáñez et al., 2006). However, numerous outbreaks, e.g. Crytosporidium, Campylobacter, and viruses, have made it clear that the presence of bacterial indicators of fecal contamination does not consistently correlate with pathogen levels (De Paula et al., 2007). Alternatively, a number of instrumental methods for the rapid determination of microorganisms based on their metabolic activity have been developed, but not really utilized, for example, impedance (Madden and Gilmour, 1995), conductance (Gibson, 1987), chemiluminescence and fluorescence (Van Poucke and Nelis, 2000). Different chromogenic and/or fluorogenic culture media have also been developed in recent years (Manafi, 2000). One is TBX agar medium, a modification of Tryptone bile agar medium where the substrate 5-bromo-4-chloro-3-indolyl-b-d-glucuronide (BCIG) is added. This substrate is cleaved by the enzyme β-d-glucuronidase (GUD) that is produced by approximately 95% of the E. coli strains investigated (Adams et al., 1990), and the released chromophore produces easy to read blue-green coloured E. coli colonies. Furthermore, the TBX agar method complies with the ISO/DIS Standard 16649 for the enumeration of E. coli in food and animal foodstuffs (Anon, 2001). In addition, different chromogenic media such as Chromocult Coliform agar (CC agar), and coli ID agar have been developed for the simultaneous detection of total coliforms and E. coli, based on the presence of chromogenic substrates for the enzymes β–galactosidase (Lac) and β-D-glucuronidase (GUD) (Geissler et al., 2000). Results from the evaluation of these two media have been reported previously (Finney et al., 2003). In spite of their advantages, the main drawback of chromogenic media is the relatively high cost for routine analysis laboratories where a great number of analysis are performed (Yáñez et al., 2006). The results obtained with the combination of the two media, Tergitol agar and TBX agar, for the enumeration of total coliforms and E. coli have been comparable to those obtained with the individual chromogenic media assayed, CC agar and coli ID (Yáñez et al., 2006). However, the combined method has the advantage of reduced analytical cost since the TBX agar is used only for total coliform-positive samples (Yáñez et al., 2006). In addition, using

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this small modification, the enumeration of E. coli in total coliforms-positive samples can be performed in only two hours, maintaining the advantages of rapid turn-around time and the specificity of the individual chromogenic media (Yáñez et al., 2006). The combination of Tergitol agar with TBX fulfils the European Drinking Water Directive where the ISO Standard 9308 is indicated for the analysis of total coliforms and E. coli (Yáñez et al., 2006). Moreover, this small modification provides a simple and comparatively cost-effective method that is as specific and rapid as the chromogenic media, and which can be used easily by laboratories dedicated to routine analysis of water (Yáñez et al., 2006).

RAPID DETECTION METHODS Many bacterial pathogens are routinely grown on selective/differential agar or broth, e.g. Shigella (Niyogi et al., 2005). Infection with E. coli O157:H7 is diagnosed by detecting the bacterium from stool samples (CDC, 2007b). About one-third of laboratories that culture stool still do not test for E. coli O157:H7, so it is important to request that the stool specimen be tested on sorbitol-MacConkey (SMAC) agar for this organism (CDC, 2007b). All individuals presenting with bloody diarrhoea should have their stool tested for E. coli O157:H7 (CDC, 2007b). However, a major limitation of enrichment methods is the length of time required to complete the testing, since at least two days are needed for the complete identification of bacterial typical colonies (Yáñez et al., 2006). To overcome this problem, new techniques are continually being developed, and in particular, molecular biology methods appear to be interesting alternatives for rapidly detecting pathogens in water samples (Yáñez et al., 2006). To maximise the useful impact of epidemiological data, it is important to be able to rapidly detect and identify low numbers of oocysts from different sample types, including water, and if possible to ascertain if they are viable (Sunnotel et al., 2006a; Sunnotel et al., 2006b). It is important to be able to accurately differentiate between nonpathogenic and pathogenic species of Cryptosporidium, and between different Cryptosporidium isolates of the same species (Sunnotel et al., 2006a; Sunnotel et al., 2006b). Also, the rapid and accurate detection and identification of E. coli O157:H7 and V. cholerae O139 pathogens is crucial for diagnosis, treatment and eventual control of the contagious disease outbreak (Jin et al., 2007). Accurate and reliable epidemiological and molecular typing techniques are crucial for tracing sources of pathogen infection, the recognition of which is important for the implementation of preventive measures (Fendukly et al., 2007). Methods include ribotyping, amplified fragment length polymorphism analysis (AFLP), pulsed field gel electrophoresis (PFGE), restriction fragment length polymorphism analysis (RFLP), restriction endonuclease analysis (REA), and arbitrary primed PCR (Fendukly et al., 2007). Nevertheless, molecular techniques are unfortunately still incompatible with most routine water laboratories because of the expense and the need for trained personnel (Angles d'Auriac et al., 2000). Disappointingly, despite the steady improvement in modern molecular biology techniques, the epidemiology of many infections remains unclear (Snelling et al., 2005b). This confusing epidemiological evidence is partly because of the lack of standard global typing methods and communication between laboratories (Wassenaar and Newell 2000). However, these

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 27 problems are being addressed via initiatives like CAMPYNET (Wassenaar and Newell 2000) and PulseNet for standard molecular typing. Nucleic acid based detection methods such as PCR and real-time PCR are more rapid and sensitive than traditional culturing techniques. However, they are limited by the number of targets that can be detected in a single assay. PCR products can be multiplexed, but typically not more than six targets can be assayed at a single time, and optimizing such a complex reaction can be challenging. For example, primers must not interact with one another to form primer-dimers, and amplification of each target must proceed with equal efficiency. Analysis of the products of the reaction requires size separation by electrophoresis, thus amplicons must be of significantly different size in order to be easily distinguished. Real-time PCR (qPCR) is more rapid as it monitors the amount of PCR products as they are being produced and does not require size separation (Kubista et al. 2006; Zhang and Fang, 2006). In addition qPCR is more sensitive than traditional PCR methods and can estimate the initial concentration of nucleic acids. As with standard PCR methods, multiplexing qPCR is limited by the difficulty in optimizing reactions so that all targets are amplified with similar efficiencies. Moreover, the number of assays that can be multiplexed is limited by the number of fluorescent dyes that can be distinguished in a single reaction (typically no more than 4, depending on the specifications of the instrument being used). Saker et al., (2007) found that PCR can be used to detect inocula for cyanobacterial populations and therefore provides a useful tool for assessing which conditions particular species can grow into bloom populations. PCR methods were particularly useful when the concentration of the target organism was very low compared with other organisms (Saker et al., 2007). The European Working Group for Legionella Infections (EWGLI) has implemented the AFLP and more recently the sequence-based typing (SBT) to define its collection of L. pneumophila serogroup 1 strains (Fendukly et al., 2007). However, the SBT has not been widely used for genotyping non-serogroup 1 L. pneumophila isolates (Fendukly et al., 2007). Of the many phenotypical, immunological and molecular typing methods which have been implemented for the characterization and epidemiological typing of L. pneumophila, only the AFLP and the SBT techniques have gained acceptance by the EWGLI. Both methods were shown to have good reproducibility and accuracy and obviated the need to submit Legionella strains between microbiological laboratories in different countries (Fendukly et al., 2007). The AFLP gel images were, however, more difficult to interpret (Fendukly et al., 2007). This method allows for comparison of the allelic profile of an isolate to previously assigned allele numbers of 6 genes in the following order: flaA, pilE, asd, mip, mompS and proA (Fendukly et al., 2007). Some enteric viruses grow poorly in cell culture, which constitutes a problem when investigating strategies for virus control and prevention (Atmar and Estes, 2001; De Paula et al., 2007). Current methods of detecting such viruses in environmental water samples rely on genome amplification using molecular techniques such as qualitative and quantitative realtime reverse-transcription polymerase chain reaction (RT–PCR) (De Paula et al., 2007). Worldwide, virus detection in environmental and potable water samples is becoming an important strategy for preventing outbreaks of infection with waterborne viruses, e.g. HAV (De Paula et al., 2007). To evaluate the public health threat posed by V. cholerae occurring in water, a rapid and accurate method for detection of toxigenic V. cholerae is essential (Chomvarin et al., 2007).

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The culture method (CM), which is routinely used for assessing water quality, has not proven as efficient as molecular methods because this notorious pathogen survives in water mostly in the viable but non-culturable state (VBNC) (Chomvarin et al., 2007). The isolation and identification of V. cholerae by CM is expensive, time-consuming, labour intensive, and unable to precisely distinguish between toxigenic V. cholerae and non-toxigenic V. cholerae (Chomvarin et al., 2007). Fluorescent monoclonal antibody combined with molecular genetic based methods can demonstrate the presence of toxigenic V. cholerae in the aquatic environment (Chomvarin et al., 2007). The last three decades have witnessed exponential progress in our understanding of the biology of pathogens. The complete sequencing of the human genome and of many microbial pathogens associated with potable water have rapidly fuelled the development of gene array technology, improved pathogen detection systems and analysis methods. A host of different molecular techniques for studying the transmission dynamics and detection of drug resistant pathogens have been developed (Katoch et al., 2007). DNA microarrays (or gene arrays) consist of segments of DNA called probes or reporters arrayed on a solid surface (Call, 2005; Lemarchand, 2004). DNA spots are typically very small, on the micrometer length scale, and can be arrayed in high density with hundreds to thousands of spots present on a single array. Detection using microarrays is based on the hybridization of fluorescently labelled nucleic acids (RNA or DNA) isolated from the test matrix to these probes, and subsequent measurement of fluorescence at each spot. Microarrays allow the simultaneous detection of thousands of targets in a single assay. Thus, a single array could be used for the detection of a wide range of pathogens, virulent strains of a particular pathogen, antimicrobial resistant strains, and even molecular typing. Microarrays have been applied to microbial quality monitoring of water (Lee et al., 2006; Lemarchand et al., 2004; Maynard et al., 2005). However, methods involving direct hybridization onto microarrays are inherently lower in sensitivity relative to PCR because of the lack of oligonucleotide amplification. For example, Lee et al. (2006) were only able to detect pathogens in wastewater at levels of 2 x 108 copies of the target gene, using direct hybridization of DNA isolated from wastewater. Straub et al. (2005) have developed a microarray-based system for analysis of RNA isolated from microbial communities. This system shows great promise, as detection of RNA could be linked to the presence of viable cells; however, the sensitivity of this system is currently too low to be useful for pathogen detection. Sensitivity can be greatly improved by PCR amplification of the target, before hybridization to the array. However, production of multiple PCR products can be labour intensive and slow. Amplification of universal genes (i.e. rRNA genes) gets around this problem as a single primer set can be used to amplify a segment from a gene that is conserved among the pathogens of interest. Species-specific sequence variability within the amplicon is detected by hybridization of the PCR products to a microarray. Using this method, with the 23s rRNA gene, Lee et al. (2006) demonstrated an exponential increase in the sensitivity of their array to 20 copies of the target gene. Maynard et al. (2005) used a similar strategy, but improved discriminatory power of their array by the inclusion of multiple genes (cpn60, 16s rRNA and wecE ). They were able to detect 100 copies of a target gene, but this detection level was decreased when the genomic DNA of interest was in a background of complex DNA mixtures. PCR amplification favors the amplification of the most abundant bacterial species and low-level targets may be underrepresented, or undetectable on the array. This is

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 29 an important problem, as pathogens are likely to represent a small proportion of the microbial communities in water supplies. Kostic et al. (2007) were able to overcome this problem to a degree by using a unique labelling method (sequence-specific end-labelling of oligonucleotides) and inclusion of competitive oligonucleotides during labelling. They were able to detect pathogens which comprised 0.1% of the total microbial community analyzed. Wu et al. (2006) used a method of total DNA amplification (multiple displacement amplification) to non-specifically amplify total genomic DNA isolated from contaminated groundwater prior to microarray hybridization. This method seems promising, as it appears to eliminate the bias of PCR based amplification, while increasing the sensitivity of the microarray-based assay. Microarrays have proven to be useful tools for monitoring microbial communities in water and environmental samples. However, there are practical limitations in the use of microarray-based approaches including the requirement of specialized equipment and trained personnel. Automation of array systems would contribute significantly to the ease of deployment of these systems. Suspension arrays, such as the Luminex 100 system, are particularly amenable to automation (Baums et al., 2007; Gilbride et al., 2006). These arrays work on similar principles to planar microarrays, but they are rapid, cost effective, flexible and provide high reproducibility. In these systems, probes are immobilized onto solid surfaces of microspheres that are labelled with fluorophores (up to 100 colours are available) to facilitate their identification. After hybridization, the beads are analyzed in a flow cytometer. A red laser identifies the bead, and a green laser registers if a target has been captured. This system was found to be effective for detecting six different fecal indicating bacteria in environmental samples (Baums et al., 2007). Straub et al. (2005) have developed a comprehensive system for sample preparation and pathogen detection called ―BEADS‖ (biodetection enabling analyte delivery system). This system incorporates immunomagnetic separation to capture cells for concentration and purification of the analytes, a flow through thermal cycler and detection of PCR products on a suspension array. Using this system with river water, Straub and colleagues were able to detect 10 cfu of E. coli. When multiplexed with Shigella and Salmonella, sensitivity was reduced, but 100 cfu of each organism could be detected. Biosensor technologies offer the promise of portable devices delivering reliable results within short periods of time. Biosensors can be defined as analytical devices incorporating a biological material such as specific antibodies or DNA probes to confer specificity, integrated within a transducing microsystem (optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical) (Lazcka et al., 2007; Gilbride et al., 2006). Biosensors can be incorporated into miniaturized microfluidic devices commonly referred to as ―lab-on-a-chip‖ devices. Typical ―Lab-on-a-chip‖ devices contain wells for samples and storage of reagents, and microchannels for distribution of the samples and reagents to reaction chambers. They are connected to an instrument for control and detection of reactions by biosensors and to a computer for data analysis. The use of such devices reduces reagent costs, increases speed of analysis, minimizes handling steps, and provides portability, capability for parallel operations and the automation of complex assays. Several biological assays have been miniaturized and automated, including PCR, qPCR, DNA electrophoresis and microarrays, and immunoassays (Chen et al., 2007). A hand held device comprising electrochemical biosensors was successfully used to screen environmental water samples for 8 pathogens, within 3-5 h (LaGier et al., 2007). The detection limit of the device was equivalent to 10 cells of Karenia

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brevis when purified genomic DNA was assayed with the device. The scale of such devices, in terms of the number of targets that can be analyzed at a time, is still limited. The incorporation of high-density microarrays into integrated microfluidic devices containing biosensors has the potential to address the important issues of sensitivity and assay automation while maintaining the high information content of a microarray. Such systems may enable the reliable real-time monitoring of microbial communities, and detection of pathogens in water systems. While current devices show promise, there is a need to improve sensitivity, to improve specificity when dealing with complex samples, to provide portability and low cost in order to gain widespread use at water treatment facilities.

CONCLUSION In most developed countries, the removal or inactivation of the majority of pathogens, e.g. Cryptosporidium oocysts, can be accomplished by conventional water treatment technology, which generally includes flocculation, coagulation, sedimentation, filtration and chlorination (Betancourt and Rose 2004). Conventional treatment consists of coagulation/flocculation, sedimentation, and filtration (Sunnotel et al., 2006a). Implementation of multiple barriers to safeguard drinking water is recommended as pathogen loads vary during the season, with peak loads during early spring and late autumn (Ferguson et al. 2003). The high occurrence of many important human pathogens in surface water sources underlines the need for frequent monitoring of the parasite in drinking water (Frost et al. 2002). The high cost of waterborne disease outbreaks should be considered in decisions regarding water utility improvements and the construction of treatment plants (Morgan-Ryan et al. 2002). The integration of watershed and source water management and protection, scientific management of agricultural discharge and run-off, pathogen or indicator organism monitoring for source and treated water, outbreak and waterborne disease surveillance is needed to reduce waterborne transmission of human diseases (Ferguson et al. 2003). Water treatment facilities employing second-line treatment practices such as UV irradiation and ozone treatment can alleviate the danger imposed by chlorine resistant pathogens, e.g. Cryptosporidium (Keegan et al. 2003). However, outbreaks of waterborne illness can still occur in developed countries, because of malfunction or mismanagement of water treatment facilities (Ferguson et al. 2003). In these cases, hazard analyses protocols (microbiological hazards based on fecal coliforms (FC) and turbidity (TBY) as indicators) for critical control points (CCPs) within each facility may help to minimise the risk of contaminated water distribution in cases of system component failure, where CCPs include raw resource water, sedimentation, filtration and chlorinedisinfection (Jagals and Jagals 2004). Without knowing the occurrence of pathogens in water it is difficult to determine what risk they present to consumers of contaminated potable water (Karanis et al., 2007). Standardized methods are required to determine the occurrence of both established and emerging pathogens in raw untreated water abstracted for potable water, water treatment systems, potable water, and also recreational waters (Karanis et al., 2007). Also, because of the potential for pathogens to interact within protozoa and/or biofilms, the total populations of potable water must be assessed and monitored, e.g. how do populations vary with seasonality

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 31 and from one country to another? An improved understanding of the microbial ecology of distribution systems is necessary to design innovative and effective control strategies that will ensure safe and high-quality drinking water (Berry, et al., 2006). Better education and increased awareness by the general public and pool operators could potentially reduce the number and impact of swimming pool and other recreational waterrelated outbreaks (Robertson et al. 2002a). Improved detection methods, with the ability to differentiate species, can be useful in the assessment of infection and identification of contamination sources. These will also provide vital data on the levels of disease burden due to zoonotic transmission (Fayer 2004; Sunnotel et al., 2006a). The roles of humans, livestock and wildlife in the transmission of microbial pathogens remain largely unclear for many different areas. The continued and improved monitoring (using appropriate molecular methods) of pathogens in surface water, livestock, wild life and humans will increase our knowledge of infection patterns and transmission of pathogens in potable water (Fayer 2004; Sunnotel et al., 2006a). For zoonotic pathogens which are spread both directly and indirectly to potable water, a wide array of interventions have been developed to reduce the carriage of foodborne pathogens in poultry and livestock, including genetic selection of animals resistant to colonisation, treatments to prevent vertical transmission of enteric pathogens, sanitation practices to prevent contamination on the farm and during transportation, elimination of pathogens from feed and water, additives that create an adverse environment for colonization by the pathogen, and biological treatments that directly or indirectly inactivate the pathogen within the host (Doyle and Erickson, 2006; Sunnotel et al., 2006a). To successfully reduce the carriage of foodborne pathogens, it is likely that a combination of intervention strategies will be required (Doyle and Erickson, 2006). Collaborative efforts are needed to decrease environmental contamination and improve the safety of produce. There is a very real need for studies that monitor the quality of the water as well as for policies and investments that focus on sanitation (De Paula et al., 2007). Unfortunately, in developed countries, the currently adopted short-term and blinkered strategy of simply detecting pathogens, e.g. a positive/negative Cryptosporidium result, without further species and isolate information impedes any significant or meaningful epidemiological analysis and/or effective implementation of intervention strategies. Because of the current necessity to minimise costs, current molecular methods (outlined previously) and future nanotechnology based methods are not being routinely employed, and thus may never fulfil their maximum potential. This situation demands significant and immediate attention and collaborative input from governments and water industries world-wide – resulting in a more concerted effort from both scientists and policy makers to standardise the global epidemiological response. Currently, due to the significant amount of work involved in sample collection and statistical analysis, there is a time lag of approximately 2-3 years in attaining up to date pathogen data (see CDC table 1). A global data base and collective goals for biological contaminant loading are vital for achieving safe potable water. More funding is needed to speed up this process, and to better educate the general public of the advantages of effective intervention strategies. Globally, rapid and accurate monitoring and species typing must be carried out routinely, not just when outbreaks occur. There is also a need for much more accurate and conclusive information on the overall microbial populations present in water and their relationships to one another symbiotic, parasitic or otherwise (Snelling et al., 2006). Once successfully

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introduced in developed countries, then and only then, can this template be effectively applied to the developing world. If successful the substantial economic cost (clinical costs and lost working hours) of water borne pathogens might, in time, be reimbursed through improved epidemiological data collection which, with proactive intervention, will dramatically reduce the health and economic burden of waterborne disease.

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In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 2

VERMICULTURE BIOTECHNOLOGY: THE EMERGING COST-EFFECTIVE AND SUSTAINABLE TECHNOLOGY OF THE 21ST CENTURY FOR WASTE AND LAND MANAGEMENT TO SAFE AND SUSTAINABLE FOOD PRODUCTION Rajiv K. Sinha1, Sunil Herat 2 , Gokul Bharambe 3, Swapnil Patil3, Uday Chaudhary3, Priyadarshan Bapat3, Ashish Brahambhatt3 David Ryan3, Dalsukh Valani3, Krunal Chauhan3, R. K. Suhane4 and P. K. Singh4 1

Visiting Senior Lecturer, School of Engineering (Environment), Griffith University, Nathan, Campus, Brisbane, QLD-4111, Australia 2 Senior Lecturer, School of Engineering (Environment), Griffith University 3 School of Engineering (Environment), Griffith University 4 Scientists, Rajendra Agriculture University, Bihar, India Keywords: Vermicomposting; Vermifiltration; Vermiremediation; Vermi-agro-production; Vermitreatment-Self-Promoted Odor-Free Process; Earthworm Gut – A Bioreactor; Earthworms – Reduce Greenhouse Gases; Earthworms Stimulate Microbial Degradation; Vermicompost – Rich in Minerals and Hormones; Vermifiltered Water - Chemicals and Pathogen Free and Nutritive.

1. INTRODUCTION A revolution is unfolding in vermiculture studies (rearing of useful earthworms species) for multiple uses in environmental management and sustainable development. (Martin, 1976; 

Principal and Corresponding Author: [email protected]

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Satchell, 1983; Bhawalkar and Bhawalkar, 1994; Sinha et.al 2002; Fraser-Quick, 2002). Vermiculture biotechnology promises to provide cheaper solutions to following environmental and social problems plaguing the civilization – 1) Management of municipal and industrial solid wastes (organics) by biodegradation and stabilization and converting them into useful resource (vermicompost) – ‗THE VERMI-COMPOSTING TECHNOLOGY‘ (VCT) ; 2) Treatment of municipal and some industrial (food processing industries) wastewater, purification and disinfection - ‗THE VERMI-FILTRATION TECHNOLOGY‘ (VFT); 3) Removing chemical contaminations from soils (land decontamination) and reducing soil salinity while improving soil properties- ‗THE VERMI-REMEDIATION TECHNOLOGY‘ (VRT); 4) Restoring and improving soil fertility and boosting crop productivity by worm activity and use of vermicompost (miracle growth promoter) while eliminating the use of destructive agro-chemicals - ‗THE VERMI-AGRO-PRODUCTION TECHNOLOGY‘ (VAPT); Vermi-composting, vermi-filtration, vermi-remediation and vermi-agro-production are self-promoted, self-regulated, self-improved and self-enhanced, low or no-energy requiring zero-waste technology, easy to construct, operate and maintain. It excels all ‗bio-conversion‘, ‗bio-degradation‘ and ‗bio-production‘ technologies by the fact that it can utilize organics that otherwise cannot be utilized by others. It excels all ‗bio-treatment‘ technologies because it achieves greater utilization than the rate of destruction achieved by other technologies. It involves about 100-1000 times higher ‗value addition‘ than other biological technologies. (Appeholf, 1997). About 4,400 different species of earthworms have been identified, and quite a few of them are versatile waste eaters and bio-degraders and several of them are bio-accumulators and bio-transformers of toxic chemicals from contaminated soils rendering the land fit for productive uses.

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2. EARTHWORMS: THE GREAT WASTE AND LAND MANAGERS ON EARTH The earthworms have over 600 million years of experience in waste and land management. No wonder then, Charles Darwin called them as the ‗unheralded soldiers of mankind‘ and ‗friends of farmers‘; and the Greek philosopher Aristotle called them as the ‗intestine of earth‘, meaning digesting a wide variety of organic materials including the waste organics, from earth. (Darwin and Seward, 1903).

Versatile Waste Eaters and Decomposers Earthworms are versatile waste eaters and decomposers. They feed lavishly on the organic waste, and also on the microorganisms (bacteria, fungi and the actinomycetes) that invade and colonize the waste biomass. Most earthworms consume, at the best, half their body weight of organics in the waste in a day. Eisenia fetida is reported to consume organic matter at the rate equal to their body weight every day.(ARRPET, 2005). Earthworm participation enhances natural biodegradation and decomposition of organic waste from 60 to 80 %. Study indicates that given the optimum conditions of temperature (20-30 C) and moisture (60-70 %), about 5 kg of worms (numbering approx.10,000) can vermiprocess 1 ton of waste into vermi-compost in just 30 days (ARRPET, 2005). Upon vermi-composting the volume of solid waste is significantly reduced from approximately 1 cum to 0.5 cum of vermi-compost. Earthworms can also treat and purify municipal wastewater (sewage) and also some industrial wastewater from the food processing industries significantly reducing the BOD and COD loads, the total dissolved and suspended solids (TDSS). It does this by the general mechanism of ‗biodegradation‘ of organics in the wastewater, ‗ingestion‘ of heavy metals and solids from wastewater and also by their ‗absorption‘ through body walls. What is more significant is that, there is no ‗sludge‘ formation in the process and the resulting vermifiltered water is clean, nutritive and disinfected enough to be reused for irrigation in farms, parks and gardens.

Bio-Accumulators of Toxic Soil Chemicals and Contaminants Earthworms have been found to bioaccumulate heavy metals, pesticides and lipophilic organic micropollutants like the polycyclic aromatic hydrocarbons (PAH) from the soil (Contreras-Ramos et. al, 2006). After the Seveso chemical plant explosion in 1976 in Italy, when vast inhabited area was contaminated with certain chemicals including the extremely toxic TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) several fauna perished but for the earthworms that were alone able to survive. Earthworms which ingested TCDD contaminated soils were shown to bio-accumulate dioxin in their tissues and concentrate it on average 14.5 fold. (Satchell, 1983). E. fetida was used as the test organisms for different soil contaminants and several reports indicated that E. fetida tolerated 1.5 % crude oil (containing several toxic organic pollutants) and survived in this environment. (OECD, 2000; Safwat et al., 2002).

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Earthworms also tolerate high concentrations of heavy metals in the environment. The species Lumbricus terrestris was found to bio-accumulate in their tissues 90-180 mg lead (Pb) / gm of dry weight, while L. rubellus and D. rubida it was 2600 mg /gm and 7600 mg /gm of dry weight respectively.(Ireland, 1983).

Earthworm Species Suitable for Waste Management (Waste Biodegradation) Long-term researches into vermiculture have indicated that the Tiger Worm (Eisenia fetida), Red Tiger Worm (E. andrei), the Indian Blue Worm (Perionyx excavatus),the African Night Crawler (Eudrilus euginae),and the Red Worm (Lumbricus rubellus) are best suited for vermi-composting of variety of organic wastes and vermifiltration of both municipal and industrial wastewater under all climatic conditions. (Graff, 1981). E. fetida and E. andrei are closely related. Our study has indicated that E. fetida is most versatile waste eater and degrader and an army of the above 5 species combined together works meticulously.

Earthworm Species Suitable for Land Remediation (Soil Decontamination) Certain species of earthworms such as Eisenia fetida, Aporrectodea tuberculata, Lumbricus terrestris, L. rubellus, Dendrobaena rubida, D. veneta, Eiseniella tetraedra, Allobophora chlorotica have been found to tolerate and remove wide range of chemicals from soil. Our study also indicate that E. fetida is most versatile chemical bio-accumulators. (Ireland, 1983). Several of above species are common to India and Australia, both nations being biogeographically very close. (Sinha et. al,.2002).

3. THE BIOLOGY AND ECOLOGY OF EARTHWORMS Earthworms are long, narrow, cylindrical, bilaterally symmetrical, segmented animals without bones. The body is dark brown, glistening and covered with delicate cuticle. They weigh over 1400-1500 mg after 8-10 weeks. On an average, 2000 adult worms weigh 1 kg and one million worms weigh approximately 1 ton. Usually the life span of an earthworm is about 3 to 7 years depending upon the type of species and the ecological situation. Earthworms harbor millions of ‗nitrogen-fixing‘ and ‗decomposer microbes‘ in their gut. They have ‗chemoreceptors‘ which aid in search of food. Their body contains 65 % protein (70-80 % high quality ‗lysine rich protein‘ on a dry weight basis), 14 % fats, 14 % carbohydrates and 3 % ash. (Gerard, 1960; ARRPET, 2005).

Enormous Power of Reproduction and Rapid Rate of Multiplication Earthworms multiply very rapidly. They are bisexual animals and cross-fertilization occurs as a rule. After copulation the clitellum (a prominent band) of each worm eject lemon-

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shaped ‗cocoon‘ where sperms enter to fertilize the eggs. Up to 3 cocoons per worm per week are produced. From each cocoon about 10-12 tiny worms emerge. Studies indicate that they double their number at least every 60 days. Given the optimal conditions of moisture, temperature and feeding materials earthworms can multiply by 28 i.e. 256 worms every 6 months from a single individual. Each of the 256 worms multiplies in the same proportion to produce a huge biomass of worms in a short time. The total life-cycle of the worms is about 220 days. They produce 300-400 young ones within this life period. (Hand, 1988). A mature adult can attain reproductive capability within 8 – 12 weeks of hatching from the cocoon. Red worms takes only 4-6 weeks to become sexually mature. (ARREPT, 2005). Earthworms continue to grow throughout their life and the number of segments continuously proliferates from a growing zone just in front of the anus. Table 1. Reproductive Capacity of Some Environmentally Supportive Worms Species

E. fetida E. eugeniae P. excavatus D. veneta

Sexual maturity time (days) 53-76 32-95 28-56 57-86

No. of cocoon. 3.8 3.6 19.5 1.6

Cocoons hatching time (days) 32-73 13-27 16-21 40-126

Egg maturity days 85-149 43-122 44-71 97-214

% hatching 83.2 81. 90.7 81.2

No. of hatchlings 3.3 2.3 1.1 1.1

Net reproduction rate/week 10.4 6.7 19.4 1.4

Source : Edwards (1988).

Sensitive to Light, Touch and Dryness Earthworms are very sensitive to touch, light and dryness. They tend to migrate away from light. Cold (low temperature) is not a big problem for them as the heat (high temperature). Their activity is significantly slowed down in winter, but heat can kill them instantly. They temporarily migrate into deeper layers when subjected to too cold or too hot situations. It seems worms are not very sensitive to offensive smell as they love to live and feed on cattle dung and even sewage sludge. However, offensive smell can persist only for a short while in any environment where worms are active. They arrest all odour problems by killing anaerobes and pathogens that create foul odour.

4. VERMICULTURE : A GLOBAL MOVEMENT The movement was started in the middle of 20th century and the first serious experiments for management of municipal / industrial organic wastes were established in Holland in 1970, and subsequently in England, and Canada. Later vermiculture were followed in USA, Italy, Philippines, Thailand, China, Korea, Japan, Brazil, France, Australia and Israel (Edward,1988). However, the farmers all over the world have been using worms for composting their farm waste and improving farm soil fertility since long time. In UK, large 1000 mt vermi-composting plants have been erected in Wales (Frederickson, 2000). The American Earthworm Technology Company started a 'vermi-

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composting farm' in 1978-79 with 500 t /month of vermicompost production (Edward, 2000). Collier (1978) and Hartenstein and Bisesi (1989) reported on the management of sewage sludge and effluents from intensively housed livestock by vermiculture in USA. Japan imported 3000 mt of earthworms from the USA during the period 1985-87 for cellulose waste degradation (Kale,1998). The Aoka Sangyo Co. Ltd., has three 1000 t /month plants processing waste from paper pulp and the food industry (Kale,1998). This produces 400 ton of vermicompost and 10 ton of live earthworms per month. The Toyhira Seiden Kogyo Co. of Japan is using rice straw, municipal sludge, sawdust and paper waste for vermicomposting involving 20 plants which in total produces 2-3 thousands tons of vermicompost per month (Edward, 2000). In Italy, vermiculture is used to biodegrade municipal and paper mill sludge. Aerobic and anaerobic sludge are mixed and aerated for more than 15 days and in 5000 cum of sludge 5 kg of earthworms are added. In about 8 months the hazardous sludge is converted into nutritive vermicompost (Ceccanti and Masciandaro, 1999). In France, 20 tons of mixed household wastes are being vermi-composted everyday using 1000 to 2000 million red tiger worms (Elsenia andrei) in earthworm tanks. (ARRPET, 2005). Rideau Regional Hospital in Ontario, Canada, vermi-compost 375 - 400 kg of wet organics mainly food waste everyday. The worm feed is prepared by mixing shredded newspaper with the food waste. (ARRPET, 2005). In Wilson, North Carolina, U.S., more than 5 tons of pig manure (excreta) is being vermi-composted every week. (NCSU, 1997). In New Zealand, Envirofert is a large vermicomposting company operating in over 70 acre site in Auckland converting thousands of tons of green organic waste every year into high quality compost. (www.envirofert.co.nz). Almost all agricultural universities in India are now involved in vermiculture and a movement is going across the sub-continent especially involving the poor rural women with dual objectives of ‗making wealth (food and fertilizer) from waste and combating poverty‘ while ‗cleaning the environment and combating pollution of land and soil‘. Earthworms have enhanced the lives of poor in India. It has become good source of livelihood for many. (White, 1994; Hati, 2001). Vermiculture is being practiced and propagated on large scale in Australia too as a part of the 'Urban Agriculture Development Program' (to convert all the municipal urban wastes into compost for local food production) and ‗Diverting Waste from Landfills Program‘ (for reducing landfills in Australia).

5. THE VERMICOMPOSTING TECHNOLOGY (VCT) Vermicomposting is a rapid biological degradation process (aided by earthworms and soil microbes) in which several kinds of organic materials are converted from ‗unstable product‘ (which is likely to decompose further, creating objectionable odors, generating greenhouse gas methane and producing environmental insanitation) to an increasingly more ‗stable product‘ whose value is upgraded as nutritive materials for the soil and that can remain in the environment without creating any environmental problem. All organic wastes by its very nature (chemical composition) is bound to disintegrate anaerobically in environment and generate greenhouse gas methane (CH4). Only if they are allowed to degrade under aerobic conditions (which is readily facilitated by earthworms) that this can be prevented.

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5.1. Wastes Suitable for Vermicomposting Earthworms can physically handle a wide variety of organic wastes from both municipal (domestic and commercial) and industrial (livestock, food processing and paper industries) streams. Earthworms are highly adaptable to different types of organic wastes (even of industrial origin), provided, the physical structure, pH and the salt concentrations are not above the tolerance level. Another matter of considerable significance is that the earthworms also partially ‗detoxify‘ (by bio-accumulating any heavy metals and toxic chemicals) and ‗disinfect‘ (by devouring on pathogens and killing them by anti-bacterial coelomic fluid) the waste biomass while degrading them into vermi-compost which is nearly sterile and odorless. (Pierre et al., 1982).

Some Industrial Wastes Suitable for Vermi-Composting Solid waste including the ‗wastewater sludge‘ from paper pulp and cardboard industry, brewery and distillery, sericulture industry, vegetable oil factory, potato and corn chips manufacturing industry, sugarcane industry, aromatic oil extraction industry, logging and carpentry industry offers excellent feed material for vermi-composting by earthworms. (Kale, et al., 1993; Kale and Sunitha, 1995; Seenappa et al.,1995; Gunathilagraj and Ravignanam, 1996; Lakshmi and Vijayalakshmi, 2000; ARRPET, 2005.) Worms Can Even Vermicompost Human Excreta (Feces) Bajsa et al. (unpublished data) studied vermicomposting of human excreta (feces). It was completed in six months, with good physical texture meeting ARMANZ (1995) requirements, odourless and safe pathogen quality. Sawdust appeared to be the best covering material that can be used in vermicomposting toilets to produce compost with a good earthy smell, a crumbly texture and dark brown colour. There was no re-growth of pathogens on storing the compost for longer period of time and the initial pathogen load did not interfere in the die off process as the composting process itself seemed to stabilize the pathogen level in the system. Waste Materials Preferably to be Avoided for Vermi-composting 1) Heavily salted products unless soaked in water for 24 hours; 2) Excess citrus (orange and lemon peels and crushes) and onions wastes (might reduce pH and impair worm activity); 3) Feces of pets (may carry viral or bacterial toxins); 4) Fresh green grasses and foliages (green waste) creates high temperature and can harm worms; 5) All non-biodegradable wastes; 6) Meat and dairy products and slaughterhouse waste are to be avoided in the initial stage till the number of earthworms become high enough in the composting bed (it may invite ‗maggots‘ and also create bad odor for sometimes). However, Nair et al (2006) studied that thermophilic aerobic composting of ‗food waste‘ for 7 days followed by vermicomposting can eliminate the need for screening off of ‗citrus‘ and other acidic wastes like ‗onion peels‘ from vermicomposting. It comprises a short period

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of high temperature (around 55 C) followed by a period of lower temperature. This also leads to reduction of pathogens in much shorter time. (See table 9 below). The Envirofert, New Zealand, which is vermicomposting thousands of tons of green waste every year, also practice putting the green waste first to a lengthy thermophilic cooking, and then to vermicomposting by worms after cooling. Cooking of green waste also help destroy the weeds and pathogens which may come from the feces of pets in grasses. They claim that each worm eat the cooked green waste at least 8 times leaving an end product which is rich in key minerals, plant growth hormones, enzymes, and beneficial soil microbes. (www.envirofert.co.nz).

5.2. Vermicomposting of Municipal Solid Waste Millions of tons of municipal solid wastes (MSW) from homes and commercial institutions, cattle and farm wastes are generated in the human society and are mostly ending up in the landfills everyday, creating extraordinary economic and environmental problems for the local government to manage and monitor them (may be up to 30 years) for environmental safety (emission of greenhouse and toxic gases). Construction of engineered landfills incurs 20-25 million US dollars before the first load of waste is dumped.

5.3. Mechanism of Worm Action in Vermicomposting Worms Reinforce Microbial Population and Act Synergistically Promoting Rapid Waste Degradation Earthworms promotes the growth of ‗beneficial decomposer aerobic bacteria‘ in waste biomass and this they do by improving aeration by burrowing actions. They also act as an aerator, grinder, crusher, chemical degrader and a biological stimulator. (Dash, 1978; Binet et al., 1998 and Sinha et al., 2002). Earthworms hosts millions of decomposer (biodegrader) microbes in their gut (as they devour on them) and excrete them in soil along with nutrients nitrogen (N) and phosphorus (P) in their excreta (Singleton et al., 2003). The nutrients N and P are further used by the microbes for multiplication and vigorous action. Edward and Fletcher (1988) showed that the number of bacteria and ‗actinomycetes‘ contained in the ingested material increased up to 1000 fold while passing through the gut. A population of worms numbering about 15,000 will in turn foster a microbial population of billions of millions. (Morgan and Burrows, 1982). Singleton et al. (2003) studied the bacterial flora associated with the intestine and vermicasts of the earthworms and found species like Pseudomonas, Mucor, Paenibacillus, Azoarcus, Burkholderia, Spiroplasm, Acaligenes, and Acidobacterium which has potential to degrade several categories of organics. Acaligenes can even degrade PCBs and Mucor can degrade dieldrin. Under favorable conditions, earthworms and microorganisms act ‗symbiotically and synergistically‘ to accelerate and enhance the decomposition of the organic matter in the waste. It is the microorganisms which breaks down the cellulose in the food waste, grass clippings and the leaves from garden wastes. (Morgan and Burrows, 1982; Xing et al. 2005) Vermicomposting of organic waste involves following actions of worms –

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1) The waste feed materials ingested is finely ground (with the aid of stones in their muscular gizzard) into small particles to a size of 2-4 microns and passed on to the intestine for enzymatic actions. The gizzard and the intestine work as a ‗bioreactor‘; 2) The worms secrete enzymes proteases, lipases, amylases, cellulases and chitinases in their gizzard and intestine which bring about rapid biochemical conversion of the cellulosic and the proteinaceous materials in the waste organics. Earthworms convert cellulose into its food value faster than proteins and other carbohydrates. They ingest the cellulose, pass it through its intestine, adjust the pH of the digested (degraded) materials, cull the unwanted microorganisms, and then deposit the processed cellulosic materials mixed with minerals and microbes as aggregates called ‗vermicasts‘ in the soil. (Dash, 1978). 3) Only 5-10 percent of the chemically digested and ingested material is absorbed into the body and the rest is excreted out in the form of fine mucus coated granular aggregates - the ‗vermicasts‘ rich in nitrates, phosphates and potash. 4) The final process in vermi-processing and degradation of organic matter is the ‗humification‘ in which the large organic particles are converted into a complex amorphous colloid containing ‗phenolic‘ materials. Only about one-fourth of the organic matter is converted into humus. The colloidal humus acts as ‗slow release fertilizer‘. (ARRPET, 2005).

5.4. Experimental Studies on Vermicomposting a). Study of Biodegradation Abilities of Individual Worm Species This study was underataken by Sinha et al. (2002) at University of Rajasthan, Jaipur, India. About 150 mixed species of composting worms Eisinia fetida, Eudrilus euginae, and Perionyx excavatus were added to 1 kg each of buffalo dung, garden and kitchen wastes in different containers. In another three containers, each of these wastes and the three individual species of worms were used separately to assess their individual feeding and biodegradation abilities. (See table 2). Table 2. Vermicomposting of Cattle Dung, Kitchen Wastes and Garden Wastes by Individual Composting Worms Waste Materials 1 kg of each Cattle dung (Week old & semi-dry) Raw food wastes (Spinach stems, cauliflower & eggplant rejects) Garden wastes (Grasses & dry leaves)

Time Taken in Vermicomposting & Complete Degradation (in days) E. fetida E. euginee P. excavatus Mixed Species 59 44 62 45

78

61

83

70

89

69

91

80

Source: Sinha et al., (2002): The Environmentalist, UK.

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Findings and Results Results clearly showed that cattle dung is accepted quickly and composted rapidly by earthworms. Dung is partially degraded materials and the worms love to feed on them as they are rich in microbes. Raw food waste and garden wastes which contain cellulosic materials are accepted slowly as they are hard to degrade. Performance of Eudrilus euginae was best among the three species followed by Eisinia fetida. b). Study of Composting with Worms (Vermicomposting) and without Worms (Conventional Aerobic Composting) to Assess the Efficiency of Two Processes This study was made by Sinha et al. at University of Rajasthan, Jaipur, India on different cooked and raw kitchen wastes. In each container, 100 mixed species of composting worms (Eisinia fetida, Eudrilus euginiae and Perionyx excavatus) were added. A control pot was also organized for conventional aerobic composting without worms. Experiments were continued throughout the year in both summer, rainy and winter seasons to assess the effect of temperature variation, humidity and climate change on worm activity. (See table 3). Table 3. Biodegradation of Raw and Cooked Kitchen Wastes With and Without Worms

Source: Sinha et al., (2002) : The Environmentalist, UK. (Data is for warmer climate in India - MayJuly 1998: Temperature Min. 20C ; Max. 42C; Values in brackets are for cold periods Dec.1998 – Feb. 1999 : Temperature Min. 8 C; Max. 18C) Key: EP = Earthworms Present (Vermicomposting); EA = Earthworms Absent (Aerobic Composting) ; NA= Never Achieved Within the Period of Study.

Findings and Results Study confirmed beyond doubt that vermicomposting by worms is most efficient process (nearly 4-6 times faster) over aerobic composting which involves only microbial decomposition. Worms act both ways – carry out enzymatic degradation and also enhance microbial decomposition. Worm activity is at still greater threshold during warmer periods than in cold. c). Study of Vermicomposting of Raw and Cooked Food Wastes to Assess the Food Preferences of Earthworms and the Time Taken in Degradation of Each Food Component The study was made by Sinha et al. (2005) and Patil (2005) at Griffith University, Brisbane, Australia. Vermi-composting was carried out in a specially fabricated plastic ‗vermiculture bin‘ (Figure 1) sold in major hardware stores all over Australia. There is adequate provisions for aeration from side walls and top is open with cover lid on it. The

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vermicomposting bed was prepared with a thick layer of wet newspaper and about 4 inches of garden soil spread over it. About 250 numbers of earthworms were released in this soil bed. Food wastes were collected from homes and restaurants. About 100 gm of each of the food waste components were used. Mixed species of Elsenia fetida, Eudrilus eugeniae and Perionyx excavatus were used. Worms ranged between 3-6 cm in length and 0.1-0.2 cm in thickness. Moisture content was maintained at around 60 % by regular spray of water. Temperature within the bin was maintained at around 22-24 C as in the glass-house.

No of Days taken for complete Biodegradation

100 90 80 70 60 50 40 30 20 10 0 E. fetida,

E. euginae,

P. excavatus

Mixed Species

Earthworm Species Used Cattle dung

Kitchen w astes

Garden w astes

Figure 1. Time Taken To Complete Biodegradation of Different Food Waste by Various Earthworm Species.

A control VC bin was organized with all above features except for the earthworms to compare the rate of waste biodegradation without worms (but by aerobic process), odor problems etc., and to exactly determine the role and efficiency of earthworms in waste biodegradation. Results of vermicomposting have been given in tables 4 and 5. 120

16

% Reduction

12 80

10

60

8 6

40

4 20

2

Raw potato cuts & peels

Crushed egg shells

Cooked chicken, lamb & beef (curry)

Indian chapatti

Cooked potato, cauliflower etc. Raw rejects of cauliflower etc.

Baked potato, tomato etc.

Apple, pear, kiwi fruit etc.

Raw cabbage, lettuce etc.

Rice, noodles & pasta

0

Bread, buns & naans

0

Food Waste components % Biodegradation With Worms

% Biodegradation WithoutWorms

Figure 2. Rate of Biodegradation of Food Waste Components.

Weeks

Duration (Weeks)

14

100

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Findings and Results Worms can eat and degrade all food components including the cooked meat wastes (except the bones) conveniently according to their food preferences. They prefer softer ones first. But they are not comfortable with egg-shells, raw onions and raw potato rejects. Table 4. Rate of Biodegradation of Raw and Cooked Food Wastes With and Without Earthworms Under Identical Climatic Conditions (Food waste components about 100 gm each with 250 worms)

Source: Sinha et al. (2005) Keys: * 100 % degradation was achieved within the study period (Week 14) though with foul odor and heavy invasion of fungus; ** 100 % degradation could never be achieved, had odor problem and badly invaded by fungus (blue, black and green moulds) and had to be terminated; *** Terminated in week 4 because of maggots, heavy fungus and offensive smell;

Table 5. Rate of Biodegradation of Fried and Oily Food Wastes With and Without Earthworms Under Identical Climatic Conditions (Food waste used was about 9.5 kg with 500 worms)

Source: Patil (2005) Key: ND = Not detectable * Mild foul odor ** Highly offensive foul odor T= Terminated.

% Biodegradation

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100 90 80 70 60 50 40 30 20 10 0 2

4

6

8

10

12

14

Time (Weeks) % Biodegradation With Worms of Crushed Food

% Biodegradation With Worms of Intact Food

% Biodegradation Without Worms of Crushed Food

% Biodegradation Without Worms of Intact Food

Figure 3. Rate of Biodegradation of Fried and Oily Food Wastes With and Without Earthworms Under Identical Climatic Conditions

Figure 4. The Vermiculture Bin

Figure 5. Fried chicken nuggets and fish, calamari rings, potato fries.

Figure 6. Worm size before vermicomposting.

Figure 7. Worm size and health after vermicomposting.

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Figure 8. Food Waste in VC Bin (Half Crushed and Half Intact) on Day 1 (30.8.05).

Figure 9. Biodegraded (Vermicomposted) food waste on Day 29 after week 8 (26.10.05).

Findings and Results Worms slowly accept the fried greasy and oily foods although reluctantly in the beginning, and then more faster, after adapting with new food products. They prefer crushed food materials.

5.4. Vermicomposting of Sewage Sludge : Some Preliminary Studies Sludge is an inevitable hazardous and odorous byproduct from the conventional water and wastewater treatment plants which eventually require safe disposal either in landfills or by incineration incurring heavy cost. When the sludge is dewatered and dried they are termed as ‗biosolids‘. Management of the biosolids remains problematic due to the high cost of installing sewage sludge stabilization reactors and dehydration systems. Vermicomposting has been successfully used for treating and stabilizing municipal (water and wastewater treatment plants) as well as industrial (paper mill, dairy and textile industry) sludge, diverting them from ending up in the landfills. (Elvira et al. 1998; Lotzof, 1999; Ndegwa and Thompson 2001; Fraser-Quick, 2002; Contreras-Ramos et al., 2005). The quality of compost is significantly improved in terms of nutritional and storage value by worms. (Klein et al., 2005). The chemical and biological composition of sewage sludge is unpredictable as they may contain chemicals and pathogens from industry and agriculture. They are potential health hazard as they have been found to contain high numbers of cysts of protozoa, parasitic ova, fecal pathogens like Salmonella, Shigella and E. coli and also heavy metals such as zinc (Zn), cadmium (Cd), mercury (Hg) and copper (Cu). However, they also contains organics and essential plant nutrients like nitrogen (N), phosphorus (P), potassium (K) and various trace elements. Sludge is stabilized to reduce or eliminate pathogens and heavy metals, eliminate offensive odor, and reduce or eliminate the potential for putrefaction. Stabilized sewage sludge can become good source of organic fertilizer and soil additive for beneficial reuse in farms. (McCarthy, 2002). Earthworms feed readily upon the sludge components, rapidly convert them into vermicompost, reduce the pathogens to safe levels and ingest the heavy

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metals. Volume is significantly reduced from 1 cum of wet sludge (80 % moisture) to 0.5 cum of vermicompost (30 % moisture). (Eastman, 1999). Contreras-Ramos et al. (2005) studied the vermicomposting of biosolids (dried sewage sludge) from various industries but mainly from textile industries and some households (municipal) mixed with cow manure and oat straw. 1,800, 1,400 and 1000 gms of aerobically digested biosolids were mixed with 800, 500 and 200 gms of cow manure and 200, 100 and 0 (zero) gms of oat straw in triplicate set up. A control was also kept with only biosolids. Cow manure was added to provide additional nutrients and the oat straw to provide bulk. 50 earthworms (weighing 40 gm live weight) were added in each sample and the species used was Eisenia fetida. They were vermicomposted at three different moisture contents – 60 %, 70 % and 80 % for two months (60 days). The best results were obtained with 1,800 g biosolids mixed with 800 g of cow manure and no (0) straw at 70 % moisture content. Volatile solids of the vermicompost decreased by 5 times, heavy metals concentrations and pathogens (with no coliforms) were below the limits set by USEPA (1995) for an excellently stabilized biosolid. Carbon content decreased significantly due to mineralization of organic matter, and the number of earthworms increased by 1.2 fold. Vermiprocessing of sludge (biosolids) from the sewage and water treatment plants is being increasingly practiced in Australia and as a result it is saving large landfill spaces every year in Australia. The Redland Shire in Queensland started vermi-composting of sludge (biosolids) from sewage and water treatment plants with the aid of Vermitech Pty. Ltd. in 1997. The facility receives 400-500 tons of sludge every week with 17 % average solid contents and over 200 tons of vermicast is produced every week by vermicomposting. (Vermitech, 1998). The Hobart City Council in Tasmania, Australia, vermicompost and stabilize about 66 cum of municipal biosolids (from sewage sludge) every week, along with green mulch diverting them from landfills. Zeolite mixed with the biosolids helps balance the pH and also in absorbing ammonia and odor. About two-third of this volume (44 cum) becomes ‗vermicompost‘ which is sold to public. The City Council is saving AU $ 56,000 per year just from avoiding landfill disposal (transport and tipping fees etc.). They are earning an equal amount as revenue from the sale of vermi-compost. (Datar et al., 1997).

5.4.1. Mechanism of Worm Action in Vermicomposting of Sludge : Worms Act as Sludge Digester The basic mechanism of worm action is same as it is in the degradation and composting of general organic waste components of MSW. However, vermistabilization of sludge involves very complex mechanical, chemical and biological transformation processes and the resultant product has higher stabilization and soil supplement value than traditional composting that relies on mechanical incorporation of sludge with green waste in large compost heaps. It decompose the organics in the sludge, mineralise the nutrients, ingest the heavy metals and devour the pathogens (bacteria, fungus, nematodes and protozoa) found in sludge making them chemicals and pathogen free ready to be reused as soil additive and organic fertilizer. Essentially, it works as a ‗sludge digester‘ which is accomplished in the following steps1) The sludge is softened by the grume excreted in the ‗mouth‘ of the earthworms and from there it goes to the ‗esophagus‘;

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2) In the esophagus the softened sludge components is neutralized by calcium (Ca) (excreted by the inner walls of esophagus) and passed on to the gizzard and the intestine for further enzymatic action.

5.4.2. Experimental Study of Vermicomposting of Sewage Sludge Brahambhatt (2006) studied the vermicomposting of sewage sludge. The sludge was obtained from the Oxley Wastewater Treatment Plant in South Brisbane and earthworms were obtained from Bunnings hardware. It contained mixed species of Eisenia fetida, Perionyx excavatus and Eudrillus eugeniae. Cow dung was obtained from cattle farm in Ipswich. Both sewage sludge and the cow manure was partially air dried for 5 days to prevent any methane and hydrogen sulfide generation that might harm the worms. Vermicompost was obtained from our vermiculture lab in the School of Environmental Engineering. Five sets of experimental bins (40 litre HDPE containers) with biosolids were prepared with one as CONTROL and were studied for morphological (color and texture), biological (pathogens) and chemical (heavy metals) changes over the 12 weeks period. Table 6. Status of Coliforms in the Untreated (Control), Vermicomposted and Microbiologically Composted Biosolids After 12 Weeks

Source: Brahambhatt (2006). Key: VC = Vermicomposting; * Microbial Composting. % of stabilisation

% of stabilisation

100.00% 80.00% 60.00% 40.00% 20.00% 0.00% Biosolids (CONTROL)

VC Biosolids by Earthw orms

VC Biosolids *Composted by Biosolids by Earthw orms + Cow Dung Cow Dung

*Composted Biosolids by Organic Soil

Nature of samples tested

Figure 10. Status of Coliforms in the Untreated (Control), Vermicomposted and Conventionally Composted (By Microbial Degradation) Biosolids after 12 Weeks.

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Findings and Results Results clearly shows that the earthworms significantly reduce or almost eliminates the pathogens from the digested (composted) sludge. Sludge treated with earthworms (with or without feed materials) only showed ‗negative results‘ by the Colilert test under the UV lamp. And this was achieved in just 12 weeks. It also infers that under the microbial composting systems the pathogens remains in the sludge for longer time even after treatment until it is completely dry with all food and moisture exhausted making them difficult to survive. Table 7. Status of Heavy Metals Cadmium (Cd) and Lead (Pb) in the Untreated (Control), Vermicomposted and Microbiologically Composted Biosolids (mg/kg of soil) in 12 Weeks

Source: Brahambhatt (2006). Key: * Conventional Composting by Microbial Degradation; NC = No Change in Value.

90 80

2.5

70

2

60 50

1.5

40

1

30 20

0.5

Lead (mg/kg of soil)

Cadmium (mg/kg of soil)

3

10

0

0 Biosolids (CONTROL)

VC Biosolids VC Biosolids *Composted *Composted by by Biosolids by Biosolids by Earthw orms Earthw orms + Cow Dung Organic Soil Cow Dung

Nature of sample tested

Cadmium Lead

Figure 11. Status of Heavy Metals Cadmium (Cd) and Lead (Pb) in the Untreated (Control), Vermicomposted and Conventionally Composted Biosolids after 12 Weeks.

Findings and Results Results clearly show that the earthworms reduce the heavy metals cadmium (Cd) and lead (Pb) from the digested sludge. There was no change in the values of heavy metals between the untreated sludge (Bin 1) and those treated by adding cow dung and by organic soil (Bins 4 and 5) to enhance microbial composting. Although biosolids can be slowly

Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al.

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stabilized by microbial degradation over a period of time, the heavy metals will remain in the system for quite sometimes after which it may leach into soil or get bound with soil organics by chemical reactions occurring in the soil. % Reduction in Cadmium

% Reduction in lead

70%

% Reduction

60% 50% 40% 30% 20% 10% 0% Biosolids (CONTROL)

VC Biosolids VC Biosolids *Composted by Earthw orms by Earthw orms Biosolids by + Cow Dung Cow Dung

*Composted Biosolids by Organic Soil

Nature of sample te ste d

Figure 12. Percentage Reduction in Cadmium and Lead in the Untreated (Control), Vermicomposted and Conventionally Composted Biosolids after 12 Weeks.

Though there was not very significant removal of heavy metals by earthworms in the 12 weeks of experiment yet their role in heavy metal removal cannot be undermined. Providing additional feed materials (Bin 3) enhanced worm activity and also their number (stimulating reproduction) and led to greater removal of heavy metals. It infers that over a period of time and with enhanced worm activity the heavy metals can be completely removed from the biosolids.

5.5. Critical Factors Affecting Optimal Worm Activity and VermiComposting 1. Moderate Temperature: In general earthworms prefers and tolerates cold and moist conditions far better than the hot and dry ones. Most worms involved in vermicomposting require moderate temperature between 20 – 30  C. Heat causes more problems in vermi-composting than the cold. Red worms are reported to become inactive above 29 C. They are at the highest levels of both waste degradation and reproduction activity as the weather cools in the fall and warms in the spring. 2. Adequate Moisture: Earthworms requires plenty of moisture for growth and survival ranging from 60 to 70 %. The bed should not be too wet as it may create anaerobic condition adversely affecting worm activity. 3. Adequate Aeration: Vermi-composting is an aerobic process and adequate flow of air in the waste biomass is essential for worm function. Worms breath through their skin and need plenty of oxygen in the surrounding areas. Although worms constantly aerate their habitat by burrowing actions, periodical turning of waste biomass can improve aeration and biodegradation.

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4. Neutral pH (7.0): Earthworms are sensitive to pH. The decomposition of organic matter produces ‗organic acids‘ that lower the pH of the bedding soil and impair worm activity. Although the worms can survive in a pH range of 4.5 to 9 but functions best at neutral pH of 7.0. Although worms can lower pH of its medium by secreting calcium (Ca), it is suggestive to add ground limestone (calcium carbonate) powder to the bedding periodically. This would serve two purposes- maintain neutral pH and also supply the much needed calcium (Ca) to the worms for its metabolism. 5. Carbon / Nitrogen (C/N) Ratio of the Feed Material: High C/N ratio above 30:1 in waste biomass can impair worm activity and vermi-composting. Although earthworms help to lower the C/N ratio of fresh organic waste, it is advisable to add nitrogen supplements such as cattle dung or pig and goat manure or even kitchen waste (which are rich in nitrogen contents) when waste materials of higher C/N ratio exceeding 40:1 such as the green cellulosic wastes (grass clippings) are used for vermi-composting. 6. Adequate Supply of Calcium (Ca): Calcium appears to be important mineral in worm biology (as calcarious tissues) and biodegradation activity. Although most organic waste contains calcium, it is important to add some additional sources of calcium for good vermi-composting. Egg shells are good source of natural calcium. Occasionally limestone powder should be added.

5.6. Advantages of Vermi-Composting Earthworms have real potential to both increase the rate of aerobic decomposition and composting of organic matter and also to stabilize the organic residues in the MSW and sludge – removing the harmful pathogens and heavy metals from the compost. The quality of compost is significantly better, rich in key minerals and beneficial soil microbes. In fact in the conventional aerobic composting process which is thermophilic (temperature rising up to 55 C) many beneficial microbes are killed and nutrient especially nitrogen is lost (due to gassing off of nitrogen). Vermicomposting by earthworms excels all other ‗bio-degradation‘ and ‗bio-conversion‘ technologies by the fact that - it can utilize waste organics that otherwise cannot be utilized by others; achieve greater utilization (rather than the destruction) of materials that cannot be achieved by others; and by the fact that it does all with ‗enzymatic actions‘ and enzymes are biological catalysts giving pace and rapidity to all biochemical reactions even in minute amounts. It also keeps the system fully aerated with plenty of oxygen available to aerobic decomposer microbes. Aerobic processes are about 10 times faster than the anaerobic.

1. Nearly Odor-free Process Earthworms create aerobic conditions in the waste materials by their burrowing actions, inhibiting the action of anaerobic micro-organisms which release foul-smelling hydrogen sulfide and mercaptans.

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2. Destroy Pathogens in the End Product (Compost) Making Them Pathogen Free The earthworms release coelomic fluids that have anti-bacterial properties and destroy all pathogens in the waste biomass. (Pierre et al.,1982). They also devour the protozoa, bacteria and fungus as food. They seems to realize instinctively that anaerobic bacteria and fungi are undesirable and so feed upon them preferentially, thus arresting their proliferation. More recently, Dr. Elaine Ingham has found in her research that worms living in pathogen-rich materials (e.g. sewage and sludge), when dissected, show no evidence of pathogens beyond 5 mm of their gut. This confirms that something inside the worms destroys the pathogens, and excreta (vermicast) becomes pathogen-free (Appelhof, 2003). In the intestine of earthworms some bacteria and fungus (Pencillium and Aspergillus) have also been found (Singelton et. al, 2003). They produce ‗antibiotics‘ and kills the pathogenic organisms in the sewage sludge making it virtually sterile. The removal of pathogens, faecal coliforms (E.coli), Salmonella spp., enteric viruses and helminth ova from sewage and sludge appear to be much more rapid when they are processed by E. fetida. Of all E.coli and Salmonella are greatly reduced. (Bajsa et al., 2003). Bajsa et al. (2004 and 2005) studied the pathogen die-off in vermicomposting of sewage sludge spiked with E.coli, S.typhimurium and E.faecalis at the 1.6-5.4 x 106 CFU/g , 7.25 x 105 CFU/g and3-4 x 10 4 CFU/g respectively. The composting was done with different bulking materials such as lawn clippings, sawdust, sand and sludge alone for a total period of 9 months to test the pathogen safety of the product for handling. It was observed that a safe product was achieved in 4-5 months of vermicomposting and the product remained the same quality without much reappearance of pathogens after in the remaining months of the test. Eastman (1999) and Eastman et. al., (2001) also studied significant human pathogen reduction in biosolids vermicomposted by earthworms. Lotzof (2000) also revealed that the pathogens like enteric viruses, parasitic eggs and E.coli were reduced to safe levels in sludge vermicast. Cardoso and Remirez (2002) reported a 90 % removal of fecal coliforms and 100 % removal of heliminths from sewage sludge and water hyacinth after vermicomposting. Contreras-Ramos et, al., (2005) also confirmed that the earthworms reduced the population of Salmonella spp. to less than 3 CFU/gm of vermicomposted sludge. There were no fecal coliforms and Shigella spp. and no eggs of helminths in the treated sludge. (Eastman et al.,2001; Kumar and Sekaran, 2004). Our studies, Brahmbhatt (2006) has also confirmed complete removal of coliforms by earthworms. In conventional aerobic composting system, which is ‗thermophilic‘ process, pathogens are killed due to high temperature (beyond 55 C). Wu and Smith (1999) studied that for efficient composting and pathogen reduction a temperature of 55 C must be maintained for 15 consecutive days. But this also kills several beneficial microbes. If this high temperature is not achieved which could be the case in small scale composting pathogen die off will not be effective. Vermicomposting is a ‗mesophilic composting‘ system where temperature does not increase beyond 30 C and the harmful microbes (pathogens) are killed selectively by the worms through biological (physiological), microbial and enzymatic actions. Nair et al. (2006) studied a combination of thermophilic followed by vermicomposting and observed that the combination of the process leads to faster reduction of pathogens than the same period of thermophilic composting (21 days) and after 2 and 3 months (Table 9). It was also noticed that 21 days of a combination of thermocomposting and vermicomposting produced compost

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with acceptable C:N ratio and good homogenous consistency of a fertilizer. However the E. coli and E. faecalis was greater than 110 MPN/g) while S. typhimurium was undetectable. The optimum period to obtain pathogen-free compost was 9 days of thermophilic composting followed by 2.5 months of vermicomposting (Nair et. al., 2006). The study also indicated that vermicomposting leads to greater reduction of pathogens after 3 months upon storage. Whereas, the samples which were subjected to only thermofilic composting, retained higher levels of pathogens even after 3 months. Table 8. Removal of Pathogens (E. coli and E. faecalis) in Thermophilic Composting Vis-a-Vis Vermicomposting Processes

Source: Nair et. al., (2006) Key: dT = days of Thermophilic composting; dV = days of Vermicomposting N.B. E.coli and E. faecalis was tested using the Most Probable Number (MPN) per gram of compost (Standards Australia, 1995 a and b respectively).

3. Vermicompost Is Free of Toxic Chemicals Several studies have found that earthworms effectively bio-accumulate or biodegrade several organic and inorganic chemicals including ‗heavy metals‘, ‗organochlorine pesticide‘ and ‗polycyclic aromatic hydrocarbons‘ (PAHs) residues in the medium in which it feeds. (Nelson et al., 1982; Ireland, 1983). 4. Low Greenhouse Gas Emissions by Vermi-composting of Waste Emission of greenhouse gases carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) in waste management programs of both garbage and sewage has become a major global issue today in the wake of increasing visible impacts of global warming. Biodegradation of organic waste has long been known to generate methane (CH4). Studies have also indicated high emissions of nitrous oxide (N2O) in proportion to the amount of food waste used, and methane (CH4) is also emitted if the composting piles contain cattle manure. N2O emission is relatively high at the beginning of the conventional aerobic composting process, but declines after two days. N2O is mainly formed under moderate oxygen (O2) concentration. High emission of N2O at the beginning might be due to the metabolism of the microbial community coming from food waste, as food waste have been found to generate N2O even stored at 40 C. (Toms et, al. 1995; Wu et, al. 1995; Wang et, al., 1997; and Yaowu et. al., 2000). In theory, vermicomposting by worms should provide some potentially significant advantages over conventional composting with respect to GHG emissions. Worms significantly increase the proportion of ‗aerobic to anaerobic decomposition‘ in the compost

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pile by burrowing and aerating actions leaving very few anaerobic areas in the pile, and thus resulting in a significant decrease in methane (CH4) and also volatile sulfur compounds which are readily emitted from the conventional (microbial) composting process.(Mitchell et al., 1980). Molecule to molecule, methane is 20-25 times more powerful GHG than the CO2. Earthworms can play a good part in the strategy of greenhouse gas reduction and mitigation in the disposal of global organic wastes as landfills also emit methane resulting from the slow anaerobic decomposition of waste organics over several years. Recent researches done in Germany has found that earthworms produce a third of nitrous oxide (N2O) gases when used for vermicomposting (Frederickson, 2007). Molecule to molecule N2O is 296 to 310 times more powerful GHG than carbon dioxide (CO2). However, analysis of vermicompost samples has shown generally higher levels of available nitrogen (N) as compared to the conventional compost samples made from similar feedstock. This implies that the vermicomposting process by worms is more efficient at retaining nitrogen (N) rather than releasing it as N2O. This needs further studies and is being done at Griffith University, Brisbane, Australia (Middleditch, 2008).

5. More Homogenous End Products Rich in Nutrient The advantages for employing vermi-composting to process and stabilize organic waste including sewage sludge over conventional composting by microbial action are that the end product are more homogenous, rich in plant nutrients and the levels of contaminants are significantly reduced. Lotzof, 1999). McCarthy (2002) asserts that vermicomposting of sewage sludge has converted them into an end product that is safe for agricultural use. Vermicompost has very ‗high porosity‘, ‗aeration‘, ‗drainage‘ and ‗water holding capacity‘ and also contains ‗plant-available nutrients‘. The resulting product appears to retain more nutrients for longer period of time and also greatly increases the water holding capacity of the farm soil (Hartenstein and Hartenstein, 1981; Appelhof, 1997). Table 9. Initial and Final Nutritional Quality of the Vermicomposted Sludge

Source: Bajsa et al., 2005 Key: SL –Sludge; LC – Lawn clippings; SD – Sawdust; S – Sand

The chemical analyses of casts showed two times more available magnesium, 15 times more available nitrogen and seven times more available potassium compared to the surrounding soil (Kaviraj and Sharma, 2003). This is an excellent biofertilizer and soil conditioner (Jensen, 1998).

6. No or Low Energy Use in Vermi-composting Process Conventional microbial composting requires energy for aeration (constant turning of waste biomass and even for mechanical airflow) and sometimes for mechanical crushing of

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waste to achieve uniform particle size. Vermi-composting do not involve such use of energy. Occasionally turning may be required for aeration which can be done manually.

7. Production of Earthworm Biomass : A Nutritive Earthworm Meal Large scale vermi-composting of sludges would result into tons of earthworms biomass every year as under favorable conditions earthworms can ‗double‘ their number at least every 60 – 70 days. Potentially large quantities of worm biomass will be available as ‗pro-biotic‘ food for the cattle and fish farming, after the first year of composting. This can be a good source of nutritive ‗worm meal‘ rich in essential amino acids ‗lysine‘ and ‗methionine‘. It is superior to even ‗fish meal‘. 8. Mineralization of Nutrients from the Sludge Earthworms accelerates the decomposition of the sludge and mineralization of the organic compounds in it. Most important is that earthworms mineralize the nitrogen (N) and phosphorus (P) in the sludge to make it bio-available to plants as nutrients. They ingest nitrogen from the sludge and excrete it in the mineral form as ammonium and muco-proteins. The ammonium in the soil is bio-transformed into nitrates. Phosphorus (P) contents increased in the vermicomposted sludge treated with earthworms but decreased in the samples without earthworms, while the nitrogen (N) content did not show much difference (Parvaresh, et. al., 2004). Elvira et, al,. (1998) reported increase in the potassium (K) content of the sludge vermicompost. 9. Decrease in Total Organic Carbon (TOC) and Lowering of C/N Ratio of Sludge This has significance when the composted sludge is added to soil as fertilizer. Plants cannot absorb and assimilate mineral nitrogen unless the carbon to nitrogen (C/N) ratio is about 20:1 or lower. Mineralization of organic matter in the sewage sludge by earthworms lead to significant decrease in total organic carbon (TOC) content thus lowering the C/N ratio. This they do by consuming and breaking carbon compounds during respiration. Elvira et al., (1998) found that vermicomposting of paper-pulp-mill sewage sludge for 40 days decreased carbon (C) content by 1.7 fold. Contreras-Ramos et al., (2005) found that carbon content decreased by 1.1 to 1.4 fold in two months. Our work (Brahambhatt, 2006) also indicated that earthworms reduce TOC from composted sludge. 10. Reduction in Volatile Solids (VS) from Sludge Maximum reduction of the volatile solids (VS) is the goal of any sludge stabilization system and reduced VS is an indicator of stabilized sludge. Study found that Elsenia fetida increases the rate of volatile solid sludge (VSS) destruction when present in aerobic sludge and this reduces the probability of putrefaction occurring in the sludge due to anaerobic conditions. (Loehr, et al,1998). Earthworms creates and maintains good aerobic conditions in the sludge due to its burrowing actions and this enhances the process of VSS destruction. Hartenstein and Hartenstein (1981) reported a 9 % reduction in volatile solids over 4 weeks of sludge vermicomposting by earthworms, which was higher than that of control by almost one-third. Fredrickson et al., (1997) found a VS reduction of 30 % in compost after 4 months of conventional composting, whereas, the reduction was 37 % after only 2 months of

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vermicomposting. Higher decrease means more stable product and earthworms plays important role.

6. THE VERMIFILTRATION TECHNOLOGY (VFT) Vermifiltration of wastewater using waste eater earthworms is a newly conceived novel technology with several advantages over the conventional systems. Earthworms body work as a ‗biofilter‘ and they have been found to remove the 5 days BOD (BOD5) by over 90 %, COD by 80-90 %, total dissolved solids (TDS) by 90-92 % and the total suspended solids (TSS) by 90-95 % from wastewater by the general mechanism of ‗ingestion‘ and biodegradation of organic wastes, heavy metals and solids from wastewater and also by their ‗absorption‘ through body walls. Suspended solids are trapped on top of the vermifilter and processed by earthworms and fed to the soil microbes immobilized in the vermifilter. The most significant part is that there is ‗no sludge formation‘ in the process as the worms eat the solids simultaneously and excrete them as vermicast. (Huges et. al., 2005). This plagues most councils in world as the sludge being a biohazard requires additional expenditure on safe landfill disposal. This is also an odor-free process and the resulting vermi-filtered water is clean enough to be reused in industries as cooling water and also highly nutritive to be reused for farm irrigation.

6.1. Vermifiltration of Municipal Wastewater (Sewage) Sewage is the cloudy fluid of human fecal matter and urine, rich in minerals and organic substances. Water is the major component (99 %) and solid suspension amounts to only 1 %. The biochemical oxygen demand (BOD) and oxygen consumption (OC) values are extremely high demanding more oxygen by aerobic microbes for biodegradation of organic matter. Dissolved oxygen (DO) is greatly depleted. Low oxygen in water leads to anaerobic decomposition of organic contents and emission of obnoxious gases like hydrogen sulfide, methane and carbon monoxide. The nitrogen (N) and phosphorus (P) contents are very high and there are heavy metals like cadmium (Cd) and significant amount of coliform bacteria. The total suspended solids (TSS) is also very high like the BOD and nutrients and this often leads to a high anaerobic microenvironment in the sediments.

BOD, COD and SS Values of Raw Sewage and the Acceptable Values of Treated Sewage The average BOD value of the raw sewage ranged between 200 – 400 mg/L, COD ranged between 116 -285 mg/L, the TSS ranged between 300 – 350 mg/L and the pH ranged between 6.9 – 7.3. There is great fluctuation in these values depending upon catchment area, flow rate and season. Sewage from industrial areas can have high COD values, very low or high pH, due to accidental mixing of industrial wastewater. The normal acceptable values for BOD in treated wastewater is 1-15 mg/L, COD is 60-70 mg/L, TSS 20-30 mg/L and pH around 7.0.

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6.2. The Mechanism of Worm Action in Vermifiltration 1) The twin processes of microbial stimulation and biodegradation, and the enzymatic degradation of waste solids by worms as discussed above simultaneously work in the vermifiltration system too. 2) Vermifilters provide a high specific area – up to 800 sq m/g and voidage up to 60 %. Suspended solids are trapped on top of the vermifilter and processed by earthworms and fed to the soil microbes immobilized in the vermifilter. 3) Dissolved and suspended organic and inorganic solids are trapped by adsorption and stabilized through complex biodegradation processes that take place in the ‗living soil‘ inhabited by earthworms and the aerobic microbes. Intensification of soil processes and aeration by the earthworms enable the soil stabilization and filtration system to become effective and smaller in size. 4) Earthworms intensify the organic loadings of wastewater in the vermifilter soil bed by the fact that it granulates the clay particles thus increasing the ‗hydraulic conductivity‘ of the system. They also grind the silt and sand particles, thus giving high total specific surface area, which enhances the ability to ‗adsorb‘ the organics and inorganic from the wastewater passing through it. The vermicast produced on the soil bed also offers excellent hydraulic conductivity of sand (being porous like sand) and also high adsorption power of clay. This is ideal for diluted wastewater like sewage. (Bhawalkar, 1995). 5) Earthworms also grazes on the surplus harmful and ineffective microbes in the wastewater selectively, prevent choking of the medium and maintain a culture of effective biodegrader microbes to function.

6.3. Critical Factors Affecting Vermifiltration of Wastewater 1. Worm Population and Density (Biomass) in Vermifilter Bed (Soil) As the earthworms play the critical role in wastewater purification their number and population density (biomass) in soil, maturity and health are important factors. This may range from several hundreds to several thousands. There are reports about 8-10,000 numbers of worms per square meter of the worm bed and in quantity (biomass) as 10 kg per cubic meter (cum) of soil for optimal function. 2. Hydraulic Retention Time (HRT) Hydraulic retention time is the time taken by the wastewater to flow through the soil profile (vermifilter bed) in which earthworms inhabits. It is very essential for the wastewater to remain in the vermifiltration (VF) system and be in contact with the worms for certain period of time. HRT depends on the flow rate of wastewater to the vermifiltration unit, volume of soil profile and quality of soil used. HRT is very critical, because this is the actual time spent by earthworms with wastewater to retrieve organic mater from it as food. During this earthworms carry out the physical and biochemical process to remove nutrients, ultimately reducing BOD, COD and the TDSS. The longer wastewater remains in the system in contact with earthworms, the greater will be the efficiency of vermi-processing and

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retention of nutrients. Hence the flow of wastewater in the system is an important consideration as it determines the retention of suspended organic matter and solids (along with the chemicals adsorbed to sediment particles). Maximum HRT can results from ‗slower rate of wastewater discharge‘ on the soil profile (vermifilter bed) and hence slower percolation into the bed. Increasing the volume of soil profile can also increase the HRT. The number of live adult worms, functioning per unit area in the vermifilter (VF) bed can also influence HRT. HRT of vermifiltration system can be calculated as – HRT = (ρ x Vs) / Q wastewater where; HRT = theoretical hydraulic retention time (hours) Vs = volume of the soil profile (vermifilter bed), through which the wastewater flow and which have live earthworms (cum) ρ = porosity of the entire medium (gravel, sand and soil) through which wastewater flows Q wastewater = flow rate of wastewater through the vermifilter bed (cum / hr) Thus the hydraulic retention time (HRT) is directly proportion to the volume of soil profile and inversely proportion to the rate of flow of wastewater in the vermifilter bed.

3. Hydraulic Loading Rate (HLR) The volume and amount of wastewater that a given vermifiltration (VF) system (measured in area and depth of the soil medium in the vermifilter bed in which the earthworms live) can reasonably treat in a given time is the hydraulic loading rate of the vermifilter (VF) system. HLR can thus be defined as the volume of wastewater applied, per unit area of soil profile (vermifilter bed) per unit time. It critically depends upon the number of live adult earthworms functioning per unit area in the vermifilter bed. The size and health of the worms is also critical for determining the HLR. HLR of vermi-filtration system can be calculated as – HLR = V wastewater / (A X t) whereLR = Hydraulic Loading Rate (m / hr) V wastewater = volumetric flow rate of wastewater (cum). A = Area of soil profile exposed (sqm). t = Time taken by the wastewater to flow through the soil profile (hr). High hydraulic loading rate leads to reduced hydraulic retention time (HRT) in soil and could reduce the treatment efficiency. Hydraulic loading rates will vary from soil to soil. The infiltration rates depend upon the soil characteristics defining pore sizes and pore size

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distribution, soil morphological characteristics, including texture, structure, bulk density, and clay mineralogy.

6.4. Some Preliminary Studies on Vermifiltration of Sewage A pilot study on vermifiltration of sewage was made by Xing et al,. (2005) at Shanghai Quyang Wastewater Treatment Facility in China. The earthworm bed which was 1m (long) x 1m (wide) x 1.6m (high), was composed of granular materials and earthworms. The worm‘s number was kept at about 8000 worms/sqm. The average chemical oxygen demand (COD) value of raw sewage used was 408.8 mg/L that of 5 days biological oxygen demand (BOD5) was 297 mg/L that of suspended solids (SS) was 186.5 mg/L. The hydraulic retention time varied from 6 to 9 hours and the hydraulic loading from 2.0 to 3.0 m3 / (m2.d) of sewage. The removal efficiency of COD ranged between 81-86 %, the BOD5 between 91-98 %, and the SS between 97-98 %. Gardner et. al.,(1997) studied on-site effluent treatment by earthworms and showed that it can reduce the BOD and COD loads significantly. Taylor et. al., (2003) studied the treatment of domestic wastewater using vermifilter beds and concluded that worms can reduce BOD and COD loads as well as the TDSS (total dissolved and suspended solids) significantly by more than 70-80 %. Hartenstein and Bisesi (1989) studied the use of earthworm for the management of effluents from intensively housed livestock which contain very heavy loads of BOD, TDSS and nutrients nitrogen (N) and phosphorus (P). The worms produced clean effluents and also nutrient rich vermicompost. Bajsa et. al., (2003) also studied the vermifiltraion of domestic wastewater using vermicomposting worms with significant results.

6.5. Experimental Study of Vermifiltration of Sewage Chaudhry (2006) studied the vermifiltration of sewage using earthworms. The raw sewage was obtained from the Oxley Wastewater Treatment Plant in South Brisbane. The study was carried out in the same vermicomposting (VC) bin which was made to work as the vermifiltration (VF) kit. This was located in PC2 lab of Griffith University, as a hazardous material (raw sewage) was to be used. The temperature in lab was maintained at 21.50C, with 50% humidity. The Vermifiltartion kit contained about 30-40 kg of gravels with a layer of garden soil on top. This formed the vermifilter bed. It has provisions to collect the filtered water at the bottom in a chamber which opens out through a pipe fitted with tap. Above the chamber lies the net of wire mesh to allow only water to trickle down while holding the gravels above. The bottommost layer is made of gravel aggregates of size 7.5 cm and it fills up to the depth of 25 cm. Above this lies the aggregates of 3.5 to 4.5 cm sizes filling up to another 25 cm. On the top of this is the 20 cm layer of aggregates of 10-12 mm sizes mixed with sand. The topmost layer of about 10 cm consists of pure soil in which the earthworms were released. The worms were given around one week settling time in the soil bed to acclimatize in the new environment. As the earthworms play the critical role in wastewater purification their number and population density (biomass) in soil, maturity and health are important factors. This may range from several hundreds to several thousands. There are reports about 8-10,000

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numbers of worms per cubic meter of the worm bed and in quantity (biomass) as 10 kg per cubic meter (cum) of soil for optimal function. (Komarowski, 2001).However, in this experiment we started with 500 worms in 0.025 cum of soil bed, which comes to about 20,000 worms per cum of soil. This means even lesser number of worms could have done the job.

The Control Kit without Earthworms: Assessing the Precise Role of Worms A control kit (exact replica of vermifilter kit but devoid of earthworms) was also organised for reference and comparison. It is important to note that the soil and sand particles and the gravels in the kit also contribute in the filtration and cleaning of wastewater by ‗adsorption‘ of the impurities on their surface. They provide ideal sites for colonization by decomposer microbes which works to reduce BOD, COD and the TDSS from the wastewater. As the wastewater passes through, a layer of microbial film is produced around them and together they constitute the ‗geological‘ and the ‗microbial‘ (geo-microbial) system of wastewater filtration. With more wastewater passing through the gravels there is more formation of ‗biofilms‘ of decomposer microbes. Hence it is important to have a control kit in order to determine the precise role of earthworms in the removal of BOD, COD and the TDSS. Experiences, however, have shown that the geo-microbial system gets ‗choked‘ after sometimes due to slow deposition of wastewater solids as ‗sludge‘ and becomes unoperational whereas, the vermifiltration system with earthworms continues to operate. The Experimental Procedures Around 5 – 6 litre of municipal wastewater (sewage) was kept in calibrated 10 litre capacity PVC drum. These drums were kept on an elevated platform just near the vermifilter kit, as shown in figure 1. The PVC drums had tap at the bottom to which an irrigation system was attached. The irrigation system consisted of simple 0.5 inches polypropylene pipe with holes for trickling water that allowed uniform distribution of wastewater on the soil surface (vermifilter bed).

Figure 13. Vermifilter Kits : Regulated flow of wastewater from storage drums determining 1–2 hrs HRT.

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Figure 14.

Figure 15.

Figure 16.

Figure 17.

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Figures 14, 15, 16. Different sizes of gravels used in preparation of the vermifilter bed. Figure 17. Soil bed with earthworms and the device of wastewater discharge and distribution through perforated PVC pipes.

Wastewater from the drums flowed through the irrigation pipes by gravity. The wastewater percolated down through various layers in the vermifilter bed passing through the soil layer inhabited by earthworms, the sandy layer and the gravels and at the end was collected in a chamber at the bottom of the kit. Next day this treated wastewater from both kits were collected and analysed for BOD5 , COD and the TSS. The hydraulic retention time (HRT) was kept uniformly between 1-2 hours in all experiments.

Key Parameters Studied in Sewage Vermifiltration a) Biochemical Oxygen Demand (BOD) The biodegradable organic matter in wastewater is expressed as BOD (Biochemical Oxygen Demand). It is the amount of oxygen needed in a specified volume of wastewater to decompose organic material by the aerobic microbes. The unit of BOD is ppm or mg / L. b) Chemical Oxygen Demand (COD) Many organic substances, which are difficult to oxidize biologically by aerobic microbes, or are toxic to micro-organisms, such as lignin, can be oxidized chemically by using strong oxidizing agent like dichromate (Cr2O7) in acidic media. The unit of COD is ppm or mg / L.

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Earthworms can ingest and remove several organic substances from the wastewater which otherwise cannot be oxidized by microbes and thus bring down the COD values significantly.

c) Total Suspended Solid (TSS) Solids in wastewater consist of organic and inorganic particles and they can either be ‗suspended‘ or ‗dissolved‘. They provide adsorption sites for chemical and biological contaminants. As suspended solids degrade biologically, they can create toxic by-products. TSS can be represented as mg/L or ppm d) Turbidity Turbidity is measured as the cloudiness of water and this is caused by suspended and colloidal particles, such as clay, slit, finely divided organic matter. Turbidity is measured in NTU ( Number of Transfer Units). e) pH Value pH is a symbol that represents negative base -10 logarithm of the effective concentration of hydrogen ions (H+) in moles per Lit. The Analytical Methods Used in Laboratory Study The untreated sewage that was fed to the vermifilter kit and treated sewage which was collected at the bottom of the kit in a chamber were analyzed to study the biological oxygen demand (BOD), chemical oxygen demand (COD), (TSS), turbidity and the pH value. Following standard methods of analysis were adopted, Table 10. Standard Methods adopted to study specified parameters SR. No. 1 2 3 4 5

Description Biological Oxygen Demand (BOD) Chemical Oxygen Demand (COD) Total Suspended Solids (TSS) Turbidity PH

Standard Methods 405.1 5220C 160.2 180.1 D1293-99

The Experimental Results and Discussion a) Removal of BOD5 Results shows that the earthworms can remove BOD (BOD5) loads by over 98 % or nearly complete at hydraulic retention time (HRT) of 1-2 hours (Chowdhary, 2006). BOD removal in the control kit (where only the geological and microbial system works) is just around 77 %. (Table 11).

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Table 11. BOD5 Removal of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT: 1- 2 hrs) Expt No

Untreated Raw Sewage BOD5 (mg/L)

Treated Sewage BOD5 (mg/L) With Worms Without Worms (Vermifiltered) (CONTROL)

1 2 3 4 5

309 260 316 328 275

1.97 4.00 6.02 2.06 4.25

86.3 45.8 63.3 83.2 63.5

% Reduction in BOD5 by Earthworms (Vermifiltered) 99.4 98.5 98.1 99.4 98.5

% Reduction in BOD5 without Earthworms (CONTROL) 72.1 82.4 80.0 74.6 76.9

% Reduction in BOD5

Av. = 98.78 % Av. = 77.2 %. Source: Chowdhary (2006). 100 90 80 70 60 50 40 30 20 10 0 1

2

3

4

5

Experim ent No

% Reduction in BOD5 by Earthworms (Vermifiltered) % Reduction in BOD5 without Earthworms (CONTROL)

Figure 18. % Reduction in BOD5 of swage Water Treated With and Without Earthworms.

b) Removal of COD Results shows that the average COD removed from the sewage by earthworms is over 45 % while that without earthworms (only the geological and microbial system in the control kit) is just over 18 %. (Chowdhary, 2006). (Table 12). COD removal by earthworms is not as significant as the BOD, but at least much higher than the microbial system. Table 12. COD Removal of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT : 1 - 2 hrs) Expt No

Untreated Raw Sewage COD (mg/L)

Treated Sewage COD (mg/L) With Worms Without Worms (Vermifiltered) (CONTROL)

1 2 3 4 5

293 280 280 254 260

132 153 128 112 139

Av. = 45.7 % Av. = 18.4 %. Source: Chowdhary (2006).

245 235 201 217 227

% Reduction in COD by Earthworms (Vermifiltered) 54.9 45.4 54.3 55.9 46.5

% Reduction in COD without Earthworms (CONTROL) 16.4 16.1 28.2 14.6 12.7

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% Reduction in COD

100 90 80 70 60 50 40 30 20 10 0 1

2

3

4

5

Experim ent No

% Reduction in COD by Earthworms (Vermifiltered) % Reduction in COD without Earthworms (CONTROL)

Figure 19. % Reduction in COD of Sewage Wastewater Treated With and Without Earthworms.

c) Removal of TSS Results shows that the earthworms can significantly remove the suspended solids from the sewage by over 90 %, which in the control kit (where geological and microbial system works together) is over 58 % only. (Chowdhary, 2006). (Table 13). Table 13. TSS Removal Efficiency of Sewage Wastewater Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT 1 - 2 hrs) Expt No

Raw Untreated Wastewater TSS (mg/L)

Treated Wastewater TSS (mg/L) With Worms Without Worms (Control)

1 2 3 4 5

390 374 438 379 407

28 24 22 27 25

116 190 184 179 168

% Reduction in TSS by Earthworms (Vermifiltered) 92.82 93.58 94.97 92.87 93.85

% Reduction in TSS without Earthworms (CONTROL) 70.25 49.19 57.99 52.77 58.72

Av. = 92.97 % Av. = 58.34 %. Source: Chowdhary (2006).

Table 14. Turbidity Removal of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT : 1- 2 hrs) Expt No

1 2 3 4 5

Untreated Raw Sewage Turbidity (NTU) 112 120 74 70 100

Treated Sewage Turbidity (NTU) With Worms (Vermifiltered) 1.5 0.6 1.1 1.2 1.1

Without Worms (CONTROL) 3.6 1.5 1.2 1.8 2.0

Av.= 98.78 % Av.= 97.88 %. Source: Chowdhary (2006).

% Reduction in Turbidity by Earthworms (Vermifiltered) 98.7 99.5 98.5 98.3 98.9

% Reduction in Turbidity Without Earthworms (CONTROL) 96.8 98.8 98.4 97.4 98.0

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% Reduction in BOD5

d) Turbidity Removal Results indicates that the average reduction in turbidity by earthworms is over 98 % while that without earthworms in the control kit is also significantly high and over 97 %. (Chowdhary, 2006). (Table 14).

100 90 80 70 60 50 40 30 20 10 0 1

2

3

4

5

Experim ent No

% Reduction in TSS by Earthworms (Vermifiltered) % Reduction in TSS without Earthworms (CONTROL) Figure 20. % Reduction in TSS of Sewage Wastewater Treated With and Without Earthworms.

% Reduction in Turbidity

100 80

% Reduction in Turbidity by Earthworms (Vermifiltered)

60 40 20 0 1

2

3

4

5

% Reduction in Turbidity Without Earthworms (CONTROL)

Experiment No

Figure 21. % Reduction in Turbidity of sewage treated with and without Earthworms.

e) Improvement in pH Value of Treated Sewage Results indicates that the pH value of raw sewage is almost neutralized by the earthworms in the vermifilter kit. pH value of treated sewage without earthworms also improved but it was not consistent in all experiments. (Table 15).

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Table 15. Improvement in pH Value of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) Expt. No

Untreated Raw Sewage pH

1 2 3 4 5

6.58 5.76 6.65 6.67 6.05

Treated Sewage pH With Worms Without Worms (Vermifiltered) (CONTROL) 7.05 6.62 7.35 6.05 7.00 7.47 7.25 6.95 7.06 7.15

Source: Chowdhary (2006).

A

B

C

Figure 22. Appearance of Sewage After and Before Treatment. Bottle A : The clear vermifiltered sewage water; Bottle B: The hazy water from the controlled kit; Bottle C : The turbid and cloudy sewage water.

6.5. Significance and Advantages of Vermi-filtration Technology Over Conventional Wastewater Treatment Systems Vermi-filtration system is low energy dependent and has distinct advantage over all the conventional biological wastewater treatment systems- the ‗Activated Sludge Process‘, ‗Trickling Filters‘ and ‗Rotating Biological Contactors‘ which are highly energy intensive, costly to install and operate and do not generate any income. Since the conventional technologies are mostly the flow-processes and have finite hydraulic retention time (HRT) it always results into a ‗residual stream‘ of complex organics and heavy metals (while only the simple organics are consumed) in the ‗sludge‘ that needs further treatment (requiring more energy) before landfill disposal. This becomes unproductive. In the vermifilter process there

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is 100 % capture of organic materials, the capital and operating costs are less, and there is high value added end product (vermicompost).

1) Vermifilter Treatment of Wastewater is an Odorless System There is no foul odor as the earthworms arrests rotting and decay of all putrescible matters in the wastewater and the sludge. They also create aerobic conditions in the soil bed and the waste materials by their burrowing actions, inhibiting the action of anaerobic microorganisms which release foul odor. 2) Vermi-filtration is Low Energy System Some energy may be required in pumping the wastewater to the vermi-filtration unit if gravity flow is not adequate. 3) Synchronous Treatment of Wastewater and the Solids : End Products Become Useful Resource for Agriculture and Horticulture Earthworms decompose the organics in the wastewater and also devour the solids (which forms the sludge) synchronously and ingest the heavy metals from both mediums. This stabilized solids is discharged in the vermifilter bed as ‗excreta‘ (vermicompost) which is useful soil additive for agriculture and horticulture. 4) Vermifiltered Wastewater is Free of Pathogens As the earthworms devour on all the pathogens (bacteria, fungus, protozoa and nematodes) in the medium in which they inhabit the resulting filtered water becomes free of pathogens. The celeomic fluid secreted by worms which has ‗anti-bacterial‘ properties (Pierre et, al., 1982) further eliminate the pathogens. 5) Vermifiltered Wastewater is Free of Toxic Chemicals (Heavy Metals and Endocrine Disrupting Chemicals) As earthworms have the capacity to bio-accumulate high concentrations of toxic chemicals including heavy metals in their tissues the resulting wastewater becomes almost chemical-free. Earthworms have also been reported to bio-accumulate ‗endocrine disrupting chemicals‘ (EDCs) from sewage. Markman et al. (2007) have reported significantly high concentrations of EDCs (dibutylphthalate, dioctylphthalate, bisphenol-A and 17  - estrdiol) in tissues of earthworms (E.fetida) living in sewage percolating filter beds and also in garden soil.

6.6. Decentralized Sewage Treatment by Vermifiltration at Source of Generation All above results were obtained with approximately 500 worms in the vermifilter bed made in about 0.032 cubic meter (cum) of soil. This comes to approximately 16,000 worms per cum of soil. The initial number of worms must have increased substantially over the period of 7-8 weeks of experiment.

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If a vermifilter bed of 0.3 cum soil is prepared with approximately 5000 worms (over 2.5 kg) to start with, it can easily treat 950 - 1000 L of domestic wastewater / sewage generated by (on an average) a family of 4 people with average BOD value ranging between 300 - 400 mg/L, COD 100 – 300 mg/L, TSS, 300 – 350 mg/L everyday with hydraulic retention time (HRT) of the wastewater in the vermifilter bed being approximately 1 - 2 hours. Given that the worms multiply and double its number in at least every 60 days under ideal conditions of temperature and moisture, even starting with this number of earthworms a huge population (biomass) of worms with robust vermi-filtration system can be established quickly within few months which will be able to treat greater amount of wastewater generated in the family. An important consideration is the peak hour wastewater generation which is usually very high and may not comply with the required HRT (1 - 2 hrs) which is very critical for sewage treatment by vermi-filtration system. To allow 1 - 2 hrs HRT in the vermifilter bed an onsite domestic wastewater storage facility will be required from where the discharge of wastewater to the vermifilter tank can be slowly regulated through flow control.

7. THE VERMIREMEDIATION TECHNOLOGY (VRT) Large tract of arable land is being chemically polluted due to mining activities, heavy use of agro-chemicals in farmlands, landfill disposal of toxic wastes and other developmental activities like oil and gas drilling. No farmland of world especially in the developing nations are free of toxic pesticides, mainly aldrin, chlordane, dieldrin, endrin, heptachlor, mirex and toxaphene. There are over 80,000 contaminated sites in Australia, over 40,000 in the US; 55,000 in just six European nations, over 7,000 in New Zealand, and about 3 million contaminated sites in the Asia-Pacific. They mostly contain heavy metals cadmium (Cd), lead (Pb), mercury (Hg), zinc (Zn) etc. and chlorinated compounds like the PCBs and DDT. Cleaning them up mechanically by excavating the huge mass of contaminated soils and disposing them in secured landfills will require billions of dollars. Earthworms in general are highly resistant to many chemical contaminants including heavy metals and organic pollutants in soil and have been reported to bio-accumulate them in their tissues. Vermiremediation (using chemical tolerant earthworm species) is emerging as a low-cost and convenient technology for cleaning up the chemically polluted / contaminated sites / lands in world. Earthworms Efficiently Remove Toxic Organic and Inorganic Chemicals from Soil: Several species of earthworms but more particularly Eisenia fetida, Lumbricus terrestris and L. rubellus, have been found to remove significant amounts of heavy metals, pesticides and lipophilic organic micropollutants like the polycyclic aromatic hydrocarbons (PAH) from the soil (Contreras-Ramos et. al, 2006). Several studies have found definite relationship between ‗organochlorine pesticide‘ residues in the soil and their amount in earthworms, with an average concentration factor (in earthworm tissues) of about 9 for all compounds and doses tested. (Ireland, 1983). Studies indicate that the earthworms bio-accumulate or biodegrade ‗organochlorine pesticide‘ and ‗polycyclic aromatic hydrocarbons‘ (PAHs) residues in the medium in which it lives. (Nelson et al., 1982; Ireland, 1983). Earthworms especially E. fetida can bio-accumulate high concentrations of metals including heavy metals in their tissues without affecting their physiology and this particularly

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when the metals are mostly non-bioavailable. Studies indicate that they can take up and bioaccumulate in their tissues heavy metals such as cadmium (Cd), mercury (Hg), lead (Pb), copper (Cu), manganese (Mn), calcium (Ca), iron (Fe) and zinc (Zn). They can ingest and accumulate in their tissues extremely high amounts of zinc (Zn) and cadmium (Cd). Of all the metals Cd and Pb appears to bio-accumulate in most species of earthworms at greater level. They can particularly ingest and bio-accumulate extremely high amounts of cadmium (Cd) which is very mobile and may be readily incorporated into soft and non-calcareous tissues of earthworms. Cadmium levels up to 100 mg per kg dry weight have been found in tissues. Contreras-Ramos et, al.,(2005) also confirmed that the earthworms reduced the concentrations of chromium (Cr), copper (Cu), zinc (Zn) and lead (Pb) in the vermicomposted sludge (biosolids) below the limits set by the USEPA in 60 days. Malley et. al., (2006) also studied bioaccumulation of copper (Cu) and zinc (Zn) in E. fetida after 10 weeks of experiment. (Table 16). Table 16. Concentration of Cu and Zn in E. fetida Tissues Initially and After 10 Weeks

Initial sample Control Dosage 1 Dosage 2 Dosage 3

Cu (mg/Kg) ±SD 17.29±2 20.34 ±4 104.58 ± 47 158.95 ± 10 213.07 ± 22

Zn (mg/Kg) ±SD 108.22±4 127.54±8 137.52±8 132.03±16 138.51±18

Source: Malley et al., (2006).

Some metals are bound by a protein called ‗metallothioneins‘ found in earthworms which has very high capacity to bind metals. The chloragogen cells in earthworms appears to mainly accumulate heavy metals absorbed by the gut and their immobilization in the small spheroidal chloragosomes and debris vesicles that the cells contain.

7.1. Mechanism of Worm Action in Vermiremediation : The Uptake of Chemicals from Soil and Immobilization by Earthworms Earthworms uptake chemicals from the soil through passive ‗absorption‘ of the dissolved fraction through the moist ‗body wall‘ in the interstitial water and also by mouth and ‗intestinal uptake‘ while the soil passes through the gut. Earthworms eat large volume of soil with microbes and organic matter during the course of their life in soil. Earthworms apparently possess a number of mechanisms for uptake, immobilization and excretion of heavy metals and other chemicals. They either ‗bio-transform‘ or ‗biodegrade‘ the chemical contaminants rendering them harmless in their bodies.

a) Biotransformation of Chemical Contaminants in Soil Heavy metals in earthworms are bound by a special protein called ‗metallothioneins‘ which has very high capacity to bind metals. Ireland (1983) found that cadmium (Cd) and lead (Pb) are particularly concentrated in chloragogen cells in L. terrestris and D. rubidus, where it is bound in the form of Cd-metallothioneins and Pb-metallothioneins respectively

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(i.e. bio-transformed) with small amounts deposited in waste nodules. The chloragogen cells in earthworms appears to mainly accumulate heavy metals absorbed by the gut and their immobilization in the small spheroidal chloragosomes and debris vesicles that the cells contain.

b) Biodegradation of Chemical Contaminants in Soil: Earthworms Promote Microbial Activity for Biodegradation Ma et al., (1995) found that earthworms biodegrade organic contaminants like phthalate, phenanthrene and fluoranthene. It may be noted that several soil microorganisms especially bacteria and fungi also biodegrade several categories of chemicals including hydrocarbons in soil. However, when earthworms are added to the soil they further stimulate and accelerate microbial activity by increasing the population of soil microorganisms and also through improving aeration (by burrowing actions) in the soil, and in totality enhance the rate of biodegradation. The earthworms excrete the decomposer (biodegrader) microbes from their gut into soil along with nutrients nitrogen (N) and phosphorus (P). These nutrients are used by the microbes for multiplication and enhanced action. Reinforcement of microbial activity by earthworms for biodegradation of chemical contaminants in soil have been reported by Binet et al., (1998). Edward and Lofty (1972) showed that the number of bacteria and ‗actinomycetes‘ contained in the ingested material by earthworms increased up to 1000 fold while passing through the gut. A population of worms numbering about 15,000 can in turn foster a microbial population of billions of millions.

7.2. Experimental Study of Vermiremediation of PAHs Contaminated Soils Ryan (2006) studied the vermiremediation of PAHs contaminated soils by earthworms. The soil was obtained from a former gas works site in Brisbane where gas was being produced from coal. The initial concentration of total PAHs compounds in the soil at site was greater than 11,820 mg/kg of soil. (Ryan, 2006). The legislative requirements for soil PAHs concentration is only 100 mg/kg for industrial sites and 20 mg/kg for residential sites. 10 Kg of contaminated soil was taken in each of the four 40 litre black HDPE containers and made into Treatments 1,2, 3 and 4. The first remained as control and no treatment was done. In Treatment 2 approximately 500 earthworms (mixed species of E. fetida, Perionyx excavatus and Eudrillus euginae) of varying ages and sizes were added to the soil. (Worms were contained in about 2 kg of primary feed materials bought from Bunning Hardware). To this was added 5 kg of semi-dried cow dung as secondary feed material. In Treatment 3, about 5 kg of kitchen waste organics were added as secondary feed material to the 500 worms. In Treatment 4, only 5 kg of organic compost was added to the contaminated soil and no worms. This was set up to assess the effect of only microbial action on the contaminated soil as any organic compost is known to contain enormous amount of decomposer microbes. In all the four treatments enough water was added time to time, to maintain the moisture content between 70-80 % and were allowed to stand for 12 weeks. They were kept under shade thoroughly covered with thick and moist newspapers to prevent any volatilization or photolysis of the PAH compounds in the soil.

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Thus, Treatments 2 and 3 had total of 17 kg materials (10 kg contaminated soil + 2 kg of primary feed materials with worms + 5 kg of secondary feed material added additionally). Treatment 4 had 15 kg (10 kg soil + 5 kg compost). Due to addition of feed materials (cow dung and kitchen waste) and compost in the contaminated soil significant dilution of PAH compounds are expected to be made and was taken into consideration while determining the impact of earthworms and the microbes in the removal of PAH compounds. The results have been shown in tables 17 and 18 and figures 23 and 24. Mauli Table 17. Removal of Some PAH Compounds from Contaminated Soil by Earthworms Provided With Different Feed Materials (10 Kg Contaminated Soil + 500 Worms* in 2 kg Feed Materials With Additional Feed Materials Cow Dung (5kg) and Kitchen Waste (5kg) for 12 Wks) and compost (5Kg)

Source: Ryan (2006). *Mixed species of worms (E.fetida, Perionyx excavatus and Eudrillus eugeniae) were used.

Table 18. Percent Removal of Some PAH Compounds from Contaminated Soil by Earthworms Provided With Different Feed Materials (10 Kg Contaminated Soil + 500 Worms* in 2 kg Feed Materials With Additional Feed Materials Cow Dung (5kg) and Kitchen Waste (5kg) for 12 Wks)

Source: Ryan (2006). (%) Values within bracket are those after taking the dilution factor (due to mixing of feed materials) into account. This is just in 12 weeks period.

Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al.

80

100.00% Set 2 Soil + Worms + Cow Dung

% Removal

80.00% 60.00%

Set 3 Soil + Worms + Kitchen w aste

40.00% 20.00%

A ve ra ge

hr ys en F B e lo en u zo ra n (k th ) e F ne lo ur a B n en th en zo e (a B en ) P zo yr en (g e ,h ,i) py re ne

C

(b )

Set 4 Soil + Compost (No Worms)

B en

B en

zo

zo

(a )

an th r

ac en e

0.00%

Extracte d PAH Com pounds

100.00% 80.00%

Set 2 Soil + Worms + Cow Dung

60.00% 40.00% 20.00%

(a )a nt hr ac en e Be C nz hr o ys (b en )F e Be lo ur nz a o nt (k he )F ne lo ur D ib an en th zo en e (a , h Be )P nz yr o en (g e ,h ,i ) py re ne Av er ag e

0.00%

Be nz o

% Removal (Considering dilution factor)

Figure 23. Percent removal of PAH from contaminated soil by earthworms, provided with different feed materials.

Set 3 Soil + Worms + Kitchen waste Set 4 Soil + Compost (No Worms)

PAH Com pounds

Figure 24. Percent removal of some PAH compounds from contaminated soil by earthworms provided with different feed materials, after taking the dilution factor (due to mixing of feed materials) into account.

Findings and Results Results confirm the decisive role of earthworms in PAHs removal which can be by both activities – bioaccumulation, and by promoting microbial activities. When microbial activity in the soil is enhanced alone (without aided by earthworms) by adding microbe rich organic compost to the soil (Treatment 4) the removal rate of PAHs are not very significant. This indicates that earthworms acts in a different manner and contributes decomposer microbes for hydrocarbons which otherwise is normally not available in soil or in ordinary compost. Singleton et al. (2003) has reported some ‗uncultured‘ bacterial flora tightly associated with

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the intestine of the earthworms. Some of them, such as Pseudomonas, Acaligenes and Acidobacterium are known to degrade hydrocarbons. Providing the earthworms with additional feed materials in the form of cow dung and kitchen waste (Treatments 2 and 3) must have played important role in raising worm activity and in its reproductive behavior, and also in stimulating the soil microbial activity. There was not much significant difference in the impact of two types of feeds. Ma et al. (1995) showed an increase in PAH loss from polluted soil when the worms were denied of any additional source of food. They concluded that earthworms increase oral intake of soil particles when driven by ‗hunger stress‘ and consequently ingested more PAHs polluted soil. While this may be a temporary phenomenon in short time (9 weeks of study by authors) it can never be a long-term strategy because any bioremediation treatment of polluted soil is time-taking – in months and years. Worms would starve and die by then. And, adding organic feed materials to the polluted soil has several other advantages. It promotes microbial activities in soil and when the worms ingest them and excrete – the excreted products are nutrient rich organic fertilizers.

7.3. Advantages of Vermiremediation Technology: Earthworms Improves Total Quality of Soil in Terms of Physical, Chemical and Biological Properties Significantly, vermiremediation leads to total improvement in the quality of soil and land where the worms inhabit. Earthworms significantly contribute as soil conditioner to improve the physical, chemical as well as the biological properties of the soil and its nutritive value. They swallow large amount of soil everyday, grind them in their gizzard and digest them in their intestine with aid of enzymes. Only 5-10 percent of the chemically digested and ingested material is absorbed into the body and the rest is excreted out in the form of fine mucus coated granular aggregates called ‗vermicastings‘ which are rich in NKP (nitrates, phosphates and potash), micronutrients and beneficial soil microbes including the ‗nitrogen fixers‘ and ‗mycorrhizal fungus‘. The organic matter in the soil undergo ‗humification‘ in the worm intestine in which the large organic particles are converted into a complex amorphous colloid containing ‗phenolic‘ materials. About one-fourth of the organic matter is converted into humus. The colloidal humus acts as ‗slow release fertilizer‘ in the soil. During the vermi-remediation process of soil, the population of earthworms increases significantly benefiting the soil in several ways. A ‗wasteland‘ is transformed into ‗wonderland‘. Earthworms are in fact regarded as ‗biological indicator‘ of good fertile soil (Neilson, 1951). One acre of wasteland when transformed into fertile land may contain more than 50,000 worms of diverse species. Bhawalkar and Bhawalkar (1994) experimented and concluded that an earthworm population of 0.2 – 1.0 million per hectare of polluted land / wasteland can be established within a short period of three (3) months.

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8. THE VERMI-AGRO-PRODUCTION TECHNOLOGY (VAPT) Vermiculture biotechnology promises to usher in the ‗Second Green Revolution‘ through ‗Organic Farming‘ doing away with the destructive agro-chemicals which did more harm than good to both the farmers and their farmland during the ‗First Green Revolution‘ of the 1960‘s. Earthworms restore and improve soil fertility and boost crop productivity by the use of their metabolic product - ‗vermicast / vermicompost‘ (Tomati and Galli, 1995). They promote soil fragmentation and aeration, and brings about soil turning and dispersion in farmlands. On an average 12 tons / hectare / year of soil or organic matter is ingested by earthworms, leading to upturning of 18 tons of soil / year, and the world over at this rate it may mean a 2 inches of fertile humus layer over the globe (Bhawalkar and Bhawalkar, 1994). Earthworms excrete beneficial soil microbes, and secrete polysaccharides, proteins and other nitrogenous compounds into the soil from their body to improve the physical, chemical and the biological properties of soil.

8.1. Earthworms Vermicast / Vermicompost : A Miracle Growth Promoter Vermicompost produced by biodegradation of MSW by earthworms is a nutritive plant food rich in NKP (2 - 3 % nitrogen, 1.85 – 2.25 % potassium and 1.55 – 2.25 % phosphorus), micronutrients, beneficial soil microbes like ‗nitrogen-fixing bacteria‘ and ‗mycorrhizal fungi‘ and are wonderful growth promoters (Buckerfield, et al.,1999). Kale and Bano (1986) reports as high as 7.37 % of nitrogen (N) and 19.58 % phosphorus as P2O5 in worms vermicast). Moreover, vermicompost contain enzymes like amylase, lipase, cellulase and chitinase, which continue to break down organic matter in the soil (to release the nutrients and make it available to the plant roots) even after they have been excreted. (Chaoui et al., 2003). Vermicompost has very ‗high porosity‘, ‗aeration‘, ‗drainage‘ and ‗water holding capacity‘ and also contains ‗plant-available nutrients‘. Vermicompost appears to retain more nutrients for longer period of time and also greatly increases the water holding capacity of the farm soil (Hartenstein and Hartenstein, 1981; Appelhof, 1997).

a) High Level of Plant-Available Nutrients Atiyeh et al. (2000) found that the conventional compost was higher in ‗ammonium‘, while the vermicompost tended to be higher in ‗nitrates‘, which is the more available form of nitrogen. The nitrogenous waste excreted by the nephridia of the worms is mostly urea and ammonia. The ammonium in the soil is bio-transformed into nitrates. Patil (1993) found that earthworm recycle nitrogen in the soil in very short time. The quantity of nitrogen recycled is significant ranging from 20 to 200 kg N/ha/year. Barley and Jennings (1959) reported that worms significantly improve soil fertility by increasing nitrogen contents. Hammermeister et al. (2004) also found that vermicompost has higher N availability than the conventional compost on a weight basis. They also found that the supply of several other plant nutrients e.g. phosphorus (P), potassium (K), sulfur (S) and magnesium (Mg), were significantly increased by adding vermicompost as compared to conventional compost to soil.

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b) High Level of Beneficial and Biologically Active Soil Microorganisms Earthworms hosts millions of beneficial microbes (including the nitrogen fixers) in their gut and excrete them in soil along with nutrients nitrogen (N) and phosphorus (P) in their excreta i.e. vermicast (Singleton et al., 2003). The nutrients N and P are further used by the microbes for multiplication and vigorous soil remediation action. The mycorrhizal fungi encouraged by the earthworms transfer phosphorus by increasing solubilisation of mineral phosphate by the enzyme phosphatase. Edward and Fletcher (1988) and Edwards (1999) showed that the number of beneficial bacteria and ‗actinomycetes‘ contained in the ingested material increased up to 1000 fold while passing through the gut. A population of worms numbering about 15,000 will in turn foster a microbial population in billions. (Morgan and Burrows, 1982). c). Enhance Seed Germination Worms castings used as a seed germinator produce rapid results with a superior strike rate of ‗unusually healthy seedlings‘

d) Rich in Growth Hormones: Ability to Stimulate Plant Growth Researches show that vermicompost further stimulates plant growth even when plants are already receiving ‗optimal nutrition‘. Vermicompost has consistently improved seed germination, enhanced seedling growth and development, and increased plant productivity much more than would be possible from the mere conversion of mineral nutrients into plantavailable forms (Edwards and Burrows, 1988). Arancon (2004) found that maximum benefit from vermicompost is obtained when it constitutes between 10 to 40 % of the growing medium. Atiyeh et al. (2000) speculates that the growth responses of plants from vermicompost appears more like ‗hormone-induced activity‘ associated with the high levels of humic acids and humates in vermicompost rather than boosted by high levels of plantavailable nutrients. This was also indicated by Canellas et al. (2002) who found that humic acids isolated from vermicompost enhanced root elongation and formation of lateral roots in maize roots. Tomati et al. (1985) had also reported that vermicompost contained growth promoting hormone ‗auxins‘ and flowering hormone ‗gibberlins‘ secreted by earthworms.

8.2. Earthworms Reduce Soil Salinity, Renew Soil Fertility and Improve Crop Productivity Earthworms not only help renew the natural soil fertility but also improve the soil pH and reduce ‗soil salinity‘. Hota and Rao (1985) reported that three tropical earthworm species viz. Perionyx millardi, Octocheaetona surensis and Drawida calebi survived in saline solutions of 9.5 gm, 8.5 gm and 7 gm NaCl per liter (L) respectively. In a study made by Kerr and Stewart (2006) at the US Department of Energy it was found that E. fetida can tolerate soils nearly half as salty as seawater i.e. 15 gm / kg of soil. (Average seawater salinity is around 35 g/L). Farmers at Phaltan in Satara district of Maharashtra, India, applied vermiculture (live earthworms) on his sugarcane crop grown on saline soils irrigated by saline ground water. The yield was 125 tones / hectare of sugarcane and there was marked improvement in soil chemistry. Within a year there was 37 % more nitrogen, 66 % more phosphates and 10 %

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more potash. The chloride content was less by 46 %. Farmer in Sangli district of Maharashtra, India, grew grapes on eroded wastelands and applied vermicasting @ 5 tones / hectare. The grape harvest was normal with improvement in quality, taste and shelf life. Soil analysis showed that within one year pH came down from 8.3 to 6.9 and the value of potash increased from 62.5 kg/ha to 800 kg/ha. There was also marked improvement in the nutritional quality of the grape fruits (Bhawalkar and Bhawalkar, 1994).

8.3. Vermicompost Protects Plants Against Various Pests and Diseases There has been considerable anecdotal evidence in recent years regarding the ability of vermicompost to protect plants against various pests and diseases either by suppressing or repelling them or by inducing biological resistance in plants to fight them or by killing them through pesticidal action. Agarwal (1999),also reported less incidence of diseases in vegetable crops grown on vermicompost. Vermicompost also contains some antibiotics and actinomycetes which help in increasing the power of biological resistance among the crop plants against pest and diseases. Pesticide spray was reduced by 75 per cent where earthworms and vermicompost were used in agriculture. The actinomycetes fungus excreted by the earthworms in their vermicast produce chemicals that kill parasitic fungi such as Pythium and Fusarium.

a) Ability to Repel Pests There seems to be strong evidence that worms varmicastings sometimes repel hardbodied pests (Anonymous, 2001; Arancon, 2004). Edwards and Arancon, (2004) reports statistically significant decrease in arthropods (aphids, buds, mealy bug, spider mite) populations, and subsequent reduction in plant damage, in tomato, pepper, and cabbage trials with 20 % and 40 % vermicompost additions. George Hahn, doing commercial vermicomposting in California, U.S., claims that his product repels many different insects pests. His explanation is that this is due to production of enzymes ‗chitinase‘ by worms which breaks down the chitin in the insect‘s exoskelton (Munroe, 2007). b) Ability to Suppress Disease Edwards and Arancon (2004), also found statistically significant suppression of plantparasitic nematodes in field trials with pepper, tomatoes, strawberries and grapes. The scientific explanation behind this concept is that high levels of agronomically beneficial microbial population in vermicompost protects plants by out-competing plant pathogens for available food resources i.e. by starving them and also by blocking their excess to plant roots by occupying all the available sites. This concept is based on ‗soil-foodweb‘ studies pioneered by Dr. Elaine Ingham of Corvallis, Oregon, U.S.(http://www.soilfoodweb.com). Edwards and Arancon (2004) reported the agronomic effects of small applications of commercially produced vermicompost, on attacks by fungus Pythium on cucumber, Rhizoctonia on radishes in the greenhouse, by Verticillium on strawberries and by Phomposis and Sphaerotheca fulginae on grapes in the field. In all these experiments vermicompost applications suppressed the incidence of the disease significantly. They also found that the ability of pathogen suppression disappeared when the vermicompost was sterilized,

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convincingly indicating that the biological mechanism of disease suppression involved was ‗microbial antagonism. More studies is required to develop this potential of vermicompost as a sustainable, non-toxic and environmentally friendly alternative to chemical pest control or at least its application in farming practices can also lead to significant reduction in use of chemical pesticides.

8.4. Vermiwash: The Nutritive Liquid Byproduct of Vermicomposting Rich in Growth Promoting and Pesticidal Properties The brownish-red liquid which collects at the base of vermcomposting unit due to high moisture content in the compost pile should be collected. This liquid partially comes from the body of earthworms too (as worm‘s body contain plenty of water) and is rich in amino acids, vitamins, nutrients like nitrogen, potassium, magnesium, zinc, calcium, iron and copper and some growth hormones like ‗auxins‘, ‗cytokinins‘. It also contains plenty of nitrogen fixing and phosphate solubilising bacteria (nitrosomonas, nitrobacter and actinomycetes). Farmers from villages near Rajendra Agricultural University in Bihar reported growth promoting and pesticidal properties of this liquid. They used it on brinjal and tomato with excellent results. The plants were healthy and bore bigger fruits with unique shine over it. Spray of vermiwash effectively controlled all incidences of pests and diseases, significantly reduced the use of chemical pesticides and insecticides on vegetable crops and the products were significantly different from others with high market value. These farmers are using vermicompost and vermiwash in all their crops since last 2 years completely giving up the use of chemical fertilizers. (Personal Communication With Farmers in Pusa, December 2006).

8.5. Experimental Studies on Agronomic Impacts of Earthworms and Vermicompost on Crop Plants 1) Potted Wheat Crops Bhatia (1998) and Bhatia et al. (2000) studied the agronomic impact of ‗earthworms‘ on potted wheat crops at University of Rajasthan, Jaipur, India. Wheat seeds (Triticum aestivum Linn) and earthworms were obtained from Rajasthan Agricultural Research Institute, Jaipur. Three identical sets of pots with ten replicates of each were prepared from uniform soil of the same stock, which was assumed to be near neutral, i.e., without any organic matter. Three sets of 10 pots each were prepared. 1) Treatment 1 was kept as a control (without any input); 2) In Treatment 2, fifty (50) worms (mixed species of Eisinia fetida, Perionyx excavatus and Eudrillus euginae) were added to each pot and 250 gm of one week old cattle dung (allowing release of methane from fresh dung) was added as feed material for the worms. 3) In Treatment 3, chemical fertilizers (104 gms urea, 0.2 gms potash and 0.75 gms single super phosphate) were added;

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4) In Treatment 4, about 250 gm of week old cattle dung was added to precisely determine the role of earthworms in growth promotion as the dung also contain nutrients and support growth after degradation and conversion into compost. Ten seeds were sown in each pot (10 x 10 = 100 in ten pots) after mixing the soil with the respective fertilizers (Urea was added in two identical doses, one at the time of sowing and another 21 days). Results are given in table 19. Table 19. Agronomic Impacts of Earthworms Compared With Chemical Fertilizers on Growth and Yield of Potted Wheat Crops (Triticum aestivum Linn.)

Parameters

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

Number of seed germinated out of 100 Root length (Av. cm) Shoot length (Av. cm) Ear length (Av. cm) Total height of plant (Av. cm) Leaf length (Av. cm) Dry weight of ears (Av. cm) Number of seed grains per ear (Average) Chlorophyll content (mg/l) Number of tillers per plant

Treatment 1 Control

Treatment 2 Soil Containing Earthworms (50 Nos.) and Cattle Dung (As feed material)

Treatment 3 Soil With Chemical Fertilizers (N=104 gm; K=0.2 gm; P=0.75 gm)

Treatment 4 Soil Containing Cattle Dung (250 gm) only

50

90

60

56

7.13 22.1 4.82

16.46 59.99 8.77

9.32 25.2 5.45

8.23 23.1 5.1

34.16

85.22

39.97

37.30

12.73

26.37

14.19

13.45

0.135

0.466

0.171

0.16

11.8

31.1

19.9

17.4

0.783 1

3.486 2-3

1.947 1-2

1.824 1-2

Scale

Source: Bhatia (1998) and Bhatia et al. (2000): Also in Sustainable Agriculture (Sinha, 2004).; Key: Av. = Average. 100 90 80 70 60 50 40 30 20 10 0 Percentage of seed germination

Root length (cm)

Shoot length Ear length (cm) Total height of (cm) plant (cm)

Leaf length (cm)

Dry w eight of ears (gm)

Number of seed grains per ear

Chlorophyll Number of content (mg/l) tillers per plant

Parameter Set Set Set Set

1 2 3 4

Control Soil with Chemical Fertilizers Soil Containing Live Earthworms & Cow Dung (as feed material) Soil Containing Cattle Dung

Figure 25. Agronomic Impacts of Earthworms Compared With Chemical Fertilizers on Growth and Yield of Potted Wheat Crops (Triticum aestivum Linn.).

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Findings and Results The potted wheat crops with earthworms and added cattle dung as feed materials (Treatment 2) made excellent progress from the very beginning of seed germination up to maturation. They were most healthy and green, leaves were broader, shoots were thicker and the fruiting ears were much broader and longer with average greater number of seed grains per year. Significantly, they were much better even as compared to those grown on chemical fertilizers (Treatment 3). Worms fed on cattle dung and excreted them as vermicast (vermicompost) which worked as the miracle growth promoter. Wheat crops added with cattle dung only (Treatment 4) were almost close to those grown on chemical fertilizers. The dung was eventually converted into compost by soil microbes which worked as growth promoter. 2) Potted and Farm Wheat Crops Reena Sharma (2001) studied the agronomic impacts of vermiculture on the potted as well as on the farm wheat crops at University of Rajasthan, Jaipur, India. In this experiment live population of earthworms and vermicompost were applied separately to pot and farm soil. Same mixed species of worms e.g. E. fetida, P. excavatus and E. euginae were used and vermicompost was also prepared by using same species by composting kitchen waste and cattle dung. The farm was divided into eight plots of 25 x 25 sqm size. Three treatments were prepared: 1) Vermicompost : In the pots 30 gm of vermicompost was used while in the farm it was used @ 2.5 tonnes / ha. 2) Chemical Fertilizers (NPK) : As urea (N), single super phosphate (P) and potash (K), in one full dose and two reducing doses for the pots and one for farm. Vermicompost (30 gm) were applied with both reduced doses of chemical fertilizers; 3) Earthworms : In each pot 50 numbers of earthworms were used, while 1000 worms were released in the farm plot of 25 x 25 sq. mt. ; and 4) The fourth farm plot was kept as control (no inputs). All the treatments were replicated twice. The wheat seed was grown @ 100 kg/ha. Irrigation schedule was maintained as recommended for wheat. Chemical nitrogen (urea) was applied in two split doses (first half at the time of sowing and second half dose after 21 days of sowing) whereas the phosphate and potash fertilizers were applied as single dose at the time of sowing. Results are given in tables 20 and 21.

Findings and Results In the pot experiments the best growth performance in respect of root, shoot and ear length and their weight, weight of 1000 grains were observed where a combination of reduced dose (3/4) of chemical fertilizer (NPK-90:75:60) and normal amount of vermicompost (30 gm) were applied. It was significantly much better than even where full doses of chemical fertilizers were used. More significantly, both vermicompost and earthworms positively influenced the total yield of the grains which is 19.2 and 19.1 grains / ear respectively.

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Table 20. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Potted Wheat Crops Treatments

1. 2. 3. 4. 5. 6.

Vermicompost Earthworms (50 Nos.) NPK (120:100:80) Full Dose NPK (90:75:60) Reduced Dose + VC NPK (60:50:40) Reduced Dose + VC CONTROL

Shoot Length (cm) 41.11 39.27 42.14 47.89 45.96 37.01

Ear Length (cm) 7.65 6.74 7.67 8.91 8.59 5.83

Root Length (cm) 15.5 8.35 16.35 20.2 18.3 7.18

Wt. Of 1000 grains (gm)

Grains/ Ear

33.38 32.41 30.2 40.58 39.29 27.28

19.2 19.1 17.1 18.67 18.91 15.4

Source : Sharma (2001); In Sustainable Agriculture (Sinha, 2004); Key : VC= Vermicompost.

Table 21. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Farm Wheat Crops Treatments

1. 3 4 5.

Shoot Length (cm) Vermicompost (@ 2.5 t / ha) 83.71 NPK (90:75:60) (Reduced Dose) + VC 88.05 (Full Dose) NPK (120:100:80) (Full Dose) 84.42 CONTROL 59.79

Ear Length (cm) 13.14 13.82

Root Length (cm) 23.51 29.71

Wt. Of 1000 grains (In grams) 39.28 48.02

Grains / Ear

14.31 8.91

24.12 12.11

40.42 34.16

31.2 27.7

32.5 34.4

Source : Sharma (2001): In Sustainable Agriculture (Sinha, 2004); Key: VC = Vermicompost. Key: VC= Vermicompost; N = Urea; P = Phosphate; K = Potash. 60 50

Scale

40 30 20 10 0 Vermicompost

Live Earthw orms NPK (120:100:80) NPK ( 90:75:60 ) + NPK (60:50:40) + (50 Nos.) VC VC

CONTROL

Parameters Shoot Length (cm) Root Length (cm) Grains/Ear

Ear Length (cm) Wt. of 1000 grains (gm)

Figure 26. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Potted Wheat Crops.

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100 90 80

Scale

70 60 50 40 30 20 10 0 Shoot Length

Ear Length

Root Length

Wt. of 1000 grains

Grains / Ear

Parameters Vermicompost

Earthworms (1000 Nos.)

NPK (90:75:60) + VC

CONTROL

Figure 27. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Farm Wheat Crops.

Findings and Results In the farm experiment the highest growth and yield was achieved where reduced doses (3/4) of chemical fertilizer (NPK- 90:75:60) were applied with full dose of vermicompost (@ 2.5 tons/ha). However, the total yield of the grain (grain / ear) as well as the ear length and the weight of 1,000 grains of crops grown on vermicompost were as good as with those grown on full doses of chemical fertilizers (NPK). 3). Farmed Wheat Crops This facility was provided by Rajendra Agriculture University, Pusa, Bihar, India under a collaborative research program with Griffith University, Brisbane, Australia. We studied the agronomic impacts of vermicompost and compared it with cattle dung compost & chemical fertilizers in exclusive application and also in combinations on farmed wheat crops. Cattle dung compost was applied four (4) times more than that of vermicompost as it has much less NPK values as compared to vermicompost. Results are given in table - 22 Table – 22: Agronomic Impacts of Vermicompost, Cattle Dung Compost & Chemical Fertilizers in Exclusive Applications & In Combinations on Farmed Wheat Crops Treatment Input / Hectare Yield / Hectare 1). CONTROL (No Input) 15.2 Q / ha 2). Vemicompost (VC) 25 Quintal VC / ha 40.1 Q / ha 3). Cattle Dung Compost (CDC) 100 Quintal CDC / ha 33.2 Q / ha 4). Chemical Fertilizers (CF) NPK (120:60:40) kg / ha 34.2 Q / ha 5). CF + VC NPK (120:60:40) kg / ha + 25 Q VC / ha 43.8 Q / ha 6). CF + CDC NPK (120:60:40) kg / ha + 100 Q CDC / ha 41.3 Q / ha ----------------------------------------------------------------------------------------------------------------------------

Suhane et. al., (2008): Communication of RAU, Pusa, Bihar, India Key: N = Urea; P = Phosphate; K = Potash (In Kg / ha)

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90

50

Control

45 40

Vermicompost (25 Q / ha)

35 30

Cattle Dung Compost (100 Q / ha)

25 20

Chemical Fertilizer (NPK 120: 60:40)

15 10

CF (Full Dose) + VC (25 Q / ha)

5 0 Yield (Quintal / hectare)

CF (Full Dose) + CDC (100 Q / ha)

Figure 28: Agronomic Impacts of Vermicompost, Cattle Dung Compost & Chemical Fertilizers in Exclusive Applications & In Combinations on Farmed Wheat Crops

Findings & Discussion Exclusive application of vermicompost supported yield comparable to rather better than chemical fertilizers. And when same amount of agrochemicals were supplemented with vermicompost @ 25 quintal / ha the yield increased to about 44 Q / ha which is over 28 % and nearly 3 times over control. On cattle dung compost applied @ 100 Q / ha (4 times of vermicompost) the yield was just over 33 Q / ha. Application of vermicompost had other agronomic benefits. It significantly reduced the demand for irrigation by nearly 30-40 %. Test results indicated better availability of essential micronutrients and useful microbes in vermicompost applied soils. Most remarkable observation was significantly less incidence of pests and disease attacks in vermicompost applied crops. 4). Potted Corn Crops Sinha & Bharambe (2007) studied the agronomic impacts of earthworms & its vermicompost on corn plants at Griffith University, Brisbane, Australia. It had two parts A & B. Part A was designed to compare the growth promoting abilities of vermicompost with chemical fertilizers and also the earthworms when significantly present in the growth medium. It had three (3) treatments with three replicas of each and a control. Treatment 1 with 25 number of adult worms only; Treatment 2, with chemical fertilizers; and Treatment 3, with vermicompost and also containing same number of worms. Soluble chemical fertilizers ‗Thrive‘ was used. Approx. 8 gm of chemicals was dissolved in 4.5 L of water. It had total nitrogen (N) 15 %, total phosphorus (P) 4 %, total potassium (K) 26 % and a combination of essential micronutrients. Three applications were made during entire growth period while the worms and vermicompost was applied only onetime. Results are given in table 23 (a).

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Table 23 (a). Agronomic Impacts of Earthworms, Worms With Vermicompost and Chemical Fertilizers on Corn Plants (Added to 4 kg of soil; Av.Growth in cm)

Parameters Studied

CONTROL (No Input)

Treatment – 1 EARTHWORMS Only (25 Nos.) (Without Feed)

Treatment – 2 Soluble CHEMICAL FERTILIZERS

Treatment – 3 EARTHWORMS + VERMICOMPOST (200 gm)

Seed Sowing Seed Germination

29th July 2007 9th Day

Do 7th Day

Do 7th Day

Do 7th Day

Avg. Growth in 4 wks

31

40

43

43

Avg. Growth in 6 wks

44

47

61

58

App. Of Male Rep. Organ (In wk 12)

None

None

46

53

87

None

None

None

48

53 (App. Of Male Rep. Organ)

88

95

None

None

None

New Corn

53

56

92

105

Pale & thin leaves

Green & thin

Avg. Growth In 12 wks App. of Female Rep. Organ (In wk 14) Avg. Growth in 15 wks App. Of New Corn (in wk 16 ) Avg. Growth in 19 wks Color & Texture of Leaves

Male Rep. Organ

Green & stout leaves

Male Rep. Organ

90 Female Rep. Organ

Green, stout & broad leaves

Source: Sinha & Bharambe (2007)

Findings and Results Corn plants in pot soil with earthworms and vermicompost (Treatment 3 – Pot D) achieved good growth after week 4, and those with chemical fertilizers (Treatment 2 – Pot C) after week 6. After that, both maintained good growth over the other two (Control & Treatment 1 – Pots A & B) and attained maturity in 11 weeks (appearance of male spike). Female reproductive organ, however, appeared in corn plants grown on vermicompost only in 14 weeks. The ‗new corn‘ appeared after 111 days (in week 16). Until week 4, corn plants in all four treatments showed almost identical growth. Ironically, the corn plants in pot soil with ‗worms only‘ (Treatment 1) could not make any significant progress. Soil being completely devoid of organics could not be used by worms to produce any vermicast. However, they were all greener and healthier than control. Another significant finding was that pot soil with vermicompost demanded less water for irrigation as compared to others.

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120

100 CONTROL

Height in cm

80 EARTHWORMS (25) Without Feed

60

Soluble CHEMICAL FERTILIZERS (NPK) 40

EARTHWORMS (25) + VERMICOMPOST (200 gm)

20

0 Avg. Growth Avg. Growth in 4 wks in 6 wks

Avg. Growth Avg. Growth Avg. Growth in 12 wks in 15 wks in 19 wks

Figure 29 : Agronomic Impacts of Earthworms, Worms With Vermicompost and Chemical Fertilizers on Corn Plants (In 19 Weeks Period)

(1). (Growth until 4 weeks)

(2). (Growth until 6 weeks)

(3). (Growth until 12 weeks) Plants with chemical fertilizers (CF) and vermicompost (VC) grew well; Male spikes appeared in both plants in week 11. Keys:

(4). (Growth until 15 weeks) Plants with CF and VC maintained excellent growth; Female reproductive organs appeared in VC only in week 14 and ‗new corn‘ in week 16.

(A) Pot soil without any input (CONTROL) (B) Pot soil with worms only (C) Pot soil with chemical fertilizers (D) Pot soil with worms and vermicompost (200gm)

Figure 6 (a). Photo showing growth of corn plants under the influence of earthworms, worms with vermicompost and chemical fertilizers.

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Part – B This study was designed to test the growth promoting capabilities of earthworms added with feed materials and ‗vermicompost‘, as compared to ‗conventional compost‘. It had three (3) treatments with three (3) replicas of each. The dose of vermicompost was ‗doubled‘ (400 gm) from previous study and same amount of conventional compost was used. Only one application of each was made. Crushed dry leaves were used as feed materials (400 gm). Results are given in table 23 (b). Table 23 (b). Agronomic Impacts of Earthworms (With Feed), Vermicompost and Conventional Compost on Corn Plants (Added to 4 kg of soil ; Av. Growth in cm)

Parameters Studied Seed Sowing Seed Germination Avg. Growth in 3 wks Avg. Growth in 4 wks App. of Male Rep. Organ (in wk 6) Avg. Growth in 6 wks Avg. Growth in 9 wks App. Of Female Rep. Organ (in wk 10) App. of New Corn (in wk 11) Avg. Growth in 14 wks Color & Texture of Leaves

Treatment – 1 Earthworms (25) with Feed (400 gm) 9th Sept. 2007 5th Day 41 49 None 57 64

Treatment–2 Conventional COMPOST (400 gm) Do 6th Day 42 57 None 70 72.5

Treatment – 3

None

None

Female Rep. Organ

None 82

None 78

Green & thick

Light green & thin

New Corn 135 Deep green, stout, thick & broad leaves

VERMICOMPOST (400 gm) Do 5th Day 53 76 Male Rep. Organ 104 120

Source : Sinha & Bharambe (2007)

160

Growth in cm

140 120

Earthworms (25) With Feed (400 gm)

100 80

Conventional COMPOST (400 gm)

60

VERMICOMPOST(400 gm)

40 20 0 Avg. Avg. Avg. Avg. Avg. Growth in Growth In Growth In Growth Growth 3 wks 4 wks 6 wks In 9 wks in 14 wks

Figure 30: Agronomic impacts of Earthworms (with feed), Vermicompost and Conventional Compost on Corn Plants (In 14 Weeks Period)

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Findings and Results Corn plants in pot soil mixed with vermicompost (Treatment 3) achieved rapid and excellent growth after week 4 and attained maturity very fast. Between weeks 6 to 11, there was massive vegetative growth with broad green leaves. Male spike appeared in week 6 while the female reproductive organs appeared in week 10 which bored the ‗new corn‘ in 11th week. Corn plants in pot soil with worms only (Treatment 1) and conventional compost (Treatment 2) could not keep pace with vermicompost. Corn plants with worms only, however, were more green and healthy and took over those plants grown on conventional compost finally in the 14th week. Female reproductive organs and ‗new corn‘ could not appear until the completion of the study by 21st November 2007.

Keys:

(A) Pot soil with earthworms only (B) Pot soil mixed with conventional compost (400 gm) (C) Pot soil mixed with vermicompost (400 gm)

Figure 6 (b). Photo showing growth of corn plants under the influence of earthworms, conventional compost and vermicompost.

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Results of both studies (Part A & B) established beyond doubt that the ‗vermicompost‘ (metabolic products of worms) works like ‗miracle growth promoter‘ and is biologically (nutritionally) much superior to the conventional compost. This has also been found by other researchers (Subler et al., 1998; Pajon (Undated); and Bogdanov, 2004). And what is most significant is that when the dose of vermicompost is doubled (from 200 grams in Part A study to 400 grams in Part B, all other conditions remaining same) it simply enhanced total plant growth to almost two-fold (from average 58 cm on 200 gm VC to average 104 cm on 400 gm VC) within the same period of study i.e. 6 weeks. Corn plants with double dose of vermicompost (400 gm) achieved maturity in much shorter time. Male reproductive organs (spike) appeared after 81 days (in week 12) in plants grown on 200 gm of vermicompost, while in those grown on 400 grams, it appeared just after 39 days (in nearly half of the time in week 6). Similarly, the female reproductive organs and eventually the ‗new corn‘ appeared after 96 days (in week 14) and 111 days (in week 16 ) respectively in plants grown on 200 grams of vermicompost, while it appeared only after 69 days (in week 10) and 75 days (in week 11) respectively, in plants grown on 400 grams of vermicompost. It is also significant to note that the corn plants with earthworms only, performed better over those grown on conventional compost, once again establishing the role of earthworms as ―growth promoters‖. Study also confirmed that ‗vermicompost‘ is superior over ‗conventional compost‘ as growth promoter & in retaining soil moisture while also helping the plants to attain maturity and reproduce faster, thus reducing the ‗life-cycle‘ of crops and also shortening the ‗harvesting time‘. Conventional compost fails to deliver the required amount of macro and micronutrients including the vital NKP (nitrogen, potassium & phosphorus) to plants in shorter time. The leaves and stems of corn plants grown on vermicompost was much greener, broader and stouter than those grown on conventional compost (Sinha and Bharambe, 2007).

8.5.1 Current Experimental Study on Potted Wheat, Corn and Tomato Plantys Under Progress: Some Striking Observations of Plants Under Vermicompost Valani, Dalsukh and Chauhan, Krunal (2008) is currently studying the agronomic impacts of vermicompost vis-à-vis conventional cow dung compost and chemical fertilizers on wheat, corn and tomato plants. Some very exciting results have started appearing in the very beginning of the study.

1). High Strike Rates of Wheat Seed Germination The striking rates of wheat seeds were very high under vermicompost. They germinated nearly 48 hours (2 days) ahead of others and the number of seeds germinated were also high by nearly 20 %. The seedlings are also much greener and healthier and have more offshoots.. 2). Rapid Growth of Corn Seedlings There was not much difference in the striking rates of seed germination but once germinated the seedlings grew at a much faster rate almost doubling in size (from 22 cm to 42 cm) in just two days. Seedlings are more greener, stouter and healthier than others.

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3). More Flowering and Rapid Growth of Tomato Fruits Flowering in tomato plants under vermicompost was delayed by 5-6 days but once started there were greater numbers of flowers (average 7-8 per plant as compared to 5-6 in chemical fertilizers) and subsequently fruit developments. Significantly, the fruits grew in size very rapidly overtaking all others and are much greener. One more striking observation is that the tomato plants on vermicompost are showing greater adaptability to higher temperature (about 30º - 35º C) in the glasshouse.

CONCLUSIONS AND REMARKS Vermiculture practices for waste and land management and for improving soil fertility to boost crop productivity has grown considerably in recent years all over the world. Vermiculture is in fact a technological revival of the traditional age-old methods practiced by the ancient farmers for disposal of farm wastes and improvement of farm fertility by earthworms. It is like getting ‗gold from garbage (solid waste) by vermicomposting‘, ‗silver from sewage (wastewater) by vermifiltration‘; ‗converting a wasteland (chemically contaminated and saline lands) into wonderland (fertile land) by vermiremediation‘; ‗harvesting green gold (crops) by using brown gold (vermicompost) – and all with the help of earthworms which are abundant in soil all over the world but most prominently in the tropical nations. The three versatile species E. fetida, E. euginae and P. excavatus performing wide environmental functions occur almost everywhere. Earthworms are justifying the beliefs and fulfilling the dreams of Sir Charles Darwin as ‗unheralded soldiers‘ of mankind. (Sinha and Sinha, 2007).

9.1. The Vermicomposting Technology (VCT) Earthworms have real potential to both increase the rate of aerobic decomposition and composting of organic matter, and also to stabilize the organic residues in them, while removing the harmful pathogens and heavy metals from the end products. Waste composting by earthworms is proving to be environmentally preferred technology over the conventional microbial composting technology and much more over the landfill disposal of wastes as it is rapid and nearly odorless process, can reduce composting time significantly and above all, there is no emission of ‗greenhouse gas methane‘ which plagues both these solid waste management options. Vermicomposting of organic waste to divert massive domestic waste from the landfills are gaining importance and many municipal council in Australia are adopting this rapid and odorless vermicomposting technology (VCT) using waste eater earthworms.(UNSW ROU, 2002(a) and 2002(b)). Poor nations cannot even afford to construct and maintain a truly engineered landfill. Developing countries needs more options for safe waste management, and also low-cost, as they have several other social and developmental priorities with limited resources. Moreover, in the rural communities of both developed and developing world, centralized waste management system is never a good option. Individual households or a cluster of homes can

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treat their wastes better and also recover some useful resources (compost). This will prove more useful in the rural areas where farms are located. The worm number and quantity (biomass) is a ‗critical factor‘ for vermi-composting of organic wastes besides the optimal temperature and moisture which determines worm activity. A minimum of about 100-150 adult worms per kg of waste would be ideal to start with for rapid biodegradation and also odor-free process. Vermicomposting process driven by the earthworms tends to become more robust and efficient with time as the army of degrader worms grows and invade the waste biomass and further proliferating several battalions of aerobic decomposer microbial army. What is of greater significance is that earthworms accept and adapt to all kinds of new food products (even the fried foods) of modern civilization (except a few) to which their ancestors were never used to in history and readily degrade them converting into vermicompost.

9.2. The Vermifiltration Technology (VFT) Vermifiltration of wastewater is a logical extension of ‗soil filtration‘ which has been used for ‗sewage silviculture‘ (growing trees) since ancient days. Healthy soil is a biogeological medium acting as an ‗adsorbent‘ of organics, inorganic, pathogens and parasites. Vermifiltration technology (VFT) can be a most cost-effective and odor-free process for sewage treatment with efficiency, economy and convenience. Any non-toxic wastewater from the households, commercial organizations or industry can be successfully treated by the earthworms and the technology can also be designed to suit a particular wastewater. It can be used in a de-centralized manner in individual industry or cluster of similar industries so as to reduce the burden on the wastewater treatment plants down the sewer system. It can treat dilute (less than 0.1 % solids) as well as concentrated wastewater. It has an in-built pH buffering ability and hence can accept wastewater within a pH range of 4 to 9 without any pH adjustment. Though significant removal of BOD, COD and the TDSS is achieved by the geomicrobial system unaided by earthworms (as shown from our study in the control kit) the system fails to work after some time as it is frequently choked due to the formation of sludge and also colonies of bacteria and fungi (in the vermifilter bed) in the absence of the earthworms which constantly keep devouring on them. Presence of earthworms in the system also improve the ‗adsorption‘ properties of the geological system (sands and soils) by grinding them in their gizzard. Vermifiltration process driven by the earthworms also tends to become more robust and efficient with time as the army of degrader worms grows, further proliferating microbial population (army of aerobic decomposers). It is also a compact biological wastewater treatment system as compared to other non-conventional system such as the ‗constructed wetland system‘ which often suffer from limitations of oxygen for the decomposer aerobic microbes to act efficiently. Wetland based technologies involve mainly treatment and low utilization of waste materials and hence can be wasteful. BOD, COD, TSS and turbidity removal efficiency generally increases with increase in HRT up to a certain limit and is positively affected by the number of earthworms (earthworms biomass) per cubic meter (cum) in the vermifilter bed (soil profile). It can reduce the small BOD loadings of sewage (200-400 mg/L) within 30 – 40 minutes of HRT. Worms

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have been found to remove very high BOD loads (10,000 – 1,00,000 mg/L often found in wastewater from food processing industries) within 4 to 10 hours of HRT. If the population of worms were to be higher, the same efficiency of BOD, COD, TSS and turbidity removal could have been achieved at lower HRT. However, in case of municipal wastewater (sewage) treatment the objectives are not only to remove BOD, COD and TDSS, but also to remove the toxic chemicals including the heavy metals and pathogens from the wastewater. The presence of ‗endocrine disrupting chemicals‘ (EDCs) in sewage which was discovered recently with the development of new instruments (Markman et. al., 2007) is causing great concerns these days. And what is the matter of more serious concern is that they cannot be removed by the conventional sewage treatment methods. They can only be removed by the reverse osmosis methods of ‗membrane filtration technology‘ (MFT) which is cost-prohibitive at present and all nations cannot afford it. The cost-effective vermifiltration technology (VFT) assumes great significance in the removal of EDCs from wastewater. Hence greater hydraulic retention time (1-2 hours) is allowed so that the worms can ingest (bio-accumulate) the toxic chemicals and also devour upon the pathogens completely. Greater interaction with wastewater components also provides better opportunity for the worms to eat all the solids and prevent any sludge formation. Vermi-filtration of wastewater must be started with higher number of earthworms, at least over 15,000-20,000 worms per cubic meter (cum) of soil in the vermifilter bed for good results. It is also important that they are mostly adult and healthy worms. In vermicomposting of solid waste, which is a continuous process (in days and weeks) the worms have to act ‗gradually‘ in phases while their population (new army of bio-degraders) keeps on building up to intensify the biodegradation process. In vermifiltration of wastewater, the worms have to act ‗instantly‘ as the wastewater flow past their body (degrading the organics, ingesting the solids and the heavy metals). That is why the wastewater has to be ‗retained‘ (HRT) in the vermifilter bed for some appropriate period time (which has to be in hours and not in days) while the worms act on the wastewater.

9.3. The Vermiremediation Technology (VRT) Earthworms have great potential in removing hydrocarbons and many other chemicals from contaminated soil, even the PAHs like benzo(a)pyrene which is very resistant to degradation. They are extremely resistant to toxic PAHs and tolerate concentrations normally not encountered in the soil. It is important to mention here that nearly 80 % removal (60-65 % if the dilution factor is taken into account) of seven important PAH compounds was achieved in just 12 weeks period and that too with only about 500 worms (of both mature and juvenile population) in 10 kg of soil (50 worms/kg of soil). And this was during the winter season in Brisbane (March – May 2006) when the biological activities of worms are the lowest ebb. Increasing the number of worms per kg of contaminated soil to about 100 mature adult worm /kg of soil, and the time of remediation up to 16 weeks could have completely (100 %) removed the PAH compounds. Contreras-Ramos et. al, (2006) studied with 10 worms / 50 gms of contaminated soil (which is equivalent to 200 worms / kg of soil) in about 11 weeks and got 50-100 % removal of some PAHs. Many soils contain abundance of pores with diameters of 20 nm or less. Such pores are too small to allow the smallest bacterium (1 um), protozoa (10 um) or root hairs (7 um) to

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penetrate and attack the chemicals. A chemical contaminant residing in such fine pores in soil is thus completely protected from attack by a microbe in the soil for biodegradation action. In other words such chemical contaminants are not ‗bio-available‘ for any biological action. Earthworms play a very important and critical role here by enlarging the pores through continuous ‗burrowing actions‘ in the soil, thus allowing the microbes to enter into the pores and act on the contaminants. It also stimulate the population of decomposer microbes to several folds for enhanced biodegradation action. The ‗gizzard‘ in the earthworms helps to grind the food very thoroughly with the help of tiny stones swallowed by the worms into smaller particles 2-4 m in size. This grinding action may serve to make PAHs or any other chemical contaminants sequestered in the soil ‗bio-available‘ to decomposer microbes for degradation. Vermi-remediation may prove very cost-effective and environmentally sustainable way to treat polluted soils and sites contaminated with hydrocarbons in just few weeks to months. With the passage of time, the remedial action is greatly intensified. As the worms multiply at an enormous rate it can quickly achieve a huge arsenal for enhanced degradation of PAHs in much shorter time. Comparing the cost incurred in mechanical treatment by excavation of contaminated soils and their safe dumping in secured landfills (as hazardous wastes), this technology is most economic. More study will be needed on the PAH removal activities of earthworms with and without additional feed materials and upon different categories and doses of organic feed.

9.4.The Vermi-Agro-Production Technology (VAPT) Earthworms when present in soil inevitably work as soil conditioner to improve the physical, chemical as well as the biological properties of the soil and its nutritive value for healthy plant growth. This they do by soil fragmentation and aeration, breakdown of organic matter in soil and release of nutrients, secretion of plant growth hormones, proliferation of nitrogen-fixing bacteria, increasing biological resistance in crop plants, and all these worm activities contribute to improved crop productivity. (Barley, 1959). Studies have found that if 100 kg of organic waste with say, 2 kg of plant nutrients (NPK) are processed through the earthworms, there is a production of about 300 kg of ‗fresh living soil‘ with 6 % of NPK and several trace elements that are equally essential for healthy plant growth. This magnification of plant nutrients is possible because earthworms produce extra nutrients from grinding rock particles with organics and by enhancing atmospheric nitrogen fixation. Earthworms activate this ground mix in a short time of just one hour. When 100 kg of the same organic wastes are composted conventionally unaided by earthworms, about 30 kg compost is derived with 3 % NPK. This usual compost thus has a total NPK of only about 1 kg. Rest one kg nutrient might have been leached or volatilized during the process of composting. (Bhawalker and Bhawalkar, 1993 and 1994). The metabolic products (vermicast / vermicompost) of worm activities (feeding and excretion) works like a ‗miracle growth promoter‘. Vermicompost is agronomically much superior to conventional microbial compost sold in the market and can play very important role in promoting growth in crop plants, even competing with chemical fertilizers as our studies have shown. It is mainly due to the growth promoting factors (hormones and enzymes) that is present in the vermicompost. Study shows that doubling the dose of

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vermicompost almost double the growth of corn plants within the same period and can help attain maturity much earlier. Worms and its vermicompost have tremendous crop growth promoting potential while maintaining soil health and fertility and significantly reducing (by over 80 %) the use of chemical fertilizers and pesticides, and in some crops can even replace them completely. This is what being termed as ‗sustainable agriculture‘. Use of vermicompost in farm soil eventually leads to increase in the number of earthworm population in the farmland over a period of time, as the baby worms grow out from their cocoons contained in the vermicast. In Argentina, farmers who use vermicompost consider it to be seven (7) times richer than conventional composts in nutrients and growth promoting values (Pajon (Undated); Munroe, 2007). No wonder, Sir Charles Darwin, the great visionary scientist of 19th Century, called the earthworms as ‗friends of farmers‘ and ‗unheralded soldiers of mankind‘ working day and night under the soil.

Tribute to the Earthworms Earthworms are justifying the beliefs and fulfilling the dreams of Charles Darwin. He wrote a book in which he emphasized that there may not be any other creature in world that has played so important a role in the history of life on earth (Bogdanov, 2004). One of the leading authorities on earthworms and vermiculture studies Dr. Anatoly Igonin of Russia has said – ‗Nobody and nothing can be compared with earthworms and their positive influence on the whole living Nature. They create soil and everything that lives in it. They are the most numerous animals on Earth and the main creatures converting all organic matter into soil humus providing soil‘s fertility and biosphere‘s functions: disinfecting, neutralizing, protective and productive‘. (Appelhof, 2003). There can be little doubt that mankind‘s relationship with worms is vital and needs to be nurtured and further expanded.

REFERENCES AND MORE READINGS Anonymous (2001): Vermicompost as Insect Repellent; Biocycle, Jan. 01: p. 19. Agarwal, Sunita (1999): Study of Vermicomposting of Domestic Waste and the Effects of Vermicompost on Growth of Some Vegetable Crops; Ph.D Thesis Awarded by University of Rajasthan, Jaipur, India. (Supervisor: Rajiv K. Sinha) ARRPET (2005): Vermicomposting as an Eco-tool in Sustainable Solid Waste Management; ; Asian Institute of Technology, Anna University, India. Appelhof, M. (1997): Worms Eat My Garbage; 2nd (ed); Flower Press, Kalamazoo, Michigan, U.S. Appelhof, Mary (2003): Notable Bits; In WormEzine, Vol. 2 (5): May 2003 (Available at (http://www.wormwoman.com) Achazi, R.K., Fleener, C., Livingstone, D.R., Peters, L.D., Schaub, K.,Schiwe, E. (1998): Cytochrome P 450 and dependent activity in unexposed and PAH-exposed terrestrial annelids; J. of Comparative Biochemistry and Physiology; Part C, Vol. 121: pp. 339-350. Arancon, Norman (2004): An Interview with Dr. Norman Arancon; In Casting Call, Vol. 9 (2); August 2004. (http://www.vermico.com)

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Atiyeh, R.M., Subler, S., Edwards, C.A., Bachman, G., Metzger, J.D. Shuster, W. (2000): Effects of Vermicomposts and Composts on Plant Growth in Horticultural Container Media and Soil; In Pedobiologia, Vol. 44: pp. 579-590. Barley, K.P. (1959): The Influence of Earthworm on Soil Fertility II : Consumption of Soil and Organic Matter by the Earthworms; Australian Journal of Agricultural Research; 10: pp. 179-185. Barley, K.P. and Jennings A.C. (1959): Earthworms and Soil Fertility III; The Influence of Earthworms on the Availability of Nitrogen; Australian Journal of Agricultural Research; Vol. 10: pp. 364-370. Belfroid, A., Meiling, J., Drenth, H.J., Hermens, J., Seinen, W., Gestel, K.V. (1995): Dietary Uptake of Superlipophilic Compounds by Earthworms (Eisenia andrei); J. of Ecotoxicology and Environmental Safety; Vol. 31: pp. 185-191. Butt, K.R. (1999): Inoculation of Earthworms into Reclaimed Soils: The UK Experience; Journal of Land Degradation and Development; Vol. 10; pp. 565-575. Bharambe, Gokul (2006): Vermifiltration of Wastewater from Food Processing Industries (Brewery and Milk Dairy) in Brisbane; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2006. (Supervisor Dr. Rajiv K. Sinha). Brahambhatt, Ashish (2006): Vermistabilization of Biosolids; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2006. (Supervisor : Rajiv K. Sinha). Bajsa, O., J. Nair, K. Mathew and G.E. Ho (2003) : Vermiculture as a tool for domestic wastewater management; Water Science and Technology; IWA Publishing; Vol. 48: No 11-12; pp. 125-132; (Viewed on 5th May 2006).<www.iwaponline.com/wst/04811/ wst048110125.htm> Bajsa O., Nair J., Mathew K. and G.E.Ho. (2004): Pathogen Die Off in Vermicomposting Process; Paper presented at the International Conference on Small Water and Wastewater Treatment Systems, Perth, 2004. Bajsa O, Nair J, Mathew K and Ho G.E (2005): Processing of sewage sludge through vermicomposting, Water and Environment Management Series; (Eds.) K Mathew and I.Nhapi IWA Publishing London, UK , ISBN: 1-84339-511-8. Binet, F., Fayolle, L., Pussard, M. (1998): Significance of earthworms in stimulating soil microbial activity; Biology and Fertility of Soils; Vol. 27: pp. 79-84. Bhawalkar, V.U. and Bhawalkar, U.S. (1993): Vermiculture: The Bionutrition System;National seminar on Indigenous Technology for Sustainable Agriculture, I.A.R.I, New Delhi, March 23-24 : 1-8. Bhawalkar, U.S and Bhawalkar, V.U. (1994): Vermiculture Eco-technology; Publication of Bhawalkar Earthworm Research Institute (BERI), Pune, India. Bhiday, M.H. (1995): Wealth from Waste : Vermiculturing; Publication of Tata Energy Research Institute (TERI), New Delhi, India; ISBN 81-85419-11-6. Bhatia, Sonu (1998): Earthworm and Sustainable Agriculture : Study of the Role of Earthworm in Production of Wheat Crop; Field Study Report of P.G. Diploma in Human Ecology, University of Rajasthan, Jaipur, India. (Supervisor: Rajiv K. Sinha). Bhatia, Sonu., Rajiv K. Sinha, and Reena Sharma (2000): Seeking Alternatives to Chemical Fertilisers for Sustainable Agriculture: A Study of the Impact of Vermiculture on the Growth and Yield of Potted Wheat Crops (Triticum aestivum Linn); International J. of

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Environmental Education and Information; University of Salford, U.K. ; Vol. 19, No.4 : pp. 295-304. Bogdanov, Peter (2004): The Single Largest Producer of Vermicompost in World; In P. Bogdanov (Ed.), ‗Casting Call‘, Vol. 9 (3), October 2004. (http://www.vermico.com). Buckerfield, J.C., Flavel, T.C., Lee, K.E. and Webster, K.A. (1999): Vermicompost in Solid and Liquid Forms as a Plant – Growth Promoter; Pedobiologia, Vol. 43: pp. 753 – 759. Canellas. L.P., Olivares, F.L., Okorokova, A.L., and Facanha, R.A. (2002): Humic Acids Isolated from Earthworm Compost Enhance Root Elongation, Lateral Root Emergence, and Plasma Membrane H+ - ATPase Activity in Maize Roots; In J. of Plant Physiology, Vol. 130: pp. 1951-1957. Chaoui, H.I., Zibilske, L.M. and Ohno, T. (2003): Effects of earthworms casts and compost on soil microbial activity and plant nutrient availability; Soil Biology and Biochemistry, Vol. 35, No. 2; pp. 295-302. Chaudhari, Uday (2006): Vermifiltration of Municipal Wastewater (Sewage) in Brisbane; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2006. (Supervisor: Rajiv K. Sinha). Chauhan, Krunal (2008): Studies in Vermiculture Biotechnology; 40 CP Honours Project, Griffith University, Brisbane, Australia (Supervisors: Rajiv K. Sinha & Sunil Herat) Collier, J (1978): Use of Earthworms in Sludge Lagoons; In: R. Hartenstein (ed.) ‗Utilization of Soil Organisms in Sludge Management‘; Virginia. USA; pp.133-137. Ceccanti, B. and Masciandaro, G. (1999): Researchers study vermicomposting of municipal and paper mill sludges; Biocycle Magazine, (June), Italy. Cardoso L, Ramirez (2002): Vermicomposting of Sewage Sludge : A New Technology for Mexico; J. of Water Science and Technology; Vol. 46; pp. 153-158. Contreras-Ramos, S.M., Escamilla-Silva, E.M. and Dendooven, L. (2005): Vermicomposting of Biosolids With Cow Manure and Wheat Straw; Biological Fertility of Soils, Vol. 41; pp. 190-198. Contreras-Ramos, Silvia M., Alvarez-Bernal, Dioselina and Dendooven Luc (2006): Eisenia fetida Increased Removal of Polycyclic Aromatic Hydrocarbons (PAHs) from Soil; Environmental Pollution; Vol. 141: pp. 396-401; Elsevier Pub. Davis, B. (1971): Laboratory studies on the uptake of dieldrin and DDT by earthworms; Soil Biology and Biochemistry, .3, pp. 221-223. Dash, M.C (1978): Role of Earthworms in the Decomposer System; In: J.S. Singh and B. Gopal (eds.) Glimpses of Ecology; India International Scientific Publication, New Delhi, pp.399-406. Datar, M.T., Rao, M.N. and Reddy, S. (1997): Vermicomposting : A Technological Option for Solid Waste Management; J. of Solid Waste Technology and Management, Vol. 24 (2); pp. 89-93. Dominguez, J., Edward, C.A. and Webster, M. (2000): Vermicomposting of Sewage sludge: Affect of Bulking Materials on Growth and Reproduction of the Earthworms E.anderi; J. of Pedobiologia, Vol. 44: pp. 24-32. Eijsackers, H., Van Gestel, C.A.M., De Jonge, S., Muijis, B., Slijkerman, D. (2001): PAH Polluted Peat Sediments and Earthworms: A Mutual Inference; J. of Ecotoxicology; Vol. 10: pp. 35-50.

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Eastman, B.R. (1999): Achieving Pathogen Stabilization Using Vermicomposting; Biocycle, pp. 62-64; (Also on Worm World Inc. Available www.gnv.fdt.net/reference/index.html. Seen on 24.7.2001). Eastman, B.R., Kane, P.N., Edwards, C.A., Trytek, L., Gunadi, B., Stermer, A.L. and Mobley, J.R. (2001): The Effectiveness of Vermiculture in Human Pathogen Reduction for USEPA Biosolids Stabilization ; Compost Science and Utilization, Vol. 9 (1); pp. 3841. Edwards, C.A. and Fletcher, K.E. (1988): Interaction Between Earthworms and Microorganisms in Organic Matter Breakdown; Agriculture Ecosystems and Environment; Vol. 24, pp. 235-247. Edward, C.A. (1988): Breakdown of Animal, Vegetable and Industrial Organic Wastes by Earthworms; In C.A. Edward, E.F. Neuhauser (ed). ‗Earthworms in Waste and Environmental Management‘; pp. 21-32; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7. Edward, C.A. (2000): Potential of Vermicomposting for Processing and Upgrading Organic Waste; Ohio State University, Ohio, U.S. Edwards, C.A. and Burrows, I. (1988): The Potential of Earthworms Composts as Plant Growth Media; In C.A. Edward and E.F. Neuhauser (Eds.) ‗Earthworms in Waste and Environmental Management‘; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7; pp. 21-32. Edwards, C.A. and N. Arancon (2004): Vermicompost Supress Plant Pests and Disease Attacks; In REDNOVA NEWS: http://www.rednova.com/display/ ?id =55938. Elvira, C., Sampedro, L., Benitez, E., Nogales, R. (1998): Vermicomposting from Sludges from Paper Mills and Dairy Industries with Elsenia anderi: A Pilot Scale Study; J. of Bioresource Technology, Vol. 63; pp. 205-211. Evans, A.C. and Guild, W.J. Mc. L. (1948) : Studies on the Relationship Between Earthworms and Soil Fertility IV; On the Life Cycles of Some British Lumbricidae; Annals of Applied Biology; 35 (4) : 471-84. Fraser-Quick, G. (2002): Vermiculture – A Sustainable Total Waste Management Solution; What‘s New in Waste Management ? Vol. 4, No.6; pp. 13-16. Frederickson, J. Butt, K.R., Morris, R.M., and Daniel C. (1997) : Combining Vermiculture With Traditional Green Waste Composting Systems; J. of Soil Biology and Biochemistry, Vol. 29: pp. 725-730. Frederickson, Jim (2000): The Worm‘s Turn; Waste Management Magazine; August, UK. Frederickson, Jim (2007): Worms are Killing the Planet; Senior Research Fellow at UK Open University Faculty of Technology ; (Viewed on http://www.content.msn.in on 18.7.2007). Ghabbour, S.I. (1996) : Earthworm in Agriculture : A Modern Evaluation; Indian Review of Ecological and Biological Society; 111(2) : 259-271. Guerero, Angelica (2005): ‗Vermicomposting of Garden Waste (Grass Clippings)‘; Project submitted for the partial fulfillment of the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2005. (Supervisor: Rajiv K. Sinha). Graff, O. (1981): Preliminary experiment of vermicomposting of different waste materials using Eudrilus eugeniae Kingberg; In: M. Appelhof (ed.) Proc. of the workshop on ‗Role

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Kristiana, R., Nair, J., Anda, M., and Mathew, K., (2005): Monitoring of the process of composting of kitchen waste in an institutional scale worm farm; Water Science and Technology; Vol. 51 (10): pp. 171-177. Komarowski, S. (2001): Vermiculture for Sewage and Water Treatment Sludge; WATER, July 2001. Kumar, A. Ganesh and G. Sekaran (2004): The Role of Earthworm, Lampito mauritii in removal of Enteric Bacterial Pathogen in Municipal Sewage sludge; Indian J. of Environmental Protection; Vol. 24, No. 2; pp. 101-105. Kale, R.D., and Bano, K. (1986): Field Trials With Vermicompost, An Organic Fertilizer; In Proc. Of National Seminar on ‗Organic Waste Utilization by Vermicomposting‘; GKVK Agricultural University, Bangalore, India. Kale, R.D., Seenappa S.N. and Rao J. (1993): Sugar factory refuse for the production of vermicompost and worm biomass; V International Symposium on Earthworms; Ohio University, USA. Kale, R.D and Sunitha, N.S. (1995): Efficiency of Earthworms (E.eugeniae) in Converting the Solid Waste from Aromatic Oil Extraction Industry into Vermicompost; Journal of IAEM; Vol. 22 (1); pp. 267-269. Kale, R.D. (1998): Earthworms : Nature‘s Gift for Utilization of Organic Wastes; In C.A. Edward (ed). ‗Earthworm Ecology‘; St. Lucie Press, NY, ISBN 1-884015-74-376. Klein, J., Hughes R.J., Nair, J., Anda, M. and G.E. Ho (2005): Increasing the quality and value of biosolids compost through vermicomposting; Paper presented at ASPIRE Asia Pacific Regional Conference on Water and Wastewater, Singapore, 10-15 July 2005. Kaviraj and S. Sharma (2003): Municipal Solid Waste Management Through Vermicomposting Employing Exotic and Local Species of Earthworms; Journal of Bioresource Technology; Vol. 90 : pp. 169-173. Kanaly, R.A., Harayama (2000): Biodegradation of High Molecular Weight PAHs by Bacteria; J. of Bacteriology, Vol. 182: pp.2059-2067. Lakshmi, B.L. and Vizaylakshmi, G.S. (2000): Vermicomposting of Sugar Factory Filter Pressmud Using African Earthworms Species (Eudrillus eugeniae); Journal of Pollution Research; Vol. 19 (3): pp. 481-483. Lotzof, M. (1999): Very Large Scale Vermiculture on Biosolids Beneficiation; What‘s New in Waste Management ? Dec.-Jan. 1998-1999; pp. 22-26. Lotzof, M. (2000): Advances in Vermiculture a New Technique for Biosolids Management : A Case Study and New Research and Development Results; Paper Presented at Watertech, Sydney, Australia.Also ‗Very Large Scale Vermiculture in Sludge Stabilization‘ (Online)www.eidn.com.au/technicalpapervermiculture.htm (Viewed: 5.8.2002). Lotzof, M. (2000): Vermiculture: An Australian Technology Success Story; Waste Management Magazine; February 2000, Australia. Loehr, R.C., Martin, J.H., and Neuhauser, E.F. (1998): Stabilization of Liquid Municipal Sewage Sludge Using Earthworms; In Edward, C.A and Neuhauser E.F. (ed). ‗Earthworms in Waste and Environmental Management‘; SPB Academic Publishing, The Netherlands; ISBN 90-5103-017-7. Martin, J.P. (1976): Darwin on Earthworms: The Formation of Vegetable Moulds; Bookworm Publishing, ISBN 0-916302-06-7.

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Mitchell, M.J., Horner, S.G., and Abrams, B.L. (1980): Decompostion of Sewerage Sludge in Drying Beds and the Potential Role of the Earthworm Eisenia fetida; Journal of Environmental Quality, Vol. 9; pp. 373-378. McCarthy (2002): Beneficial Reuse of Sewage Sludge Vermicompost in Action; Water, March 2002; pp. 72-76. Masciandaro, G., Ceccanti, B. and Garcia, C. (2000): ‗In-Situ‘ Vermicomposting of Biological Sludges and Impacts on Soil Quality; J. of Soil Biology and Biochemistry; Vol. 32 (7): pp. 1015-1024. Morgan, M., Burrows, I., (1982): Earthworms/Microorganisms interactions; Rothamsted Exp. Stn. Rep. Ma, W.C., Imerzeel and Bodt, J. (1995): Earthworm and Food Interactions on Bioaccumulation and Disappearance of PAHs : Studies on Phenanthrene and Flouranthene; J. of Ecotoxicology and Environmental Safety; Vol. 32: 226-232. Markman, Shai., Irina, A. Guschinna., Sara Barnsleya, Katherine L. Buchanana, David Pascoea and Cartsen T. Mullera (2007): Endocrine Disrupting Chemicals Accumulate in Earthworms Exposed to Sewage Effluents; Cardiff School of Biosciences, Cardiff University, Cardiff, U.K.); J. of Chemosphere; Vol. 70 (1): pp. 119 – 125. Middleditch, Richards (2008): Estimation and Analysis of Greenhouse Gases (GHG) from Composting (Aerobic, Anaerobic and Vermicomposting) of Waste; Honours Project, Griffith University, Brisbane. (Supervisors: Andrew Chan and Rajiv K. Sinha). Malley, Christopher, Jaya Nair and Goen Ho (2006). Impact of heavy metals on enzymatic activity of substrate and on composting worms Eisenia fetida; Journal of Bioresource Technology, Vol 97: pp. 1498-1502. Munroe, Glenn (2007): Manual of On-farm Vermicomposting and Vermiculture; Pub. of Organic Agriculture Centre of Canada; 39 p. Neuhauser, E.F., Loehr, R.C. and Malecki, M.R. (1988): The Potential of Earthworms for Managing Sewage Sludge‘; In Edward, C.A and Neuhauser E.F. (ed). ‗Earthworms in Waste and Environmental Management‘; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7. Nair, Jaya., Vanja Sekiozoic and Martin, Anda (2006): Effect of pre-composting on vermicomposting of kitchen waste, Journal of Bioresource Technology, 97(16):20912095. Nair, Jaya., Kuruvilla Mathew and Goen, Ho (2007): Earthworms and composting wormsBasics towards composting applications; Paper at ‗Water for All Life- A Decentralised Infrastructure for a Sustainable Future‘; March 12-14, 2007, Marriott Waterfront Hotel, Baltimore, USA. Ndegwa, P.M. and Thompson, S.A. (2001): Integrated Composting and Vermicomposting in the Treatment and Bioconversion of Biosolids; J. of Bioresource Technology, Vol. 76 (2); pp. 107-112. Nelson, Beyer W.,Chaney, R.L. and Mulhern, B. (1982): Heavy Metals Concentrations in Earthworms from Soil Amended with Sewage Sludge; J. of Environmental Quality; Vol. 11 (3); pp. 382-385. Neilson, R.L. (1951): Earthworms and Soil Fertility; In Proc. Of 13th Conf. Of Grassland Assoc., New Plymouth, U.S; pp. 158 – 167. Neilson, R.L. (1965). Presence of Plant Growth Substances in Earthworms, Demonstrated by the Paper Chromatography and Went Pea Test; Nature, (Lond.) 208 : 1113-1114.

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NCSU (1997): Large Scale Vermi-composting Operations – Data from Vermi-cycle Organics, Inc.; North Carolina State University, U.S. OECD (2000) : Guidelines for Testing Organic Chemicals, Proposal for New Guidelines : Earthworms Reproduction Tests (E.fetida andrei); Organization for Economic Cooperation and Development. (www.oecd.org). Pajon, Silvio (Undated): ‗The Worms Turn - Argentina‘; Intermediate Technology Development Group; Case Study Series 4; (Quoted in Munroe, 2007). (http://www.tve.org./ho/doc.cfm?aid=1450andlang=English) Parish, Z.D., White J.C., Asleyan, M., Gent, M.P.N., Lannucci-Berger, W., Eitzer, B.D., Kelsey, J.W., and Mattina, M.I. (2005): Accumulation of Weathered PAHs by Plant and Earthworms Species; J. of Chemosphere, Vol. (N.A.): pp. (N.A.). Patil, B.B. (1993): Soil and Organic Farming; In Proc. Of the Training Program on ‗Organic Agriculture‘; Institute of Natural and Organic Agriculture, Pune, India. Patil, Swapnil (2005): Vermicomposting of Fast Food Waste; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; November 2005. (Supervisor : Rajiv K. Sinha). Piearce, T.G. and Piearce, B. (1979): Responses of Lumbricidae to Saline Inundation; Journal of Applied Ecology, Vol. 16 (2): pp. pp. 461 – 473. Pierre, V, Phillip, R. Margnerite, L. and Pierrette, C. (1982): Anti-bacterial activity of the haemolytic system from the earthworms Eisinia foetida andrei; Invertebrate Pathology, 40, pp. 21-27. Parvaresh, A. et, al., (2004): Vermistabilization of Municipal Wastewater Sludge With E.fetida; Iranian J. of Environmental Health, Science and Engineering; Vol. 1(2): pp. 4350. Palanisamy, S. (1996): Earthworm and Plant Interactions; Paper presented in ICAR Training Program; Tamil Nadu Agricultural University, Coimbatore. Ryan, David (2006): Vermiremediation and Other Bioremediation Options for PAHs Contaminated Soil; Project Report submitted for the partial fulfillment of the degree of Bachelor of Environmental Engineering, School of Engineering, Griffith University, Brisbane. (Supervisor : Rajiv K. Sinha). Riggle, D. and Holmes, H. (1994): New Horizons for Commercial Vermiculture; Biocycle, Vol. 35 (10); pp. 58-62. Singleton, D.R., Hendrix, B.F., Coleman, D.C., Whitemann, W.B. (2003): Identification of uncultured bacteria tightly associated with the intestine of the earthworms Lumricus rubellus; Soil Biology and Biochemistry; Vol. 35: pp. 1547-1555. Saxena, M., Chauhan, A., and Asokan, P. (1998): Flyash Vemicompost from Non-friendly Organic Wastes; Pollution Research, Vol.17, No. 1; pp. 5-11. Satchell, J. E. (1983) : Earthworm Ecology- From Darwin to Vermiculture; Chapman and Hall Ltd., London; pp.1-5. Seenappa, S.N. and Kale, R. (1993): Efficiency of earthworm Eudrillus eugeniae in converting the solid wastes from the aromatic oil extraction units into vermicompost; Journal of IAEM; Vol. 22; pp.267-269. Seenappa, S.N., Rao, J. and Kale, R. (1995): Conversion of distillery wastes into organic manure by earthworm Eudrillus euginae; Journal of IAEM; Vol. 22; No.1; pp.244-246. Sinha, Rajiv. K., Sunil Herat, Sunita Agarwal, Ravi Asadi, and Emilio Carretero (2002): Vermiculture Technology for Environmental Management : Study of the action of the

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earthworms Eisinia fetida, Eudrilus euginae and Perionyx excavatus on biodegradation of some community wastes in India and Australia; The Environmentalist, U.K., Vol. 22, No.2. June, 2002; pp. 261 – 268. Sinha, Rajiv K. (2004): Sustainable Agriculture; Surabhee Publication, Jaipur, India; ISBN 81-86599 – 60 – 6; p.370. Sinha, Rajiv K., Sunil Herat, P.D. Bapat, Chandni Desai, Atul Panchi and Swapnil Patil (2005): Domestic Waste - The Problem That Piles Up for the Society: Vermiculture the Solution; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, Canada; pp. 55-62. Sinha, Rajiv K. and Rohit Sinha (2007). Environmental Biotechnology (Role of Plants, Animals and Microbes in Environmental Management and Sustainable Development); Aavishkar Publisher, Jaipur, India; ISBN 978-81-7910-229-9; p. 315. Sinha, Rajiv K. and Gokul Bharambe (2007): Studies on Vermiculture Technologies (Vermicomposting, Vermi-desalinization and Vermi-agroproduction); Center for Environmental Systems Research (CESR) Project, Griffith University, Nathan Campus, Brisbane, Australia. Sharma, Reena (2001) : Vermiculture for Sustainable Agriculture : Study of the Agronomic Impact of Earthworms and their Vermicompost on Growth and Production of Wheat Crops; Ph.D. Thesis, submitted to the University of Rajasthan, Jaipur, India. (Supervision: Dr. Rajiv K. Sinha). Standards Australia (1995 a): Australian StandardsTM Method 6; Thermotolerant Coliforms and Escherichia coli – Estimation of Most Probable Number (MPN), AS 4276.6. Sinha, Rajiv K. (2004): Sustainable Agriculture; Surabhee Publication, Jaipur, India; ISBN 81-86599 – 60 – 6; p.370. Standards Australia (1995 b): Australian StandardsTM Method 8; Water Microbiology – Faecal streptococci - Estimation of Most Probable Number (MPN), AS 4276.8. Standards Australia (1995 c): Australian StandardsTM Method 14; Water Microbiology – Salmonellae- Estimation of Most Probable Number (MPN), AS 4276.14. Safawat, H., Hanna, S., Weaver, R.W. (2002): Earthworms Survival in Oil Contaminated Soil; J. of Plant and Soil; Vol. 240: pp. 127-132. Sims, R.C. and Overcash, M.R. (1983): Fate of Polynuclear Aromatic Hydrocarbons (PNAs) in Soil-Plant Systems; Residue Reviews, Vol. 88: pp. 2-68. Sharma, Reena (2001) : Vermiculture for Sustainable Agriculture : Study of the Agronomic Impact of Earthworms and their Vermicompost on Growth and Production of Wheat Crops; Ph.D. Thesis, submitted to the University of Rajasthan, Jaipur (Supervised by Dr. R.K. Sinha). Sadhale, Nailini (1996) : (Recommendation to Incorporate Earthworms in Soil of Pomogranate to obtain high quality fruits); In Surpala‘s Vrikshayurveda, Verse 131. (The Science of Plant Life by Surpala, 10th Century A.D.); Asian Agri-History Bulletin; No. 1. Secunderabad, India. Subler, Scott., Edwards, Clive., and Metzger, James (1998): Comparing Vermicomposts and Composts; Biocycle, Vol. 39: pp. 63-66. Suhane, R.K. (2007): Vermicompost; Publication of Rajendra Agriculture University, Pusa, Bihar, India. Suhane, R.K, Sinha, Rajiv.K. & Singh, Pancham.K. (2008): Communication of Rajendra Agriculture University, Pusa, Bihar, India.

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Tang, J., Liste, H., Alexander, M. (2002): Chemical Assays of Availability to Earthworms of PAHs in Soil; J. of Chemosphere, Vol.48: pp. 35-42. Tomati, V., Grappelli, A., Galli, E. (1983) : Fertility Factors in Earthworm Humus; In Proc. of International Symposium on ‗Agriculture and Environment : Prospects in Earthworm Farming; Rome, pp. 49-56. Tomati, V.; Grappelli, A. and Galli, E. (1987) : The Presence of Growth Regulators in Earthworm - Worked Wastes; In Proceeding of International Symposium on ‗Earthworms‘; Italy; 31 March- 5 April, 1985; pp. 423-436. Tomati, V. and Galli, E. (1995): Earthworms, Soil Fertility and Plant Productivity; Acta Zoologica Fennica; Vol. 196, pp. 11-14. Taylor et. al (2003): The treatment of domestic wastewater using small-scale vermicompost filter beds; Ecological Engineering; Vol. 21; pp. 197-203. Toms, P. Leskiw, J., and Hettiaratchi, P. (1995): Greenhouse Gas Offsets : An Opportunity for Composting; Presentation at the 88th Annual Meeting and Exhibition, June 8-12, 1995, San Antonio, Texas, USA; pp. 18-23. UNSW, ROU (2002 a): Vermiculture in Organics Management- The Truth Revealed; (Seminar in March 2002) University of New South Wales Recycling Organics Unit; Sydney, NSW, Australia. UNSW, ROU (2002 b): Best Practice Guidelines to Managing On-Site Vermiculture Technologies; University of New South Wales Recycling Organics Unit; Sydney, NSW, Australia; (Viewed on December 2004) www.resource.nsw.gov.au/data/ Vermiculture%20BPG.pdf USEPA (1995): A Guide to the Biosolids Risk Assessment for the EPA; Part 503 Rule EPA/B32-B-93-005; US Environmental Protection Agency Office of Wastewater Management, Washington, D.C. Valani, Dalsukh (2008): Studies in Vermiculture Biotechnology; 40 CP Honours Project, Griffith University, Brisbane, Australia (Supervisors: Rajiv K. Sinha & Sunil Herat) Vermitech (1998): Successful Biosolids Beneficiation With Vermitech‘s Large-Scale Commercial Vermiculture Facility in Redlands; Waste Disposal and Water Management in Australia; Vol. 25 (5); September-October, 1998. White, S. (1997): A Vermi-adventure in India; J. of Worm Digest; Vol. 15 (1): pp. 27-30. Wang, Y.S., Odle, WSI, Eleazer, W.E., and Baralaz, M.A (1997): Methane Potential of Food Waste and Anaerobic Toxicity of Leachate Produced During Food Waste Decomposition; Journal of Waste Management and Research; Vol. 15: pp. 149-167. Wu, XL., Kong, HN, Mizuochi, M., Inamori, Y., Huang, X., and Qian, Y. (1995): Nitrous Oxide Emission from Microorganisms; Japanese Journal of Treatment Biology, Vol. 31 (3): pp. 151-160. Wu, N., and Smith, J.E., (1999): Reducing Pathogen and Vector Attraction for Biosolids; Biocycle; Vol. (November 1999); pp. 59-61. Xing M., Yang, J. and Lu, Z. (2005): Microorganism-earthworm Integrated Biological Treatment Process – A Sewage Treatment Option for Rural Settlements; ICID 21st European Regional Conference, 15-19 May 2005; Frankfurt; Viewed on 18 April 2006. <www.zalf.de/icid/ICID_ERC2005/HTML/ERC2005PDF/Topic_1/Xing.pdf> Yaowu, He., Yuhei, Inamori., Motoyuki, Mizuochi., Hainan, Kong., Norio, Iwami., and Tieheng, Sun (2000): Measurements of N2O and CH4 from Aerated Composting of Food Waste; J.of the Science of The Total Environment, Elsevier, Vol. 254: pp. 65-74.

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Dr. Rajiv Sinha with his vermiculture team at Griffith University, Australia.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 3

HUMAN WASTE - A POTENTIAL RESOURCE: CONVERTING TRASH INTO TREASURE BY EMBRACING THE 5 R‘S PHILOSOPHY FOR SAFE AND SUSTAINABLE WASTE MANAGEMENT Rajiv K. Sinha1, Sunil Herat2, Gokul Bharambe3, Swapnil Patil3, Pryadarshan Bapat3, Krunal Chauhan3 and Dalsukh Valani3 1

School of Engineering (Environment), Griffith University, Nathan Campus, Brisbane, QLD-4111, Australia 2 School of Engineering (Environment), Griffith University 3 School of Engineering (Environment), Griffith University

Keywords: Culture of Consumerism; Culture of Disposables; Packaging Culture; Australians and Americans as Superconsumers and Waste Generators; Waste – A Misplaced Resource; Traditional Societies – Recycling Societies; Modern Society – Throwaway Society; Waste – Source of Greenhouse Gases; Vermiculture Movement for Efficient and Cost-effective Waste Management.

1. INTRODUCTION Waste is being generated by the human societies since ancient times. Ironically waste was not a problem for the environment when men were primitive and uncivilized. Waste is a problem of the modern civilized society. Materials used and waste generated by the traditional societies were little and ‗simple‘ while those by the modern human societies are large and ‗complex‘. With modernization in development drastic changes came in our consumer habits and life-style and in every activity like education, recreation, traveling, feeding, clothing and housing we are generating lots of wastes. The world today generate 

Corresponding Author: [email protected]

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about 2.4 billion tones of solid waste every year in which the Western World alone contributes about 620 million tones / year. Discarded products arising from all human activities (cultural and developmental) and those arising from the plants and animals, that are normally solid or semi-solid at room temperature are termed as solid wastes. Municipal solid waste (MSW) is a term used to represent all the garbage created by households, commercial sites (restaurants, grocery and other stores, offices and public places etc.) and institutions (educational establishments, museums etc.). This also includes wastes from small and medium sized cottage industries. We are facing the escalating economic and environmental cost of dealing with current and future generation of mounting municipal solid wastes (MSW), specially the technological (developmental) wastes which comprise the hazardous industrial wastes, and also the health cost to the people suffering from it. Developmental wastes poses serious risk to human health and environment at every stage – from generation to transportation and use, and during treatment for safe disposal. Another serious cause of concern is the emission of greenhouse gases methane and nitrous oxides resulting from the disposal of MSW either in the landfills or from their management by composting. Dealing with solid household waste in more sustainable ways involves changes not only to everyday personal habits, consumerist attitudes and practices, but also to the systems of waste management by local government and local industry and the retailers. This chapter reviews the causes and consequences of escalating human waste, the increasing complexity of the waste generated, and the policies and strategies of safe waste management. It also provides ‗food for thought‘ for future policy decisions that government of nations may have to take to ‗reduce waste‘ and divert them from ending up in the landfills, drawing experiences from both developed nation (Australia) and a developing nation (India).

2. CITIES AS THE CENTERS OF MOUNTING MUNICIPAL WASTES Cities have become major ‗centers of consumption and waste generation‘ all over the world. In fact a city ‗consumes‘ as well as ‗produce‘. This is called ‗urban metabolism‘. City use some 75 % of world resources and release a similar proportion of wastes. According to UN Population Fund Report (1990), a city with one million population consumes 2000 tones of food and 9,500 tones of fuel, generating 2000 tones of solid wastes (garbage and excreta) and 950 tones of air pollutants; consumes 6,25,00 tones of pure water and secrete 5,00,000 tones of sewage. (UNEP, 1996). United Nation Environment Program (UNEP) worked out the urban metabolism of London city. Greater London with a population of 7 million consumed 2,400,000 tones of food; 1,200,000 tones of timber; 2,200,000 tones of paper; 2,100,000 tones of plastics; 360,000 tones of glass; 1,940,000 tones of cement; 6,000,000 tones of bricks, blocks, sand and tarmac; 1,200,000 tones of metals every year and produced 11,400,000 tones of industrial and demolition wastes; 3,900,000 tones of household, civic and commercial wastes and 7,500,000 tones of wet, digested sewage sludge. Everyday London dispose off some 6,600 tones of household wastes. (UNEP, 1996).

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3. MODERN CULTURE OF CONSUMERISM: THE ROOT OF WASTE PROBLEM The root of waste problem is the ‗culture of consumerism‘ and is directly proportional to the affluency of the human societies. To this has added the ‗culture of disposables‘. Large number of goods in the society are being manufactured for only ‗one time use‘, and to be discarded as waste after use. Modern urban culture of using ‗canned and bottled foods‘, ‗frozen foods‘, ‗take-away foods‘, exchange of ‗greeting cards‘ on all occasions, using ‗disposable‘ home equipments (spoons, cups, plates, tumblers and safety razors), medical instruments (syringes, sharps and needles), office equipments (writing pens and utilities), and plastic bags in all grocery shopping has escalated the solid waste problems. People all over the world consume food and the 5 P‘s (paper, power, petrol, potable water and plastics) in their daily life without realizing the environmental consequences and costs of the waste generated in their production, distribution, and consumption. Every commodity processed from natural resources, and every consumer product, from ‗shampoo to champagne‘ has an environmental cost and generally causes some damage to the environment, ‗before use‘, as a source of pollution during production, and ‗after use‘, as waste (Eklington and Hailes, 1989).

3.1. Waste Generation Is Proportional to Resource Consumption and the Way We Use Resources Consuming resources and generating wastes are ‗two sides of the same coin‘. The way we use resources to maintain our ‗quality‘ of life, assumes as much significance in waste generation as the sheer amount of resources that we use. For instance, one kilogram of steel might be used in a construction that lasts hundreds of years or in the manufacture of several cans thrown away just after a few uses. A few kilograms of PVC plastic materials might be molded into durable home and office furniture, water and sewer pipes, and remain useful for decades or might be used to manufacture plastic bags to be used just once or twice and then thrown away as enduring waste.

Packaging Culture Proliferate Waste Generation Packaging materials have become part of our modern culture and generate huge amounts of waste. Manufacturers and retailers see packaging as a way to attract purchasers. Everything needs fine packaging today, from cornflakes to computers, from gifts to garments, from flowers to foods. Life today cannot be imagined without plastic bags, glass bottles, paper boxes, tins and cans. Plastics are versatile, convenient and light weight — good packaging material — but ultimately end up as a non-biodegradable waste in the landfills to remain intact for centuries. On average each European Union citizen is currently responsible, directly or indirectly, for the generation of some 172 kg of ‗packaging waste‘ every year. Packaging waste generation increased by 10 % in the EU between 1997 and 2002. Per capita consumption of plastics increased by almost 50 % from 64 kg / year in 1990 to 95 kg / year in 2002. Only UK

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managed to actually reduce, and Austria stabilize the generation of packaging waste since 1997. (GEO, 2006). Plastic consumption in Australia has increased from negligible quantities in the early 1940s to enormous quantities today. Plastics made up around one-third of all rubbish collected on Clean Up Australia Day (Clean Up Australia 2004). Even though Australians reduced their use of plastic shopping bags by around one-fifth between 2002 and 2004, each person was still using almost one bag per day (EcoRecyle 2006). Australians use 6 billion plastic bags every year much of that end up in landfills. These bags form litter, infest and block waterways, kill animals.

The ‘Ecological Footprint’ of Global Human Population The Measure of Resource Consumption and Waste Generation. An ecological footprint is an estimate of the average area of productive land and water required to maintain a given population‘s resource consumption and waste generation. (Table 1). In this instance we simply use it to indicate the comparable cost and sheer significance of waste generation. Australia with a small population of just 19.5 million, is part of the North developed world, and Australians are ‗super-consumers‘ and ‗super-waste makers‘. Table 1. Ecological Footprints of Human Population on Earth Reflecting Resource Consumption Vis-à-Vis Waste Generation (2002) Total Population (millions) World High income˚ countries Middle income countries Low income countries Australia United Kingdom China Asia Pacific (regional) Canada USA

6 225.0 925.6 2 989.4 2 279.8 19.5 59.3 1302.3 3448.4 31.3 291.0

Total Ecological Footprint (global ha/person) 2.2 6.4 1.9 0.8 7.0 5.6 1.6 1.3 7.5 9.7

Total Energy Footprint (global ha/person) 1.2 4.1 0.9 0.3 4.0 3.6 0.7 0.6 4.6 6.3

Total Biocapacity* (global ha/person) 1.8 3.4 2.1 0.7 11.3 1.6 0.8 0.7 15.1 4.7

Source: Global Footprint Network (2005). *Biocapacity includes cropland, grazing land, forest and fishing ground. ˚High income countries includes Australia.

In developing Asian countries, consumption and waste generation is growing at an unprecedented pace and populous countries like China and India are becoming consumerist. The ‗consuming class‘ in India is estimated at around 100 million, and in China at 200 million; traditional conservative Indians believing in modesty, simple living and saving, are gradually giving way to rich generations highly influenced by the consumerist North (UNEP, 2006). China manufactures, packages, transports, distributes nationally and for export over 60 billion pairs of disposable wood chopsticks which use up over 32 million trees, harvested

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unsustainably, indeed China risks being without forests within ten years.().

4. WASTE GENERATED IN THE RICH VIS-A-VIS IN THE POOR SOCIETIES OF WORLD The United Nations Environment Program (1992) carried out a study of per capita waste generation in the low, middle and the high-income countries and found it to be 0.5, 1.5 and 3.5 kg. per day per person respectively. The solid wastes generated in the rich affluent societies of the developed nations are exceptionally large in quantity and varied in quality (components). Of these, a considerable part is hazardous waste.

4.1. Waste Generated in the Rich Developed Countries of World The World Watch Institute (1991), Washington, reported that 14 out of 16 members of OECD countries showed increase in generation of MSW per person between 1980 – 85. In the US, each sunset sees a new mountain of nearly 410,000 tones of garbage. (Toth, 1990). The countries of the European Community (EC) throws away an estimated 2 billion tones of solid waste each year. Only Japan and West Germany produced less waste, but after unification MSW in Germany skyrocketed. Americans, Canadians and Australians are great waste makers. They generate roughly twice as much garbage per person as West Europeans or Japanese do. In his /her lifetime an average American wear and discard 250 shirts and 115 pairs of shoes; use and discard 27,50 newspapers, 3900 weekly magazines and 225 pounds of phone directories; consume 12,000 paper grocery bags, use 28,627 aluminum cans weighing 1022 pounds, use 69,250 pounds of steel and 47,000 pounds of cement. The Scandinavian nations generate much less waste than the Americans and Europeans. (WWI, 1991; UNEP, 1996). Table 2. Average Per Capita Municipal Solid Waste (MSW) Generated by Some Developed Nations Country Waste Generation Per Day (in Kg) Country USA Japan France Singapore Germany Italy

Waste Generation Per Day (in Kg) 1.80 – 2.60 1.38 - 2.10 1.10 – 1.90 0.87 – 1.37 0.75 – 1.85 0.69 – 1.75

Source: WWI (1990):‗State of the World‘ (These are 1990 Values which must have increased).

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Urban Waste Generated in Some Developed (Rich) Countries Country Annual Generation (In tones) USA 20,00,00,000 Canada 1,26,00,000 Australia 1,00,00,000 Spain Netherlands 89,28,000 54,00,000 Belgium Sweden 30,82,000 25,00,000 Switzerland 21,46,000 Denmark 20,46,000 Norway New Zealand 17,00,000 15,28,000 Finland 12,00,000 Source: Dorling Kindersley ‗Blueprint for Green Planet‘ ;London (1987).

4.2. Waste Generated in the Poor Developing Countries of World In the low and middle income developing countries of Asia, Asia-Pacific and Africa, waste is a luxury, only produced by the wealthy minority which of course is increasing with the growing economy and the exploding population. What is most concerning is that the waste is not regularly collected by the municipal authorities and often becomes a horrible site of piled and rotting waste on street corners with stray animals (dogs, pigs and cows) feeding on the scraps. Domestic waste heaps often becomes sites of defacation and discharge of human excreta and illegal dumping of hazardous wastes by scrupulous industries with potential health threats. Municipal waste services often swallow between a fifth and a half of city budgets, yet much solid waste is not removed. Even if municipal budgets are adequate for collection, safe disposal of collected wastes often remains a problem. (Holmes, 1984). Another ugly feature in these countries are that waste is often picked up by poor people called ‗rag-pickers‘ for whom waste reuse and recycling is a way of life, and many poor societies survive here by scouring the garbage of the rich for valuable scraps. They collect recyclable wastes (mainly papers, plastics, glasses and metals) from the street corners and even the dumpsites and sell them to the recycling industries to earn for their livelihood. Table 4. Average Per Capita Municipal Solid Waste (MSW) Generated by Some Developing (Poor) Nations Country Waste Generation Per Day (in Kg) Country Pakistan Indonesia India Nigeria

Waste Generation Per Day (in Kg) 0.25 – 0.60 0.33 – 0.55 0.15 – 0.51 0.16 – 0.46

Source:WWI (1990);‘State of the World‘ (These are 1990 Values which must have increased).

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4.3. Typical Waste Components in the MSW Generated in Both Rich Developed and Poor Developing and Underdeveloped Nations Several waste components have been identified in the municipal solid waste (MSW) of both the rich and poor societies of world. It ranged from twelve (12) to fourteen (14) and a considerable portion of this waste is ‗Organic‘ while others are ‗Inorganic.‘ Ironically, there is relatively high amount of food waste in poor developing and underdeveloped nations as compared to the rich developed nations. In contrast, there is high amount of paper and garden wastes in rich nations and low in poor nations. However, very little (or none) food waste finally reach the waste dump-sites in poor nations as they are scoured and scavenged by stray animals – pigs, dogs and cattle and even by the poor street beggars. Paper, cardboard, plastics, leather, wood and metals also do not reach the dump-sites and are picked up by ‗rag-pickers‘. (WHO, 1976). Table 5. Solid Waste Components in MSW of Low, Middle and High Income Countries (In % age) Waste Components Organic Food Waste Paper Cardboard Plastics Textiles Rubber Leather Garden Wastes Wood Inorganic Glass Tin cans Aluminum Other Metals Dirt, Ash etc.

Low-Income

Middle-Income

High-Income

40 – 85 1 – 10 1–5 1–5 1–5 1–5 1 – 10 1–5 1 – 40

20 – 65 8 – 30 2–6 2 – 10 1–4 1 – 10 1 – 10 1–5 1 – 30

6 – 30 20 – 40 5 – 15 2–8 2–6 0–2 0–2 10 – 20 1–4 4 – 12 2–8 0–1 1–4 0 – 10

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995). Low Income = Underdeveloped African, Asian and Pacific Nations; Middle Income = Developing Asian, African, Pacific and South American Nations. High Income = Developed European and North American Nations and Australia.

Table 6. Typical Solid Waste Components in the MSW of an European Society Waste Components Percentage (%) Waste Components 1. Paper and paper products 2. Metals 3. Glass 4. Plastics

Percentage (%) Germany 19.9 8.7 11.6 6.1

Switzerland 26.6 5.6 11.5

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5. Textiles 6. Minerals 7. Wood, leather, bones, rubber 8. Compounded materials 9. Sieving fractions (0-12 mm) 10. Sieving fractions (12-50 mm) 11. Residue 12. Total unidentified fraction

1.5 2.9 2.3 0.8 8.6 15.6 26.8 51.0

2.8 1.1 3.1 0.7 9.2 8.1 27.1 44.4

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Table 7. Typical Solid Waste Components in the MSW in U.S. Society (in % age) Organic 1 Food Waste 2. Paper 3. Cardboard 4. Plastics 5. Textiles 6. Rubber 7. Leather 8. Yard waste

9.0 34.0 6.0 7.0 2.0 0.5 0.5 8.5

Inorganic 10. Glass 11. Tin cans 12. Aluminum 13. Other Metal 14. Dirt, Ash etc.

8.0 6.0 0.5 3.0 3.0

Total = 100.00

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995)

5. CHANGING CHARACTER OF THE MSW IN MODERN SOCIETY: INCREASING AMOUNTS OF TOXIC MATERIALS The character and composition of the municipal solid wastes (MSW) are changing in the modern human society. Significant changes have occurred in the composition of municipal solid waste (MSW) ever since the technological revolution of the 20th century. They are no longer only ‗organic‘ waste, as it used to be in the earlier societies. The technological development which mainly influenced the character of the MSW was the fossil fuel driven ‗industrial revolution‘ and the agro-chemicals driven ‗green revolution‘. Waste components that have an important influence on the composition of the MSW are food waste, paper and plastic wastes, the white goods and the hospital wastes. What is the matter of more serious concern is that all ‗living organisms‘ including the human beings have become exposed to chemicals for which there has been no evolutionary adaptation and experience. The chemicals in the hazardous wastes mixed up with the MSW are completely ‗foreign‘ to living organism. (Sinha and Sinha, 2000).

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5.1. Changing Quality and Quantity of Food Wastes in the MSW The quantity and quality of residential food waste has changed significantly over the years as a result of technical advances in food growing (use of agro-chemicals) and food processing and packaging (use of chemical preservatives), and public attitudes towards food procurement i.e. relying more on processed and packed takeaway foods rather than cooking food at home from raw materials. However, due to education and awareness about the nutritive values of home cooked food, now people are moving back again towards home cooking. Two technological developments that have had a significant effect on generation of food waste are the development of the ‗food processing and packaging industries‘ and the ‗use of kitchen food waste grinders‘ in modern homes. Because of kitchen grinders the grinded food wastes are delivered directly into the sewer systems rather than being disposed as MSW. In these modern homes, the percentage of food wastes, by weight, has decreased from about 14 % in the early 1960s to about 9 % in 1992. In the packed and takeaway food culture, the generation of food wastes in homes have reduced, but it has increased significantly in the food processing industries and the food outlets. In homes, and the food outlets there are more paper and plastic wastes due to over-packaging of processed foods, than food wastes itself.

5.2. Proliferating Plastic Wastes in the MSW Percentage of plastics in MSW has also increased tremendously during the last 50 years. The use of plastic has increased from almost non-measurable quantities in the 1940s to between 7 - 8 %, by weight, in 1992. It is anticipated that the use of plastics will continue to increase, but at a slower rate than during the past 25 years. (UNEP, 1996).

5.3. Escalating Electronics Waste in MSW: Heading for E-waste ‗Tsunami‘? Electronic waste is a growing concern as technology changes and new generations of electronic products and equipments more sophisticated, improved and upgraded versions continue to invade the market and the minds of consumer‘s. The unfortunate part is that the price of the new models and upgraded versions is continuously falling giving more temptation to the consumers for discarding the old ones and replacing with the new. The UNEP working group on Sustainable Product Design described the e-waste essentially as a chemical waste. Electronics industry uses several hazardous chemicals including toxic heavy metals (lead, cadmium, mercury, chromium, barium etc.), acids and plastics, chlorinated and brominated compounds in production process. Developers of electronic products are introducing chemicals on a scale which is totally incompatible with the scant knowledge of their environmental or biological characteristics. (O, Rourke, 2004). More than 2 million tons of e-waste ends up in landfills every year and there is serious threat of leaching lead (Pb) and other heavy metals that may seep into groundwater supplies. Incineration results into emission of dangerous dioxins and furans as e-waste contain considerable amounts of plastics, brominated and chlorinated compounds. (Sinha, et. al., 2006).

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Recent study indicate that e-waste make up approximately 1 % of the MSW waste stream in all developed nations and mercury (Hg) from the e-waste has been cited as the main source of this heavy metal in the general MSW. E-waste in MSW is creating serious health and environmental problems for the MSW landfills and the waste workers, and for the MSW incinerators. In Europe the e-waste is growing at three times the rate of other MSW and mixing with it. ‗USA today is virtually sitting on a mountain of obsolete PCs‘. A report produced by the Silicon Valley Toxics Coalition (a grassroots coalition that performs research and advocacy on health and environmental issues related to electronics industries in the U.S.) in 2001 suggest that if all the consumers decided to throw out their obsolete computer at the same time, the country would face a ‗tsunami‘ of e-waste scraps between 2006 and 2015. The report called ‗Poison PCs and Toxic TVs‘ was released by another grassroots organization California Against Waste (CAW). (Anonymous, 1999).

Developing Countries as the Electronic Junkyards of U.S. and Industrialized Nations : An Untenable Choice Between Poverty and Poison There are reports about Asia, mainly India, China, Taiwan, Vietnam, Singapore and Pakistan, being made as the high-tech dumping ground of U.S. An estimated 20 million computers become obsolete each year in the U.S. and an estimated 200 tons of these computers end up in these countries in the name of ‗reuse‘ and ‗recycling‘. Low labor cost and weak environmental regulations have made these countries dumping grounds of e-waste destined for recycling and final disposal in landfills. A pilot program that collected electronic scrap in San Jose, California estimated that it was10 times cheaper to ship CRT monitors to China than it was to recycle them in the U.S. It is still legal in the U.S., despite international law (The Basel Convention, 1989) to the contrary, to allow export of hazardous waste without controls. (It may be recalled that U.S. has not yet signed the Basel Convention (1989) which prohibits trans-boundary movement of hazardous wastes). Industry insiders indicate that about 80 % of the e-waste goes to Asia and of that 90 % ends up in China. (Puckett et. al, 2002) A report by Basel Action Network and the Silicon Valley Toxics Coalition ‗Exporting Harm: The Techno-Trashing of Asia‘ asserts that 50 to 80 % of e-waste collected for recycling in the U.S. is exported to developing nations. BAN produced a film on the report which shows the Guiyu village in Guangdong province in China as ‗electronics junkyard‘. Some 100,000 men, women and children make US $1.50 a day dismantling e-waste by bare hands to retrieve the valuable metals and materials. Circuit boards are melted over coal grills to release valuable metals giving highly toxic dioxin fumes. Riverbank acid baths are used to extract gold. Lead-containing cathode ray tubes from monitors and televisions are not of much market value and hence are dumped in some wastelands. Toner cartridges are pulled apart manually, sending clouds of toner dust into the air. Soil and drinking water at Guiyu are contaminated by lead much above WHO limits- soil by 200 times and water by 2,400 times. Water has to be trucked from 30 km away. At one point of time both China and India were willing to take the e-waste for almost free. For poor countries of the world it is an untenable choice between ‗poverty and poison‘. (Puckett et. al, 2002) In November, 2002 officials from eight Asian nations met in Tianjin, China, under the auspices of Basel Convention (1989) to prevent their nations from being made the dumping grounds of hazardous e-waste in the name of free trade (export and import) for recycling discarded electronic products. It was represented by India, Malaysia, the Philippines,

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Singapore, Sri Lanka, Thailand, and Vietnam. Resource persons came from Canada, Japan, the U.S. and the Secretariat of the Basel Convention. Financial support was provided by Australia, Japan and Canada. China has now banned and India also follows. (Sinha, et. al., 2006).

5.4. The White Goods and Bulky Items in MSW There are ‗bulky items‘ which include large worn-out or broken household, commercial or industrials goods such as furniture, lamps, bookcase, filing cabinets, etc. There are ‗consumer electronics‘ wastes which include worn-out, broken, and no longer wanted items like stereos, radios, computer and television sets. There are ‗white goods‘ as waste which include worn-out and broken household, commercial and industrial appliances like stoves, refrigerators, dishwashers, cloth washers and driers. The rejected auto-parts like tires, batteries and accessories also constitute important constituents of MSW. About 230 to 240 million rubber tires are disposed off annually in landfills or in tire stockpiles. (UNEP, 1996).

5.5. The Biomedical Wastes in MSW The biomedical waste from hospital and clinics and slaughterhouses contain about 85 % as general refuse, but 10 % is hazardous wastes contaminated with infectious pathological agents, and 5 % is non-infectious but potentially toxic (chemicals) and radioactive and hence hazardous. Dressing and swabs contaminated with blood and body fluids; syringes, needles and sharps; surgically removed placenta, tissues, tumors, organs or limbs are potentially infected wastes from hospitals. There are several ‗disposable‘ items made of PVC and thermocol now being used in medical organizations. Hospital wastes are of special category and require special care for final disposal. WHO has provided strict guidelines for their safe disposal. (WHO, 1976).

6. THE COMPLEX SYNTHETIC WASTES INVADING HUMAN ENVIRONMENT Human ingenuity has created some ‗new and synthetic materials‘ in the wake of technological revolution. They contain both organic and inorganic chemicals and resins and creates more ‗complex‘ type of waste after being discarded. Nature do not possess any organism and mechanism to biodegrade them. The processing of some new materials discovered by technology such as ‗semiconductors‘, ‗optical fibers‘, new class of ‗ceramics‘, and ‗composites‘ requires the use of large amount of toxic chemicals which eventually ends up as hazardous wastes in the MSW. These technological wastes are often toxic and are posing danger not only for the environment, but also for the human health. It has already caused several accidents, deaths and disabilities among the municipal waste workers.

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6.1. The Non-Biodegradable Wastes: Potential to Remain Long in Human Ecosystem The synthetic wastes are ‗non-biodegradable‘ because they cannot be decomposed and can remain in the human ecosystem for years and decades polluting the environment. Common examples are all forms of plastics, x-ray films, celluloid films, cells and batteries, several chemicals and all synthetics. What nature cannot do, human beings are trying to do through the knowledge of environmental biotechnology. Genetically tailored bacteria are being created which would possess the necessary enzymes to degrade the synthetic wastes. Some strains of bacteria and fungi have been identified in nature too, which has the arsenal to degrade some of the complex organic chemicals. Efforts are also being made to create ‗biodegradable synthetics‘ using ‗starch‘ as the raw material. They can be degraded in 4-6 weeks. (Sinha and Sinha, 2007).

6.2. The Hazardous Wastes: Permeating the Human Society Wastes containing toxic chemicals, radioactive substances and infectious materials which poses potential risk to human health and environment are categorized as ‗hazardous wastes‘. Toxicity, radioactivity, flammability, chemical reactivity, corrosivity, nonbiodegradability, carcinogenicity, mutagenicity, infectiousness, oxidizing and leachating are some of the characteristics of hazardous wastes. (WHO, 1983). Many primary and manufacturing industries using toxic chemicals generate hazardous waste (solid or liquid) in the production process. They can be referred as ‗industrial hazardous wastes‘ (IHW). Many of our favorite cultural activities depend on products the manufacture of which creates industrial hazardous waste. Glaring examples are ‗glass and metal‘, ‗paper and plastic‘, ‗leather and textile‘, ‗painting and dyeing‘, ‗printing and publication‘ and ‗photography and dry cleanings‘. Consumer industries today use a variety of chemicals to produce ‗consumer goods‘ a number of which are now ‗disposables‘. When these items are consumed and discarded by society, they eventually end up as hazardous wastes in our homes. This can be referred as ‗household hazardous waste‘ (HHW). Prime examples are torch dry-cells and batteries, pesticides / disinfectant cans and bottles, fluorescent tubes and electric bulbs, detergents and shampoos, lead-acid car batteries, auto tires and waste oils, and the expired medicines. As we enjoy the benefits of consumer goods (furniture and fixtures, white goods, electrical and electronic goods, automobiles, processed and packed food and drinks etc.) produced by consumer industries, we also generate considerable amount of hazardous wastes as by-products when we discard them after use or change the ‗old‘ version with ‗new‘. They can be referred as ‗consumer hazardous waste‘ (CHW). Industries producing products that sustain our modern life-style and living habits generate tremendous amount of hazardous wastes. Glaring examples are the ‗agro-chemical industries‘ (to boost our food production) and the ‗petroleum industries‘ (which drives our automobiles). (Raghupati, 1994; Sinha and Herat, 2004).

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Household Hazardous Wastes (HHW): The Poison in Our Homes Household hazardous wastes are either solids, semi-solids, or liquids. In addition trace chemical compounds can exist as a solute within a liquid solvent, as a gas adsorbed onto a solid, or as a component of the gaseous emissions from MSW. Plastics contain organochlorine compounds, organic solvents in PVC; paints contain heavy metals, pigments, solvents and organic residues; home pesticides contain organochlorine and organophosphates compounds; oil and gasoline contain phenols and organic compounds, heavy metals, salt acids, ammonia and caustics; textiles may have metal dyes and organochlorine compounds; carpets can contain chemical stain resisters, pesticides, solvent-heavy glues, VOCs laden underlayer; curtain treated with stain resisters and backing can give off VOCs; dry-cleaned clothes give off VOCs; chip board and plywood furniture releases formaldehyde; fibre-glass based insulation of ceiling cavity gives off VOCs; window sealants give off toxic fumes; bathroom air fresheners release VOCs; and the carcinogenic benzene can be formed in the garage from the car exhaust. Virtually all of the mercury in MSW is due to the disposal of household dry cell batteries (mercury, alkaline, and carbonzinc types). A smaller amount of mercury may come from the disposal of broken home thermometers. (Sinha, et. al., 2005 a) Several toxic chemicals are commonly used today in modern homes that also results into generation of HHW. They are usually mixed and disposed with the MSW. Some glaring examples of toxic chemicals used in modern homes that contributes in the generation of household hazardous wastes (HHW) are1) Perfumes and cosmetics used by the women contain some 884 ‗neurotoxic‘ chemical compounds. (Report of National Institute of Occupational Safety and Health in the US). 2) Paper whitener is toxic. The chemical used in it has potential to kill. 3) Cadmium is present in food processing equipment, kitchenware enamels, pottery glazes and plastics and relatively high levels in the sea foods. 4) Lead is present in paints and dyes, toys and newspapers, solder and batteries, lead water pipe; 5) The highly toxic polychlorinated biphenyls (PCBs) are added to paints, copying and printing paper inks, adhesive and plastics to improve their flexibility. Fish food contain generally higher levels of PCB‘s. High levels of PCB‘s were reported from the breakfast cereals in Sweden and Mexico as a result of contamination by ‗packaging materials‘. 6) Containers of paints and enamels used in homes and in automobiles. They contain dangerous chemicals like glycol, ether, ammonia, benzene and formaldehyde and continue to give out toxic fumes at least for 7 years. 7) Containers of pesticides, insecticides, herbicides and fungicides are available in modern homes to eradicate pests and insects in garden plants, cockroaches and spiders in kitchens and storerooms. 8) Many relatively innocuous items, such as plastics, glossy magazines, and flashlight batteries used in homes, contain metallic elements. 9) Metals like cadmium (Cd), chromium (Cr), mercury (Hg), and lead (Pb) are present in several household items. After combustion with MSW metals are either emitted as

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Very little is known about the amount of HHW generated in various countries. UNEP (2006) reported that The Netherlands generate 41,000 tonnes of HHW every year. The University of Arizona, US, made a survey and found that about 100 hazardous items (containers) are discarded per household each year. Australian study made in Melbourne in 1990-92 also found 89,576 kg of hazardous wastes from households. (CSIRO, 1996).

Mercury in the MSW from Household Wastes Has the Potential to Kill : A Case Study from U.S. It is interesting to note that if the tons of ‗household batteries‘ generated in California is calculated for whole of U.S., the total amount would be 160,000 tons per year. Given that a typical household battery weighs 50 grams, the corresponding number of batteries is 2,910,000,000. It is estimated that more than 2,700,000,000 battery units were purchased in the US in 1990. If half the household batteries were ‗alkaline‘, and assuming that each battery contained about 1200 mg of mercury (Hg), then, based on the data reported above, 1923 tons of mercury (Hg) would enter the environment each year in California alone. This mercury is enough to kill 8,730,000,000 people based on a lethal dose of 200 mg per person (Tchobanoglous et. al., 1993). Such situation exist in all metropolitan cities of world, in both developingand the developed countries. Clearly, proper disposal of these household batteries in the MSW is an important issues that must be addressed. If not collected separately, all these household batteries get mixed up with MSW and is disposed in the ‗ordinary sanitary landfills‘ instead of the ‗secured landfills‘. The Hazards of Disposable Baby Nappies in the MSW More than 20 billion disposable baby nappies (equivalent to some 2.7 million tons of solid waste) are ending up in the landfills every year the world over. They contain hazardous chemical ‗sodium polyacrylate‘ (a super water absorbent) responsible for several medical conditions in infants, including hampering of genital growth in male child. On an average disposable nappies occupy landfill space of 0.40 m2 per child per year in Australia. They are 2 % by weight in total solid waste but occupy 3.5 % of total landfill space. They are also disturbing the natural microbial biodegradation processes of organic wastes in the closed landfills by absorbing all water internally. (Brahambhatt and Saeed, 2005).

6.3. The Nuclear Waste : Radioactive Substances Invading the Human Environment Nuclear wastes are the result of our urge to generate nuclear energy without emission of greenhouse gases (which is in fact a myth as it requires 18 years of CO2 producing fossil fuel energy (in uranium mining and enrichment, building reactors etc.) to produce one calories of nuclear generated energy. (Report of Friends of Earth, 2000). Nuclear waste are produced regardless of whether nuclear fission is controlled (such as for energy generation in reactors),

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or occurs explosively, as in the atom bomb. The resulting fission products, isotopes of approximately 30 elements, have mass numbers in the range of 72 to 162, are for the most parts solids, and emit beta particles, together with electromagnetic reaction (gamma rays) which are exceedingly penetrating. The chemical separation of fission products and their conversion to nuclear fuel are the most important sources of radioactive wastes. The radioactive wastes can be in all the three forms – solid, liquid and gaseous and two categories of radioactive wastes are mostly encountered- the Low Level Radioactivity Waste (LLRW) and the High Level Radioactive Wastes (HLRW). (IAEA, 1991). Eight tons of liquid radioactive waste result per year from the typical average-size, nuclear reactor. The ‗nuclear reactors‘ mostly generate HLRW in the form of plutonium-239 (Pu239). Other two most significant fission products are strontium-90 (Sr90) and cesium-137 (Cs137) with half-life of 19.9 and 33 years respectively. They are routinely emitted from the reactors and continue to release radiation energy over long periods of time (several generations of the human race). Even dismantling (decommissioning) of retiring nuclear reactors produce enormous amount of radioactive wastes and contaminate vast land area. There are 439 nuclear power reactors in operation around the world mostly in France and Japan. They would all retire in years to come. Uranium mining and processing (enrichment) produces huge amount of solid and liquid radioactive wastes which is highly hazardous. The extraction of uranium from the earth crust leaves vast quantities of wastes as ‗tailings‘ which contain up to 80% of the original radioactivity of the extracted ore. After mining uranium is further enriched to produce ‗nuclear fuel‘. Depleted uranium hexafluoride (DU) is a radioactive waste by-product of enrichment. For every 1000 tones of processed uranium fuel , 100,000 tones of mined wastes as tailings and 3,500,000 liters of liquid waste is produced. They migrate into the environment through air, soil and water. Processing of uranium ores produces considerable volumes of alpha emitters, mainly radium-226. The half-life of Ra226 is 1600 years and gives rise to a toxic gas ‗radon‘. (IAEA, 1991).

7. WASTE : POTENTIAL SOURCE OF GREENHOUSE GASES (METHANE AND NITROUS OXIDE) So far, not much attention was paid towards this aspect of waste generation. Marked increases in the amount of waste generated has also contributed to emission of greenhouse gases carbon dioxide, methane and nitrous oxides. A major issue of concern today is emission of greenhouse gas methane (CH4) resulting from disposal of MSW in landfills and this may be between 45 – 60 %. This is mainly due to anaerobic degradation of the organic waste components in the landfills as oxygen becomes deficient due to compaction. Methane is 2025 times more powerful GHG than carbon dioxide (CO2) in absorbing the infrared solar radiation. Studies have also indicated high emissions of nitrous oxide (N2O) in proportion to the amount of food waste. N2O is mainly formed under moderate oxygen (O2) concentration. (Yaowu et. al., 2000). Molecule to molecule N2O is 296 times more powerful GHG than carbon dioxide (CO2). Yaowu et. al., (2000) has studied the emission of both methane and nitrous oxides from aerated food waste composting.

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In fact, improved recycling of waste can significantly contribute to abatement of GHG emissions. However, convention microbial composting (biological recycling) of organic wastes also emits methane (Wang et, al., 1997) due to ‗anaerobic sites‘ appearing in the inner layers of compost piles. However, it can be reduced significantly by improving ‗aeration‘ in the waste biomass by periodical turning or through mechanical aerating systems. (Toms et. al., 1995). Significantly, vermicomposting of waste by waste eater earthworms decrease the proportion of ‗anaerobic to aerobic decomposition‘, resulting in a significant decrease in methane (CH4). Earthworms can play a good part in the strategy of greenhouse gas reduction and mitigation in the disposal of global organic wastes. Currently we are studying the potential of GHG emissions by various systems of biodegradation of wastes (Aerobic & Anaerobic Composting & Vermicomposting by Earthworms). (Chauhan & Valani, 2008).

8. SAFE MANAGEMENT OF WASTE : A TECHNO-ECONOMIC PROBLEM Both rich developed and the poor developing nations of world have become conscious towards safe waste management in the changing situation where the human waste is no longer ‗simple and organic‘ to be salvaged by nature in course of time but getting more complex and even hazardous, and threatening to remain in the human ecosystem for long time. Safe management of all waste becomes imperative for the safety and security of the society and it require the input of knowledge of diverse disciplines of material science, political science, economics, geography, sociology, demography, urban planning, public and environmental health, communication, conservation, and civil and mechanical engineering. Waste management has been termed as ‗Cradle- to- Grave‘ management i.e. from point of generation to final disposal, involving safe storage, transport and treatment- and in all these steps, the generator owes the main responsibility. The collection and disposal of waste involves huge expenditures in the development of landfills, waste collecting vehicles, precious fuel (petrol and diesel) and labour costs.

8.1. The Nature‘s Technology at Work: Salavaging the Organic Wastes The organic wastes in the MSW are ‗biodegradable‘ and are decomposed in nature by diverse microorganisms – bacteria, fungi, actinomycetes and the protozoa. Among the biodegradable wastes some are ‗rapidly‘ degraded while others are ‗slowly‘ degraded over time. It may take time from few days to several months and years to degrade the organic wastes, but it does happen ultimately. Some organic materials in the waste decomposes rapidly (3 months to 5 years), while others slowly (up to 50 years or more). The relative ease with which an organic waste is biodegraded (decomposed) depends on the ‗genetic makeup‘ of the microorganisms present and the ‗chemical makeup‘ of the organic molecules (mainly carbon structure that the organisms use as a source of energy and biodegrade in the process). Carbons in sugars, lipids and proteins are easily decomposed than the carbon in lignin, while the carbon in plastics are not at all biodegraded.

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The biodegradability depends to a large extent on the lignin content (present in wood fibers) of the waste. Lesser the lignin content, rapid will be the biodegradation rate of that organic material. Nature has those ‗decomposer microorganisms‘ (mainly bacteria and fungi) in soil, air and water which perform the task. This is how the natural ecosystems on earth has been operating since life evolved. Had there been no decomposer organisms, the earth would have been full of animal and human excreta, animal carcasses and vegetable matters (leaves and twigs) and dirt, and life impossible. The biodegradable waste include all plant, animal and human products, the kitchen waste in every home and restaurants, wastes from the agriculture farm, food processing industries, slaughter houses, fish and vegetable markets, and paper and cotton wastes. All these wastes mainly contain organic matters. The process of biodegradation (decomposition) in nature can be enhanced to several times by introducing decomposer organisms such as the earthworms or even the bacterial biomass directly into the waste biomass. This is being done these days to dispose the mounting organic wastes rapidly. The process is called ‗composting‘ and the byproduct is NKP rich biofertilizer. Table 8. Rapidly and Slowly Biodegradable Organic Constituents in MSW Rapidly Biodegradable Food Waste Newspaper Office Paper Cardboard Yard Waste (Leaves and Grass Trimmings)

Slowly Biodegradable Textiles Rubber Leather Yard Waste (Woody portions) Wood Misc. Organics

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Table 9. Biodegradability of Some Organic Components in the MSW Component Food Waste Newspaper Office Paper Cardboard Yard Waste

Lignin Contents (% of volatile solid) 0.4 21.9 0.4 12.9 4.1

Biodegradability (% of vs) 82 22 82 47 72

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

8.2. Sanitary Landfills - The Ultimate Graveyard for Wastes: An Economic and Environmental Burden Sanitary landfills constitute the ultimate graveyard for the safe burial of all human waste in the womb of ‗mother earth‘. There is an enormous range of materials found in landfills. ‗Today‘s landfill is tomorrow‘s time capsule,‘ writes Blatt (2005). Experience have shown that modern landfills although made with great engineering skills are unable to contain the toxic ‗landfill gases‘ and the ‗leachate discharge‘ into the environment. They are proving to

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be a techno-economic burden and a curse in disguise. The cost involved in landfill construction is not only up-front, but also in its monitoring and maintenance, for controlling landfill gases and the leachate collection etc., which is long term, often up to 30 to 50 years. The up-front development costs for new landfills in U.S. varied from US $ 10 million to $ 20 million in 1992, before the first load of waste was placed in the landfill. This must have gone up substantially by now. (Tchobanoglous, 1995).

Health and Environmental Concerns of Waste Landfills : The Uncontrolled Release of Greenhouse and Toxic Gases into the Environment Methane (CH4), carbon dioxide (CO2), carbon monoxide (CO), hydrogen (H2), oxygen (O2) and nitrogen (N2) are the principal landfill gases. Methane and carbon dioxide are known greenhouse gases and molecule to molecule methane (CH4) is 20 – 25 times more powerful greenhouse gas than carbon dioxide in absorbing the infrared solar radiation. Methane can cause explosion when present in air in concentrations between 5 and 15 %. Ammonia (NH3), hydrogen sulfide (H2S) and the Volatile Organic Compounds (VOCs) are in trace amounts. They are products of biochemical reactions occurring in the landfills. Landfill gases migrate horizontally and migration distances greater than 300 m have been observed with 40 % concentration. Gas samples collected from over 66 landfills in UK showed the presence of 116 organic compounds, many of which are classified as VOCs. (UNEP, 1996). VOCs are mostly evolved from the newly placed MSW and specially which also contains hazardous wastes (HW). The increasing quantities of ‗household hazardous wastes‘(HHW) in the modern society and their disposal along with the MSW, is posing a serious problem for the landfill engineers. Table 9. Typical Hazardous Chemical Constituents Found in MSW Landfill Gases Chemical Constituents Methane Carbon dioxide Nitrogen Oxygen Sulfides, disulfides, mercaptans, etc. Ammonia Hydrogen Carbon monoxide Trace constituents (In VOCs)

% (By Dry Volume Basis) 45 – 60 (Greenhouse gas) 40 – 60 (Greenhouse gas) 2–5 0.1 – 1.0 0 – 1.0 0.1 – 1.0 0 – 0.2 0 – 0.2 0.01 – 0.6

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Table 10. Concentrations of Toxic Trace Compounds Found in MSW Landfill Gases Compound Acetone Benzene Chlorobenzene Chloroform

Mean Value in Parts Per Billion (ppb) by Volume 6, 838 2, 057 82 245

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Table 10.- Continued Compound 1,1 Dichloroethane Dichloromethane 1,1 Dichlorethene Diethylene chloride trans- 1,2-Dichloroethane Ethylene dichloride Ethyl benzene Methyl ethyl ketone (MEK) 1,1,1-Trichloroethane Trichloroethylene Toluene 1,1,2,2-Tetrachloroethane Tetrachloroethylene Vinyl chloride Styrenes Vinyl acetate Xylenes

Mean Value in Parts Per Billion (ppb) by Volume 2, 801 25, 694 130 2, 835 36 59 7, 334 3, 092 615 2, 079 34, 907 246 5, 244 3, 508 1,517 5,663 2,651

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Some hazardous chemicals like ‗vinyl chloride‘ is going to the landfills by way of plastic bags. Residents are throwing their kitchen wastes mostly packed in grocery plastic bags. Earlier, there was also a practice to dispose some ‗industrial solid wastes‘ (ISW) with MSW in the landfills, which have now been banned. However, to minimize the emission of VOCs, a vacuum is applied and air is drawn through the completed portions of the landfill. Trace gases although present in small amounts, can be toxic and pose grave risk to public health and environment. Trace compounds may carry ‗carcinogenic‘ and ‗teratogenic‘ compounds into the surrounding environment. Half-lives of various trace compounds in the VOCs have been found to vary from fraction of a year to over a thousand years. (Heath, 1983).

The Uncontrolled Release of Leachate and the Threat of Contamination of Groundwater and Surface Water The liquid (waste juice) that collects at the bottom of the landfill is known as ‗leachate‘. It is the result of percolation of precipitation, uncontrolled runoff, and irrigation water into the landfill. Leachate can also include water initially contained in the waste as well as infiltrating groundwater. Leachate seeps downward to the base of the landfill by gravity and poses a potential health risk to public as it can percolate into the groundwater aquifer and contaminate it. Concern is growing worldwide about wastes leaching heavy metals that may seep into groundwater supplies. Leaching into soil and groundwater will occur regardless of whether the landfill is sealed or not. It has become a common knowledge that all landfills leak. Even the best ‗state of the art‘ landfills are not completely tight throughout their lifetimes and a certain amount of chemicals and metal leaching will occur.

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Landfill leachate contains a variety of chemical constituents including heavy metals (Pb, Cu, Ni, Cr, Zn, Cd, Fe, Mn, Hg, Ba, Ag), arsenic, cyanide, fluoride and selenium, and organic acids derived from the solubilization of the materials deposited in the landfill and from the products of chemical and biochemical reactions occurring in the landfills. (Tchobanoglous et, al.,1995). Disposal of consumer electronics mixed with MSW accounts for 40 % of lead (Pb) in the landfills. Mercury (Hg) will leach when circuit breakers are destroyed, PCBs will leach when condensers are destroyed. When plastics with brominated flame-retardants or cadmium containing plastics are landfilled, both PDBE and cadmium (Cd) may leach into the soil and groundwater. (Miller, 2004).

8.3. Thermal Destruction (Incineration) of Waste High temperature incineration of wastes especially the hazardous chemical and biomedical wastes is considered to be the safest remedy to get rid of it. It enables detoxification of all combustible carcinogens, mutagens and teratogens. Destruction is done in stages. In the first stage the waste is thermally decomposed at 800 C in a refractory lined chamber to produce ashes and volatiles. The ashes are removed and the volatiles with gases are led to second stage where they are heated to around 1200C with additional air. It is the only environmentally acceptable means of disposing some complex organic wastes like chlorinated hydrocarbons, polychlorinated biphenyls (PCBs) and dioxins. These chemicals are persistant, non-biodegradable and highly toxic. However, the conventional incinerators emit dangerous ‗dioxins‘, ‗furans‘ and other highly toxic pollutants when inorganic chemical dyes in plastics are incinerated. Dangerous dioxins may also be formed if some organic materials are incinerated at too low temperatures. But, the modern, well-regulated incinerators have dramatically reduced such toxic emissions. It has been observed that injection of lime and activated carbon significantly remove the ‗dioxins‘ and ‗mercury‘ from the gases by 95 %. However, people in most countries are opposing incinerators and landfills in their neighborhood.

Plasma Arc Incineration System High temperature incineration using ‗plasma arc furnaces‘ is a growing technology for the management of hazardous chemical and biomedical wastes. In plasma arc system a thermal plasma field is created by directing an electric current through a low-pressure gas stream. Plasma fields can reach temperature from 5000 C to 15,000 C. This intense high temperature zone can be used to dissociate the wastes into its atomic elements in the combustion chamber. Heat generated from the plasma torch can melt and vitrify solid wastes. Organic components can be vaporized and decomposed by the intense heat and ionised by the air used as the plasma gas. Oxygen may also be added in the primary chamber to enhance combustion. Metal-bearing solids are vitrified into a monolithic non-leachable mass. (Hasselriss, 1995). The system is hermetically sealed and operated below atmospheric pressure to prevent leakage of process gases. Dioxin formation is prevented. The vented gas is held in the tank and recycled into the furnace. The clean gases are released into the atmosphere through an exhaust stack. The destruction and removal efficiency (DREs) of organic compounds are

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greater than 99.99 %. There is less public opposition to such incineration system. Plasma technology‘s inherent ability to eliminate risks of future liabilities from waste disposal through a single step treatment is an added advantage. A mobile plasma arc system would also reduce or eliminate public health risks associated with the hazards of accidents or spills resulting from the transportation of toxic or hazardous wastes by road or rail. Plasma Arc Technology has proved to be an ideal and economically viable method to successfully treat even the carcinogenic asbestos waste, the nuclear power plant wastes and for safe disposal of arms and military waste worldwide. Temperatures in the order of 1,000 C are necessary to irreversibly transform asbestos into a non-hazardous material. Plasma treatment transformed the asbestos waste into a harmless vitrified slag. Japan has been using the plasma arc technology on large scale to vitrify its municipal solid wastes (MSW) to reduce the volume of its MSW going to the landfills. Land starved Japan cannot afford to have large number of landfills on its island. The slag produced by the plasma arc pyrolysis is recycled into glassy bricks used as construction materials in buildings. General cost of waste treatment by plasma arc technology range between US $ 400 and $ 2,000 per ton, depending on the characteristics of the waste. PAT is however, a capital intensive technology due to its initial equipment costs. A new plasma waste processing plant can cost between US $ 3 million and $ 12 million depending on size, the hazardous nature of the waste and the complexity of the treatment process. (Hasselriss, 1995).

Combining Incineration with Energy Generation : Killing two Birds in One Shot Although waste incineration has been discredited worldwide particularly due to emission of ‗dioxins‘ and ‗furans‘, most European nations have waste incinerator plants combined with energy recovery plants, and several categories of wastes (with high calorific value) including the hazardous wastes are used as fuel to achieve the dual objectives of waste disposal and electricity generation. The ‗waste-to-energy‘ movement started with the ‗oil crisis‘ in the Middle East and the increased cost of oil in the 1970s. Denmark and Sweden are leaders. They incinerate 65 % and 55 % of their MSW respectively and also produce thermal electricity from steam generation. Of the 12 MSW incinerator plants in Netherlands, 5 generate thermal electricity. Some large German cities operate combined incinerator plants providing up to 5 – 10 % of the total electricity demand. U.S. has few incinerators and incinerates 16 % of its MSW currently, but recovers energy from almost 80 % of the plants. The U.S. National Energy Strategy (1991), projected 7 fold increase in the electricity generation from MSW incineration plants by 2010. Nearly three quarters of the Japan‘s 1900 MSW incinerators, recover energy. (UNEP, 1996).

9. EMBRACING THE 5 R‘S PHILOSOPHY OF WASTE MANAGEMENT THROUGH WASTE AND CONSUMER EDUCATION As the generation of waste has increased in volume and also become more complex in nature (due to mixing of non-biodegradable plastic wastes and household hazardous wastes) traditional waste management systems have proved inadequate and inappropriate. Landfills are not the lasting solution to the waste problem. Worldwide consensus is emerging to discourage incineration and landfill disposal of wastes. In accordance with the EU Landfill

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Directive of 2001, the UK government is now phasing out the landfills for waste disposal. Several U.S. states have banned landfill disposal of e-waste.

9.1. Waste and Consumer Education for the Wasteful Modern Society Environmental education about consumption and waste is a kind of ‗re-education‘ of the already educated but ignorant modern wasteful society. Waste generation involves the entire population, so broad cooperation is necessary for sustainable and efficient waste management. Partnerships are required between people and governments to deal with waste collection and disposal. Waste and consumer education aims to raise public awareness about the stresses on municipal councils and enable communities to understand their role and share in the financial responsibilities needed for efficient management of solid wastes. Educational programs must involve consciousness-raising on these fundamentals: · · ·

·

Waste is an inevitable by-product of all individual and social activities and is proportionate to daily consumption. Waste is a potential resource and every individual needs to commit to help recover waste by reuse and ‗recycling‘. Waste generation can be minimized — it can never be eliminated — through changing the patterns of production and consumption of goods and materials either used most and/or that are used once then end up as waste. Judicious consumption of food and the 5 P‘s — paper, plastic, power, petrol and potable water — can help minimize waste generation on a large scale.

However, there are series of issues that consumers have no control over and that require government interventions at the level of production and distribution, involving manufacturers and retailers.

The Consumer Power of Acceptance / Rejection Can Positively Influence Consumer Industries Counteracting Vested Commercial Interest : India Showed the Way in 1940s Consumer power can change things provided they are aware. By accepting or rejecting consumer goods for sale, buyers can positively influence the production process, conveying messages to industries and retailers to reduce waste at source both in quantity as well as in quality (hazardous). Consumers can also influence government and industrialists‘ policies to manufacture goods that are durable, and also re-useable and recyclable after one use. Consumer boycotts, which represent the kind of non-violence preached by the great Indian saint, Mahatma Gandhi, back in the 1940s, counteract the vested commercial interest of manufacturing industries promoted by media for commercial gain. Consumer Education: Women Holds the Key Consumerism is a growing human culture all over the world. Shopping is a key activity in market based modern societies and grocery shopping for domestic consumption is done mostly by women all over the world who are keenly aware of the various shelf products. They

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also observe the invasion of diverse consumer products in the market through media and assess their values for family consumption. However, buying less and narrowing choices are seen as infringements on people‘s rights. Individuals and householders who care about the environment can feel powerless. Therefore policy makers, program coordinators, and educators need to address a series of complex issues associated with educating about consumerism and waste generation. A news poll survey conducted in mid-2005 found that five out of ten women compared with three out of ten men were saying ‗No‘ to plastic bags and only two in ten women but more than a third of the men surveyed preferred to use plastic bags rather than reusable ones (Clean Up Australia 2006) ; (Also on ).

International Efforts and Role of UNEP, WWF and WWI The United Nation Environment Program (UNEP) and the World Wide Fund for Nature Conservation (WWF) and the Washington based World Watch Institute (WWI) are playing important roles in educating people at an international level, creating mass awareness about waste problems as well as other environmental issues. UNEP sponsored the first Clean Up the World Campaign, 17-19 September 1993. Several million people, from 79 countries, actively participated in that campaign, which combined dual objectives, environmental sanitation and resource generation. The campaign followed up on the first ‗World Clean Up Movement‘ in Sydney, Australia, initiated by Australian yachtsman, Ian Kiernan, on 8 January 1986. Kiernan gathered around 40,000 Sydney siders to collect more than 5000 tons of rubbish from different parts of the city. The first Clean Up Australia Day, 1990, involved almost 300,000 volunteers. Programs have been developed to influence consumers and to encourage them to rethink about their patterns of consumption which can reduce waste. The Japanese Environmental Agency (JEA) has launched a scheme called ‗Household Eco-Account Books‘ that encourages the citizens to live sustainabily in an environmentally friendly manner and also save money by consuming resources judiciously and by embracing the philosophy of 3 R‘s for waste reduction, reuse and recycling. In Norway NGOs run a program that builds householders awareness on environmental impacts of consumer products, especially on its contribution to piling municipal solid waste (MSW) and also the household hazardous wastes (HHW) as several consumer products in modern homes may contain hazardous chemicals and materials.

9.2. Educating about the Golden Rules of 5 R‘s: Refusal, Reduction, Reuse, Recycling and Responsible Behavior People and policy makers need to embrace the ‗5 R‘s environmental philosophy for sustainable waste management. In the waste management hierarchy ‗waste refusal and reduction‘ is the best option, ‗waste reuse and recycling‘ the better option than the ‗waste disposal‘ in landfills. (Sinha et. al. 2005 c). It is imperative to educate the masses to 1. Refuse to accept articles and materials that generate / create more waste, especially those that generate toxic and hazardous wastes;

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Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al. 2. Reduce waste at source by consuming resources as little as possible and which is enough to enjoy a minimum quality of life; 3. Reuse articles and materials several times and as much as practicable before discarding them as waste and insist on buying durable, reusable and recyclable articles and products and ‗repair‘ household goods before rejection as waste; 4. Recycle / recover / retrieve new materials / energy from discarded waste products and assist the recycling industries by separating the recyclable waste from the nonrecyclables faithfully at source; and to behave with 5. Responsibility (for both consumer societies and producer industries) with regard to judicious use of resources and reduction in waste generation in everyday activities.

If people (society) and producers (industries) embrace the 5 R‘s golden rule majority of the waste will be taken care of and very little will be left for ‗treatment‘ or to ‗contain‘ them and finally to dispose them in landfills. The environmental organization ‗EcoRecycle‘ of Victoria, Australia (2006) proposed a ‗waste management hierarchy‘ which emphasizes that waste ‗reduction / avoidance‘ should be the first option and ‗disposal‘ the last. Waste reuse and recycling comes after reduction. If this management plan is sincerely implemented, there will be very little waste left to be disposed finally. (Figure 1).

Figure 1. Source: EcoRecycle 2006.

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The Power of Refusal : It Can Prevent Misuse of Resources In the 1980s, the Women Environmental Network (WEN) of the UK campaigned against over-packaging of food products and refused to buy those products that were heavily wrapped with plastics and papers. Women groups went into supermarkets, ripping off all the unnecessary extra layers of paper and plastics and handed them back to the managers. WEN has campaigned against several environmentally unfriendly consumer products such as paper handkerchiefs and toilet rolls made from newly produced paper refusing to accept them. (Belamy 1995). Way back in the 1940‘s the great Indian philosopher and educator M.K. Gandhi, used this ‗power of refusal‘ against the mighty British Empire (although in a different context) to generate awareness among the masses. Box 1. Responsibility of Consumers: Check Before Shopping and Refuse to Accept Articles That Can Generate More Waste Upon Use 1. 2.

3.

Refuse to accept non-biodegradable plastic bags for grocery and other shopping — force manufacturers and retailers to offer environmentally friendly alternatives. Refuse to accept any plastic or paper bag for small or just a few articles that you can carry instead in your pockets or personal bags — always carry easily tucked away cloth or alternative bags for shopping. Refuse to accept articles over-packed in plastic and paper, which have no eco-label (are not produced through clean methods of production), and have the potential to generate more waste when used.

The Wisdom of Waste Reduction / Prevention: It Conserves Material Resources This is the best option in the waste management hierarchy. Preventing waste is like preventing a ‗social disease‘ to occur. Waste reduction means conserving resources, that otherwise constitute waste, with further economic and ecological benefits, including: conserving valuable raw geo-chemical and biological resources, water and energy (fossil fuels) used in the manufacture of products; saving money otherwise spent on constructing and maintaining waste landfills; reducing health and environmental impacts of air, water and soil pollution and global warming. Science and technology, the industry and the society all have to play critical role towards waste reduction. If people judiciously use and reduce the consumption of 5 P‘s (paper, plastic, power, petrol and potable water) in daily life, it would dramatically reduce all the three wastes- the solid, liquid and the gaseous from the environment. Society has to begin the process of reducing waste at source – the home, office, or factory- so that fewer materials will become part of the disposable solid wastes of a community. Efforts must be made to reduce the quantity of materials used in both packaging and obsolescent goods. Waste reduction may also occur at the household, commercial, or industrial facility through ‗selective and judicious buying and consuming‘ patterns and the reuse of products and materials. Source reduction is an option that will conserve resources and also has economic viability.

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Technology Has Improved Efficiency of Resource Use and Reduced Waste Generation by Consumers at Source Technological advancement has undergone a process of ‗dematerialization‘ for reducing the consumption of resources (metals, plastics, glasses etc.) in manufacturing products, with consequent reduction in waste generation. Many packaging items (cans and bottles), consumer ‗electronic goods‘ and even the ‗automobiles‘ have become lighter, slikker and smaller. Since 1977, the popular 2-liter PET plastic soft drinks bottles have been reduced from 68 grams each to 51 grams, a 25 % reduction in material used per bottle. One hundred 12 fluid ounce aluminum cans which weighed 4.5 pounds in 1972, only weighed 3.51 pounds in 1992, a 22 % reduction in material use. Steel beverage cans have also been downsized and are now 40 % lighter than they were in 1970. This means that people can still enjoy a good quality of life while consuming smaller amounts of resources from the environment and generating lesser amount of waste. Lesser resource use would also mean ‗lesser energy consumption‘ and ‗lesser waste generation‘, thus benefiting the environment in every way. (WWI, 1991). The Wisdom of Waste Reuse: It Extends the Life of Material Resources There are several articles like bottles and jars made of glasses and tough plastic materials, large tins, metallic cans and cansisters which can remain in our economy and ecosystem for very long time (if not discarded as waste) just by simple cleaning and washing. Box 2. Recipe and Responsibility of Consumers to Reduce and Re-use Waste in Daily Life 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Buy in bulk and concentrates. Buy durable not disposable products. Buy products packed in reusable and refillable containers. Chose products without or as little packaging as possible. Take Your Own Bags, Box or Basket (TYOB). Insist on reusable cotton bags or durable synthetic bags for grocery shopping; keep a few used shopping bags in your car. Select paper bags/wrap only if needed and avoid plastic bags/wrap. Reuse paper/envelopes only printed/photocopied on one side printed. Use rechargeable batteries. Use refillable ink fountain pens and biros. Avoid using greeting/invitation cards. Avoid wrapping, or sparsely wrap, presents etc. Avoid paper napkins — carry cotton handkerchiefs. Save electronic copies of data or print and photocopy on both sides of paper. Prepare meals at home (not take-away/convenience foods). Avoid frequent use of canned, bottled, packed, processed and preserved foods and eat raw, fresh foods (good for human and environmental health). Avoid peeling fruits and vegetables to reduce kitchen wastes (and preserve nutrients for digestion) and compost any food scraps and leftovers. Carry food and edibles to school/workplace/friends‘ place in reusable boxes. Repair clothes and garments, toys, tools and appliances. Go to garage sales rather than throwing away old things or buying new ones. Use safe retreated car tyres.

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Box 3. Responsibility of Producers (Industries) to Reduce Waste 1.

2.

3.

Producers of consumer goods and products MUST embrace the philosophy and principles of ‗cleaner production‘, which emphasize producing ‗more with less‘, thus conserving resources and reducing waste. Producers MUST embrace the ethical principle of producing durable, re-usable and recyclable goods, which can remain in use for longer periods of time even through changes in the purposes of their uses before being discarded. Producers MUST inform consumers about the recycling potential of their products and be committed to ‗take back‘ products after use for recycling, to recover maximum useful materials from them to reduce the amount of final waste.

It do not involve any complex industrial processing, use of chemical and energy for their reuse. They might have been originally made for some other use, but now can be reused for different purpose- especially for storing and packaging. It should rather be termed as ‗resource reuse‘.

The Wisdom of Waste Recycling : Retrieving New Materials / Energy from Waste and Conserving Virgin Raw Materials and Natural Resources Waste recycling is a technological process to reuse waste materials involving physical, chemical and biological processing to recover useful materials from them. It converts waste into a resource, conserves primary and virgin raw materials from environment (geo-chemical and bio-chemical natural resources), saves tremendous amount of water and energy and protect the environment. Hidden environmental protection values of recycled goods, include the energy and water saved, the pollution and deforestation prevented. Government and People’s Support is Paramount for Recycling to Succeed Recycling combines social, economic, and ecological values. The opportunity to recycle provided by local and state governments has seen an almost fourfold increase in the Victorian recycling industry in Australia in the years 1993–2003 (EcoRecycle, 2006). EcoRecycle estimates that re-processors have saved: water equivalent of filling 17,500 Olympic sized swimming pools; greenhouse gas equivalent to that produced by 580,000 cars; and, ‗enough energy to power every household in Victoria (Australia) for 7 months‘ (EcoRecycle, 2006). Consumers can support recycling by buying goods which bear ‗recycled‘ stickers from their manufacturers. People can indirectly support and promote recycling industries by separating the recyclable from non-recyclable wastes and can directly participate by recycling domestic, e.g. kitchen, wastes through composting. Policy makers could encourage or regulate for all recycled goods to bear a tag recording the origin and life history of the goods, i.e. identifying from which waste it was produced and how it has saved damage to the environment. Such initiatives allow consumers to confidently reuse and recycle products. A demand for recycled goods in society can be created by education.

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Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al. Box 4. Responsibilities of Consumers (Society) in Promoting Waste Recycling 1.

2. 3.

Separate recyclable wastes — glass bottles and jars, aluminum and steel cans, plastic bottles, milk cartons, paper, cardboard, magazines and phone books — from other household wastes faithfully for collection by councils or deliver them to recycling centers / industries. Buy recycled goods. This will encourage promotion of recycling efforts by the government and industries. In order to make recycling an effective strategy, people must understand more about the qualities of materials in everyday items. For example, several vitreous materials that look like, and shatter like, glass are not recyclable. Even a minute amount (such as 5 grams per ton) of these vitreous materials found in some ceramic mugs and plates, cups and crockery, mirror, broken drinking glasses, flower vases, light globes and laboratory glass, can contaminate a load of recyclable glass and render it useless.

Box 5. Responsibilities of Producers (Consumer Industries) Towards Waste Recycling 1.

2.

3.

Producer industries MUST produce / manufacture consumer goods that are ‗potentially recyclable‘. 2. Producers should have a policy to ‗take back‘ their own products to recycle them; Producers should provide necessary information to its prospective buyers on the matter of using, handling, conservation, disposal and recycling potentialities of its products. In designing new products, the industry must assess its potential and even suspected adverse impact on its consumers health and the environment.

10. WASTE RECYCLING : A GROWING GLOBAL BUSINESS WHERE ‗WASTE‘ (TRASH) IS TURNED INTO ‗RESOURCE‘ (TREASURE) Waste is no longer considered as a discarded product to be disposed off from the human ecosystem. Wastes are in fact now considered as a ‗misplaced resource‘ to be brought back into the human ecosystem through the reuse and recycling technologies. Recycling means that otherwise wasted items are returned back to the country‘s economy to make either the same product again, another product or other products. This saves cost on landfill disposal, save landfill space, and prolongs the life of the primary resources used to make the product. (Fairlie, 1992). Recycling can involve mechanical / biological / chemical / thermal processing of waste using some energy, water and chemicals to effectively and efficiently reconvert the waste into ‗secondary raw materials‘ to manufacture new products or recover ‗energy‘ from them in the form of fuel gas or heat. Several of the items of domestic, industrial (non-hazardous) and commercial wastes viz. paper, leather, rubber, cotton rags, metals and glasses, wood and plastics can be recycled in the material recovery and reprocessing industries to get valuable new products. Science and technology has provided a tool in the hands of mankind to renew all those ‗non-renewable resources‘ on earth which otherwise cannot be ‗renewed‘ by nature‘s

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mechanism. This would also save tremendous energy, preserve forest, prevent soil erosion and pollution and above all arrest emissions of greenhouse gases (GHG‘s) and reduce global warming. Some economies in the developed nations of Europe and America, and also in Australia, is retrieving back 80 to 85 % of the materials from the waste as useful products for societal consumption or to be reused in developmental activities and only less than 20 % are going to the landfills for final disposal. (Goldoftas, 1989).

10.1. Global Trade and Exchange for Recycling The old saying ‗one persons trash is another person‘s treasure‘ is becoming true as ‗waste trade and exchange‘ between nations for recycling is entering into new era both economically, ecologically and politically. Japan, Germany, France, Hong Kong, U.K., U.S. and also Australia is recycling their wastes on large scales. Japan recycles 65 % of its MSW into usable products and 40% of its waste paper into high quality paper. A computerized ‗waste exchange register‘ has been prepared to link ‗waste producers‘ with potential ‗waste users‘ and diverse waste items like discarded aluminum cans, paper and card-boards, wooden crates and off-cuts, steel plate off-cuts, plastic products, saw dust, eggshells, caustic soda and chemicals have been listed in this register. The OECD countries have established a central system for moving the non-hazardous ‗recyclable wastes‘ across international borders under the rules of normal trade goods listed in the ‗green list‘ of Basle Convention (1989). In these countries several C and F agents have come up with list of ‗waste available‘ and ‗waste wanted‘. The buyers benefit by the reduced cost of raw materials, while the sellers benefit by the reduced cost of treatment and disposal of wastes. Trade in waste between two countries is economically and politically justified if it is based on ethics. Faced with rising cost of safe waste disposal and recycling at home, many industrialized nations prefer to pass their hazardous wastes along with the ‗recyclable wastes‘ on to the poor Third World countries where there are facilities, cheaper manpower and infrastructure for waste recycling. This has happened greatly in case of hazardous electronics wastes. U.S. has dumped huge pile of e-waste in China and India for recycling.

Trade-in Program for Recycling Electronic Waste Recycling is a good option for the extremely old generation computers such as the PrePentium generation, or the computers (specially the monitors) which are broken. According to the International Association of Electronics Recyclers (IAER) more than 1.5 billion pounds of electronics equipment are recycled annually and is likely to grow by a factor of 4 or 5 by the end of this decade. Eleven countries currently have ‗mandatory‘ electronics recovery laws on the books. These are Denmark, The Netherlands, Norway, Sweden, Switzerland, Japan, Belgium, Taiwan, Portugal and South Korea. Some EU nations have very strong system for ewaste collection, such as the SWICO system in Switzerland and the Netherlands Association for Disposal of Metalectro Products (NVMP). NVMP collect 80 % of e-waste. About 77 % of TVs and 64 % of other small brown goods are recovered for reuse and recycling. (Cui and Forssberg, 2003) Most major computer manufacturers in world e.g. Dell, Hewlett-Packard (HP), Compaq, Gateway have begun to address e-waste problems with their own end-of-life management

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programs which offers a combination of trade-in, take-back and recycling programs. Dell and Gateway lease out their products thereby ensuring they get them back to further upgrade and lease out again. Dell offers both reuse and recycling programs. Dell purchases it computers for a nominal $15 fee. They will recycle both Dell and non-Dell computers, but for corporate customers only. Dell Europe, however, recycles individual consumer‘s computers. Gateway provides $25-50 cash refund for new PC and donate the customer‘s used computers. HP and Compaq‘s trade-in program provides a refund cheque for the value of the used computer if it is traded-in on the purchase of a new HP/Compaq computer. HP has been very active in recycling programs. Since 1987, it has recycled over 500 million pounds of materials from the e-waste and pledge to achieve 1 billion pounds by 2007. HP entered into a joint venture with Micro Metallics in 1996 to recycle materials recovered internally and to recover parts from products returned by customers and reported US $ 72.3 billion as revenue from the recycling program. IBM accepts any type of PC (even other brands) but would charge $ 29.99 to take it back. (Cui and Forssberg, 2003).

10.2. Economics of Recycling: Value Addition When Garbage Becomes Gold Any waste has negative economic and environmental value and is a big techno-economic problem for the local government and involves an economic and environmental cost in safe disposal. But if the same waste is converted into an useful ‗new material‘ its economic and environmental value goes to the positive. (Sinha, 1994).The Brisbane City Council in Queensland, Australia process over 60,000 tones of recyclable materials every year and add $ 20 million to their economy. (BCC, 2002). What was the value of flyash before technology founds its use to reconvert it into building material ? It was an environmental a hazard. What is the value of food scraps and the human excreta ? It is a human waste to be safely disposed off every day and involves cost. Composting technology can convert them into a high value end product i.e. compost (a nutritive organic fertilizer for the farms). Biogas technology can now retrieve ‗methane‘- a clean burning fuel for power generation. (Sinha, 1994; Goldstein, 1995). Recycling also reduces the cost of construction and siting of new ‗landfills‘ and closing it after use. It reduces the economic and environmental cost of incineration of several categories of wastes. Recycling economy is a closed loop in which consumers, manufacturers and waste collectors and haulers, all have a critical sustaining role to play. Many waste articles in the society are ‗potentially recyclable‘, but they may not actually be recycled unless there is a practical way to do so and there is a demand / market for the ‗recycled goods‘. The waste haulers would be encouraged to collect the recyclable wastes if there is a demand by the recycling industries, and the recycling industries will buy these wastes (as secondary raw materials for processing) only if it is less expensive (economically cheaper) than the primary (virgin) raw material. The consumers will buy recycled items only if it is as good as the product made from virgin materials and still less expensive. None of them are bothered about the high ‗environmental cost‘ of the procurement of the primary raw materials from earth or the products made from them. When recycled materials have a high ‗social and economic value‘ despite the cost of collecting and processing, they find a ready market. Much of the gold, silver and other precious metals that were ever mined and extracted from their primary ores centuries ago is

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still circulating (recycling) in our economy (human ecosystem). Materials with low social and economic value relative to the cost of collecting and processing do not find a ready market. The grocery and kitchen wastes, agriculture, dairy and slaughter house wastes and the sewage sludge, with greater organic contents can be biologically recycled to get ‗fuel (biogas) and fertilizer‘ (compost). Even the ‗human hair‘ which are protein materials can be recycled for getting ‗amino acids‘ and making newer and cheaper proteins. It is like getting ‗gold from the garbage‘ and ‗silver from the sewage‘. Recycling of hazardous industrial wastes also minimizes the cost and risk of transporting, storing, treating and disposing of hazardous wastes to distant places. US and Japan recycles a great part of its hazardous wastes. Several recyclable hazardous wastes are ‗exchanged‘ among nations under strict rules of Basel Convention (1989). The recycling potential of wastes from the pharmaceutical industries is 95 %, paints and allied products 40 %, organic chemicals 25 %, petroleum refinery 10 % and small industrial machinery 20 %. Used and discarded products from the automobiles e.g. the lead-acid batteries (LABs), the waste oil and the auto tires are also recycled. In some cases the waste may have to undergo some modifications, such as ‗dewatering‘, in order to become recyclable and salable product. An aluminum die-casting firm developed a market for a by-product of their production process- the ‗fumed amorphous silica‘. After much researches into uses for the product it was found to be a valuable additive to concrete. The firm marketed the waste and now sells all the fumed amorphous silica it generates to cement plants. This is bringing an income of US $ 1 million every year for the company and also saves the enormous cost of disposal. (Noll et. al., 1985). An x-ray film manufacturer in U.S. generates a salable waste product. The company installed equipment that flakes and bales waste polyester –coated film stock which is sold as raw material input to another firm. Over 9 million kg of film stock is exchanged each year. This saves US $ 200,000 annually which would have incurred in collection, transport, and disposal cost. Above that, there is annual profit of US $ 150,000 to the x-ray film manufacturing firm from the sale of the recyclable materials. (Noll et. al., 1985).

10.3. The Environmental Significance of Recycling Nature possess tremendous capacity of recycling of several categories of wastes by ‗biodegradation‘ (biological recycling) aided by the decomposer organisms on earth. The earth provides all the necessary ‗resources‘ for development of mankind and also ‗assimilate‘ all the wastes generated by them in the process of those developmental activities. This is defined as the ‗carrying capacity‘ of Earth. Unfortunately, due to the growing consumerism of resources and the consequent increase in the quantity and concentration of human wastes on earth, this carrying capacity is threatened to be jeopardized with dangerous consequences for the global ecological balance and grave risk of poisoning of the life support systems on earth. The addition of ‗non-biodegradable technological wastes‘ has aggravated the problem. They can remain in the human environment for centuries as nature has not evolved the mechanism to degrade and recycle these new man-made materials.

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Waste Recyled 1. Iron & Steel Scraps 2. Aluminum Cans 3. Papers Wastes 4. Glass Wastes

Energy Savings 60-70 % 90-95 % 60-65 % 30-32 %

Pollution Control

Reduction in Solid Waste

Water Savings

Forest Protection

30 % 95 % 95 % 20 %

95 % 100 % 100 % 60 %

40 % 46 % 58 % 50 %

100 % 100 % 100 % -

Source: WWI (1984) ‗State of the World‘.

By resorting to ‗recycling technologies‘ we can actually ‗renew and sustain‘ the natural ‗carrying capacity‘ of earth ecosystems and not only the natural human wastes can be reconverted into a resource, but also the ‗ man-made synthetic and hazardous wastes‘. Recycling of some municipal and industrial wastes such as papers, metals, glasses can accrue several environmental benefits by way of water and energy saving (consequent reduction of greenhouse gas emission), control of air pollution and forest protection.

Figure 2. Explaining the Economic and Environmental Significance of Recycling.

10.4. Need for Appropriate Recycling Technologies Several municipal as well as industrial solid wastes have potential to be recycled. They only need appropriate technologies for recycling. New recycling techniques are being developed constantly to maximize recovery of useful materials and minimize the amount of waste going to the landfills. Essentially two types of recycling technologies are being developed for waste processing to get valuable end products – mechanical and biological. Mechanical technology involves thermal and chemical processing of waste (both organic and inorganic fractions), while biological technology involves biological processing of waste

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(organic fraction only) by microbes and earthworms. While mechanical technologies incur expenditure of energy, the biological technologies may generate energy.

10.5. Biological Recycling of Wet Organic Wastes into Fertilizer and Fuel: Diverting the Major Part (70- 80 %) of MSW from Landfills Biological recycling involves processing of wet and dry organics such as the food waste from homes and commercial institutions, green garden and farm wastes, cattle and farmyard wastes and the waste organics from the food processing industries). The traditional composting methods are essentially a biological recycling technology which is being revived and improved with new knowledge in microbiology and environmental biotechnology. Other biological recycling methods developed are technology to retrieve ‗biogas‘ and ‗bio-alcohol‘ (cleaner energy sources) from organic wastes. MSW contain 70-80 % by weight of organic materials and the waste biomass is rich in carbon (C) and nitrogen (N) with other valuable minerals like phosphorus (P) and potassium (K) and have potential to be biologically recycled to recover fertilizer and fuel (energy). However, waste with high organic components should preferably be recycled to produce fertilizers (composts) and not fuel. (White, 1996). Table 11. Useful Materials Recovered from Waste by Recycling Technologies Recyclable Materials from MSW

Useful Materials Recovered / Typical Uses

WET RECYCLABLES Organic Fraction of MSW (70-80 %) Food waste, yard & garden waste etc.

Recovery of fertilizer (compost) and fuel (methane and ethanol) ;

DRY RECYCLABLES Paper Old Newspaper Corrugated Cardboard High-grade Paper Plastics Polyethylene Terephthalate (PET) High-density Polyethylene (HDPE) Polyvinyl Chloride (PVC) Low-density Polyethylene (LDPE) Polypropylene (PP)

Polystyrene (PS)

Newsstand and home-delivered newspaper Bulk packaging, fiberboard & roofing material Computer paper, white ledger paper Soft drink bottles, salad dressing and vegetable oil Bottles; photographic film Milk jugs, water containers, detergent and cooking oil bottles. Home landscaping, irrigation pipes, some food packaging, and bottles Thin-film packaging and wraps; dry cleaning film bags; other film material Closures and labels for bottles and containers, battery casings, bread and cheese wraps, cereal box liners Packaging for electronic and electrical component,

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Multilayer and other mixed plastics Glass Metals Ferrous Metals (Iron & Steel) Non-ferrous Metals Textiles Construction & Demolition Wastes

foam cups, fast-food containers, tableware and microwave plates; Packaging, ketchup and mustard bottles Clear, green and brown glass bottles & containers; Tin cans, white goods, reinforcing bars Aluminum cans, copper and lead products Wiping rags Soil, asphalt, concrete, wood, drywall, shingles

HAZARDOUS WASTES Auto Tires Household Batteries Automobile Batteries Incinerator Residues Engine Oil and Lubricants

Road building materials, paving and tire-derived fuel; Recovery of zinc, mercury and silver Recovery of acid, plastic and lead Concrete, road construction material Refined Oil

Fertilizer Production : Retrieving Nutrients from Organic Wastes i) Compost from Waste (Getting Gold from Garbage) There is always greater economic as well as ecological wisdom in converting as much ‗waste into compost‘ (a nutritive fertilizer for farms), so that less and less waste (the noncompostable and non-recyclable residues) finally go to the landfills. The organic fraction of MSW (70-80 %) is rich in nitrogen, potash and phosphorus (NKP) and all macro and micronutrients. This can be easily recycled through microbial biodegradation – process called composting, to retrieve the nutrients from them. If residents all over the world, practice ‗home composting‘ and ‗backyard composting‘ of their ‗kitchen and garden wastes‘, this will significantly reduce burden on the councils and very little MSW will be left for the landfills. Even councils should practice composting of waste on commercial scale instead of sending them to the landfills, earn money by selling them to the farmers, rather than spending money on their landfill disposal. The components that constitute the organic fraction of MSW are food wastes, paper, cardboard, plastics, textiles, rubber, leather, yard wastes, and wood. Yard waste may contain even higher percentage of organic matter. All of these waste materials can be recycled, either separately or as a commingled waste. Certain industrial wastes such as those from the ‗food processing‘, ‗agricultural‘ and ‗paper-pulp industries‘ are mostly organic in composition and can also be composted. Controlling the environmental conditions i.e. the biological, physical and the chemical factors can significantly improve and enhance the composting process without the emission of foul odor and pollution of the environment and without the loss of essential nutrients from the compost. (Epstein, 1997). The conventional composting technology has now been significantly improved with our modern scientific knowledge in ‗microbiology‘ and ‗biotechnology‘ to ‗biodegrade‘ all kinds of organic wastes including the ‗municipal solid wastes (MSW)‘containing sufficient organic components under a completely controlled environmental conditions. We have innovated

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some new, cost-effective and more efficient methods of converting organic waste into compost which besides providing the macro and micronutrients also provide ‗beneficial soil microorganisms‘ to the soil and work as ‗soil conditioner‘ to prevent soil erosion. (Haug, 1993; Epstein, 1997). The relative ease with which an organic material is biodegraded (composted) depends on the ‗genetic makeup‘ of the microorganisms present and the ‗chemical makeup‘ of the organic molecules. Carbons in sugars, lipids and proteins are easily decomposed than the carbon in lignin. A small portion of the carbon is converted to new microbial cells, while a significant portion is converted to carbon dioxide and lost to the atmosphere. The new cells that are produced become part of the active biomass (decomposer microbes), to further enhance and multiply the biodegradation and composting process and on death ultimately become part of the compost. European cities and societies are developing and adopting ambitious technologies for recycling and composting of wastes to divert them from landfills. Some cities have installed sophisticated equipments for composting and resource recovery. Austrian, French and Swiss cities have taken the lead in installing waste recycling and composting systems. Twenty seven (27) composting plants with a combined annual capacity of 60,000 tones of compost from city trashes are currently under construction in German towns and cities (UNEP, 2004).

Vermicomposting: Using Waste Eater Earthworms for Rapid and Odorless Composting of Municipal and Industrial Organic Wastes Vermicomposting is rapid and odorless process triggered by earthworms through enzymatic breakdown of waste organics and also enhancing the microbial degradation by proliferating the microbial population in the waste biomass. Vermicompost is completely free of pathogens but rich in beneficial decomposer microbes including nitrogen fixing bacteria, mycorrhizal fungi and actinomycetes. It is rich in NKP, trace elements, enzymes, growth promoting hormones (gibberlins and auxins) and readily works as ‗soil conditioner‘. Long-term researches into vermiculture have indicated that the Tiger Worm (Elsenia fetida), Red Tiger Worm (E. andrei), the Indian Blue Worm (Perionyx excavatus),the African Night Crawler (Eudrilus euginae),and the Red Worm (Lumbricus rubellus) are best suited for vermi-composting of variety of organic wastes including some of the hazardous wastes like the ‗sewage sludge‘ (biosolids) from the sewage treatment plants and the ‗fly-ash‘ from the coal power plants. Vermiculture was started in the middle of 20th century for management of municipal / industrial organic wastes in Holland in 1970, and subsequently in England, and Canada. Later vermiculture were followed in USA, Italy, Philippines, Thailand, China, Korea, Japan, Brazil, France, Australia and Israel. However, the farmers all over the world have been using worms for composting their farm waste and improving farm soil fertility since long time. In UK, large 1000 mt vermi-composting plants have been erected in Wales. The American Earthworm Technology Company started a 'vermi-composting farm' in 1978-79 with 500 t /month of vermicompost production. Japan imported 3000 mt of earthworms from the USA during the period 1985-87 for cellulose waste degradation. The Aoka Sangyo Co. Ltd., has three 1000 t /month plants processing waste from paper pulp and the food industry. This produces 400 ton of vermicompost and 10 ton of live earthworms per month. The Toyhira Seiden Kogyo Co. of Japan is using rice straw, municipal sludge, sawdust and paper waste for vermicomposting involving 20 plants which in total produces 2-3

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thousands tons of vermicompost per month. In Italy, vermiculture is used to biodegrade municipal and paper mill sludge. Aerobic and anaerobic sludge are mixed and aerated for more than 15 days and in 5000 cum of sludge 5 kg of earthworms are added. In about 8 months the hazardous sludge is converted into nutritive vermicompost. In France, 20 tons of mixed household wastes are being vermi-composted everyday using 1000 to 2000 million red tiger worms (Elsenia andrei) in earthworm tanks. Rideau Regional Hospital in Ontario, Canada, vermi-compost 375 - 400 kg of wet organics mainly food waste everyday. In Wilson, North Carolina, U.S., more than 5 tons of pig manure (excreta) is being vermi-composted every week. (Edward, 1998; Frederickson, 2000; Fraser-Quick, 2002; Sinha et. al., 2005 b).

ii) Fertilizer Pellets from Sludge Cake (Getting Silver from Sewage) A technology has been developed in U.S. by Massachusetts Water Resources Authority to recycle the dewatered ‗sewage sludge‘ into ‗fertilizer pellets‘. The sludge is shipped from sewage treatment plant and pumped directly from barges into the storage tank and then transferred to belt-filter press where water is mechanically squeezed out. The sludge cake is moved through the conveyer belts to rotating heat dryers and heated to convert into small hard pellets. This also destroys the foul smell and harmful bacteria. The pellets are low-grade fertilizers, but if blended with other synthetic nutrients can form a complete fertilizer. Cities all over the US is operating sludge processing plant like MWRA. Fuel Production : Retrieving Cleaner Sources of Energy from Organic Waste The organic municipal solid wastes enriched with biomass and materials with high calorific value (combustible) can be recycled to yield either gaseous fuel ‗methane‘ (biogas) or liquid fuel ‗ethanol‘ by fermentation. The wastes can be directly incinerated and the heat liberated is used for steam generation and electricity production. The new idea is to use the wastes a source of fuel in ‗cement kilns‘ in cement industries to replace the costly fossil fuels. The emission problems accompanying incineration is also minimized to a great extent. But only wastes with high calorific value is useful for energy recovery. (Parker and Roberts, 1985; Porter and Roberts, 1985). i) Biogas There is always greater ecological wisdom in generating ‗biofuel‘ (biogas and bioethanol) from the MSW rather than generating ‗thermal energy‘ from them by combustion technology, with accompanying release of toxic gases especially ‗dioxins‘ and residual ‗ashes‘ which needs landfill disposal. The biogas technology by the use of anaerobic ‗metanobacteria‘ utilizes the organic wastes rich in cellulosic materials with high carbon and nitrogen (C/N) ratio. It produces both fuel and fertilizer. Each ton of organic waste by dry weight yields about 36 cum of biogas and 350 kg of biomanure. Methane is a clean burning substance and on combustion yields 550 BTU of heat per cft of its volume. Even the sewage sludge rich in organic matter and high C/N ratio is efficiently recycled to yield methane. ii) Bio-diesel Bio-diesel is emerging as an alternative cleaner fuel for diesel engines brewed from waste organic feed-stocks, such as animals waste fats (tallows), lard and waste cooking oils. This is

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being produced in Australia and other countries on commercial scales. Bio-diesel is also being produced from offal at a turkey-processing plant in the U.S. Any vegetable oil can form the feedstock to produce bio-diesel. It can produced by recycling the waste oil from fast-food restaurants and the deep friers of French fries which generate huge amount of waste vegetable oils.It can now be produced from household waste and used auto tires. Bio-diesel is completely non-toxic and biodegradable, almost free of sulfur and aromatics, and have lower CO2 emissions than the mineral oil derived diesel. It can be used in existing fuel engines without any modifications. The emissions from bio-diesel are 100 % lower in sulfur, 96 % lower in total hydrocarbons (HC), 80 % lower in polycyclic hydrocarbon (PAH), 45 % lower in carbon monoxide (CO) and 28 % lower in suspended particulate matters (SPM). (UNEP, 2006).

iii) Bio-alcohol (Ethanol) Waste biomass rich in starch and cellulosic materials provides good raw material for ethanol production by enzymatic fermentation carried out by organisms ‗yeast‘ and some bacteria. Bagasse, the waste from sugarcane industry is most appropriate raw material. 6000 kg of bagasse upon fermentation yields 1000 litres of ethanol of 95 % strength. It is a cleaner auto-fuel and Brazil is already using it as an ‗auto-fuel‘ since 1975. New bioconversion technologies could open the gate to the cost-effective use of a wide variety of feed-stocks including agricultural waste products like corn stalks, rice and wheat straws and perennial grasses to produce bio-fuel ethanol iv) Coal Briquetts Technology to recycle the agricultural wastes into a non-polluting fuel has been developed. The dried biomass is crushed and pre-heated to a temperature of 100-120 C and then compacted. v) Fuel Pellets A technology has been developed for converting garbage into non-polluting fuel pellets. The garbage is first shredded and blown dry in rotary kiln. It is then blended with combustible wastes like saw dust and is then pelletized. The pellets have calorific value around 4,000 kcal /kg and there is no harmful emission upon combustion.

10.6. Mechanical Recycling of Dry Recyclables into Original or New Products Mechanical recycling involves processing of dry organic and inorganic recyclables such as papers and cardboards, plastic cans and bottles, metal cans, glass jars and bottles. Mechanical methods include thermal and chemical processing of waste materials and also consumes considerable amounts of water and energy. Now new mechanical technologies is being developed to recycle even hazardous industrial wastes. Metals, glasses, papers and plastics, wood and rubbers are some common domestic and commercial wastes which have high recycling potential to recover goods of mass consumption through mechanical technologies. (WWI, 1984).

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Recycling Potential of Metallic Wastes Iron, aluminum, lead, tin and copper are metals of mass consumption by the society. Their production from their virgin ores by mining activities requires huge amount of energy and water and also cause blatant environmental pollution, solid waste generation (as tailings) and deforestation. Metals like iron and aluminum can be effectively recycled for ever with least impact on environment while conserving huge amount of energy. (AEN, 2000). a) Ferrous Metals (Iron and Steel) The ferrous scraps include autos, household appliances, equipment, bridges, cans and other iron and steel products. The largest amount of recycled steel has traditionally come from large items like road unworthy cars and appliances. It can be reclaimed from the automobile bodies and engines, disused household or industrial equipment and building materials. The shipyards, railyards and the automobile junkyards offer vast amount of waste metallic products to be recycled. They can alone meet more than 50 % of the world‘s metal requirements. Marine ship break is a continuous process throughout the world as the ships lose sea worthiness in 25 to 30 years. Recycling of steel cans is also becoming very popular. They can easily be separated from the mixed recyclables or MSW using large magnets. To protect them from corrosion, all steel cans are coated with a thin layer of ‗tin‘ that must be removed in the recycling process. Immersing the sheets of steel in alkaline bath and transmitting an electric current completes the ‗detinning‘ process. Iron and steel has high recycling potential and can be recycled again and again without reducing the quality of the end products. Nearly half of the iron and steel which has already entered into the human ecosystem is now being used through recycling. World steel production alone consumes as much energy annually as Saudi Arabia produces. Making steel from recycled materials uses only a quarter of the energy needed to make steel from iron ores. (AEN, 2000). Iron scraps costs little more than iron ore but can be converted into steel with much lower economic and environmental cost. Using coke for iron ore reduction produce copious particulate matters including carcinogenic benzopyrene. Recycling of iron reduces this emission by 11 kg/metric tons of steel produced and also cuts iron ore waste and coal mining wastes by 1100 kg/metric tons recycled. Iron and steel scraps are baled into bricks at the material recycling facility (MRF) and melted at 1700 C in the smelters to produce ingots. b) Non-ferrous Metals Non-ferrous scrap metals include aluminum, copper, lead, tin, and precious metals. Recyclable nonferrous metals are recovered from common household items (outdoor furniture, kitchen cookware and appliances, aluminum cans, ladders, tools, hardware); from construction and demolition projects (copper wire, pipe and plumbing supplies, light fixtures, aluminum siding, gutters and downspouts, doors, windows); and from large consumer, commercial and industrial products (appliances, automobiles, boats, trucks, aircraft, machinery) Aluminum: It has high recycling potential. The amount of aluminum which has already entered into the human ecosystem is sufficient to cater the needs of the society through recycling and there is no need to process it from the virgin ores. Aluminum cans are baled into bricks and melted at 700 C in rotary furnaces. The molten aluminum is cast into ingots

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and remade into cans or processed into other aluminum products like saucepans and homewares. Copper: It can be recycled from the boilers of hot water systems, old car radiators and copper pipes. Electric cabling and wiring contains copper and aluminum which can be recycled. Lead: Lead can be recycled from old car batteries and old lead pipes. Lead is recycled in high rate because it is highly toxic and processing from its ore is highly damaging to both man and environment. Silver can also be recovered from silver-plating industries through recycling. A ‗silverplating plant‘ in the US spends about US $ 120,000 a year on waste treatment, of which US $ 60,000 is returned as credit for ‗silver‘ recovered from the waste. Silver and even gold is recovered from electrical industries. Table 12. Metal Consumption Procured through Recycling in USA (1). Lead (4). Aluminum (7). Tungsten

73 % 45 % 29 %

(2). Copper 60 % (5). Zinc 43 % (8). Nickel 26 %

( 3). Iron & Steel (6.) Tin (9). Chromium

56 % 38 % 21 %

Source : WWI, Washington D.C. ‗State of the World‘ (1989).

Environmental and Economic Benefits of Recycling Metallic Wastes World steel production alone consumes as much energy annually as Saudi Arabia produces. Making steel from recycled materials uses only a quarter of the energy needed to make steel from iron ores. Iron scraps costs little more than iron ore but can be converted into steel with much lower economic and environmental cost. Using coke for iron ore reduction produce copious particulate matters including carcinogenic benzopyrene. Recycling of iron reduces this emission by 11 kg/metric tons of steel produced and also cuts iron ore waste and coal mining wastes by 1100 kg/metric tons recycled. Recycling aluminum uses only 5 % of the energy needed to produce new aluminum from its ore ‗bauxite‘. Recycling one aluminum beverage can, saves energy enough to run TV for 3 hours. The energy needed to make 1 ton of virgin aluminum from bauxite could be sufficient to recycle 20 tons of aluminum from its scrap. It takes about 4 tons of bauxite to produce 1 ton of finished aluminum. Recycling aluminum reduces air pollution including the toxic ‗fluoride‘ by 95 %, and cut millions of tones of greenhouse gases (mainly CO2). (AEN, 2000). Recycling Potential of Paper and Cardboard Wastes About 30 % of the paper products which we use today are made from recycled papers and cardboards. Three common grades of paper recycled are corrugated cardboard, high grade office paper and old newsprint. Waste papers and card boards make excellent pulp for making different grades of paper to be used for stationary, magazine and newspapers, game boards, ticket stubbs, cereal and cake mix boxes, grocery bags, tissue papers, paper towels, egg boxes, cards and packaging materials. If cotton rags are mixed with them they make excellent pulp for making other kinds of papers too. Office papers are recycled to manufacture computer papers, writing and printing papers.

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However, infinite recycling of paper is not possible because the fibers become shorter and shorter and the quality of papers declines. Also the tissue papers and the wax coated papers cannot be recycled. The printed papers also need to be de-inked before recycling as it may contaminate the entire fibre stock.

Environmental and Economic Benefits of Recycling Paper Wastes Recycling of waste papers saves green trees, large amount of energy and water and prevent the use of chemicals. Recycling 1 ton of waste paper saves 13 trees, 2.5 barrels of oil, 4100 kWh of electricity and 31,780 litres of water. About 64 % less energy and 58 % less water is needed to make papers from recycled fibers than to make from virgin pulp obtained from the plants. Recycling half of the papers used in the world today would meet almost 75 % of the demand for the new paper and would liberate 8 mha of forest from clear-felling for plantation for paper production. According to one estimate the energy required to produce one ton of paper from the virgin wood pulp is 16, 320 kWh, while only 6000 kWh is needed to obtain the same amount of paper through recycling from paper waste. Reduction in energy use (oil or electricity generated from fossil fuels) proportionately reduce the emissions of greenhouse gas CO2 and other pollutants which would have occurred otherwise. (UNEP, 2004). Recycling of Paper Industry Waste All paper mills recycle and recover their intermediate and untreated effluents called ‗white water‘ (water passing through a wire screen upon which paper is formed) and thus reduces the volume of spray and wash water it uses. Other wastes like ‗sulfite waste-liquor‘ by-product have been found to have multiple uses. Their complete evaporation produces fuel and other salable by-product used in making core binder, road-binder, road bank stabilizer, cattle fodder, fertilizer, insecticides and fungicides, linoleum cement, ceramic hardener, insulating compounds, boiler-water additives, flotation agents, and in the production of alcohol and artificial ‗vanillin‘. The liquor may be ‗fermented‘ to produce ethyl alcohol. About 40 liters of alcohol can be produced per ton of dry solids. Acetone and butyl alcohol can also be produced from the liquor waste. Another product of fermenting the liquor is yeast for cattle feed. The ‗spent cooking liquor‘ produced in the sulfate process (kraft mills) contain recoverable quantities of sodium salts, resins and fatty acids. Caustic soda (Na2CO3) is recovered from them. The resin and fatty acids are further refined and have a variety of applications in industry. In chemical precipitation with lime as coagulant, ‗lignin‘ is precipitated which is used both as a fuel and in the manufacture of plastics, production of tannins, as an anti-scale or antifoam agent in boilers. Wastewater from the paper mills have also been found to be good for ‗irrigation‘ of number of crops. Recycling Potential of Glasses Glasses are 100 % recyclable and can be effectively recycled for ever. The recyclable glasses in MSW are container glass (the clear, green and brown bottles and jars for food and beverages), flat glass (e.g. window panes), and pressed amber or green glass. Glasses to be recycled is often separated by color into categories of clear, green and amber. Broken glasses are known as ‗cullet‘ which are valuable raw material in the production of new glasses.

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Manufacturer adds about 40 tons of cullet to every 100 tons of raw materials silica sand to produce glass. However, several vitreous materials may look and shatter like glasses, but are NOT recyclable. They do not melt like glass. Even a minute amount of these materials can contaminate the whole load of recyclable glasses and render them useless. These are ceramic mugs and plates, cups and crockery such as pyrex and corning ware, mirror, broken drinking glass and flower vase, light globes and laboratory glass. Contamination as little as 5 grams per ton is harmful. One tiny fragment of ceramic material in a load of glass ‗cullet‘ can cause a weakness in the new glass which may crack or even explode when the bottle is filled or opened. Recycled glasses are being used as raw material in cement production. (Wong, 2000). A British firm is building an industry to recycle the used wine bottles into ‗green sand‘ to be used for filtering drinking water and purify sewage. When the recycling plant is fully developed it can save the quarrying of high quality sand and use all the waste wine bottles. The European Union and the UK Department of Environment, Food and Rural Affairs are funding the project. (www.drydenaqua.com).

Environmental and Economic Benefits of Recycling Glass Wastes The cullet (broken glass materials) melt at low temperature than the primary raw materials and hence require 25-30 % less energy on addition and also extends the life of melting furnaces. Every ton of cullet used saves the equivalent of 30 gallons of oil and replaces 1.2 tons of virgin raw materials and proportionately reduce the emissions of greenhouse gas CO2 and other pollutants which would have occurred upon use of oil and other energy sources in the extraction of virgin materials from nature. Recycling Potential of Plastic Wastes Recycling of plastic wastes is not really a good option and is rather a hazard for the human health and the environment. It gives out ‗toxic‘ fumes like ‗dioxins‘ during melting. Technologies are not available for recycling of all categories of plastics. Plastics are recycled by codes. Currently only plastic bottles made of PET (Code 1: soft drink, juice and water bottles and some plastic jars); HDPE (Code 2: milk bottles, cream containers and juice bottles); PVC (Code 3: detergent, shampoo and cordial bottles); PP (Code 5: ice cream containers, takeaway food containers flower pots etc.); PS (Code 6: polystyrene cups, glasses and meat trays), and bottles with ‗R‘ or ‗please recycle‘ symbols are accepted for recycling. Two types of plastics most commonly recycled in world are PET (Polyethylene Terephthalate) and HDPE (High Density Polyethylene). PET is recycled to make soft drink bottles, deli and bakery trays, carpets, fibrefill and geotextile liners for the waste landfills. HDPE is recycled to manufacture plastic milk, disinfectant and detergent bottles, recycling bins, sleeping bags and pillow stuffing, roadside guide posts, irrigation pipes, eskies, water meter boxes, air-conditioning and recycled layer in new PET plastic bottles, agricultural water pipes, bags and motor oil bottles. LDPE (Low Density Polyethylene) is recycled to make new plastic bags and films. PVC (Poly Vinyl Chloride) is recycled to manufacture drainage pipes, non-food bottles and fencing posts. Recycled polypropylene (PP) is used in auto-parts, carpets and geo-textiles. Recycled polystyrene (PS) is used to manufacture wide range of office accessories, video-cassettes and cases.

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Plastic grocery bags made of LDPE or very thin HDPE cannot be recycled as they clog the system. Some supermarkets take back plastic shopping bags, recycling them into bin liners, and hospital waste bags. Other plastics like motor oil containers, plastic takeaway food containers and disposable nappies also cannot be recycled. Also different plastics cannot always be recycled together. The recycled plastic is often hard and brittle. To overcome this problem a ‗compatibilser‘ molecule that sticks together the different plastic molecules have been developed. Such commingled plastic with much gloss and sturdiness as the originals can be used to make ‗car bumpers‘ and fence posts.

Environmental and Economic Benefits of Recycling Plastic Wastes Recycling of plastics at least diverts thousands of tons of plastic materials from ending up in landfills every year and besides saving landfill space also arrest emissions of some very toxic gases and leachate discharge from the landfills. Recycling Potential of Waste Wood It is a waste from the timber industries, forestry and agricultural activities and from building construction and demolitions. Technology found its use in the manufacture of ‗plywood‘ and ‗medium density fiber board‘ (MDF). They have properties like natural wood with additional advantage of not being flammable and absorbing moisture, resistant to pest attack and low cost. They are also shredded and processed as wood chips for fuel or landscaping cover. Recycling Potential of Construction and Demolition (CD) Wastes CD wastes results from construction, renovation, and demolition of buildings; road repaving; bridge repairs; and the cleanup after natural disasters. Typically they are made of about 40-50 % rubbish (concrete, asphalt, bricks, blocks, and dirt), 20-30 % wood and related products (pallets, stumps, branches, forming and framing lumber, treated lumber, and shingles), and 20-30 % miscellaneous wastes (painted or contaminated lumber, metals, tarbased products, plaster, glass, white goods, asbestos and other insulation materials, plumbing, heating and electrical parts). The principal materials that are now recovered from the CD wastes are asphalt, concrete, wood, drywall, asphalt shingles, and ferrous and non-ferrous metals. Reinforcing steel used in foundations, slabs, and pavements are usually recovered and sold as scraps for recycling. Concrete and asphalt are processed for road base, aggregate in asphalt pavement, and as substitute for gravel aggregate in new concrete. Many landfills use the rubbles for road building and as ‗daily cover‘ material for the compacted waste in the landfill. (Hamilton, 2000).

10.7. Recycling Potentials of Some Hazardous Industrial Wastes (IHW) Technologies have been developed to recycle several categories of hazardous industrial wastes. Several hazardous industrial wastes are now finding new use and applications in construction industries through recycling. (Noll et. al., 1985).

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New Use of Phosphogypsum from Phosphatic Fertilizer Industry Radioactively contaminated calcium sulphate slurry called ‗phosphogypsum‘ (CaSO4.2H2O) is a waste byproduct of these industries. For every ton of phosphoric acid produced, 5 tons of phosphogypsum (PG) must be removed. It is now being recovered and reused to manufacture partition panel, ceiling tiles/boards, walling blocks etc. They save cement and steel and minimize the use of timber. Direct reuse of PG, however, presents the potential problem of incorporating radioactivity into building or road products. Several firms in the US and UK is processing the PG into useful cement or plaster. The quality of cement compares favourably with limestone-based cement and is used in all classes of building construction and civil engineering. It possess a ‗compressive strength‘ 3 to 4 times that of Portland cement (1100 kg / cm2 as compared to 300-400 for Portland cement). Moreover the cost of cement production from PG was US $10 / ton as compared to $ 30- $ 40 / ton for Portland cement. The PG is chemically processed into ‗hemihydrate powder‘ to use in cement manufacture. Some South African fertilizer plants disposes about 25 % of its PG as soil conditioner, cement clinker and as cement retarder. (Noll et. al., 1985). Any recovery and reuse of phosphogypsum (PG) would also free up reclaimable land resources for productive purposes by the industry or privately. New Use of Fly-ash from Waste Slurry in Coal-fired Power Plants Fly-ash is a waste byproduct of coal combustion in coal-fired power plants generally consisting of very fine particles. It pollutes water bodies. The wastewater usually contain 6750 gm / liter of fly-ash. A common method of fly-ash removal from the steam power-plant flue gases is by the use of ‗wet scrubbers‘, ‗electrostatic precipitators‘, or ‗cyclone separators‘. Major chemical component of fly-ash are silica (30-50 % by weight as SiO2) and alumina (20-30 % by weight as Al2O). Other materials are sulfur trioxide (SO3), carbon (C) and boron (B). It is now being reused to manufacture clay bonded bricks / blocks, fly-ash ceramics, aerated light weight cellular blocks and slabs, precast blocks for footpath, butiminous concrete for road surfacing and cement concrete etc. Fly-ash bricks have replaced baked earthen bricks and have saved million tones of fertile soil from getting eroded. Technology has found several new uses for fly-ash: 1) 2) 3) 4) 5) 6) 7) 8)

As a pozzolana ingredient in Portland cement; As a pozzolana in soil stabilization; As a soil conditioner in agriculture; As a grout in oil wells; In asphalt roofing and siding materials; As a cement replacement in concrete; As a mineral filler in asphalt pavement; As a coagulant in sewage treatment.

New Use of Red Mud from Aluminum Industry ‗Red Mud‘ is a waste from the aluminum industry being generated in million tones every year. It is a bauxite residue and clay like silt consisting of undissolved minerological components. It is being reused in cement industries both as component of cement raw mix as

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well as additives. Red mud light roofing sheets has been developed as an alternative to the dangerous ‗asbestos‘ roofs. New Use of Metallic Slag from Iron and Steel Industries It is a waste from the Iron and Steel industries and an excellent secondary raw material for cement production.

10.8. Thermal Recycling of Hazardous Wastes to Retrieve Thermal Energy Several hazardous industrial wastes such as paint thinners, degreasing solvents, solvents from ink and printing industries, dry-cleaning fluids, chemical by-products from pharmaceutical industries, waste papers, waste oils and waste auto tires, sewage sludge and municipal solid waste can be used as fuel in industries. Cement Kiln Using Hazardous Industrial Wastes as Fuel and Replacing Fossil Fuels The manufacture of cement from limestone require high kiln temperatures (about 1500 º C) and long residency times. This create an excellent opportunity for hazardous waste to be used as fuel and also get destroyed in the process. As much as 40 % of the fuel requirement of a well operated cement kiln is saved by the use of hazardous wastes.. It is like ‗killing two birds in one shot‘. Further, as the environment inside the kiln is alkaline due to the presence of lime, the acidic gases and the hydrogen chloride generated from the chlorinated wastes (which is poses problem in conventional incinerators) are neutralized. Combustion of wastes in a commercial incinerator also produces ‗ash‘ which needs to be disposed off. Here there is no ash and the only by-product is the ‗dust‘ which is recycled. Any incombustible material such as metals in the waste, becomes vaporized and incorporated in the product. Up to 95-99 % of the chloride and over 99 % of the lead entering the kiln are retained by the process solids. When the kilns are operating properly ‗dioxins‘ and ‗furans‘ and the particulate matters (which are emitted in the conventional incinerators) are significantly cut down and there is no risk to human health and the environment. U.S. uses about 1 million tons of hazardous wastes as a fuel in cement kilns every year. Reduction in use of fossil fuels as the source of energy in cement plants proportionately reduce the emissions of greenhouse gas CO2 and other pollutants which would have occurred otherwise. (Jones and Herat, 1984).

10.9. Recycling Potential of Some Hazardous Consumer Wastes (CHW) Appropriate technologies are now available to recycle several categories of hazardous wastes including the hazardous electronics wastes (e-waste) generated by society.

Recycling of E-Waste – A Hazardous and Costly Process: Reuse – A Better Option Millions of tones of e-waste all over the world are ending up in landfills. According to the Silicon Valley Toxics Coalition in the U.S. cost of recycling obsolete and discarded computers is estimated to be at US $ 10 to $ 60 per unit. And if poorly handled during the clean-up cost of toxic materials from the e-waste, it could go higher. SVTC estimates the minimum costs for recycling and proper disposal of the remaining non-recyclable e-waste in US will reach US $ 10.8 billion by 2015 (Schneiderman, 2004). This could be the approximate cost of recycling of e-waste in all developed nations including the European

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Union, Canada and Australia where labor cost is high. Local governments and councils in these nations have neither the technical ability nor the financial resources to tackle this gigantic techno-economic problem at their own. ‗Reuse‘ is a better option for several categories of e-waste. The term ‗reuse‘ would mean using any old and obsolete electronic product or equipment with or without minor repair and reasonable upgrading, if possible. The best way of reuse is that computers can be sold to the employees of the organizations or the students of institutions at very reasonable price or donated to charitable organizations, schools, orphanage centers, old people homes, women asylums etc. Institutions and organizations in the rich developed nations (where computer models are changing fast) should develop a system based on ethics for donating their old computers to the needy organizations in the developing countries. The term ‗recycling‘ of e-waste would mean to dismantle the equipment or a product and retrieve the valuable components / materials from it for their reuse in other equipment or remanufacture a new equipment / product. The difficulty with electronic waste and many other end of life electronic products is that they are made from a huge range of component materials that are useless for further manufacture until the product is dismantled and the component materials are separated – often a very difficult and expensive process. Recycling may be a good option for the extremely old generation computers such as the Pre-Pentium generation, or the computers (specially the monitors) which are broken. According to the International Association of Electronics Recyclers (IAER) more than 1.5 billion pounds of electronics equipment are recycled annually and is likely to grow by a factor of 4 or 5 by the end of this decade. Eleven countries currently have ‗mandatory‘ electronics recovery laws on the books. These are Denmark, The Netherlands, Norway, Sweden, Switzerland, Japan, Belgium, Taiwan, Portugal and South Korea. Some EU nations have very strong system for e-waste collection, such as the SWICO system in Switzerland and the Netherlands Association for Disposal of Metalectro Products (NVMP). NVMP collect 80 % of e-waste. About 77 % of TVs and 64 % of other small brown goods are recovered for reuse and recycling. (Monchamp, 2000; Cui and Forssberg, 2003; Mathew et. al., 2004)

Recycling Potential of Lead-Acid Car Batteries Billions of lead-acid car batteries are used and replaced every year across the world, 70 to 80 millions in US alone. The average battery contains about 18 pounds of recoverable lead and the worldwide recycling rate is now 90 %. Batteries are crushed and then the lead, plastic, and sulfuric acid are separated. In an innovative recycling process developed in Italy in the 1980s, batteries are crushed in a hammer mill and the components are separated on a vibrating screen. The acid / lead – paste slurry is neutralized, the lead oxide is separated, and the reusable sodium hydroxide and sulfuric acid are recovered from the solution by electrodialysis. Lead oxides are reduced by electrolysis and combined with metallic components, then melted at 400-500 C and cast into ingots. (Vaysgant, et. al., 1995). Recycling Potential of Household Batteries Billions of household dry-cell batteries are used and discarded worldwide, 2.5 billion in the US alone. These batteries are discarded with the general household waste. These batteries contain mercury, cadmium, lead, and other metals, which become toxic contaminants in the landfill leachate or in the air emissions if MSW are incinerated. Study reveals that household

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batteries are the source of more than 50 % of the mercury and cadmium in the MSW. Recycling of these batteries are difficult. Cylindrical 6-volt and 9-volt alkaline and carbonzinc batteries are not recyclable. Only nickel-cadmium cylindrical cells or mercuric oxide and silver oxide button cells can be recycled. Mixed button batteries are difficult to sort out and may present a storage hazard in the MSW waste bin due to mercury vapor emissions.

Recycling Potential of Auto Tires Several millions of auto tires are rejected every year all over the world which turn as hazardous wastes. Earlier they were reused after ‗retreading‘, but with the advent of steelbelted radials and cheaper new tires most are discarded as waste. They cannot be landfilled as they occupy large volume and tend to come to the surface. Auto tires present problem for safe recycling as they are made of ‗hazardous materials‘. The best option is to reuse them as ‗fuel‘ resource in cement kilns. About 70 % of the waste auto tires in the US is being used as source of energy in cement kilns thus also reducing use of coal and emission of greenhouse gases. Power plants, paper and pulp mills and the cement kilns commonly use the shredded tires as fuel. Whole tires are also used to create ‗artificial reefs‘ for erosion control and as highway crash barriers. Split and punched tires are used to make muffler hangers, belts, gaskets, and floor mats. Recycling Potential of Waste Oils and Lubricants from Automobiles Industries Billions of gallons of petroleum-derived waste oil are produced worldwide mostly from the automobiles and some from the industries. Automotive oils include crancase oil, diesel engine oil, transmission, brake and power steering fluids. Waste oil often contains metals like arsenic, cadmium, barium, chromium, and zinc; chlorinated solvents and organic compounds like benzene and naphthalene. Used lubricants can be recycled in two ways- by reprocessing and by re-refining. Reprocessing is done by water and bottom sediment removal of suspended material and ash by gravity settling or chemical treatment to produce partially cleaned fuel oil. Heat is sometimes used to decrease viscosity and improve gravity settling. Distillation is also done to evaporate light fuel fractions. Re-refining produces a clean oil but it is very expensive affair. This is done by solvent treatment / vacuum distillation / hydrotreatment; by acid clay, chemical cleaning / demetalling etc. Recycled lubricating oils are used as transformer oil, equipment oil, motor oil, cutting oil, soluble oil, diesel oil, gasoline, antifreeze, brake fluids and hydraulic oils. Waste oils are mainly recycled to be used as fuel in cement kilns, in commercial, industrial, and marine boilers, and for space heating. Waste oils can also be used as fuel in cement kilns, in commercial, industrial, and marine boilers, and for space heating.

11. SOME POLICY MEASURES ON WASTE MANAGEMENT BY GLOBAL SOCIETY Considerations of environmental sanitation, health and hygiene, contributing to safety and security of society are key factors which has prompted to enact suitable legislation for management of all kinds of waste by the global society. Safe waste management is not only a

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technological pursuit, but also require legal and administrative, economic and ecological planning. Governments can develop policies to encourage and support the manufacture of ‗durable‘ and ‗recyclable‘ goods as well as ‗take-back programs‘ by producers to recycle their products. If required to ‗take-back‘ and recycle their products, manufactures will be compelled to produce durable and recyclable items, thus reducing waste for consumers.

11.1. The European Union‘s Packaging and Packaging Waste (PPW) Directive (1994) On average each EU citizen is currently responsible, directly or indirectly, for the generation of some 172 kg of ‗packaging waste‘ every year. (UNEP, 2006). As early as in 1994 it issued this directive to ‗prevent‘ packaging waste. Yet, the packaging waste (PW) generation increased by 10 % in the EU-15 between 1997 and 2002, in close line with the 12.6 % growth in GDP. Per capita consumption of plastics increased by almost 50 % from 64 kg / year in 1990 to 95 kg / year in 2002. Only UK managed to actually reduce, and Austria stabilize the generation of PW since 1997. The review of the EU-15 PPW in 2005 however, showed that the both the producers (companies) and the consumers (society) made good progress in recycling PW. The EU target of recycling 25 % of packaging waste in 2001 significantly increased to 54 % in 2002 in the 15 member countries then. (UNEP, 2006).

11.2. EU Directives on ‗Extended Producer / Manufacturer Responsibility‘ (EPR) for Reducing Electronic Waste (2001) In Europe, e-waste is projected to reach 12 million metric tones by 2010 (Schneiderman, 2004). In 2001, the European Union adopted a system called ‗Extended Producer / Manufacturer Responsibility‘ that requires the electronics manufacturers to ‗take-back‘ their used products and assume full responsibility for the production of cleaner electronics items, phasing out of hazardous materials in production process, and also dismantling the e-waste products more easily for recycling at the end of their useful life by trading-in the products for recycling. In January 2003, the EU parliament enacted two directives. The first - ‗Waste Electrical and Electronics Equipment‘ (WEE) is based on the concept of Extended Producer Responsibility (EPR) which requires the industries to ‗take-back‘ all their used and obsolete electronic products for safe recycling. The second directive ‗Restriction on Hazardous Substances‘ (RoHS), called for phasing out of heavy metals Hg, Cd, Pb and Cr VI in all electronics items by July 2006 (with a number of exemptions) to reduce hazardous waste when they are discarded. This has now come into effect. (Adam, 2005).

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11.3. The International Legal Instruments for Combating the Problem of Hazardous Wastes at Global Level The United Nations General Assembly initiated to review the international environmental laws in 1981 at Montevideo. The laws were to cover among other things, the transport, handling and disposal of toxic and hazardous wastes. Real negotiations to impose curb on the production, storage and transport of hazardous chemicals and wastes began soon after the Basel Disaster (Switzerland) in 1986 which resulted into the adoption of ‗Basel Convention‘ in 1989.

The Basel Convention on the Control of Transboundary Movement of Hazardous Wastes and Their Safe Disposal (1989) The Convention was adopted in 1989 unanimously by 116 States in the Swiss city of Basel, to reduce the global generation of hazardous wastes and chemicals to a minimum and to prohibit / regularize the illegal traffic in hazardous wastes and to fix the responsibilities of the parties involved. It entered into force on 5 May, 1992 and now have 131 Parties to the Convention except the U.S. It outlines the general obligations of the hazardous waste generating nations for moving their wastes across their boundaries either for disposal or for exchange for recycling. It also outlines the principles of international co-operation to improve and achieve environmentally sound management of all kinds of hazardous wastes. Trade in hazardous waste that does not comply with the terms of the Basel Convention or its control system is illegal, and considered to be criminal. Parties are obliged to enact stringent national laws to prevent and punish the illegal trafficators in hazardous waste. The Convention also cooperates with the Interpol over illegal traffic. The first 10 years of the Convention (1989 – 1999) concentrated on consolidating its control system, legal framework and operation through improving the classification of wastes and refining the work on their hazard characterization. Cooperation to Developing Countries Under the Convention international cooperation is to be extended to developing countries in: 1. Transferring technology and management systems for hazardous waste; 2. Developing and implementing new environmentally sound low-waste technologies, and improving existing ones to eliminate the generation of waste, as far as practicable, and studying the economic, social and environmental effects of adopting the new technologies; 3. Developing and promoting environmentally sound management of hazardous and other wastes; 4. Promoting public awareness about hazardous waste.

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12. SOME INNOVATIVE IDEAS TOWARDS SUSTAINABLE WASTE MANAGEMENT 12.1. The Australian Innovations on Landfill Gas Utilization for Energy Generation While Reducing Emission of Greenhouse Gases An innovative technology to convert the MSW landfills into a bioreactor is being experimented in Australia. In the traditional landfills the ‗anaerobic decomposition‘ of waste is highly retarded and rather inhibited, as the waste has very little moisture. Daily covering and heavy compaction of waste exclude the moisture content. Normal waste typically contains 10-30 % moisture. The rate of waste biodegradation is a function of moisture content. Bioreactor technology significantly reduces the decomposition time and the production of gas begins soon. Bioreactor is a large ‗anaerobic digester‘, in a specially designed void in landfills which receives municipal solid wastes and the also the biosolid (sludge) from the sewage treatment plants. In the landfill bioreactor, the waste is not heavily compacted and sufficient moisture content (45 – 60 %) is maintained through extensive networks of pipes that re-circulate the nutrient rich leachate and inject additional water such as the stormwater and the run-off. Additional microbial inoculum is added to promote rapid anaerobic microbial decomposition and biogas (60 % methane and 40 % carbon dioxide) production is augmented with 98 % collection efficiency and conversion to energy. Each kilogram of waste can generate up to 220 liters of methane. In case there is more paper content in the waste, the methane production is 365 litre / kg. A typical 4000,000 tones per year ‗landfill bioreactor‘ will produce about 7,500 cum of methane per hour, with a generating capacity of 25 MW. This will also save greenhouse gas equivalent to taking 175,000 cars off the road. When biogas generation in the bio-cell declines, the residual partially degraded organic materials can be excavated and used as a ‗soil conditioner‘ or feedstock for composting. Besides, generating biofuel and biofertilizer, the recycling technology will reduce the emission of greenhouse gas (methane) from the landfills. Fugitive emissions of VOCs is decreased to as low as 0.7 %. It would also reduce the environmental risk period of landfills to 5-10 years from 30-50 years.

Power Generation from Landfill Gas The ReOrganic Energy Swanbank in Ipswich, Brisbane, Australia is generating electricity from the landfill biogas. Natural biodegradation processes in the landfills are enhanced in a special configuration to accelerate the production of biogas (methane). This is supplied to the Swanbank Power Plant for electricity generation displacing coal. By end of April 2002, some 20,000 cubic meters per day of biogas was produced and utilized and over 2,300 MWh of electricity generated. It is envisaged that over this period beginning from 2002 to 2016, some 90,000 cubic meters of biogas per day will be utilized, 500,000 MWh of electricity will be generated, while some 250,000 tones of coal will be displaced and approximately 3 million tones of carbon dioxide equivalent greenhouse gas (methane and carbon dioxide) will be arrested from going to the atmosphere. The $ 4 million project took some 20 months to complete.

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After successful utilization of landfill gases for power generation at Ipswich, another $ 5 million landfill gas power generating facility was launched at Rochedale Landfill site in Brisbane in 2004, generating 3 MW of power. This is expected to reduce greenhouse gases by 20,000 tones a year and also supply electricity to 5000 homes. Since the closure of the landfill at Roghan Road, Fitzgibbon in March 2000, BCC has also started generating 2 MW electricity from the methane produced in the old landfill. New waste landfills in Brisbane are now purpose designed to be used as ‗bioreactors‘ for biogas generation and electricity production, killing two birds in one shot (www.thiess-services.com.au).

12.2. Molok Waste Bins: Odor Free, Less Space, Holds More Waste and Emptied Less Often: An Australian Innovation The Molok Pty. Ltd. Of NSW in Australia has invented a new waste collection bin (40 x 120 L size) 60 % of which lies underground and only 40 % is visible. It is installed to a depth of 1.5 meter in ground and takes 80 % less surface area than the conventional waste bins. It comes in 300 L, 1300 L, 3000 L and 5000 L sizes suiting to all locations in residential and commercial areas. The containers are water tight and the waste drop hole opening is approximately 1.1 m above ground level. Within the PVC container is suspended the lifting waste bag made of double layered textile material. While emptying the bag is simply lifted by the hydraulic lifting arm and hoisted into the truck container. Emptying 5000 L Molok bin takes about 3 mins and is one man job. The key advantage of the vertical bin is that gravity forces the old waste to compact as the new waste is added, and the oldest waste materials at the bottom of the container is kept cool because the earth underground is naturally cool due to evaporation. The lowering of temperature at the bottom reduces microbial activity arresting any odor problem. It is also likely that there will be lesser emission of greenhouse gas methane.(www.molok.com.au).

12.3. The Innovative ‗Bio-Bin‘ System of Cleanway for Kerbside Composting of Green Waste Cleanaway‘ is an enterprise of Brambles Industries Limited established in 1970. It operates across Australia and around the world. It provides services in waste recycling, safe hazardous waste disposal, site remediation, and landfill operations. It operates 8 waste recycling plants in four Australian states. It‘s co-mingled bin recycling system services over 770,000 Australian households and has participation rate over 85 %. ‗Cleanaway‘ has introduced a new and innovative concept of ‗Bio-insert‘ system. The ‗Bio-Bins‘ break down ‗green organic waste‘ on the kerbside and reduces the weight of the waste. It promotes oxygen-flow around and through the organic waste, triggering the aerobic decomposition process. Problems like odor, leachate production and greenwaste sticking to the bottom of the bin are eliminated because of oxygenation. The contents of the bio-bins are less compacted, drier, lighter and more uniform than those in the standard bins. It forms good feed-stock material for the ‗compost facility operators‘. A collection frequency of 4 weeks is recommended for green organics, while 2 weeks for the food organics. It is also inexpensive

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to integrate the bio-bin system into the existing waste management services and would save the city councils considerable expenses in collection and landfill costs. Bio-Bins are widely in use in Europe and North America for the past ten years. A trial of bio-bins is being conducted in the City of Mooney Valley, Melbourne, Victoria with very encouraging results.

12.4. The Innovative DiCOM ‗Aerobic-Anaerobic-Aerobic‘ Hybrid System of Bioconversion of MSW into Clean Biofuel and Biofertilizer A new and innovative hybrid biological system for composting of MSW was developed in Australia in 2000 by AnaeCo which process the MSW and produces a finished product in less than half the time required for normal aerobic process of microbial composting without any odor problem. The end products are ‗green energy‘ (biogas methane) and ‗compost‘. This has been termed as DiCOM bioconversion process. The facility was installed at the cost of $ 5 million and is situated in Perth, Western Australia. It receives commingled municipal solid wastes (MSW) from the local councils, removes the non-compostable inert materials (the dry recyclable) such as the metals, glasses and plastics from the waste and then subject the organic materials to a ‗Three (3) Stage Processing‘ that involves an ‗Anaerobic Digestion Phase‘ in between the initial and final ‗Aerobic Composting‘ period. The methane (CH4) gas generated from the anaerobic digestion phase is captured and used as biogas fuel for electricity generation at the facility. The separated recyclable materials are sent to MRF. (www.anaeco.com). The company has patented this technology. The initial bioconversion capacity was 17,000 tons of MSW per annum which increased to 55,000 tons per annum by 2005. The whole plant has small ‗ecological foot print‘ requiring small operational area and with potential for decentralization of waste treatment to multiple sites close to the sources of community waste generation, thus significantly reducing the cost of waste transportation and pollution.

12.5. The Innovative Total Waste Management System‘ (TWMS) in Australia: No Waste to Landfills Berrybank Piggery in Victoria Shows the Way Charles Integrated Farming Enterprises Private Limited company in Victoria developed an innovative concept of ‗Total Waste Management System‘ (TWMS) for its Berrybank Piggery. in Victoria, Australia. The piggery produces on an average 275,000 L of effluents (sewage) a day with ‗solid contents‘ approximately 2 %. The company found that the ‗traditional farming philosophy‘ of ‗wasting nothing‘ and ‗waste from one part of a farm is the input in another‘ makes a good business sense. On regular inventory program the management of piggery found that half of the feed consumed by the animals is actually utilized, and the remaining half goes as waste. The company considered this generation of waste as ‗poor return on investment‘. The TWMS is a seven-stage process which salvages all the waste byproducts in the form of fuel, fertilizer and flush water. Since the introduction of TWMS, the Berrybank Piggery now daily produces –

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Seven (7) tones of waste solids which are utilized as ‗fertilizer‘ for farms; 100,000 L of flush water (recycled water) to be used for flushing toilets; 100,000 L of mineralized water rich in essential micro and macro-nutrients which is utilized as ‗liquid fertilizer‘ for farm irrigation; and 1,700 cum of biogas fuel (methane) which is used for power generation on farm with daily output of 2,900 kW of electricity.

The capital cost of the Berrybank Farm project was approximately AU $ 2 million which was paid back in about 6 years by way of fuel, fertilizer and usable water. From a $ 2 m investment made in the TWMS for the Berrybank Piggery, the Charles IFE brings in AU $ 435,000 return every year. Other environmental benefits was that the odor problem and the risk of groundwater contamination (due to sewage) was completely eliminated and it dramatically reduced the consumption of freshwater (www.environment.gov.au/settlement/industry/corporate/eecp/casestudies/charlesife.html)

12.6. The Indian Innovation on Dumpsite Composting of Commingled MSW: A Cheaper and Safer Alternative to Costly Landfills? An innovative technology to compost the un-segregated municipal solid waste (MSW) biomass on ordinary waste dump-site was developed in India in the 1990‘s and is giving excellent results for managing the solid wastes. No prior segregation of commingled waste is required. Segregation of commingled wastes at source or before composting imposes the biggest obstacle in any composting technology because it is highly labour intensive job to segregate the often sticky and wet biodegradable (compostable) matters from the dry nonbiodegradable ones. It becomes much easier after composting. The technology was developed by Ms. Excel Industries, Mumbai, India and can suit to all countries and is also flexible for 150-700 MT of waste per day. The largest plant with an installed capacity to bioprocess 500 tones /day of MSW is operating in Mumbai. The process can recycle all organic wastes from the households, restaurants and hotels, dairy, agriculture and agro-processing industries, brewing industries and slaughter houses. (Ranjwani, 1996;.Sinha and Herat, 2002 b).

The Bioconversion Process and the Composting Mechanism The dump site is leveled and either cemented or paved with bricks on the bottom to prevent leachate and for easy movement of waste carrying vehicles. Long windrows, about 5 meters wide and 2-3 meters high (deep) are erected and the MSW is then stacked and heaped in the windrows. A ‗microbial consortium‘ slurry containing active decomposer bacteria and enzymes are then added to the windrows to initiate rapid aerobic decomposition of the waste biomass. The slurry is spread on the surface of the garbage and inside the heaps in the windrows with help of probes, so that it reaches deep and in every pocket of the heap. Microbial culture of active decomposer bacteria is prepared from the ‗sewage sludge‘ which contains active decomposer bacteria in millions/gram of the sludge. (Ranjwani, 1996).

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The microbial culture is known as ‗Celrich Substrate DF BC-01. It is prepared after analyzing the composition of the waste and identifying the predominant materials such as celluloses, hemicelluloses, lignins, proteins and fats etc. The microbes produce enzymes such as cellulase, lipase, amylase, protease, pectinase and phospholipase to breakdown the long chain complexes of the substrates. About 1 kg of the consortium in the colloidal emulsion form is mixed with 20 litres of water to be used for spraying on about 3 cubic meter of solid waste and for one ton of waste 200 litres of water is needed. Recycled water can also be used. The waste heaps are turned around once in 7 to 10 days for proper aeration and the inoculant slurry is sprayed in each turning to enhance decomposition and maintain the proper moisture level which is usually 45 - 55 %. The process is ‗exothermic‘ and the windrows reach a temperature of 70-75 C within 24-36 hours, killing the harmful pathogens and repelling all birds, stray animals, flies and mosquitoes from the dump site. (Ranjwani, 1996).

Recovery of Compost The entire process of aerobic decomposition of garbage is completed within 4-6 weeks and as the decomposition is complete the temperature comes down to normal. It recovers over 90 % of the organic matter in the form of compost which may be 25-30 % of the raw waste on dry weight basis. Recovery of compost depends upon the presence of organic matter in the garbage. There will be greater recovery of compost in developed countries as much higher amount of organic wastes reaches the dump-sites (tips) in every city. Retrieving the Dry Recyclables The decomposed waste biomass is passed through rotary and vibratory screens to sieve out the compost. The soft decomposed powdery materials gets easily separated from the plastics, metals, stones and pebbles. About 20-25 % are dry recyclable materials and the rest about 20-25 % are inert materials which are disposed in ordinary land-fills. Same Land for Dumpsite Can be Reused and No Need of Engineered Landfill The same dump-sites can be used again and again after excavating the biodegraded mass (compost) and there is no need of additional land for making more dump-sites. Also the need for ‗engineered land-fills‘ are greatly reduced because very little is left to be disposed off after retrieving the compost and the recyclable materials. Low Emission of Greenhouse Gas Methane The problem of emission of landfill gas methane (CH4) is significantly minimized as the waste is turned constantly and the waste biomass is thoroughly aerated throughout the composting period. Foul Odor at Waste Dumpsite Disappear Soon Giving Relief to Waste Workers Emission of ammonia and hydrogen sulfide which are mainly responsible for foul odor at the waste dump-site (tips), and the leachate discharge is also greatly reduced. The foul odor at the waste dump-site disappear within 2-3 days of sanitization by the microbial culture giving great relief to the waste workers and the local residents.

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13. CONCLUSION AND RECOMMENDATIONS An assessment of waste generation not only involves the production and distribution of commodities and services but also its actual history of use/reuse/recycling. How people act as consumers, re-users and recyclers is as important as how a thing is made and sold to consumers. Any government policy, any waste reducing and recycling technology and strategy cannot succeed unless every member of society is aware and behave responsibly. Sometimes policing becomes essential to change societal behavior. Random check of waste bins by councils and ‗refusal‘ to pick up waste not disposed according to council directives, can force people to behave. The strategy has worked well in some Canadian cities. Economic instruments also seem to affect human behavior. ‗Landfill taxes‘ in Denmark, Austria, Ireland, Italy and the UK, and charging people for plastic bags in supermarkets in Denmark and Ireland (and in France from late 2005 onward) have changed the human behavior in these nations. Manufacturers of consumer goods hold great responsibility in reducing waste in modern consumerist society. Governments of nations need to come out with a policy of ‗durable and recyclable goods‘ by manufacturers, and also ‗take back policy‘ of their products for recycling. It will also be a ‗disincentive‘ for them to change their product versions too frequently to lure people. Society has to play very critical role in all waste management programs. The traditional societies were basically ‗recycling societies‘. They made best use and reuse of all materials several times before discarding them. The modern society is basically a ‗throwaway society‘ which discard most materials after short use. Modern human societies have to revive some of the ‗traditional cultures‘ of resource conservation, resource recycling and their ethical use. Waste recycling is essential for economic stability, ecological sustainability, environmental safety and survival of the global human society. Human society has to begin the process of recycling at the source – the home, office, or factory- so that fewer materials will become part of the disposable solid wastes of a community. A ‗consumer education‘ program is needed to educate the society about the hidden ‗environmental value‘ (the energy and water it has saved, the pollution and deforestation it has prevented) of the recycled goods. All recycled goods should have ‗recycled tag‘ telling about the origin and life history of the goods (from which waste it was produced) and how it has saved the environment. It would develop a sense of ‗civic pride‘ among the consumers that he / she is helping the environment. There is always greater economic and ecological wisdom in reducing waste at source, and diverting more and more of the waste to the ‗mechanical recycling, ‗biological recycling‘ (composting) and ‗thermal recycling‘ (combustion) processes to recover some useful materials and energy from them and reduce their volume, so that less and less waste is left for final disposal in landfills. Government, industries, science and society all have to join hands in fighting the menace of mounting wastes. Industries producing consumer goods have to play more responsible role in waste management program because they have potential to generate waste twice in the lifecycle of the goods produced. First at the ‗production level‘ when they process the virgin raw materials and generate ‗industrial wastes‘. Second at the ‗consumption level‘ when the goods produced by them are used and discarded by the society as ‗municipal solid waste‘ (MSW). Policy makers have carrot and stick options to encourage or enforce industry to contribute to

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reduce waste. Industrialists also owe moral obligation to provide necessary information to its prospective buyers on the matter of using, handling, conservation, disposal and recycling potentialities of its products. In designing new products, the industry must assess its potential and even suspected adverse impact on its consumers health and the environment. We need more and more waste to be converted into resource (by recycling) to sustain our growing population as the several natural resources are either on decline or becoming scarce or are unavailable and beyond our capability to exploit them sustainabily with present technology and within the ecological limits. Government must encourage and promote the recycling industries using waste as raw materials by way of reduced taxation, reduced cost of water and electricity supply etc. Given current technology, not all the municipal or industrial wastes can be readily recycled. Nor do all the waste materials have qualities that currently make them a valuable commodity in the recycling marketplace. Hazardous waste is growing in U.S. industries and very little is being done to reduce or recycle them at source. Most hazardous wastes are being exported to poor developing countries either for dumping or for recycling. (Duke, 1994). Wrong policy decisions of the U.S. government has aggravated the problem. Study made by an environmental organization in the U.S. indicates that subsidies given to the timber, mining, oil, energy, and waste disposal industries undermine and discourage waste recycling industries. These subsidies lower the cost of products made from new and virgin materials, giving them a competitive advantage over those made from recyclable materials. Fifteen government subsidies given to these industries in the U.S. amounted to as much as US $ 13 billion over the next 5 years. It was a great setback for the waste recycling industries in the U.S. They include indirect subsidies such as cheap water and energy supply to these industries.(UNEP, 2006). Life-cycle assessments is also important to determine the recycling potential of a waste product. A number of life-cycle assessments have found that fully recycled paper is not always the most environmentally friendly choice. In some countries paper produced from local agricultural wastes, such as rice straw, may be environmentally more sustainable than that produced from recyclable paper wastes shipped from overseas or transported from distant locations in the same country. One study in Australia found that if the recyclable paper waste is transported more than about 20 km by road, the energy balance (fuel used and pollution generated during transport) is not in favor of recycling. Safe disposal of the radioactive wastes which are accumulating exponentially in the human environment cannot be guaranteed at all even after huge expenditure. A huge pile of radioactive wastes (most in the U.S., France and Japan) remains to be disposed safely. It was just 84,000 tones in 1990 and must have crossed 100,000 tones limit by now. According to the celebrated geologist Konrad – ‗No scientist or engineer can give an absolute guarantee that radioactive waste will not some day leak in dangerous quantities from even the best of repositories‘. The word ‗safe‘ seems to be incompatible with radioactive wastes. However, ‗waste prevention‘ through ‗cleaner production‘ is considered to be even more wiser and sustainable idea of waste management so that any treatment or recycling is not at all needed and tremendous cost can be saved. Waste prevention would also reduce the cost of construction and maintenance of secured landfills because some residual wastes are always left after treatment or recycling which has to be safely dumped in the landfills. Educating the ‗environmentally illiterate‘ society is a big challenge. People need to understand the importance of their activities in the context of global consequences, for instance, the ‗links between waste generation, greenhouse gas emission and climate change‘.

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Waste education needs to take place in a continuous way through schools and universities, through mass and diffuse media and community education programs run by governments at all levels as well as non-government organizations, businesses and industries. Waste education of society should become an integral part of all waste management programs. We have to mend our ways, change our behavior and attitude of life, re-order our priorities, simplify our life-style, and then only the gigantic problem of mounting solid waste, which literally threatens to bury the mankind alive, can be overcome. Only a ‗resource conserving, waste reducing and waste recycling‘ society would be the ‗sustainable human societies‘ of the future and the ‗throwaway wasteful societies‘ would perish.

REFERENCES AND ADDITIONAL READINGS Anonymous (1990): Characterization of the Municipal Solid Wastes in the U.S. : 1990 Update; US EPA, Office of Solid Waste and Emergency Response, Washington, D.C. Anonymous (1999): USA Today, 22nd June, 1999. AEN (2002): Metals Recycling Conserves Energy; Australian Energy News, Issue 24, June 2002; pp. 12-13. Adam Teva V‘Din (2005): Models for E-Waste Management- The European Union Leads the Way; The Israel Union for Environmental Defense; www.softwarelabs.com Viewed February 2005. BCC( 2002): Newsletter ‗Your City Your Say‘; Published by Brisbane City Council, Queensland, Australia; April 2002. Bellamy, Carol (1995): Women and the Environment; Our Planet; Vol. 7 No. 4; Publication of United Nation Environment Program (UNEP), Nairobi, Kenya. Blatt, Harvey (2005): America‘s Environmental Report Card; Are We Making the Grade? MIT Press. Cambridge (Mass.), London. Brahmbhatt, Ashish and Saeed Parshin (2005): Environmental Impact of Disposable Baby Diapers on Landfills; Research Project on Solid Waste Management (7095 EVE); Griffith University, Brisbane, October 2005.(Supervisor Dr. Rajiv Sinha). CSIRO (1996): State of the Environment in Australia; Council of Scientific and Industrial Research Organization, Canberra, ACT. Connor, M.A., Evans, D.G. and Hurse T.J. (1995): Waste Flows in the Australian Economy; Waste Disposal and Water Management in Australia; June 1995, pp. 9-28. Cointreau, S. J. (1982): Environmental Management of Urban Solid Waste in Developing Countries: A Project Guide; Urban Development Technology Papers, No.5, World Bank, Washington D.C. Cave, Shane and Jacquie Ashton (1993): Clean Up the World; Our Planet; Vol. 5, No. 6; UNEP Publication. Cui, J. and Forssberg, E. (2003): Mechanical Recycling of Waste Electric and Electronic Equipment : A Review; Journal of Hazardous Materials; Lulea, Sweden. Chandra, R., Majumdar., A. Roy, S., Joshi, P., Kumar, V., and Marda, V. (2005), E-Waste : The Indian Context; Indian Institute of Management; Kozhikode. Viewed Online April 2005http://www.indiainfoline.com/bisc/ari/ewas.pdf

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Chauhan, Krunal & Valani, Dalsukh (2008): Studies into Aerobic, Anaerobic and Vermicomposting Systems (Part of 40 CP Honours Project – ‗Studies in Vermiculture Biotechnology‘), Griffith University, Brisbane, Australia.(Supervisors: Rajiv K. Sinha & Sunil Herat). Courier Mail (2002): Newspaper Daily; Brisbane, Queensland, 9th May, 2002. Datar, M.T., Rao, M.N. and Reddy, S. (1997): Vermicomposting : A Technological Option for Solid Waste Management; J. of Solid Waste Technology and Management, Vol. 24 (2); pp. 89-93. Dorling, Kindersley (1987): Blueprint for Green Planet; London. Duke, L. Donald (1994): Hazardous Waste Minimization: Is it Taking Roots in U.S. Industry?; Waste Management; Vol.14, No.1; Pergamon Press; pp. 49-59. Edward, C.A. (1988): Breakdown of Animal, Vegetable and Industrial Organic Wastes by Earthworms; In C.A. Edward, E.F. Neuhauser (ed). ‗Earthworms in Waste and Environmental Management‘; pp. 21-32; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7. Eklington, John and Julia Hailes (1989): The Green Consumer Guide – From Shampoo to Champagne: High Street Shopping for Better Environment; Victor Gollancz Ltd. London. Epstein, E (1997): The Science of Composting;Flintoff, F. (1976): Management of Solid Wastes in Developing Countries; WHO Report, pp. 245. Eawag (2008): Global Waste Challenge : Situation in Developing Countries; Swiss Federal Institute of Aquatic Science & Technology (www.sandec.ch) Fairlie, Simon (1992): Long Distance Short Life, Why Big Business Favors Recycling; The Ecologist; Vol. 22: No. 6; pp. 276-282. Fraser-Quick, G. (2002): Vermiculture – A Sustainable Total Waste Management Solution; What‘s New in Waste Management ? Vol. 4, No.6; pp. 13-16. Frederickson, Jim (2000): The Worm‘s Turn; Waste Management Magazine; August, UK. Gottas, H.B. (1956): Composting ; World Health Organization Monograph, Geneva; p. 25. Global Footprint Network (2005): National Footprint and Biocapacity Accounts. Available from and accessed 15 March 2006 — http://www.footprintnetwork.org. Goldoftas, Barbara (1989): Making Waste Work; SPAN Magazine, United States Information Service (USIS) Publication, New Delhi, India; July 1989. GOI (1989) : The Hazardous Wastes ( Management and Handling Rules) 1989; Department of Environment and Forest, Government of India, New Delhi. GOI (1997): Biomedical Wastes (Management and Handling Rules) 1997; Department of Environment and Forest, Government of India, New Delhi. Goldstein, J. (1995): Recycling Food Scraps into High End Markets; Residuals Biocycle; Vol. 36 (8); pp. 40. Hamilton, John (2000): Construction Waste Recycling at SENT Landfill; Green Valley Landfill Ltd. Proceedings of the Conference on ‗Recycle 2000‘, Hong Kong. Haug, R.T. (1993): The Practical Handbook of Compost Engineering; Lewis Publishers, Boca Raton. Holmes, John, R. (1984): Managing Solid Waste in Developing Countries; John Wiley and Sons; New York. Harding, Ronnie (ed) (1998) Environmental Decision-Making : The Roles of Scientists, Engineers and the Public. Federation Press, Annadale/Leichhardt.

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Heath, C.W., Jr., (1983): Field Epidemiologic Studiesof Population Exposed to Waste Dumps; Environmental Health Perspectives; Vol. 48 : pp. 3-7 Hasselriss. Floyd (1995) : Medical Waste Incineration ; Technical Monitor; US.Menon, Subhadra (1977): Drowning in Trash; India Today; Bi-Monthly Magazine Published in India; May 15, 1977; pp. 78-83. IAEA (1991): Nuclear Power, Nuclear Fuel Cycle and Waste Management: Status and Trends; Report of International Atomic Energy Agency, Vienna, Austria; Part C; pp. 73. Jones, P.H. and Herat, Sunil (1994) : Use of Cement Kilns in Managing Solid and Hazardous Wastes : Implementation in Australia; Journal of the Institution of Water and Environmental Management; Vol. 8; No. 2, April 1994, UK. Komarowski, S. (2001): Vermiculture for Sewage and Water Treatment Sludge; WATER, July 2001. Mathew, J. Realff, Michele Raymonds, and Jane C. Ammons (2004): E-waste : An Opportunity; Materials Today; Georgia Institute of Technology, Atlanta, U.S.; January 2004, pp. 40-45. Miller, C. (2004): E-Waste: Time to Address It? , MAXUS Technology, Inc., Canada. (Viewed online April 2005) http://www.municipalsuppliers.com/MagazineIndex/ 2003/cpwe2003_page18.asp Monchamp, Amanda (2000), The Evolution of Materials Used in Personal Computers; 2nd OECD Workshop on ‗Environmentally Sound Management of Wastes Destined for Recovery Operations‘; Vienna, Austria, September 28-29, 2000. Naidu, Ravi (2004): Report on Hazardous Waste Dump Sites in Australia by Center for Environmental Risk Assessment and Remediation, University of South Australia. In ‗The Australian‘, March 2004. Noll, K.E., C.N. Haas, C. Schmidt and P. Kodukula (ed) (1985): Recovery, Recycle and Reuse of Industrial Waste; Industrial Waste Management Series, Lewis Publishers Inc., Chelsea, Michigan; 196 pp. O‘Rourke, Morgan (2004), Killer Computer: The Growing Problem of E-Waste; Journal of Risk Management; New York; Vol. 51; Issue 10; pp.12-17. Puckett, J., Byster L., Westervelt, S. Gutierrez, R., Davis, S., Hussian, A., and Dutta, M. (2002), Exporting Harm : The High-Tech Trashing of Asia; Basel Action Network and Silicon Valley Toxics Coalition; (Viewed Online April 2005). http://www.ban.org/Ewaste/technotrashfinalcomp.pdf Parker, C, and Roberts, T. (1985): Energy from Waste; Elsevier Applied Science, NY. Porter, R., and J. Roberts (1985): Methods of Recovering Material and Energy from Refuse; In Porter and Roberts (ed.) ‗Energy Savings by Waste Recycling‘; Elsevier Applied Science, NY. Ranjwani, P.U. (1996): City Waste Treatment and Its Bioconversion into Organic Fertilizer; Paper by Ms. Excel Industries Ltd., Mumbai for all Municipal Corporations of India. Rao, Jayshree (1998) Management of Municipal Urban Wastes : Some Innovative Strategies; PhD Thesis, University of Rajasthan, Jaipur, India. (Supervised by Rajiv K. Sinha). Raghupaty, L. (1994): Hazardous Wastes: Management and Minimization; Paper Presented at International Seminar on ‗Solid Waste Management and Training‘; Bangalore, September 19-24, 1994. (Sponsored by Ministry of Environment, New Delhi, India). Schneiderman, Ron (2004), Electronic Waste: Be Part of the Solution; Journal of Electronic Design; Cleveland; Vol. 52; Issue 8, pp. 47-51.

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Sinha, Rajiv K. (1991): Ecological Management of Urban Solid Wastes for Human Survival; Journal of the Institute of Public Administration; University of Lucknow (India), Vol. 6 Nos. 1-4. Sinha, Rajiv K and Ratna Rawat (1991): Waste recycling and reutilisation, essential for environmental safety and sustainable development : Case study of paper and cotton rags recycling industries in Jaipur, India : Journal of Ecobiology : Vol. 11: pp 193-198. Sinha, Rajiv K. (1994): Ecological Economics of Waste Management: Value Addition on Waste, Waste Imports and Waste Exchange for Recycling; Paper Presented at International Seminar on ‗Solid Waste Management and Training‘; Center for Environment Education (South India), Bangalore, September, 19-24, 1994. (Sponsored by IUCN and WWF – Indian Chapter as Resource Person) Sinha, Rajiv. K (1996): Waste Bomb : The Threat to Bury the Humanity Alive; In Self (ed.): Environmental Crisis and Humans at Risk; INA Shree Publication, India; pp. 118-126. Sinha, Rajiv K. and A.K. Sinha (2000): Waste Management: Embarking on the 3R‘s Philosophy of Waste Reduction, Reuse and Recycling; Inashree Publication, India; ISBN 81-86653-32-5. Sinha, Rajiv. K., Sunil Herat, Sunita Agarwal, Ravi Asadi, and Emilio Carretero (2002 a): Vermiculture Technology for Environmental Management : Study of the action of the earthworms Elsinia foetida, Eudrilus euginae and Perionyx excavatus on biodegradation of some community wastes in India and Australia; The Environmentalist, U.K., Vol. 22, No.2. June, 2002; pp. 261 – 268. Sinha, Rajiv K. and Sunil Herat (2002 b): A Cost-effective Microbial Slurry Technology for Rapid Composting of Municipal Solid Wastes on the Waste Dump Sites in India and the Feasibility of its Use in Australia: The Environmentalist, U.K. Vol..22; No.1; pp. 9-12. Sinha, Rajiv K. and Sunil Herat (2004): Industrial and Hazardous Wastes : Health Impacts and Management Plans; Pointer Publishers, Jaipur (India); ISBN 81-7132-365-0; pages 365. Sinha, Rajiv K., Sunil Herat, P.D. Bapat, Chandni Desai, Atul Panchi and Swapnil Patil, (2005 a): Household Hazardous Waste : The Hidden Danger in Every Home : Regulating Their Management; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, CANADA; pp. 45-54. Sinha, Rajiv K., Sunil Herat, P.D. Bapat, Chandni Desai, Atul Panchi and Swapnil Patil, (2005 b): Domestic Waste - The Problem That Piles Up for the Society : Vermiculture the Solution; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, CANADA; pp. 55-62. Sinha, Rajiv K., Jayraman, V.B., and Michael Noronha (2005 c): Waste and Consumer Education for Society about the 3 R‘s and 5 P‘s Holds the Key to Sustainable Waste Management at Source; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, Canada: 288-293. Sinha, Rajiv K., Sunil Herat, P.D. Bapat and Chandini Desai (2006): Electronics Wastes : Landfill Disposal, Reuse and Recycling; Indian Journal of Environmental Protection; Vol. 26(7): pp. 577-584; ISSN 0253-7141; Regd. No. R.N. 40280/83; Indian Institute of Technology, BHU, Varanasi, India. Sinha, Rajiv K. and Rohit Sinha (2007): Environmental Biotechnology; Pointer Publisher, India. (Under Publication) Tchobanoglous, George; Theisen, Hilary; and Vigil, Samuel (1993):

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Useful Websites Clean Up Australia : Accessed 15 March 2006. EcoRecycle : : Accessed 15 March 2006

Human Waste - A Potential Resource: Converting Trash into Treasure … Womens Environment Network: Accessed 15 March 2006. UNEP Magazine ‗Our Planet‘ : < http://www.ourplanet.com> Global Waste Challenge; www.sandec.ch

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In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 4

EFFECTIVE REMOVAL OF LOW CONCENTRATIONS OF ARSENIC AND LEAD AND THE MONITORING OF MOLECULAR REMOVAL MECHANISM AT SURFACE Yasuo Izumi Department of Chemistry, Graduate School of Science, Chiba University Yayoi 1-33, Inage-ku, Chiba 263-8522, Japan

ABSTRACT New sorbents were investigated for the effective removal of low concentrations of arsenic and lead to adjust to modern worldwide environmental regulation of drinking water (10 ppb). Mesoporous Fe oxyhydroxide synthesized using dodecylsulfate was most effective for initial 200 ppb of As removal, especially for more hazardous arsenite for human's health. Hydrotalcite-like layered double hydroxide consisted of Fe and Mg was most effective for initial 55 ppb of Pb removal. The molecular removal mechanism is critical for environmental problem and protection because valence state change upon removal of e.g. As on sorbent surface from environmental water may detoxify arsenite to less harmful arsenate. It is also because the evaluation of desorption rates is important to judge the efficiency of reuse of sorbents. To monitor the low concentrations of arsenic and lead on sorbent surface, selective X-ray absorption fine structure (XAFS) spectroscopy was applied for arsenic and lead species adsorbed, free from the interference of high concentrations of Fe sites contained in the sorbents and to selectively detect toxic AsIII among the mixture of AsIII and AsV species in sample. Oxidative adsorption mechanism was demonstrated on Fe-montmorillonite and mesoporous Fe oxyhydroxide starting from AsIII species in aqueous solution to AsV by making complex with unsaturated FeOx(OH)y sites at sorbent surface. Coagulation mechanism was demonstrated on double hydroxide consisted of Fe and Mg from the initial 1 ppm of Pb2+ aqueous solution whereas the mechanism was simple ion exchange reaction when the initial Pb2+ concentrations were as low as 100 ppb.

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[email protected]

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Yasuo Izumi

INTRODUCTION Recently, global environment induces even serious debate, e.g. the Novel Prize 2007 for Piece to "An Inconvenient Truth" by Al Gore [1]. The environmental problem is not only the global warming as the major claim in this movie/book by Gore. Contamination of water and soil is one of traditional, major environmental problems and directly affects the human health, e.g. carcinogenic risk via drinking water [2 – 4]. Cadmium contaminated in rice [5], copper contaminated in environmental water from mine, mercury contaminated in fish [6 – 8], and arsenic contaminated in powdered milk are most notorious environmental tragedies occurred in Japan between 1890 and 1960. These accidents are all related to contamination of water derived from human activity (industry) [2]. The health risk of poisonous elements in water has been studied and is becoming clear. Recent environmental regulation sets the minimum level of Mn, Cu, Cd, Pb, Cr, As, and Hg to 400, 125, 5, 10, 50, 10, and 0.5 ppb, respectively. Among these elements, lead is less focused and less intensively studied to adjust to the regulation. Arsenic can be contaminated in ground water not only anthropogenically but from the earth naturally [2 – 4]. Unfortunately, mines containing As mainly distribute in contact with the ground water in developing countries, e.g. Bangladesh, East Bengal, Argentina, Chile, and Vietnam [9]. Especially in Bangladesh, the shallow ground water with arsenic concentrations up to 1 ppm is often used pumped from tube wells without adequate treatment for drinking and thus leading to serious health problem. The arsenic release into ground water is related to microbial metabolism of organic matter [10]. The high capital and maintenance costs of piped water supply are still not acceptable in Bangladesh, especially most affluent villages compared to present individual tube wells [10]. Lead was used as gasoline additive and automobile tail pipes and may be included in drinking water from water supply pipes made of lead, soil contamination, and toys/tools made in developing countries [11, 12]. This chapter reviews recent development of sorbents for low concentrations of Pb and As to adjust to modern environmental regulation for drinking water and monitoring of the molecular removal process by selective spectroscopy in water [13]. The monitoring of surface uptake of Pb [14] and As includes local coordination structure and electronic structure changes in the inner sphere reaction.

METHODS This chapter focuses on the removal of Pb and As by sorption because sorption is economic process to be applicable in developing countries and the reuse is possible by desorption. The concentrations of Pb and As in test aqueous solutions were set between 55 ppb and 32 ppm in this chapter. Various sorbents were evaluated to maintain the concentrations of Pb or As less than 10 ppb or not. Fe-montmorillonite was prepared by mixing 0.43 M ferric nitrate solution with Namontmorillonite (Kunipia F; Na1.5Ca0.096Al5.1Mg1.0Fe0.33Si12O27.6(OH)6.4) [15]. A 0.75 M sodium hydroxide solution was added dropwise to the mixture until the molar ratio Fe3+ added and hydroxide reached 1:2. Iron cations and/or FeOx(OH)y nanoparticles were inserted between negatively-charged montmorillonite clay layers. Recently, some chemical forms of

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FeIII species formed between montomorillonite layers were spectroscopically analyzed [16]. Monomeric and/or dimeric FeIII species was active in oxidative dehydrogenation of propane. In contrast, polymeric FeOx(OH)y nanoparticles were effective for arsenic sorption and unselective propane combustion. FeOx(OH)y porous material was prepared by mixing 0.10 M ferrous chloride with 0.070 M sodium dodecylsulfate followed by the addition of 0.25 M H2O2 [17]. Obtained FeOx(OH)y material was mixed with 0.050 M sodium acetate in ethanol for anion exchange or with pure ethanol for washing. The micro/mesoporous FeOx(OH)y material was characterized by X ray diffraction (XRD), specific surface area measurements and pore volume determination by N2 adsorption/desorption, high-resolution transmission electron microscope (TEM), Fouriertransformed infrared absorption (FT-IR), inductively coupled plasma (ICP) combined with optical emission spectroscopy (OES), electron probe microanalysis (EPMA), thermogravimetric differential thermal analysis (TG-DTA), and Fe K-edge X-ray absorption fine structure (XAFS). Based on these analyses, detailed structural transformation was clarified for the sorbents as depicted in Figure 8 of Ref [17]. Hydrotalcite-like (pyroaurite) layered double hydroxide Mg6Fe2(OH)16(CO3)•3H2O was synthesized via the procedure described in Ref 18. The carbonate anions are sandwiched between positively-charged [Mg3Fe(OH)8]+ layers. To monitor low concentrations of Pb and As, XAFS spectroscopy is most appropriate technique. For several spectroscopic techniques of structural analysis in the application to nanotechnology, the advantage and drawback were summarized in Table 1 [19]. For noncrystalline or hybrid samples, EXAFS (extended X-ray absorption fine structure) gives direct structural information for X-ray absorbing local element sites. XANES (X-ray absorption near-edge structure) is a part of EXAFS spectrum near the X-ray absorption edge region ranging up to 100 eV and gives electronic and (indirectly) structural information [20]. Thus, XAFS spectroscopy (EXAFS, XANES) is essentially single technique for local structure analysis accompanied with valence and coordination symmetry information of nanoparticles and micro/mesoporous materials. The Pb and As adsorbed from low concentrations of aqueous solutions in this chapter are typical examples of nanoparticles and micro/mesoporous material samples. Further, this chapter combines X-ray fluorescence (XRF) spectrometry with the XAFS spectroscopy [21 – 24]. Simply, XRF spectra support valence state information deduced from XAFS. Essentially, high-energy-resolution XRF spectrometry is able to discriminate valence state of Pb and As. In this chapter, the XRF signals originating from PbII, AsIII, and AsV were monitored independently in the XAFS measurements to obtain each coordination structure of PbII, AsIII, and AsV (state-selective XAFS). The experimental setup and measurement conditions for state-selective XAFS were depicted and described in Refs 21, 23, and 24. In brief, XRF spectra and state-selective XAFS spectra measurements were performed at Undulator beamline 10XU of SPring-8 (Sayo, Japan) by utilizing a homemade high-energyresolution Rowland-type fluorescence spectrometer equipped with a Johansson-type Ge(555) crystal (Saint-Gobain) and NaI(Tl) scintillation counter (Oken). The monochromator of beamline used Si(111) double crystal and the X-ray beam intensity in front of sample was monitored using ion chamber (Oken) purged with N2 gas.

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Table 1. Various Analytical Methods for Nano Structure Classified Based on Directness of the Information and the Target to Be Analyzed Method

Directness

Target

TEM (Transmission electron microscope)

Direct

Local

XRD (X-ray diffraction)

Direct

Local

EXAFS (Extended X-ray absorption fine structure)

Direct

Bulk

XANES (X-ray absorption near-edge structure)

Indirect

Bulk

Raman

Indirect

Bulk

Small angle scattering

Indirect

Bulk

NMR (Nuclear magnetic resonance)

Indirect

Local

Mössbauer

Indirect

Local

SPM (Scanning probe microscope)

Indirect

Local (surface)

Reflectivity

Indirect

Local (surface)

RESULTS AND DISCUSSION Arsenic Problem. Adsorption isotherms of arsenite and arsenate at 290 K for 12 h on Femontmorillonite in batch setup are depicted in Figure 1. The Fe-montmorillonite was superior to -FeO(OH) (göthite > 95%) both for arsenite and arsenate sorptions. Fe-montmorillonite consisted of 2-dimensionally distributed Fe3+ ions and FeOx(OH)y nanoparticles between clay layers [16]. The saturated amount of As adsorbed was evaluated to 8.0 and 76 mgAs gsorbent–1 for arsenite and arsenate, respectively, on Fe-montmorillonite. The equilibrium adsorption constant was 1.4×106 ml gAs–1 for arsenite on Fe-montmorillonite. Even better adsorption capacity was found on acetate-exchanged microporous FeOx(OH)y as depicted in Figure 2 for arsenite. The saturated amount and the equilibrium adsorption constant of As adsorbed were evaluated to 21 mgAs gsorbent–1 and 1.0×107 ml gAs–1, respectively. The high specific surface area of microporous FeOx(OH)y (230 m2 g–1) was advantageous compared to Femontmorillonite (100 m2 g–1) with as much as 14 wt% of Fe. Utilizing template synthesis technique for microporous and mesoporous materials, lower coordination FeOx sites were effectively exposed to surface to complex with AsIII(OH)3 [17, 26]. The acetate-exchanged and ethanol-washed FeOx(OH)y consist of 3-dimensionally distributed wormholes exposed with coordinatively unsaturated FeO(OH) sites.

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Figure 1. Adsorption isotherms of arsenite (A) and arsenate (B) at 290 K on Fe-montmorillonite (14.0 wt% Fe) (circles) and -FeO(OH) (triangles). Batch tests for 12h. Observed data were plotted as points and the fits to first-order Langmuir equations were drawn as lines.

The surface uptake mechanism of most toxic arsenite was monitored by XRF and XAFS spectroscopy. Arsenic was adsorbed on Fe-montmorillonite from 200 ppb test aqueous solution of arsenite. The As K1 emission spectrum was depicted in Figure 3. The peak energy position suggested that the adsorbed As state changed to V, not remained at III, compared to the data for KH2AsVO4 and AsIII2O3 [15]. As K-edge XANES spectra for standard inorganic compounds of As0, AsIII, and AsV consist of broad peak feature (Figure 4a – c) and it is complicating to evaluate each valence contribution to a spectrum for sample of mixed valence. In order to demonstrate directly the oxidative adsorption of arsenite suggested above, the author of this chapter observed the uptake of low concentrations of arsenite on Fe-montmorillonite by means of state-selective XAFS. Note that the energy resolution of fluorescence spectrometer (1.3 eV; Figure 3) was smaller than the core-hole lifetime width of As K level (2.14 eV) [27].

Figure 2. Adsorption isotherms of arsenite at 290 K on acetate-exchanged FeOx(OH)y previously heated at 423 K (circles), ethanol-washed FeOx(OH)y previously heated at 423 K (squares), Fe-montmorillonite (14.0 wt% Fe; diamonds), and -FeO(OH) (triangles) [25]. Batch tests for 12h. Observed data were plotted as points and the fits to first-order Langmuir equations were drawn as lines.

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Figure 3. Arsenic K1 emission spectrum for As adsorbed on Fe-montmorillonite (14.0 wt% Fe) from 200 ppb test solution of arsenite (points). A fit to data with pseudo-Voigt function (solid line) and the energy resolution of fluorescence spectrometer (dotted line) were also drawn. The intensity ratio of the Lorentzian and Gaussian components was fixed to 1:1 for the pseudo-Voigt function.

Figure 4. XANES spectra measured at 290 K in transmission mode for As metal (a), AsIII2O3 (b), and KH2AsVO4 (c). Arsenic K1-selecting As K-edge XANES spectra measured at 290 K for As adsorbed on Fe-montmorillonite (14.0 wt% Fe) (d – f) from aqueous test solutions of 16 ppm of KH2AsVO4 (d), 16 ppm of AsIII2O3 (e), and 200 ppb of AsIII2O3 (f). Tune energy of fluorescence spectrometer was 10544.3 eV for spectra d – f.

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Based on the theory discussed in the Appendix section of Ref 24, the As K1-selecting As K-edge XANES spectrum with the energy resolution of 1.3 eV would be shaper and more resolved. The energy values of K absorption edge and first strong peak after the edge were essentially identical for As adsorbed on Fe-montmorillonite from 200 ppb – 16 ppm of AsIII solutions (Figure 4e, f) and from 16 ppm of AsV solution (d). Thus, oxidative adsorption of 200 ppb – 16 ppm of arsenite on Fe-montmorillonite was confirmed based on As K1selecting XANES and As K1 emission spectrum. Proposed molecular surface uptake mechanism was illustrated in Figure 5 over acetate-exchanged microporous FeOx(OH)y. The reaction formula was AsIII(OH)3 + FeO(OH)  (FeO)2AsV(OH)2 + H2O. In summary, oxidative adsorption of low concentrations (200 ppb – 16 ppm) of arsenite was found on coordinatively unsaturated FeOx(OH)y nanoparticles or micro/mesoporous FeOx(OH)y partially covered with acetate anions. The oxidation to arsenate seems to be due to lower coordination of surface FeOx(OH)y species. The lower coordination was also the reason to make the equilibrium sorption constant greater for acetate-exchanged FeOx(OH)y and Femontmorillonite [15, 17]. Lead Problem. Sorption tests for low concentrations (55 ppb) of lead in flow setup were depicted in Figure 6 [18]. The superiority of Mg6Fe2(OH)16(CO3)•3H2O was clearly demonstrated to maintain the Pb2+ concentration less than modern environmental regulation (10 ppb) compared to commercially available activated carbon. Lead L1 emission spectrum for Pb adsorbed on Mg6Fe2(OH)16(CO3)•3H2O from 100 ppb Pb2+ test solution was depicted in Figure 7. The peak energy was identical to that for standard PbII compounds. The energy resolution of fluorescence spectrometer in this measurement condition was 5.0 – 10.1 eV dependent on the measurement conditions of fluorescence spectrometer (Figure 7) [24, 28]. Because the core-hole lifetime widths are 5.81 and 2.48 eV for Pb L3 and M5 levels, respectively [27], the width for L1 is 8.29 eV comparable to the energy resolution of fluorescence spectrometer. Thus, sharper, more resolved spectral feature was expected in Pb L1-selecting Pb L3-edge XANES spectrum if the energy resolution of fluorescence spectrometer was smaller than 5.81 eV, similar to the case of As K1-selecting XANES in previous section.

Figure 5. Proposed adsorption mechanism of low concentrations of arsenite on acetate-exchanged FeOx(OH)y previously heated at 423 K.

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Figure 6. Results of sorption on Mg6Fe2(OH)16(CO3)•3H2O and on activated carbon from 55 ppb of Pb2+ aqueous test solution. The space velocity was 150 min–1.

Figure 7. Lead L1 emission spectrum for 0.12 wt% of Pb adsorbed on Mg6Fe2(OH)16(CO3)•3H2O. The solid line is the experimental data, and (wider) dotted line is a fit with a pseudo-Voigt function. The intensity of the Lorentzian and Gaussian components was fixed to 1:1. The narrower dotted line is the energy resolution of fluorescence spectrometer (10.1 eV).

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Pb L1-selecting XANES spectra are shown in Figure 8a – c. The spectral pattern of b and c resembled each other. The two samples contained 0.30 – 0.12 wt% of lead adsorbed from 100 ppb test aqueous Pb2+ solution. The Pb contents in samples were determined by ICP. Compared to XANES spectra for standard inorganic Pb compounds (Figure 8d – i) and supported Pb species on zeolite [29] or on Fe2O3 [30], the spectra b and c resembled most spectrum d measured for ion-exchanged PbY zeolite (d). Thus, surface uptake mechanism via ion exchange reaction was proposed on Mg6Fe2(OH)16(CO3)•3H2O from relatively low concentration of 100 ppb divalent lead solutions (Figure 9). The reaction formula is [Mg3Fe(OH)8]+ + Pb2+  [Mg3Fe(OH)7(OPb)]2+ + H+. Pb L1-selecting XANES spectrum for Pb adsorbed from 1.0 ppm test Pb2+ solution is depicted in Figure 8a. Compared to XANES spectra for standard inorganic Pb compounds (Figure 8d – i), the spectrum a resembled most spectrum g measured for 2PbCO3•Pb(OH)2. Thus, coagulation uptake mechanism was proposed on Mg6Fe2(OH)16(CO3)•3H2O from relatively high concentration of 1 ppm Pb2+ test solution. Thus-identified reaction formula was Pb2+ + nCO32– + 2(1 – n)OH– ⇄ nPbCO3•(1 – n)Pb(OH)2.

Figure 8. Lead L1-selecting Pb L3-edge XANES spectra measured at 290 K for Pb adsorbed on Mg6Fe2(OH)16(CO3)•3H2O (a – c). Tune energy of fluorescence spectrometer was 10551.5 eV. The Pb content was 1.0 wt% adsorbed from 1.0 ppm Pb2+ aqueous test solution (a) and 0.30 (b) and 0.12 wt% (c) from 100 ppb Pb2+ test solution. XANES spectra measured in transmission mode (d – i) for PbY zeolite (d), PbO (e), Pb(NO3)2 (f), 2PbCO3•Pb(OH)2 (g), Pb6O4(OH)4 (h), and PbCO3 (i).

With closer look of Figure 8a – c, a shoulder feature appeared at 13049 eV. Similar shoulder feature can be found in spectra for PbO, 2PbCO3•Pb(OH)2, and Pb6O4(OH)4 (spectra

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e, g, and h, respectively). However, intense peak at 13060.1 – 13060.5 eV for PbO or Pb6O4(OH)4 did not appear in spectra a – c. Thus, minor coagulation uptake mechanism was suggested from 100 ppb Pb2+ solutions in addition to major ion exchange process (Figure 9).

Figure 9. Pb2+ adsorption mechanism on Mg6Fe2(OH)16(CO3)•3H2O from Pb2+ 1.0 ppm and 100 ppb aqueous solutions.

In summary, Pb2+ uptake mechanism on Mg6Fe2(OH)16(CO3)•3H2O exhibited a switchover from coagulation to (major) ion exchange reactions as the Pb2+ concentration decreased from 1.0 ppm to more environmentally plausible 100 ppb [28].

CONCLUDING REMARKS AND FUTURE PROSPECTS This chapter focused on the removal of low concentrations (55 – 200 ppb) of arsenite and lead by utilizing Fe-montmorillonite, micro/mesoporous FeOx(OH)y effectively porous due to carboxyl-exchange method, and layered double hydroxide consisted of Fe and Mg (pyroaurite). It is still open to discuss the systematic survey of the removal process of other dilute toxic elements, e.g. Cr, Cu, Zn, Cd, or Hg. It is important to formulate the efficiency of surface uptake with respect to critical factors, i.e. pH, chemical species of toxic elements (naked cations, oxyanions, or oxyhydroxyl anions), initial concentrations (10 – 100 ppb) and space velocity of aqueous solutions to be processed, and chemical combination of surface versus chemical species (e.g. FeIIIO(OH) versus As(OH)3 and OH–(clay surface)/CO32– (between layers) versus Pb2+). In the analytical point of view to monitor the destiny of low concentrations of toxic elements, selective XAFS technique, with which the author of this chapter has also

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investigated surface catalytic sites of gold, platinum, tin, and vanadium [31 – 35], needs to be combined with other technique with nanoscale spacial resolution. Spectroscopy with nanoscale spacial resolution is under investigation, but not established to be applicable to nanotechnology (Table 2). At present, spacial resolution of X-ray microscopy is 1 m [36]. Several types of X-ray microscopy/imaging are under investigation, e.g. microbeam XAFS, photoemission electron microscope (PEEM), or phase contrast imaging [37, 38]. The spacial resolution of TEM is already smaller than 1 nm if the sample nanoscopic condition matches to the high-resolution measurement. To monitor the sorption between 2-dimensinal layers (e.g. montmorillonite, hematite), in 2-dimensional mesopores (e.g. FSM-16, MCM-41), and in 3-dimensional micro/mesopores (e.g. ZSM-5, acetate-exchanged FeOx(OH)y [17]), 3-dimensional TEM images would be very helpful by taking series of TEM snapshots from various angles to sample and organizing 3-dimensional image on computer [39]. Scanning probe microscope (SPM), especially scanning tunneling microscopy (STM) and atomic force microscope (AFM), has an advantage of atomic resolution for well-defined surface [40]. To utilize SPM technique to monitor the sorption from low concentrations of toxic elements, the combination with element specific spectroscopy, e.g. XPS, XAFS, is essential to describe the surface chemical mechanism. The author of this chapter is developing this combination (AFM and XPS) based on temporal electron trap phenomena in the metal nano-dots in the front of the AFM tip [41, 42]. Table 2. Spectroscopy Needed to Be Developed to Give Direct Spacial Information of Surface Uptake Mechanism from Low Concentrations (10 – 100 ppb) of Toxic Metal Elements Probe

Method

Factor to be developed for this application

Refs

X-ray Microscope

Smaller X-ray beam (< 10 nm)

[36 – 38]

Electron Microscope

3-dimensional information

[39]

Tip Microscope

Scanning probe microscope to discriminate

[41, 42]

the kind of element X-ray Diffraction

Surface-sensitivity between nano-layers or

[43]

in micro/mesospace X-ray XAFS/XRF

On-site analysis in the field

[44, 45]

Surface-sensitive XRD may be applicable to monitor the application of water purification [43]. The breakthrough is selectivity to internal surface of layered and micro/mesoporous materials used as sorbents. Portable XRF/XAFS apparatus that has been investigated for space science [44, 45] will be applicable to environmental on-site monitoring of the fate of toxic elements in the field.

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ACKNOWLEDGMENTS This article is part 26 in the series of state-sensitive XAFS. The X-ray experiments were performed under the approvals of the SPring-8 Program Review Committee and of the Photon Factory Proposal Review Committee. The works included in this chapter were financially supported by grants from the Grant-in-aid for Encouragement of Young Scientists (B14740401, A12740376) and the Grant-in-aid for Basic Scientific Research (B13555230, C17550073) from the Ministry of Education, Culture, Sports, Science, and Technology, Yamada Science Foundation (2000), Toray Science Foundation (98-3901), and Research Foundation for Opto-Science and Technology (2005 – 2006).

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[23] Izumi, Y.; Kiyotaki, F.; Nagamori, H.; Minato, T. J. Electro. Spectrsc. Relat. Phenom. 2001, 119(2/3), 193 – 199. [24] Izumi, Y.; Nagamori, H.; Kiyotaki, F.; Masih, D.; Minato, T.; Roisin, E.; Candy, J. P.; Tanida, H.; Uruga, T. Anal. Chem. 2005, 77(21), 6969 – 6975. [25] Dixit, S.; Hering, J. G.; Environ. Sci. Technol. 2003, 37, 4182 – 4189. [26] Izumi, Y.; Masih, D.; Aika, K.; Seida, Y. Micropor. Mesopor. Mater. 2007, 99, 355. [27] Zschornack, G. Handbook of X-Ray Data; Springer: Berlin, 2007. [28] Izumi, Y.; Kiyotaki, F.; Minato, T.; Seida, Y. Anal. Chem. 2002, 74(15), 3819 – 3823. [29] Huang, F. T.; Jao, H. J.; Hung, W. H.; Chen, K.; Wang, C. M. J. Phys. Chem. B 2004, 108(52), 20458 – 20464. [30] Pinakidou, F.; Katsikini, M.; Paloura, E. C.; Kalogirou, O.; Erko, A. J. Non-Cryst. Solids 2007, 353(28), 2717 – 2733. [31] Izumi, Y.; Obaid, D. M.; Konishi, K.; Masih, D.; Takagaki, M.; Terada, Y.; Tanida, H.; Uruga, T. Inorg. Chim. Acta 2008, 361(4), 1149 – 1156. [32] Izumi, Y.; Masih, D.; Roisin, E.; Candy, J. P.; Tanida, H.; Uruga, T. Mater. Lett. 2007, 61(18), 3833 – 3836. [33] Izumi, Y.; Masih, D.; Candy, J. P.; Yoshitake, H.; Terada, Y.; Tanida, H.; Uruga, T. "XRay Absorption Fine Structure 13th International Conference", Hedman, B.; Pianetta, P. Eds., AIP Conference Proceedings 2007, Vol. 882, 588 – 590. [34] Izumi, Y.; Konishi, K.; Obaid, D. M.; Miyajima, T.; Yoshitake, H. Anal. Chem. 2007, 79(18), 6933 – 6940. [35] Izumi, Y.; Kiyotaki, F.; Yagi, N.; Vlaicu, A. M.; Nisawa, A.; Fukushima, S.; Yoshitake, H.; Iwasawa, Y. J. Phys. Chem. B 2005, 109(31), 14884 – 14891. [36] Hokura, A.; Kitajima, N.; Terada, Y.; Nakai, I. SPring-8 Research Frontiers 2006, 120 – 121. [37] Tuohimaa, T.; Otendal, M.; Hertz, H. M. Appl. Phys. Lett. 2007, 91(7), 074104. [38] Koshikawa, T.; Guo, F. Z.; Yasue, T. SPring-8 Research Frontiers 2005, 52 – 53. [39] Midgley, P. A.; Thomas, J. M.; Laffont, L.; Weyland, M.; Raja, R.; Johnson, B. F. G.; Khimyak, T. J. Phys. Chem. B 2004, 108(15), 4590 – 4592. [40] Marti, O.; Möller, R. Eds. Photons and Local Probes; Kluwer Academic Publishers: Dordrecht, 1995. [41] Klein, L. J.; Williams, C. C. Appl. Phys. Lett. 2001, 79(12), 1828 – 1830. [42] Klein, L. J.; Williams, C. C.; Kim, J. Appl. Phys. Lett. 2000, 77(22), 3615 – 3617. [43] Takahasi, M. SPring-8 Research Frontiers 2006, 56 – 57. [44] Robinson, A. L. Science 1980, 208, 163 – 164. [45] Frierman, J. D.; Bowman, H. R.; Perlman, I.; York, C. M. Science 1969, 164, 588.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 5

ON THE REDISTRIBUTION OF TISSUE METAL (CADMIUM, NICKEL AND LEAD) LOADS IN MINK ACCOMPANYING PARASITIC INFECTION BY THE GIANT KIDNEY WORM (DIOCTOPHYME RENALE) Glenn H. Parker and Liane Capodagli Department of Biology, Laurentian University, Sudbury, Ontario, Canada, P3E 2C6

ABSTRACT Patterns of metal uptake and accumulation in mink living under conditions of environmental pollution and simultaneously inflicted with the invasive giant kidney worm (Dioctophyme renale) parasite have not been examined, nor is the combined effect of these dual insults on the health and physical condition of the animal known. Using animals collected within the influence of the long-active ore-smelters at Sudbury, Ontario, an examination was made of toxic metal (Cd, Ni and Pb) levels and their tissue distributions within adult male mink bearing different intensities of parasite infection. Higher metal burdens were indicated within infected specimens than those uninfected. Combined renal and hepatic nickel and lead burdens were highest for mink with multiple worm infections, although only lead accumulations reached statistical significance. Cadmium accumulated to the greatest extent in the hypertrophied left kidney and liver, whereas nickel and lead were deposited more readily in the bony spicule of the parasitized right kidney cyst. The relative distribution of cadmium among renal, hepatic and renal cyst tissues (cast, spicule, worms) remained unchanged subsequent to D. renale infection, while the proportions of nickel and lead deposited in hepatic tissue were reduced. Metal burdens in female D. renale were three-fold higher than those of male worms, with the difference being attributable to the substantially greater size of the females. Canonical Correlation Analyses of condition measures and body metal burdens failed to indicate a direct relationship between infection intensity and body fat deposits but did confirm a positive association between metal loads and increased fat levels, along with enhanced gonad weights, neck circumference and reduced spleen weights. Such associations may be productive aspects for future investigation into the combined effects of increased metal loads and parasitic infection on the host system.

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INTRODUCTION Investigating metal contaminant levels in parasites has received considerable attention of recent, with the goal of most studies being to identify useful bioindicators and/or biomonitors of environmental pollutants. The use of parasites as indicators of metal contaminants within fish and aquatic environments has been studied extensively, and has shown that acanthocephalans and to a lesser extent cestodes, can accumulate extremely high concentrations of metal pollutants (Sures et al., 1994 a,b,c; Sures and Taraschewski, 1995; Sures et al., 1997 a,b,c; Siddall and Sures, 1998; Sures et al., 1999; Tenora et al., 1999a; Turcekova and Hanzelova, 1999: Zimmermann et al., 1999; Tenora et al., 2000; Barus et al., 2001b; Turcekova et al., 2002; Palikova and Barus, 2003; Sures, 2003; Williams and Mackenzie, 2003). By comparison, studies focused on bird-parasite systems are relatively few (Barus et al., 2001a; Tenora et al., 2001; Tenora et al., 2002). Virtually no work of this nature has been done on nematode parasites in mammals, with the exceptions of Szefer et al. (1998) who studied the bioaccumulation of trace elements in lung nematodes of harbour porpoises, Greichus and Greichus (1980) and Sures et al. (1998) who reported on roundworms of the pig and Tenora et al. (1999b) who examined metal levels in either gender of Toxocara canis and Protospirura muricola specimens and their respective hosts. The extent to which parasites affect metal uptake and distribution in host tissues is largely unknown and, for the most part, was not investigated in the above studies. It is known from the literature, however, that helminth infestation may affect the hosts‘ sensitivity to toxic metals (Pascoe and Cram, 1977; Poulin, 1992; Sures and Siddall, 1999). Since the giant kidney worm parasite, Dioctophyme renale, produces pathophysiological changes in both the liver through which it migrates and the kidney which it ultimately occupies, and these two organs represent the primary sites of metal accumulation in the vertebrate body, one might expect measurable changes in the tissue distribution of metals within the host animal following infection. The development and life cycle of the giant kidney worm have been summarized by Anderson (2000), and are depicted in Figure 1. Mink (Mustela vison) are the preferred definitive host for D. renale, although other carnivorous species have occasionally been infected (including otter Lontra canadensis, marten Martes americana, short-tailed weasel Mustela erminea, long-tailed weasel M. frenata, wolverine Gulo luscus and Gulo gulo, coyote Canis latrans, wolf C. lupus, dog C. familiaris, red fox Vulpes vulpes, bear Ursus americanus, raccoon Procyon loto, and coati Nasua nasua). Adult kidney worms typically occupy the right kidney (Figs. 2a and 2b) but on occasion have been observed unrestricted within the abdominal cavity or entwined within the liver of their host (Figure 2d). At least one male and one female worm (Figure 2f) must be present in the same kidney for a fertile infection to occur and for the life cycle to continue. Fertilized eggs are released from the female worm, passed down the ureter (which remains open post-infection) to the urinary bladder and voided with the urine. If the eggs are released into water and reach appropriate temperatures (approximately 14 to 30 ºC), they embryonate and are frequently consumed by the aquatic oligochaete Lumbriculus variegatus. The larvae moult twice in this intermediate host, reaching the infective third larval stage. Suitable paratenic hosts (frogs Rana catesbeiana, R. clamitans and R. septentrionalis, pumpkinseed fish Lepomis gibbosus, brown bullheads Ictalurus nebulosus and black bullheads I. melas) eat the parasitized oligochaete, but no futher development of D. renale larvae takes place in these paratenic hosts.

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Definitive Host (final moult and growth to adult worm occupying right kidney)

Incidental direct transfer via infected drinking water

Eggs released with urine into water

Unembryonated Eggs

Paratenic Hosts

Intermediate Host (Lumbriculus variegatus) (eggs hatch and develop to infective 3rd stage larvae)

Figure 1. Development and transmission of the giant kidney worm, Dioctophyme renale.

Although it is possible for mink to become infected through the incidental consumption of parasitized oligochaetes during feeding or drinking, the above-mentioned paratenic hosts are frequent items in the mink‘s diet, and thus typically serve as the vectors through which the parasite is transferred to mink. Once ingested, third-stage larvae penetrate the stomach wall of the definitive host and develop to the adult stage before migrating through the liver. The adult worms make their way to the right kidney, which they penetrate and ultimately excavate by destroying the functional cortical and medullary tissues as they grow to full size. Only a tough thickened capsule or cast (Figure 2c) remains of the right kidney, serving to encase the mature D. renale worms and in most cases a bony spicule (or ‗staghorn‘). The parasitized kidney with its occupants and component structures is commonly referred to as a kidney cyst. The spicule is usually embedded in the lumenal surface of the dorsal wall of the kidney cast; although size, shape and colour have been shown to vary considerably (Dhaliwal and Taylor, 2000), the basic form is a flat plate on the dorsal side, with finger-like projections or spicules radiating ventrally (Figure 2c). Histological examination of the spicule has revealed osteoblasts within lacunae and canaliculi making up the Haversian system of true replacement bone (McNeil, 1948; Mace, 1976). The plate edges and spicule projections have been found to contain osteoclasts and consist of hyaline cartilage (McNeil, 1948). The left kidney of the infected animal hypertrophies (50 to 100% by weight; Figure 2e) to compensate for the loss in function of the right kidney.

190

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Figure 2. Dioctophyme renale infection in mink: a) ventral view of kidneys showing right renal cyst of infected animal b) cyst opened showing several worms present c) cast with bony spicule (‗staghorn‘) embedded, after removing worms d) ‗free-floating‘ abdominal infection showing portions of 3 worms present among visceral organs e) hypertrophied left kidney of infected animal (on right) vs control f) 3 male worms (top) and 3 female specimens (below) removed from renal cyst (bottom). Magnifications: a), b) and d) = 0.75X actual size; c) and e) = 1.25X; f) = 0.38X.

In the Sudbury-area, the prevalence of D. renale infection in mink is approximately 50% (N. Schaffner and G. Parker, unpublished data) and, due to local ore-mining/smelting operations over the past 125 years, wildlife living within the influence of the area-wide smelters are exposed to significantly elevated metal levels in their environment (Wren et al., 1988; Capodagli and Parker, 2007). Patterns of metal uptake and accumulation in mink living under these conditions of environmental pollution and additionally inflicted with the invasive giant kidney worm parasite are currently unknown. As part of an on-going program examining toxic metals and endoparasites in Ontario wildlife species, the opportunity arose to investigate patterns of metal distribution in wild Sudbury-area mink populations infected with the parasite. The objectives of this investigation were as follows:

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1) to determine the effects of D. renale infection (at high and low intensities) on the uptake and renal-hepatic distribution of toxic metals (cadmium, nickel and lead) in wild field-trapped mink. 2) to quantify the extent to which these toxic metals are accumulated within the right kidney cyst and its component structures (namely the parasitic worms, bony spicule and cast tissues) of the infected animal. 3) to compare the extent of metal uptake and accumulation in male versus female specimens of the parasite. 4) to examine the extent to which metals are accumulated in the osseous spicule of the infected animal relative to concentrations occurring in skeletal (femur) bone. 5) to determine the extent to which renal-hepatic metal levels and D. renale infection, either singly or in concert, may influence the health and physical fitness of mink, through an assessment of the effects of these perceived insults on selected morphometric measures and fat reserves of the animal.

MATERIALS AND METHODS Mink carcasses were obtained from local fur trappers in the Sudbury District of Northern Ontario during the October to December trapping seasons of 1997, 1998 and 1999. Metal fallout and environmental contaminant conditions prevailing within the Sudbury Basin, home to intensive mining-smelting operations over the past 125 years, have been described elsewhere (Capodagli and Parker, 2007). A total of 30 adult male specimens were selected based on the intensity of giant kidney worm (Dioctophyme renale) infection present. Three groups were formed; non-infected mink (n=14), those infected with a single worm (n=8) and those infected with 4-6 worms (n=8). Assignment to the adult (>1 yr) male cohort was based on visual inspection of the genitals/gonads and the pattern of temporal muscle coalescence on the dorsal aspect of the skull (E. Addison (pers. comm.). Prior to dissection, the animals were completely thawed and morphometric measurements taken, including total peltless mass (to nearest 0.1 g), body length (from nose to tail base), total body + tail length (nose to tip of tail) and neck circumference (all to nearest 0.5 cm). Subcutaneous fat deposits in the groin and scapular regions were subjectively rated as high (3), medium (2) or low (1). During necropsy, the liver, heart, spleen, kidneys, gonads, omental fat, thoracic postcardinal fat deposit and abdominal mid-ventral fat deposit were removed and weighed (to nearest 0.1 g) immediately upon removal to minimize the effects of dehydration. Livers, femurs and kidneys (or their component parts in the case of infected animals) were retained for determination of metal content. In the case of infected subjects, the right kidney cyst was opened and each individual worm was removed, counted, weighed (to the nearest 0.1 g) and measured (to the nearest mm). Individual D. renale worms were sexed by the presence/absence of the prominent bell-shaped bursa at the caudal end and according to differences in body length and diameter (females ranged from 16-50 cm in length and were approximately 0.5 cm in diameter, while males ranged from 8-17 cm in length and were 0.25 cm in diameter) (Fyvie, 1971). Bony spicules were carefully dissected from the kidney cast of infected mink and kept in small airtight plastic cups at –20 ºC. Kidneys, liver, femur, kidney

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cast and worms were immediately packaged in individual polyethylene (Whirlpak-Nasco) bags and restored at –20 ºC until metal analysis could be performed. All glassware was washed using SparkleenTM dish detergent, rinsed, passed through a 20% HCl bath followed by two deionized distilled water baths and allowed to air dry before use. The right kidney, left kidney, liver, kidney cast, shaft of the right femur, individual whole worms and bony spicule were dehydrated in petri dishes in a drying oven at 60 ºC until constant weight was attained (approximately 3 days). A 2.0 (± 0.005) g sub-sample of dried liver, the shaft of the right femur and the entire sample of each of the remaining tissues were weighed into ceramic crucibles and placed in a muffle furnace. The temperature in the furnace was increased at a rate of 0.7 ºC/minute to 150 ºC and held for 10 hours, and then raised by 0.3 ºC/minute to 500 ºC, where it was held for 15 hours. The samples were allowed to cool to room temperature before digestion began. The ash was then transferred to sterile 15 ml polypropylene centrifuge tubes and a three ml aliquot of 2.5 N reagent grade HNO3 was added to the ash. The centrifuge tubes were capped and vortexed, and the samples then placed in an oven at 60 ºC to digest for 20 hours. The samples were then removed from the drying oven, vortexed again to break up any precipitate and left to stand overnight to ensure complete digestion. The samples were then centrifuged at full speed (approximately 5000 G) for five minutes and the supernatant was extracted with disposable glass Pasteur pipettes and transferred into acid-washed test tubes. Where necessary, the samples were diluted (5 fold) with 20% reagent grade HCl before being analyzed by flame atomic absorption spectrophotometry for Cd, Ni and Pb content using a Perkin Elmer Spectrophotometer (Model 703). Procedural blanks were included in each sample run to control for metals introduced by the digestion process. Metal concentrations were calculated and expressed as µg•g-1 dry weight. Tissue metal burdens (expressed as µg) were derived by multiplying metal concentration by the total tissue dry weight. To determine recovery rates and assess the reliability of the analytical procedures, certified reference materials (oyster tissue from the National Institute of Standards and Technology and citrus leaves from the National Bureau of Standards) were also subjected to the above procedure. As recovery rates (112 to 131%; n=3 per element) were generally deemed acceptable, tissue concentrations were left unadjusted. Where violations of normal distribution and/or homogeneity of variance in the data could not be rectified with standard transformations, the data were statistically evaluated using both parametric and non-parametric procedures. Organ weights and fat deposit measures were standardized for body size using the formula: Standardized measure = organ (or fat) weight x individual body length / mean body length

RESULTS Body and Organ Weights Dried weights of the primary organs of metal accumulation as well as standard body weights for the three infection groups (uninfected mink, mink infected with one kidney worm and mink infected with 4-6 worms) are reported in Table 1.

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Table 1. Body weights and dried organ weights (g) in mink infected with D. renale at different intensities (0, 1 or 4-6 worms). Values are means followed by standard error in parenthesis Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

10.55

(0.43)

a

10.01

(0.35)

a

10.25 (0.72)

a

Left kidney

0.78

(0.02)

a

1.23

(0.08)

b

1.32

(0.05)

b

Right kidney/cyst

0.73

(0.03)

a

0.88

(0.15)

a

2.26

(0.23)

b

a) cast

0.34

(0.03)

a

0.75

(0.06)

b

b) spicule

0.10

(0.04)

a

0.19

(0.06)

a

c) worm(s)

0.44

(0.13)

a

1.32

(0.17)

b

Cyst components:

Body weight

746.68 (24.43) a

766.12 (22.70) a

715.27 (43.39) a

Within organs, mean values bearing the same letter are not significantly different (p > 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test.

There were no significant differences in mean dry liver weights or overall body weights among the three infection groups. However, substantial weight differences occurred among the kidneys. The left kidneys of mink infected with either 1 worm or 4-6 worms were approximately 58% and 69% heavier, respectively, than the left kidney of uninfected mink. The right kidney cysts that developed in infected mink were likewise heavier than the intact right kidney of non-infected animals. This increase in weight amounted to 21% and 210% in the single worm and 4-6 worm infections, respectively. Despite the 2.6-fold weight difference between single and multiple worm cysts, the contributions of individual structural components were relatively uniform: cast tissue comprised 38.6 versus 33.2%, spicule 11.4 versus 8.4%, and worms 50.0 versus 58.4%, respectively.

Cadmium Levels Cadmium burdens and concentrations in the liver, kidneys, kidney cast, spicule and giant kidney worms of mink with varying intensities of D. renale infection are presented in Table 2. There was an overall increase in cadmium burden in infected mink over uninfected mink. Together renal and hepatic burdens approximated 7 µg in uninfected mink whereas infected mink averaged 13 to 17 µg. The elevated cadmium burdens in parasitized mink were manifested in both the liver and the uninfected left kidney (Figure 3). On average, liver burdens were elevated 2-fold and left kidney burdens by 3.6 fold over values seen in noninfected animals. Cadmium burdens present in the right kidney (non-infected animals) or its component structures (cast, spicule and worms) in infected animals did not differ significantly among the groups (Table 2). Thus the amount of cadmium that was present within the

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confines of the renal cyst appeared to remain unchanged from that present in the right kidney prior to infection, and was distributed among the component tissues/structures (Figure 3). Table 2. Cadmium levels in body tissues of mink infected with D. renale at different intensities. Values are means followed by standard error in parenthesis CADMIUM BURDEN (µg) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

4.74

(0.55)

a

10.98

(2.61)

b

7.07

(1.08)

a,b

Left kidney

1.23

(0.23)

a

4.89

(1.41)

b

4.02

(0.88)

b

Right kidney/cyst

1.15

(0.21)

a

1.07

(0.18)

a

1.63

(0.25)

a

a) cast

0.40

(0.10)

a

0.35

(0.05)

a

b) spicule

0.18

(0.04)

a

0.26

(0.05)

a

c) worm(s)

0.48

(0.15)

a

1.02

(0.21)

a

Cyst components:

All above organs/components 7.29 (0.99) a 16.94 (4.06) b 12.75 (2.43) a,b combined Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test. CADMIUM CONCENTRATION (µg•g-1) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

0.47

(0.07)

a

1.14

(0.30)

b

0.70

(0.10)

a,b

Left kidney

1.59

(0.33)

a

4.10

(1.30)

b

3.03

(0.66)

a,b

Right kidney/cyst

1.62

(0.31)

b

1.66

(0.54)

b

0.72

(0.09)

a

a) cast

1.43

(0.57)

b

0.47

(0.07)

a

b) spicule

4.33

(1.36)

a

1.82

(0.23)

a

c) worm(s)

1.73

(0.55)

a

0.80

(0.14)

a

Cyst components:

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test.

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Liver

20

Left kidney Right kidney/cyst Cyst components:

CADMIUM BURDEN (µg)

15

Cast Spicule Worm(s)

D

10

D

C

5

B A, B

A

0

0 worms

1 worm

4-6 worms

INFECTION STATUS

Figure 3. Cadmium burden (µg) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean composite burden. 6

Liver Left kidney

CADMIUM CONCENTRATION (µg • g-1)

5

Right kidney/cyst Cyst components: 4

Cast Spicule Worm(s)

3

B

A

2 A,B A

1

A B

B B

B

0

A A,B

A 0 worms

1 worm

A

A

A

4-6 worms

INFECTION STATUS

Figure 4. Cadmium concentration (µg•g-1 dry wt) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean.

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While cadmium burdens of the left kidney increased 3 to 4-fold as a result of infection (Table 2), organ hypertrophy (58 - 69%; Table 1) reduced mean tissue concentrations to values of 3 - 4 µg•g-1 (Table 2). Nevertheless these values were significantly (1.9 to 2.6-fold) higher than concentrations present in the left kidneys of non-infected subjects. Within the right kidney cyst of infected animals, cadmium was most concentrated in the bony spicule (mean = 4.33 ± 1.36 and 1.82 ± 0.23 µg•g-1; Figure 4). Worm concentrations averaged 1.73 ± 0.55 and 0.80 ± 0.14 µg•g-1 in the single and multiple worm groups, while cast tissues averaged 1.43 ± 0.57 and 0.47 ± 0.07 µg•g-1 in these two respective groups. For all tissues examined, mean cadmium concentration values tended to be higher among animals infected with single versus multiple worms; however, only in the case of the right kidney cyst and cast tissue did these differences reach statistical significance (p = 0.007 and 0.012, respectively).

Nickel Levels Tissue nickel burdens and concentrations for the same three groups of mink are reported in Table 3. Similar to cadmium, nickel burdens were likewise greater in the combined renal and hepatic tissue of infected mink compared to non-infected mink. Unlike cadmium however, the liver was not responsible for any of the increase in nickel burdens of the parasitized animal. The elevated nickel burden was distributed within the renal tissues of the animals, primarily the parasitized right kidney cyst. Approximately twice the nickel burden was present in the left kidney of infected mink, whereas 4 and 8 times more nickel were observed in the right kidney cyst of mink with 1 and 4-6 worm infections respectively, over burdens present in respective uninfected kidneys (Figure 5). Table 3. Nickel levels in body tissues of mink infected with D. renale at different intensities. Values are means followed by standard error in parenthesis NICKEL BURDEN (µg) Infection Status Organ

0 worms (n=14)

1 worm (n=7)

4-6 worms (n=8)

Liver

4.47

(0.28)

a

5.14

(0.67)

a

4.63

(0.50)

a

Left kidney

0.52

(0.07)

a

1.03

(0.21)

b

0.98

(0.10)

b

Right kidney/cyst

0.52

(0.08)

a

2.10

(0.50)

a,b

4.08

(0.51)

b

a) cast

1.16

(0.38)

a

1.44

(0.26)

a

b) spicule

0.71

(0.20)

a

1.03

(0.21)

a

c) worm(s)

0.60

(0.19)

a

1.78

(0.78)

b

7.71

(1.13)

a,b

10.19

(0.86)

b

Cyst components:

All above organs/components 5.49 combined

(0.33)

a

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test.

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197

NICKEL CONCENTRATION (µg•g-1) Infection Status Organ

0 worms (n=14)

1 worm (n=7)

4-6 worms (n=8)

Liver

0.43

(0.02)

a

0.51 (0.05)

a

0.45

(0.03)

a

Left kidney

0.65

(0.08)

a

0.83 (0.14)

a

0.75

(0.08)

a

Right kidney/cyst

0.72

(0.10)

a

2.21 (0.36)

b

1.88

(0.19)

b

a) cast

3.73 (1.50)

b

1.95

(0.32)

a

b) spicule

11.47 (2.30)

a

7.78

(1.21)

a

c) worm(s)

0.97 (0.19)

a

1.47

(0.22)

a

Cyst components:

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. 12

Liver Left kidney 10

Right kidney/cyst Cyst components: Cast Spicule Worm(s)

NICKEL BURDEN (µg)

8

6

C C

B 4

A 2

0

A

0 worms

A

1 worm

4-6 worms

INFECTION STATUS

Figure 5. Nickel burden (µg) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Oneway Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean composite burden.

Mean hepatic nickel concentrations ranged between 0.43 ± 0.02 and 0.51 ± 0.05 µg•g-1, and did not differ significantly among the three infection groups (Table 3). Similarly, concentration values in the left kidney were comparable across the three infection groups, indicating that the infection-induced increase in nickel loads within this organ were

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proportional to the degree of hypertrophy undergone. Within the right kidney cyst, the greatest concentration of nickel occurred within the spicules (mean = 11.5 ± 2.3 and 7.8 ± 1.2 µg•g-1 in single worm and multiple worm infections respectively; Figure 6). 14

Liver Left kidney

NICKEL CONCENTRATION (µg • g-1)

12

Right kidney/cyst Cyst components:

10

Cast Spicule Worm(s)

8

A 6

A

4

A A

2

A

A

A

A A

B B

A A

A B

0

0 worms

1 worm

4-6 worms

INFECTION STATUS

Figure 6. Nickel concentration (µg•g-1 dry wt) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean.

Worm concentrations averaged 0.97 ± 0.19 and 1.47 ± 0.22 µg•g-1 while cast concentrations averaged 3.7 ± 1.5 and 2.0 ± 0.3 µg•g-1 respectively in the two infection groups. Relative to non-infected animals, tissue nickel levels (burdens and concentrations) in infected animals were more variable with few statistically significant differences occurring between single-worm and multiple-worm groups.

Lead Levels Lead burden and concentration values in mink from the three infection intensity groups are presented in Table 4. Total lead burdens followed the same trend seen for both cadmium and nickel in that combined kidney and liver lead loads were greater in mink infected with D. renale when compared to uninfected mink. Elevations averaged 21% higher in mink infected with 1 worm and 69% in those with 4-6 worms. As seen for nickel, the infection-induced increase in lead burden was attributable to enhanced deposition in the renal tissues. For the left kidney, lead burdens were elevated 1.6-fold (one worm infections) and 1.9-fold (4-6 worm infections) higher than values seen in the non-parasitized animal. The majority of the increase in total lead burden arose from the right kidney components, which showed

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elevations of more than 3-fold for 1-worm infections and 7-fold for 4-6 worm infections (Figure 7). Although noticeably higher, lead concentrations in the liver and left kidney followed the same trend as nickel in that mean tissue concentrations failed to differ significantly across the three treatment groups (Table 4). Table 4. Lead levels in body tissues of mink infected with D. renale at different intensities. Values are means followed by standard error in parenthesis LEAD BURDEN (µg) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

10.28 (0.60) a

10.23

(0.44)

a

11.72

(1.18)

a

Left kidney

0.95

(0.05) a

1.54

(0.07)

b

1.83

(0.17)

b

Right kidney/cyst

0.93

(0.07) a

3.37

(0.73)

b

6.73

(1.03)

c

a) cast

0.54

(0.07)

a

1.11

(0.14)

b

b) spicule

2.19

(0.68)

a

3.44

(0.77)

a

c) worm(s)

0.64

(0.14)

a

2.15

(0.30)

b

Cyst components:

All above organs/components 12.50 (0.61) a 15.13 (0.87) b 21.15 (1.13) c combined Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test. LEAD CONCENTRATION (µg•g-1) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

0.98

(0.04) a

1.02

(0.02)

a

1.13

(0.07)

a

Left kidney

1.19

(0.05) a

1.26

(0.05)

a

1.40

(0.14)

a

Right kidney/cyst

1.29

(0.10) a

4.07

(0.71)

c

2.93

(0.29)

b

a) cast

1.68

(0.27)

a

1.43

(0.10)

a

b) spicule

40.50

(12.08) a

24.08

(2.84)

a

c) worm(s)

1.78

(0.19)

1.95

(0.10)

a

Cyst components:

a

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test.

Glenn H. Parker and Liane Capodagli

200 Liver Left kidney 20

Right kidney/cyst

LEAD BURDEN (µg)

Cyst components: Cast Spicule Worm(s)

15

C B

C

A

A

10

A 5

0

1 worm

0 worms

4-6 worms

INFECTION STATUS

Figure 7. Lead burden (µg) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Oneway Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean composite burden. Liver 50

Left kidney Right kidney/cyst Cyst components:

LEAD CONCENTRATION (µg • g-1)

40

Cast Spicule Worm(s)

30

20

A A 10

C A

A

A

A

A

A

A

B A

A

A

A

0

0 worms

1 worms

4-6 worms

INFECTION STATUS

Figure 8. Lead concentration (µg•g-1 dry wt) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean.

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

201

Among components of the right kidney cyst, the bony spicule concentrated lead to the greatest extent with mean concentrations reaching 40 ± 12.1 and 24 ± 2.8 µg•g-1 for 1 worm and 4-6 worm infections respectively (Figure 8). Concentrations in cast tissues and in worms averaged less than 2 µg•g-1. Although burden values in the right kidney cyst were substantially higher in the more heavily infected group, concentration values in the structural components of the cyst did not differ significantly between 1 worm and 4-6 worm infection groups.

Proportional Deposition among Tissues To better control for variability in levels of uptake among individuals, the relative distribution of body metal burdens between the liver and renal tissues/structures was calculated and expressed on a percentage basis for both infected and non-infected animals (Table 5). Data available from two mink infected with a single D. renale worm in the abdomen (free-floating infection) and four mink with a single worm in the abdomen and one in the right kidney (double infection) have been included in Table 5 to represent two additional infection groups. The proportions of cadmium, nickel and lead deposited in renal and hepatic tissues of mink with a single abdominal infection were similar to the proportions deposited in non-infected mink. Of the total metal burden in mink with a free-floating infection and in non-infected mink, liver tissue accumulated 67 and 68% of the cadmium, 79 and 82 % of the nickel and 83 and 85% of the lead, respectively. Sixteen percent of the total cadmium, between 8 and 9% of the total nickel and 7-8% of the total lead was deposited in each kidney of non-parasitized mink and mink with an abdominal infection. Although there was a two-fold increase in total cadmium burden in mink infected with 1 worm over uninfected mink (Figure 3), the proportion deposited in the liver remained constant at 65 to 68% (Table 5). Slightly less cadmium (59% and 57%) was deposited in the livers of mink with double and 4-6 worm infections. Approximately 30% of the total body cadmium was found in the functioning renal tissue, regardless of infection status. In noninfected and abdominally infected mink, this was equally distributed between the right and left kidneys, while in mink parasitized in the right kidney, all 30% accumulated in the lone functioning left kidney, regardless of the number of worms present. In single worm and double worm renal infections, only 8 and 9% of the total cadmium was found in the resultant kidney cyst whereas 14% was accumulated in the cyst of animals harbouring 4-6 worms. A higher proportion of cadmium was deposited in worms from multiple worm infections (8%) versus single worms (3%). At all infection intensities, 3% was deposited in the kidney cast and 1-3% was sequestered in the bony spicule. When present, abdominal worms contained only 1-2% of the cadmium burden. In contrast to cadmium, nickel burdens in the liver did not differ with varying degrees of infection intensity (Figure 5). However, the proportion of total nickel burden deposited in the liver did change. In the non-parasitized group, approximately 82% of the total nickel burden accumulated in the liver. Subsequent to infection, the proportion of total nickel found in the liver dropped to 62% in mink infected with 1 renal worm, 44% in mink with a double infection and 46% in mink infected with 4-6 worms.

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Table 5. Relative distribution of metals among tissues, expressed as a percentage of total renal-hepatic burden, in mink infected with D. renale at different intensities Metal

Infection Status 0 worms Organ

(n=14)

1 worm Abdomen (n=2)

2 worms

4-6 worms

Right kidney Abdomen + right Right kidney (n=8) kidney (n=4) (n=8)

Cd Liver

68 b

67 a,b

65 a,b

59 a,b

57 a

Left kidney

16 a

16 a

27 b

31 b

29 b

Right kidney/cyst

16 c

15 c

8a

9 a,b

Cyst components a) cast

-

-

3a

3a

3a

b) spicule

-

-

1a

3a

2a

c) worm(s)

-

-

3a

3a

8b

Abdominal worm

-

1

-

2

-

82 c

79 c

62 b

44 a

46 a

14 b,c

Ni Liver Left kidney

9 a,b

9 a,b

12 b

7a

10 a,b

Right kidney/cyst

8a

8a

25 b

47 c

44 c

Cyst components a) cast

-

-

10 a

27 b

14 a

b) spicule

-

-

10 a

7a

11 a

c) worm(s)

-

-

5a

13 a,b

19 b

Abdominal worm

-

4

-

2

-

85 b

83 b

68 a

67 a

58 a

Pb Liver Left kidney

8 a,b

7a

10 c

9 b,c

9 b,c

Right kidney/cyst

8a

7a

21 b

21 b

32 b

Cyst components a) cast

-

-

4a

3a

5a

b) spicule

-

-

13 a

14 a

17 a

c) worm(s)

-

-

4a

3a

11 b

Abdominal worm

-

4

-

3

-

Within organs, values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test.

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

203

The relative proportion of nickel deposited in the left kidney increased only slightly in one worm infections, while that accumulating in the infected right kidney cyst reached as much as 25% and 44% of the body burden in 1 worm and multiple worm animals respectively. The left kidney of animals with an abdominal worm plus a right kidney worm did not differ from that noted for non-infected mink, however 47% of the total nickel burden was deposited in the right kidney cyst. In the case of single worm infections, 10% of the total nickel was deposited in the kidney cast and 5% in the developing worm, whereas in mink with 4-6 worms 14% was observed in the right kidney cast and 19% was taken up by the worms. Mink with both an abdominal and right kidney worm accumulated 27% of the total nickel in the cast and 13% in the right kidney worm. The spicule accounted for 10, 7 and 11 % of body nickel burdens in single, double and 4-6 worm infections respectively. Relative proportions of lead deposited in renal and hepatic tissues paralleled those observed for nickel. Eighty-five percent of the total body burden of lead was deposited in the liver of uninfected mink, while only 68%, 67% and 58% were accumulated in the hepatic tissue of mink with 1, 2 and 4-6 worm infections respectively. In response to infection, proportions in the left kidney increased only slightly whereas total body proportions accumulating in the infected right kidney cyst reached as much as 21% for both single and double infections and 32% for the 4-6 worm infected animals. In each group, more than half of the lead in the right kidney cyst was sequestered within the bony spicule. The worms accounted for 4%, 3% and 11% and the cast tissues for 4%, 3% and 5% of the total lead burden present in single worm, double worm and 4-6 worm infections respectively.

Gender Comparisons A comparison of metal levels in male versus female D. renale taken from mink with varying infection intensities is presented in Table 6. Dry weights for male and for female worms did not vary with infection status as indicated by One-way Analyses of Variance (males p = 0.46 and females p = 0.14). For multiple worm infections, metal levels in male and in female worms occupying the same cyst were averaged and entered in a 2-way Analysis of Co-Variance to assess the effects of infection status and worm gender while covarying for the effects of total worm weight in the infected cyst. Total worm weight in each cyst was not significantly correlated with levels of any of the three metals examined (p > 0.05), thus indicating that competition between male and female parasites within the same cyst had not affected metal uptake by the resident worms. For all three metals, overall mean burdens (i.e. in all worms combined regardless of infection status) tended to be higher in female compared to male worms by a factor of approximately 3 (Table 6). When differences in body mass were factored in by calculating concentration values, significant gender differences were observed only for lead, with males showing approximately 50% higher levels.

Femur versus Spicule Levels The geometric means for metal concentrations in femur sub-samples and in the spicules of parasitized animals were examined for the three infection groups and are presented in Table 7.

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Table 6. Metal levels in male and female D. renale taken from mink with varying infection intensities. Values are means followed by standard error in parenthesis Element

Infection Status Variable

1 worm (♂ n=4, ♀ n=4)

4 worms a 5–6 worms a (♂ n=3, ♀ n=4) (♂ n=4, ♀ n=3)

Sex

All groups combined (♂ n=10, ♀ n=11)

Cd Burden (µg) Male

0.29 (0.09) *

Female

0.07 (0.01) **

0.15 (0.04) *

0.18 (0.04) *

0.69 (0.26)

0.34 (0.03)

0.57 (0.14)

0.53 (0.10)

Male

2.27 (0.85)

0.60 (0.13)

0.97 (0.25)

1.34 (0.37)

Female

1.20 (0.70)

0.62 (0.10)

1.14 (0.17)

0.97 (0.25)

0.11 (0.04)

0.16 (0.03)

0.27 (0.03)

0.19 (0.03)

Concentration (µg•g-1)

Ni Burden (µg) Male

** Female

**

0.78 (0.20)

0.55 (0.06)

0.55 (0.16)

0.63 (0.09)

Male

0.82 (0.35)

1.36 (0.24)

1.94 (0.38)

1.43 (0.23)

Female

1.08 (0.25)

0.96 (0.10)

1.10 (0.28)

1.04 (0.11)

Male

0.29 (0.02)

0.29 (0.03)

0.35 (0.07)

0.31 (0.02)

Female

0.99 (0.09)

0.81 (0.07)

0.62 (0.006)

0.82 (0.06)

2.14 (0.17)

2.43 (0.21)

2.20 (0.24)

2.24 (0.12)

Concentration (µg•g-1)

Pb Burden (µg)

**

*

**

Concentration (µg•g-1) Male

* Female a

1.41 (0.20)

1.42 (0.11)

** 1.30 (0.14)

1.38 (0.08)

In multiple-worm infections, values represent the average for all worms of same sex present within the cyst. Gender differences assessed by 2-way Analysis of Variance while covarying for total worm weight within cyst; p < 0.05 *; p ≤ 0.01 **.

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

205

Table 7. Metal concentrations (µg•g-1 dry wt) in femurs and spicules of mink infected with D. renale at different intensities. Values are geometric means (presented as antilogs of the log10 transformed data) followed by standard error in parenthesis Tissue Metal Infection Group Cd

Ni

Pb

(n)

Femur

Spicule

0 worms

(12)

0.87

(+0.035, -0.033)

1 worm

(8 and 6)

0.94

(+0.038, -0.036)


80% of viable population size.

Good: functioning within its range of acceptable variation; it may require some human intervention Fair: lies outside its range of acceptable variation and requires human intervention

The extent of likely or most likely suitable habitats can accommodate 60-80% of viable population size.

Poor: restoration or preventing extirpation practically impossible

The extent of likely or most likely suitable habitats can accommodate 2 km2) are surrounded by intact natural vegetation ( >60% of intact forest within 2 km). Moderately connected, the suitable habitats (patch > 2 km2) are surrounded by moderately intact natural vegetation (> 4060% of intact forest within 2 km). Moderately fragmented, the suitable habitats (patch > 2 km2) are surrounded by altered vegetation (> 20-40% of intact forest within 2 km). Highly fragmented, the suitable habitats (patch > 2 km2) are entirely or almost entirely surrounded by altered vegetation and human-induced land use (< 20% of intact forest within 2 km).

Remarks: 1/ described by TNC (2000); 2/ defined by the planning team and wildlife experts

3.4. Delineate the Congregation Areas of Target Species The current congregation areas were simply derived from combining the likely and most likely suitable habitats of all target species. The output grid map shows the current location of suitable habitats of focal conservation target species. On the other hand, the extend of suitable habitats for each species to desired level of population viability was obtained from expanding the existing suitable habitat to meet the expected size based on the probability values as defined in the previous step. Later, all desired suitability maps were aggregated as done for the current status.

4. RESULTS AND DISCUSSION 4.1. Selected Target Wildlife Species Five wildlife species were selected by wildlife scientists and a planning team as the conservation targets. These species were tiger, Asian elephant, gaur, banteng, and sambar based on the selection criteria and appropriate observations to run a logistic model (Pattanavibool et al., 2003; Vanichbancha, 2001). The observations for elephant, sambar, gaur, tiger, and banteng were 960, 700, 641, 224, and 131 points, respectively.

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Tiger, elephant and banteng are classified as endangered species by IUCN (2000). According to Lekakul and McNeely (1977), tigers are found in a very wide range of habitat types; they only require sufficient prey species, water and shelter from the sun and occupy a large home range (50-200 km2), the average size of which in Thung Yai is approximately 80 km2 (Prommakul, 2003). They also require good protection from tiger and prey poaching pressures (Karanth et al., 2004). In addition, Simcharoen et al. (2007) used photographic capture-recapture sampling to estimate tiger density in the core area of Huai Kha Khaeng. The sample area yielded a density estimate of 3.98 tigers per 100 km2. Elephant is a top herbivore and found in a variety of forested areas. The home range sizes of elephant herds are between 105 and 320 km2 (Sukumar, 1989). Similarly, banteng lives in loose herds of 2-20 individuals. Prayurasiddhi (1997) reported that the annual home range size was 44 km2 for banteng herds in Huai Kha Khaeng. Gaur is classified as a vulnerable species by IUCN (2000). Gaur live in herds of 3-40 individuals and the home ranges of gaur herds are between 29.9 and 52.1 km2 (Conry, 1989). Sambar still exist in many protected areas in Thailand and make a significant contribution to the long-term integrity and conservation values of the WEFCOM. It is a preferred prey species of several carnivores, including tiger.

4.2. Suitable Habitat Models The results of logistic multiple regressions indicated that three physical factors and two anthropogenic factors were significantly related to the distributions of elephant, sambar, gaur, tiger, and banteng. The environmental factors were elevation, slope, distance to stream, distance to village, and distance to ranger station. The logistic regression models and overall accuracy at cut-off of 0.5 are shown below. Z

sambar

=

1.5191 – 0.0009Alt -0.0002Rst - 0.0136Slp –

0.0003Str+0.0003Vil; overall accuracy 68.06% Z

banteng =

-1.3795 - 0.0018Alt -0.0008Rst - 0.0939Slp –

0.0005Str + 0.0020Vil; overall accuracy 91.97% Z

gaur

=

-3.3434 - 0.0042Alt -0.0001Rst - 0.0946Slp –

0.0002Str + 0.0003Vil; overall accuracy 83.13% Z

=

elephant

1.4603 - 0.0013Alt -0.0001Rst -0.1109Slp –

0.0003Str +0.0002Vil; overall accuracy 83.32% Z

tiger

=

1.1335 + 0.0024Alt -0.0003Rst - 0.1327Slp –

0.0003Str + 0.0003Vil; overall accuracy 84.18% where

Alt = altitude (m);

Rst = distance to ranger station (m);

Assessing Population Viability of Focal Species Targets in the Western Forest …

293

Slp = slope (%); Str = distance to stream (m); Vil = distance to village (m). The GIS habitat models indicated that most species prefer to inhabit low altitude, close to ranger stations, low slope, close to streams and far from villages. These areas are safe from illegal poaching and other human disturbances with near year-round water sources. The overall accuracies for all species except sambar, were greater than 83%, being especially high for banteng because its habitat is concentrated in small patches. For sambar, the model indicates low accuracy because it uses various vegetation types. In addition, the pseudoabsent data may contain occurrence locations. Figure 2 shows the likelihood of habitat uses of the five focal species in the WEFCOM, while Table 2 presents the coverage of each suitable class. The distributions of each species are detailed below. Suitable habitats of sambar (likely and most likely) are found in deciduous forest and open areas in the core area and in the southern part of the WEFCOM, covering approximately 8,032 km2 or 43% (Figure 2a). Sambar is unlikely to be present in densely populated areas and the steep terrain mainly in the northern and western parts of WEFCOM. Banteng or wild ox is now restricted to small and fragmented populations congregated along Huai Kha Khaeng stream. Other possible patches are in Khao Laem and Salakpra (Figure 2b). The suitable habitats cover less than 4% of the complex. The main threats to this species are poaching, habitat destruction and human encroachment, and overgrazing by domestic cattle (Prayurasiddhi, 1997). Besides, the habitats of banteng have been degraded in recent years due to the RFD and Department of national Park, Wildlife and Plant Conservation (DNP) have implemented the policy of 100% forest fire prevention nationwide, especially in protected areas. This misunderstood concept influenced vegetation community dynamics and dependent fauna. Parts of open deciduous forests and grassland which are favorable habitats for ungulate species have been invaded by pioneer species such as Cratoxylum spp. and Eupatorium odoratum. Table 2. Predicted suitable habitats for selected wildlife species in the WEFCOM

Species Sambar Banteng Gaur Elephant Tiger 1/

Extent of Suitability Class (%/km2) Unlikely Less Likely 37.9 19.2 7,099 3,596 89.4 7.3 16,731 1,362 41.1 17.1 7,699 3,211 26.6 21.7 4,951 4,058 63.6 16.7 11,922 3,126

= Likely suitability + most likely suitability.

Likely 14.6 2,736 2.6 492 12.8 2,390 18.3 3,425 9.3 1,736

Most Likely 28.3 5,296 0.7 140 29.0 5,427 33.6 6,292 10.4 1,944

Suitable habitat 1/ 42.9 8,032 3.4 632 41.7 7,817 51.9 9,717 19.7 3,680

Figure 2. Current suitable habitats of selected species in the WEFCOM based on probability values.

Figure 3. Comparison of congregation areas of suitable habitats for all species in current condition and desired condition.

Table 3. Assessing population viability in WEFCOM

1

Species target Sambar

Banteng

Good

Very good

Current Rating 2/

Desired Rating 3/

3,0004,500

4,5006,000

6,000-7,500

Very good

Very good

60%

Very good

NA 5/

(25 r2S)T and u 1ug per dot, whereas a planar microarray immobilizes 10 to 20 pg per spot (Cho and Tiedje, 2002). As mentioned above, gel-pad microarrays have a higher probe binding capacity than planar arrays due to their three-dimensional nature. Gel-pad arrays have been used to immobilize 3 pmol of oligonucleotide probe per gel pad (Urakawa et al., 2002), which corresponds to approximately 18 ng for a typical 20 base pair probe. Therefore, gel-pad microarrays should result in an increase in sensitivity over planar arrays, but no direct comparisons of the sensitivity of these two formats are available in the literature. Several design and methodological approaches can also be used to increase sensitivity, such as increasing probe length and reducing the stringency of the hybridization and/or wash conditions. Several studies have demonstrated that increasing probe length results in increased sensitivity of microarray hybridization (Zhou , 2003). In addition, a number of studies have demonstrated higher hybridization signals at lower stringency: Guschin et al.

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369

(1997) showed higher hybridization signals at lower hybridization temperatures, and several studies have reported higher hybridization signals at lower denaturant (formamide) concentrations (Urakawa et al., 2002; Zhang et al., 2007). However, increasing probe length and reducing stringency also have the drawback of decreasing hybridization specificity, so it is necessary in any microarray application to find an appropriate balance between specificity and sensitivity. Increasing probe concentration is another approach that has been shown to increase sensitivity (Guschin et al., 1997), and this approach has the advantage of not decreasing specificity. Several groups have demonstrated that microarrays can be designed with adequate sensitivity to detect nucleic acids directly from environmental samples. As mentioned above, ribosomal RNA is a good target for hybridization because active cells contain thousands to tens of thousands of copies of ribosomal RNA per cell (Amann and Ludwig, 2000). Several groups have demonstrated successful microarray-based detection of ribosomal RNAs from a variety of environmental samples: Small et al. (2001) detected Geobacter chapellei 16S rRNA directly from a total-RNA soil extract; El Fantroussi et al. (2003) successfully detected Acidobacteria as well as Alpha, Beta, and Gamma Proteobacteria by hybridizing RNA extracted directly from estuarine sediments to a microarray containing 16S rRNA targeted oligonucleotide probes; and Kelly et al. (2005) detected nitrifying bacteria by hybridizing RNA extracted directly from a wastewater treatment plant aeration tank to a microarray containing 16S rRNA-targeted oligonucleotide probes. Detection of bacterial DNA in environmental samples via direct hybridization is more challenging since most bacterial cells contain only a single chromosome which generally includes one or perhaps a few copies of each gene. Nevertheless, Wu et al. (2001) successfully hybridized DNA isolated from soil and sediment samples to a microarray using large PCR products (0.76 kb) as hybridization probes. These very large probes enabled high sensitivity (1 ng of target DNA), but low specificity (genes had to be at least 15% to 20% different in sequence in order to be discriminated). Zhou (2003) used much smaller oligonucleotide probes (50 mer) and successfully hybridized DNA isolated from soil. These shorter probes resulted in improved specificity (genes that were at least 10% to 14% different in sequence were discriminated), but lower sensitivity (8ng of target DNA). However, Zhou (2003) suggested that this level of sensitivity should be sufficient for many studies in microbial ecology since DNA yields from soil and sediment samples typically range between 10 and 400 g of DNA per gram of soil dry weight.

AMPLIFICATION OF TARGETS PRIOR TO MICROARRAY HYBRIDIZATION Although the studies cited above have shown some success in detecting bacteria in environmental samples by direct hybridization of either RNA or DNA, the sensitivity of these approaches may not be adequate to detect nucleic acids in low biomass systems or to detect nucleic acids that represent a small fraction of the total nucleic acid pool in a sample. For example, microarrays may not be sensitive enough to directly detect organisms whose rRNA makes up a small fraction of the total community rRNA pool either because the organisms are present in low numbers or because their cellular rRNA content is low due to a low level of

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John J. Kelly

metabolic activity. In addition, functional genes present in low concentrations may not be detectable by direct hybridization. For this reason, several groups have used PCR amplification prior to microarray hybridization to increase target concentration. Because highly conserved universal primers for amplifying rRNA genes are available, PCR amplification can be run with universal 16S primers to simply increase the overall 16S rRNA gene concentration prior to hybridization with a microarray containing specific 16S rRNA probes. This approach was used by Loy et al. (2002) to detect sulfate-reducing prokaryotes in a hypersaline cyanobacterial mat, by Wang et al (2002) to detect a variety of human intestinal bacteria in fecal samples, and by Loy et al. (2005) to detect members of the betaproteobacterial order ―Rhodocyclales‖ in activated sludge from an industrial wastewater treatment plant. This approach has the advantage of increasing sensitivity, and general amplification of all 16S rRNA genes requires only a single PCR reaction. PCR can also be run with group-specific primers to selectively amplify the 16S rRNA genes from specific phylogenetic groups. For example, Siripong et al. (2006) improved their detection of ammonia oxidizing bacteria (AOB) in wastewater treatment plant samples by selectively amplifying the 16S rRNA genes of beta-proteobacterial AOB with specific PCR primers prior to microarray hybridization, and Loy et al. (2005) found that the selective amplification of ―Rhodocyclales‖ 16S rRNA genes prior to microarray hybridization allowed the detection of rare ―Rhodocyclales‖ groups in activated sludge. Group-specific PCR amplification has the advantage of improving detection of the targeted group, but it does require separate PCR reactions for each group, which can limit the number of groups detected in a particular experiment. Several groups have also used PCR amplification prior to microarray hybridization to detect functional genes in environmental samples. For example, Taroncher-Oldenberg et al. (2003) amplified nirS genes from sediment samples using general nirS primers and detected specific nirS variants by hybridization of amplicons to a microarray containing a set of 64 nirS-specific probes. Zhang et al. (2007) used a similar approach to detect nifH variants in roots of wild rice. One limitation of PCR amplification of functional genes prior to microarray hybridization is that each functional gene targeted by the array must be amplified via a separate PCR reaction. This was not a problem for the Zhang et al. (2007) study, as their array targeted only one functional gene, nifH, so universal nifH primers were used to amplify all nifH sequences in the samples followed by hybridization of amplicons to a set of 56 specific oligonucleotide probes on the microarray. However, the need for separate PCR reactions for each targeted gene could be problematic for microarrays targeting a large number of different functional genes. For example, the microarray used by TaroncherOldenberg et al (2003) included probes targeting four distinct functional genes (amoA, nifH, nirK, and nirS), each of which would have required a separate PCR reaction prior to microarray hybridization. Running multiple PCR reactions prior to microarray hybridization is not desirable for a number of reasons: 1) it will significantly increase the time and labor involved in microarray analysis, 2) it could introduce experimental error due to sample splitting, and 3) it limits the main advantage of DNA microarrays, namely the potential for highly parallel analysis of a high number of targets. One way to avoid multiple PCR reactions is via Multiplex PCR, in which multiple primer sets are included in a single PCR reaction. Several groups have utilized multiplex PCR amplification prior to microarray hybridization. Panicker et al. (2004) demonstrated detection of Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of four gene

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371

targets followed by microarray hybridization. Gonzalez et al. (2004) detected five marine fish pathogens by multiplexing nine primer sets and subsequently hybridizing PCR products to a microarray that included nine specific oligonucleotide probes. However, multiplex PCR is restricted in the number of targets that can be amplified simultaneously due to primer-primer interactions (Edwards and Gibbs, 1994). For example, Panicker et al. (2004) were able to multiplex four primer pairs targeting Vibrio parahaemolyticus, but were unable to multiplex ten primer pairs. Pemov et al. (2005) developed a unique approach to multiplex PCR called multiplex microarray-enhanced PCR (MME-PCR) that avoids some of the issues associated with standard multiplex PCR. MME-PCR enables PCR amplification of multiple targets via specific primers immobilized within a gel-pad based microarray. MME-PCR prevents primerprimer interactions because each primer pair is immobilized within a separate gel-pad, so each primer pair is physically separated, and the specific amplification occurs within the gel pad. Amplification within each gel pad is detected by subsequent hybridization of fluorescently labeled, gene-specific probes. Pemov et al. (2005) demonstrated successful, specific on-chip amplification via MME-PCR of six genes from Bacillus subtilis using six different, gene-specific primer pairs. This approach could also be useful for many microbial ecology applications. For example, our lab is currently designing a MME-PCR chip for the detection of specific variants of nitrogen cycling functional genes. Another way to avoid multiple PCR reactions prior to microarray hybridization is through a process known as multiple displacement amplification (MDA). The MDA reaction uses DNA polymerase and random primers to amplify entire genomes (Raghunathan, 2005). MDA has been used to amplify whole genomes from pure cell cultures (Raghunathan, 2005), and Wu et al. (2006) recently developed an MDA method for the amplification of entire genomes from mixed microbial communities, which they termed whole-community genome amplification (WCGA). Wu et al. (2006) used WCGA to amplify all of the genes in a microbial community prior to hybridization to an oligonucleotide (50 mer) functional gene array in order to improve sensitivity. They compared direct hybridization of genomic DNA isolated from groundwater to WCGA amplification of dilutions of this DNA prior to hybridization, and they found that WCGA-assisted microarray hybridization with as little as 1 ng of community DNA produced results that were highly similar to direct DNA hybridization. These results suggest that WCGA may be a useful method for improving the sensitivity of microarray-based assays for low biomass samples. As demonstrated above, the addition of an amplification step to a microarray detection protocol can increase sensitivity and enable the detection of low abundance targets, but the incorporation of an amplification step does create some challenges as well. For example, double stranded nucleic acids such as PCR amplicons do not hybridize as effectively as single stranded nucleic acids (i.e. ssDNA or ssRNA) due to the potential for double stranded DNAs to reanneal instead of hybridizing to the microarray immobilized probes (Zhang et al., 2007). Several studies have avoided this problem by removing one of the DNA strands prior to hybridization via amplification with a 5‘-biotin-labeled reverse primer and subsequent removal of this strand via streptavidin-coated paramagnetic beads (Peplies et al., 2003; Zhang et al., 2007). Another potential challenge related to amplification of targets prior to microarray hybridization is the fact that amplification may introduce biases, as was discussed above for PCR-based assays. Since an amplification step may not amplify all targets in an

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unbiased manner, amplification prior to microarray hybridization may interfere with the ability to extract quantitative information from microarray data.

CHALLENGES FOR MICROARRAY TECHNOLOGY: QUANTIFICATION The value of microarrays as a tool for microbial ecologists would be greatly enhanced if microarrays could not only detect target nucleic acids but also provide quantitative information on their abundance. In theory, it should be possible to extract quantitative information from microarray hybridization if the amount of hybridization to a probe (i.e. the fluorescent intensity) correlates to the amount of target present in the sample. Several studies have confirmed linear relationships between target concentrations and signal intensities for specific probes: Wu et al (2001) found a log-linear relationship between the concentration of target genomic DNA and the hybridization signal for a 760 bp probe, and Rhee et al. (2004) found strong log-linear relationships between signal intensity and DNA concentrations for a large set of 50-mer oligonucleotide probes. Subsequently, Cho and Tiedje (2002) developed a method that involved co-immobilization of functional gene probes (500-900 bp) with a control probe (500 bp) and simultaneous hybridization of differentially labeled target genes and control DNA, and they achieved good log-linearity between signal ratio and DNA concentration ratio for three gene targets. These data confirmed that a correlation exists between the hybridization intensity for a microarray immobilized probe and the concentration of that probe‘s target. However, the challenge for microbial ecologists is that quantification of distinct bacterial populations in a complex community would require the comparison of hybridization signals from multiple probes with a microarray. This is a challenge because different probes can vary significantly in hybridization potential due to differences in probe length (Wu et al., 2001) and G+C content (Siripong et al., 2006) among other factors. These differences in hybridization potential have significant implications. Several studies have shown that redundant probes targeting the same organism often do not produce identical signal intensities when hybridized with RNA from the target organism (e.g. Peplies et al., 2003; Guschin et al., 2007). For example, Loy et al. (2005) demonstrated that probes targeting different regions of the 16S rRNA gene of the same organism can vary by a factor of 240 in signal intensity when hybridized with DNA from that organism. In addition, different probes can yield dramatically different hybridization signals even when hybridized to equal amounts of their respective targets (Ward et al., 2007). These situations make it extremely challenging to extract quantitative information on relative target abundance from multiple probes within a microarray. Since linear relationships have been shown to exist between target concentration and hybridization signal (Wu et al., 2001; Rhee et al., 2004), a standard curve could be developed for each probe on an array (Cho and Tiedje, 2002), as is routinely done for dot-blot membrane hybridizations. However, having to develop a standard curve for every probe on a microarray would be extremely labor intensive, and would limit the main advantage of microarrays, their high probe capacity. Therefore, attempting to determine the relative abundance of distinct bacterial populations in a complex community based on microarray hybridization is a very difficult task, and further work is needed to determine if microarrays can provide this type of information.

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MICROARRAY-BASED MONITORING OF GENE EXPRESSION IN MIXED MICROBIAL COMMUNITIES Functional gene arrays in which the presence of specific functional genes is detected via either direct hybridization of DNA extracted from an environmental sample (e.g. Zhou 2003) or via hybridization of functional genes amplified by PCR (e.g. Taroncher-Oldenberg et al., 2003; Zhang et al., 2007) can provide valuable information on the distribution of specific functional guilds in the environment as well as insight into the functional potential of microbial communities. However, the presence of a functional gene in an environmental sample does not necessarily mean that the gene is being expressed. One way to determine expression is by detection of gene transcripts (mRNAs). As discussed above, microarrays have been used extensively to investigate gene expression patterns in eukaryotic cells, such as human cell lines (DeRisi et al., 1996) and yeast (Lashkari et al., 1997), via analysis of mRNAs. Microarray analysis of mRNAs has been less extensively applied to prokaryotic cells, due to difficulties in priming cDNA synthesis from bacterial mRNA (Dennis et al., 2003). In addition, most microarray studies examining prokaryotic mRNA have been conducted under controlled laboratory conditions on single cell lines or pure bacterial cultures (e.g. de Saizieu et al., 1998; Methé et al., 2005) due to the difficulties associated with obtaining sufficient quantities of high quality mRNA from environmental samples (Parro et al., 2007). One of the first studies to demonstrate the detection of bacterial mRNAs from mixed communities used a microarray containing near-full length amplicons from 25 catabolic genes involved in the degradation of chlorinated aromatic compounds (Dennis et al., 2003). The steps used in this study were extraction of total RNA, synthesis of cDNAs via reverse transcription, labeling of cDNAs, and hybridization of labeled cDNAs to the microarray. This approach demonstrated the induction of catabolic genes in response to substrate additions in pure cultures, in an artificial six member microbial community, and in sludge-fed pulp mill effluent (Dennis et al., 2003). Zhang et al. (2007) used a similar approach to detect expression of nifH gene variants in roots of wild rice samples with a microarray containing short (15-25 mer) oligonucleotide probes. Recently Parro et al. (2007) took a slightly different approach and built a DNA microarray containing a gene library from a single target organism, Leptospirillum ferrooxidans. They used this microarray to assess the relative expression of L. ferrooxidans genes in two habitats with low bacterial diversity that differed in salt and oxygen contents. The results demonstrated increased expression of genes required for adaptation to each of the two habitats: for example, genes related to halotolerance were preferentially expressed in the high salinity environment (Parro et al., 2007). One of the challenges associated with microarray detection of bacterial mRNAs in environmental samples such as soil and sediments is the isolation of sufficient quantities of high quality mRNA for analysis (Parro et al., 2007). Several recent studies have addressed this challenge by developing strategies for amplification of low quantity bacterial mRNAs prior to microarray hybridization. Moreno-Paz and Parro (2006) used random-primed reverse transcription coupled to in vitro transcription as a method for total bacterial RNA amplification, and they demonstrated their ability to amplify as little as 250 ng of total bacterial RNA from a pure culture and successfully hybridize the product to a microarray. Gao et al. (2007) utilized a similar approach, which they termed whole-community RNA

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amplification (WCRA), to amplify RNA from mixed bacterial communities. The WCRA approach used fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis, followed by second strand synthesis and in vitro transcription. This approach resulted in representative microarray detection from as little as 50 to 100 ng total RNA, and Gao et al. (2007) demonstrated that bacterial mRNAs could be detected in groundwater samples via WCRA amplification followed by hybridization to an oligonucleotide (50-mer) functional gene array. The publications described above illustrate the potential for microarrays to monitor bacterial gene expression in environmental samples, which would provide extremely valuable insights into bacterial community function. The success of these pioneering studies should lead to further applications of this approach.

PARALLEL ANALYSES OF MICROBIAL COMMUNITY COMPOSITION AND FUNCTION WITH MICROARRAYS One of the key goals in microbial ecology research is the elucidation of the relationship between the composition of a microbial community (i.e. the species present) and the function of that community, but assessing this relationship for complex microbial communities in environmental samples has always been extremely challenging. As described above, numerous studies have demonstrated the ability of microarray technology to assess microbial community composition, via hybridization of either 16S rRNAs, 16S rRNA genes, or functional genes, and a few studies have demonstrated the ability of microarrays to assess microbial community function through the detection of mRNAs (Dennis et al., 2003; Gao et al., 2007; Parro et al., 2007). Several recent studies have expanded upon this work and developed highly innovative, microarray-based approaches to examine both microbial community composition and function in parallel. Adamczyk et al. (2003) developed an isotope array approach. In this approach, a microbial community is incubated with a 14C-labelled substrate, and after an incubation period, total RNA is extracted from this community, fluorescently labeled, and hybridized with a microarray containing 16S targeted oligonucleotide probes. The array can then be scanned for fluorescence as well as for radioactivity. For each probe on the array, fluorescence indicates the presence of the target organism in the community, and radioactivity indicates that the target organism has incorporated 14C into RNA, indicating active growth and utilization of the labeled substrate. Adamczyk et al. (2003) demonstrated this approach using a microarray containing oligonucleotide probes targeting 16S rRNA genes of ammoniaoxidizing bacteria. In this study, activated-sludge samples were incubated with 14C-labelled bicarbonate, and detection of radioactivity for specific probes on the array was taken as an indication of CO2 fixation by the targeted organisms. This study demonstrated the potential use of an isotope array for the simultaneous assessment of community composition and function. Another novel approach to examining community composition and function was developed by Zhang et al. (2007). They assessed both the presence and expression of different variants of nifH in roots of wild rice using a microarray containing 56 oligonucleotide probes (15-25 mers) targeting nifH variants. In this study, DNA and RNA were coextracted from

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plant roots, and separate aliquots were treated with either DNase I or RNase A to yield pure RNA and DNA samples, respectively. DNA was amplified prior to microarray hybridization via PCR with universal nifH primers. RT-PCR with a universal nifH primer was used to convert mRNA to cDNA, and PCR with universal nifH primers was then used to amplify the RT-PCR products prior to microarray hybridization. In this creative approach, hybridization of amplified DNA indicated the presence of specific nifH variants, and hybridization of amplified mRNA indicated the expression of specific nifH variants. The results of this study demonstrated that only a small subset of the nifH-containing organisms found in the roots were expressing the nifH gene (Zhang et al., 2007). This approach has tremendous potential for simultaneous exploration of microbial community composition and function, which could provide fascinating insights into the functioning of microbial communities in the environment.

APPLICATIONS OF DNA MICROARRAYS TO MICROBIAL ECOLOGY Since the pioneering work of Guschin et al. (1997) the use of microarray technology in microbial ecology research has increased rapidly (Figure 3). The vast majority of microarray studies published to date in the field of microbial ecology have focused on demonstrating the capabilities of microarrays and developing the technology, but recently a growing number of studies have been applying microarrays as tools in studies asking ecological questions.

Figure 3.Number of publications per year that include application of microarray technology to microbial ecology. Numbers are based on a search of the Web of Science database on February 1, 2008 using the following search string: (TS=microarray* OR TS=microchip*) AND (TS=microbial ecology OR TS=microbial community OR TS=microbial detection OR TS=microbial identification). This approach was based on Wagner et al.(2007).

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Some examples include the following: Taroncher-Oldenberg et al. (2003) used a microarray to reveal differences in the distribution of nirS variants in sediment samples from two stations in the Choptank River that differed in salinity, inorganic nitrogen, and dissolved organic carbon. Loy et al. (2002) used a 16S rRNA gene-based oligonucleotide microarray to detect sulfate-reducing prokaryotes in an unusual habitat, a low-sulfate acidic fen, and they found differences in the distribution of these sulfate-reducers in fen samples from different locations. Rhee et al. (2004) demonstrated that the community composition of naphthalenedegraders in soil microcosms differed depending on incubation conditions based on analysis with a functional-gene microarray containing oligonucleotide probes. Sanguin, Remenant, et al. (2006) used a 16S rRNA-based microarray to reveal a significant maize rhizosphere effect on soil bacterial community composition. Ward et al. (2007) demonstrated variations in the composition of ammonia oxidizing communities across a freshwater/marine transect extending from the Choptank River through the Chesapeake Bay and out into the Sargasso Sea using an oligonucleotide (70 mer) functional gene microarray targeting amoA genes. This study also revealed correlations between amoA guilds and environmental parameters, suggesting that different amoA-containing organisms occupy different ecological niches within the estuarine/marine environment (Ward et al., 2007). All of the studies cited above are excellent illustrations of the effective use of microarrays in microbial ecology, and based on these and other successes it is likely that the uses of microarrays in this field will continue to expand rapidly.

CONCLUSIONS Microarrays have the potential to revolutionize the field of microbial ecology via high throughput analysis of microbial community structure, function, and population dynamics. There are significant challenges to the use of microarrays in microbial ecology studies, including optimization of specificity and sensitivity and quantification of targets. However, as reviewed above, the last decade has seen tremendous progress in addressing these challenges, and this progress suggests that microbial ecologists are now on the verge of finally being able to realize the tremendous potential of microarray technology.

ACKNOWLEDGMENTS JJK‘s efforts in preparing this review were supported by a Research Support Grant from Loyola University Chicago and by USDA Grant 2005-35107-16098.

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Pozhitkov, A, B Chernov, G Yershov, PA Noble (2005) Evaluation of gel-pad oligonucleotide microarray technology by using artificial neural networks. Appl. Environ. Microbiol. 71: 8663–8676. Proudnikov, D, A Mirzabekov (1996) Chemical methods of DNA and RNA fluorescent labeling, Nucleic Acids Res. 24: 4535–4542. Purkhold, U, A Pommerening-Röser, S Juretschko, MC Schmid, H-P Koops, M Wagner (2000) Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys Appl. Environ. Microbiol. 66: 5368–5382. Raghunathan, A, HR Ferguson, CJ Bornarth, W Song, M Driscoll, RS Lasken (2005) Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71: 3342–3347. Raskin, L, BE Rittmann, DA Stahl (1996) Competition and coexistence of sulfate-reducing and methanogenic populations in anaerobic biofilms. Appl. Environ. Microbiol. 62: 3847–3857. Rees, WA, TD Yager, J Korte, PH von Hippel (1993) Betaine can eliminate the base pair composition dependence of DNA melting. Biochem. 32: 137–144. Rhee, S-K, X Liu, L Wu, SC Chong, X Wan, J Zhou (2004) Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-Mer oligonucleotide microarrays. Appl. Environ. Microbiol. 70: 4303-4317. Rich, JJ, RS Heichen, PJ Bottomley, K Cromack, DD Myrold (2003) Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils. Appl. Environ. Microbiol. 69: 5974–5982. Rooney-Varga, JN, R Devereux, RS Evans, ME Hines (1997) Seasonal changes in the relative abundance of uncultivated sulfate- reducing bacteria in a salt marsh sediment and in the rhizosphere of Spartina alterniflora. Appl. Environ. Microbiol. 63: 3895-3901. Rosch, C, H Bothe (2005) Improved assessment of denitrifying, N2-fixing, and totalcommunity bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Appl. Environ. Microbiol. 71: 2026–2035. Rotthauwe, JH, KP Witzel, W Liesack (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl. Environ. Microbiol. 63: 4704-4712. Sanguin, H, A Herrera, C Oger-Desfeux, A Dechesne, P Simonet, E Navarro, TM Vogel, Y Moënne-Loccoz, X Nesme, GL Grundmann (2006) Development and validation of a prototype 16S rRNAbased taxonomic microarray for Alphaproteobacteria. Environ. Microbiol. 8: 289–307. Sanguin, H, B Remenant, A Dechesne, J Thioulouse, TM Vogel, X Nesme, Y MoënneLoccoz, GL Grundmann (2006) Potential of a 16S rRNA-based taxonomic microarray for analyzing the rhizosphere effects of maize on Agrobacterium spp. and bacterial communities. Appl. Envir. Microbiol. 72: 4302-4312. Schena, M, D Shalon, RW Davis, PO Brown (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467-470. Siripong, S, JJ Kelly, DA Stahl, BE Rittmann (2006) Impact of pre-hybridization PCR amplification on microarray detection of nitrifying bacteria in wastewater treatment plant samples. Env. Microbiol. 8: 1564-1574.

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Ward, BB, D Eveillard, JD Kirshtein, JD Nelson, MA Voytek, GA Jackson (2007) Ammoniaoxidizing bacterial community composition in estuarine and oceanic environments assessed using a functional gene microarray. Environ. Microbiol. 9: 2522–2538. Weber, S, S Stubner, R Conrad (2001) Bacterial populations colonizing and degrading rice straw in anoxic paddy soil. Appl. Environ. Microbiol. 67: 1318-1327. Wick, LM, JM Rouillard, TS Whittam, E Gulari, JM Tiedje, SA Hashsham (2006) On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes. Nucleic Acids Res. 34: e26. Wilson KH, WJ Wilson, JL Radosevich, TZ DeSantis, VS Viswanathan, TA Kuczmarski, GL Andersen (2002) High-density microarray of small subunit ribosomal DNA probes. Appl. Environ. Microbiol. 68: 2535–2541. Wu, L, X Liu, CW Schadt, J Zhou (2006) Microarray-based analysis of subnanogram quantities of microbial community DNAs using whole-community genome amplification. Appl. Environ. Microbiol. 72: 4931–4941. Wu L, D Thompson D, G Li, RA Hurt, JM Tiedje, J Zhou (2001) Development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl. Environ. Microbiol. 67: 5780-5790. Yershov G, V Barsky, A Belgovskiy, E Kirillov, E Kreindlin, I Ivanov, S Parinov, D Guschin, A Drobishev, S Dubiley, A Mirzabekov (1996) DNA analysis and diagnostics on oligonucleotide microchips. Proc. Nat. Acad. Sci. 93: 4913-4918. Zehr, M, MT Mellon, S Zani (1998) New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. Appl. Environ. Microbiol. 64: 3444-3450. Zehr, JP, BD Jenkins, SM Short, GF Steward (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ. Microbiol. 5: 539– 554. Zhang, L, T Hurek, B Reinhold-Hurek (2007) A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms. Microb. Ecol. 53: 456-470. Zhou, JZ (2003) Microarrays for bacterial detection and microbial community analysis. Curr. Opin. Microbiol. 6: 288-294.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 14

FOOD SAFETY IN INDIA: CHALLENGES AND OPPORTUNITIES Wasim Aktar* Pesticide Residue Laboratory, Department of Agricultural Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, India

1. INTRODUCTION Rising incomes and urbanization, an expanding domestic consumer base concerned about food quality and safety, and rapidly growing agricultural exports have been important drivers for the increased attention to food safety in India. But the development of effective food safety systems is hampered by a number of factors, including: restrictive government marketing regulations, weak policy and regulatory framework for food safety, inadequate enforcement of existing standards, a multiplicity of government agencies involved, weak market infrastructure and agricultural support services. The small farm structure further limits farmer capacity to meet increasing domestic and export food safety and SPS requirements. Addressing food safety concerns in India will require adoption of appropriate legislation, strengthening capacity to enforce rules, promoting adoption of good agricultural, manufacturing and hygiene practices, greater collective action, and some targeted investments. Implementing these actions will require joint efforts by the government and the private sector. Developing countries are paying increased attention to food safety, because of growing recognition of its potential impact on public health, food security, and trade competitiveness. Increasing scientific understanding of the public health consequences of unsafe food, amplified by the rapid global transmission of information regarding the public health threats associated with food-borne and zoonotic diseases (e.g. E. coli and salmonella, bovinespongiform encephalopathy (BSE), severe acute respiratory syndrome (SARs) and H5N1 avian flu) through various forms of media and the internet has heightened consumer awareness about food safety risks to new levels globally (Lindsay 1997, Unnevehr 2003, *

Correspondence to: [email protected]

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Buzby and Unnevehr 2003, Kafersteing 2003, Ewen et al. 2006, Bramhmbatt 2005). Increased understanding of the impact of mycotoxins, which can contaminate dietary staples such wheat, maize, barley and peanuts, has further raised food security and public health concerns in many developing countries (Dohlman 2003, Bhat and Vasanthi 2003, Unnevehr 2003). As developing countries seek to expand agricultural exports especially to OECD countries, many are receiving a wake-up call on the challenges of meeting both government and private sanitary and phyto-sanitary (SPS) standards in export markets (Otsuki et al. 2001, Henson 2003, Unnevehr 2003, World Bank 2005a). Private standards or supplier protocols have grown in prominence over the past decade as a means to further ensure compliance with official regulations, to fill perceived gaps in such regulations, and/or to facilitate the differentiation of company or industry products from those of competitors. Trends in private standards increasingly tend to blend food safety and quality management concerns (i.e. the recent creation of ISO 22000), or to have protocols which combine food safety, environmental, and social (child labor, labor conditions, animal welfare) parameters (Willems et al. 2005, World Bank 2005). At the same time, increasing globalization of trade introduces greater risks of cross-border transfer of food-borne illnesses. Recent cases of disease episodes in the United States resulting from imported food produce, such as cyclospora from raspberries, hepatitis A from strawberries and salmonella from cantaloupe (Calvin 2003), illustrate to developing countries the potential food safety challenges that can arise in a more globalized market. Weaknesses in food safety systems can have a high cost to society and the global economy. The World Health Organization (WHO) estimates that 2.2 million people worldwide die from diarrheal diseases caused by a host of bacterial, viral and parasitic organisms, which are spread by contaminated water (WHO 2006a). In India, it is estimated that 20% of deaths among children under five are caused by diarrheal disease (WHO 2006b). The SARs outbreak in 2003 in East Asia is estimated to have caused an immediate economic loss of about 2% of the Region‘s GDP in the second quarter of that year, even though only 800 people died from the disease (Brahmbatt 2005).1 The Lowy Institute for International Policy (2006) estimates that a mild global outbreak of the avian flu can cost the world 1.4 million lives and close to 0.8% of GDP (US$330 billion) in lost economic output. At the same time, country reactions to protect its citizens from food safety risks can also have large consequences for exporting countries. Otsuki et al (2001) examined the projected impact of the EU‘s new harmonized aflatoxin standard on the value of trade flows to 15 European countries from 9 African countries and found that it could decrease African exports by 64% (US$670 million). Food safety concerns are getting widespread attention in India. The country‘s rural development strategy, for which a key element is the promotion of increased agricultural exports as a means to foster rural growth and poverty reduction, is coming up against tightening food safety and SPS standards in prospective markets (World Bank 2006a, 2006b). From a domestic perspective, the large national market of 1.2 billion people is undergoing rapid change. Increasing incomes, a growing middle class, increased urbanization and 1

The large economic impact resulted primarily from uncoordinated efforts of individuals to avoid becoming infected, contributing to a contraction in services sectors (tourism, mass transportation, retail, hotel and restaurant sales) and workplace absentiism (Brahmbatt 2005).

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literacy, and a population highly tuned to international trends fueled by the information technology boom are creating a large consumer base giving increasing value to food quality and safety. Improving food safety systems, to meet domestic and export requirements, however, face a number of policy, regulatory, infrastructural and institutional obstacles.

2. OBJECTIVES (i) To review the main drivers for the increased priority to addressing food safety risks in India in both the export and domestic markets, (ii)To examine the nature and effectiveness of government and private responses to the food safety challenges, with special focus on high value agriculture; (iii)To identify the constraints to more effective responses; (iv) To examine the implications for policy; v) To review food safety with special relation to Pesticides; and vi) To discuss briefly about the food safety from consumer point of view.

3. TYPES OF FOOD SAFETY RISKS

Figure 1. Food Supply Chain: Potential Sources of Food Safety Hazards

Food safety risks, as they relate to human health, arise from of a number of factors. These include: (i) microbial pathogens (bacteria, viruses, parasites, fungi and their toxins); (ii) pesticide residues, food additives, livestock drugs and growth hormones; (iii) environmental toxins such as heavy metals (e.g. lead and mercury); (iv) persistent organic pollutants (e.g.

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dioxins); and (v) zoonotic diseases (e.g.Avian flu, Japanese encephalitis, tuberculosis) (Buzby and Unnevehr 2003, Ewen et al. 2004).2 The health risks associated with these agents impact the whole food supply chain, starting from input supply to the farm to the consumer table (Figure 1). Common use of pesticides in modern farming inevitably leaves some residues on food crops.

Potential food safety hazards at HOME can be divided into three categories:

2

There remains considerable debate regarding the food safety risks associated with genetically modified organisms (GMOs). The paper will not be covering issues relating to GMOs.

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2. Chemical

3. Physical

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While all the above type of hazards are important from viewpoint of prevention, the focus here will be on the microbiological hazards and in that on foodborne bacteria, which can lead to illness if the food is mishandled, particularly for those more at risk -- the very young, the elderly and the immuno-compromised. Certain processes or handling practices by consumers in the home have been identified as being essential or critical in preventing foodborne illness. These practices, which prevent or control the "meals" microbial contamination associated with foodborne illness, are under the direct control of the consumer, from food acquisition through disposal. They are purchasing, storing, pre-preparation, cooking, serving, and handling leftovers. Failure to take appropriate action at these critical points could result in foodborne illness.

4. PESTICIDES AND FOOD SAFETY Fruits, vegetables and cereal crops treated with pesticides are perceived by some as a health risk, and this belief along with affordability, and time pressures may all play a role in limiting consumption of plant foods, such as cereal grains, fruit and vegetable consumption of consumers in Asia. The World Health Organisation (WHO), the World Cancer Research Fund (WCRF) and many other national and inter-governmental agencies recommend that adults consume at least 400g of fruit and vegetables per day and 25-30 grammes of dietary fibre per day, but analysis of current dietary patterns around the world indicate that many consumer are not achieving these dietary goals, particularly those who are less affluent. AFIC‘s Short Briefing on Pesticides, Food Safety and Health is intended to provide a science-based factual overview of the issue, to enable consumers to make better informed choice about their diet, in particular fruit, vegetables and grains consumption, and allay unwarranted anxieties and concerns.

Definition of Pesticide The Food and Agriculture Organisation (FAO) defines a pesticide as ‗any substance or mixture of substances intended for preventing, destroying, attracting, repelling, or controlling any pest including unwanted species of plants or animals during the production, storage, transport, distribution, and processing of food, agricultural commodities, or animal feeds or which may be administered to animals for the control of ectoparasites‘

Natural Toxins Substances that are capable of causing cancer are virtually everywhere, even in natural compounds. The FDA estimates that the intake of carcinogens from man-made pesticide residues is extremely small compared to carcinogenic residues that plants produce naturally. According to Bruce Ames, a professor of molecular biology and biochemistry at the University of California, more than 99.99 percent of the pesticides Americans ingest are "nature's pesticides" or "natural toxins" (Hotchkiss, 1992; Moore, 1989).

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Natural toxins are present in all plants and such food products as beans, lettuce, apple juice, wine, black pepper, spinach, peanut butter and many others. Of the known natural toxins, which concentrate in parts per thousand versus parts per billion in synthetic pesticides, none has been shown to cause cancer (Hotchkiss, 1992; Moore, 1989).

Reasons of Pesticide Residues in Food Pesticide residues may be present in food because of the following reasons: 1) Direct use of pesticides on food crops; 2) Animal feeding on pesticide treated feed; 3) Environmental contamination

Pesticide Use on the Farm Many of today's food producers are taking an Integrated Pest Management (IPM) approach to preventing, reducing or eliminating pest problems. Growers and processors must make complicated decisions prior to planting, during the growing season, and during postharvest handling. Scientific IPM strategies give the grower economic incentives for sustaining long-term crop protection with minimal disruption to the environment. The agricultural community typically will use pesticides judiciously as part of the IPM strategy whenever proven alternatives are not available for pest control. Growers are hiring professional crop consultants with increasing frequency for advice on maintaining or increasing production through the utilization of IPM programs structured toward their specific agronomic situations.

Integrated Pest Management It is an ecological approach to pest management in which all available control techniques are consolidated into a unified program so that pest populations can be managed in such a manner that economic damage is avoided and adverse side effects are minimized. Practices used as a part of this management philosophy include the following: 1) destruction of crop debris, 2) having pests feed and concentrate on trap crops, 3) crop rotation, 4) selectivity of planting and harvest dates, 5) soil test analysis for crop nutrient needs, 6) planting crop species adapted for local conditions, 7) using genetically improved crop varieties with resistance to specific pests, 8) using biological control, 9) predicting pest outbreaks with computers, 10) pheromones for trapping pests, 11) scouting and monitoring for pests, 12) economic thresholds as guides to pest control, 13) better timing and application of pesticides, 14) use of biological insecticides, 15) improved pesticide application efficiency, 16) adapting promising technology, including the use of infrared scanners, satellite photos, gene-splicing biotechnology, and new pesticide delivery systems that incorporate farm-specific information on tractor mounted computers.

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Pesticide Limits and Regulation Approval for use of any pesticide in a country is subject to its safety evaluation. Safety levels for any pesticide are calculated over a number of formal assessments. The Codex Alimentarius Commission is an international body which sets international guidelines on many elements of food safety, including pesticides residues on food. These guidelines are not mandatory, but many countries in Asia use these guidelines, sometimes with additional scientific data determined by their national regulatory agencies to establish limits on use and also acceptable residue levels at point of sale.

Acceptable Daily Intake One of the most important tools in the safety evaluation of pesticide use on food crops is the calculation of what is an Acceptable Daily Intake (ADI). The ADI for any given pesticide is a measure of the quantity of a particular chemical in food that can be consumed daily over a lifetime without any known risk to health. It is expressed in relation to bodyweight. ADI is derived by first conducting diet trials on laboratory animals and observing the maximum level of pesticide that can be consumed by the animal with no observable adverse effect on health. This level expressed as percentage of body weight is known as the No Observable Adverse Effect Level (NOAEL or NOEL), The investigations include checks for birth defects, cancer, reproductive changes, damage to the nervous system, harm to organs such as the kidney or liver, and many other measurable health indicators. A safe level for human consumption is estimated by dividing the NOAEL on humans by an uncertainty factor (usually 100) to allow for the possibility that humans may more sensitive than the animals used for testing and also to account for possible variation in sensitivity to the pesticide between human individuals, for example adults and children. These results in an ADI for humans which is 100 times lower than the NOAEL consumption rate established from trials on laboratory animals.

Acute Reference Dose Safety evaluation of all pesticides also requires an estimate of the acute refrence dose (ARfD). The ARfD is an estimate of the amount of a substance in food or drinking water expressed as percentage of body weight, that can be consumed over a short period of time, usually one meal or one day, without any known effect on health. This figure is also expressed as a percentage of body weight.

Maximum Residue Levels A maximum reside levels (MRL) is the maximum permissible quantity of pesticide that may still be present on the crop at point of sale. It is derived from an assessment of the residues found when the crop is treated according to good agricultural practices. The MRL is

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the maximum concentration of a pesticide residue that is legally permitted in, or on, a food commodity, and is set by national governments if the approval is given for the use of the pesticide on specified crops. MRLs are set to determine legal trading limit, and are not an indicator of risk to health. MRLs are set at levels which would result in consumption of any residue at a level substantially lower than the ADI or the ARfD for the pesticide, and any pesticide whose MRL could result in dietary intake which might exceed the ADI or ARfD would not receive approval.

Pesticide Residue Monitoring Under FFDCA, the Food and Drug Administration (FDA) and USDA share responsibility for monitoring levels of pesticide residues on foods. FDA enforces pesticide tolerances for all domestically produced food shipped in interstate commerce and in imported foods, except for meat, poultry and some egg products, which are monitored by USDA. Many agriculturallyintensive states such as California and Florida also conduct extensive pesticide residue monitoring programs. FDA uses three approaches for pesticide residue monitoring: 1) incidence/level monitoring, 2) regulatory monitoring, and 3) Total Diet Study (FDA, 1994).

Total Diet Studies To assess potential health problems from contaminants, both natural and man-made in the food supply, the WHO recommends total diet studies (TDS) as the one of the most costeffective means for assuring that people are not exposed to unsafe levels of toxic chemicals through food. TDS provides an additional tool to assess whether or not any pesticides may be present in the diet at levels which might pose a risk to health. A TDS is conducted by purchasing through standard retail outlets a typical selection of foods commonly consumed in the country or region. The ‗basket‘ of foods is processed and prepared as if for normal consumption and then analysed in the laboratory to measure total levels of the substances of interest, for example pesticides. Drinking water and water used in cooking are also included in the assessments. The TDS provides a measure of the average amount of the pesticide consumed by different age/sex groups living in a country. See box for an example of an actual TDS and results for estimate of pesticide consumption.

Risk Calculation Risk = exposure x toxicity. Risk of harm from a chemical depends on both the level of exposure to the chemical and on the toxicity of the chemical (Chaisson et al., 1991). Therefore, to quantify potential risks from consuming minute quantities of a particular chemical residue in food, scientists consider the toxicity of the chemical, the residue content of foods and the amounts of these foods eaten by population subgroups. Population subgroups such as infants, children, women, women of child-bearing age and ethnic subgroups may be considered in risk assessments in addition to the total population. The groups considered

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depend on the toxicologic characteristics of a particular chemical. Risk assessments that consider regional and seasonal variations also are performed.

Exposure = residue concentration in food x amount of food consumed. Potential exposure to a chemical in a specific food is assessed by multiplying the residue concentrations in food times the amount of food consumed by each person in the population. This exposure is expressed as milligrams of residue per kilogram of body weight per day (mg/kg BW/day). Potential dietary exposure to a chemical is assessed by adding together residue intakes from all foods. Different assumptions regarding residue concentrations in food may be used to assess exposure. A worst-case exposure scenario may be calculated using tolerance levels for pesticides in food. This exposure assessment is the theoretical maximum residue contribution. Exposure may also be calculated using anticipated residue levels (Chaisson et al., 1991; California Agriculture,1994).

5. FOOD SAFETY AND THE INDIAN DOMESTIC MARKET Increasing incomes, urbanization, and literacy, improved infrastructure and closer ties to global trends, especially during the last decade, are driving changes in consumer demand and preferences in India. Sustained economic growth (6.0% per year in real terms from 1990/91 to 2003/04) resulted in GDP per capita increasing by about 70%, from about US$315 in 1990 to US$538 in 2004 (constant 2000 dollars). National poverty rates (headcount) declined from 38.9% (Central Statistical Organization 2002) in 1987/88 to 28.5% in 1999/00 (Deaton and Dreze 2002).3 The middle class, which now accounts for about 15% of the 1.2 billion people in India, is the fastest growing income group and is a major force shaping the diet revolution that is occurring (Landes and Gulati 2003). 3

There continues to be a debate on the headcount poverty rate in 1999/00, arising from the adjustment in the design of the 1999/00 National Sample Survey. Depending on the methodology used, the poverty estimates range from 26.1% (Planning Commission) to 28.9% (Sundaram and Tendulkar) (Virmani 2006).

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Figure 2. Diversification on Food Consumption Expenditures

These structural changes are reshaping consumer demand. The Indian food consumption basket is diversifying away from cereals towards higher value and more perishable products, such as fruits and vegetables, dairy, meat and fish (Figure 2). Increasing female participation in the work force and higher disposable incomes to spend on non-home cooked foods are driving growth in demand for prepared and semi-prepared foods, and thus the growth of the processed food industries (Pingali and Khwaja 2004). These trends bring increased attention to safety concerns in the handling, processing and marketing of foods. In addition, growing consumer preference for shopping convenience, increased exposure to the media (TV, cable and the internet) and ownership of durables such as refrigerators and cars are fostering the growth of modern retailing (i.e. supermarkets and hypermarkets), which in turn demand greater efficiency and food quality and safety standards in the supply chain Mukherjee and Patel 2005, Chenggapa, et al 2005). Increased vigilance by NGOs, consumer groups, and local research institutes is also raising awareness and spurring action among consumers and policy makers to address food safety risks. Findings of high levels of pesticides in bottled water and soft drinks in 2003 by the Centre for Science and Environment (CSE), an NGO, shook the country and forced the Government of India (GOI) to take swift action (Mathur et al 2003, CSE 2004). The CSE tested 30 bottled water brands from the major cities of Delhi and Mumbai in Maharashtra and found that all except one contained pesticide residues. The Delhi brands on average contained pesticide residues 36.4 times the maximum pesticide residues stipulated by the European Union standards for bottled water (CSE 2004). Shortly thereafter, Mathur et al. (2003) tested 12 brands of soft drinks sold in Delhi for 16 organochlorine and 12 organophosphorus pesticides and 4 synthetic pyrethroids commonly used in agricultural fields and homes in India. Their analysis found that all brands exceeded the EU maximum pesticide residue limit of 0.0005 ppm (Figure 3).

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Figure 3. Pesticide Residues in Soft Drinks in India, 2003

To deal with the back-to-back crises, the GOI established a special Joint Parliamentary Committee on ―Pesticide Residues in and Safety Standards for Soft Drinks, Fruit Juice and Other Beverages‖ in August 2003 to investigate the allegations. Two GOI Laboratories were instructed to conduct tests on the 12 brands (but using different samples) and their findings showed that 9 of the 12 samples exceeded the EU limits (Hindu Business Line 2003). Weak regulations and inadequate standards were major causes of these high profile food safety crises. In the case of bottled water, while the existing norm set out by the Bureau of Indian Standard (BIS) required that ―no pesticides should be detectable,‖ the prescribed methodology could only detect pesticides at extremely high levels. Consequently, GOI issued a notification revising the standards for pesticide residues on bottled water, adopting the EU single residue limit of 0.0001 ppm and multiple residue limit of 0.0005 ppm (CSE 2004). In the case of soft drinks, the BIS only had voluntary standards, not mandatory standards for pesticide residues. To address the problem, BIS constituted a 39 member committee, consisting of representatives from the soft drinks industry, government scientists, NGOs and consumer groups to formulate the new BIS standards. The outcome was the Indian Ready to Serve Non-Alcoholic Beverages Specifications, which established the limits for 16 pesticides in the finished product (0.0001 mg/l for individual pesticides and total pesticide residue limit of 0.0005 mg/l) (CSE 2004). Even the government-sponsored Mid-day Meals program encountered serious food safety incidents. The National Program for Nutritional Support to Primary Education (NPNSPE), more popularly known as the Mid-Day Meals Scheme, aims to improve child enrollment in primary school and encourage regular attendance by providing supplementary feeding, while improving their nutritional status. It covers children enrolled in classes I to IV in government

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and government-aided schools in the whole country (Jha and Umali-Deininger 2003). In June 2006, 85 students from a Chennai primary school were admitted to the hospital because of food poisoning after consuming food prepared under mid-day meal scheme.4 In February 2004, 281 children attending municipal schools in Delhi fell ill and were admitted to the hospital after consuming their mid-day meal.5 There have been many other cases, despite quality norms being established for the mid-day meal program. While issues related to pesticides in bottle water and carbonated drinks, and out-breaks of food-borne illnesses received wide media attention, there are other serious domestic food safety concerns that have been identified including heavy metal contamination in foods. Marshall, et al. (2003), tested fresh cauliflower, okra, and spinach — common vegetables in the Indian diet — in 5 production sites around the Delhi region and in Delhi‘s Azadpur wholesale market from May 2001 to June 2003. They found that 72% of the 222 spinach samples exceeded the Indian MRLs for lead of 2.5 mg/kg, and 100% exceeded the Codex MRL of 0.3 mg/kg. They attributed the high lead content to a number of possible causes, including contamination of the irrigation water by sewage and industrial effluent and industrial pollution.6 Contamination was exacerbated by their locations—the production sites and market were in peri-urban and urban areas. When tested for zinc, 21% of samples exceeded both the Indian and international standards. Currently, however, no regular testing for heavy metals in vegetables is undertaken by government agencies in India. Tests undertaken by the Indian Council for Agricultural Research found pesticide residues above the MRL in 5.3% of 666 samples of vegetables in 2003 and 15% of 468 samples of milk tested in 2001 (Directorate of Plant Protection and Quarantine 2006). The long term use of pesticides in agriculture and for disease control (e.g. DDT for malaria control) is manifesting itself in the blood, human milk and fatty tissue in the population in many states. Table 1 presents the results of micro-research studies in selected states in India from 1980 to 2005. Table 1. Level of DDT and HCH Content in Human Blood Samples in Selected States in India. Location

Year

Number of Samples

Total DDT (ppm)

Total HCH (ppm)

Lucknow, Uttar Pradesh Delhi Lucknow, Uttar Pradesh Delhi Ahmedabad, Gujarat (rural) Ahmedabad, Gujarat (urban) Punjab (rural)

1980 1982 1983 1985 1992 1997 2005

25 340 48 50 31 14 20

0.020 0.710 0.028 0.301 0.048 0.032 0.0652

00.022 0.049 0.075 0.148 0.039 0.057

Note: HCH - Hexachlorocyclohexane Source: ICMR 2001, Mathur et al. 2005. 4

http://www.newkerala.com/news3.php?action=fullnews&id=11595 http://www.hindu.com/2004/02/27/stories/2004022713760300.htm 6 Potential sources of industrial pollution include emissions from vehicles, industrial plants, coal power generation plants, and diesel generator sets and re-suspended road dust. Marshall et al. found that washing the spinach twice reduced the lead contamination by 50% indicating that a large proportion of the lead was air-borne. 5

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6. FOOD SAFETY CONCERNS IN INDIAN EXPORTS Increased globalization and liberalization of markets, facilitated by the World Trade Organization (WTO), are opening new export markets for Indian agricultural products, both fresh and processed. Indian agricultural exports grew at an average annual rate of 7.2% from 1990/91 to 2003/04. In response to these new opportunities, India‘s agriculture exports diversified from traditional exports of tea, spices, and coffee to include horticultural, fish and livestock products. Between the triennium ending (TE) 1991/92 and TE 2003/04, the value of fresh and processed fruit and vegetable exports rose from US$84 million to US$394 million in real terms (1993/94 dollars) while marine product exports rose from US$516 million to US$1.5 billion during the same period (Figure 4). As Indian agricultural exports diversified, and the value of exports to high income countries increased, India has had to confront new food safety challenges. Concerns over numerous rejections of Indian agro-food exports on food safety grounds have spilled over domestically, generating greater domestic attention to pervasive food safety problems in the supply chain including high levels of pesticide residues, presence of heavy metals in food, and micro-biological contamination. The following section describes recent food safety challenges in Indian horticultural, spice and fisheries exports.

Figure 4. Trend in Agricultural Exports, Triennium Ending (TE) 1990/91 to TE 2003/04

Horticultural Exports In 2004, India exported US$575 million of fresh and processed fruits, vegetables and flowers. Traditionally India‘s fresh fruit and vegetables exports were targeted to markets in neighboring South Asian countries, to the Middle East and to East Asia. Since the early 1990s India achieved some success in exporting fresh horticultural produce to Western Europe. India has been quite proud of its penetration into the U.K, Netherlands and German fresh

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grape markets. Grapes are a highly seasonal crop and Indian exporters have been targeting a crucial March to April window in the European market, which falls at the end of the main southern hemisphere production season (in South Africa and Chile) and before Egypt and Turkey enter the market. Virtually all of India‘s grape exports are of the Thompson Seedless variety. The Indian grape export crisis in May 2003 was a pivotal wake-up call to Indian exporters concerning the costs of failing to meet food safety standards. In the midst of a commercial dispute with an Indian grape exporter, a Dutch importer had samples of the Indian grapes tested by a private laboratory. On finding that the grapes contained residues of the insecticide methomyl in excess of the EU maximum residue limit (0.05 microgram/kg.), the importer placed an advertisement in the local paper warning that grapes from this Indian supplier contained ―poison‖ (World Bank, 2006b). Dutch authorities, who were alerted about the finding, tested samples from the 28 containers of Indian grapes then in Rotterdam port and found that about 75% of the samples exceed the MRLs for methomyl and/or acephate.7 The problem was reported on the EU Rapid Alert system, causing not only significant short term economic losses, but also considerable longer term reputation damage. The price of Indian grapes dropped sharply, and the Indian grape shippers incurred losses, either in Dutch sales or by diverting the shipments to other markets.

Spice Exports India is the world‘s largest consumer and producer of spices and is also a significant exporter of spices (Jaffee, 2005). In 2004/05, India‘s spice exports totaled US$399 million. India, however, has encountered a number of food safety problems in its spice exports including high pesticide residues, aflatoxin contamination and the use of prohibited food colorants. In the mid-nineties, Indian dry chili exports faced several rejections including rejections in Spain due to pesticide residue in excess of permissible MRLs, and in the United States because residues of quinalphos, a pesticide not registered in the United States (Jaffee, 2005). Between 1998 and 2000, Indian dry chili exports also faced rejection in Germany, Italy, Spain and the U.K. due to the presence of aflatoxin.8 More recently, exports of chili and curry powder faced problems due to the use of the prohibited red dye Sudan 1 (Jaffee, 2005). In February 2005, a massive recall of some 600 food products took place in the UK because of the detection of Sudan 1 in Worcester sauce. This was the largest ever food recall in the U.K. and it affected all major retailers as well as large numbers of food manufacturers and food service companies, as the Worcester Sauces had been used in the preparation of a large number of different products. It is estimated that this recall, and associated expenses, cost the U.K. and other European food manufacturers some 200 million Euros (Jaffee, 2005). The source of the Sudan 1 dye in the Worcester sauce was traced to chili powder imported from India in 2002.

7

Of the twenty Indian samples with violative levels of methomyl, six exceeded the MRL by ten times, but most of the others were also far in excess of the MRL (Schee 2004). 8 Aflatoxin may emerge in dried chilies as a result of improper dying (Jaffee, 2005).

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Fish and Fish Product Exports Fish and fish products are one of India‘s largest agricultural export earners, totaling US$1.3 billion in 2004/05. Over the years, India has encountered several food safety problems with its fish and fish product exports. Most prominent, in 1997, the European Commission found the industry to be non-compliant in maintaining hygiene standards in fish processing plants. In May 1997 the European Commission banned Indian exports of fresh crustaceans and cephalopods and imposed border testing for Salmonella and Vibrio spp. for frozen products (Henson, Saqib and Rajasena, 2005). Because of continued detection of salmonella, all exports of fish and fishery products to the EU from India were banned in 1997. While India has for the most part been able to address the hygiene-related problems plaguing its export of fishery products in the late nineties, Indian exports are now under scrutiny because of problems related to antibiotic residues and bacterial inhibitors (antibiotics, preservatives and chlorine) (Henson, Saqib and Rajasena, 2005). It is widely acknowledged that in the future, heavy metals and other contaminants could be an emerging issue particularly because of the increased attention to heavy metals in the EU. Surveillance of fisheries products for heavy metals has already begun in the U.K. Although India has been able to broadly comply with food safety requirements for each of the export commodities mentioned above, it continues to face problems across a range of agro-food exports. Evidence of continuing trouble is clearly apparent from Import Refusal Reports issued each month by the USFDA for food and drug imports into the United States. Most recently, in both April and May 2006, India had one of the highest rejections among all countries exporting to the USA; India faced 176 rejections in May, 2006 and 211 rejections in April, 2006.9 While a significant number of the 176 rejections were issued for drugs and cosmetics, the grounds for rejection among the various food items included salmonella and/or filth in raw peeled shrimp, prepared Indian breads (paratha, roti), basmati rice, sesame seeds, pepper, coriander and chili powder; pesticide residues in lentils; failure to declare the color additive FD & C Yellow No. 5 in banana chips; and unsafe coloring in cream biscuits. The number of rejections and the range of problems reveal extensive safety problems in Indian food products. It is also reasonable to assume that the extent of the problems faced by domestic consumers is far more serious as there many more micro, small and medium enterprises that cater to domestic consumers and generally pay less attention to food safety issues. By contrast, exporters are likely to be more well-established and larger firms with better technology and relatively more cognizant about food safety concerns.

7. CHALLENGES TO IMPROVE FOOD SAFETY IN INDIA Improving food safety in India, whether for the domestic market or for export trade, is hampered by a number of structural, policy, institutional, technical and cultural barriers.

9

India had the most rejections of any country in May and the second highest number of rejections, behind Mexico, in April, 2006. http://www.fda.gov/ora/oasis/5/ora_oasis_cntry_lst.html.

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Policy and Regulatory Environment A number of policies and regulations governing agricultural marketing and food processing complicate the implementation of food safety measures by the government and by the private sector. Two critical marketing regulations are the State level Agricultural Produce Marketing (Development and Regulation) Acts and the Small Scale Industry Reservation Policy. Almost all states in India have an Agricultural Produce Marketing (APM) Act, which gives state governments the sole authority to establish and manage wholesale markets.10 The Act, adopted by most states in the 1960s and 1970s, prescribes the setting up of a network of state controlled ―regulated markets‖ or mandis and the establishment of Market Committees to operate each. All ―notified‖ agricultural commodities grown in areas surrounding the market are required by law to be sold only through these markets, with the number of notified commodities varying by state and market. Implementation of the Act and its enforcement vary considerably by state. In 2005, there were nearly 8,000 regulated markets in the whole country.11 The requirement that all agricultural commodities be channeled through the regulated markets not only increases transactions costs, but is also a major obstacle to preserving produce quality and traceability. In 2003, the GOI formulated a model Agricultural Produce Market Act for state governments to adopt, which removes the restrictions on farmer direct sales and permits entities outside of government to establish and operate wholesale markets. To date only 10 of the 28 states and Union Territories have adopted the model Act.12 The Small Scale Industry (SSI) Reservation restricts the processing of certain commodities to the small scale sector. Although the list of commodities subject to this restriction has been reduced significantly during the last decade, several processed agricultural products are still subject to SSI reservation, such as rapeseed, mustard and ground nut oil,13 bread, pastry, pickles and chutneys, and hard boiled sugar candy (Department of Small Scale Industries 2006). The SSI reservation imposes constraints on enterprises‘ ability to undertake the necessary investments (e.g. HACCP) and certifications required to meet the domestic and international food safety and SPS requirements.14 There is a complex web of laws governing the processed food sector which complicate implementation of food safety measures. These laws are enforced by 8 different ministries. Some of the most critical are: Prevention of Food Adulteration Act 1954 implemented by the Ministry of Health and Family Welfare; Milk and Milk Products Order 1992 and Agricultural Produce Grading and Marking Act 1937 implemented by the Ministry of Agriculture; the Essential Commodities Act 1955, Standards of Weights and Measures Act 1976, Consumer Protection Act 1986, and Bureau of Indian Standards Act 1986 implemented by the Ministry of Food, Consumer Affairs and Public Distribution; the Fruit Products Order 1955 10

The states of Kerala, Jammu and Kashmir, Manipur, Andaman and Nicobar Islands, Dadra and Nagar Haveli, and Lakshadweep do not have the regulation. 11 In 2003, there were 7,383 wholesale markets in the country of which 7,360 were regulated markets. In addition, there were 27,294 rural periodic markets (Ministry of Agriculture as cited in www.indiastat.com). 12 The states that adopted the model Act include : Punjab, Madhya Pradesh, Andhra Pradesh, Orissa, Maharashtra, Rajasthan, Chhattisgarh, Himachal Pradesh, Sikkim and Nagaland. 13 Exceptions are rapeseed, mustard, and ground oil through solvent extraction and those processed by growers cooperatives and state agro-cooperatives (Ministry of Small Scale Industries 2005) 14 This issue is more serious for domestic consumers since food processing units exporting more than 50% of production are not subject to the SSI reservation.

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implemented by the Ministry of Food Processing Industries; import and export regulations implemented by the Ministry of Commerce; Trade in Endangered Species Act implemented by the Ministry of Forest and Environment; Atomic Energy Act 1962/Control of Irradiation of Food Rule 1991 implemented by the Ministry of Science and Technology; and Infant Milk Substitutes, Feed Bottles and Infant Foods (Regulation of Production, Supply and Distribution) Act 1992 implemented by the Ministry of Human Resource Development (Patnaik 2005). These laws also authorize several agencies to lay down standards for food products: (i) Bureau of Indian Standards (BIS) of the Ministry of Food, Consumer Affairs and Public distribution under the BIS Act, (ii) Ministry of Food Processing Industry under the Fruit Products Order, (iii) Ministry of Agriculture under ―Ag Mark‖ and the FPO, (iv) Ministry of Health and Family Welfare (MOHFW) under the PFA Act; (v) Export Inspection Council under the Export-Import Policy, and (vi) the Defense Ministry for their own purchases. These laws and associated regulations in some cases prescribe contradictory or differing standards. For example, while the Fruit Products Order (FPO) allows the use of artificial sweeteners in fruit products, the Prevention of Food Adulteration (PFA) Act bans it. Mandatory declaration labels required by the PFA differ from those of the Packaged Commodity Regulation Rules (1977) under the Standard Weights and Measures Act. The emulsifier and stabilizers permitted for use in jams and chutneys under the PFA differ from those allowed under the FPO. In 1998, the GOI began the process of rationalizing the legal and regulatory framework for food and food processing. The Prime Minister‘s Council on Trade and Industry established a Task Force on Food and Agro-Industries Management Policy to recommend options for rationalizing the various policies and regulations. The outcome was a new Food Safety and Standards Bill, which was submitted to Parliament in August 2005 and is awaiting approval. The Bill aims to consolidate the laws relating to food. The key provisions of Bill include: (i) the repeal of a number of Acts and Orders;15 (ii) the establishment of a Food Safety and Standards Authority of India; (iii) definition of the standards for food additives, contaminants, genetically modified and organic foods, packaging and labeling, and food imports; (iii) accreditation of laboratories, research institutions and food safety auditors; (iv) licensing and registration of food business and setting penalties for offenses; and (v) establishment of a Food Safety Adjudication Tribunal (Ministry of Food Processing Industries 2005). Approval of the Bill will be an important milestone in strengthening food safety systems in India. There are a large number of government agencies involved in agricultural marketing activities, more broadly or with respect to specific commodities, which complicates effective implementation of a coherent food safety strategy for the country. As in the case of the soft drink contamination, the multiple laws and agencies added to the confusion. The BIS was charged with setting the standards for pesticides in soft drinks, while the MOHFW is charged with setting the pesticide standards for bottled water.

15

The laws and orders repealed are the: Prevention of Food Adulteration Act 1954 (37 of 1954), Fruit Products Order 1955, Meat Food Products Order 1973, Vegetable Oil Products (Control) Order 1947, The Edible Oils Packaging (Regulation Order) 1998, Solvent Extracted Oil, De-oiled Meal and Edible Flour (Control) Order 1967, Milk and Milk Products Order 1992, and other orders under the Essential Commodities Act 1955 (10 pf 1955) relating to food.

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Smallholder Agriculture The current structure of the farm sector in India constrains farmer capacity to meet domestic and international food safety standards. Farming in India is dominated by small farmers — the average farm size in 1990/00 was 1.8 ha (NABARD 2002). Most farmers face credit constraints (World Bank 2004), and literacy rates are low.16 These constraints impose limits on the number of farmers capable to adopt more sophisticated farm practices and undertake the necessary investments (e.g. land improvements, obtaining necessary certifications, cold storage) to meet more stringent food quality and safety requirements. They increase the cost of transacting business and monitoring compliance with food safety standards. Stringent land policies, e.g. land ceilings and restrictions on land rental, limit possibilities for greater land amalgamation (World Bank 2006c). International experience indicates, however, that farm size constraints may be overcome through innovative interventions such as organizing farmers into producer groups, establishing collection centers (by supermarkets and exporters), using contract farming arrangements, and by creating public-private partnerships to assist farmers in a variety of ways, including help in obtaining the capital required to make on-farm improvements and other investments (e.g. grading or cooling facilities), developing and improving farming skills through joint extension provision, and assistance in acquiring the required national and international certifications (Berdegué et al. 2003, Boselie et al. 2003, Dries et al 2004, Reardon and Swinnen 2004, Reardon and Timmer 2005a, 2005b). In order to address various food safety concerns in both the spices and fresh and processed fruit and vegetable sectors, some exporters initiated contract farming operations or ―vendor screening‖ programs. One industry that has been especially successful in establishing contract farming arrangements and meeting stringent food safety and quality standards is the pickled gherkin industry. The industry, consisting of some 42 companies and nearly 50,000 smallholder outgrowers, is concentrated in Karnataka, Andhra Pradesh, and Tamil Nadu. The leading gherkin exporting companies each have several thousand farmers under contract. The companies provide intensive oversight and maintain extensive records of farmer practices, especially related to pesticide use. At least one company began the process of getting outgrowers certified under EurepGAP (World Bank 2006b). Contract farming has worked relatively well in the case of gherkins as almost the entire production from India is exported and there is no local market. Hence contract enforcement has not been a major challenge as in the case of other commodities where the export intensity is much lower and the majority of production is consumed domestically. Until recently, contract farming was illegal in India as per the provisions of the APM Act. The only way entrepreneurs can legally enter into contract farming with farmers is to obtain a special waiver from the APM Act from the State Government. The new model APM Act provides the legal framework and guidelines for contract farming. The provisions in the model Act allow contract buyers to directly purchase commodities from farmers under individual contracts or from farmers‘ markets. It also allows the direct sale of farm produce at the farmers‘ fields without having them routed through regulated markets. Adoption of the

16

The rural literacy rate in 1999/2000 was 50% http://www.indiastat.com/india/ShowData.asp?secid=16611 &ptid=367635&level=5

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model Act by state governments will therefore facilitate not only more efficient marketing, but also improved food safety and the adoption of improved agricultural practices.

Weak Extension Systems The public agricultural extension systems at the state level are very weak and have not effectively caught up to the changing needs of farmers and the market (World Bank 2005b). In view of the GOI‘s earlier concentration on food self-sufficiency, the state-level Department of Agriculture (DoA) extension systems generally focused on cereals, particularly rice and wheat, with an emphasis on the transfer of improved varieties and management practices. The weak coordination between the state DoAs and the other line departments (e.g. Departments of Irrigation, Horticulture, Livestock, Marketing, etc) and the limited staff capacity beyond the Department of Agriculture also often translated to limited extension activities beyond cereals, limiting its impact on agricultural and market diversification trends. The weak coordination with research at the central level further increased the difficulty of ensuring effective research-extension-farmer linkages at the state level. In many states, tight fiscal constraints contributed to the breakdown of the state extension machinery (Hanumantha Rao 2003). Private extension provision (fee for service) is emerging. There are an increasing number of input suppliers, traders, contract buyers, supermarkets, and exporters which provide extension services to farmers as an integral part of their trading arrangements (World Bank 2005b). However in the national context, private extension remains limited. Table 2. Farmer Sources of Information.

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The findings of a World Bank agricultural marketing survey, covering 1,579 farmers producing high value crops (tomatoes, potatoes, mangoes, maize and tumeric) in four states in India (Orissa, Tamil Nadu, Uttar Pradesh, and Maharashtra) conducted during February to May 2005, confirm the limited effectiveness of the national extension system. Farmers primarily depended on personal observation or on other farmers for information about crop prices, post harvest practices, irrigation, fertilizer and pesticide use (Table 2). Although food safety concerns have not been a major focus in the extension program, it is partly addressed through the increased Ministry of Agriculture (MoA) priority to integrated pest management (IPM). MoA established the National Center for Integrated Pest Management in1988 to develop and promote IPM technologies. Notably there has been a decline in total pesticide consumption in India from 75,000 mt in 1990/91 to 48,400 mt in 2003/03 (Directorate of Plant Protection and Quarantine 2006).

Poor Infrastructure and Services in the Marketing System Reducing food safety risks from the farm to domestic and export markets is constrained by inadequate infrastructure and facilities, particularly at the wholesale markets. The World Bank Agricultural Marketing Survey also collected information on the operations of 78 wholesale markets in the four states. The survey found that the infrastructure and facilities in these markets are limited and rudimentary. Overall, Maharashtra and UP had slightly better infrastructure than the other two states. About 83% of markets had covered shops, but only 18% had paved roads within the market and 51% had public toilets. Access to warehouses is limited, except in Maharashtra (85%). Less than 40% of markets had a drying area and no markets in Orissa or Uttar Pradesh had cold storage facilities (compared to 5% in Tamil Nadu and 20% in Maharashtra). Waste management and pest control in the markets are very weak. Officials working in the wholesale markets were asked how the spoiled produce and waste products were disposed off. Fifty-four percent responded that market employees or contracted firms handled garbage disposal and waste management; 29% reported that they were just left to rot in the market, while 13% reported that they were left for the animals to eat. Market officials were also asked about the pest control measures they undertake. Fifty-nine percent indicated that no particular control measure for rats and insects are implemented in their market, 32% indicated it was up to the individual shop owners to take care of their rat problems. Only 8% reported the market management or association or a subcontracted firm took care of rat problems. Reducing food safety risks will require significant public and private investments to upgrade the market infrastructure and services. For regulated markets, this will also require improving the operational and fiduciary management to ensure that more resources are re-invested back into the markets.

Cultural Issues Religious beliefs further constrain the kinds of food safety measures that could be adopted in India. The sacred value attached to cattle imposes limits on disease control

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measures to address food safety and public health (BSE, foot and mouth disease), such as culling to limit disease spread or to create disease free zones.

Inadequate Grades and Standards for the Domestic Market and Poor Enforcement The Directorate of Marketing & Inspection under the Department of Agriculture and Cooperation is responsible for enforcing and implementing the Agricultural Produce (Grading and Marking) Act. Its mandate includes promoting standardization and grading of agricultural products. Grades and standards have been prescribed for 164 commodities under the APM Act for domestic trade, for export trade and for grading at the producer‘s level. The AGMARK grades are primarily voluntary grades covering aspects such as size, variety, weight, color, and moisture levels. For certain items they also cover parameters such acceptable levels of organic and inorganic foreign matter (in pulses, for example) and other chemical properties such as specific gravity for essential oils. Different grades and standards are laid out under AGMARK for domestic consumption versus exports. The Directorate provides third party certification under the AGMARK quality certification scheme. The ‗AGMARK‘ seal is supposed to ensure quality and safety. Any consumer, trader or manufacturer can have products tested at one of the 23 regional AGMARK laboratories for designated commodities. Typically, testing is only carried out for adulteration prone commodities such as oils, ghee, whole and ground spices, honey, and whole and milled food grains. Blended edible vegetable oils and fat spreads are compulsorily required to be certified under AGMARK. The Prevention of Food Adulteration Act also sets standards for food products including aspects such as permissible food colorings, preservatives, pesticide residues, packaging and labeling. As illustrated by the bottled water and soft drink pesticide residue incidents, inadequate standards and weak enforcement remain a problem. The grades specified under AGMARK and standards as laid out in the PFA are designed to facilitate trade as well as ensure food safety. The food safety standards under the PFA in general need to be aligned with international standards. However there are many commodities that are not grown or consumed outside of India. For these commodities it may not be possible to align domestic standards with international standards because there are no established international standards. In these instances it is important for research to be conducted in India to set appropriate standards for the domestic market.

Lack of Pro-Activity in Addressing Food-Safety Issues Domestic food safety scares and the more notable food-safety problems faced by Indian agro-exports, reveal the overall absence of any pro-activity in addressing food safety concerns in India. Several factors contribute to this. In the case of exports, many if not most of the emerging SPS and international standards are widely viewed as not scientifically based and as representing unfair ―barriers to trade‖ (World Bank, 2006b). These measures are viewed as efforts to protect foreign farmers or processors from competition, or are being fueled by

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unreasonable consumer fears in high income countries and improved technologies for detecting hazards. Consequently, the approach of the government and private sector has been to try to negotiate away the problems with trading partners and, failing that, addressing the various measures in international standard-setting or dispute flora. As a consequence, insufficient attention is devoted to monitoring the requirements of official and private standards, interpreting their implications for Indian agriculture and using current and anticipated requirements as catalysts to upgrade existing operations and strengthen supply chain management (World Bank 2006b). This absence of pro-activity has meant that India has either had to adopt a ―defensive‖ strategy avoiding markets with more stringent food safety and agricultural health standards or launch into a fire-fighting mode when it faces potential disruption or loss of trade due to noncompliance with standards.17 The absence of pro-activity is well illustrated through examples of problems faced with exports of fishery products in the late nineties and the more recent troubles with grape exports to Europe. In both cases, although there were signs of potential problems for a considerable period of time, the food safety problems were not given serious attention until India was faced with a crisis. In the case of exports of fish and fishery products, necessary monitoring and enforcement measures for ensuring that exports complied with food safety concerns were not put in place until the loss of EU markets in 1997 (Henson, Saqib and Rajasena, 2005). This was despite the fact that India had continually faced rejections because of failure to meet hygiene standards and other food safety requirements since the 80s, and in spite of regulatory reforms to provide safety assurance for fish and fishery products undertaken in 1995 (Henson, Saqib and Rajasena, 2005). Similarly, in the case of grape exports to the EU, pesticide residue problems had surfaced since the late nineties. During this period, some limited testing was done for pesticide residues in export-oriented grapes. Testing was made mandatory in 2000, but most of the available testing equipment was not up to date, could not test to the same level of detection as was common in Europe and was unable to detect certain heat-sensitive chemicals such as acephate and methomyl (World Bank, 2006b).18 Only after EU Rapid Alerts were issued in 2003 did the Government and industry step into action to address the problem. In general India has not viewed complying with food safety and agricultural health standards as a means to both improve its competitive position and to enhance the effectiveness of its negotiations on particular technical and commercial matters, which is in stark contrast to the approach of leading agro-food exporting countries (World Bank, 2006b). A consequence of the lack of pro-activity and the crisis management mode of operation has been the adoption of very rigorous and strict controls for commodities threatened with the loss or disruption of trade. This has led to extremely high costs of compliance in some cases (e.g. grapes) (World Bank, 2006b) or rather onerous requirements (e.g. requirements for processing facilities exporting fishery products) (Henson, Saqib and Rajasena, 2005). In the case of grapes, the Government of India (GOI) Agricultural and Processed Food Products Export Development Authority (APEDA), formulated an integrated system of intensive grape supply chain oversight that included: 17

An example of a defensive strategy is the existing trend where many of India‘s mango pulp exporters are forced to sell to less remunerative markets because they are not HACCP compliant. 18 As reported in Buurma et al 2001.

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A requirement that all farms growing grapes for export to Europe have to register with the Department of Agriculture. About 6200 growers registered for the 03/04 season; Three field inspections (for registered exporters) during the crop cycle by a newly constituted cadre of horticultural field inspectors. Some 244 such officers were initially appointed and trained. There are now 291 such officers; The inspection and registration of all grape export packinghouses by APEDA. Mandatory pesticide residue testing from each registered field of export grapes. Testing would be done prior to harvest and only if the tests were passed would authorization be given for harvesting for export. Grapes from fields with failed results would need to be sold in other markets or re-tested. Every consignment would be checked by AGMARK to ensure conformity with EU quality specifications for grapes. AGMARK would issue certificates. Obtaining a phytosanitary certificate issued by Plant Protection, Quarantine and Storage for every consignment; and Later, in 2005, another procedure was added whereby National Research Center for Grapes would take a 5% sample of ex-packhouse grape consignments to re-test for pesticide residues.

The extensive system of checks and controls primarily focused on end-of-the-pipeline solutions. In addition to the protocols that potential exporters to the EU have to follow, the government also invested heavily in upgrading laboratory testing equipment, training field inspectors, subsidizing packhouse upgrades, and strengthening the National Research Centre for Grapes. Overall, it is estimated that the cost of this control system for pesticide residues (to government and the private sector) is about US$1.2 million, equivalent to 7.9% of the FOB value of India‘s grape trade to Europe in 2005 (Table 3). If all other costs associated with the oversight of the grape supply chain are added to the costs of pesticide residue testing, SPS compliance costs are estimated to account for 13% of this FOB value. Table 3. Estimated Annual Cost of Meeting EU SPS Standards—2005 US $

While it is arguable that there are many spillovers and important lessons that have been learned from the handling of the pesticide residue problem with grape exports, and that these measures have been ―successful‖ in that they have not resulted in further alerts or rejections, the heavy handed approach with which the problems were addressed, and the costs involved,

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clearly suggest that it is not a strategy that should be replicated. Although India has not faced further rejections of exports to the EU, routine laboratory testing still reveals violative residues, indicative of the continuing need to focus on improving overall agricultural practices to assure food safety.

Lack of Good Agricultural, Manufacturing and Hygiene Practices In addition to constraints that arise due to small farm sizes, the lack of good agricultural, manufacturing and hygiene practices remain a major challenge for improving food safety both for the domestic and export market. It is only recently that efforts are being made to promote good practices. For example, Marine Products Export Development Authority (MPEDA) promoted codes of good practice, particularly with regards to addressing antibiotic use. To this extent the organization was involved in monitoring antibiotic usage levels, providing training and disseminating information (Henson, Saqib and Rajasena, 2005). In the spices sector, the Spices Board (SB) undertook measures to address problems with regards to pesticide residues and aflatoxin. The SB, in conjunction with State Departments of Agriculture and various NGOs, supported measures to promote integrated pest management (IPM) and the production of organic spices (Jaffee, 2005). They helped address the aflatoxin concern by promoting better drying practices. The Ministry of Food Processing Industries and APEDA have both been promoting adoption of HACCP and ISO certification among processed food manufacturers through a range of training initiatives and private sector investment grant for upgrading processing plants to obtain HACCP/ISO certification. However, the adoption of good practices remains limited. Much remains to be done in improving practices with regards to the manufacture and use of pesticides and improving post harvest techniques. Although there have been some limited spillovers from the export sector into the domestic market, in terms of improving production practices, for most commodities, including spices and fresh fruit and vegetables, farmers do not necessarily see any advantages or necessity for altering their production practices since the vast majority of production is consumed in the domestic market. Until domestic consumer awareness and willingness to pay for improved food safety becomes more widespread, it is unlikely that addressing food safety concerns will become standard practice nationally. Similarly, significant measures are needed to improve the safety of processed foods. In the food processing sector there are a growing number of firms with modern factories and good quality assurance systems, but this segment co-exists with large numbers of small and older firms that would need to make significant upgrades to implement HACCP and other quality assurance systems.19 In the short term, developments in the food retail sector in India are likely to bring about improvements in food safety. International experience shows that modernization of the food retail sector is an important driver for change not only in the structure of production and wholesale marketing of produce, but also in fostering adoption of improved grades and food safety standards (Berdegué et al 2003, Reardon and Timmer 2005a, 2005b). Despite the ban on foreign direct investments in food retailing, the supermarket industry is growing rapidly,

19

For instance, in recent work on the mango pulp sector in India one company reported costs of $35,000 to put it in a position to implement a proper HACCP system (World Bank, 2006b).

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driven by investments from the Indian corporate sector.20 Many of the modern retail outlets are beginning to undertake direct procurement from individual farmers or farmers‘ associations. In some cases farmers or associations supplying these outlets are required to follow a code of practice to meet quality and safety requirements of their buyers. The retail outlets are also involved in disseminating new agricultural techniques and information to their suppliers as well as providing training on quality control of produce handling, grading and packaging. There are also efforts by the public sector to promote good agricultural practices among producer groups and to help establish linkages with the organized food retail sector.21 The Government of India and State governments are working closely with the supermarket industry (with support from USAID) to develop an India Good Agricultural Practice standard for agricultural produce (INDIA-GAP), which will in turn also provide the framework for government extension support to farmers.

Need for More Collective Action International experience highlights the importance of collective action within the private sector to promote awareness of SPS matters, find solutions to emerging challenges, promote good agricultural and manufacturing practices, and otherwise provide a degree of selfregulation, which in turn reduces the need for government agencies to play enforcement roles. While there are some examples of successful collective action in both the spice and fishery export industries in India, it has been lacking in many other sectors, notably in horticulture (World Bank, 2006b). For example, the Seafood Exporters Association of India (SEAI) has developed a model to provide a number of pre-processing units with common water, ice and effluent infrastructure. SEAI in collaboration with MPEDA has also been involved in developing a system to ensure traceability for shrimp from aquaculture in order to address quality problems (Henson, Saqib and Rajasenan, 2005). In the Spices sector, the All India Spice Exporters Forum has been an important player in trying to influence standards for pesticides in spices grown under tropical conditions and in finding solutions to address food safety concerns in its export markets (Jaffee, 2005).

20

Corporate manufacturers such as Hindustan Lever Ltd, International Tobacco Company, Godrej, Bharti,Reliance, DCM Sriram Conolidated, RPG Group, Pantaloon Group are setting up or have set hypermarkets, supermarkets and retail outlets in rural areas, recognizing the huge untapped potential (World Bank 2006a). Gasstation stores are also another growing retail outlet. Petroleum companies like Hindustan Petroleum Corporation Limited, Indian Oil, and Bharat Petroleum have introduced branded outlets like Speedmart (around 60-65 in number), ConveniO‘s (around 150), and In&Out Stores (around 100) which sell food items (Singh 2004). 21 The Marashtra Agricultural Agricultural Marketing Board in collaboration with USAID is trying to promote good agricultural practices among mango farmers in the state and link these farmers with various supermarkets and other retail outlets that are interested in procuring better quality and safer fruit.

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8. CONSUMER CONTROL POINTS Minimizing Possible Consumption of Pesticide Residues For those consumers who wish to take additional measures to reduce any possible pesticide residues on their foods, here are some tips from AFIC. 1) Raw foods should be washed thoroughly before cooking and/or consumption. Washing in dilute vinegar solution, or solution of sodium bicarbonate, then rinsing with clean water will help to remove any chemical residues and also any soil or other foreign matter on the produce. 2) Many chemicals applied to crops to protect from insects and disease are sprayed onto external surfaces, so peeling outer layer or skin when preparing fresh fruit and vegetables will remove any surface residues. 3) Look out for the many of the quality assurance schemes, which guarantee chemical treatment of produce has strictly followed manufacturers recommendations and residues levels at point of harvest are either zero or very low. There are also an increasing number of retailers and growers offering ‗organic‘ produce, however, be aware that ‗organic‘ farming often uses some pest control substances, approved by the various associations established to promote this form of cultivation. 4) Do not consumer berries, leaves or other edible plant material picked from roadsides or other public areas, as these plants as it is not possible to know if these plants have been sprayed intentionally or unintentionally contaminated with pesticides or other substances and will not be subject to safety restrictions of designated food crops. Certain processes or handling practices by consumers in the home have been identified as being essential or critical in preventing foodborne illness. These practices, which prevent or control the "meals" microbial contamination associated with foodborne illness, are under the direct control of the consumer, from food acquisition through disposal. They are purchasing, storing, pre-preparation, cooking, serving, and handling leftovers. Failure to take appropriate action at these critical points could result in foodborne illness.

Critical Point 1: Purchasing  



Purchase as far as possible perishables on a daily basis. Procure processed food items only after checking properly the `Best Before date ‗ validity and prefer processed branded food items with product certification marks viz. ISI, FPO & AGMARK. Procure milk and milk products, fish, seafood, poultry, meat and meat products, eggs and other perishables last when out on purchasing and keep packages of raw meat and meat products separate from other foods, particularly foods that will be eaten without further cooking. Consider using plastic bags to enclose individual packages of raw meat and meat products.

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Make sure milk and milk products, fish, seafood, poultry, meat and meat products, eggs and other perishables are refrigerated as soon as possible after purchase. Plan to drive directly home after purchases from the grocery store. Canned food items, if purchased, should be free of leaks, dents, cracks or bulging lids.

Critical Point 2: Home Storage 

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

 

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Verify the temperature of your refrigerator and freezer with an appliance thermometer - refrigerators should run at 5°C or below; freezers at -18°C. Most foodborne bacteria grow slowly at 5°C, a safe refrigerator temperature. However, freezer temperatures of -18°C stops bacterial growth. Keep High risk products such as fish, seafood, poultry, meat and meat products in the freezer cabinet of refrigerator immediately on reaching home. Keep the milk and milk products in the designated racks in the refrigerator or on the first topmost rack just under the freezer cabinet. To prevent raw juices drippings & pieces of meat and meat products from falling on cooked to other foods kept in the refrigerator. Keep them in shelves below the stand cooked food. Use plastic bags or place meat and poultry on a plate. Wash hands with soap and water for 20 seconds before and after handling any raw fish, seafood, poultry, eggs, meat and meat products. Store canned foods in a cool, clean dry place and as per the labeling directions of the manufacturer. Avoid extreme heat or cold which can be harmful to canned food items. Never store any foods directly under a sink and always keep foods off the floor, on a rack or pallet and completely separate from cleaning supplies.

Critical Point 3: Pre-Preparation 



 

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The importance of hand washing cannot be overemphasized. This simple practice is the most economical, yet often forgotten way to prevent contamination or crosscontamination. Wash hands with soap and water thoroughly (for 20 seconds): before beginning preparation; after handling raw meat, poultry, seafood or eggs; after touching animals; after using the toilet/bathroom; after changing diapers; or after blowing the nose. Don't let juices from raw meat, poultry or seafood come in contact with cooked foods or foods that will be eaten raw, such as fruits or salads. Wash hands, kitchen slabs / tables, equipment, utensils, and cutting boards with soap and water immediately after use. Kitchen slabs / tables, equipment, utensils and cutting boards can be sanitized with a chlorine solution of 1 teaspoon liquid household bleach per quart of water. Let the solution stand on the board after washing, or follow the instructions on sanitizing products. Before processing frozen fish, seafood, poultry, meat and meat products, thaw the same properly in the refrigerator but NEVER ON THE KITCHEN SLAB / TABLE.

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

It is also safe to thaw in cold water in an airtight plastic wrapper or bag, changing the water every 30 minutes till thawed. Thawing may also be carried out in the microwave and followed immediately by cooking. Marinate foods in the refrigerator and NEVER ON THE KITCHEN SLAB / TABLE.

Critical Point 4: Cooking 

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



Make sure to cook food thoroughly, particularly the meat products, Depending upon the type of dish made, thoroughly cooking would be signified by colour, texture and taste. For example boiled/steamed rice shall have grains double the size of original rice grains, with each grain separate; Fried item viz cutlets, pokoras etc should be brought to golden brown coloir at medium flame, which ensures thorough cooking upto the core of the fried item. If harmful bacteria are present, only thorough cooking will destroy them (core temperature of product to be higher than 75 °C) ; remember freezing or rinsing the foods in cold water is not sufficient to destroy bacteria. Avoid interrupted cooking. Never refrigerate partially cooked products to later finish cooking on the grill or in the oven. Fish, seafood, poultry, meat and meat products must be cooked thoroughly the first time and then they may be refrigerated and safely reheated later. When microwaving foods, carefully follow manufacturers instructions. Use microwave-safe containers, cover, rotate, and allow for the standing time, which contributes to thorough cooking.

Critical Point 5: Serving:  

 

Wash hands with soap and water before serving or eating food. Serve cooked products on clean plates with clean utensils and clean hands. Never put cooked foods on a dish that has held raw products unless the dish is washed with soap and hot water. Hold hot foods above 60°C and cold foods below 5°C. Never leave foods, raw or cooked, at room temperature longer than 2 hours.

Critical Point 6: Handling Leftovers      

Wash hands before and after handling leftovers. Use clean utensils and surfaces. Divide leftovers into small units and store in shallow containers for quick cooling. Refrigerate within 2 hours of cooking. Discard anything left out too long. Never taste a food to determine if it is safe. When reheating leftovers, reheat thoroughly (temperature of 75 °C) until the dish is hot and steamy. Bring soups, sauces and gravies to a rolling boil. If in doubt, throw it out.

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Ten Steps to a Safe Kitchen: Step 1 Keep your refrigerator at 4° C or less.A temperature of 4° C or less is important because it slows the growth of most bacteria. The fewer bacteria there are, the less likely you are to get sick from them. Step 2 Refrigerate cooked, perishable food as soon as possible but within two hours after cooking. A temperature of 4° C or less is important because it slows the growth of most bacteria. The fewer bacteria there are, the less likely you are to get sick from them. Date leftovers so that they can be used within one day. If in doubt, throw it out! Step 3 Sanitize your kitchen dishcloths and sponges regularly. Wash with a solution of one teaspoon (5 ml) chlorine bleach to one litre of water, or use a commercial sanitizing agent, following product directions. Step 4 Wash your cutting board with soap and hot water after each use to prevent any subsequent contamination in food during preparation. Never allow raw meat, poultry, and fish to come in contact with each other as they have generally high bacteria count including pathogens which cause food poisoining .Washing with only a damp cloth will not remove bacteria. Periodically washing in a bleach solution is the best way to prevent bacteria from remaining on your cutting board. Step 5 Cook meats , seafood and poultry products thoroughly so as to ensure that cooked food is free from harmful bacteria. Cooking meat products on a law/medium flame such that the core temperature reaches at least 75° C usually protects against foodborne illness. Welldone meats reach that temperature. Step 6 Don't consume raw or lightly cooked eggs as they may contain the harmful Salmonella bacteria. Always cook the eggs thoroughly before eating them. Step 7 Clean kitchen counters and other surfaces that come in contact with food with hot water and detergent or a solution of bleach and water. Bleach and commercial cleaning agents are best for getting rid of pathogens. Hot water and detergent are good, but may not kill all strains of bacteria. Keep sponges and dishcloths clean because, when wet, these materials harbor bacteria and may encourage their growth. Step 8 When washing dishes by hand, it‘s best to wash them with warm water and detergents all within two hours--before bacteria can begin to form. Allow dishes and utensils to air-dry in order to eliminate re-contamination from hands or towels. Step 9 Wash hands with soap and warm water immediately after handling raw meat, poultry, or seafood. Wash for at least 20 seconds before and after handling food, especially raw meat. If you have an infection or cut on your hands, wear rubber or plastic gloves. Step 10 Defrost frozen meat, poultry and fish products in the refrigerator, microwave oven, or cold water that is changed every 30 minutes. Cook microwave-defrosted food immediately after thawing. Changing water every 30 minutes when thawing foods in cold water ensures that the food is kept cold, an important factor for slowing bacterial growth on the outside while inner areas are still thawing.

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CONCLUSION The Indian experience illustrates the many challenges faced by developing countries in addressing food safety concerns in domestic and export markets. Despite a large number of food safety incidents in the past, it is only in the past five years or so that food safety issues have begun to receive greater attention. As elaborated in this paper, this has partly been due to greater consumer awareness arising from campaigns led by NGOs, increased coverage of food-safety incidents in the media, wider access to media and the internet, and the problems encountered with agro-food exports in high income markets. Despite this, considerable efforts are still needed to give the issue of food safety the attention it warrants. Because of low consumer awareness, the private sector engaged in agriculture, food processing and the food retail industry in India, for the most part, has not taken the necessary steps to improve the quality and safety of food products. In most cases, the responsibility of ensuring food safety fell into the hands of government through enacting and enforcing legislation and setting standards. While government has taken actions in instances where there have been immediate public health scares or disease outbreaks, less attention has been given to food safety concerns whose impact is only apparent in the medium to long-term. One of the positive results of globalization and the emergence of modern food retailing in India is the increased attention to quality and safety issues. As incomes are increasing, consumers are also more willing and able to pay for better quality and safer food. Addressing food safety issues in India will require the adoption of more appropriate legislation and their enforcement (Table 4). Parliamentary approval of the Food Safety and Standards Bill will be critical to removing the uncertainty arising from, and the associated additional cost of dealing with, overlapping and conflicting food safety regulations. Broad based adoption of the model APM Act and the removal of the remaining agricultural commodities from the SSI reservation will foster both increased market efficiency and facilitate adoption by firms of appropriate food safety measures. Joint efforts by the government and the private sector will be needed in a number of areas. These include better risk management, the promotion and adoption of good agricultural, manufacturing and hygiene practices, greater collective action and some targeted public investments. Responsibilities for these functions need to be shared between the private and public sectors. While there are many critical regulatory, research and management functions that are normally carried out by governments, the private sector also has an important role in the actual compliance with food safety requirements. Quality grades should be voluntary for fruits, vegetables and for most other fresh produce, since they are set primarily to facilitate trade and are not a regulatory instrument. Yet, for matters of food safety, standards should be mandatory rather than voluntary. These standards would apply for pesticide residues, heavy metal and other forms of environmental contamination, and especially for microbiological contaminations for which there could be acute health risks. A coordinated program of food safety product surveillance can be used to highlight the nature and scope of pertinent problems and also be used as a basis for developing consumer and supply chain awareness and good practice promotion. Overall there is a large role for extension service providers to promote good practices in order to ensure that farmers follow recommended dosages for agro-chemicals and observe appropriate pre-harvest

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intervals. Soil and water testing should also be routinely conducted through the extension apparatus (World Bank, 2006a). Table 4. Role of Public and Private Sector in Enhancing Food Safety Capacity. Role of Public Sector Policy and Regulatory Environment • Adopt domestic food safety legislation and standards suited to local risk conditions and preferences and consistent with India‘s WTO and other treaty obligations. Risk Assessment and Management: • Strengthen national or state-level systems of pest and animal disease surveillance and market surveillance programs to gauge the incidence of various food safety hazards in the domestic agrofood system. • Find solutions to animal health constraints that limit domestic (for imports) and foreign (for exports) market access. This might entail, product inspection, agreed development of disease-free areas. etc Awareness building and promoting good practices: • Consumer awareness campaigns about food safety risk and improve hygiene in the home • Raise stakeholder awareness about and promote good agricultural, hygiene, and manufacturing practices and quality management. Incorporate these areas into curricula of public agricultural/ technical institutes and universities. • Incorporate food-safety focused practices in extension program, including through public-private partnerships • Accredit private laboratories and conduct reference/consistency testing. • Facilitate technical, administrative and institutional change and innovation within the private sector for example through publicprivate partnerships Public Expenditures • Investments in water supply and sanitation, marketing facilities, to reduce food safety hazards • Support research to address food safety and agricultural health concerns International Trade Diplomacy: • Undertake continuous dialogue and periodic negotiations to address emerging constraints or opportunities.

Source: Adapted from World Bank 2006a.

Role of Private Sector ‗Good‘ Management Practices: • Implement appropriate management practices to minimize food safety risks. Examples include ‗good‘ agricultural, hygiene, and manufacturing practices and HACCP principles. • Where commercially valuable, gain formal certification for such adopted systems. • Develop incentives, advisory services and oversight systems to induce the similar adoption of the above ‗good practices‘ by supply chain partners. Traceability: • Develop systems and procedures to enable the traceability of raw materials and intermediate and final products in order to identify sources of hazards, manage product recalls or other emergencies, etc. Develop Training, Advisory, and Conformity Assessment Services: • On a commercial basis provide support services to agriculture, industry, and government related to quality and food safety management. Invest in the needed human capital, physical infrastructure and management systems to competitively supply such services. Collective Action and Self-Regulation: • Work through industry, farmer, and other organizations to share the costs of awarenessraising and systems improvement, alert government to emerging issues, advocate for effective government services, and provide a measure of self-regulation through the adoption and oversight of industry ‗codes of practice‘

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The use of agricultural chemicals in relation to food safety will continue to be a complex subject. Some studies have shown that pesticides may affect ground-water, wildlife and occupational workers if the chemicals are not used in accordance with the law. But the future looks promising as food scientists, research-ers, government officials and manufacturers search for methods to improve agricultural techniques while reducing pesticide-related risks. Today's consumers can feel confident that they can choose from an abundant and safe food supply for themselves and their families. There is also a need for regular inspection of health and sanitary conditions at certain types of food premises that may be associated with more severe consumer health risks, (abattoirs, for example). Inspection should not be random, but should be targeted based upon risk assessments that government may do on different types of food establishments to help pinpoint areas requiring particular attention, not only in the form of inspection, but also including awareness-raising, training, periodic licensing, etc. The challenges for ensuring food safety in the domestic market and in its food exports remain large. India has made some progress in the last decade to strengthen food safety measures at home and in meeting food safety and SPS standards abroad. The challenge for the future will be to adopt a more strategic, rather than crisis management approach. This will be essential to ensuring the sustainability and cost effectiveness of these efforts.

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In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 15

IMPACT OF PESTICIDE USE IN INDIAN AGRICULTURE THEIR BENEFITS AND HAZARDS Wasim Aktar* Pesticide Residue Laboratory, Department of Agricultural Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, India

1. INTRODUCTION The term pesticide covers a wide range of compounds including insecticides, fungicides, herbicides, rodenticides, molluscicides, nematicides, plant growth regulators and others. Among these, organochlorine (OC) insecticides, used successfully in controlling a number of diseases, such as malaria and typhus, were banned or restricted after the 1960s in most of the technologically advanced countries. The introduction of other synthetic insecticides – organophosphate (OP) insecticides in the 1960s, carbamates in 1970s and pyrethroids in 1980s and the introduction of herbicides and fungicides in 1970s - 1980s contributed greatly in pest control and agricultural output. Ideally a pesticide must be lethal to the targetted pests, but not to non-target species, including man. Unfortunately, this is not, so the controversy of use and abuse of pesticides has surfaced. The rampant use of these chemicals, under the adage, ―if little is good, a lot more will be better‖ has played havoc with human and other life forms.

1.1. Production and Usage of Pesticide in India The production of pesticides started in India in 1952 with the establishment of a plant for the production of BHC near Calcutta, and India is now the second largest manufacturer of pesticides in Asia after China and ranks twelfth globally[9]. There has been a steady growth in the production of technical grade pesticides in India, from 5,000 metric tonnes in 1958 to 102,240 metric tonnes in 1998. In 1996-97 the demand for pesticides in terms of value was *

Correspondence to: [email protected]

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estimated to be around Rs. 22 billion (USD 0.5 billion), which is about 2% of the total world market. The pattern of pesticide usage in India is different from that for the world in general. As can be seen from Figure 1, in India 76% of the pesticide used is insecticide, as against 44% globally.[9] The use of herbicides and fungicides is correspondingly less heavy. The main use of pesticides in India is for cotton crops (45%), followed by paddy and wheat.

Figure 1. Consumption pattern of pesticides

2. BENEFITS OF PESTICIDES 2.1. Improving Productivity Tremendous benefits have been derived from the use of pesticides in forestry, public health and the domestic sphere - and, of course, in agriculture, a sector upon which the Indian economy is largely dependent. Food grain production, which stood at a mere 50 million tonnes in 1948-49, had increased almost fourfold to 198 million tonnes by the end of 1996-97 from an estimated 169 million hectares of permanently cropped land. This result has been

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achieved by the use of high-yield varieties of seeds, advanced irrigation technologies and agricultural chemicals.[1] Similarly outputs and productivity have increased dramatically in most countries, for example, wheat yields in the United Kingdom, corn yields in the USA. Increases in productivity have been due to several factors including use of fertiliser, better varieties and use of machinery. Pesticides have been an integral part of the process by reducing losses from the weeds, diseases and insect pests that can markedly reduce the amount of harvestable produce. Warren (1998) also drew attention to the spectacular increases in crop yields in the United States in the twentieth century. Webster et al. (1999) stated that "considerable economic losses" would be suffered without pesticide use and quantified the significant increases in yield and economic margin that result from pesticide use. Besides this, most of the pesticides, in environment, undergo photochemical transformation to produce metabolites which are relatively non-toxic to the human beings as well as environment.[47]

2.2. Protect Crop Losses/Yield Reduction In medium land rice even under puddle conditions during the critical period warranted an effective and economic weed control practice to prevent a reduction in rice yield due to weeds that ranged from 28 to 48% based on comparisons that included control (weedy) plots[43].Weeds reduce yield of dry land crops[43] by 37-79%. Severe infestation of weeds particularly in early stage of crop establishment ultimately accounts for a yield reduction of 40%. Herbicides provided an economic and labour benefit too.

2.3. Vector Disease Control Vector-borne diseases are most effectively tackled by killing the vectors. Insecticides are often the only practical way to control the insects that spread deadly diseases such as malaria that results in an estimated 5000 deaths each day (Ross, 2005). In 2004, Bhatia wrote that malaria is one of the leading causes of morbidity and mortality in the developing world and a major public health problem in India.

2.4. Quality of Food In the countries of first world, it is now observed that a diet containing fresh fruit and vegetables far outweigh potential risks from eating very low residues of pesticides in crops [27]. Increasing evidence (Dietary Guidelines, 2005) shows that eating fruit and vegetables regularly reduces the risk of many cancers, high blood pressure, heart disease, diabetes, stroke, and other chronic diseases. Lewis et al (2005) discussed the nutritional properties of apples and blueberries in the US diet and concluded that their high concentrations of antioxidants act as protectants against cancer, heart disease. Lewis attributed doubling in wild blueberry production and subsequent increases in consumption chiefly to herbicide use that improved weed control.

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2.5. Other Area-Transport, Sport Complex, Building The transport sector makes extensive use of pesticides, particularly herbicides. Herbicides and insecticides are used to maintain the turf on sports pitches, cricket grounds and golf courses. Insecticides protect buildings and other wooden structures from damage by termites and wood boring insects.

3. HAZARDS OF PESTICIDES 3.1. Direct Impact on Human Being If the credits of pesticides include enhanced economic potential in terms of increased production of food and fibre, and amelioration of vector-borne diseases, then their debits have resulted in serious health implications to man and his environment. There is now overwhelming evidence that some of these chemicals do pose potential risk to humans and other life forms and unwanted side effects to the environment [17-19]. No segment of the population is completely protected against exposure to pesticides and the potentially serious health effects, though a disproportionate burden is shouldered by the people of developing countries and by high risk groups in each country.[20] The world-wide deaths and chronic illnesses due to pesticide poisoning number about 1 million per year[21]. The high risk groups exposed to pesticides include the production workers, formulators, sprayers, mixers, loaders and agricultural farm workers. During manufacture and formulation, the possibility of hazards may be more because the processes involved are not risk free. In industrial settings, the workers are at increased risk since they handle various toxic chemicals including pesticides, raw materials, toxic solvents and inert carriers. In India, the first report of poisoning due to pesticides was from Kerala in 1958, where over 100 people died after consuming wheat flour contaminated with parathion.[2] This prompted the Special Committee on Harmful Effects of Pesticides constituted by the ICAR to focus attention on the problem[3]. Further, Carlson in 1962 warned that OC compounds could pollute the tissues of virtually every life form on the earth, the air, the lakes and the oceans, the fishes that live in them and the birds that feed on the fishes.[4] Later, the US National Academy of Sciences stated that the DDT metabolite, DDE causes eggshell thinning and that the bald eagle population in the United States declined primarily because of exposure to DDT and its metabolites[5]. Certain environmental chemicals including pesticides termed as endocrine disruptors are known to elicit their adverse effects by mimicking or antagonising natural hormones in the body and it has been postulated that their long-term, low-dose exposure are increasingly linked to human health effects such as immunosuppression, hormone disruption, diminished intelligence, reproductive abnormalities and cancer[6-8]. A study on workers (N=356) in four units manufacturing HCH revealed neurological symptoms (21%) which were related to the intensity of exposure[22]. The magnitude of the toxicity risk involved in the spraying of methomyl, a carbamate insecticide, in field conditions was assessed by the National Institute of Occupational Health (NIOH) [24]. Significant changes were noticed in the ECG and the levels of serum LDH and ChE activities in the spraymen indicating the cardiotoxic effects of methomyl.

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Observations confined to health surveillance in male formulators engaged in production of dust and liquid formulations of various pesticides (malathion, methyl parathion, DDT and lindane) in industrial settings of the unorganised sector revealed a high occurrence of generalized symptoms (headache, nausea, vomiting, fatigue, irritation of skin and eyes) besides psychological, neurological, cardiorespiratory and gastrointestinal symptoms coupled with low plasma cholinesterase (ChE) activity [23]. Data on reproductive toxicity were collected from 1,106 couples when the males were associated with the spraying of pesticides (OC, OP and carbamates) in cotton fields.[25] A study in malaria spraymen was initiated to evaluate the effects of a short term (16 week) exposure in workers (N=216) spraying HCH in field conditions.[26]

3.2. Impact through Food Commodities The UK Pesticide Residue Committee annual report (2002) showed that over 70% of the food in the UK contained no pesticide residues at all and only 1.09% contained residues above the statutory maximum residue levels (MRLs). It concluded that ―none of these residues caused concern for people's health‖. Yet these very small quantities of chemicals in our food, detected at ever lower levels due to increasingly sensitive laboratory equipment, are now easy targets for the media. In India, a study revealed that 50% of the vegetable samples taken from farm gate were found contaminated with various pesticides (0.01-2.23 ppm) of which 16% were above MRL.[48]

3.3. Impact on Environment Pesticides can contaminate soil, water, turf, and other vegetation. In addition to killing insects or weeds, pesticides can be toxic to a host of other organisms including birds, fish, beneficial insects, and non-target plants. Insecticides are generally the most acutely toxic class of pesticides, but herbicides can also pose risks to non-target organisms.

3.3.1. Surface Water Contamination Pesticides can reach surface water through runoff from treated plants and soil. Contamination of water by pesticides is widespread. The results of a comprehensive set of studies done by the U.S. Geological Survey (USGS) on major river basins across the country in the early to mid- 90s yielded startling results. More than 90 percent of water and fish samples from all streams contained one, or more often, several pesticides.45 Pesticides were found in all samples from major rivers with mixed agricultural and urban land use influences, and 99 percent of samples of urban streams.[28] The USGS also found that concentrations of insecticides in urban streams commonly exceeded guidelines for protection of aquatic life 41. Twenty-three pesticides were detected in waterways in the Puget Sound Basin, including 17 herbicides. According to USGS, more pesticides were detected in urban streams than in agricultural streams. [29]

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3.3.2. Ground Water Contamination Pesticides, including herbicides, can and do leach to contaminate ground water. According to the USGS, at least 143 different pesticides and 21 transformation products have been found in the ground water, including pesticides from every major chemical class. Over the past two decades, detections have been found in the ground water of more than 43 states[30]. Contamination of ground water is of concern because ground water supplies 50 percent of the U.S. population with Drinkingwater.[31] During one survey in India it has been found that 58% of drinking water samples drawn from various hand pumps and wells around Bhopal are contaminated with Organ Chlorine pesticides above the EPA standards.[46] Once ground water is polluted with toxic chemicals, it may take many years for the contamination to dissipate or be cleaned up. Cleanup may also be very costly and complex, if not impossible [32-34]. 3.3.3. Soil Contamination Pesticides have various characteristics that determine how they act once in soil. Mobility refers to how much a pesticide will move around in the soil. The half life of a pesticide refers to the length of time it takes for half of the pesticide to degrade. Persistence refers to the length of time until all measurable residues of a pesticide are gone. 3.3.4, Effect on Soil Fertility (Beneficial Soil Microorganisms) One spoonful of healthy soil has millions of tiny organisms including fungi, bacteria, and a host of others. These microorganisms play a key role in helping plants utilize soil nutrients needed to grow and thrive. Microorganisms also help soil store water and nutrients, regulate water flow, and filter pollutants.[38] The heavy treatment of soil with pesticides can cause populations of beneficial soil microorganisms to decline. Sometimes pesticides have a negative impact on the available NPK from soil.[49] According to soil scientist Dr. Elaine Ingham, ―If we lose both bacteria and fungi, then the soil degrades. Overuse of chemical fertilizers and pesticides have effects on the soil organisms that are similar to human overuse of antibiotics. Indiscriminate use of chemicals might work for a few years, but after awhile, there aren‘t enough beneficial soil organisms to hold onto the nutrients.‖ [40]. 3.3.5. Contamination of Air, Soil, and Non-Target Vegetation Pesticide sprays can directly hit non-target vegetation, or can drift or volatilize from the treated area and contaminate air, soil, and non-target plants. Some pesticide drift occurs during every application, even from ground equipment.[35] Drift can account for a loss of 2 to 25% of the chemical being applied, which can spread over a distance of a few yards to several hundred miles. There are thousands of reported complaints of off target spray drift each year in the U.S. [36]. Many pesticides can volatilize (that is, they can evaporate from soil and foliage, move away from the application, and contaminate the environment.)[38]. As much as 80-90 percent of an applied pesticide can be volatilized within a few days of application[39]. Despite the fact that only limited research has been done on the topic, studies consistently find pesticide residues in air. According to the USGS, pesticides have been detected in the atmosphere in all areas of the USA sampled.[40] Nearly every pesticide investigated has been detected in rain, air, fog, or snow across the nation at different times of

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the year.[41] Many pesticides have been detected in air at more than half the sites sampled nationwide.

3.3.6, Non-Target Organisms: Pesticides are found as common contaminants in soil, air, water and on non-target organisms in our urban landscapes. Once there, they can harm plants and animals ranging from beneficial soil microorganisms and insects, non-target plants, fish, birds, and other wildlife.[37]

CONCLUSION Pesticides are often considered a quick, easy, and inexpensive solution for controlling weeds and insect pests in urban landscapes. However, pesticide use comes at a significant cost. Pesticides have contaminated almost every part of our environment. Pesticide residues are found in soil and air, and in surface and ground water across the nation, and urban pesticide uses contribute to the problem. Pesticide contamination poses significant risks to the environment and non-target organisms ranging from beneficial soil microorganisms, to insects, plants, fish, and birds. Contrary to common misconceptions, even herbicides can cause harm to the environment. In fact, weed killers can be especially problematic because they are used in relatively large volumes. The best way to reduce pesticide contamination (and the harm it causes) in our environment is for all of us to do our part to use safer, nonchemical pest control (including weed control) methods.

REFERENCES [1] [2] [3]

[4] [5] [6]

[7]

Employment Information: Indian Labour Statistics 1994. Chandigarh: Labour Bureau, Ministry of Labour, 1996. Karunakaran, (1958), C.O. The Kerala food poisoning. J Indian Med Assoc, 31: 204. Eds. A.M. Wadhwani and I.J. Lall. (1972) Harmful Effects of Pesticides. Report of the Special Committee of ICAR, Indian Council of Agricultural Research, New Delhi, p. 44. Carlson, R. (1962) Silent Spring. Houghton-Mifflin Co, Boston. Liroff, R.A. (2000) Balancing risks of DDT and malaria in the global POPs treaty. Pestic Safety News 4: 3. Crisp, T.M., Clegg, E.D., Cooper, R.L., Wood, W.P., Anderson, D.G., Baeteke, K.P., Hoffmann, J.L., Morrow, M.S., Rodier, D.J., Schaeffer, J.E., Touart, L.W., Zeeman, M.G. and Patel, Y.M. (1998) Environmental endocrine disruption: An effects assessment and analysis. Environ Health Perspect, 106: 11. Hurley, P.M., Hill, R.N. and Whiting, R.J. (1998) Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumours in rodents. Environ Health Perspect 106: 437.

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Wasim Aktar Brouwer, A., Longnecker, M.P., Birnbaum, L.S., Cogliano, J., Kostyniak, P., Moore, J., Schantz, S. and Winneke, G. (1999) Characterization of potential endocrine related health effects at lowdose levels of exposure to PCBs. Environ Health Perspect 107:639. Mathur, S.C. (1999) Future of Indian pesticides industry in next millennium. Pesticide Information;XXIV(4):9-23. Warren, G.F. (1998) Spectacular Increases in Crop Yields in the United States in the Twentieth Century, Weed Technology, Vol. 12, P.752. Webster, J.P.G., R.G. Bowles, and N.T. Williams (1999) Estimating the Economic Benefits of Alternative Pesticide Usage Scenarios: Wheat Production in the United Kingdom, Crop Production, Vol. 18, P.83. MAF (Ministry of Agriculture and Forestry) New Zealand. Motivation for Growing Organic Products (available at http://www.maf.govt.nz/mafnet/rural-nz/sustainableresourceuse/organic-production/organic-farming-in-nz/org30004.htm). Oerke, E.C. and Dehne, H.W. (2004) Safeguarding Production - Losses in Major Crops and the Role of Crop Protection, Crop Protection, Vol. 23, P.275. Ross, G., (2005) Risks and benefits of DDT, The Lancet, Vol. 366, No.9499, P.1771November. Lewis, Nancy M., Jamie Ruud, (2005) Blueberries in the American Diet, Nutrition Today, Vol. 40, No.2, P.92March-April. Dietary guidelines for Americans (2005). U.S. Department of Health and Human Services U.S. Department of Agriculture. Forget, G. (1993) Balancing the need for pesticides with the risk to human health. In: Impact of Pesticide Use on Health in Developing Countries. Eds. G. Forget, T. Goodman and A. de Villiers, IDRC, Ottawa, p. 2. Igbedioh, S.O. (1991) Effects of agricultural pesticides on humans, animals and higher plants in developing countries. Arch Environ Health 46: 218. Jeyaratnam, J. (1985) Health problems of pesticide usage in the third world. BMJ 42: 505. WHO. Public Health Impact of Pesticides Used in Agriculture. World Health Organization, Geneva, p. 88, (1990). Environews Forum. Killer environment. Environ Health Perspect 107: A62, (1999). Nigam, S.K., Karnik, A.B., Chattopadhyay, P., Lakkad, B.C., Venkaiah, K. and Kashyap, S.K. (1993) Clinical and biochemical investigations to evolve early diagnosis in workers involved in the manufacture of hexachlorocyclohexane. Int Arch Occup Environ Health 65: S193. Gupta, S.K., Jani, J.P., Saiyed, H.N. and Kashyap, S.K. (1984) Health hazards in pesticide formulators exposed to a combination of pesticides. Indian J Med Res, 79: 666. Saiyed, H.N., Sadhu, H.G., Bhatnagar, V.K., Dewan, A, Venkaiah, K. and Kashyap, S.K. (1992) Cardiac toxicity following short term exposure to methomyl in spraymen and rabbits. Hum Exp Toxicol, 11: 93. Rupa, D.S., Reddy, P.P. and Reddy, O.S. (1991) Reproductive performance in population exposed to pesticides in cotton fields in India. Environ Res 55: 123. Gupta, S.K., Parikh. J.R., Shah, M.P., Chatterjee, S.K. and Kashyap, S.K. (1982) Changes in serum exachlorocyclohexane (HCH) residues in malaria spraymen after short term occupational exposure. Arch Environ Health 37: 41.

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[27] Brown, Ian UK Pesticides Residue Committee Report (2004) (available online http://www.pesticides.gov.uk/uploadedfiles/Web_Assets/PRC/PRCannualreport2004 .pdf also available on request). [28] Bortleson, G. and D. Davis. (1987-1995). U.S. Geological Survey & Washington State Department of Ecology. Pesticides in selected small streams in the Puget Sound Basin. pg. 1-4. [29] US Department of the Interior. (1995). Pesticides in ground water: current understanding of distribution and major influences. U.S. Geological Survey. National Water Quality Assessment. Factsheet number FS-244-95. [30] Waskom, R. (1994). Best management practices for private well protection. Colorado State Univ. Cooperative Extension (August). http://hermes.ecn.purdue.edu:8001/cgi/. [31] O‘Neil, W. and Raucher, R. (1998). Groundwater public policy leaflet series #4: The costs of groundwater contamination. Wayzata, MN:Groundwater Policy Education Project. http:// www.dnr.state.wi.us/org/water/dwg/gw/costofgw.htm (Aug). [32] US EPA. (2001). Managing small-scale application of pesticides to prevent contamination of drinking water. Water protection practices bulletin, Washington, DC: Office of Water (July). EPA 816-F-01-031. [33] Johnson, J. and Ware, W.G. (1991). Pesticide litigation manual 1992 edition. Clark Boardman Callaghan Environmental Law Series, New York, NY. 65. US EPA. 1999. Spray drift of pesticides. Washington, DC: Office of Pesticide Programs (December). http:// www.epa.gov/pesticides/citizens/spraydrift.htm#1. [34] US EPA. (1999). Spray drift of pesticides. Washington, DC:Office of Pesticide Programs (December). http://www.epa.gov/pesticides/citizens/spraydrift.htm#1. [35] Glotfelty and Schomburg. (1989). Volatilization of pesticides from soil in Reactions and Movements of organic chemicals in soil. Eds. BL Sawhney and K. Brown. Madison, WI: Soil Science Society of America Special Pub. [36] Que, S. et al. (1975). Factors effecting the volatility of DDT, dieldrin, and dimethylamine salt of (2,4-dichlorophenoxy) acetic acid (2,4-D) from leaf and glass surfaces. Bull. Environ. Contam. Toxicol. 13(3):284-290. [37] USGS. (1995). Pesticides in the atmosphere: current understanding of distribution and major influences. Fact Sheet FS- 152-95. http://water.wr.usgs.gov/pnsp/atmos/ [38] Marx, J et al. (1999). The relationship between soil and water, how soil amendments and compost can aid in salmon recovery. Soils for Salmon 1-18. [39] Majewski, M. and P. Capel. (1995). Pesticides in the atmosphere: distribution, trends, and governing factors. Volume one, Pesticides in the Hydrologic System. Ann Arbor Press Inc. pg. 118. [40] Savonen, C. (1997). Soil microorganisms object of new OSU service. Good Fruit Grower. http://www.goodfruit.com/archive/ 1995/6other.html. [41] U.S. Geological Survey. (1999). The quality of our nation‘s waters – nutrients and pesticides. Circular 1225. Reston VA: USGS. http://water.usgs.gov/pubs/circ/circ1225/ [42] Bhatia, Mrigesh R., Fox-Rushby, J and Mills, M. (2004) Cost-effectiveness of malaria control interventions when malaria mortality is low: insecticide-treated nets versus inhouse residual spraying in India. Soil Science and Medicine, Vol. 59, p-525. [43] Behera, Basudev, Gauri Shankar Singh. (1999) Studies on Weed Management in Monsoon Season Crop of Tomato. Indian Journal of Weed Science, Vol. 31, No.1+2, p67.

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[44] Porwal, M.K. (2002) Relative Economics of Weed Management Systems in Winter Sweet Potato (Ipomoea batatus L.) in Command Area of Southern Rajasthan. Indian Journal of Weed Science, Vol. 34, No.1+2, P.88. [45] Kole R.K., Banerjee H. and Bhattacharyya A. (2001) Monitoring of market fish samples for Endosulfan and Hexachlorocyclohexane residues in and around Calcutta. Bull. Envir. Contam. Toxicol 67: 554-559. [46] Kole R.K. and Bagchi M.M. (1995) Pesticide residues in the aquatic environment and their possible ecological hazards. J. Inland Fish. Soc. India. 27(2): 79-89. [47] Kole R.K., Banerjee H., Bhattacharyya A., Chowdhury A. and AdityaChaudhury N. (1999) Photo transformation of some pesticides. J. Indian Chem. Soc. 76:595-600. [48] Kole R.K., Banerjee H. and Bhattacharyya A. (2002) Monitoring of pesticide residues in farm gate vegetable samples in west Bengal. Pest. Res. J. 14(1): 77-82. [49] Sardar D. and Kole R.K. (2005) Metabolism of Chlorpyriphos in relation to its effect on the availability of some plant nutrients in soil. Chemosphere 61: 1273-1280.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 16

OZONE DECOMPOSITION BY CATALYSTS AND ITS APPLICATION IN WATER TREATMENT: AN OVERVIEW J. Rivera-Utrilla, M. Sánchez-Polo, J. D. Méndez-Díaz Inorganic Chemistry Department, Faculty of Science, University of Granada, Granada, Spain

ABSTRACT Ozone has recently received much attention in water treatment technology for its high oxidation and disinfection potential. The use of ozone brings several benefits but has a few disadvantages that limit its application in water treatment, including: i) low solubility and stability in water, ii) low reactivity with some organic compounds and iii) failure to produce a complete transformation of organic compounds into CO2, generating degradation by-products that sometimes have higher toxicity than the raw micropollutant. To improve the effectiveness of ozonation process efficiency, advanced oxidation processes (AOPs) have recently been developed (O3/H2O2, O3/UV, O3/catalysts). AOPs are based on ozone decomposition into hydroxyl radicals (HO·), which are high powerful oxidants. This chapter offers an overview of AOPs, focusing on the role of solid catalysts in enhancing ozone transformation into HO· radicals. Catalytic ozonation is a new way to remove organic micropollutants from drinking water and wastewater. The application of several homo- and heterogeneous ozonation catalysts is reviewed, describing their activity and identifying the parameters that influence the effectiveness of catalytic systems. Although catalytic ozonation has largely been limited to laboratory applications, the good results obtained have led to investigations now under way by researchers worldwide. It is therefore timely to provide a summary of achievements to date in the use of solid materials to enhance ozone transformation into HO· radicals.

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1. INTRODUCTION Demographic development and an exponential increase in industrial activity have largely been responsible for the large rise in the demand for water for domestic, public, and industrial use, and for the high volume of effluents discharged into waters. Legislation has been introduced to reduce permissible contamination levels. However, conventional treatment systems cannot completely remove a large amount of organic and inorganic contaminants present in waters, because most cannot be metabolized by microorganisms as carbon source and can even inhibit the activity of these microorganisms, leading to their bioaccumulation in the food chain. Hence, there is an increasing demand for more effective treatments to reduce the potential environmental impact of effluents and to comply with increasingly strict legislations. Successful water treatment requires the use of more sophisticated methods, including: 





  



Biological systems for nitrogen removal: Ammonium can be transformed into nitrate by using nitrifier microorganisms in aerobic medium. Nitrate can be removed in a subsequent stage under anaerobic conditions, when it is transformed by denitrifying bacteria into molecular nitrogen. Advanced oxidation processes for removal of toxic organic compounds, chromophore compounds, and other non-biodegradable organic compounds. These are based on the use of highly oxidizing agents, e.g., ozone or hydrogen peroxide. A greater effectiveness is observed when these oxidizing agents are used in the presence of UV radiation. Ionic exchange for ion removal. This is highly effective for removing cations and anions from the aqueous phase but transfers the problem to the solid phase by concentrating the pollutant in the adsorbent medium. Adsorption on activated carbon for removal of metals, organic compounds, etc. This has the same drawback as described above for ionic exchange. Chemical precipitation for phosphorus removal: Chemical agents (Al2(SO4)3, Fe2(SO4)3 or FeCl3) are used to precipitate phosphorus. Distillation for removal of volatile organic compounds. This is only appropriate when there are high concentrations of the contaminant and its recovery brings economic benefits. Liquid-liquid extraction. This is also only useful under the above conditions.

Chemical oxidation processes currently play a very important role in water treatment. Table 1 lists the most common chemical oxidants used in water treatments and their corresponding reduction potentials [1]. Oxidants can be used to remove both inorganic pollutants and toxic organic compounds (pesticides, hydrocarbons, toxins, etc.) [2,3]addition, oxidants are widely used to degrade compounds responsible for odor, color or taste [4,5,6] Ozone, due to its high oxidation and disinfection potential, has recently received much attention in water treatment technology. Despite several advantages of using ozone, it has a few disadvantages that limit its application in water treatment, including: i) low solubility and stability in water, ii) low reactivity with some organic compounds, and iii) failure to produce a complete transformation of organic compounds into CO2, generating degradation by-

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products that sometimes have a higher toxicity than the raw micropollutant. To improve the effectiveness of ozonation, advanced oxidation processes (AOPs) have recently been developed (O3/H2O2, O3/UV, O3/catalysts). AOPs are based on the decomposition of ozone into hydroxyl radicals (HO·), a very powerful oxidant. This chapter offers an overview of the role of different solid catalysts in enhancing ozone transformation into HO· radicals. Catalytic ozonation is a new way to remove organic micropollutants from drinking water and wastewater. The application of several homo- and heterogeneous ozonation catalysts is reviewed, describing their activity and identifying the parameters that influence the effectiveness of catalytic systems. Although catalytic ozonation has largely been limited to laboratory applications, the good results obtained have led to investigations now under way by researchers worldwide. It is therefore timely to provide a summary of achievements to date in the use of solid materials to enhance ozone transformation into HO· radicals. Table 1. Reduction potentials of different oxidants used in water treatments. Oxidant Ozone

Reduction semireaction O3 (aq) + 2H+ + 2e-  O2 (aq) + H2O

E0 (V) 2.08

Permanganate

2MnO4- + 8H+ + 6e-  2MnO2 (s) + 4H2O

1.68

-

-

Chlorine dioxide

ClO2 + e  ClO2

Hypochlorous acid

HOCl + H+ + 2e-  Cl- + H2O -

+

-

0.95 1.48

-

Hypochlorite ion Dichloramine

ClO + 2H + 2e  Cl + H2O NHCl2 + 3 H+ + 4e-  2Cl- + NH4+

1.64 1.34

Oxygen Hydroxyl radical

O2 (aq) + 4H+ + 4e-  2H2O HO· + H+ + e-  H2O

1.23 2.85

Hydrogen peroxide

H2O2 + 2H+ + 2e-  2H2O

1.78

2. OZONE Ozone, O3, discovered by Schönbein in 1840 [7], is an allotrope of oxygen consisting of three atoms. It is a diamagnetic compound that is an unstable gas at room temperature with a characteristic sharp odor. Experimental results show a bond angle of 116.8 ± 0.5º and an interatomic distance of 127.8 pm between central oxygen and each terminal [8]. Fig 1 depicts the two resonant forms of the ozone molecule according to the valence bond theory:

+ ·· O

:O :

··

·· O+ :O :

Figure 1. Chemical structure of ozone.

:O :

O : :

··

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Due to its configuration, ozone is a highly oxidant compound (Eº = +2.08 V), and its natural tendency is to transfer an oxygen atom and release O2. Applications of ozone applications in water treatment can be grouped into three categories: i) as disinfectant or biocide, ii) as oxidant for the removal of organic pollutants and, iii) as pre- or post-treatment in another procedure (coagulation, flocculation, sedimentation, biological oxidation, adsorption on activated carbon, etc.). Ozone began to be used as bactericide agent to treat drinking waters in Nice (France) at the beginning of the 20th century. As a result of the large amount of resources invested in the study of ozone, other advantages of its use in treatment plants [9] are now widely known, including: i) removal of compounds that produce odor, taste or color in water, ii) oxidation of inorganic chemical compounds, e.g., iron and manganese, iii) removal of algae and other aquatic microorganisms, iv) oxidation of organic micropollutants, v) absence of the increase in the presence of organochlorinated compounds found when chlorine is used for the treatment, and vi) enhanced performance of adsorption and coagulation processes. Nevertheless, its large-scale application to treat industrial liquid waste did not become widespread due to the high economic costs involved and the chemical complexity of industrial effluents. However, ozone has now become an attractive option for the treatment of these effluents because conventional systems are inadequate to reduce the toxic organic compounds they contain to levels required by new environmental legislation. O3 + OH-

O2-· + HO2·

Start 2HO2·

O3 + H2O HO2·

H+ + O2-·

O2-· + O3

O2 + O3-·

(2) (3) (4)

HO· + O2

(5)

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(6)

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HO· + O3

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HO2· + O2

(8) (9)

HO2· + HO2·

H2O2 + O2

HO2· + O2-·

HO2- + O2

(10)

2HO- + 2O2

(11)

O3-· + O2-· + H2O

Figure 2. Mechanism of ozone decomposition in aqueous medium.

Over the past two decades, there has been a notable increase in research into the reaction between ozone and numerous organic and inorganic compounds, especially aromatic compounds [10,11,12,13,14]. Because it is highly reactive, ozone can interact with a large number of organic and inorganic substances by direct oxidation/reduction reaction, cycloaddition-substitution, or nucleophilic addition [15,16,17]. The direct reaction between

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ozone and a given compound is not the only pathway by which ozone can act to degrade pollutants, since ozone is very unstable in aqueous solution and spontaneously decomposes by a complex chain mechanism (Fig 2) in which different radical species participate [18]. The radicals generated are of great interest for water treatment because some of them, e.g., the hydroxyl radical HO·, are even more reactive than ozone and play an essential role in removing pollutants in solution [19]. These systems will be described in greater depth in next section, which is devoted to AOPs. Despite its high efficacy in some systems, ozone has inadequate capacity to degrade surfactants and tensioactive substances, among other pollutants. Narkis et al. [20] observed that ozone treatment favored the biodegradation of non-ionic surfactants but was unable to remove them. Other authors also reported the lack of reactivity of saturated cationic surfactants [21]. Regarding anionic surfactants, although some researchers achieved a high degradation of alkylbenzene-sulphonates with ozone [22], the reaction rate constant values were subsequently determined to be low [23], suggesting that an indirect mechanism was largely responsible for the degradation.

3. ADVANCED OXIDATION PROCESSES BASED ON OZONE Polluted waters can generally be effectively treated by biological, adsorbent or conventional chemical approaches (chlorination, ozonation or oxidation with permanganate). However, as stated earlier, these methods are sometimes inadequate to degrade pollutants to levels required by law or necessary for subsequent utilization of the effluent. AOPs have proven highly effective in the oxidation of numerous organic and inorganic compounds and are based on the generation of free radicals, notably hydroxyl radical HO·. These free radicals are highly reactive species that can successfully attack most organic and inorganic compounds with very high reaction rate constants (106-109 M-1s-1). The numerous systems that can be produced by these radicals (Table 2) account for the high versatility of AOPs. Advanced oxidation processes based on the use of ozone are briefly described below, highlighting catalytic ozonation (also see section 4). Table 2. Water treatment technologies based on advanced oxidation processes.



Non-photochemical processes Oxidation in sub/supercritical water





Fenton‘s reagent (Fe2+/H2O2)



Photochemical processes Photolysis of water with vacuum UV (VUV) UV/hydrogen peroxide



Electrochemical oxidation



UV/ozone

    

Radiolysis Non-thermal plasma Ultrasound Ozonation in alkaline medium (O3/OH-) Ozonation in the presence of hydrogen peroxide (O3/H2O2) Catalytic ozonation

 

Photo-Fenton Heterogeneous photocatalysis

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3.1. Ozonation in Alkaline Medium Based on studies by Hoigné et al.[10,11,12], Aieta et al. [24] published an illustrative diagram in 1988 describing ozone in aqueous solution. Figure 3 summarizes the two reaction pathways of molecular ozone: i) direct reaction of the substrate with molecular ozone, which is selective but slow or null with some species, and ii) decomposition and generation of HO· hydroxyl radicals, which attack rapidly but not selectively. Direct oxidation of the substrate

O3

Products

Slow / Selective

OHRadical formation

HO·

Radical oxidation Fast / Non-selective

Products

Figure 3. Reaction pathways of ozone.

Moreover, hydroxyl radicals react rapidly with molecular ozone, contributing towards autocatalytic decomposition of the ozone. Several researchers have studied the mechanism and reaction kinetics involved in ozone decomposition in aqueous phase [18,25,26,27,28,29,30,31,32,33]. Ozone stability largely depends on the aqueous matrix, especially the pH, type of organic matter present, and alkalinity [34]. The water pH is especially important, because hydroxyl ions considerably increase the ozone decomposition rate [18]. Thus, according to equations 12 and 13, ozone decomposition spontaneously accelerates with an increase in the solution pH, leading to an AOP. However, it must be taken into account that a high pH increase can have a negative effect on the degradation, depending on the composition of the water, because of the inhibiting action of HO· radical scavengers, e.g., bicarbonate or carbonate ions [35]. O3 + OH-  HO2- + O2 k = 70 M-1s-1 (13) O3 + HO2-  HO· + O2·- + O2 k = 2.8 · 106 M-1 s-1

(12)

3.2. Ozonation in the Presence of Hydrogen Peroxide The addition of hydrogen peroxide favors the ozonation of organic compounds in the medium. The combination of ozone and hydrogen peroxide is largely used to oxidize pollutants that require high ozone consumption. Hydrogen peroxide is a weak acid (pKa = 11.6), a powerful oxidant (See Table 1), and an unstable compound that easily dismutes (equations 14-16). H2O2  HO2- + H+ (14) H2O2 + 2e- +2H+  2 H2O

(15)

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439

(16)

The mechanism by which H2O2 favors free radical generation was described by Forni et al. [25], who demonstrated that the conjugated base of hydrogen peroxide initiates ozone decomposition in aqueous phase via an electronic transference reaction. HO2- + O3  HO2 + O317) Taking advantage of the capacity of H2O2 to initiate ozone decomposition in aqueous phase (Figure 4), numerous researchers have used this process for a faster and more effective oxidation of organic matter [36,37,38,39,40,41,42,43. The presence of H2O2 in the system favors oxidation, although the possibility that added H2O2 is consumed in reactions with other contaminants has hampered application of this method to treat industrial effluents. Fernández et al. [44] compared the efficacy of the O3/H2O2 system and photolysis to remove linear alkylbenzene-sulphonates (LAS), observing the complete degradation of the mixture of surfactants after 20 min of ozonation. HO2- + H+

H2O2

O3 O3O2- *

HO2*

HR+ HO3 RHO2* Degradación HO* HR* O2

HRH

Figure 4. Mechanism of oxidation of an organic compound (HRH) by means of O3/H2O2.

3.3 Ozonation in the Presence of UV Radiation Ozonation coupled with UV radiation is one of the most effective chemical oxidation techniques to treat polluted waters. This process is capable of oxidizing organic substances at room temperature and generates products that are innocuous to the environment. As in the O3/H2O2 system, the UV radiation of O3 generates hydroxyl radicals in solution [45]. The reactions involved in this process are: O3 + h (30 kb; for example from fosmid and BAC clones) using shotgun strategies. Bioinformatic support is offered by MGF- Edinburgh, including preliminary sequence trace processing, assembly and annotation. For EST projects, a suite of in-house developed sequence annotation tools can also be applied to sequence datasets using programs such as PartiGene (databasing tool for EST datasets) [7], trace2dbEST (for processing EST sequencing traces) [7], prot4EST (for prediction of peptides from neglected genome sequence datasets) [8], and annot8r (annotation of peptide or nucleotide sequences with GO, KEGG and EC tags based on sequence similarity). The Advanced Genomic Facility (AGF) based at the University of Liverpool offers access to the ultra-high throughput pyrosequencing Genome Sequencer FLX system (GS FLX, Roche), which at present can generate in excess of 100 Mb of genome sequence in a single run with a mean length of 250 bases. The FLX pyrosequencing platform incorporates single-molecule amplification and parallel sequencing of up to ~400,000 individual molecules for 250 bases, making large-scale scientific projects feasible and more affordable. By mid2008 this instrument is expected to yield up to 1 Gb with read lengths approaching 500 bp. FLX pyrosequencing is suited to deep surveying of transcriptomes, complete sequencing of large-insert clones and smaller genomes, resequencing of larger genomes, and metagenomic analyses. AGF-Liverpool also has the latest computational tools and trained staff to deliver bioinformatic services for data handling and analysis, in particular for assembling sequence reads (Newbler assembly) and sequence alignment (FlowMapper). Sequencing by synthesis is also massively parallel, but generates ~30 million reads of ~35 bases per run, generating 1 Gb of sequence. This technology is ideal for resequencing of complex genomes, metagenomic surveys of mesocosms, digital transcriptomics and de novo sequencing of smaller genomes. MGF-Edinburgh also offers a sequencing-by-synthesis service using an Illumina Genome Analyser instrument. The production of raw sequence data is only the start of the process: the genomic or transcriptomic sequence must be correctly assembled and annotated in order to provide

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meaningful information that can be used at the next stage for microarray design. As shown above, both facilities have their own particular expertise in certain areas. However, in addition to the bioinformatic facilities offered by MGF-Edinburgh and AGF-Liverpool, additional bioinformatic support for genomic annotation is offered by the NERC Environmental Bioinformatics Centre (NEBC). The bioinformatic support provided by MGFSheffield is focused on population genetic data analyses, including genomic mapping.

MICROARRAY DESIGN AND FABRICATION The key requirement for generating a DNA array is access to suitable genomic resources. For arrays printed in-house using amplified DNA, this comprises cloned cDNAs or largeinsert genomic fragments isolated from libraries [9]. Alternatively, arrays can be printed from oligonucleotides synthesised in bulk with defined sequences as probes against known DNA targets of interest. Both techniques are expensive in time (for cloning and amplification) and costs (for oligonucleotide purchase). However, for situations where sequence data already exist it is now preferable to design probes in silico against all known targets for a particular species using one of several design algorithms, followed by on-chip synthesis by one of several manufacturers, notably Agilent. Advancing fabrication techniques now makes this a relatively inexpensive yet highly effective approach to array provision. The design of oligonucleotide probes from a known target DNA sequence can be achieved using any one of several open source or commercial packages [10]. The MGFLiverpool uses a commercial package (ArrayDesigner, Premier Biosoft) as an alternative to the widely used open source package OligoArray2 [11]. Design algorithms continue to advance, and one algorithm might suggest different probe designs to another. Commercial providers of oligonucleotides or arrays offer a design service (e.g. Agilent‘s eArray system), but the precise algorithms employed remain confidential to the company and so one cannot be totally confident of design quality or the criteria used in its execution. It is also worth noting that the outputs of any design algorithm do not necessarily provide for optimal function as a hybridisation probe, since they are based on thermodynamic criteria that are surrogates of the hybridisation process. This is why the MGF-Liverpool uses an optimisation approach, comparing the properties of several competing probe designs generated from a single algorithm against stringent performance criteria [12] in order to define the best performing design. All of these options are available at the MGF-Liverpool which has access to both contact and non-contact (piezo) printing robots for array fabrication, has service centre status for both Affymetrix and Agilent platforms, and has access to the Illumina BeadStation, this being the third major commercial platform. As a central array facility, it has negotiated special rates for purchasing large numbers of synthesised oligos and all of the other necessary consumables, including catalogue arrays. It is thus well able to provide support to users with all aspects of microarray design, construction and commissioning. In the past few years MGF-Liverpool has constructed cDNA arrays for a wide range of species (e.g., common carp, flounder, rainbow trout, ground squirrel and eel) all of which were preceded by large-scale cDNA sequencing projects. It has also been involved in a series of microbial metagenomics projects printing commercially synthesised oligoarrays directed against rRNA targets defined by the

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user. MGF-Liverpool has also some experience in constructing oligoarrays for environmental model species directly from public EST data, taking advantage of the rapidly increasing capacity of new platforms. Thus, an oligoarray directed against 22,000 BLAST-identified genes in the rainbow trout was fabricated by Oxford Gene Technology to a MGF-Liverpool design, and this has been expanded to 44,000 probes [12]. This design was optimised in the sense that multiple predicted probes were quantitatively compared using several different tests and the best performing probe identified. The resulting arrays displayed a dynamic range and sensitivity that far exceeded that of conventional cDNA arrays [12] .

ANALYSING THE RESULTS OF MICROARRAY EXPERIMENTS Once a microarray has been designed and fabricated the actual experimental biology can begin. The first important issue is the statistical design of the array experiment. since the shift from the early reference-based designs to more complex ANOVA-based loop designs [13], and the need to account for multiple sampling corrections in such large-scale statistical comparisons, makes this the realm of specialist statistical advice. Only rarely do biologists who develop skills in laboratory technique have the numerical and statistical skills for highlevel data analysis and pattern searching. The staff of the MGF-Liverpool and NERC Environmental Bioinformatics Centre (NEBC) provide specialist advice to define the most appropriate and cost-effective experimental plan to success. The researcher also needs to understand the very fine definition offered by array experiments to ensure that the experiment is adequately controlled and that sufficient independent biological replicates are generated for detailed statistical analysis. Dedicated microarray bioinformaticians at both the MGFLiverpool and at NEBC keep abreast of the latest developments and issues with microarray analysis and are best positioned to advise on correct experimental design. The MGFLiverpool also supports all of the stages of array usage, including sample preparation, hybridisation protocols, imaging/data acquisition and interpretation of the resulting data. This includes the training of research students and staff from NERC-funded laboratories. The fundamental responsibility for sample acquisition and for extraction of DNA or RNA lies with the client. Indeed, sample processing, hybridisation and data acquisition can be performed at the researchers own laboratory if facilities are available. Alternatively the entire programme can be performed as a service by MGF-Liverpool staff or the latter can provide the necessary training for clients to undertake the work. Emphasis is placed on the researcher remaining in control of the experiment, but with the full benefit of state-of-the-art protocols and equipment. MGF-Liverpool offers several scanning platforms for image acquisition including Agilent, Perkin-Elmer and Axon. It also advises on the critical issue of interpreting the image file in order to quantify the distribution of fluorescence intensities across the population of probes. Once microarray images have been processed and quantified, it is strongly recommended that that the researcher interacts further with the MGF-Liverpool or the NEBC if they require help progressing the statistical analysis. NEBC offers access to the commercial GeneSpring (Agilent) and the open-source maxD analysis platforms, and both the NEBC and the MGF-Liverpool routinely use algorithms from within the Bioconductor package written in the R statistical language. This combined provision has three main advantages. Firstly, the

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researcher is directed to well established statistical and data visualisation algorithms. Second, he/she can be trained in analytical technique through interactions with the facilities. Third, he/she can be exposed to objective approaches for drawing inferences both in terms of which genes are responding to experimental treatment, but also how this relates to the biological pathways and processes, and to the questions being addressed. This avoids biasing the analysis to favoured genes or to genes that fit with particular hypotheses. Bioinformaticians at any NERC facility can explain the data output, its strengths and its limitations. However, it is entirely up to the researcher to interpret and publish the data. This involves the use of gene ontology profiling tools such as GOMatrix, GoMiner, Reactome etc, as well as the newer gene set analysis techniques originated in the Broad Institute, MIT. At the final stage of publication, it is necessary for all microarray data to be submitted to a public repository such as GEO or ArrayExpress [14, 15]. This is aided by the development of the Biolinux based maxdload2 data submission program, developed by NEBC partners at the University of Manchester, which ensures it is MIAME-compliant [16]. Indeed, in response to the issues associated with environmental microarrays, the NEBC has developed its own guidelines for MIAME for environmental projects, designated MIAME/Env [17]. Researchers funded by NERC are also required to submit their data to the NERC-supported database EnvBase (http://nebc.nox.ac.uk/).

DATA ANALYSIS AND LONG-TERM MANAGEMENT While each of the sequencing facilities have specialist informatics services focused on specific sequencing platforms, the NEBC is focused on delivering multi-omic approaches to analysis and data management. NEBC was formed in 2002 to specifically provide support for the NERC Environmental Genomics Programme, and subsequently for the sister PostGenomics and Proteomics thematic programme. NEBC‘s primary remit is to archive all NERC-funded data generated under these programs subscribing to the NERC data policy of spending up to 15% of the programme budget on data management and archiving activities. This was one of the earliest data policies operated by a major UK funding agency and its wider implementation is mediated by a set of seven designated data centres that receive core funding from NERC for long-term data management. To guide the data management of the programme, and the stewardship of ‗omic data in NERC as a whole, NEBC has also produced an extension of the NERC data policy to cover ‗omic data. This policy includes very specific mechanisms for submission of data. All data are catalogued in the EnvBase portal and must be submitted to an appropriate public repository where one exists. EnvBase contains discovery-level information about all listed projects, and for each data-holding includes a list of relevant hyperlinked accession numbers leading to databases such as ArrayExpress [14, 15]. EnvBase also provides a mechanism to house all data for which a recognized database does not exist. NEBC was designed to build informatic capacity within the newly forming NERC environmental genomic community. NEBC is therefore staffed by bioinformaticians and computer scientists, in addition to data managers, who provide direct consultation to the wider community. NEBC was established in part to realize the vision of providing the entire Environmental Genomics community with a powerful and uniform computing environment

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based on Linux. This system, called Bio-Linux, is one of a growing suite of highly customized Linux distributions [18]. It contains over 60 software packages and is distributed free of charge to all labs supported by the program. The Bio-Linux system has been used to build teaching labs and computer clusters in several locations across the UK and is used by the core facilities and researchers to coordinate research. The primary focus of NEBC efforts under these programs has been support for research that is generating microarray and EST data. Microarrays were a focus because of their widespread use among funded researchers, the expense and effort of tooling up to produce and interpret them for new users, and the added complexity that several NERC-funded groups aimed to build custom arrays for novel species and sets of genes. In order uphold its remit to provide stewardship over the collective data holdings of these programmes; the centre has become progressively more involved in standardization activities. While at first, this simply meant monitoring emerging standards and compliance activities, over time this led to active participation and leadership in standardization activities that expressly benefit the environmental ‗omics community and that have made submission of data to public repositories possible. The NEBC spearheaded a standardization project in response to the difficulties NERC researchers were facing in complying to the MIAME standard, a requirement for submission to ArrayExpress [19]. The MIAME/Env project emerged out of work aimed at simplifying this process for researchers across the board. This initiative extended the core MIAME specification to capture additional information about the environmental context of samples (for example geographical location, biological interactions, and environmental conditions) [17]. Work on MIAME/Env has since been extended into the metabolomics and genomics domains [20]. Because of participation in such projects, and its focus on multi-omic data sets, NEBC is also a founding member of the ―Minimum Information about a Biological and Biomedical Investigation‖ (MIBBI) community [21].

CONCLUSION The UK has developed a powerful network of specialist facilities to promote the use of high-density microarrays that help researchers overcome the myriad challenges of working with non-model but environmentally-focussed organisms. Depending on the resources available to them and their level of expertise, environmental researchers can selectively choose the facilities while having as much or as little control in the development, construction and processing of the microarrays as they are comfortable with. The nature of the organisms involved can vary dramatically, as can the type of experiments undertaken, but help is available at every stage of the process. This has had a strong beneficial effect on the UKbased environmental community by promoting the use of microarrays and driving environmental sciences into the 21st century. To illustrate this, the following are two case studies, typical of the type of work that has been undertaken, by researchers in the environmental genomics community.

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NON-MODEL ORGANISM CASE STUDY 1: THE COCCOLITHOVIRUS MICROARRAY The Science Emiliania huxleyi is a marine coccolithophorid with worldwide distribution capable of forming vast blooms that can cover up to 10 000 km2 [22, 23]. Its production of calcium carbonate coccoliths and its role in CO2 cycling and dimethyl sulphide (DMS) production makes E. huxleyi an important species with respect to past, present and future marine primary productivity. Viruses have been shown to be a major cause of bloom termination [24-26]. Emiliania huxleyi virus 86, EhV-86, is a giant virus that was originally isolated from a bloom in the English Channel off the south coast of England in 1999 [27]. EhV-86 is the type species of the genus Coccolithovirus within the family Phycodnaviridae and is currently its largest fully sequenced member [28]. The 407,339 bp genome is the second largest virus genome sequenced and is predicted to encode around 472 protein coding sequences (CDSs) [29]. The majority of EhV-86 CDSs exhibit no similarity with proteins in the public databases; a mere 21% of the CDSs contain protein–protein basic local alignment search tool (BLAST) results that matched an E value lower than 0.01 [29]. The large size (and availability) of the EhV-86 genome and its highly unknown nature easily justified the construction of a microarray to aid in the molecular characterisation of this unique virus family.

The Array The EhV-86-based coccolithovirus microarray has been through 2 stages of development. Version 1 was dominated by probes for the 472 predicted CDSs of EhV-86 and contained only a handful of E. huxleyi probes, and consisted of around 1,600 features. As the work has progressed and developed, and the nature of the questions asked of the array changed, so the latest version 2 of the array (printed at MGF-Liverpool) has around 25,000 features and contains probes for multiple coccolithovirus strains and for ESTs from the host species E. huxleyi (developed with the aid of MGF-Edinburgh).

The Research The majority of the EhV-86 genome is composed of genes of which the function is totally unknown [29]. Indeed, unusually among sequenced genomes, a number of predicted genes fail to find any matches whatsoever in the public databases [30]. This novelty of the genome makes the task of annotation difficult: without functional information it is difficult to assess whether what is predicted to be a gene is actually a gene. Thus, the first use to which the coccolithovirus array was put was simply to detect the presence of viral transcripts. The likelihood of an open reading frame being a functional coding sequence and gene is vastly increased by detecting the presence of a mRNA transcript. This approach was used effectively

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during the annotation of the EhV-86 genome, where message for 65% of the 472 predicted genes of EhV-86 was confirmed from a lytic phase total RNA extraction [29]. Following the lytic phase transcriptional work the microarray was used to outline the transcriptional profile generated by EhV-86 during the early stages of infection. Using a highly sensitive amplification method, the precise expression profile of the EhV-86 infection of E. huxleyi was determined over the first 4 h of infection [31]. This strategy allowed the designation of EhV-86 CDSs into three groups on the basis of when their transcripts are first detected (1, 2 or 4 h post infection). The results obtained suggested two distinct phases to the infection process: a primary phase dominated by a group of CDSs localized to a sub-region of the genome, which have no database homologues and are associated with a unique promoter element; and a secondary phase during which CDSs are transcribed from the remainder of the genome [30-32]. Since the majority of EhV-86 CDSs are unknown, the designation of CDSs into transcriptional categories is a significant step forward in determining their function. The distinctive transcriptional profile has answered and raised many interesting questions about the infection strategy and life style of the virus, in particular the role and function of the virally encoded RNA polymerase. It is important to note that a microarray based on sequence data for a particular strain is not restricted for sole use with that strain. The Plymouth Virus Collection (PVC) contains 12 virus strains all capable of infecting E. huxleyi [33]. These strains were collected over the 4year period between 1999 and 2003 from sampling sites in the English Channel and in a Norwegian Fjord [25, 27, 34]. The construction of the coccolithovirus microarray allowed the genetic diversity of the viruses in the PVC to be assessed. A major disadvantage of using a specific strain/species microarray for this purpose is that the microarray can only tell you what is highly variable or not present in a particular genome. Genomic additions go undetected since there is no probe on the array to detect it. However, the use of the array for screening for genomic content can provide a wealth of information on genetic diversity within a family of closely related species. At least 70 genes found in EhV-86 are absent or highly variable from one or more of the genomes of the coccolithoviruses found in the PVC [33]. The distinctive pattern of genetic content displayed by each of the strains (essentially a genomic fingerprint) suggested a complicated series of gene loss/gain events. In particular, a high frequency of deleted or variable genes was found in a 100 kb region of unknown function suggesting this section of the genome may be under a high selection pressure [30, 33]. Thus the microarray method is a relatively cheap, efficient and quick way to glean an insight into the genetic content of a family of closely related species. Genes of interest that are conserved among all strains or are lacking and/or variable in some strains can be identified rapidly for further downstream analysis. In summary, over a relatively short period of time, a predominantly uncharacterized virus has had its genome annotated, the kinetic profile of its transcripts determined and the diversity within its family assessed through the use of microarrays [29, 31, 33, 35]. The results have indicated a plethora of new and novel genes, a distinctive biphasic transcriptional pattern during the infection process and an unexpectedly large degree of genomic content variability among the coccolithoviruses. The construction of the microarray rapidly increased the understanding of the virus and its infection strategy and serves an excellent model for other researchers to follow.

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CASE STUDY 2: GENE DISCOVERY AND SYSTEM-WIDE OVERVIEWS OF ENVIRONMENTAL STRESS RESPONSES IN THE COMMON CARP The Science Abiotic environmental stress, such as heat and cold, hypoxia, UV and pollution pose major problems for all forms of life, and organisms respond to stress by both protecting themselves from the damaging effects of extreme stress, but also by adjuting themselves to allow life processes continue unaffected by the change in environmental conditions. Animals from temperate climatic zones and which inhabit small freshwater ponds are routinely exposed to seasonal fluctuations of temperature and daily fluctuations of environmental oxygen concentrations, and survival depends upon the existence of powerful stress-coping mechanisms [36-38]. Understanding the underpinning mechanisms of stress responses and adaptation is important not only academically but practically in agriculture and in human society [39]. Cold presents special problems both in the form of reduced biochemical activity but also from the damaging effects of freezing [36]. Our understanding of response patterns to chronic cold in complex animals has been transformed by microarray analyses, from one composed of a few key or ‗candidate‘ genes to one where the system-wide effects on largescale networks of genes and proteins can be appreciated [40, 41]. Since 2000 NERC has funded a large-scale analysis of response patterns at the transcript level in the tissues of the common carp, Cyprinus carpio L..

The Array Given that there were no genomic resources for the carp, it was first necessary to generate a collection of cDNA clones, representing genes, all of which was accomplished by the MGFLiverpool. Fish were exposed to the full range of environmental treatments under investigation, including hypoxia, and RNA isolated, normalised to rebalance representation the representation of genes, and then cloned directionally into a suitable vector. The inserts were sequenced from the 5‘ end, clustered clones into groups containing overlapping sequences and then their identity established by homology searching. The inserts of 13,500 clones were amplified by PCR and the amplicons were used to print a microarray on glass slides using a contact-printing robot. More recently, another 20,000 clones have been added to the collection which were ‗cherry-picked‘ to reduce redundancy. The new, more representative collection consists of 26,000 clones, which have been printed using a noncontact, piezo-deposition robot. The evolution of the carp array has been detailed in Williams et al (2008) [42]

The Research Our first experiment [9] followed changes in the expression of genes in 7 different tissues of the carp during long-term transition from warm (30°C) to cold conditions (10°C). The fish

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were cooled over a 3-day period and then held at 3 cooled temperatures (23, 17 and 10°C) for up to 21 days. On each sampling interval 6-8 animals were killed and the tissues dissected into a plastic (‗sushi) tray for long-term freezing at -80°C. Samples of these tissues were used to generate target cDNA, which were hybridised to over 400 high-density cDNA arrays. The data was subjected to refined statistical analysis to reveal a very complex picture of gene responses [9]. Over 3000 probes were found to be significantly different between warm and cooled specimens in at least one tissue. The first focus was on genes that were differentially expressed (DE) in all 7 tissues, which generated a list of 260 genes of which 180 were identified. This list was composed of genes that were largely involved in cell homeostasis, with the greatest and most consistent responses being for the ∆9-desaturase and CIRBP. The former gene is widely invoked in cold responses as being the key gene in transforming lipids from saturated to more unsaturated, thereby fluidising the cold-ordered lipids in cell membranes [43, 44]. The latter gene is an RNA-binding protein of unknown function, but its prominent responses indicate that it is important to all tissues during cold adaptation. A large number of genes involved in protein turnover were found, indicating strong up-regulation in all tissues. The second focus was on the remaining ~1,700 identified genes that were regulated in one or more tissues. Clustering of this data using unsupervised techniques generated 23 groups of genes with characteristic tissue-specific responses. Each cluster was profiled by statistical analysis of the Gene Ontology annotations [45] and this pointed directly at the nature of gene responses in relation to tissue function. For example, cluster 5 was composed of genes that were up-regulated exclusively in the intestinal mucosa. This cluster included an unusually large number of transport-associated genes, which is entirely consistent with the role of the intestinal epithelium in nutrient absorption and ion/pH regulation. This gene profiling technique allows the broader gene expression properties across treatment space to be interpreted within the context of tissues function. Comparing between clusters allowed the identification of major effects in the pathways of intermediary metabolism [9] [46]. Thus, it was found that genes of glycolysis were generally up-regulated in heart and brain, but down-regulated in the other tissues, indicating a shift between tissues in the patterns of energy provision and storage. Consistent responses were identified in all of the major tissues, despite them being mediated by different genes or isoforms. Thus brain, liver, heart and liver all showed a very high representation of genes involved in energy pathways and carbohydrate metabolism in the responding gene clusters, which is consistent with a cold-induced modification of the substrates and sources of energy metabolism, and of the pathways involved. A second major experiment involved the exposure of carp to chronic hypoxia [47]. Carp is unusually tolerant to hypoxia which allows it to prosper in natural ponds. This experiment involved over 700 microarrays, comprising responses to hypoxia in 7 tissues but also at two environmental temperatures, 17°C and 30°C. Again this revealed strong patterns within and between tissues which were surprisingly different between the two exposure temperatures. Curiously, a transcript corresponding to myoglobin was substantially up-regulated by hypoxia in carp liver. Myoglobin is an oxygen-binding protein which was famous for its role in protecting oxidative muscle cells including cardiac myocytes. Expression of this gene in nonmuscle tissues is linked to the presence of myoglobin protein in the liver of hypoxia-treated fish [48] and that the amount of protein increases in carp exposed to chronic hypoxia. It was also found that carp possess a second isoform of this important gene, whose expression was

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limited to the brain, this being the only known vertebrate species possessing two myoglobin isoforms. This fact is likely to be linked to a whole genome duplication event some 15MYA in a clade of fish that includes the goldfish and crucian carp [49]. This discovery of non-muscle expression of this gene was an entirely serendipitous and unexpected finding, arising directly from an open microarray screen that was directed at the discovery of both genes and their environmental responses. It is notable because it flies in the face of conventional respiratory physiology of vertebrates animals. Similar myoglobin expression has been found in zebrafish and the mouse system is now being explored. This phenomenon is likely to have major implications for understanding hypoxia responses in vertebrate animals generally but particularly in humans where hypoxia injury caused by ischaemia and infarct are major clinical conditions in the developed world. Table 1. Integration of different UK research facilities in the microarray research What? DNA sequencing Sample acquisition Sequencing Annotation Microarray Construction Oligo design Oligo synthesis cDNA clones and EST sequencing Microarray printing or submission of in silico oligoprobes to 3rd part fabrication Microarray experiment Experimental design Sample acquisition Array hybridisation Image acquisition and quantification Data analysis Interpretation Publication

Researcher   

Who? MGF- MGF-Liverpool MGF-Liverpool NEBC Edinburgh (AGF) (LMF)  

 

 

   









   

  

  

  





  

The combination of data from both cold and hypoxia treated carp constitutes a very large data set which can be used to explore the correlation of gene expression properties between pairs of genes. This allows the regulatory relationships between genes to be explored, as whether the correlated profiles between genes are conserved between species. More than 400 gene pairs have been identified whose close expression relationships are conserved between carp and human (Li, W and AR Cossins, to be published), and thus by extension across all vertebrate animals. Finally, the expression data collected on such a large scale and across several different tissues and stress modalities, constitutes another resource with which to differentiate the identities of closely-related members of gene families, which completes the

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more usual sequence-based approaches. This is a particular problem in recently duplicated genomes, such as the common carp [49] and the salmonid fish. Indeed, genes given a common identity by homology alignment proved to have distinguishable expression properties. On further investigation these expression forms have been found to constitute separate genes.

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[6] [7] [8] [9]

[10] [11]

[12]

[13] [14] [15]

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[16] Hancock, D., et al., maxdLoad2 and maxdBrowse: standards-compliant tools for microarray experimental annotation, data management and dissemination. BMC Bioinformatics, 2005. 6(1): p. 264. [17] Morrison, N., et al., Annotation of environmental OMICS data: application to the transcriptomics domain. Omics, 2006. 10(2): p. 172-8. [18] Field, D., et al., Open software for biologists: from famine to feast. Nat Biotechnol, 2006. 24(7): p. 801-3. [19] Brazma, A., et al., Minimum information about a microarray experiment (MIAME)toward standards for microarray data. Nat Genet, 2001. 29(4): p. 365-71. [20] Sansone, S.A., et al., The metabolomics standards initiative. Nat Biotechnol, 2007. 25(8): p. 846-8. [21] Taylor, C.F., Standards for reporting bioscience data: a forward look. Drug Discov Today, 2007. 12(13-14): p. 527-33. [22] Westbroek, P., et al., A Model System Approach to Biological Climate Forcing - the Example of Emiliania huxleyi. Global and Planetary Change, 1993. 8(1-2): p. 27-46. [23] Westbroek, P., et al., Emiliania huxleyi as a key to biosphere-geoshere interactions., in The Haptophyte Algae., J.C. Green and B.S.C. Leadbeater, Editors. 1994, Clarendon Press: Oxford. p. 321-334. [24] Jacquet, S., et al., Effects of inorganic and organic nutrient addition on a coastal microbial community (Isefjord, Denmark). Marine Ecology-Progress Series, 2002. 228: p. 3-14. [25] Schroeder, D.C., et al., Coccolithovirus (Phycodnaviridae): Characterisation of a new large dsDNA algal virus that infects Emiliania huxleyi. Archives of Virology, 2002. 147(9): p. 1685-1698. [26] Wilson, W.H., G. Tarran, and M.V. Zubkov, Virus dynamics in a coccolithophoredominated bloom in the North Sea. Deep-Sea Research Part Ii-Topical Studies in Oceanography, 2002. 49(15): p. 2951-2963. [27] Wilson, W.H., et al., Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel. Journal of the Marine Biological Association of the UK., 2002. 82: p. 369-377. [28] Wilson, W.H., et al., Family: Phycodnaviridae., in Virus Taxonomy, VIIIth ICTV Report, C.M. Fauquet, et al., Editors. 2005, Elsevier/Academic Press: London. p. 163175. [29] Wilson, W.H., et al., Complete Genome Sequence and Lytic Phase Transcription Profile of a Coccolithovirus. Science, 2005. 309(5737): p. 1090-2. [30] Allen, M.J., et al., Evolutionary History of the Coccolithoviridae. Mol Biol Evol, 2006. 23(1): p. 86-92. [31] Allen, M.J., et al., Locus-specific gene expression pattern suggests a unique propagation strategy for a giant algal virus. J Virol, 2006. 80(15): p. 7699-705. [32] Allen, M.J., D.C. Schroeder, and W.H. Wilson, Preliminary characterisation of repeat families in the genome of EhV-86, a giant algal virus that infects the marine microalga Emiliania huxleyi. Arch Virol, 2006. 151: p. 525–535. [33] Allen, M.J., et al., Use of microarrays to assess viral diversity: from genotype to phenotype. Environ Microbiol, 2007. 9(4): p. 971-982. [34] Schroeder, D.C., et al., Virus Succession Observed during an Emiliania huxleyi Bloom. Applied and Environmental Microbiology, 2003. 69(5): p. 2484-2490.

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[35] Allen, M.J. and W.H. Wilson, The coccolithovirus microarray: an array of uses. Brief Funct Genomic Proteomic, 2006. 5(4): p. 273-9. [36] Cossins, A.R. and K. Bowler, The Temperature Biology of Animals. 1987, London: Chapman & Hall. 339. [37] Wendelaar Bonga, S., The stress response in fish. Physiological Reviews, 1997. 77: p. 591-625. [38] Bickler, P.E. and L.T. Buck, Hypoxia tolerance in reptiles, amphibians, and fishes: Life with variable oxygen availability. Annual Review of Physiology, 2007. 69: p. 145-170. [39] Feder, M., Key issues in achieving an integrative perspective on stress. J. Biosciences, 2007. 32: p. 433-440. [40] Gracey, A.Y. and A.R. Cossins, Application of microarray technology in environmental and comparative physiology. Annual Reviews of Physiology, 2003. 65: p. 231-259. [41] Cossins, A.R. and D.L. Crawford, Opinion - Fish as models for environmental genomics. Nature Reviews in Genetics, 2005. 6(4): p. 324-333. [42] Williams, D., et al., Genomic resources and microarrays for the common carp (Cyprinus carpio L). J. Fish Biology 2008: p. In press. [43] Tiku, P.E., et al., Cold-induced expression of ∆9-desaturase in carp by transcriptional and posttranslational mechanisms. Science, 1996. 271: p. 815-818. [44] Polley, S.D., et al., Differential expression of cold- and diet-specific genes encoding two carp liver delta 9-acyl-CoA desaturase isoforms. American Journal of Physiology; Regulatory, Integrative and Comparative Physiology, 2003. 284(1): p. R41-50. [45] Ashburner, M., et al., Gene Ontology: tool for the unification of biology. Nature Genetics, 2000. 25: p. 25-29. [46] Cossins, A., et al., Post-genomic approaches to understanding the mechanisms of environmentally induced phenotypic plasticity. Journal of Experimental Biology, 2006. 209: p. 2328-2336. [47] Nikinmaa, M. and B.B. Rees, Oxygen-dependent gene expression in fishes. Am J Physiol Regul Integr Comp Physiol, 2005. 288(5): p. R1079-90. [48] Fraser, J., et al., Hypoxia-inducible myoglobin expression in nonmuscle tissues. Proceedings of the National Academy of Sciences USA, 2006. 103: p. 2977-2981. [49] David, L., et al., Recent duplication of the Common Carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci. Molecular Biology and Evolution, 2003. 20: p. 1425-1434.

Reviewed by William H. Wilson of Bigelow Laboratory for Ocean Science Research, Maine, USA.

INDEX A abatement, 126, 458 absorption, x, 43, 64, 77, 173, 175, 176, 179, 192, 212, 320, 322, 476 abstraction, 326, 335, 353 abuse, xv, 423 accessibility, 237, 238, 242, 244, 245, 249, 254, 255 acclimatization, 235 accounting, 21, 212 accreditation, 403 accuracy, 27, 290, 292, 293, 351, 352, 353 acetic acid, 431 acid, 17, 19, 27, 33, 120, 122, 141, 144, 153, 155, 156, 192, 222, 226, 227, 228, 233, 235, 236, 362, 368, 369, 377, 379, 431, 435, 438, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 455, 456, 457, 458, 459, 460, 461, 462, 463 acidity, 446 activated carbon, 130, 179, 180, 434, 436, 445, 449, 450, 451, 452, 461, 462, 463 activation energy, 449, 457 active centers, 446 active site, 212, 446, 447 adaptability, 96 adaptation, 118, 373, 475, 476 additives, 31, 150, 154, 387, 403 adjudication, 403 adjustment, 97, 265, 395 adsorption, x, 18, 65, 68, 70, 97, 173, 175, 176, 177, 179, 182, 436, 442, 445, 446, 447, 449, 450, 451, 452, 459, 461, 462 advantages, xiii, 25, 31, 61, 62, 64, 81, 221, 357, 364, 378, 410, 434, 436, 470 advocacy, 120 aerobic bacteria, 48

aerogels, 450, 463 aflatoxin, xv, 386, 400, 410 AFM, 183 Africa, 5, 17, 20, 35, 116, 400 agar, 25, 26, 34, 222 agencies, xiv, 385, 391, 393, 398, 403, 411 aggregation, 221 aging process, 259, 260, 265, 266, 267 agricultural exports, xiv, xv, 385, 386, 399 agricultural market, 402, 403, 406 agricultural sector, 309, 310, 312 agriculture, 54, 75, 84, 100, 127, 141, 153, 162, 220, 307, 308, 309, 310, 311, 312, 315, 319, 387, 398, 399, 408, 416, 417, 424, 475 AIDS, 24, 25 air emissions, 155 air pollutants, 112 alanine, 227, 233 aldehydes, 448 algae, 24, 36, 436 algorithm, 469 allele, 27 alters, 379 aluminum oxide, 460 amino acid, 227, 235 amino acids, x, 63, 85, 141, 219, 224, 227, 228, 233 ammonia, 55, 82, 123, 163, 227, 358, 359, 365, 366, 370, 374, 376, 377, 378, 380, 382 ammonium, 63, 82, 377, 462 amphibians, 480 amplitude, 259 amylase, 82, 163 anaerobic bacteria, 60 anaerobic sludge, 46, 146 anatase, 446 anger, 468

482

Index

animal disease, 358, 417 animal welfare, xiv, 386 anionic surfactants, 437, 455 annealing, 383 annotation, 468, 469, 473, 479 annual rate, 399 ANOVA, 470 anoxia, 478 antagonism, 85 anthrax, 358, 380 antibiotic, 401, 410 antibody, 28 antioxidant, 280 anus, 45 apples, 425 aquaculture, 411 aquatic habitats, 320 aquatic life, 427 aqueous solutions, 174, 175, 182, 224, 453, 454, 457, 458, 459, 460, 461, 463 aquifers, xii, 16, 325, 326, 327, 328, 331, 333, 334, 335, 337, 338, 339, 350, 352, 353, 354 architecture, 278 Argentina, 100, 107, 174 Aristotle, 43 aromatic compounds, 373, 436, 441, 454 aromatic hydrocarbons, 43, 61, 76, 454 aromatics, 147 arrest, 45, 139, 152 arrests, 75 ARs, xiv, 385 arsenic, ix, x, xiii, 130, 156, 173, 174, 175, 325, 326, 327, 328, 338, 353 arthritis, 20, 38 arthropods, 84 asbestos, 131, 152, 154 aseptic, 16 Asia, xv, 17, 20, 76, 105, 114, 116, 120, 168, 287, 289, 386, 391, 393, 399, 419, 420, 421, 423 asian countries, 114, 399 assessment, xii, 5, 6, 31, 164, 191, 213, 254, 285, 289, 299, 304, 318, 324, 366, 374, 382, 383, 393, 395, 429 assimilation, 319 atmosphere, vii, 3, 130, 428 atmospheric pressure, 130 atomic force, 183 atomic force microscope, 183 atoms, 435 atypical pneumonia, 24

Austria, 37, 114, 157, 164, 168 authorities, 100, 116, 309, 310, 314, 400 automation, xiii, 29, 357 automobiles, 122, 123, 136, 141, 148, 156 avoidance, 134 awareness, xii, xiv, 31, 119, 132, 133, 135, 158, 305, 306, 310, 319, 320, 385, 396, 410, 411, 416, 417, 466

B BAC, 463, 468 Bacillus subtilis, 222, 371 bacteria, viii, xiii, 12, 13, 19, 20, 23, 24, 25, 29, 32, 36, 39, 43, 48, 55, 60, 64, 75, 78, 82, 83, 85, 97, 99, 107, 122, 126, 127, 145, 146, 147, 162, 222, 357, 358, 359, 361, 362, 363, 365, 367, 369, 370, 374, 377, 379, 381, 382, 383, 387, 391, 413, 414, 415, 428, 434 bacterial infection, 24 bacterium, 19, 25, 26, 98, 358, 382 Bangladesh, vi, xiii, 174, 325, 326, 327, 328, 353, 354, 355 banks, 240, 316, 323 barium, 119, 156 barometric pressure, 338 barriers, 30, 156, 401, 407 base pair, 367, 368, 382 basicity, 446 baths, 120, 192 batteries, 121, 122, 123, 124, 136, 141, 149, 155 bauxite, 149, 153 beef, 18, 222 behaviors, 255, 447 Beijing, 323 Belgium, 116, 139, 155 beneficial effect, 472 benefits, xv, 122, 142, 162, 242, 252, 255, 424, 430, 433, 468 benzene, 123, 129, 156, 461, 462 benzo(a)pyrene, 98 beta particles, 125 beverages, 150 bias, 29, 360, 377, 381 bicarbonate, 374, 412, 438 bile, 25 bioaccumulation, 77, 80, 188, 434 biochemistry, 391 bioconversion, 147, 161 biodegradability, 122, 127

Index biodegradable wastes, 47, 126 biodegradation, viii, x, 42, 43, 49, 51, 58, 59, 64, 65, 78, 82, 97, 98, 99, 108, 124, 126, 127, 141, 144, 145, 159, 169, 219, 220, 221, 222, 223, 224, 225, 227, 233, 234, 235, 382, 437 biodiversity, 286, 289, 306, 320, 322, 323, 360 biofuel, 146, 159 biogas, 146, 159, 160, 161, 162 bioindicators, 188, 216 bioinformatics, xvi, 465, 466, 467 biological control, 392 biological stability, 13 biological systems, xi, 257, 258, 264, 265, 466 biomarkers, 268 biomass, vii, 3, 7, 43, 45, 47, 48, 58, 59, 60, 62, 63, 65, 67, 76, 97, 105, 126, 127, 143, 145, 146, 147, 162, 163, 226, 235, 236, 369, 371 biomonitoring, xi, 271, 272 bioremediation, 81 biosensors, 29, 36 biosphere, 100, 258, 479 biosynthesis, 282 biotechnology, viii, 42, 82, 122, 143, 144, 392 biotic, 63, 290 biotin, 371 birds, xi, 154, 160, 163, 217, 271, 272, 273, 274, 275, 277, 279, 281, 282, 323, 324, 426, 427, 429 bisphenol, 75 blood pressure, 425 body fat, x, 187 body fluid, 121 body size, 192, 207 body weight, 43, 192, 193, 206, 208, 393, 395 boilers, 149, 150, 156 bonds, 453 bone, 189, 191, 205, 212 bones, 44, 52, 118, 220 boundary conditions, 352 brain, 16, 476, 477 Brazil, 45, 145, 147 breakdown, 99, 145, 163, 213, 405 breeding, xi, 271, 274, 275, 279 brevis, 30 bridges, 148 Britain, 242, 255 Brno, 215 buffalo, 49 buildings, 131, 152, 241, 307, 426 Bulgaria, 257, 271 Burma, 289

483

burn, 264 bursa, 191 butyl ether, 447 buyers, 132, 138, 139, 165, 404 by-products, xv, 70, 122, 154, 433, 435, 441, 447, 450, 460

C cabbage, 84 cabinets, 121 cadmium, x, 54, 57, 64, 76, 77, 119, 123, 130, 155, 156, 187, 191, 193, 196, 198, 201, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 calcium, 56, 59, 77, 85, 153, 473 calcium carbonate, 59, 473 campaigns, 416, 417 cancer, 17, 378, 391, 392, 393, 425, 426 candidates, 307 capillary, 468 capital intensive, 131 capitalism, 252 capsule, 127, 189 carbohydrate, 233, 476 carbohydrate metabolism, 476 carbohydrates, 44, 49 carbon, vii, 3, 4, 7, 11, 33, 35, 61, 62, 63, 64, 123, 125, 126, 128, 130, 143, 145, 146, 147, 153, 156, 159, 179, 180, 220, 234, 376, 434, 436, 449, 450, 451, 452, 453, 454, 457, 459, 461, 462, 463 carbon dioxide, 61, 62, 125, 128, 145, 159, 220 carbon materials, 461 carbon monoxide, 64, 128, 147 carboxylic acids, 448, 459 carcinogenicity, 122 Caribbean, 242 carotenoids, 279, 282 carp, 467, 469, 475, 476, 477, 480 cartilage, 189 case study, 251, 255, 323, 377 cash, 140 casting, 141 catalysis, 442, 445, 447, 458, 461 catalyst, 440, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 456, 458, 459, 460, 461, 462 catalytic activity, 440, 445, 446, 447, 448, 449, 450, 451, 463 catalytic effect, 443 catalytic reaction, 449, 450 catalytic system, xvi, 433, 435

484

Index

category a, 121 cation, 224, 443 cattle, 21, 45, 48, 50, 56, 59, 61, 63, 85, 86, 87, 89, 90, 117, 143, 150, 235, 293, 361, 406 CBS, 280 cDNA, 373, 375, 378, 469, 475, 476, 477 cell culture, 27, 371 cell line, 25, 362, 373 cell membranes, 476 cellulose, 46, 48, 49, 145, 222 cement, 21, 112, 115, 141, 146, 150, 151, 153, 154, 156 cement plants, 141, 154 census, xi, 237, 238, 239, 240, 241, 242, 247, 255 ceramic, 138, 150, 151, 192, 222 certificate, 409 certification, 407, 410, 412, 417 cesium, 125 cestodes, 188, 216 charitable organizations, 155 cheese, 143 chemical, viii, 17, 19, 21, 42, 43, 44, 46, 48, 54, 55, 56, 58, 62, 67, 70, 76, 77, 78, 81, 82, 85, 87, 89, 90, 91, 92, 95, 96, 99, 100, 119, 122, 123, 124, 125, 126, 130, 135, 137, 138, 142, 144, 145, 147, 150, 153, 154, 156, 174, 182, 183, 222, 235, 236, 259, 260, 378, 393, 394, 395, 407, 412, 428, 429, 434, 436, 437, 439, 445, 446, 449, 450, 463 chemical kinetics, 259 chemical properties, 236, 378, 407 chemical reactions, 58 chemical reactivity, 122 chemical stability, 446 chemicals, viii, ix, 42, 43, 44, 54, 55, 61, 66, 75, 76, 77, 78, 82, 84, 90, 98, 118, 119, 121, 122, 123, 129, 130, 133, 138, 139, 141, 158, 394, 408, 412, 416, 418, 426, 427, 428, 431 chemiluminescence, 25 chemoreceptors, 44 Chicago, 302, 376 chicken, 53, 281 child labor, xiv, 386 childhood, 34 Chile, 174, 400 China, 45, 67, 114, 120, 139, 145, 289, 322, 323, 423 chitin, 21, 84 chlorinated hydrocarbons, 130 chlorination, viii, 12, 13, 19, 30, 36, 437 chlorine, viii, 12, 21, 30, 401, 413, 415, 436, 452 chlorobenzene, 447, 460

chloroform, 274 cholera, 20, 23, 35 cholinesterase, 427 chromatography, 454 chromium, 77, 119, 123, 156 chromosome, 369 chronic diseases, 260, 267, 425 chronic hypoxia, 476 chronic illness, 426 chronology, 377 circulation, vii, 3, 209, 210, 212, 214 cities, 12, 124, 131, 145, 396 citizens, xv, 133, 386 city, 11, 55, 112, 140, 161, 166, 168, 240, 421 civilization, viii, 42, 97 class, 114, 121, 211, 290, 293, 427, 428 classes, 153, 290, 397 classification, 5, 6, 158, 252 cleaning, 46, 68, 76, 136, 143, 154, 156, 413, 415, 456 cleanup, 152 clients, 470 climate, vii, 3, 4, 5, 6, 7, 50, 165, 307, 308, 313 climate change, 5, 7, 50, 165 clitellum, 44 clone, 358, 359, 360, 366 cloning, 282, 359, 360, 378, 469 closure, 160 clusters, 472, 476 coagulation process, 436 coal, 78, 120, 145, 148, 149, 153, 156, 159, 398 coastal management, 322 cobalt, 457, 458 cocoon, 45 coding, 381, 473 coffee, 399 coke, 148, 149 colonization, 24, 31, 39, 68 color, 56, 150, 222, 282, 328, 401, 407, 434, 436, 441, 449 colour patches, 272 combined effect, x, 187 combustion, vii, 3, 123, 130, 146, 147, 153, 164, 175 commercial crop, 221 commodity, 113, 165, 394 communication, 26 community, xvi, 13, 24, 29, 32, 38, 39, 40, 61, 108, 135, 161, 164, 166, 169, 252, 286, 293, 335, 359, 360, 361, 364, 369, 371, 372, 373, 374, 375, 376,

Index 377, 378, 379, 380, 381, 382, 383, 384, 392, 452, 465, 466, 467, 471, 472, 479 competition, 203, 279, 364, 407, 467 competitive advantage, 165 competitiveness, xiv, 385 competitors, xiv, 386 complaints, 428 complement, 466 complementary DNA, 382 complexity, ix, 112, 131, 258, 259, 436, 451, 452, 472 compliance, xiv, 386, 404, 408, 409, 416, 472 complications, 19 composites, 121 composition, 46, 54, 118, 144, 163, 221, 222, 224, 227, 228, 233, 242, 243, 247, 290, 312, 313, 355, 360, 364, 366, 374, 376, 377, 379, 382, 384, 438 compost, 43, 46, 47, 54, 55, 59, 60, 61, 62, 63, 78, 79, 80, 82, 85, 86, 87, 89, 90, 93, 94, 95, 97, 99, 102, 105, 126, 136, 140, 141, 143, 144, 145, 146, 160, 161, 162, 163, 222, 227, 235, 236, 431 composting, viii, ix, 42, 43, 44, 45, 47, 49, 50, 55, 57, 58, 59, 60, 61, 62, 63, 87, 96, 97, 98, 99, 105, 106, 107, 108, 112, 125, 126, 127, 137, 143, 144, 145, 159, 161, 162, 163, 164, 222, 234, 236 compounds, xv, 17, 62, 63, 76, 78, 79, 80, 82, 98, 119, 123, 128, 129, 130, 150, 156, 177, 179, 181, 220, 221, 226, 227, 231, 367, 373, 391, 423, 426, 433, 434, 436, 437, 438, 440, 441, 442, 443, 445, 446, 448, 449, 450, 451, 452, 453, 454, 455, 456, 460, 462 compression, 220 computing, 471 conceptual model, 253 conditioning, 151, 319 conductance, 25, 34 conduction, 333 conductivity, 65, 221, 258, 327, 331, 340, 341, 342, 352, 355 conference, 104 configuration, 159, 436 conflict, 309, 313 conformity, 409 connective tissue, 210 connectivity, xiii, 287, 307, 321, 325, 338, 353 consciousness, 132 consensus, 131 consent, 312 conservation, 126, 138, 164, 165, 286, 287, 289, 290, 291, 292, 301, 302, 304, 305, 307, 308, 309,

485

310, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324 constant rate, 332, 350 constitution, 286 construction, 30, 113, 128, 131, 140, 144, 145, 148, 152, 153, 165, 307, 308, 311, 316, 319, 351, 353, 467, 469, 472, 473, 474 consumer choice, 254 consumer demand, 395, 396 consumer education, 132, 164 consumer electronics, 121, 130 consumer goods, 122, 132, 137, 138, 164 consumers, 13, 30, 114, 119, 120, 132, 133, 137, 138, 140, 157, 164, 165, 310, 391, 396, 401, 402, 412, 416, 418 consumption, vii, 11, 16, 17, 20, 22, 33, 38, 64, 112, 113, 114, 132, 133, 135, 136, 139, 147, 148, 157, 162, 164, 189, 213, 224, 391, 393, 394, 396, 406, 407, 412, 425, 438, 447, 449 contact time, 17 contaminant, 31, 99, 188, 191, 434, 443, 444, 446, 447, 450, 452 contaminated sites, 76 contaminated soil, 78, 79, 80, 98 contaminated soils, ix, 42, 43, 76, 78, 99 contaminated water, xiv, 13, 24, 30, 327, 386 contamination, viii, xiii, 12, 13, 16, 17, 25, 31, 35, 123, 162, 174, 211, 263, 272, 325, 326, 354, 391, 392, 398, 399, 400, 403, 412, 413, 415, 416, 428, 429, 431, 434 content analysis, 306 contract enforcement, 404 control measures, 406, 407 controversies, 314 convention, 126 cooking, 48, 119, 143, 146, 150, 391, 394, 412, 414, 415 cooling, 48, 64, 404, 414 coordination, 174, 175, 176, 179, 260, 365, 379, 405 copper, 54, 77, 85, 144, 148, 149, 174 copulation, 44 corporate sector, 411 correlation, 208, 279, 372, 477 correlation coefficient, 208 correlations, 268, 376 corrosion, 148 corrosivity, 122 cosmetics, 123, 401 cost, viii, ix, xiii, xiv, xvi, 12, 13, 22, 25, 29, 30, 32, 40, 54, 76, 96, 97, 98, 99, 112, 113, 114, 120, 128,

Index

486

131, 138, 139, 140, 141, 145, 147, 148, 149, 152, 153, 154, 161, 162, 165, 357, 359, 386, 394, 400, 404, 409, 416, 418, 429, 440, 465, 466, 470 cost effectiveness, 418 costs of compliance, 408 cotton, 127, 136, 138, 149, 169, 424, 427, 430 covering, 47, 159, 286, 287, 293, 300, 306, 312, 388, 406, 407, 466 coxsackievirus, 17 cracks, 413 crew, 221 crisis management, 408, 418 critical period, 425 critical value, 248 criticism, 311 crop production, 221 crops, 18, 39, 84, 85, 87, 89, 90, 95, 96, 100, 150, 221, 235, 388, 391, 392, 393, 394, 406, 412, 424, 425 cross-fertilization, 44 crude oil, 43 crust, 125 cryptosporidium, 37 crystalline, 175 cues, 279 cultivation, 221, 231, 232, 308, 311, 377, 412 cultural barriers, 401 culture, 25, 26, 27, 28, 36, 37, 65, 113, 119, 132, 162, 163, 219, 221, 222, 223, 229, 230, 231, 232, 233, 234, 235, 236, 358, 359, 373, 468 culture media, 25, 37 cumulative frequency, 247, 248 cumulative percentage, 248 current limit, 380 curricula, 417 customers, 140 cuticle, 44, 212 cyanide, 130, 456 cycles, 7, 19, 21, 38, 449, 450 cycling, 7, 366, 367, 371, 383, 473 cyst, x, 24, 39, 187, 189, 190, 191, 193, 194, 196, 197, 198, 199, 201, 202, 203, 204, 208, 209, 210, 211 cystathionine, 280, 283 cysteine, xii, 227, 233, 272, 280, 283

D damages, 260, 264 danger, 30, 121

Darwin, Charles, 43, 96, 100 data analysis, 29, 351, 353, 355, 466, 470 data collection, 32, 338 data set, 344, 345, 472, 477 database, 5, 239, 240, 241, 375, 378, 471, 474, 478 Dead Sea, 308 deaths, xv, 18, 121, 386, 425, 426 decay, 75, 244, 245 decentralization, 161 decision makers, 310, 318, 319, 320 decomposition, vii, xv, 3, 5, 6, 7, 43, 48, 50, 59, 61, 63, 64, 96, 126, 127, 159, 160, 162, 163, 235, 236, 433, 435, 436, 438, 439, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 456, 457, 458, 460, 461, 463 defects, 393 deficiencies, 306, 312, 317, 319, 320 deficiency, 221, 231, 252, 279, 283 deforestation, 137, 148, 164, 466 degradation, viii, xv, 17, 42, 46, 49, 50, 52, 55, 58, 59, 65, 86, 98, 99, 125, 145, 338, 360, 373, 377, 378, 433, 434, 437, 438, 439, 440, 441, 443, 446, 451, 455, 456, 457, 459, 460, 461, 463 degradation process, 46 Degussa, 440, 441, 442 dehydration, 22, 54, 191 dematerialization, 136 demographic data, 302 demography, 126 denitrification, 365, 366 denitrifying, 236, 377, 382, 434 Denmark, 20, 116, 131, 139, 155, 164, 479 Department of Agriculture, 405, 407, 409, 421, 430 Department of Energy, 83 Department of Health and Human Services, 33, 430 Department of the Interior (DOI), 355, 431 dependent variable, 208, 289 deposition, 6, 68, 198, 209, 212, 213, 214, 279, 323, 327, 475 deposits, x, 40, 187, 191, 206, 207, 212, 214 depression, 331, 333, 346 deprivation, xi, 237, 238, 242, 243, 247, 249, 250, 251, 252, 255 derivatives, 440 desorption, x, 173, 174, 175 destination, 244, 245 destiny, 182 destruction, viii, 42, 59, 63, 130, 213, 293, 392, 458, 459, 460, 466

Index detection, xiii, 17, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 39, 40, 272, 273, 357, 362, 363, 364, 365, 366, 367, 369, 370, 371, 373, 374, 375, 377, 378, 379, 380, 381, 382, 383, 384, 400, 401, 408, 441, 466 detection system, 28 detergents, 122, 415 detoxification, 130, 209, 210 developed countries, vii, 11, 12, 17, 21, 22, 30, 31, 32, 124, 163 developed nations, 115, 117, 120, 139, 154, 155 developing countries, vii, xiv, 11, 17, 19, 20, 21, 116, 155, 158, 165, 174, 386, 416, 426, 430 developing nations, 76, 120, 126 deviation, 245 diabetes, 425 diagnosis, 26, 268, 430 diamonds, 177 diaphragm, 210 diarrhea, 18, 38, 421 diesel engines, 146 diet, xii, 189, 209, 212, 272, 273, 279, 281, 391, 393, 394, 395, 398, 425, 480 dietary intake, 209, 210, 394 diffraction, 175, 176 diffusion, 258, 365, 444, 445 digestion, 23, 136, 161, 192, 441, 456 dimorphism, 211, 281 dioxin, 43, 120 dioxins, 119, 130, 131, 146, 151, 154, 388 direct investment, 410 directives, 157, 164, 318, 320 dirt, 127, 152 disability, 242 disadvantages, xv, 433, 434 discharges, 330 discriminant analysis, 274 discrimination, 366, 367, 383 disinfection, viii, xv, 12, 13, 21, 24, 30, 34, 36, 37, 42, 433, 434, 458 dispersion, 82, 449 displacement, 29, 248, 371 disposable income, 396 dissipative structure, 258 dissociation, 367, 368, 379, 380, 384 dissolved oxygen, 222, 224 distillation, 156 distilled water, 192 disturbances, 259, 273, 286, 293, 303 diversification, 405

487

diversity, xiii, 13, 252, 286, 306, 323, 357, 358, 359, 360, 373, 377, 379, 380, 381, 382, 383, 384, 474, 479 DNA, vi, xiii, 28, 29, 32, 37, 273, 277, 357, 359, 360, 361, 362, 365, 368, 369, 370, 371, 372, 373, 374, 375, 377, 378, 379, 380, 381, 382, 383, 384, 466, 468, 469, 470, 477, 478 DNA extraction, 360, 381 DNA polymerase, 371 DNA sequencing, 466, 468, 477 DNase, 375 DOC, 454 dogs, 21, 116, 117 domestic markets, 387 DOP, 317 dosage, 283 double bonds, 453 draft, 255 drainage, 62, 82, 151, 306, 307, 310, 311, 312, 316, 319, 324 drawing, ix, 112, 471 drinking water, viii, ix, xv, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 120, 151, 173, 174, 393, 428, 431, 433, 435, 436, 452, 453, 454, 460 drosophila, 466, 478 drug resistance, 20 drugs, 387, 401 drying, 192, 308, 406, 410 dumping, 99, 116, 120, 165, 220 duodenum, 210 dyeing, 122 dyes, 27, 123, 130, 362, 455, 457, 458

E E.coli, 18, 60, 61 early warning, 38 earthworms, viii, ix, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 107, 108, 126, 127, 143, 145, 146, 169 East Asia, xv, 386, 399 Eastern Europe, 419 ecology, xiii, 13, 31, 32, 303, 322, 323, 324, 357, 359, 360, 369, 371, 374, 375, 376, 377, 379, 380, 381, 383 economic consequences, 13

488

Index

economic development, 317 economic growth, 395 economic incentives, 392 economic independence, 315 economic losses, 22, 400, 425 economic problem, 140, 155 economy, 97, 116, 136, 138, 140, 141, 309, 424 ecosystem, xii, 122, 126, 136, 138, 141, 148, 278, 285, 286, 289, 302, 313, 321, 322, 466 editors, 38, 282 education, 31, 119, 132, 166 educators, 133 efficiency, 50, 72, 105, 107, 136, 462 effluent, 18, 67, 220, 373, 379, 398, 411, 437, 440, 441, 443, 455, 456 effluents, xii, 36, 46, 67, 150, 161, 220, 305, 306, 307, 308, 378, 434, 436, 439, 440, 443, 451, 455, 460, 462 egg, 21, 52, 139, 149, 394 Egypt, 400 electric charge, 260 electric conductivity, 327 electric current, 130, 148, 258 electricity, 131, 146, 150, 159, 160, 161, 162, 165 electrolysis, 155 electromagnetic, 125, 259 electromagnetism, 258 electron, 175, 176, 183 electronic structure, 174 electrons, 441 electrophoresis, 26, 27, 29, 360, 378, 380, 381 elongation, 83, 227, 229 elucidation, 374 emission, ix, 48, 61, 64, 96, 112, 119, 124, 125, 129, 131, 142, 144, 146, 147, 148, 149, 156, 159, 160, 163, 165, 175, 177, 178, 179, 180 emitters, 125 employment, 242 encephalitis, 16, 388 encephalopathy, xiv, 385 encoding, 358, 380, 480 endangered species, 289, 292 endocrine, 75, 98, 426, 429, 430, 467 endonuclease, 26 energy, viii, xi, 42, 62, 74, 75, 124, 125, 126, 131, 134, 135, 136, 137, 142, 143, 146, 148, 149, 150, 151, 154, 156, 164, 165, 177, 178, 179, 180, 181, 209, 213, 235, 257, 259, 260, 261, 262, 268, 358, 449, 457, 476 energy consumption, 136

energy recovery, 131, 146 energy supply, 165 energy transfer, 260 enforcement, xiv, 309, 314, 385, 402, 404, 407, 408, 411, 416 engineering, 126, 127, 153 England, vi, xi, 37, 39, 45, 145, 237, 238, 239, 252, 254, 473 enlargement, 283 enrollment, 397 enterovirus, 16, 33 entrepreneurs, 404 entropy, 258, 264, 265 environmental awareness, 319, 320 environmental change, 273, 466 environmental conditions, xi, 21, 144, 252, 268, 271, 280, 466, 472, 475 environmental contamination, 31, 211, 416 environmental degradation, 17 environmental effects, 158 environmental factors, 292, 379 environmental impact, 133, 135, 434 environmental influences, xi, 257 environmental issues, 120, 133, 319 environmental management, viii, 41, 320 environmental policy, 305, 324 environmental protection, 137 Environmental Protection Agency (EPA), 13, 22, 109, 166, 428, 431 environmental quality, 252, 313 environmental regulations, 120 environmental temperatures, 34, 476 enzymatic activity, 106 enzymes, 25, 48, 49, 59, 81, 82, 84, 99, 122, 145, 162, 163, 365, 382 eosinophils, 210 epidemic, 17 epidemiology, 22, 26, 32, 40 epithelial cells, 280 epithelium, 476 EPR, 157 equilibrium, 176, 179, 269, 332, 335, 340, 342, 367, 368, 380, 384 equilibrium sorption, 179 equipment, 29, 123, 131, 139, 141, 148, 155, 156, 224, 364, 408, 409, 413, 427, 428, 470 equity, xi, 237, 238, 252, 253, 254, 255, 256 erosion, 6, 139, 145, 156, 221, 327 esophagus, 55, 56 EST, 466, 468, 470, 472, 477

Index ethanol, 143, 146, 147, 175, 176, 177 ethics, 139, 155 ethnic groups, xi, 237, 242, 247, 248, 249, 252, 255 ethnicity, 237, 238, 242, 252 ethyl alcohol, 150 etiology, 22, 23, 37, 380 eukaryotic cell, 373 European Commission, 401 European Community, 115 European Union (EU), xv, 113, 131, 139, 151, 155, 157, 166, 386, 396, 397, 400, 401, 408, 409, 410, 418 evaporation, 150, 160 EXAFS, 175, 176, 184 excretion, 77, 99 execution, 469 exercise, 253 expenditures, 126, 255 experiences, ix, 112 experimental condition, 440, 445, 446, 448 experimental design, 467, 470 expertise, 466, 467, 468, 469, 472 exploitation, xii, 305, 306 exploration, 255, 257, 274, 375 exporter, 400 exports, xiv, xv, 385, 386, 399, 400, 401, 407, 408, 409, 416, 417, 418 exposure, 12, 17, 209, 211, 213, 394, 395, 396, 426, 427, 430, 476 expressed sequence tag, 468, 478 external environment, 21 extinction, 287 extraction, 47, 107, 125, 151, 212, 360, 373, 381, 402, 434, 468, 470, 474

F fabrication, 466, 469, 477 factor analysis, 22 factories, 220, 410 false negative, 302 false positive, 302 famine, 479 farm size, 404, 410 farmers, 43, 45, 82, 85, 96, 100, 144, 145, 404, 405, 406, 407, 410, 411, 416 farmland, 76, 82, 100 farms, 43, 54, 97, 140, 144, 162, 235, 241, 409 fat, x, 187, 191, 192, 205, 206, 207, 212, 214, 407 fatty acids, 150

489

faults, 312, 317 fauna, 43, 293 FDA, 391, 394 fears, 408 feces, 18, 20, 47, 48 feedback, 253, 260, 261, 262 femur, 191, 192, 203, 205, 212 fencing, 151, 241 fermentation, 146, 147, 220, 224, 236 ferrous ion, 457, 458 fertility, viii, 42, 45, 82, 83, 96, 100, 145, 220, 227, 233, 307 fertilization, 44, 221, 222, 231, 235 fertilizers, x, 81, 85, 86, 87, 89, 90, 91, 92, 95, 96, 99, 100, 143, 146, 219, 221, 223, 224, 226, 227, 228, 229, 230, 231, 232, 233, 307, 428 fever, 16, 18 fiber, 152, 450, 463 fibers, 121, 127, 150 fibrin, 210 fibrosis, 378 field trials, 84 films, 122, 151, 457 filters, 40 filtration, viii, 18, 25, 30, 42, 65, 66, 68, 74, 75, 76, 97, 98, 449 financial resources, 155 financial support, xii, 285 Finland, 18, 40, 116 fish, x, 53, 63, 127, 174, 188, 212, 215, 216, 219, 220, 221, 222, 233, 234, 235, 240, 279, 371, 379, 396, 399, 401, 408, 412, 413, 415, 427, 429, 432, 467, 475, 476, 478, 480 fisheries, 220, 235, 399, 401 fishing, 114 fission, 124, 125 fitness, 191 fixation, 99, 361, 366, 374, 381 flame, 130, 192, 414, 415 flammability, 122 flatness, 332 flexibility, 123, 309 flocculation, 30, 436 flooding, 306, 313, 335 flora, 48, 80, 408 flotation, 150 flour, 426 fluctuations, xiii, 308, 325, 335, 338, 353, 475 flue gas, 153 fluid, 20, 47, 64, 75, 136, 463

Index

490

fluorescence, 25, 28, 175, 177, 178, 179, 180, 181, 361, 362, 374, 470 fluorophores, 29 foams, 222 food additives, 387, 403 food industry, 46, 145 food poisoning, 398, 429 food production, 46, 122 food products, 20, 54, 97, 135, 392, 400, 401, 403, 407, 416 food safety, xiv, xv, 385, 386, 387, 388, 393, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 416, 417, 418, 419 food security, xiv, 386 foodborne illness, 18, 391, 412, 415 foreign direct investment (FDI), 410, 420 formaldehyde, 123 formamide, 367, 369 formula, 179, 181, 192, 223, 245, 332, 338, 346 fouling, viii, 12, 13 foundations, 152 fragments, 378, 468, 469 framing, 152 France, 45, 46, 115, 125, 139, 145, 146, 164, 165, 436 free radicals, 261, 437, 443, 454 free trade, 120 freezing, 414, 475, 476 frequencies, xi, 209, 259, 271, 278 frequency distribution, 247 freshwater, xiii, 38, 162, 216, 358, 359, 376, 377, 475 fruits, 84, 85, 96, 108, 136, 221, 396, 399, 413, 416 funding, 31, 151, 256, 302, 466, 471 fungi, 24, 43, 60, 78, 82, 83, 84, 97, 122, 126, 127, 145, 235, 387, 428, 442 fungus, 52, 55, 60, 75, 81, 84 fusion, 24, 25, 374

G gamma rays, 125 garbage, ix, 61, 96, 112, 115, 116, 141, 147, 162, 163, 308, 406 gastroenteritis, 16, 18, 20, 22, 32, 37 gastrointestinal tract, 20, 24, 211 GDP, xv, 157, 386, 395 GDP per capita, 395 gel, 26, 27, 360, 364, 365, 367, 368, 371, 378, 380, 381, 382

gender differences, 203 gene arrays, 28, 373, 384 gene expression, xiii, 280, 357, 362, 373, 374, 378, 380, 382, 476, 477, 478, 479, 480 genes, 24, 27, 28, 272, 358, 359, 360, 362, 363, 365, 366, 367, 369, 370, 371, 372, 373, 374, 376, 377, 378, 379, 380, 381, 382, 383, 384, 470, 471, 472, 473, 474, 475, 476, 477, 478, 480 genetic diversity, 474 genetic information, 260, 378 genetic mutations, 265 genetics, 289, 302 genome, 27, 28, 40, 261, 371, 378, 379, 380, 383, 384, 466, 468, 473, 474, 477, 478, 479, 480 genomics, xvi, 465, 466, 468, 472, 480 genotype, 479 geography, 126 geology, 355 Georgia, 168, 255 Germany, 62, 115, 117, 139, 224, 400 germination, 83, 87, 95, 223, 226, 229, 231, 232, 235, 442 gizzard, 49, 56, 81, 97, 99 glasses, 116, 136, 138, 142, 147, 150, 151, 161 global climate change, 5 global consequences, 165 global economy, xiv, 386 globalization, xiv, 386, 399, 416 glucose, 33 glutamate, 280 glutathione, 280, 281, 283 glycine, 227, 233, 280 glycol, 123 glycolysis, 476 gonads, 191 Gore, Al, 174 governance, 309 government intervention, 132 government policy, 164 governments, 31, 132, 137, 155, 166, 394, 402, 405, 411 GPS, 289 grades, 149, 407, 410, 416 gradient formation, 234 grading, 290, 404, 407, 411 granules, 280 graph, 347, 348, 351, 353 grass, 48, 59 grasses, 47, 48, 147 grassroots, 120

Index gravity, 21, 69, 75, 129, 156, 160, 407 grazing, 114 Greece, 38 green revolution, 82, 118 greenhouse gases, vii, ix, 3, 61, 112, 124, 125, 128, 139, 149, 156, 160 greening, 323 groundwater, xiii, 22, 29, 32, 40, 119, 129, 130, 162, 325, 326, 328, 329, 331, 333, 334, 335, 338, 342, 352, 353, 354, 355, 371, 374, 431, 453, 454 group activities, xi, 237 grouping, 244 growth factor, 234 growth hormone, 48, 85, 99, 387 growth rate, 361 Guangdong, 120 guidelines, 4, 121, 314, 317, 393, 404, 427, 430, 471

H habitats, xii, 24, 32, 285, 287, 289, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 301, 305, 306, 307, 310, 314, 316, 320, 358, 360, 361, 373 hair, 141 half-life, 125 hammer, 155 hardener, 150 hardness, 280, 281 harvesting, 35, 95, 96, 409 hazardous materials, 156, 157, 454 hazardous waste, 120, 122, 154, 157, 158, 160, 256 hazardous wastes, 99, 116, 118, 120, 121, 122, 123, 124, 128, 131, 133, 139, 141, 142, 145, 154, 156, 158, 165 hazards, 30, 35, 131, 388, 391, 408, 417, 426, 430, 432 HDPE, 56, 78, 143, 151, 152 headache, 427 Health and Human Services, 430 health care, 13, 24 health effects, 23, 38, 426, 430 health problems, 394 health status, xi, 257, 259, 272, 279 heart disease, 425 heavy metals, 43, 47, 54, 55, 56, 57, 58, 59, 61, 64, 74, 75, 76, 77, 78, 96, 98, 106, 119, 123, 129, 130, 157, 216, 217, 234, 261, 265, 387, 398, 399, 401, 442 height, 86, 224, 330, 331 hematite, 183

491

hemisphere, 5, 6, 400 hepatitis, xiv, 17, 386 herbicide, 425 heterogeneous catalysis, 442, 445 high blood pressure, 425 highlands, 287 HIV, 20 homeostasis, xi, 257, 259, 260, 476 homocysteine, 280 homogeneity, 192 Hong Kong, 139, 167, 170, 236 host, x, xiv, 20, 24, 28, 31, 33, 37, 39, 187, 188, 189, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 386, 427, 428, 473 hotel, 386 hotels, 162 house, 281 housing, ix, 111, 242, 252, 317, 323 hub, 216 human activity, 174 human behavior, 164 human capital, 417 human genome, 28 human health, ix, 112, 121, 122, 151, 154, 174, 387, 426, 430 human immunodeficiency virus, 33 human milk, 398 humoral immunity, 261 humus, 49, 81, 82, 100, 222 Hunter, 19, 35, 272, 281 hunting, 273, 279, 283 hyaline, 189 hybrid, 161, 175 hybridization, xiii, 28, 29, 357, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 378, 380, 381, 382, 383 hydrocarbons, 76, 78, 80, 98, 99, 130, 147, 434, 449, 454 hydrogen, 56, 59, 64, 70, 128, 154, 163, 358, 434, 437, 438, 439, 453, 454, 455, 457, 460, 461, 462 hydrogen chloride, 154 hydrogen peroxide, 434, 437, 438, 439, 453, 454, 455, 457, 460, 461, 462 hydrolysis, 222, 462 hydroponics, 221, 235 hydroquinone, 448 hydroxide, ix, x, 155, 173, 174, 175, 182, 453 hydroxyl, xv, 433, 435, 437, 438, 439, 440, 443, 445, 446, 451, 453 hygiene, xiv, 156, 385, 401, 408, 410, 416, 417

Index

492 hypertrophy, 196, 198, 213 hypoxia, 467, 475, 476, 477

I ice, 151, 411 ideal, 65, 68, 76, 97, 131, 238, 449, 468 identity, 367, 475, 478 ideology, 322 image, 183, 287, 308, 470 image interpretation, 287 images, 27, 183, 470 immigration, 316, 319, 320, 321, 322 immobilization, xiii, 77, 78, 357, 372 immune memory, 261 immune reaction, 261 immune response, 260, 261 immune system, 260 immunity, 261 immunocompromised, 13 immunodeficiency, 33 immunosuppression, 426 impacts, xii, 61, 87, 89, 90, 93, 95, 214, 305, 306, 307, 308, 317, 319, 323 imports, 401, 403, 417 impregnation, 446 impurities, 68 incidence, viii, 12, 17, 20, 36, 37, 84, 90, 259, 394, 417 income support, 242 incomplete combustion, vii, 3 incubation period, 374 independence, 315, 319 independent variable, 208, 289 India, vi, ix, xiv, xv, 41, 44, 46, 49, 50, 83, 85, 87, 89, 100, 101, 102, 104, 105, 107, 108, 109, 112, 114, 116, 120, 132, 139, 162, 167, 168, 169, 289, 302, 303, 355, 385, 386, 387, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 406, 407, 408, 409, 410, 411, 416, 417, 418, 419, 420, 421, 423, 424, 425, 426, 427, 428, 430, 431, 432 Indians, xi, 114, 237 individual differences, 211 Indonesia, 116 induction, 373 industrial processing, 137 industrial revolution, 118 industrial wastes, ix, 112, 141, 142, 144, 147, 152, 154, 164, 165 inequality, 248, 255, 265

inequity, 249 infants, 124, 394 inferences, 471 inflammation, 17 information exchange, 258 information technology, xv, 387 infrastructure, xiv, xvi, 139, 307, 311, 312, 313, 324, 385, 395, 406, 417, 465, 467 ingest, 49, 54, 55, 63, 70, 75, 77, 81, 98, 391 ingestion, 12, 13, 17, 43, 64 initiation, 319, 453 inoculum, 159 insecticide, 400, 424, 426, 431 insects, 84, 123, 406, 412, 425, 426, 427, 429 inspections, 409 inspectors, 409 institutional change, 417 institutions, ix, 112, 155, 310, 312, 313, 314 insulation, 123, 152 integration, 30 intelligence, 426 intercepts, 347 interface, 38, 306 interference, x, 173 International Atomic Energy Agency, 168 international law, 120 international standards, 398, 407 intervention, xii, 31, 32, 291, 305 intervention strategies, 31 intestinal malabsorption, 21 intestine, 43, 48, 49, 56, 60, 81, 107 intoxication, 260 invertebrates, 22 investments, 31, 402, 404, 406, 411 ion-exchange, 181 ionic strength, 445 ions, 70, 176, 365, 438, 453, 456, 458 Iowa, 215 Ireland, 11, 40, 44, 61, 76, 77, 104, 164 iron, 77, 85, 148, 149, 326, 338, 436, 457, 460 irradiation, 30, 455 isolation, 25, 28, 373 isotherms, 176, 177 isotope, 7, 374, 376 Israel, vi, xii, 45, 145, 166, 305, 306, 307, 308, 309, 312, 313, 314, 315, 316, 317, 319, 320, 321, 322, 323, 324 Italy, 43, 45, 46, 102, 109, 115, 145, 146, 155, 164, 400

Index

J Jammu and Kashmir, 402 Japan, 45, 46, 115, 121, 125, 131, 139, 141, 145, 155, 165, 173, 174, 175, 282, 456 Jordan, 308 Jordan River, 308 jurisdiction, 310, 311, 312, 313, 315, 316 juveniles, 209, 277, 279

K Kenya, 166, 170 keratin, 21, 280, 281 keratinocytes, 282 kidney, x, 187, 188, 189, 190, 191, 192, 193, 194, 196, 197, 198, 199, 201, 202, 203, 206, 208, 209, 210, 211, 212, 213, 214, 215, 393 kidneys, 18, 190, 191, 193, 196, 201, 208, 209, 210 kill, 21, 45, 84, 114, 124, 415 kinetic constants, 450 kinetic model, 454, 455 kinetics, 258, 259, 360, 367, 438, 441, 443, 452, 455, 457, 460 Korea, 45, 139, 145, 155, 219, 220, 223, 224, 233

L labeling, 365, 373, 376, 380, 382, 403, 407, 413 lack of confidence, 13 lakes, viii, 12, 22, 38, 379, 426 landfills, ix, 46, 48, 54, 55, 62, 76, 96, 99, 112, 113, 114, 119, 120, 121, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 139, 140, 142, 144, 145, 151, 152, 154, 159, 160, 164, 165, 316 landscape, xii, 6, 285, 286, 287, 289, 290, 299, 300, 301, 306, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319, 321, 322, 323, 324 landscapes, xii, 300, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 429 Latin America, 17 leaching, 119, 129, 447, 448, 449 lead, ix, x, 18, 44, 57, 63, 76, 77, 85, 119, 120, 123, 130, 144, 145, 149, 154, 155, 173, 174, 179, 181, 182, 187, 198, 201, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 280, 309, 313, 374, 387, 391, 398 lead content, 398

493

leadership, 472 leakage, xiii, 130, 325, 326, 332, 335, 338, 340, 341, 353 leaks, 413 legislation, xiv, 156, 306, 309, 311, 312, 315, 319, 320, 385, 416, 417, 436 leisure, vii, 11, 12, 240, 254, 307 lens, 280, 282, 342 lesions, 210 Lewis acids, 446 liberalization, 399 liberation, 17 life cycle, 188 lifetime, 115, 177, 179, 393 ligand, 449, 461 lignin, 69, 126, 127, 145, 150, 455 limestone, 59, 153, 154 limitations, 360 lipases, 49 lipids, 21, 126, 145, 476 liquid chromatography, 454 liquid phase, 224, 445 liquids, 123 listeria monocytogenes, 23 literacy, xv, 387, 395, 404 literacy rates, 404 litigation, 431 liver, x, 17, 187, 188, 189, 191, 192, 193, 196, 198, 201, 203, 206, 209, 210, 211, 214, 215, 393, 476, 480 liver cancer, 17 liver damage, 214 livestock, 12, 18, 19, 20, 22, 31, 34, 46, 47, 67, 387, 399 living environment, 242 local authorities, 314 local government, ix, 48, 112, 140 localization, 274 logging, 47 low temperatures, vii, 11, 130 lubricants, 156 lubricating oil, 156 lupus, 188 lymphocytes, 210 lysine, 44, 63, 227, 233, 362 lysis, 23 lysosome, 24

Index

494

M machinery, 141, 148, 405, 425 macromolecules, 258, 260, 261, 265 macronutrients, 221 macrophages, 25, 210 magazines, 115, 123, 138 magnesium, 62, 82, 85 magnetic resonance, 176 magnets, 148 magnitude, 359, 426 Maine, 480 majority, xiii, xvi, 5, 12, 21, 30, 134, 198, 240, 308, 357, 375, 404, 410, 440, 465, 473, 474 malabsorption, 21 malaria, xv, 398, 423, 425, 427, 429, 430, 431 Malaysia, 120, 235, 302 management, viii, ix, xii, xiv, 30, 35, 41, 43, 45, 46, 61, 67, 96, 101, 112, 126, 130, 131, 132, 133, 134, 135, 139, 145, 156, 158, 161, 164, 165, 166, 285, 286, 303, 308, 309, 312, 313, 314, 320, 321, 322, 324, 326, 386, 392, 405, 406, 408, 410, 416, 417, 418, 431, 466, 471, 479 mandarin, 421 mandible, xi, 271, 272, 273, 274, 275, 276, 277 manganese, 77, 326, 436, 457, 458, 459, 461 manpower, 139 manufacture, 113, 122, 132, 135, 138, 149, 150, 151, 152, 153, 154, 155, 157, 410, 426, 430 manufacturing, xiv, 47, 122, 132, 136, 141, 220, 221, 365, 385, 410, 411, 416, 417, 426 manure, 18, 19, 46, 55, 56, 59, 61, 107, 146, 220, 234, 235 mapping, 256, 469, 478 marine environment, 376 markers, 361, 377 market access, 417 marketing, xiv, 385, 396, 402, 403, 405, 406, 410, 417 marketplace, 165 marsh, 382 Marx, 431 Maryland, 324 mastitis, 20 mathematical methods, 339 matrix, xii, 28, 285, 300, 438 meat, 20, 52, 151, 394, 396, 412, 413, 414, 415 mechanical engineering, 126 media, xiv, 23, 25, 34, 37, 69, 132, 133, 166, 355, 385, 396, 398, 416, 427, 454

median, 243, 247, 249, 287 medicines, 122 Mediterranean, 307, 308, 316, 324 Mediterranean climate, 308 MEK, 129 melanin, xii, 272, 280, 282 melt, 130, 151 melting, 151, 190, 211, 214, 215, 366, 367, 382 melting temperature, 366 membrane separation processes, 234 membranes, 24, 476 memory, 261 meningitis, 16 mercury, 54, 76, 77, 119, 120, 123, 124, 130, 144, 155, 174, 212, 387 mesoporous materials, 175, 176, 183, 445 messages, 132 meta-analysis, 303 metabolic disturbances, 269 metabolic pathways, 226, 279 metabolism, 59, 61, 112, 174, 214, 224, 236, 261, 281, 476 metabolites, 227, 425, 426 metal oxides, 445, 446, 447, 448, 459 metals, 43, 44, 47, 54, 55, 56, 57, 58, 59, 61, 64, 74, 75, 76, 77, 96, 98, 106, 112, 116, 117, 119, 120, 123, 129, 130, 136, 138, 140, 142, 148, 152, 154, 155, 156, 157, 161, 163, 188, 190, 191, 192, 202, 203, 205, 208, 209, 210, 212, 214, 215, 216, 217, 234, 261, 265, 387, 398, 399, 401, 434, 442, 443, 445, 446, 447, 448, 450, 452, 457 meter, 65, 68, 75, 97, 98, 151, 160, 163, 326 methanol, 449 methodology, 252, 255, 306, 395, 397 metropolitan areas, 12 Mexico, 102, 123, 401 mice, 25, 214, 215, 269 microarray detection, 371, 373, 379, 382 microarray technology, xiii, 37, 358, 364, 366, 374, 375, 376, 380, 382, 466, 467, 478, 480 microbial cells, 145, 377 microbial communities, xiii, 24, 28, 29, 30, 34, 357, 359, 360, 361, 363, 365, 366, 367, 371, 373, 374, 375, 377, 378, 379, 382, 383 microbial community, 13, 29, 40, 61, 359, 360, 361, 364, 371, 373, 374, 375, 376, 377, 379, 381, 383, 384, 479 microbial metagenomics, 469 microcosms, 376 micrometer, 28

Index micronutrients, 81, 82, 90, 95, 144, 145 microorganism, 17 microscope, 175, 176, 183, 361, 362 microscopy, 183, 362, 363 microspheres, 29 middle class, xv, 386, 395 Middle East, 131, 399 migration, 128, 210 military, 131 milligrams, 395 mineral water, 34 mineralogy, 6, 67 miniaturization, 362 mining, 76, 124, 125, 148, 149, 165, 190, 191, 211, 214, 215 Ministry of Education, 184 minorities, 238, 253 misconceptions, 429 missions, vii, 3, 5, 139, 147, 152 mixing, 46, 64, 79, 80, 86, 120, 131, 174, 175 modeling, 290, 301, 302, 458 modelling, 244, 455 models, vii, 3, 6, 24, 119, 155, 254, 287, 290, 292, 293, 301, 480 modern society, 128, 164 modernization, ix, 111, 410 modification, 25, 34, 267, 274, 346, 450, 476 moisture, 43, 45, 55, 57, 58, 76, 78, 85, 95, 97, 152, 159, 163, 407 moisture content, 55, 78, 85, 159 molecular biology, 26, 257, 391, 467 molecular weight, 209, 227, 455 molecules, 126, 145, 152, 259, 269, 365, 446, 454, 468 monitoring, viii, 12, 28, 29, 30, 31, 35, 37, 128, 174, 183, 225, 272, 278, 313, 328, 329, 335, 342, 361, 366, 380, 382, 392, 394, 404, 408, 410, 472 monoclonal antibody, 28 morbidity, vii, 11, 20, 22, 425 morphology, 361 morphometric, 191, 206 mortality rate, 20 mosquitoes, 163 mRNA, 362, 373, 375, 473 mRNAs, 362, 373, 374 mucoid, 19 mucosa, 476 mucous membrane, 24 mucus, 49, 81 multiple regression, xii, 285, 289, 290, 292, 301

495

multiplication, 23, 48, 78, 83 muskrat, 213, 215 mutation, 287, 378 mutations, 265, 362, 379 Myanmar, 287, 300, 301 mycobacteria, 13, 19, 23, 24, 38 mycorrhiza, 235 mycotoxins, xiv, 386 myocarditis, 16 myoglobin, 476, 477, 480

N NaCl, 83, 222 NAD, 355 nanoparticles, 174, 175, 176, 179 nanotechnology, 31, 175, 183 naphthalene, 156, 376, 450 National Bureau of Standards, 192 national parks, xii, 285, 287, 308, 315, 319 national policy, 313 National Research Council, 11 national security, 319 natural disasters, 152 natural habitats, xii, 305 natural resources, 113, 137, 165, 286 nausea, 427 necrosis, 213 needy, 155 negative relation, 213 neglect, 308, 311, 346 nematode, 188, 211 nervous system, 393 Netherlands, 103, 105, 106, 116, 124, 131, 139, 155, 167, 354, 355, 399 neural network, 382, 383 New England, 37, 39 New South Wales, 109 New Zealand, 46, 48, 76, 116, 430 next generation, 252 NGOs, 133, 396, 397, 410, 416 nickel, x, 156, 187, 191, 196, 197, 198, 201, 203, 205, 207, 208, 210, 211, 212, 213, 214 Nigeria, 116 nitrate, 174, 434, 441, 446, 451, 462 nitrates, 49, 63, 81, 82 nitrification, 366 nitrifying bacteria, 358, 363, 369, 379, 381, 382 nitrobenzene, 461

Index

496

nitrogen, 44, 48, 54, 59, 62, 63, 64, 67, 78, 81, 82, 83, 85, 87, 90, 95, 99, 128, 143, 144, 145, 146, 221, 222, 234, 235, 236, 358, 366, 367, 371, 376, 377, 381, 383, 384, 434, 453, 456 nitrogen fixation, 99, 366, 381 nitrogenase, 384 nitrogen-fixing bacteria, 82, 99 nitrous oxide, ix, 61, 62, 112, 125 NMR, 176 noble metals, 448 nodes, 467 nodules, 78, 358 nonequilibrium, 258 nonequilibrium systems, 258 non-renewable resources, 138 normal aging, 267 normal distribution, 192 North America, 21, 117, 161, 252, 323 North Sea, 479 Northern Ireland, 11, 40 Norway, 116, 133, 139, 155 nuclear energy, 124 nuclear power, 125, 131, 455 nucleic acid, xiii, 17, 27, 28, 33, 357, 362, 364, 365, 366, 368, 369, 371, 372, 377, 380 nucleotides, 361, 365, 374 nuisance, 252, 253 null, 438 nutrients, 20, 23, 48, 54, 55, 62, 63, 64, 65, 67, 78, 82, 83, 85, 86, 99, 100, 136, 144, 146, 162, 209, 220, 221, 226, 227, 228, 231, 428, 431, 432 nutrition, 83, 221, 267

O obstacles, xv, 387 oceans, 384, 426 offenders, 314 oil, 43, 47, 76, 85, 107, 123, 131, 141, 143, 147, 150, 151, 152, 153, 156, 165, 220, 363, 379, 402 oligonucleotide arrays, 378, 379, 381 omentum, 210 omission, 302 open spaces, xi, 237, 240, 253, 305, 314, 316 operating costs, 75 operations, 29, 190, 191, 214, 314, 404, 406, 408 operon, 358 opportunities, 32, 307, 399, 417 optical fiber, 121 optimization, xiii, 225, 357, 376, 381, 446, 451, 452

oral cavity, 383 oral health, 383 ores, 125, 140, 148, 149 organ, 30, 91, 192, 193, 196, 197, 206, 208 organic chemicals, 122, 141, 431 organic compounds, xv, 63, 123, 128, 130, 156, 433, 434, 436, 438, 440, 442, 446, 448, 451, 452, 454, 455, 456, 462 organic food, 403 organic materials, 43, 46, 75, 126, 130, 143, 159, 161 organic matter, vii, 3, 6, 43, 48, 49, 55, 59, 63, 64, 66, 69, 70, 77, 81, 82, 85, 96, 99, 100, 127, 144, 146, 163, 174, 220, 226, 227, 233, 234, 236, 378, 438, 439, 442, 443, 446, 451, 460, 461 organic solvents, 123 organism, xi, 26, 27, 29, 30, 118, 121, 257, 260, 263, 271, 358, 363, 372, 373, 374, 468 organizing, 183, 404 organochlorine compounds, 123 osmosis, 98 overgrazing, 293 overlap, 289, 311, 313, 318, 320 overlay, 247 oversight, 404, 408, 409, 417 ownership, 396 oxidation, xv, 179, 222, 225, 358, 365, 433, 434, 436, 437, 439, 440, 442, 443, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 oxidation rate, 451 oxidative stress, 280, 282 oxygen, 58, 59, 61, 64, 67, 69, 70, 97, 125, 128, 160, 222, 224, 235, 280, 373, 381, 435, 436, 459, 475, 476, 480 oxygen consumption, 64, 224 oyster, 192 oysters, 40 ozonation, xv, 433, 435, 437, 438, 439, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 ozone, xii, xv, 21, 30, 272, 279, 280, 433, 434, 435, 436, 437, 438, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 ozone reaction, 462 ozonization, 458, 460 ozonolysis, 456

Index

P Pacific, 16, 40, 76, 105, 114, 116, 117, 304, 377 pain, 400 paints, 123, 141 pairing, 273, 279 Pakistan, 116, 120, 355 palladium, 461 palpation, 210 parallel, 29, 335, 362, 370, 374, 380, 441, 455, 466, 468 paralysis, 16 parasite, viii, x, 12, 30, 34, 187, 188, 189, 190, 191, 208, 210, 211, 212, 213, 214, 215, 216, 272, 273 parasites, 21, 188, 203, 212, 213, 215, 216, 217, 281 parasitic infection, x, 187, 209, 211, 213, 214 parliament, 403 partition, 153 pathogens, viii, 11, 12, 13, 16, 18, 19, 20, 21, 22, 23, 24, 26, 28, 29, 30, 31, 32, 34, 36, 37, 38, 40, 45, 47, 48, 54, 55, 56, 57, 59, 60, 61, 75, 84, 96, 97, 98, 145, 163, 371, 379, 387, 415 pathology, 32 pathophysiology, 281 pathways, 226, 279, 438, 450, 471, 476 peat, 459 penalties, 403 peptides, 17, 459, 468 percentile, 248 percolation, 66, 129, 333, 337 performance, 35, 87, 308, 330, 430, 436, 440, 441, 442, 443, 445, 447, 450, 469 peri-urban, 398 permeability, 335, 340 permit, 324 peroxide, 434, 435, 437, 438, 439, 448, 453, 454, 455, 457, 460, 461, 462 person-to-person contact, 19 Perth, 101, 161, 244 pest populations, 392 pesticide, xv, 61, 76, 387, 391, 392, 393, 394, 396, 397, 398, 399, 400, 401, 403, 404, 406, 407, 408, 409, 410, 412, 416, 418, 423, 424, 425, 426, 427, 428, 429, 430, 432 pesticides, xv, 43, 76, 85, 100, 122, 123, 307, 388, 391, 392, 393, 394, 395, 396, 397, 398, 403, 410, 411, 412, 418, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 440, 457 pests, xv, 84, 85, 90, 123, 221, 392, 423, 425, 429 PET, 136, 143, 151

497

petroleum, 122, 141 phagocytosis, 24 PHB, 236 phenol, 274, 441, 446, 447, 448, 450, 456, 459, 460, 461, 462 phenotype, 479 Philippines, 45, 120, 145, 421 phosphates, 49, 81, 83, 236, 446 phosphorus, 48, 54, 63, 64, 67, 78, 82, 83, 90, 95, 143, 144, 221, 231, 236, 434 phosphorylation, 282 photocatalysis, 437, 442, 456, 457 photodegradation, 454, 455 photoemission, 183 photolithography, 362 photolysis, 78, 439, 454 phylogenetic tree, 365 phylum, 363 physical activity, 253, 254 physical environment, 254 physical exercise, 253 physical fields, 269 physical fitness, 191 physical properties, 220, 234 physics, 258, 259, 263, 268 physiology, 76, 221, 358, 467, 477, 478, 480 phytoplankton, 467 pigs, 116, 117 pilot study, 67 pioneer species, 293 pitch, 241 placenta, 121 plants, ix, xiii, 21, 30, 45, 54, 55, 63, 83, 84, 85, 90, 91, 92, 94, 95, 96, 97, 99, 112, 123, 131, 141, 145, 150, 153, 154, 156, 159, 160, 220, 221, 222, 224, 226, 227, 228, 234, 235, 306, 316, 357, 362, 391, 392, 398, 401, 410, 412, 427, 428, 429, 430, 436, 449, 455 plasmid, 378 plastic products, 139 plasticity, 480 plastics, 112, 113, 116, 117, 119, 122, 123, 126, 130, 135, 136, 138, 144, 147, 150, 151, 152, 157, 161, 163 platform, 68, 317, 367, 468, 469 platinum, 183, 462 PLS, 5 plutonium, 125 pneumonia, 24 point mutation, 379

498

Index

poison, 120, 400 policy makers, 31, 133, 396 polio, 16, 33 pollutants, 76, 130, 150, 151, 154, 188, 387, 434, 436, 437, 438, 440, 445, 446, 448, 454, 455, 458, 463 polluters, 314 pollution, x, xii, 25, 46, 113, 135, 137, 139, 142, 144, 148, 149, 161, 164, 165, 184, 187, 190, 208, 211, 216, 217, 221, 234, 252, 272, 305, 306, 308, 313, 318, 398, 475 polychlorinated biphenyls (PCBs), 48, 76, 123, 130, 430 polycyclic aromatic hydrocarbon, 43, 61, 76 polymerase, 27, 36, 40, 371, 381, 474 polymerase chain reaction (PCR), xiii, 26, 27, 28, 29, 33, 35, 36, 39, 274, 281, 282, 357, 359, 360, 361, 366, 369, 370, 371, 373, 375, 377, 378, 379, 380, 381, 382, 383, 468, 475 polymerization, 280 polymers, 365 polymorphism, 26, 38, 360, 377, 380, 381, 382, 383 polymorphisms, 362, 379, 380 polypeptide, 366 polypropylene, 68, 151, 192 polystyrene, 151 pools, vii, 3, 21, 22, 137 population density, 65, 67 population group, xi, 237, 248, 249 population size, 242, 286, 291, 300, 303 porosity, 62, 66, 82, 220, 342, 353, 365 porous media, 355 portability, 29 Portland cement, 153 Portugal, 139, 155 positive relationship, 205, 212 potassium, 54, 62, 63, 82, 85, 90, 95, 143, 221 potato, 39, 47, 52, 53, 234, 235 poultry, 18, 31, 34, 394, 412, 413, 414, 415 poverty, xv, 46, 120, 386, 395 poverty reduction, xv, 386 power plants, 145, 153, 316, 455 pozzolana, 153 precipitation, 129, 150, 434, 447 predation, 17 predators, 24 prejudice, 252 preparation, 29, 69, 317, 359, 400 prevention, 22, 27, 165, 235, 293, 391 primary school, 397

priming, 210, 373 prioritizing, 308 private investment, 406 private sector investment, 410 probability, 63, 248, 250, 251, 274, 278, 290, 291, 294 probability distribution, 248, 250, 251 probe, 175, 176, 183, 361, 363, 364, 366, 367, 368, 372, 374, 378, 469, 470, 474 process control, 34, 225 process gas, 130 procurement, 119, 140, 411 producers, 134, 139, 157, 392 productivity, viii, 5, 6, 22, 42, 82, 83, 96, 99, 213, 425, 473 profit, 141 project, xii, 151, 159, 162, 239, 254, 285, 287, 300, 301, 314, 378, 467, 468, 472 prokaryotes, 370, 376, 380 prokaryotic cell, 373 proliferation, 33, 60, 99, 209 promoter, viii, 42, 87, 95, 99, 374, 449, 474 propagation, 479 propane, 175 prophylaxis, 25 proteases, 49 protected areas, 286, 287, 292, 293, 300 proteins, 21, 49, 63, 82, 126, 141, 145, 163, 234, 265, 279, 383, 448, 473, 475 prototype, 382 Pseudomonas aeruginosa, 23 public awareness, 132, 158 public health, xiv, 12, 27, 34, 38, 40, 129, 131, 253, 310, 385, 407, 416, 424, 425 public interest, 289 public investment, 416 public parks, xi, 237, 238, 244, 252, 255 public policy, 323, 431 public sector, 411, 416 public-private partnerships, 404, 417 pulp, 46, 47, 63, 144, 145, 149, 150, 156, 373, 408, 410, 460 pumps, 335, 428 pure water, 112 purification, viii, 16, 29, 42, 65, 67, 183, 381, 458 PVC, 68, 69, 113, 121, 123, 143, 151, 160, 474 pyrolysis, 131

Index

Q quality assurance, 410, 412 quality control, 378, 411 quality of life, 134, 136 quality standards, 404 quantum mechanics, 258, 263 quartile, 249 Queensland, 55 quinone, 280

R race, 125, 255 racism, 255 radial distance, 326, 346, 347 radiation, vii, xii, 3, 125, 128, 272, 279, 280, 434, 439, 440, 454, 455 radical mechanism, 447 radical reactions, 452, 453 radicals, xv, 261, 433, 435, 437, 438, 439, 443, 445, 448, 450, 451, 452, 453, 454, 462 radioactive waste, 125, 165 radium, 125 radius, 330, 333, 338, 344, 346, 354 radon, 125 rain forest, 289 rainfall, 6, 309, 335 rash, 16 raw materials, 119, 137, 138, 139, 140, 151, 164, 165, 417, 426 RDP, 378 REA, xii, 26, 285, 289 reaction mechanism, 441, 442, 443, 444, 446, 447, 460 reaction order, 442, 449 reaction rate, 437, 440, 441, 448, 450 reaction rate constants, 437, 440, 450 reactions, xv, 27, 29, 58, 59, 128, 130, 182, 220, 224, 280, 370, 371, 386, 439, 440, 445, 449, 451, 452, 453, 457, 461, 462 reactive arthritis, 20, 38 reactive oxygen, 280 reactivity, vii, xv, 3, 122, 262, 433, 434, 437 reading, 289, 473 reagents, 29, 442, 445 real terms, 395, 399 reality, 252, 253, 331 recall, 400

499

recognition, xiv, 17, 26, 385 recommendations, 273, 412 recovery plan, 131 recovery processes, 259, 260, 262, 267 recreation, ix, xii, 111, 240, 253, 305, 307, 308, 310, 313, 314, 316 recurrence, 310, 312 recycling, 116, 120, 126, 132, 133, 134, 137, 138, 139, 140, 141, 142, 143, 145, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157, 158, 159, 160, 164, 165, 166, 169, 220 red blood cells, 18, 210, 213 Red List, 302 redistribution, 210 redundancy, 475 reflectivity, 273 reforms, 408 regeneration, 448, 450 regression, xii, 285, 289, 290, 292, 301 regression method, 301 regression model, 289, 290, 292 regulatory framework, xiv, 385, 403 rehabilitation, xii, 305, 306, 310, 314 rehydration, 20 rejection, 134, 400, 401 relevance, 466 reliability, 192, 281, 352 relief, 6, 163 remediation, viii, 42, 81, 83, 98, 99, 160 repair, 134, 155, 260 reparation, 460 replacement, 153, 189 replication, 25, 39 reporters, 28 reprocessing, 138, 156 reproduction, 45, 58 reproductive organs, 92, 94, 95 reputation, 400 requirements, 34, 78, 209, 401, 408, 411 research facilities, 477 research institutions, 403 researchers, xvi, 95, 363, 433, 435, 437, 438, 439, 440, 443, 445, 451, 465, 466, 467, 470, 472, 474 reserves, 191, 241, 303, 308, 319 residuals, 13 residues, 59, 61, 76, 96, 123, 144, 220, 236, 387, 388, 391, 392, 393, 394, 396, 397, 398, 399, 400, 401, 407, 408, 409, 410, 412, 416, 425, 427, 428, 429, 430, 432 resins, 121, 150

Index

500

resistance, viii, 12, 13, 20, 25, 84, 99, 221, 261, 262, 265, 272, 326, 392 resolution, 175, 177, 178, 179, 180, 183, 242, 243, 290, 363, 368 resources, 84, 96, 112, 113, 133, 134, 135, 136, 137, 138, 141, 153, 155, 165, 220, 253, 286, 308, 309, 310, 316, 355, 406, 436, 466, 469, 472, 475, 480 respiration, 63, 221, 236, 358 restaurants, ix, 51, 112, 127, 147, 162 restriction enzyme, 382 restriction fragment length polymorphis, 26, 360, 377, 380, 381, 382 retail, 386, 394, 410, 411, 416 retardation, 22 revenue, 55, 140 ribosomal RNA, 358, 359, 361, 369, 378, 379 rights, 133, 324 rings, xv, 53, 433 risk assessment, 394, 418 risk factors, 39 risk management, 416 risks, xiv, xv, 32, 115, 131, 385, 386, 387, 388, 394, 406, 425, 427, 429 river basins, 427 river systems, 327 RNA, 28, 358, 359, 361, 363, 369, 372, 373, 374, 377, 378, 379, 380, 381, 382, 470, 474, 475, 476 robotics, 362 rodents, 429 room temperature, ix, 112, 192, 414, 435, 439 root hair, 98 roundworms, 188 rowing, 409 rubber, 118, 121, 138, 144, 415 rubbers, 147 rules, 139, 141 runoff, 129, 427 rural areas, 97, 411 rural development, xv, 386 rural women, 46 Russia, 100

S salinity, viii, 42, 83, 306, 326, 327, 373, 376 saliva, 363 salmon, 431 salmonella, xiv, 385, 386, 401 salts, 150, 220, 443 sanctuaries, xii, 285, 287

Saudi Arabia, 148, 149 sawdust, 46, 60, 145 scarcity, 308, 309 scattering, 176 scavengers, 438, 449, 450, 451 scientific knowledge, 144 scientific understanding, xiv, 385 screening, 47, 404, 474 sea level, xiii, 325, 327, 338 seafood, 220, 234, 412, 413, 414, 415 sea-level, 328, 334 seasonal changes, 360, 378 seasonality, 30, 242 secrete, 49, 82, 112 secretion, 24, 99 sediment, 7, 66, 156, 352, 359, 369, 370, 376, 377, 381, 382 sedimentation, 30, 436 sediments, viii, xiii, 5, 6, 7, 12, 64, 307, 327, 328, 352, 358, 363, 369, 373, 377 seed, 83, 86, 87, 95, 223, 229, 231, 232 seedlings, 83, 95 segregation, 162 selectivity, 183, 392, 449 selenium, 130 self-organization, 258 self-regulation, 411, 417 semiconductors, 121 sensitivity, xiii, 7, 28, 29, 30, 183, 188, 357, 368, 369, 370, 371, 376, 393, 470 sequencing, xvi, 28, 39, 359, 360, 362, 366, 378, 383, 465, 466, 467, 468, 469, 471, 477, 478 serum, 426, 430 service provider, 416 settlements, 300, 315, 319 severe acute respiratory syndrome, xiv, 385 sewage, 18, 36, 43, 45, 46, 54, 55, 56, 60, 61, 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 96, 97, 98, 101, 112, 141, 145, 146, 151, 153, 154, 159, 161, 162, 234, 306, 307, 308, 398 sex, xi, 204, 271, 273, 278, 281, 394 sexual dimorphism, 211, 281 shade, 78 shape, 189, 242, 331 shear, 13, 36 sheep, 21 shellfish, 22, 370, 381 shelter, 13, 292 shingles, 144, 152 ships, 148

Index shoot, 87, 264 shortage, 231, 308 shrimp, 401, 411 side effects, 392, 426 signals, 175, 272, 279, 365, 368, 372 signs, 16, 289, 408 silica, 141, 151, 153, 445, 448 Silicon Valley, 120, 154, 168 silver, 96, 140, 141, 144, 149, 156, 460 Singapore, 105, 115, 120, 121 single-nucleotide polymorphism, 379, 383 sintering, 446 skin, 24, 58, 412, 427 slag, 131 Slovakia, 217 sludge, 43, 45, 46, 47, 54, 55, 56, 57, 59, 60, 62, 63, 64, 68, 74, 75, 77, 97, 98, 101, 102, 105, 112, 141, 145, 146, 154, 159, 162, 234, 236, 370, 373, 374, 447, 460 social activities, 132 social change, 309 social conflicts, 319 social group, 238, 252, 253 social justice, 238 social problems, viii, 42 socioeconomic status, 254 sociology, 126 sodium, 35, 124, 150, 155, 174, 175, 412, 455, 463 sodium hydroxide, 155, 174 software, 367, 472, 479 soil erosion, 6, 139, 145 soil particles, 81, 307, 360 soil pollution, 135 solid phase, 434, 445 solid surfaces, 29 solid waste, viii, ix, 18, 42, 43, 48, 96, 98, 107, 112, 113, 115, 116, 117, 118, 124, 129, 130, 131, 132, 133, 135, 142, 144, 146, 148, 154, 159, 161, 162, 163, 164, 166 solubility, xv, 224, 433, 434, 448 solvents, 123, 154, 156, 426 sorption, 174, 175, 179, 180, 183 South Africa, 153, 400 South Asia, 399, 419, 420, 421 South Korea, 139, 155 Southeast Asia, 287, 289 sovereignty, 309 SPA, 378, 381 Spain, 116, 400, 433 specialists, 289

501

speciation, 234 species richness, 323 specific gravity, 21, 407 specific surface, 65, 175, 176 specifications, 27, 409 spectrophotometry, 192 spectroscopic techniques, 175 spectroscopy, x, 173, 174, 175, 177, 183 speculation, 210, 213 spicule, x, 187, 189, 190, 191, 192, 193, 194, 196, 197, 199, 201, 202, 203, 205, 208, 209, 211, 212, 214, 215 spiders, 123 spillovers, 409, 410 spleen, x, 187, 191, 206, 207, 208, 213, 214 splenomegaly, 213 spreadsheets, 354 Spring, 282, 429 Sri Lanka, 121 SSI, 402, 416 stabilization, viii, 42, 54, 55, 63, 65, 153, 227, 233, 236, 316 stabilizers, 403 stakeholders, xii, 285, 286, 300, 301 standard deviation, 245 standard error, 194, 196, 199, 204, 205, 206 standardization, 407, 472 starch, 122, 147, 222 state control, 402 State Department, 410, 431 statehood, 315 states, 132, 310, 311, 313, 394, 398, 402, 406 statistics, 13, 238, 240, 242, 243, 248, 249, 250, 267, 282, 467 steel, 113, 115, 138, 139, 148, 149, 152, 153, 156 sterile, 47, 60, 192, 223 stimulus, 210 STM, 183 stoichiometry, 443, 444, 457 stomach, 189 storage, xiii, 29, 54, 61, 68, 76, 126, 146, 156, 158, 209, 210, 212, 234, 326, 330, 331, 332, 333, 335, 340, 342, 343, 345, 352, 353, 354, 355, 391, 404, 406, 476 stormwater, 159 stoves, 121 stratification, 234, 331 streams, viii, 12, 47, 220, 235, 293, 307, 308, 309, 340, 427, 431 streptococci, 108

502

Index

stroke, 425 strontium, 125 structural changes, 396 structural gene, 382 style, ix, 111, 122, 309, 474 subgroups, 248, 394 subjective judgments, 351, 353 submarines, 221 substitution, 436, 449, 461 substitution reaction, 461 substrates, 25, 163, 476 Sudan, 400 sugar beet, 220 sugarcane, 47, 83, 147 sulfate, 150, 225, 235, 358, 366, 376, 449 sulfur, 62, 82, 147, 153, 227, 233, 366, 367 sulfuric acid, 155 Sun, 109, 170, 283, 379, 456 supermarkets, 135, 152, 164, 396, 404, 405, 411 supplier, xiv, 386, 400 suppliers, 411 supply chain, 388, 396, 399, 408, 409, 416, 417 support services, xiv, 385, 417 suppression, 84 surface area, 65, 160, 175, 176, 364, 448, 450 surface chemistry, 449, 450, 451 surfactant, 453, 463 surplus, 65, 335 surrogates, 32, 469 surveillance, 16, 30, 416, 417, 427 survey, 124, 133, 182, 239, 242, 243, 244, 245, 255, 288, 289, 301, 406, 428, 468 survival, vii, 11, 13, 24, 35, 58, 164, 475 susceptibility, 208, 221 suspensions, 456 sustainability, 164, 314, 326, 418 sustainable development, viii, 41, 169, 326 Sweden, 36, 37, 116, 123, 131, 139, 155, 166 Switzerland, 116, 117, 139, 155, 158, 224, 235, 302, 303 symmetry, 175 symptoms, 16, 17, 19, 426, 427 synchronization, 260 syndrome, xiv, 18, 385 synergistic effect, 456 synthesis, xii, 176, 260, 261, 272, 280, 283, 373, 374, 445, 451, 468, 469, 477

T tags, 361, 468, 478 Taiwan, 120, 139, 155, 235 tanks, 46, 146 tannins, 150 tapeworm, 215, 217 tar, 152 target, xv, 27, 28, 29, 157, 289, 290, 291, 296, 297, 298, 299, 300, 301, 361, 363, 364, 365, 366, 367, 368, 369, 370, 372, 373, 374, 380, 423, 427, 428, 429, 469, 476 taxation, 165 taxonomy, 40 technical support, 466 technological developments, xvi, 119, 465 technological revolution, 118, 121 technologies, viii, xiii, 29, 42, 59, 74, 97, 142, 145, 147, 154, 220, 259, 357, 408, 425, 437, 467 technology, xiii, xv, 37, 64, 76, 96, 97, 98, 99, 119, 121, 130, 131, 138, 140, 142, 143, 144, 146, 147, 158, 159, 162, 164, 165, 357, 362, 364, 366, 374, 375, 380, 382, 387, 401, 433, 459, 462, 466, 468, 478, 480 TEM, 175, 176, 183 temperature, vii, ix, 3, 4, 16, 43, 45, 47, 48, 50, 58, 59, 60, 67, 76, 96, 97, 112, 124, 130, 147, 151, 160, 163, 170, 192, 222, 223, 258, 259, 272, 306, 366, 367, 368, 413, 414, 415, 435, 439, 445, 462, 475 territory, 327 test data, 278, 331, 341, 351, 352, 354, 355 testing, 26, 393, 398, 401, 407, 408, 409, 410, 417 tetrachlorodibenzo-p-dioxin, 43 textiles, 123, 144, 151 textural character, 451 texture, 5, 47, 56, 67, 414, 449, 450 Thailand, vi, xii, 33, 45, 121, 145, 285, 286, 287, 288, 289, 292, 302, 303, 304 therapy, 268 thermal analysis, 175 thermal energy, 146 thermodynamics, 258, 259, 263, 265, 269 thermometer, 413 thin films, 457 thinning, 426 Third World, 139 threats, xiv, 116, 293, 385 threonine, 234 thyroid, 429

Index time periods, 264 time pressure, 391 time use, 113 tin, 148, 183 tissue, x, 149, 150, 187, 188, 192, 193, 196, 198, 199, 201, 203, 205, 208, 209, 210, 211, 212, 213, 214, 215, 398, 476 titanium, 456, 459 TLR, 238 toluene, 380 tones, ix, 83, 112, 115, 116, 125, 140, 145, 149, 153, 154, 157, 159, 160, 162, 163, 165 total energy, 260 tourism, 286, 314, 316, 386 toxic effect, 227, 229 toxic gases, 48, 146, 152 toxic metals, 188, 190, 191, 209, 212, 215 toxic waste, 76, 97 toxicity, xv, 215, 379, 394, 426, 427, 430, 433, 435, 440, 441, 447, 456 toxin, 18, 20 toys, 123, 136, 174 trace elements, 54, 99, 145, 188, 216 tracks, 289 trading partner, 408 training, 37, 242, 260, 302, 409, 410, 411, 418, 466, 470 traits, xi, 271, 273, 467 transactions, 402 transcription, 27, 362, 373 transcriptomics, 468, 479 transcripts, 373, 473, 474 transference, 439, 448 transformation, xv, 20, 55, 175, 420, 425, 428, 432, 433, 434, 435, 443, 445, 448, 451, 452, 454, 462 transformation processes, 55 transformation product, 428 transformations, 192 transition metal, 443, 457 translocation, 20 transmission, xiv, 16, 17, 19, 21, 22, 28, 30, 31, 35, 36, 38, 156, 175, 178, 181, 189, 215, 385 transport, 6, 55, 126, 141, 158, 165, 209, 238, 391, 426, 476 transportation, ix, 31, 112, 131, 161, 315, 317, 386 trauma, 260 treatment methods, 98 trial, 161 trickle down, 67 triggers, 319

503

tropical forests, 302 trucks, 148 tsunami, 120 tuberculosis, 20, 38, 358, 388 tumors, 121 tumours, 429 tunneling, 183 Turkey, 400 Turks, 308 turnover, vii, 3, 4, 476 typhoid, 18 typhoid fever, 18 typhus, xv, 423 typology, xi, 237, 240, 252, 253 tyrosine, 228, 280

U U.S. Geological Survey, 354, 355, 427, 431 Ukraine, 271, 282 UNESCO, 289, 303 uniform, 63, 68, 85, 160, 193, 211, 252, 330, 331, 471 united, xiv, 11, 14, 16, 18, 24, 33, 38, 112, 114, 115, 133, 158, 166, 167, 170, 322, 380, 386, 400, 401, 421, 425, 426, 430, 466 United Kingdom (UK), vi, xi, xvi, 11, 21, 45, 49, 50, 101, 103, 113, 114, 128, 132, 135, 145, 151, 153, 157, 164, 167, 168, 237, 238, 239, 254, 255, 400, 420, 425, 427, 430, 431, 465, 466, 467, 471, 472, 477, 479 United Nations (UN), 112, 115, 158, 354 universities, 46, 166, 417 uranium, 124, 125 urban area, xi, 237, 251, 252, 253, 320, 398 urban population, 252 urbanization, xiv, xv, 385, 386, 395 urea, 82, 85, 87 ureter, 188 ureters, 210 urinary bladder, 188 urine, 20, 64, 188, 279, 283 USDA, 376, 394, 418, 419 USSR, 316 UV irradiation, 30, 455 UV light, 279, 440 UV radiation, 280, 434, 439, 440, 455

Index

504

V vacuum, 129, 156, 437 valence, ix, 173, 175, 177, 435 validation, 377, 382, 478 valuation, 321 vanadium, 183 vapor, 156 variables, 35, 208, 289, 445, 449, 450, 451 variations, 211, 254, 273, 274, 276, 317, 338, 353, 366, 376, 395 varieties, 392, 405, 425 vector, 426, 475 vegetable oil, 47, 143, 147, 407 vegetables, 18, 20, 136, 221, 391, 396, 398, 399, 410, 412, 416, 425 vegetation, xii, 285, 289, 290, 291, 293, 300, 304, 306, 335, 427, 428 vehicles, 17, 126, 162, 398 vein, 274 velocity, 180, 182, 342, 353 ventilation, 223 versatility, 437 vertebrates, 22, 215, 477 vertical transmission, 31 video, 151 Vietnam, 120, 121, 174 vinasse, 456 vinyl chloride, 129 violence, 132 viral meningitis, 16 virus infection, 33, 37 viruses, 13, 16, 17, 25, 27, 32, 60, 387, 467, 474, 479 viscosity, 156, 331 vision, 273, 279, 281, 283, 312, 314, 317, 320, 471 visual impression, 249 vitamins, 85 VOCs, 123, 128, 129, 159 volatility, 431 volatilization, 78 vomiting, 427 vulnerability, 307

W waiver, 404 Wales, 45, 109, 145 walking, 254

Washington, 109, 115, 133, 149, 166, 170, 302, 303, 304, 323, 377, 418, 419, 420, 421, 431, 455 waste disposal, 131, 132, 133, 139, 160, 165 waste incineration, 131 waste incinerator, 131 waste management, ix, 61, 96, 112, 126, 131, 132, 133, 134, 135, 156, 161, 164, 165, 166, 406 waste treatment, 131, 149, 161, 443, 452 wastewater, viii, x, xv, 13, 28, 34, 36, 42, 43, 44, 47, 54, 64, 65, 66, 67, 68, 69, 70, 74, 75, 76, 96, 97, 98, 101, 104, 109, 153, 219, 220, 225, 233, 234, 235, 236, 363, 369, 370, 378, 379, 382, 433, 435, 453, 455, 456, 460, 462, 463 water policy, 308, 309, 315 water quality, viii, 12, 13, 17, 25, 28, 33, 38, 40, 309, 313, 316, 326, 330 water resources, 220, 308, 309, 310 water supplies, 12, 17, 18, 22, 24, 29, 326, 428, 452 watershed, 30, 311, 313, 318, 320 waterways, 114, 427 weakness, 151 wealth, 46, 252, 466, 474 wear, 115, 415 web, 402, 418 websites, 170 weight changes, 208, 215 weight loss, 22 welfare, xiv, 386 wells, xiii, 29, 153, 174, 325, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 342, 343, 348, 350, 352, 353, 354, 355, 428 Western Europe, 309, 399 wetlands, 323 wholesale, 398, 402, 406, 410 wild animals, viii, 12, 13, 17 wildlife, xii, 12, 18, 20, 31, 38, 190, 240, 285, 287, 289, 290, 291, 293, 301, 303, 418, 429 windows, 148 withdrawal, xiii, 325, 326, 328, 333, 335, 338, 340 wood, 114, 117, 127, 138, 144, 147, 150, 152, 426 workers, 120, 121, 163, 418, 426, 427, 430 working hours, 32 workplace, 136, 386 World Bank, xiv, xv, 166, 386, 400, 404, 405, 406, 407, 408, 410, 411, 417, 419, 420, 421 World Health Organisation, 40, 391 World Trade Organization (WTO), 399, 417 worldwide, ix, xiv, xvi, 6, 12, 13, 16, 21, 36, 129, 131, 155, 156, 173, 386, 433, 435, 473

Index worms, x, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 75, 76, 78, 79, 81, 82, 83, 84, 85, 87, 90, 91, 92, 94, 95, 97, 98, 99, 100, 106, 145, 146, 187, 188, 189, 190, 191, 192, 193, 194, 196, 197, 198, 199, 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 212, 214, 216

X x-axis, 267 XPS, 183 x-ray, x, 173, 175, 176, 183, 184 x-ray diffraction (XRD), 175, 176, 183

505

Y Yale University, 269 yeast, xiii, 147, 150, 222, 357, 362, 373, 466, 478 yield, 83, 87, 89, 90, 146, 220, 228, 234, 245, 326, 330, 351, 353, 372, 375, 425, 468

Z zeolites, 448 Zimbabwe, 104 zinc, 54, 76, 77, 85, 123, 144, 156, 398 ZnO, 440, 455