Advances in Marine Biology, Volume 37

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Advances in MARINE BIOLOGY VOLUME 37

This Page Intentionally Left Blank

Advances in MARINE BIOLOGY Edited by

A. J. SOUTHWARD Marine Biological Association, The Laboratoty, Citadel Hill, Plymouth, England, UK

I? A. TYLER School of Ocean and Earth Science, University of Southampton, England, UK

and

C . M. YOUNG Harbor Branch Oceanographic Institution, Fort Pierce, Florida, USA

ACADEMIC PRESS A Horcourt Science and Technology Company

San Diego San Francisco New York Boston London Sydney Tokyo

This book is printed on acid-free paper Copyright 0 2000 by ACADEMIC PRESS Chapter entitled 'Population Structure and Dynamics of Walleye Pollock, Theragru chalcogrumma' is a US Government work in the public domain and not subject to copyright. All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press 24-28 Oval Road, London NW1 7DX, UK http:llwww.hbuk.co.uWap/ Academic Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:llwww.apnet.com ISBN 0-12-026137-5 A catalogue record for this book is available from the British Library

'Ehpeset by Keyset Composition, Colchester, Essex Printed in Great Britain by MPG Books Ltd., Bodmin, Cornwall

99 0001 02 03 04 MP 9 8 7 6 5 4 3 2 1

K . M . BAILEY,Resource Assessment and Conservation Engineering Division, Alaska Fisheries Science Center, 7600 Sand Point Way NE, Seatde W A 98115, USA R. BEIRAS, Area de Ecoloxia, Universidade de Vigo, 36200 Galicia, Spain P. BEmzm, Marine Molecular Biology Laboratory, School of Fisheries, University of Washington, Seattle W A 98195, USA W. S. GRANT,Conservation Biology Division, Northwest Fisheries Science Center, 2725 Montlake Blvd., Seattle W A 98112, USA E. H I S ,IFREMER, Quai Silhouette, 33120 Arcachon, France T. J. QUINN 11, Juneau Center, School of Fisheries and Ocean Sciences, University of Alaska Fairbanks, 1120 Glacier Highway, Juneau A K !?98018677, U S A M . N . L. SEAMAN, Institute of Marine Research, 24105 Kiel, Germally


CONTENTS

CONTRIBUTORS TO VOLUME 37...........................................

v

The Assessment of Marine Pollution .Bioassays with Bivalve Embryos and Larvae E. His. R. Beiras and M. N. L. Seaman 1. Introduction .................................................................. 3 2. Relevant Aspects of Bivalve Biology....................................... 10 3. Bioassay Methodology ....................................................... 40 4. Testing the Toxicity of Marine Pollutants to Bivalve Embryos and Larvae 87 5 . Assessing Marine Environmental Quality with Bivalve Embryo and Larval Bioassays..................................................................... 125 130 6. Summary and Discussion.................................................... 138 Acknowledgements ........................................................... References .................................................................... 139

Population Structure and Dynamics of Walleye Polllock. Theragra chalcogramma K. M. Bailey. T. J. Quinn II. I? Bentzen and W. S. Grant 1. 2. 3. 4. 5.

Introduction .................................................................. Background The Fishery. Life History and Ecosystem Interactions ....... Population Ecology.......................................................... Population Structure ......................................................... Management Implications ................................................... Acknowledgements ........................................................... References ....................................................................

Taxonomic Index .............................................................. Subject Index .................................................................. Cumulative Index of Titles .................................................... Cumulative Index of Authors .................................................

180 184 189 206 238 242 242 257 261 269 278


The Assessment of Marine Pollution . Bioassays with Bivalve Embryos and Larvae E. His'. R . Beiras' and M . N. L . Seaman3

'IFREMER. Quai Silhouette. 33120 Arcachon. France 2 h e a de Ecoloxia. Universidade de Ego. 36200 Galicia. Spain 31nstitute of Marine Research. 24105 Kiel. Germany Correspondence to: Edouard His. IFREMER. Quai Silhouette. 33120 Arcachon. France. Fax: 05 56 83 89 80. Tel. 05 56 83 78 17. e-mail: [email protected]

1. Introduction ......................................................................... 3 1.1. Generalities on pollution ...................................................... 3 1.2. Bioassays: advantages and limitations ...................................... 4 6 1.3. Bivalve larvae and pollution .................................................. 2. Relevant Aspects of Bivalve Biology ............................................. 10 2.1. Species used in bioassays ................................................... 10 2.2. Reproduction .................................................................. 16 2.3. Larval rearing in the laboratory ............................................. 25 3. Bioassay Methodology ............................................................ 40 3.1. General methods .............................................................. 40 50 3.2. Bioassay procedures ......................................................... 3.3. Bioassay applications: toxicity tests and environmental bioassays ....... 74 82 3.4. Statistical methods ........................................................... 4. Testing the Toxicity of Marine Pollutants to Bivalve Embryos and Larvae .... 87 87 4.1. Pollutants ...................................................................... 117 4.2. Intrinsic (biological) factors affecting toxicity ............................... 4.3. Extrinsic (environmental) factors affecting toxicity ......................... 121 122 4.4. Interactions between different toxicants .................................... 5. Assessing Marine Environmental Quality with Bivalve Embryo and Larval 125 Bioassays ........................................................................... 5.1. Algal and bacterial toxins .................................................... 125 127 5.2. Urban and industrial effluents ............................................... 5.3. Receiving waters .............................................................. 128 129 5.4. Sediments ..................................................................... 6. Summary and Discussion ......................................................... 130

ADVANCES IN MARINE BIOLOGY VOL. 37 ISBN 0-12-026137-5

Copyright 0 1999 Academic Press All rights of reproduction in any form reserved

2

E. HIS, R. BEIRAS AND M. N. L. SEAMAN

6.1. Sensitivity of bioassay organisms ........................................... 131 6.2. Assessment of the toxicity of various contaminants ....................... 132 6.3. Bivalve embryo and larval bioassay methodology ........................ 134 6.4. Perspectives in future research on bivalve larval bioassays .............. 137 6.5. Concluding remarks .......................................................... 138 Acknowledgements ................................................................... 138 References ............................................................................. 139

Tens of thousands of synthetic substances are in existence today and hundreds of new compounds are being introduced every year. Because of the complexity of the physico-chemical interactions between pollutants and the marine environment, the potential toxicity of contaminants can be assessed adequately only by means of bioassays with living organisms. From a practical point of view, a bioassay needs to be sensitive and scientiJically valid, yield rapid results at moderate cost, and the organism in question must be readily available. Ecotoxicological bioassays with bivalve embryos and larvae fulfil these criteria better than most other tests. They have increasingly come into use during the past three decades and are now commonly employed to ascertain the biological effects of pure chemicals, as well as to determine the quality of efluents, coastal waters and sediments sampled in the field. There do not appear to be very great differences between bivalve species with regard to larval sensitivity to toxicants. The principal species f o r bioassays are oysters (Crassostrea gigas and C. virginica), and mussels (Mytilus edulis and M . galloprovincialis). Bioassays are conducted with gametes and larvae of all ages: sperm and unfertilized eggs, embryos, young D-larvae, intermediate umboned larvae, and pediveligers towards the end of the pelagic period. Embryos are usually the most sensitive stage. Recent advances now also permit bioassays on metamorphosing pediveligers, a method particularly suited to investigate the effects of adsorbate-contaminated surfaces. There are various criteria for the assessment of toxic effects, including embryogenesis success (abnormalities), larval growth, mortality, physiology (e.g. feeding or swimming activity), and metamorphosis success. Chronic toxicity studies may be carried out over periods of several weeks, but larval rearing in the laboratory requires considerable effort (e.g. cultivation of algal food). The method of choice f o r investigations of acute toxicity and for routine monitoring studies is the embryo bioassay because it is very sensitive, relatively simple, and produces results within 24 or 48 hours. The data obtained by different investigators are often dificult to compare, however, because of differences in methodology. There is no firmly established procedure, and further simplification and standardization of techniques is required. In bioassays with a single pollutant, the effective toxic concentration may

THE ASSESSMENT OF MARINE POLLUTION

3

span several orders of magnitude, depending on bioassay procedures, larval stage and choice of response. Tributyl-tin (TBT) is the most toxic compound ever bioassayed with bivalve larvae, with effective concentrations (EC,,) as low as a few nanograms per litre (i.e. lo--’ ppb). Heavy metals (particularly mercury, silver and copper) are next in order of toxicity, with ECSovalues between a few micrograms per litre (ppb) and several hundred ppb. Chlorine and some organochlorine pesticides may also have ECso values of less than 100 ppb, while detergents and petroleum products are generally less toxic.

1. INTRODUCTION

1.1. Generalities on pollution

Although human activities have always impacted on coastal areas, it is only within the last two centuries that the effects of industrialization, intensive agriculture and coastal engineering (including tourism) have seriously begun to threaten marine life. Some of these impacts lead to environmental pollution, i.e. the introduction by man into the environment of substances or energy which may put human health or living resources at risk (Holdgate, 1979). Pollutants are defined as substances present in the natural environment which are (at least in part) of anthropogenic origin, and which may have deleterious effects on living organisms (Moriarty, 1990). According to this definition it is necessary to distinguish between contaminants, which encompass all substances of anthropogenic origin introduced into the environment, and pollutants, which consist of those contaminants with presumably negative biological effects. Moriarty recognizes, however, that the term “pollutant” is generally applied in the sense of “contaminant”. The assessment of marine pollution is not restricted to the study of the water. Most of the pollutants - heavy metals and hydrophobic organic compounds in particular - have a tendency to adsorb to sediment particles, and their concentrations in marine sediments may be several orders of magnitude higher than in the water column (Livett, 1988). The association between pollutants and sediments can be of very long duration and it may have deleterious effects on organisms living on, and within, the bottom. The assessment of the toxicity of marine bottom sediments has, therefore, also come into focus since the late 1970s. According to Moriarty (1990) some 63000 chemical products are presently in use, and 3000 of these constitute 90% of the total mass of

4


compounds being produced industrially. In addition, between 200 and 1000 new synthetic compounds are being introduced every year. It would obviously be desirable to screen their possible ecological effects. As already pointed out by Cunningham (1979), ecotoxicological studies require several levels of investigation. Depending on the mode of action of the pollutants in question, these may be at the subcellular, cellular, organismic, population or ecosystem level (see also Bayne, 1985; Haux and Forlin, 1988). 1.2. Bioassays: advantages and limitations

Anthropogenic impacts on the aquatic environment may be viewed from a physical, chemical or biological perspective. The biological effects of pollutants in the environment are more important than the mere presence of pollutants; with regard to environmental quality criteria, the data from chemical analyses of pollutants can only be interpreted within a biological context. It is therefore logical to use biological systems for the assessment of environmental quality (Anonymous, 1989). The following points apply to ecotoxicological bioassays in general:

1. Detection of new pollutants: reliance on chemical analyses alone would presuppose that the potentially important pollutants are known and already being monitored. Examples such as tributyl-tin (TBT) demonstrate that this is not always the case and that biological systems provide the means to detect and identify the presence of new or unexpected pollutants. 2. Bioavailability: chemical data often do not reflect the bioavailability of pollutants, e.g. owing to the speciation of organic compounds or the bonding state of metals. By definition, biological systems can only respond to what is effectively available to them, and test organisms therefore provide the best indication of bioavailable pollutants exceeding toxicity thresholds. 3. Integration of toxic effects: toxic contaminants typically do not occur singly and environmental quality is generally determined by their combined effects. Biological systems respond to the totality of environmental pollution, thus providing an integrated response to the totality of pollutants present, as well as to their interactions. 4. Cost: the continuous increase in the number of contaminants being introduced entails a constant rise in the cost of chemical monitoring programmes. This underscores the importance of using biological techniques as reconnaissance systems, because they help to focus the effort of chemical analysis on situations of demonstrated biological


5

relevance. Therefore, “bioassays ... may be used before any other testing commences as a cost-effective screening tool” (Chapman and Long, 1983, p. 83). Any bioassay used for routine monitoring purposes should meet a variety of criteria (Moore, 1966; Butler et al., 1971; Bryan et al., 1980, 1985; Stebbing et al., 1980): it should be easy to learn, affordable, and not require very sophisticated equipment it should be of short duration (a few hours or days, rather than weeks) the organisms employed should, if possible, be available for bioassay purposes year-round the organisms should be of ecological or economic importance the organisms, whether they originate from laboratory cultures or from field sampling, should be identical as far as possible, in order to reduce variability resulting from age, size etc. genetically homogeneous, or cloned, organisms would be preferable, to reduce the effects of genetic variability the data should be of a type that can be analysed by standard statistical methods. Most bivalves of commercial interest generally fulfil these criteria, particularly those living in the marine and brackish water environment, such as oysters of the genus Crassostrea and mussels of the genus Mytilus. They are eurytypic organisms with higher tolerance to environmental fluctuations and therefore better suited for studies on the evaluation of environmental quality (Bayne, 1985), than stenotypic organisms with narrow tolerance. The different ontogenetic stages in marine species may differ in their sensitivity to pollutants (Coglianese and Martin, 1981). Among the many methods employed in bioassays, those using meroplanktonic stages (such as sea urchin plutei or bivalve veligers) appear to be the most promising to obtain reliable biological responses with regard to coastal water quality (Klockner et al., 1985). Many authors have proposed the use of early life stages of bivalves for marine toxicological studies, because it is clearly established that they are more sensitive to toxic substances than are the adults (Wisely and Blick, 1967; Granmo, 1972; Brereton et al., 1973; Hrs-Brenko et al., 1977; Coglianese and Martin, 1981; Bourne et al., 1981; His and Robert, 1981, 1982; Ringwood, 1991). The success of a species depends on its performance during passage through successive life stages, and it is therefore realistic to use the most sensitive stage for the purpose of testing environmental quality (Stebbing et al., 1980; Calabrese, 1984).


1.3. Bivalve larvae and pollution

Bivalve molluscs are an exclusively aquatic taxon of extremely wide distribution in fresh, brackish and marine waters around the globe. They are particularly abundant in the highly productive marine coastal areas. They have been exploited for food since prehistoric times and they have been cultivated systematically in various parts of the world for many centuries. The first problems in bivalve culture with regard to pollution were encountered at the beginning of the twentieth century. In studies on the American oyster, Crassostrea virginica,Prytherch (1924) asserted that “the rapid decline of this valuable industry has been brought about by a constant depletion of the oyster beds from various factors, such as pollution . . .” (p. 1). Prytherch was also the first to attempt, and to succeed in, the “artificial reproduction” of oysters. Besides intending to obtain larvae for culture purposes and thus offset the irregularities of natural recruitment, he also intended to conduct basic studies on larval biology and “to produce larvae for tests in regard to the effect upon them of various chemicals in solution” (op. cit., p. 2). It was already evident from Prytherch’s studies that bivalve larvae (veligers) are particularly suited for investigating environmental impacts, because in oyster cultivation areas the first environmental effects to be observed concerned the species’ natural reproduction. The same observation was made again 60 years later by Stanley and DeWitt (1983) who declared that the absence of a population of Mercenaria mevcenaria in the species’ normal area of distribution is an ecological indicator of environmental disturbance. The efforts of Loosanoff and his associates at the Milford Laboratory in Connecticut led to the development of practical and reliable methods for the culture of bivalve larvae (reviewed by Loosanoff and Davis, 1963) and the first toxicological bioassays with such larvae. A serious constraint in working with oyster larvae, however, was the limitation to the species’ reproductive period, from June to September. The invention of “conditioning”, i.e. techniques to obtain ripe adults at all seasons (Loosanoff, 1945) made it possible to study larval nutrition throughout the year. The work conducted by Cole (1937,1939) in Great Britain, made it possible to study the nutritional value of marine unicellular algae, first isolated by Parke at Plymouth; certain species, especially Isochrysis galbana, permitted the rearing of larvae to metamorphosis (Davis, 1953; Loosanoff, 1955; Walne, 1956; Davis and Guillard, 1958). It thus became possible to grow veligers in the laboratory as a matter of routine, and to study their principal environmental requirements. This was followed by studies on the effects of micropollutants on growth and survival in Crassostrea virginica larvae (Davis, 1961; Calabrese and Davis, 1967).


7

At the same time, the concept of the “biological quality” of seawater was introduced by Wilson (1951) and Wilson and Armstrong (1961), who found that pluteus larvae of the sea urchin Echinus esculentus were capable of developing in seawater obtained from the Celtic Sea, but not in water from the vicinity of Plymouth. Subsequently Woelke (1961, 1966, 1967, 1972), Okubo and Okubo (1962) and Dimick and Breese (1965) suggested the use of oyster embryos and larvae to study the general effects of pollutants in water samples from the natural environment, and not just for toxicological laboratory investigations of specific pollutants. This ecotoxicological approach, i.e. the study of samples obtained from the natural environment, has since been expanded to include assays with marine sediments (Chapman and Morgan, 1983; Swartz, 1989; Phelps and Warner, 1990). Most bivalve embryo-larval bioassays can be conducted within a relatively short time period (24 to 48 hours after fertilization). Assessments of sublethal toxic effects of pollutants are based on the percentage of normal D-shaped larvae obtained at the end of embryogenesis (determination of the concentration that inhibits larval development in 50% of the fertilized eggs). These types of studies have been conducted by Woelke and coworkers (Woelke, 1960a,b, 1961, 1966, 1967, 1968, 1972; Cardwell et al., 1977a,b), Davis and coworkers (Davis, 1961; Calabrese and Davis, 1967), and many others. Experiments of longer duration, based on the assessment of larval growth, have also been conducted in order to study the effects of sublethal micropollutant concentrations (e.g. Davis and Hidu, 1969a; His and Robert, 1981 and 1982; Watling, 1982). These types of studies represent a better simulation of conditions prevailing in the natural environment, but they also necessitate the cultivation of algae to feed the larvae during the experiment, which may last several days, or several weeks if it is continued until the end of the veliger’s pelagic life stage. Finally, recent work has focused on the use of eyed larvae several days old, in order to investigate the effects of pollutants on the pediveliger stage and on metamorphosis, because these stages are considered to be especially sensitive (Stafford, 1913; also Watling, 1978, 1983; Sheffrin, 1982; Sheffrin et al., 1984; Beiras and His, 1994, 1995a; His et al., 1997b). Most bioassays with bivalve larvae and embryos have been conducted with various species of oysters (57% of the publications listed in Table l), followed by mussels (22%). Only two other species have been used regularly in larval bioassays - the quahog Mercenaria (Venus) rnercenaria and the coot clam Mulinia lateralis. The first step in bioassays with bivalve embryos and larvae is to obtain spawning adults and rear the fertilized gametes. We will therefore review the present state of knowledge on bivalve reproduction, methods to obtain


8

Table 1 Bivalve species used in embryo and larval bioassays. Mytilus

Crassosfrea

c gigas Beiras and His 1994, 1995b Beiras ef al., 1998 Bourne ef al., 1981 Boyden el al., 1975 Brereton el ol., 1973 Butler el ni., 1992 Cardwell, 1976,1978 Cardwell ef al., 1976, 1977a.b. 1979a.b Chang ef al., 1996 Chapman and Morgan, 1983 Chapman ef al., 1991, 1992 Chien and Chou, 1989 Cleary ef al., 1993 Coglianese, 1982 Coglianese and Martin, 1981 Connell ef al.. 1997 Coon era[., 1990 Crecelius, 1979 Eertman ef 01.. 1W3 Rtt el al., 1990 Garland el al., 1986 Geffard, 1997 Glickstein. 1978 Helm ef al., 1974 Hi%1996 His and Robert, 1980, 1981,1982,1987b His and Seaman, 1993 His ef al., 1983, 1996 Klockner ef al., 1985 Konar and Stephanson, 1995 Le Gore, 1974 Lourens el a/., 1995 McFadzen, 1992 McFadzen and Cleary. 1994 Maimstone el al., 1989 Martin ef al., 1981 Nelson ef al., 1983 Okubo and Okubo, 1962 Pbelps and Warner, 1990 Renard, 1991 Renzoni, 1973a,b Robert and His, 1981, 1985 Robert ef al., 1986 Smith, 1968 Stewart and Blogoslawski, 1985

C virginica Baker and Mann, 1992. 1994a.b Brown. 1981 Brown and Roland, 1984 Butler and Lowe, 1978 Calabrese, 1972 Calabrese and Davis, 1964,1%7,1970 Calabrese ef a[.. 1973, 1977a.b Capuzzo, 1979 Chapman et nl., 1987 Davis 1958,1960,1961 Davis and Calabrese,

1964 Davis and Hidu, 1%9a,b Dim, 1973 Hidu, 1965 Hidu el ni., 1974 Ho and Zubkoff, 1979, 1980 Mann and Rainer, 1990 Mchnes, 1981 McInnes and Calabrese, 1978,1979 Nelson ef al., 1983 Noyes ef al., 1978 Phelps and Mihursky, 1986 Phelps and Warner, 1990 Prytherch, 1931, 1934 Reuzoai, 1915 Richardson ef ol., 1982 Ringwood and Brouwer, 1995 Roberts 1980,1987 Roberts and Casey, 1985 Roberts and Gleeson, 1978 Roberts ei al., 1975,1977 Roesijadi ef al., 19% Roosenburg ef ~ l .1980b , Sigler and Leibovitch, 1982 Stewart er al., 1979 Stiles-Jewel],1994 Stiles and Blogoslawski, 1993 Tagatz and hey, 1981 Ukeles and Sweeney, 1969 Widdows ef al., 1989 Wikfors and Ukeles 1982 Wirtb eta/., 1996 Wolfe ef al., 1993

M. edulis Akberali ef a/., 1985 Armstrong and Millemann, 1974 Beaumont and Budd, 1982,1984 Beaumont and Tserpes, 1984 Beaumont el al., 1981 Breese eral., 1963 Brunetti ef a/., 1989 Butler ef al., 1990 Chapman and Long, 1983 Chapman ef al., 1993, 1996 Courtright el a/., 1971 Dimick and Breese, 1965 Dixon and Prosser, 1986 Granmd, 1972 Granmo and Jorgensen, 1975 Granmo ef 01.. 1988, 1989 Hansen ef 111.. 1997 Hoare er al., 1995a,b Johnson, 1988 Knezovicb ei al., 1996 Lapota er al., 1993 Le Pennec and Prieur, 1972 Le Pennec ef al., 1973 Long ef al., 1990 Magnusson el al., 1996 Martin ef d., 1981 Mitchell ef al., 1985 Morgan ef al., 1986 Okubo and Okubo, 1962 Pavicic, 1980 Sheffrin and Williams, 1984 Sheffrin ef al., 1984 Stewart ef al., 1%7 Strdmgren and Nielsen, 1991 main, 1983 Wisely and Blick, 1967 Wolfe er al.. 1995 Zhadan er al., 1992

M.galloprovincialis Beiras and Hq 1995a Brunetti ef ai., 1989 Bucaille and Kim, 1979 His and Beiras, 1995 €in-Brenko er a[., 1977 Ix Pennec and Le Roux, 1979 Le ROUK,1977 Lucu ef a/., 1980 Pagano ef ol., 1996 Pavicic, 1976 Pavicic and Pihlar,1982 Pavidc ef al., 1984a, 1994a,b Renzoni, 1973a Robert and His, 1981 Seaman el al., 1991

9


Table 1 -(Continued). Crnsroslrea

C. gigas Slewart el al., 1967,1983, 1991 Thain and Watts, 1987 Thain eral., 1990 Utting and Helm, 1985 Van den Hurk, 1994 Van den Hurk el al., 1997 Wang ef 01.. 1985 Warder al., 1992a,b Watling, 1978,1981,1982, 1983 Wikfors el al., 1993 Williams el 01.. 1986 Woelke, 1960,1961,1967, 1968, 1972 Wolfe er el., 19% Zhadan ei a/., 1992

Mytilus

C. virginica

M. edulis

M. galloprovincialis

Wright el al., 1983 Zaroogian and Morrison, 1981

Other species Argopeclen irradians: Nelson and Siddall, 1988; Wright el al., 1983 Callisfa brevisiphonata: Zhadan el a/., 1992 Cerastodema edule: Tunmemans el al., 19% Chlamys asperrima: Krassoi, 1995; Krassoi el 01.. 1996, 1997: Pablo el al., 1997; Stauber er a/., 1996 Clinocardium nultalli: Stewart el al., 1967 Crassoszreu ungulata: Renzoni, 1973a Crassosrrea nrccullaru: Watling, 1981,1982 Crassosrrea iredalei: Ramachandran ef a/., 1997 Crassostrea margaritacea: Watling, 1981, 1982 Crassosrrea rhizophorae: Chung, 1980; Nascimento, 1989; Pereira er al., 1998 Isognomon californicurn: Ringwood, 1990,1991,1992a.h, 1993 Macoma balthica: Tunmemans el al., 1996 Macrru chinensix Zhadan el a/., 1992 Mercenaria mercenaria: Brown, 1974;Byme and Calder, 197R Calabrese, 1972; Calabrese and Davis 1966,1970; Calabrese and Nelson, 1974; Calabrese el al., 1977a.b Davis, 1958, 1%0; Davis and Calabrese, 1964; Davis and Hidu, 1969a.b:

Hidu, 1965;Huntington and Miller, 1989; Laughlin el a/., 1988.1989; Miller, 1989; Pavicic. 1980, Roberts, 1987; Roberts 1983; Wright el a/.. 1983 Meretrix lusoria: Tzong-Shean and Chen, 1993 Mizuhopecfen yessoensix Karaseva and Medvedeva, 1993; Zhadan era/., 1992 Mulinia larerdix Burgess and Momson, 1994, Calabrese, 1970b; Calabrese and Rhodes 1974; Diaz 1975; Gormly e1 al., 19%; Hall el a/., 1995; Ho and Zubkoff, 1980, 1982, 1983; Mann el al., 1991; Morrison and Petrocelli, 1990 Pelletier el al., 1997; Renzoni, 1975; Roberts 1980:Wright eral., 1983 Mya arenaria: Roosenburg el al., 1980a Mytilus califomianus: Cherr er al., 1990; Spangenberg and Cherr. 1996 Mytilus lrossulus: Karaseva and Medvedeva, 1993 Osrreu edulix Connor, 1972 Davis 1961;Davis and Hidu. 1969b Helm. 1971; Millar and Scott, 1968, Nottage and Birkbeck, 1987a.b Renzoni. 1973b: Smith, 1968; Thain. 1983 Osfreaplicalula: Xu el al., 1994 Peaen marimus: Beaumont and Budd, 1982; Beaumont el al., 1987 Prolothaca staminea: Cardwell el a/., 1979b Rangiu cuneala: Mann el al., 1983 Saccosfrea commercialis (= Crassostrea commercialis): Krassoi. 1995; Nell and Holliday, 1986; Wilson and Hynes 1997; Wiseley and Blick, 1967 Scrobicularia plma: Ruiz er al., 1994,1995a.b.c Spisula sukhalinensis: Zhadan el a l , 1992 SpisuIu solidissima: Thurberg er al., 1975; Calabrese et a/., 1982: Eyster and Morse, 1984; Mann et al., 1983; Wright er a/., 1983 Tapesphilippinarum ( = bjaponica): Cardwell e1 al., 1979b: Cleary el al., 1993; McFadzen, 1992; McFadzen and Cleary, 1994 Tresus capar and 7: nuttalli: Cardwell. 1976; Cardwell el a/., 1978, 1979a el al., 1975; Robinson,

10

E. HIS, R. BEIRAS AND M . N. L. SEAMAN

and fertilize the gametes, and cultivation of the larvae to metamorphosis, with emphasis on the principal species used to date in toxicological assays and environmental monitoring.

2. RELEVANT ASPECTS

OF BIVALVE BIOLOGY

2.1. Species used in bioassays

The species used most often in bioassays is the Pacific or Japanese oyster Crassostrea gigas (30%), followed by the American or Eastern oyster C. virginica (23%), the blue mussel Mytilus edulis (15%), the quahog or hard clam Mercenaria mercenaria (13%), the Mediterranean mussel Mytilus galloprovincialis (7%), the European oyster Ostrea edulis (3%), and the coot clam Mulinia lateralis (3%) (Table 1). The biology of these species is reviewed below. 2.1 .l. The Japanese oyster Crassostrea gigas The Pacific or Japanese oyster originates from Eastern Asia, where it is distributed from the Sea of Okhotsk southward along the coasts of Korea and Japan to China (Ahmed, 1975; Andrews, 1980; Ventilla, 1984). Following the studies by Imai and Sakai (1961) four varieties or races are generally recognized; these are, distributed from north to south, Hokkaido, Miyagi, Hiroshima and Kumamoto (see Quayle, 1969). They are characterized, among other points, by different spawning temperatures. The Miyagi variety has repeatedly been introduced to the Pacific coast of North America since the beginning of the twentieth century, and it has formed an important natural population which spawns irregularly, but intensively, in British Columbia. Kumamoto oysters have been introduced in smaller numbers to Washington State (Quayle, 1969; Andrews, 1980). Owing to the scarcity of naturally setting oysters, the spat for aquaculture in British Columbia and Washington State are mostly produced by hatcheries. Miyagi, but also Hiroshima and Kumamoto oysters, have been introduced to Australia, and now form important natural populations on the southwest coast and around Tasmania (Thompson, 1951; Summer, 1980a,b). They have spread accidentally to the coast of New South Wales, where oyster farmers consider them a nuisance (Medcof and Wolf, 1975). They were also introduced accidentally to New Zealand in the 1960s, where they quickly formed abundant natural populations (Dinamani, 1971, 1987).


11

In Europe (discounting the probable introduction centuries ago of Crassostrea angulata, often considered the same species as C. gigas, but never used in bioassays, except by Renzoni (1973a,b), Pacific oysters were first introduced to Great Britain in the form of hatchery-produced spat from British Columbia in 1972 and 1975 (Gosling, 1981). Natural spatfall has been observed in Britain after warm summers (Askew, 1972). After the decline of the stocks of C. angulata in France, massive imports of Miyagi spat from Japan, as well as spat and spawners from British Columbia, quickly led to the formation of important natural stocks in the early 1970s (His, 1976). C. gigas reproduces very successfully in most oyster culture areas of southwest France, and occasionally in Brittany. Natural spatfall has also led to the establishment of significant wild populations in the Netherlands (Andrews, 1980) and Germany. The latter probably originated from spawning by the Dutch stocks (Seaman and Ruth, 1997), because successful local reproduction of the German stocks appears improbable (Neudecker, 198l), even though very ripe adults are found in some years. Finally, Japanese oysters also reproduce in the Adriatic part of the Mediterranean from Triest (Valli et al., 1979) to the Canal of Lim, where spawning occurs from July to October (Filic and Krajnovic-Ozretic, 1978; Hrs-Brenko, 1982). Andrews (1980) considers C. gigas to be the only species of oyster to have been introduced as a successful member of coastal communities around the world; “this oyster has adapted well to a wide range of environmental conditions and is probably the most globally widespread and ubiquitous oyster species in the world” (Chew, 1990). This explains why it is one of the species used most frequently in marine ecotoxicology. Gametogenesis in C. gigas is activated when the water temperature attains 14°C and continues as temperatures rise; Mann (1979) showed that not only are temperatures of 15°C to 18°C required, but that gametogenesis also depends on the duration at different temperatures (day-degrees). In addition, a temperature of at least 18°C to 20°C is necessary for spawning to occur (Fujiya, 1970; Mann, 1979). Buroker (1985), on the other hand, mentions a spawning temperature of 19°C to 24”C, and a salinity of 11 to 32 psu. With respect to the reproductive cycle in the natural environment, Imai and Sakai (1961) showed the differences between the four varieties in transplantation experiments at Miyagi province. The Hokkaido and Miyagi oysters spawned in late August to early September, those from Hiroshima in early or mid-September; those from Kumamoto spawned in late July when water temperatures attained 22°C to 23°C and began to mature again, with mature gametes still present in November and December. More generally, Koganezawa (1978) reports that Pacific oysters in Japan spawn from June to August. On the Pacific coast of Siberia (Vostok Bay,

12


Sea of Japan), they generally spawn in July (Yakovlev, 1978). In the United States there are no natural populations except at Washington State, where they spawn in July and August (Katkansky and Sparks, 1966; Perdue and Erickson, 1984). In Australia the oysters spawn from midJanuary or mid-February on, after water temperatures exceed 22°C (Thompson, 1951). In New Zealand the reproductive season may be very extended, with light spawning in mid-spring and successive spawnings in summer and early fall (Dinamani, 1987). In Europe, spawning is occasional in British, Dutch and German waters, occurring only in exceptionally warm summers (Askew, 1972; Andrews, 1980; Neudecker, 1981). Pacific oysters spawn intensively on the Atlantic coast of France from the Bidassoa estuary on the French-Spanish border, to the Loire estuary, which is the region with the most important centres of oyster culture. Further north, in Brittany and Normandy, the oysters attain sexual maturity, but spawn only in unusually warm summers; spatfall is light, except in the nearly closed bay of the Rade de Brest. Gametogenesis begins at the end of winter and spawning takes place after the water temperature reaches 20°C or more. At Arcachon the oysters spawn every 2 or 3 weeks from mid-June to mid-August with intermittent periods of gonad restoration (His, 1976); from mid-August to the end of September, bioassays can be conducted here without laboratory conditioning by using unspawned ripe oysters from Brittany and Normandy. 2.1.2. The Eastern oyster Crassostrea virginica The Eastern oyster has its natural distribution on the Atlantic coast of North America from the Gulf of St Lawrence to the Gulf of Mexico and the West Indies (Ahmed, 1975; Hayes and Menzel, 1981; Buroker, 1983 and 1985; Kennedy, 1983). It has repeatedly been introduced for aquaculture purposes to the Pacific coast of North America (Andrews, 1980) and to the Baltic coast of Denmark and Germany since the late nineteenth century (Seaman and Ruth, 1997), but has never become established in Europe. It has, however, been introduced successfully to Hawaii. Loosanoff and Engle (1942) already suspected the existence of different physiological races, because of variations in spawning behaviour. Stauber (1950) and Loosanoff and Nomejko (1951) subsequently established the existence of three different races in the northern, central and southern areas of its distribution, with spawning thresholds at 17"C, 20°C and 25"C, respectively. Similarly, C. virginica from Long Island Sound may mature at 12"C, whereas those from Florida do not ripen at 18°C (Loosanoff, 1968). The spawning season is in late June and early July in the Gulf of St Lawrence, and from mid-June to early August at New Haven (Stiles and


13

Longwell, 1973). In Chesapeake Bay it may last until the end of August (Kennedy and Krantz, 1982), and in Florida it lasts from March to October (Ingle, 1951). In Hawaii, C. virginica reproduces from February to November (Sakuda, 1966). 2.1.3. The European oyster Ostrea edulis The European oyster was originally distributed from Norway to Morocco and the Mediterranean (Walne, 1965; Ahmed, 1975; see also Korringa, 1941, 1958; Cole, 1941, 1942). It was introduced experimentally in 1949 to various estuaries in Maine (United States), where it reproduced, notably establishing itself at Boothbay Harbor (Loosanoff, 1955). It was also transplanted to the western coast of the United States with moderate success (Matthiessen, 1970). European oysters generally reproduce at temperatures of 15°C to 20°C (Buroker, 1985). According to the differences in spawning temperature, various physiological races exist (Korringa, 1958), but their distribution is not necessarily linked to geographic latitude. In Galicia (northwest Spain) some stocks spawn at temperatures as low as 12°C to 13”C, whereas in certain Norwegian fjords (where a superficial freshwater layer creates a greenhouse effect) the European oyster spawns at 25°C (Yonge, 1960; Wilson and Simons, 1985). Along the Atlantic coast the spawning season may last from early spring to November or December (Yonge, 1960). In the Adriatic, its larvae are encountered from April to October (HrsBrenko, 1982). 2.1.4. The mussels Mytilus edulis and M. galloprovincialis The genus Mytilus is widely distributed throughout the world (see Figure 1.6 in Gosling, 1992); its taxonomic status has often been investigated and discussed (Seed, 1971, 1976; Gosling, 1984, 1992; Lubet et al., 1984; Brock, 1985; MacDonald et al., 1991). Mytilus edulis and M . galloprovincialis are often sympatric, characterized by considerable morphological variability. They frequently interbreed, giving rise to intermediate phenotypes; where they coexist, the former is more frequently found in exposed, and the latter in more protected areas (Lubet, 1959). Their spawnings rarely coincide (Lubet, 1973; Seed, 1976). According to Gosling (1984) morphological, cytological, immunological, electrophoretic and hybridization studies show that M. galloprovincialis is “a form or ecotype” of M . edulis. There are no major differences between the chromosomes of the two forms (Dixon and Flavell, 1986). For reasons of convenience, however, they are still often referred to as separate species in the literature.

14

E. HIS,

R. BEIRAS AND M. N. L. SEAMAN

Both forms are very widely distributed. M. edufis is boreal, but eurythermic (Lubet, 1973), and is distributed from northern Norway (where summer temperatures hardly surpass 4°C to 5°C) to the Bay of Biscay (where summer temperatures may reach 23°C at Arcachon). It is found on both coasts of the North Atlantic, including the North and Baltic Seas (Gosling, 1992), but it is rare in Spain and Portugal, and very infrequent in the Mediterranean (Lubet et af.,1984; McDonald et af.,1991; Gosling, 1992). M. galloprovincialis is distributed from southern Britain to the northern coast of Africa, and the Mediterranean and Black Seas. According to Gosling (1992) M. trossufus,another ecotype of M. edufis, has a more northerly distribution than either M. edufis or M. gaffoprovincialis, and is found only in the northern hemisphere. The range of M. cafifornianus, considered to be a true species distinct from the M. edufis group, is restricted to the northeast Pacific, but is continuous over a latitudinal range of about 30 degrees. M. trossufus and M. cafifornianusare almost never used in bioassays. In many areas mussels may maintain sexual maturity and spawn repeatedly throughout the year. It is generally accepted that the reproductive period is shorter in cooler regions, and that it is more extended in M . galloprovincialis than in M . edulis (Seed, 1971, 1976). Spawning may generally be obtained in M. galloprovincialis throughout the year, except in unusually cold winters, when reproductive activity may cease altogether. The spawnings of greatest relevance for recruitment are in early spring and in fall, although the fall spawning may be late, or lacking altogether, in the northern range of its distribution (Lubet, 1973). In M. edufis on the coasts of Europe, the principal spawnings take place from March to early June. This is followed by a non-reproductive period during the summer, and gametogenesis resumes in October or November and continues through the winter (Bayne et af., 1975). Mussels from warmer waters generally spawn earlier, although this was not found to be the case in seven M. edulis populations on the east coast of the United States; Bayne’s rule on day-degrees did not apply either (Newel1 et af., 1982). 2.1.5. The hard clam Mercenaria mercenaria The northern quahog, or hard clam, is distributed on the Atlantic coast of North America from the Gulf of St Lawrence to the northern Gulf of Mexico, and it is particularly abundant from Maine to Virginia. In the southern part of its range, it is sympatric with M. campechiensis, with which it may interbreed. M. mercenaria has been introduced to the coast of California (Ansell, 1968; Stanley and DeWitt, 1983). It has also been introduced to Europe, forming wild stocks in the areas of Portsmouth and Southampton in Britain (Mitchell, 1974), but despite various introductions


15

to France, significant populations have only become established in the Marennes-OlCron area and in Brittany. Small stocks also exist on the Dutch and Belgian coasts (Tebble, 1966). The reproductive cycle of this species has been reviewed by Stanley and DeWitt (1983), with additional descriptions by Keck et al. (1975), Dalton and Menzel (1983), Knaub and Eversole (1988), Manzi et al. (1985), and Walker and Heffernan (1995). The sexual cycle has been described for populations of Long Island Sound (Loosanoff, 1937a,b), Delaware Bay (Keck et al., 1975), North Carolina (Porter, 1964), South Carolina (Eversole et aE., 1980), and northwest Florida (Dalton and Menzel, 1983; Eversole, 1989). M . rnercenaria generally has two breeding peaks in North and South Carolina, where spawning occurs at 27°C to 30°C (Porter, 1964; Eversole et al., 1980). This bimodal pattern is not found at Delaware Bay, where spawning takes place at 25°C to 27°C (Keck et al., 1975), nor at Long Island Sound, where the temperature for spawning is 23°C to 25°C (Loosanoff, 1937a,b). In Britain the species has adapted to the colder climate and spawns at 18°C to 20°C (Mitchell, 1974). In contrast, Dalton and Menzel (1983) describe a trimodal spawning pattern in northern Florida, with peaks in autumn, winter and spring. Laughlin et al. (1988) assert that reproduction occurs year-round and that mature spawners are available without laboratory conditioning throughout the year. The existence of different spawning temperatures for different stocks has led Porter (1964) and Keck et al. (1975) to hypothesize the existence of different races. In summary, M. mercenaria does not seem to have a sexually inactive season, and individuals at various stages of gametogenesis can be found at all times, although the proportions vary greatly between locations. 2.1.6. The coot clam Mulinia lateralis The coot clam is also distributed on the eastern coast of North America from Canada to the eastern Gulf of Mexico (Kennedy and Mihursky, 1971; Calabrese and Rhodes, 1974; Morrison and Petrocelli, 1990; Burgess and Morrison, 1994). The species is not very abundant, except at certain favourable locations (Calabrese, 1969a,b, 1970a). According to the presence of the larvae in the plankton, the reproductive period is from mid-July to early December at Prince Edward Island, Canada (Sullivan, 1948); the larvae are found from May to November at Tred Avon River, Maryland (Shaw, 1965). At Long Island Sound, gametogenic activity continues throughout the year, with principal spawning peaks in late July and mid- to late August, the first peak being at temperatures near 20°C (Calabrese, 1970a; Calabrese and Rhodes, 1974).

16

E. HIS, R. BEIRAS AND M. N.

L. SEAMAN

This confirms the observations of Sullivan (1948) and Loosanoff et al. (1966).

2.1.7. Other species At least 25 other bivalve species have also been used in bioassays (see Table l), but most of them do not fully satisfy the criteria required (Stebbing et al., 1980). Although some of these species are of economic importance, their geographic distribution is generally limited. This applies in particular to the mussels Mytilus californianus and M . trossulus, the oysters Crassostrea angulata, C. cuccullata, C. margaritacea, C. rhizophorae, Saccostrea commercialis and Ostrea plicatula, as well as to the Hawalan species, Zsognomon californicum, which has been studied in detail by Ringwood (1990, 1991, 1992a,b, 1993) with regard to the micropollutant sensitivity of adults and larval stages.

2.2. Reproduction

Experiments with larvae depend first of all on the availability of spawning individuals of both sexes. The bivalve species commonly in use for bioassays have a very wide geographical distribution, but the availability of mature adults may nevertheless be highly variable. Following the studies by Orton (1927) on Ostrea edulis, Coe (1932a,b) on Crassostrea virginica and Loosanoff (1937a,b) on Mercenaria mercenaria, there have been numerous publications on bivalve reproduction (reviews by Galtsoff, 1964; Raven, 1964; Purchon, 1968; Sastry, 1975,1979; Seed, 1976; Andrews, 1979; Mackie, 1984; Gosling, 1992). 2.2.1. Gametogenesis The reproductive anatomy of bivalves is rather simple. The gonad consists of a mass of follicles which develop fully at the period of sexual maturity, at which time the sexual products make up a significant part of the body. The sexual products grow within genital ducts, the diameters of which increase progressively during gametogenesis; the various small ducts converge on larger gonoducts, through which the gametes are expelled into the exhalent part of the pallial cavity at the time of spawning. The simplicity of the reproductive organs, notably the absence of specialized structures (such as a penis in the male or accessory glands in the female) facilitates the change of sex observed in many bivalve species. In oysters, during the winter, when there is no reproductive activity, the


17

gonad mass is replaced by a mass of connective tissue with vesicle cells containing lipids and glycogen. Embedded in this and close to the internal organs is a duplicate system of branching tubules, one on each side, beginning near the anterior end of the body, uniting into a single tube on either side and ending in the genital pore. These pores open into the suprabranchial chamber at the posterior base of the adductor muscle, in close proximity to the opening of the urinal ducts (Quayle, 1969). During the breeding season the reproductive organs form at least 50% of the body volume. In a fully ripe oyster, the gonadal tubules, small in diameter at the anterior end of the oyster and thickening as they approach the genital opening, may be clearly seen on the surface of the soft body of the oyster. At this time, the two gonadal systems are almost completely inseparable, except at the genital pores. In mussels the gonad extends in a diffuse manner throughout the mantle. It is made up of a multitude of acini which are grouped around tubules, which are in turn clustered around the gonoducts. These fuse to form the terminal gonoducts on either side of the body, which then unite and open into the genital papilla near the posterior adductor muscle. In venerid clams such as Mercenaria mercenuria, the gonad is very diffuse and located around the digestive gland, sometimes covering it entirely and extending into the foot by the time maturity is attained. The genital pores open into the exhalent part of the pallial cavity and the gametes are released by the exhalent siphon. In Muliniu lateralis, the gonad of ripe animals forms a uniform and continuous mass around the digestive tube and gland (Calabrese, 1970a, b). Most lamellibranchs (96%) are gonochoric, i.e. they have separate sexes (Coe, 1943), but there are various hermaphroditic species, including oysters. With regard to the hermaphrodites, the following classifications have been proposed (Table 2). Crussostrea gigus and C. virginica are both alternative hermaphrodites (Amemiya, 1929; Coe, 1932a,b), changes in sex occurring irregularly during the course of life. Most individuals only emit sexual products corresponding to one sex in the course of the reproductive season, but there is a small proportion of simultaneous hermaphrodites (Loosanoff, 1965a). Crassosfreu is usually protandric, about 70% of the individuals male in their first year, and about half in the second year, with females dominant in the older age groups. In Ostreu edulis, which is larviparous, protandric sex changes are the rule (Sparks, 1925; Orton, 1927). The time lag between the maturation of spermatocytes and oocytes is small, however, and European oysters may alternately function as males and females in the course of one reproductive season. The frequency of sex changes increases with temperature, so

18


Table 2 nYo systems of classifying hermaphroditisms in Bivalvia (from Fretter and Graham, 1964).

Coe Functional or simultaneous Consecutive Rhythmical consecutive Alternative

Bacci

Simultaneous (with synchronous ripening) Successive (with asynchronous ripening) or consecutive Successive (with asynchronous ripening) or alternate Successive (with separate ripening) or alternate

it is related to latitude (Cole, 1942). The change from the female sex to the male is more rapid than vice versa. Mytilus edulis and M. galloprovincialis are gonochoric, even though rare cases of simultaneous hermaphroditism have been reported by Coe (1943) and Lubet (1959). According to the latter, hermaphroditism occurs in about 0.02 to 0.1% of the population. Changes of sex by the same individual have never been observed. Mercenaria rnercenaria is a protandric hermaphrodite (Eversole, 1989), but the simultaneous development of oocytes and spermatocytes is common during the first year of life (Loosanoff, 1937a,b), and a small percentage of simultaneous hermaphrodites is always present in the population. Nevertheless, 98% of the individuals are male during the first year, but the sex ratio becomes even with advancing age. Mulinia lateralis is gonochoric. The reproductive cycle is very brief, the egg-to-egg cycle being only 39 to 135 days with an average generation time of 60 days (Calabrese, 1970a). Individuals as small as 2.7 mm in length are already capable of spawning (Calabrese and Rhodes, 1974). Another practical aspect of this species is that the sexes are readily distinguishable, because the orange-red female gonad and the whitish male gonad are discernible through the shell near the umbo. Galtsoff (1964), Bayne (1976) and Mackie (1984) have described the structure of bivalve gametes in detail. In the males the chromosomal reduction (first order spermatocytes with 2n to second order spermatocytes with n chromosomes) is achieved by an equal division during spermatogenesis. In the female, however, the gametes are spawned at the germinal vesicle stage and only develop to the metaphase of the first meiotic division: meiosis is arrested until the egg is activated by sperm. Penetration of the ovum by the sperm is made possible by the acrosomal reaction (i.e. dissolution of the egg membrane by the sperm’s acrosome), after which the sperm cytoplasm fuses with the egg cytoplasm; this occurs within about 5 minutes in Mytilus edulis. Meiosis in the egg (Figure 1)then


19

Figure 1 Normal development in the bivalve egg. For simplification only one pair of chromosomes is shown. (a) egg at release at metaphase of meiosis I, activation by sperm; (b) meiosis I is complete, first polar body extruded, sperm nucleus has entered egg; (c) meiosis I1 completed, second polar body extruded, male and female pronucleus unite; (d) first cleavage perpendicular to point of polar body extrusion. (From Beaumont and Fairbrother, 1991.)

continues with the formation of the first and second polar bodies, after which the egg’s chromatin forms the chromosome vesicle. This is followed by the fusion of the male and female chromosome vesicles and the formation of the 2n pronucleus (Longo and Anderson, 1969a,b; Bayne, 1976; Mackie, 1984; Cherr et al., 1990). 2.2.2. Sexual maturation in the field Spawning in marine invertebrates may occur year-round in regions with little seasonal variability (Sanders and Hessler, 1969, cited after Dalton and Menzel, 1983). According to Rand (1973), cool climates are characterized by species with a single annual spawning season, temperate zones by species with two separate spawning periods, and tropical areas by species with year-round spawning. The determination of sexual cycles has been based on direct observation of spawning activity in the natural environment, on determinations of the state of maturation of the gonad, on the appearance in the plankton of larvae of the species in question, and on the settlement of the juveniles (Seed, 1976). The reproductive cycle of bivalves can generally be divided into three phases: gametogenesis and vitellogenesis, spawning and fertilization, and larval growth and development (Newel1 et al., 1982). With regard to the maturation state of the gonad, a variety of scales have been proposed by Chipperfield (1953), Lubet (1959), Seed (1969) in mussels, Loosanoff (1942) and Kennedy and Battle (1964) in C. virginica, and Keck et al. (1975) and Eversole et al. (1980) in Mercenaria mercenaria. 2.2.2.1. Environmental factors influencing gametogenesis. Mackie (1984) has reviewed the principal factors governing sexual maturation in bivalves. They are either exogenous, such as temperature, lunar cycle, and,

20

E. HIS, R. BEIRAS AND

M. N.

L. SEAMAN

particularly, nutritional factors, or endogenous, such as genetic or hormonal factors. Among the external factors, Orton (1920) considered temperature the most important influence. Loosanoff and Nomejko (1951) were the first to advance a hypothesis on the existence of physiological races in Crassostrea virginica with different temperature requirements for spawning. The issue was taken up again by Ahmed (1975) with respect to oysters. As will become apparent in the next section, laboratory “conditioning” of tachydictic species, i.e. the possibility of inducing sexual maturation out of season, largely depends on an elevation of the temperature. Even though this applies to most bivalve species used in bioassays, it must be emphasized that mussels, to the contrary, reproduce at low temperatures. Bayne (1975) established a day-degree function for the relationship between temperature and duration of gametogenesis in Mytilus edulis. The same observation was made by Mann (1979) with regard to C. gigas, and by Knaub and Eversole (1988) in Mercenaria rnercenaria. In eurythermic species with high temperature tolerance, the duration of the reproductive cycle often varies with geographical latitude. Nutritional factors are also of paramount importance. Various authors have shown that bivalves accumulate nutritional reserves in their connective tissue at the time of sexual inactivity, in order to mobilize them towards the formation of sexual products during gametogenesis (see Mackie, 1984). The nutritional needs of mussels increase during gonad maturation (Bayne, 1975); stress and bad nutritional conditions, on the other hand, are accompanied by a reduction in fertility, with a decrease in the number of gametes produced and a modification of their biochemical composition (Bayne et al., 1975,1978; Bayne, 1985). A rise in temperature and stress from a simultaneous decrease in available food, and the influence of pollutants such as hydrocarbons also lead to lower gamete quality and larval viability. In C.virginica starvation leads to a decrease in the proportion of females (Bahr and Hillman, 1967). An increase in the proportion of males in a population of oysters may therefore be an indication of environmental disturbance (Kennedy, 1983). Among the endogenous factors, neurosecretion was first shown to be important in mussels by Lubet (1959), and in oysters by Nagabhushanam (1963). Genetic factors may also be important (Lannan et al., 1980). 2.2.2.2. Environmental factors influencing spawning behaviour. Male oysters are more sensitive to stimulation and usually spawn first. The first males induce spawning in their male neighbours, then the first females follow suit, and finally the entire population spawns simultaneously. This results in the formation of “spawn streaks” described by Quayle (1969), milky masses of water several hundred metres in length spreading in the channels of bays and estuaries. Among the environmental factors which


21

induce spawning behaviour are the thermal stimulation resulting from abrupt differences in temperature between the water masses of the falling and the rising tide, changes in salinity during the tidal cycle, mechanical agitation by waves and currents, and differences in pressure between high and low water and during the falling tide. Spawning in the field is never provoked by one single stimulus; when an appropriate combination of stimuli occurs, spawning is most common during the 3 hours following high tide (58% of all spawnings), and it is more frequent (69%) and more prolonged (76% of the duration of all spawnings) at spring tides (His, 1976). In flat oysters, “swarming” (i.e. the release of larvae incubated within the mother’s pallial cavity) is also related to the lunar cycle: from July to September larvae are most abundant in the plankton following a full moon (Orton, 1926), or 10 days after a full or new moon (Korringa, 1941; Knight-Jones, 1952).

2.2.3. Cleavage, embryogenesis and larval development Bioassays may be conducted with gametes and larvae of all stages, from sperm and unfertilized eggs to metamorphosis of the pediveliger at the end of pelagic life. Many researchers indicate the time passed between fertilization and the fertilized gametes’ first exposure to the substance being investigated, as this may have a bearing on the interpretation of the results. The rates of development and cleavage, and the duration of the successive embryonic and larval stages differ widely between the various bivalve species, and they also depend largely on environmental factors, notably on the temperature. The development of bivalves consists of an embryonic phase followed by a larval phase, and has been described by various authors (Erdmann, 1935 in 0. edulis; Cahn, 1950 in C. gigas; Galtsoff, 1964 in C. virginica; Lubet, 1973 and Bayne, 1976 in M. edulis). More recent work by Elston (1980) on C. virginica and by Waller (1981) on 0. edulis and C. gigas have provided additional details of larval morphology, particularly demonstrating the complexity of various larval organs. The common terminology employed for the various development stages is shown in Figure 2. 2.2.3.1. Embryonal stages. The fertilized egg (see Figure 1) completes meiosis by expelling the first and second polar bodies, and then begins to divide. The first division is unequal, giving rise to the blastomeres AB and CD, as well as a polar lobe which fuses with CD. The second division (4-cell stage) leads to the formation of three blastomeres (A, B and C) at the animal pole and one very large blastomere (D) at the vegetal pole. During the third division, the cleavage becomes spiral and subsequent

22


TERM EGG BLASTULA GASTRULA TROCHOPHORE VELIGER 0-SHAPED STAGE UMBO STAGE PEDIVELIGER SPAT

PRESHELLED PRODISSOCONCH I STAGE (less (about 7 days) than 1 day)

DISSOCONCH (remainder of life)

PRODISSOCONCH II (10-15 days)

--

--...

...

--...

....

....-

-

Relationship of stages of development of the prodissoconch to other common terms used to describe the larval shell and body of Ostrea edulis (dashed lines indicate uncertainty or transition; duration of stages may be highly variable). (From Waller, 1981.)

Figure2

divisions lead to the formation of the morula. Cilia appear, and the embryo becomes motile, gyrating slowly. Gastrulation begins as the micromeres of the animal pole cover the blastula, followed by a slight invagination that gives rise to the archenteron and to a small blastopore. 2.2.3.2. Larval stages. The first larval stage is the trochophore, which is covered by short cilia; in mussels and some other species it also has a flagellum. A dorsal thickening of the ectodermis is secreted by the shell gland, forming the initial organic cuticle which eventually spreads to cover the entire body. A circular band of cilia, the prototroch, also forms, enveloping the apex. The second larval stage is the veliger, which is formed 24 hours after fertilization in Crussostrea, and after 48 hours in Mytilus. It has a straight dorsal hinge giving the larva the characteristic shape of a capital letter D (hence the synonymous terms “straight-hinge larva” and “D-larva”), and it is some 60 to 70 pm in size in most bioassay species (see below). At this stage the larva begins to feed. The shell, consisting of two valves, begins to calcify, forming the prodissoconch I. The prototroch has continued its development to form the velum. As the larva grows beyond 100 pm, secretion of the secondary shell (the prodissoconch 11) begins and the umbo begins to form, extending over the dorsal hinge (hence the terms “veliconcha” and ‘‘urnboned larva”). In oysters and mussels, as the larva grows to a size of 250 or 300 pm, an “eye spot” appears within the shell and the foot is formed (hence the terms “eyed larva” and “pediveliger” for this stage). As the ciliated foot grows and becomes functional, the larva becomes capable of both pelagic and benthic


23

modes of life, either swimming by use of the velum (e.g. to feed), or creeping along a hard substrate with its foot (e.g. to explore for a suitable site for settlement). Once it has attained this stage it is ready to metamorphose. All of the various stages mentioned above are used in bioassays. 2.2.3.3. Settlement and metamorphosis. The first photographs of settlement and metamorphosis in Crassostrea virginica were made by Prytherch during the 1930s, and some of his images were reproduced by Medcof (1961). The fixation of pediveligers of Ostrea edulis has been described by Cranfield (1973). The first attachment stage after metamorphosis is the plantigrade or juvenile (Figure 3). Oysters settle by glueing their shell to the substratum with a proteinaceous cement produced by the pallial gland (Figure 4). Mussels and clams attach themselves to the substratum with the help of byssal threads produced by the byssal gland at the base of the foot. During metamorphosis the velum disappears, and in oysters the foot is resorbed as well; in mussels and clams, the foot continues to develop and specialize to the adult form. Labial palps and gills are developed to replace the larval feeding apparatus, the velum, and the final shell, the dissoconch, is secreted. The anatomical changes of metamorphosis may make mussel larvae unable to feed for 1 to 3 days (Bayne, 1976). In Crassostrea virginica, Baker and Mann (1994b, p. 239) reported that “velar feeding occurred during the searching and crawling stages, but not during cementation”, and subsequently, Baker and Mann (1994a, see Figure 1, p. 94) identified four separate phases of metamorphosis: settler, prodissoconch postlarva, dissoconch postlarva and juvenile. Various studies have been devoted to the problems of settlement and metamorphosis in bivalves (see Ritchie and Menzel, 1969;Lutz et al., 1970; Hidu and Haskin, 1971; Keck et al., 1971; Veitch and Hidu, 1971; Andrews, 1979; Mackie, 1984; Baker and Mann, 1994a,b). Prytherch (1924,1931 and 1934) found that copper induces metamorphosis in larvae of C. virginica. Since then, a great number of publications have documented natural and artificial chemical substances capable of inducing metamorphosis (see Hadfield, 1984; Pawlick, 1992). The special role of neuroactive compounds (L-DOPA, dopamine, serotonin, epinephrine, norepinephrine) has been shown by Coon and Bonar (19861, Coon et al. (1985, 1986, 1990), Shpigel et al. (1989) and Beiras and Widdows (1995), and the importance of biofilms has been demonstrated by Weiner et al. (1989). In oysters, neurotransmitters may induce metamorphosis without fixation, producing “cultchless spat” which are anatomically identical to naturally set spat (see photographs in Coon et al., 1985, p. 217; Coon and Bonar, 1986). These advances are of considerable practical significance in toxicological bioassays with pediveligers (see Section 3.2.4.5).

24

E. HIS, R. BEIRAS AND M .

N. L. SEAMAN

Figure 3 Crassostrea gigas pediveliger metamorphosing (a), and metamorphosed post-larva (b). Gr: gill rudiment; F foot; P d prodissoconch shell; D: dissoconch shell. (Experiments by His et al., 1997b.) Metamorphosis was induced by epinephrine.

25


2 Larva begins swimlcrawl

Larva searches for surface cue

Larva detects

Cementation to substratum

Metamorphosis to juvenile oyster

Figure 4 Two-cue model of microbial induction of oyster settlement (1-3) and metamorphosis (45). A soluble cue induces searching behaviour and then a surface cue induces attachment. (From Weiner et al., 1989.)

2.3. Larval rearing in the laboratory

2.3.1. Laboratory conditioning of spawners To conduct routine bioassays with bivalve embryos and larvae it is desirable to be able to obtain ripe adults year-round. In some species, mature adults can be found in the field during many months, and in others it is possible to condition them out of season. This was first discovered by Loosanoff (1945), when he attempted to clear Eastern oysters from a growth of sponges by keeping them in running seawater at elevated temperatures and found that by doing so he had induced gametogenesis in winter. He subsequently demonstrated that the duration of conditioning can be prolonged or shortened by manipulating the temperature, and that the technique can be applied to Mercenaria mercenaria as well (Loosanoff and Davis, 1950, 1951, 1952, reviewed 1963). In the case of Crassostrea virginica,gonad maturation can be obtained in the winter by gradually raising the water temperature to 20°C or 25°C; it is even possible to transfer oysters directly from a temperature of 5°C in the field to 20°C in the laboratory. The duration of conditioning depends on the temperature, as long as it is maintained within the range of 15°C to 30°C. Prolonged incubation at 27°C to 28°C can, however, inhibit or delay spawning (Ruddy et al., 1975). At the end of the reproductive season, after the adult oysters have used up their reserves, it is difficult or impossible to induce them to mature

26


again immediately. To avoid this inconvenience, Loosanoff and Davis (1963) took maturing oysters from Long Island Sound and transferred them to the cooler waters of Maine, where their maturation was arrested. They were subsequently able to induce final maturation and spawning by manipulating the temperature in the laboratory. Spawning can be delayed this way for 6 to 8 weeks; beyond this time limit, progressive resorption of the gonad makes spawning impossible. To circumvent the problem, Loosanoff and Davis induced oysters to spawn early and then transferred them also to Maine, where they resorbed the residual sexual products and reconstituted their glycogen reserves, thus beginning a new reproductive cycle and becoming susceptible to conditioning again. This two-sided approach to conditioning (acceleration and inhibition of gametogenesis) can be applied to C. virginica and M. mercenaria (Davis and Chanley, 1955a; Loosanoff and Davis, 1963), and also to C. gigus, and it is of great practical relevance to toxicological bioassays. In Mufinia faterafis it is equally possible to obtain mature adults throughout the year by delaying or accelerating sexual maturation with the same manipulations of temperature (Calabrese and Rhodes, 1974); apparently it works even better with the coot clam than with the other species (Burgess and Morrison, 1994). Conditioning has also been achieved with Ostrea edufis (Dannevig, 1951; Abou-Ela, 1960), where adults can be transferred from environmental temperatures of 1°C to 5°C to conditioning tanks at 21°C (Walne, 1966). Supplementary feeding with cultured algae (Tetrasefmis suecica) during conditioning improves the subsequent survival of the larvae (Helm et af., 1973). It is equally possible to delay spawning in mussels after initiation of gametogenesis by transferring them to 4°C or 5°C one month before the time of spawning and by supplementing their nutrition with cultured phytoplankton (Bayne, 1965, 1975; Riisgard et af., 1980; Dixon and Prosser, 1986). Mussels have been thermally conditioned by Mitchell et al. (1985) by maintaining them for 3 weeks in unfiltered seawater at 14°C. Spawning was obtained by Bayne (1975) one month early in adults which had initiated gametogenesis by keeping them at 15°C (5°C higher than the ambient temperature) and by feeding them with Tetrasefmis suecica (at least 2.2% of the meat weight per day). The time required for gonad maturation depends on the stage of maturation at the onset of conditioning as well as on the conditioning temperature. On the other hand, stress and sub-optimum rearing conditions (particularly with respect to nutrition) reduce the fertility of mussels, as well as the quality of the gametes and larvae (Bayne, 1975, 1985). The methods for conditioning scallops, oysters and clams in hatcheries have been reviewed by Utting and Millican (1997): “of particular

27


Table 3 The fecundity of bivalves used in bioassays. Species

Crassostrea gigas Crassostrea virginica Mytilus edulis

Mytilus galloprovincialis Mercenaria mercenaria

Mulinia lateralis Ostrea edulis

Fecundity (eggs per female; x106) 15 to 114 15 to 114.8 10 to 66.4 (M = 28.8) 10 >0.5 1.2 to 7.6 2 no data 0.38 to 18.83 0.6 to 13.2 (A4 = 6.3) 8 to 39.5 ( M = 24.6) 1.4 >7 (M = 3 to 4) 0.09 to >1 0.616 to 1.155 0.1 (1year old) to 1.5 (7 years old)

Reference Galtsoff, 1964 Galtsoff, 1964 Davis and Chanley, 1955a Lubet, 1959 Bayne, 1975 Bayne et al., 1978 Thompson, 1979 Lubet, 1973 Ansell, 1967 Bricelj and Malouf, 1980 Davis and Chanley, 1955a Knaub et al., 1987 Calabrese, 1969a,b Cole, 1941 Millar, 1961 Walne. 1974b*

M: mean value; *estimated from the number of incubated larvae.

importance is the optimization of the quantity and the quality of microalgae diets that are provided during the broodstock conditioning”. Flow-through systems are generally better than recirculation systems, because “natural phytoplankton is a valuable component of the diet during broodstock conditioning” (p. 46). Gametogenesis is improved by preconditioning in high food regimes before the actual conditioning at elevated temperature. Egg quality and larval viability depend largely on an adequate accumulation of lipid reserves in the ova. 2.3.2. Spawning

To have a suitable number of replicates, or to be able to study a range of concentrations in toxicity tests, a great number of larvae are often required in bioassays. Most of the bivalves used in bioassays are characterized by high fertility. Flat oysters are an exception; being larviparous, not only are they less fecund, but they also have much larger oocytes (100 to 150pm in diameter in Ostrea edulis) than the other species. The data on fecundity (Table 3) are incomplete, especially with regard to Mytilus galloprovincialis. The data on oysters have been reviewed by Galtsoff (1964), those on Mercenaria mercenaria by Davis and Chanley

28


(1955a) and by Eversole (1989), Mytilus edulis by Thompson (1979), and Mulinia lateralis by Calabrese (1969a,b). The number of eggs released by any individual always depends on its size and physiological condition, as evidenced by significant correlation between size and number of eggs spawned in Crassostrea virginica and M. mercenaria. Stress and pollution, however, are known to reduce the fertility in mussels (Bayne, 1972; Zaroogian et al., 1979; Zaroogian and Morrison, 1981). The reproductive physiology of Crassostrea virginica has been studied in detail by Galtsoff (1938a,b, 1940, 1964). He determined that spawning in female oysters involves complex mechanisms with the participation of the nervous system, the gills, the mantle and the adductor muscle. The genital pore is situated near the anus. Oocytes are expelled into the exhalent part of the pallial cavity, then pass through the gills and accumulate between the gills and the mantle before being expelled by muscular action through the inhalant part of the pallial cavity into the surrounding water. The shell movements associated with spawning are “so characteristic that they cannot be mistaken for any other type of muscular activity” (Galtsoff, 1964), and they can serve to monitor the frequency and duration of reproduction in the field (His, 1975, 1976). Ova are violently expelled at regular intervals, forming whitish clouds near the spawning female. In the male, however, the sperm leave as a steady stream with the exhalent current, almost without contraction of the adductor muscle (Galtsoff, 1938a,b). In the water column, the gametes emit pheromones (termed “fertilizins” by Galtsoff, 1938a,b, 1940) which induce spawning in members of the opposite sex. Sperm also release a nucleoprotein termed diantline which facilitates spawning in both sexes by relaxing the adductor muscle, enlarging the gill ostiae, augmenting ciliary activity, and thus increasing the pumping rate (Nelson and Allison, 1940). These observations probably apply to all oysters of the genus Crassostrea (Galtsoff, 1964). The presence of similar pheromones has been demonstrated in mussels by Lubet (1959). In Ostrea edulis the ova also pass through the gills into the inhalant part of the pallial cavity, but this does not give rise to any visible change in behaviour (Yonge, 1960). The ova are fertilized by sperm inhaled by the female and are subsequently retained within the pallial cavity for about one week, after which they are released (a phenomenon termed “swarming” by Dutch and German authors). In Mercenaria mercenaria and in Mytilus, sperm are liberated with the exhalent current in a whitish thread-like stream which rapidly dissipates and gives the water a milky appearance. The ova are similarly liberated by the females without particular valve movements. In the laboratory, spawning can be induced in mature bivalves by a


29

variety of physical and chemical stimuli (Table 4, after Le Pennec, 1981; see also Mackie, 1984). In the case of Eastern oysters from South Carolina and Florida it is not possible to induce spawning in the laboratory by the usual methods (thermal stimulation and addition of sperm suspension), because they are often subjected to these stimuli for extended periods in their habitat (Maurer and Price, 1968). Gametes may be obtained from individuals that refuse to spawn naturally by “stripping” (i.e. teasing the gonad with a forceps), however this is not generally recommended (Woelke, 1961, 1966; ASTM, 1989; Widdows, 1993). These methods are discussed in Sections 3.1.3.2 and 6.3.2. 2.3.3. Fertilization In the oviparous species, with the notable exception of Mercenaria mercenaria, the eggs are somewhat irregular in shape at the time of release, becoming spherical after a few minutes. Egg sizes (Table 5) do not vary much among species, with diameters of 50 to 55 pm in Crassostrea, 60 to 70 pm in Mytilus, 70 to 73 pm in M. mercenaria, and about 50 pm in Mulinia lateralis (Lubet, 1959; Yonge, 1960; Loosanoff and Davis, 1963; Galtsoff, 1964; Fretter and Graham, 1964; Calabrese and Rhodes, 1974; Purchon, 1968; Mackie, 1984; Eversole, 1989). In M. mercenaria the egg has a gelatinous envelope which swells to a diameter of 163 to 170 pm,and which may persist to the blastula stage (Loosanoff and Davis, 1950). Fertilization is practically instantaneous when the egg meets the spermatozoa (Allen et al., 1988). According to Lu (1986, in Allen et al., 1988) in Crassostrea gigas the second polar body is extruded 50 minutes after fertilization at a temperature of 18”C, after 43 minutes at 2VC, and after 32 minutes at 25°C. 2.3.3.1. Polyspermy. Galtsoff (1964, p. 343) states, “A few seconds after the sperm head touches the egg surface a thin transparent fertilization membrane is elevated”. Alliegro and Wright (1983) have, however, questioned the existence of this phenomenon: “It is possible that the fertilization envelope reported by Galtsoff can be attributed to the refractile nature of the fertilized egg surface seen with light microscopy.” Galtsoff also notes that two or more sperm (“supernumeraries”) may enter the egg before the barrier is developed when sperm suspensions are thick; this leads to polyspermy and results in an abnormal embryo (Turner, 1958; Loosanoff and Davis, 1963; Galtsoff, 1964). Determination and use of appropriate sperm to egg ratios significantly enhance the yields of viable larvae (Stephano and Gould, 1988). According to Stiles and Longwell (1973), in C. virginica the number of sperm has to approach 20 per egg before the incidence of chromosome and division abnormalities increases significantly, but this has been questioned by

30


Table 4 Experimental methods used to induce spawning in marine bivalves (completed after LE Pennec, 1981). Method

Species

Reference

Stimulation due to transport

Crassostrea gigas

Imai, 1967

Unspecified temperature fluctuations

Crassostrea virginica C. virginica Mercenaria mercenarin Mulinia lateralis

Prytherch, 1924 Wells, 1927 Loosanoff, 1937b Calabrese, 1969b

Specified temperature fluctuations

Mytilus edulis C. gigas Mytilus galloprovincialis M. edulis

Bayne, 1965 Imai, 1967 Masson, 1975 Lutz and Hidu, 1979

Temperature fluctuations and addition of gametes

C. virginica C. virginica, M. mercenaria M. mercenaria several bivalves M. lateralis M. lateralis

Davis, 1953

Constant temperature and addition of gametes

C. virginica, C. gigas

Galtsoff. 1930

Salinity fluctuations

tsognomon califomicum

Ringwood, 1990,1991,1992

Electrical stimulation

M. edulis M. edulis M. edulis

Aboul-Ela, 1960 Iwata, 1950 Sugiura, 1962

M. edulis M. edulis

Iwata, 1951a,b Morse et al., 1977; Garland et al., 1986 Loosanoff and Davis, 1963 Loosanoff and Davis, 1963 Castagna et al., 1985

Chemical stimulation KCI 0.5 M

HzOz NbOH Injection of serotonin Pricking the adductor muscle

M. mercenaria C. virginica M. mercenaria, M. edulis M. edulis

Loosanoff, 1954 Chesnut et al., 1957 Loosanoff and Davis, 1963 Kennedy et al., 1974 Calabrese and Rhodes, 1974


Hrs-Brenko and Calabrese, 1969 Masson, 1975

Use of "Kraft mill effluent"

M. edulis

Breese et al., 1963

Addition of algae

M. edulis, Mytilus californianus, C. gigas, M. rnercenaria M. californianus

Breese and Robinson, 1981 Smith and Strehlow, 1983

Table 5 Sue of egg, size of early veliger, sue at time of metamorphosis and duration of larval development under laboratory conditions.

Species Crassostrea gigas Crassostrea virginica Mytilus edulis Mytilus galloprovincialis Mercenaria mercenaria Mulinia lateralis Ostrea edulis

Temperature ("C) 25 24 22 20 30-32.5 16 (11)

Egg diameter

Size of early veliger

Size at time of metamorphosis

(Pm)

(Pm)

(CLm)

-

76.8 68 68-75

303-320 310 275-315

96 100-120 76

260 215-300 298 ? 23 258

50-55

?

?

18 24*

77,8 ? 4

18 ?

20-25 20-22 18-20

-

73-75 70-73 ? 50 114-126

86 60 ?

170-190*** 208

175-240 21Cb230 20fA220 240-350 280-300

Duration of larval development (4 1619 17-21 12** 36-40 10-12 1 6 2 0 (34-38) ? ?

17-19 16 (6-8**) 6 6-8 10-16 15-16

Reference Gerdes, 1983b Beiras and His, 1994 Loosanoff and Davis, 1963 Davis and Calabrese, 1964 Davis and Calabrese, 1964 Bayne, 1965 Loosanoff and Davis, 1963 Sprung, 1984 Beiras and His, 1995a Aguirre, 1979 Loosanoff and Davis, 1963 Loosanoff and Davis, 1963 Calabrese and Rhodes, 1974 Walne, 1974b Loosanoff and Davis, 1963

*egg incubation at 20°C; **33"C. Data from references in italics. ***larval sue at liberation. Mytilus edulis (Bayne, 1965): temperature and corresponding duration of pelagic life in parenthesis.

32


Staeger and Horton (1976) in C. gigas and Alliegro and Wright (1983) in C. virginica. In the Japanese oyster, the mean percentages of larvae developing to the D-shape stage increased until 7.3 X 10’ s p e d 1 0 0 eggs were used (Staeger and Horton, 1976), and in C. virginica the number of sperm entering eggs is restricted to one per fertilized egg at a sperm:egg ratio as high as 1OOO:l (Alliegro and Wright, 1983). In both cases gametes were obtained by stripping the gonads. In mussels, viable larvae can be obtained with variable sperm to egg ratios. The recommended sperm to egg ratio is in the order of 103:1in M. edulis (Sprung and Bayne, 1984) and M . galloprovincialis (Sedan0 et al., 1995). In M . californianus it varies between 25 and 200 sperm per egg, depending on the physiological state of the adults of both sexes (Cherr et al., 1990). In Mercenaria mercenaria the optimum ratio is between 1.3 and 2.5 X 103:1 (Bricelj and Malouf, 1980). Finally, Calabrese (1984) states that polyspermy is to be avoided in Mulinia lateralis, but without speclfying sperm densities. In their study on polyspermy in C. gigas Stephano and Gould (1988) found that, compared to eggs spawned naturally, those obtained by stripping are very susceptible to polyspermy upon immediate fertilization. This susceptibility decreases greatly when the eggs are held in seawater for 90 minutes before fertilization. The authors conclude that stripped eggs lack a “sperm block” present in naturally spawned eggs. Togo et al. (1995) obtained normal (monospermic) fertilization in Mytilus edulis when the eggs were fertilized at a sperm ratio of 5 X 103:1 within 30minutes after spawning. They recognized three mechanisms for blocking polyspermy in mussels. The first depends on a rapid depolarization of the egg’s plasma membrane, the second consists of the suppression of the acrosomal reaction, and the third is the blocking of contact or fusion between the plasma membranes of sperm and egg. 2.3.3.2. Ageing of gametes affer spawning. One of the prerequisites for bivalve embryo and larva bioassays is to begin with the best possible biological material, i.e. with a proportion of normal larvae in the controls as close to 100% as possible. “Oyster eggs undergo aging and lose their ability to be fertilized” (Galtsoff, 1964). Except in Mulinia lateralis (see Section 2.1.6), it is impossible to know in advance to which sex the individual adult belongs. Successful fertilization requires simultaneous spawning of at least one member of each sex; in general, however, one or more males spawn first, and only very rarely does a female spawn before any of the males has done so. According to Galtsoff (1964), concentrated sperm (presumably obtained by stripping) of C. virginica conserves its fertilizing capacity for at least 24 hours at lO”C,whereas diluted sperm at room temperature [probably 18°C to 20°C] loses its capacity within 4 or 5 hours. The number of dividing eggs drops to 60% after 5 hours, and to 20% after 10 hours. In C. gigas the sperm

33

THE ASSESSMENT OF MARINE POLLUTION 6-

I

0

I

20

I

I

40

L

I

I

M)

I

80

I

I

100

Hours from fertilization

Figure 5 The rates of cleavage and early development of embryos of Mytilus edulis at different temperatures. A,8°C (Bayne, 1965); 0, 18°C (Bayne, 1965); X , 20°C (Field, 1922, cited by Bayne, 1976); U, 19-22°C (Rattenbury and Berg, 1954, cited by Bayne, 1976). Stages of development as follows: 1, first polar lobe; 2, first cilia; 3, trochophore; 4,appearance of velar cilia; 5, appearance of shell gland; 6, prodissoconch I. (From Bayne, 1976.)

remain very motile for 1.5 hours after release at temperatures of 21°C to 25°C (Stephano and Gould, 1988);however, "if fertilization was delayed for more than 60 to 90 minutes after gamete liberation, the proportion of larvae which developed was greatly reduced" (Walne and Helm, 1974, p. 1; Helm and Millican, 1977, p. 2). In Mytilus edulis, diluted sperm lose their motility within 1 or 2 hours at room temperature, but maintain their fertilizing capacity for several hours at 15°C;fertilization of eggs is possible after 4 to 6 hours at 18"C, but at 15°C the fertilization rate drops to 40% within 6 to 11 hours after spawning (Sprung and Bayne, 1984). In M. galloprovincialis, fertilization was still possible within 8,7 and 4 hours after spawning at 10°C, 14°C and 18"C, respectively (Sedan0 et al., 1995).

2.3.4. Physical requirements of bivalve larvae The range of environmental conditions over which embryogenesis is possible will depend on the location of the adult population (Loosanoff, 1954; Bayne, 1976). The duration of the embryonal and larval phases depends mainly on temperature (Figure 5, Tables 5 and 6), and also on salinity and other environmental factors, including food availability

Table 6 Embryonic and larval development event times of oviparous marine bivalves. Crassostrea gigas, 25°C (Cahnn, 1950); in parenthesis, 20°C (Tazawa et al., 1985). Crassostrea virginica, 23-25°C (Galtsoff, 1964). Mytilus edulis, 17°C and 2 5 0 ~(Armstrong and Millemann, 1974). Mytilus galloprovincialis, 10-17°C (Aguirre, 1979); in parenthesis, 20°C (Masson, 1975). Mercenaria mercenaria, 22°C (Loosanoff and Davis, 1950). Mulinia lateralis, 20-25°C (Calabrese and modes, 1974).

Developmental stage

Crassostrea gigas

Crassostrea virginica

Mytilus edulis

Fertilized egg 1st polar body 2nd polar body + polar lobe 2-cell 4-cell 8-cell 16-cell 32-cell 64-cell ciliated blastula trochophore veliger

0 min 50-60 min 70 rnin

Omin 25-52 rnin 40-65 min

20 min

100 min

45-72 min 52-120 rnin 55-195 min

(420) 480 min 24h (15) 48 h

390 min 8-9 h 24 h

Omin

Mytilus galloprovincialis

Mercenaria mercenaria

Mulinia lateralis

0 min (80 min)

0 min

Omin

90 min 180-210 min

45 min 90 min

(24 h) 48 h

360 rnin 12h 24-36 h

40 min

65 min 90 min 120 min 150 rnin 180 min 220 min 450 min 16-19 h 40 h

9h 15h

Table 7 Effects of temperature, salinity and combined effects of temperature and salinity on survival and growth of bivalve 1arvae. Salinity (psu)

Temperature ("C) Species

Min.

Max.

Crassostrea gigas

15"

34"

Crassostrea virginica

17.7"

Mytilus edulis

10" 14" 5"

Opt.

Min.

Max.

15

39

30" 30-32.5"

15

15-20'

15 24 15

Opt. 25 19-27 30 2&27

T/S opt.

30"/30 30"/18-35

Mytilus galloprovincialis

22" 20" 20"


20" 12.5" 25-27.5" 2CL25" 10.5-15 25-30" 12.5"

Mulinia lateralis

20"

30"

27.5"

30"

30" 25-27"

20"

Ostrea edulis 17.5"

40 33 40

30-32 25-30 25-30 35 30-35 27

20

30

7

33

32.5 25-30

20

22.5-27

18"/27 20"/25-30 20'135 20°/30 21.5-33'122-31 22.5-27.5"/20-35 20-26'123-32 25"130

References Helm and Millican, 1977 Nell and Holliday, 1988 His et al., 1989 Davis and Calabrese, 1964 Lough, 1975 Bayne, 1965 Lough, 1974 Hrs-Brenko and Calabrese, 1969 His et al., 1989 Hrs-Brenko, 1977 Davis and Calabrese, 1964 Lough, 1975 Calabrese, 1969c Lough, 1975 Morrison and Petrocelli, 1990 Robert et al., 1989 Davis and Calabrese, 1969 Davis and Ansell, 1962

36

E. HIS, R . BEIRAS AND

M. N. L. SEAMAN

(Calabrese, 1969c; Mackie, 1984; Widdows, 1991). The time required to attain the D stage in different species is shown in Table 6. It must be pointed out that the data of Cahnn (1950) are not very accurate, because C. gigas attains the veliger stage within 24 hours, the same as C. virginica, at least at 25°C (see also Loosanoff et al., 1966; Helm and Millican, 1977; Tazawa et al., 1985). Moreover, it should be kept in mind that in bivalves, particularly in oysters, development is not highly synchronous (Stephano and Gould, 1988). The influence of various environmental effects on Crussostrea virginica larvae has been the subject of a modelling study by Dekshenieks et al. (1993). 2.3.4.1. Temperature. Embryos are generally more sensitive to environmental factors than larvae (see Section 4.2.2). Thus with regard to temperature the cleavage stages in M . edulis require a slightly narrower temperature range than the shelled larvae (Bayne, 1976; see Sastry, 1979); similar observations have been made concerning Mercenaria mercenaria (Loosanoff, 1954; Kennedy et al., 1974). In Mytilus galloprovincialis the embryos do not develop normally above 20"C, although this temperature represents the optimum for larval growth (Hrs-Brenko, 1977; His et al., 1989). In general, the rate of development increases with temperature as long as it remains within the range of tolerance (Sastry, 1979). 2.3.4.2. Salinity. As in the case of temperature, development of the eggs requires a narrower salinity range than survival and growth of the larvae (Bayne, 1976,1983). In the case of C. virginica the minimum salinity at which the eggs will develop is determined by the salinity at which the broodstock was kept prior to spawning (Davis, 1958; Davis and Calabrese, 1964). Salinity tolerance also depends on genetic factors (Newkirk et al., 1977; Newkirk, 1978; Widdows, 1991) and on interactions with temperature (Table 7). Studies on the combined effects of temperature and salinity show that, compared to nutrition and temperature, salinity has relatively little effect on larval growth and development. Significant interactions occur only at the extreme limits of the tolerance ranges of temperature and salinity. Within these limits, growth depends on temperature and food (Widdows, 1991). 2.3.4.3. Oxygen. The respiration of bivalve larvae has been studied by Gerdes (1983b), Tazawa et al. (1985) and Hoegh-Guldberg and Manahan (1995) in Crassostrea gigas, by MacInnes and Thurberg (1973), Widdows et al. (1989), and Baker and Mann (l992,1994a,b) in Crassostrea virginica, by Riisgard et al. (1980, 1981), and Wang and Widdows (1991) in Mytilus edulis, and by Morrison (1971) in Mercenaria mercenaria. According to Morrison (1971), hypoxia does not affect larval growth except at oxygen concentrations below 4 mg 1-I. Hyperoxia, on the other hand, negatively affects growth at 13.7 mg 1-I (180% saturation) in larvae


37

of Mercenaria mercenaria (Huntington and Miller, 1989). Settlement was reduced significantly in hypoxic treatments, as compared to normoxic treatments, and no settlement took place in anoxic conditions in Crassostrea virginica larvae (Baker and Mann, 1992, 1994a,b). In mussels, both embryos and early prodissoconch larvae developed and grew normally at Po, values greater than or equal to 3.16 kPa (Wang and Widdows, 1991; see also Widdows, 1991, p. 158). Aeration in the presence of an antibiotic (1 mg 1-' erythromycin) increased larval survival in Crassostrea rhizophorae larvae at high temperature (30"C), but reduced individual growth and total biomass (Lemos et al., 1994). Hoegh-Guldberg and Manahan (1995) found that the conditions to which bivalve larvae are subjected in the small respirometers used in respiration studies negatively affect larval metabolism. From a practical point of view, artificial oxygenation of the small chambers used in these tests is not recommended, because resulting turbulence inhibits larval growth and survival (Helm and Spencer, 1972; see also Loosanoff and Davis, 1963, p. 38). 2.3.4.4. Turbidity. The effects of turbidity on bivalve larvae have been studied by Davis (1960), Robinson (1983), and Huntington and Miller (1989) in Mercenaria mercenaria; by Davis and Hidu (1969b) and Dekshenieks el al. (1993) in C. virginica; by Davis and Hidu (1969b) in Ostrea edulis; and by Seaman et al. (1991, and unpublished data) in Mytilus edulis, M. galloprovincialis, Crassostrea gigas and 0. edulis. Natural turbidity values, up to a few hundred mg 1-' tend to be beneficial to larval growth (excepting polluted sediments), because adsorption and desorption processes on the surface of suspended particles may buffer and stabilize the water (Koke, 1993). In bioassays, however, the water used is usually filtered (mesh of 1pm, or less), and turbidity is not a factor of influence in most experiments (excepting sediment bioassays). 2.3.4.5. p H . The data on pH requirements of larvae are scarce. The pH should not be less than 6.75 for C. virginica, and 7 for Mercenaria mercenaria, and both species are unable to reproduce when the pH remains above 9. M. lateralis requires a pH between 7.25 and 8.25 for reproduction (Calabrese and Davis, 1966,1970; Calabrese, 1970b). Krassoi et al. (1996) maintained the pH between 7.8 and 8.4 in tests on Chlamys asperrima.

2.3.5. Nutritional requirements of bivalve larvae Nutrition plays a decisive role in the development of bivalve larvae, even though they are capable of surviving a few days without food (Loosanoff, 1954; Bayne, 1965; Millar and Scott, 1967; Ukeles, 1975; His and Seaman, 1992). With regard to the combined effects of salinity, temperature and

38

E. HIS, R. BEIRAS AND M.

N. L.

SEAMAN

nutrition, the last has the greatest effect, explaining 85 to 88% of the variance in growth in Ostrea edulis larvae (Robert et al., 1989), 64 to 75% in Mytilus galloprovincialis, and 54 to 70% in Crassostrea gigas (His et al., 1989). A great number of studies have been devoted to the nutrition of bivalve larvae (reviews by Ukeles, 1975; Bayne 1976, 1983; Sastry, 1979; Widdows, 1991; Boidron-Metarion, 1995). As far as bivalve larval bioassays are concerned, nutrition is a topic of paramount importance, because although it is not difficult to maintain adequate temperature and salinity ranges for an experiment, providing adequate food may be more difficult to put into practice. Imai et al. (1950) and Imai and Sakai (1961), for instance, succeeded in rearing larvae of C. gigas to metamorphosis in tanks by feeding them Monas sp., a colourless flagellate, at Milford. However, Loosanoff (1969) was unable to obtain any growth in larvae fed with the strain he received from Imai, and this species is no longer used in larval culture. 2.3.5.1. Phytoplankton. Marine bivalve larvae feed mostly on nanoplankton and are thus termed planktotrophic (Thorson, 1950). Veligers pass through three different trophic stages: the endotrophic period, during which nutrition depends exclusively on vitelline reserves; the mixotrophic period, in which both vitelline reserves and exogenous resources are used; and the exotrophic period with exclusive use of external food sources (Lucas et al., 1986; Boidron-Metairon, 1995). Despite studies with other feeds, it has become clear since the pioneering work by Loosanoff and Davis (1963) and Walne (1963) that phytoplankton is the principal food source for bivalve larvae; no food other than unicellular algae has been found to be entirely satisfactory for bivalve cultures (Webb and Chu, 1981). Food algae for culture of veligers must meet three criteria: adequate size (limited by the diameter of the mouth and oesophagus of the larvae), good nutritional quality and ease of cultivation. Some 50 species of algae have been tested for the purpose but only a dozen are generally used in bivalve larval culture (ChrCtiennot-Dinet et al., 1986). Not only does the nutritional quality vary among different algal species, but food quality can also vary during cultivation, and some may produce substances which are toxic to the larvae (Davis and Chanley, 1955b). For instance, Nannochloris cells may excrete a growth-inhibiting substance in great concentrations during the stationary phase of algal growth (Bayne, 1965). Methods to feed bivalve larvae were developed at Milford by Davis (1953) and Davis and Guillard (1958), and reviewed by Loosanoff and Davis (1963). The food consisted of a mixture of live algal flagellates (Calabrese and Davis, 1966) given at a rate of 0.01 ml packed cell volume per litre culture per day (Hidu, 1965). According to Calabrese


39

(1970a) the algae used were Zsochrysis galbana, Monochrysis Zutheri and ChZoreZZa sp. Veligers are capable of feeding selectively (Davis, 1953). The work of Davis and Guillard (1958) and Walne (1963) has shown that bivalve larvae grow best when they are fed a mixed diet of two or more algal species (see also Bayne, 1983). Their quantitative and qualitative dietary requirements may change in the course of development (Davis and Guillard, 1958 Loosanoff and Davis, 1963). Veligers of Crassostrea virginica, for instance, are incapable of using Chlorella sp. during the first days of life (Babinchak and Ukeles, 1979), although they do utilize them at the age of about 1 week, at a size of 110pm (Loosanoff and Davis, 1963). Oyster larvae of the genus Crassostrea have particularly narrow requirements, and the number of algal species suitable as food for them is limited, Mytilus and Mercenaria are rather tolerant and Ostrea is intermediate. The methods of rearing larvae of Crassostrea gigas most frequently used nowadays follow Walne and Helm (1974), Helm and Millican (1977), and Utting and Spencer (1991). These methods may be used for rearing most species of bivalve larvae used in ecotoxicological bioassays. The best results were obtained by rearing D-larvae without aeration at densities of about 10 per ml in 1 litre of filtered and ultraviolet-sterilized seawater, and feeding them with an algal mixture of Zsochrysis galbana Parke and Chaetoceros calcitrans (Paulsen) Takano (50 cells of each per pl of larval incubation volume). The water is changed every 2 days and larvae up to a size of 120 pm are retained on a 45 pm nylon mesh-based PVC sieve, rinsed with freshly filtered seawater and put back into incubation. After the umbo has developed (usually at the age of 6 to 8 days) the cultivation densities may be reduced to 5000 or 6000 per litre and the feeding regime is changed to 3.3 cells per pl of culture of Tetraselmis suecica (Kylin) Butch plus 33 cells per p1 each of 1. galbana and C. calcitrans (representing approximately equal volumes of all three species of algae). At Arcachon we follow the same methods, but we have found that ultraviolet sterilization of the water is not indispensable, and we use stainless steel sieves which are sterilized at 180°C. 2.3.5.2. Bacteria and microencapsulated diets. Davis (1953), Hidu and Tubiash (1963), Millar and Scott (1967), Mengus (1978) and Prieur (1981), among others, have studied the importance of bacteria for larval nutrition. Widdows (1991, p. 151) concluded “there is little evidence that they play a significant role in meeting the nutritional requirements during larval growth”. Douillet (1993) found that oyster larvae fed on a bacterial strain received only 41% of their carbon requirements. There have also been experiments with inert foods (see Robert and Trintignac, 1997). The first investigations by Ukeles (1975) were not very encouraging. Chu er al. (1982, 1987) have been able to rear larvae of Crassostrea

40


virginica to metamorphosis, but in their experiments only 2 to 25% of the larvae attained the eyed stage, and 2 to 20% achieved metamorphosis. The development of a reliable artificial diet would represent an enormous progress in bivalve larval culture, because it would vastly reduce the time and effort required (Ukeles, 1975), while standardizing feeding of the larvae between laboratories. 2.3.5.3. Dissolved organic matter. Finally, bivalve embryos and larvae are also able to take up dissolved organic substances (review by Widdows, 1991). This may well be beneficial, but Widdows (1991) concludes that there is no evidence that larvae are able to grow and develop solely on dissolved organics. The issue of the uptake of dissolved organic matter is important with regard to bioassays because it may also be a factor in the action of various pollutants.

3. BIOASSAY METHODOLOGY As Calabrese (1984) has pointed out, the various techniques and methods of ecotoxicological investigation with embryos, larvae and adult marine bivalves have resulted from advances in aquaculture. There are various manuals and reviews describing in detail the technical precautions necessary for conducting ecotoxicological tests with seawater (types of material, cleaning and sterilization, preparatory steps, etc.). Recommended literature on bioassays with bivalve larvae includes the work of Woelke (1972), the standards of ASTM (1980, 1989), Calabrese (1984), and more recently Widdows (1993) and Krassoi et al. (1996). The general principle is to eliminate the influence of toxic materials and any type of hazardous conditions, which could invalidate the bioassay, other than the condition under study. A broad theoretical and practical knowledge of larval rearing and larval biology is an important prerequisite for performing embryo and larval bioassays. Valid ecotoxicological studies and proper interpretation of their results presuppose an understanding of the normal development of the test species and a mastery of the technical procedures for optimal laboratory rearing. 3.1. General methods

3.1.1. Seawater quality 3.1.1.1. Natural seawater. One common element for all types of toxicity tests is the need for seawater of excellent quality. The first studies with marine bivalve embryos and larvae were carried out at biological


41

laboratories specialized in shellfish aquaculture and located on bays and estuaries where running seawater was obtained from an area in which oyster populations reproduced naturally. This was notably the case at Milford (Connecticut), where bioassays were pioneered by Davis (1961), Loosanoff and Davis (1963), and Hidu (1965). As pointed out by Loosanoff and Davis (1963) and Woelke (1972), the source of seawater must be located at a site which is not contaminated in any way, and where the salinity is stable year-round (or can be kept stable by pumping water at appropriate periods of the tidal cycle). In addition, materials that might release toxic substances must be scrupulously avoided. Most coastal areas are nowadays increasingly subject to anthropogenic impacts resulting in a deterioration of the “biological quality” of the seawater. One of the first examples was noted by Millar and Scott (1968) at the laboratory of Millport (U.K.), where an unknown toxic substance slowed the growth of Ostrea edulis larvae. Shortly thereafter, Helm (1971, p. 8) remarked that “at Conway, sea water varies considerably in quality both in the short term and seasonally”, and the biological quality of the seawater was improved by adding 1mg 1-l of EDTA (Utting and Helm, 1985). Brereton et al. (1973) and Boyden et al. (1975) showed that the poor growth of Crassostrea gigas larvae and the irregularities in oyster recruitment resulted from the presence of zinc in the water, the source of which was mines in the catchment area that drain into the bay at which the biological station was located. This corroborated the observations of Wilson (1951) and Wilson and Armstong (1961) concerning the variability of the “biological quality” of coastal waters (cf. Section 1.3). The volume of the vessels employed in bioassays has been greatly reduced and can now be as small as 3ml (see 3.2.3), so that large quantities of seawater are no longer required and bioassays are not restricted to laboratories located on the seashore. In addition, seawater may be obtained offshore beyond the region of anthropogenic impact in order to obtain it “from an area known to support a healthy naturally reproducing population of bivalves” (ASTM, 1989, p. 339). Seawater filtration. Following the work of Loosanoff and Davis (1963) and Walne (1966, 1974b), most authors agree that bioassays should be conducted only with filtered seawater sterilized with ultraviolet light (Beaumont and Budd, 1982; Beaumont and Terpes, 1984; ASTM, 1989; Hoare et al., 1995a,b; Krassoi et al., 1996). The porosity of the filter is usually equal to, or less than, 1 pm. The ASTM proposes 0.45 pm, but many authors prefer 0.2 pm (Utting and Spencer, 1991; Ringwood, 1991; Hoare et al., 1995a,b; studies by His and coworkers at Arcachon). Filters of 0.1 pm have been used as well (Lemos et al., 1994). Simple filtration systems may prove equally useful. For example, Armstrong and

42


M. N. L. SEAMAN

Millemann (1974) were able to cultivate various larval stages of Mytilus edulis in seawater which had merely been passed through a sand filter. In contrast to almost all other workers, Garland et al. (1986) found that 0.2 pm filtered seawater led to poor growth in bivalve larvae. lbenty years of experience with bivalve embryos and larvae of Crassostrea gigas, Ostrea edulis and Mytilus galloprovincialis at Arcachon have shown clearly, however, that 0.2 pm Htration of seawater is perfectly adequate for toxicity bioassays of 24 or 48 hours duration, as well as for tests of larval growth over periods of several weeks, and for studies of metamorphosis, as long as the seawater is obtained from adequate sites. Filtration of the water should be carried out immediately prior to the incubation, no more than 1 or 2 hours before the test is initiated. In the case of monitoring of in situ water quality, on the other hand, “ideally, filtration is not recommended, because it may remove some of the dissolved contaminants” (Thain, 1991, p. 4), and because it will definitely remove fine suspended particles to which pollutants are likely to adsorb. In such cases good results can be obtained by simply sieving the water through a 32 pm stainless steel sieve in order to eliminate the largest suspended particles and possible predators of the larvae (His and Beiras, 1995). Bacteria and antibiotics. Because the comparatively small volume of the incubation vessels (usually 1 litre at most) favours bacterial growth, and the bacterial populations eliminated by filtration are rapidly reconstituted (Walne, 1958; Lemos et al., 1994), many authors have added antibiotics to the seawater even after filtration and ultraviolet sterilization (cf. Davis and Chanley, 1955b; Walne, 1958). Hidu and Tubiash (1963) recommended the systematic addition of antibiotics (dihydrostreptomycin-streptomycin sulphate = Combistrep at 2 ppm) in larval cultures of Crassostrea virginica and Merceaaria mercenaria. They thought (p. 25, op. cit.), “an antibioticinduced bacterial flora. . . may be utilized by larvae as a food source” (see also Section 2.3.5.2). Similarly, Calabrese and Davis (1967, p. 12) used sulfamethazine soluble powder at 0.33 ppm, Brereton et al. (1973) added 0.3g1-1 of penicillin to the seawater, and Lemos et al. (1994) added 1mg 1-’ of erythromycin. The latter, however, found that although growth is thus improved by antibiotics in Crassostrea rhizophorae cultured at high temperatures and low salinity (30°C and 12 to 20 psu), this is later followed by increases in mortality. Chloramphenicol (5 mg 1-1 of 1pm filtered seawater) was used in larval cultures of Spisula solidissima by Thurberg et al. (1975). Le Pennec and coworkers (Le Pennec and Prieur, 1972; Le Pennec et al., 1973) have shown that antibiotics (aureomycin, erythromycin, chloramphenicol and sulfamethazine) may influence the metabolism and growth of bivalve larvae. This may affect the accuracy of


43

bioassays designed to test a pollutant’s possible reduction of larval growth. Although Woelke (1966) stated that the seawater should be used within 3 hours of sampling, we have obtained excellent results at Arcachon with seawater sampled offshore and stocked for 24 hours at ambient temperature (18°C to 19OC), but filtered immediately before use (see above). On the other hand, Thain (1991) apparently did not obtain good results with seawater sampled offshore during winter and stocked at -18°C in acid-washed bottles, as he states that “even 60 percent abnormalities - in controls - is acceptable” (Thain, 1991, p. 7). We shall see below that most workers do not accept such high levels of abnormalities in the controls. Klockner et al. (1985) also encountered high levels of abnormality in the controls when they reared larvae in “aged (4years, filtered - Seitz K 15) and pasteurized (8OOC for 1 h) sea water” (p. 2). 3.1.1.2. Artificial seawater. Seawater quality problems can be overcome with artificial seawater made by dissolving the mineral constituents of oceanic water in distilled water. This also prevents the complex interactions between pollutants and organic matter that may affect toxicity. Contrary to the opinion of Thain (1991), artscial seawater is perfectly suited for bivalve embryo bioassays (e.g. Krassoi, 1995; His et al., 1997a). The seawater formula of Zaroogian et al. (1979), used successfully by Calabrese et al. (1973), Calabrese and Nelson (1974), Utting and Helm (1985) and recommended by ASTM (1989), gives excellent results in embryo-larva bioassays of 24 hours duration in Crassostrea gigas, or 48 hours in Mytilus galloprovincialis. It may be prepared using reagent-grade chemicals and deionized water aerated before conducting the test. EDTA, which chelates metals and some pollutants, may be omitted (His et al., 1997b, p. 352). Besides Zaroogian’s formulation, various other seawater media have been used to rear bivalve larvae (Table 8), and Krassoi (1995) has recently used several different formulations to grow larvae of Chlamys asperrima and Saccostrea commercialis. Kester et al. (1967), who proposed a modification of the formula by Lyman and Fleming, stressed that artificial seawater should be made up with reagents of known composition; this is not always the case, as the composition of the salt used by Courtright ef al. (1971) is unknown. The formula used by Chang et al. (1996) is a simplification of the medium of Zaroogian et al. (1979), containing only six mineral salts (NaC1, KC1, CaCl,, MgCl,, MgS04 and NaHCO,), instead of eleven. 3.1.2. Broodstock Spawners in good physiological condition are indispensable; they must be able to mature in an unpolluted environment and in excellent nutritional

Table 8 Artificial seawater (ASW) formulations used for the rearing of bivalve larvae.

Artificial seawater Lyman and Fleming, in Sverdrup et al. (1949) Wood (1961) Zaroogian et al. (1969)

Leslie coarse hide salt Instant Ocean sea salt mixture Instant Ocean Aquarium Systems, Mentor OH Different ASW formulations

Bivalve species Ostrea edulis 0. edulis Crassostrea virginica Mercenaria mercenaria Crassostrea gigas C. gigas Mytilus galloprovincialis C. virginica Mytilus edulis C. virginica C. gigas C. virginica Chlamys asperrima Saccostrea commercialis

Reference Helm (1971) Millar and Scott (1968) Calabrese et al. (1973) Calabrese and Nelson (1974) Utting and Helm (1985) His et al. (1997a) His et al. (1997a) La Roche et al. (1970) Courtright et al. (1971) Sigler and Leibovitz (1982) Chang et al. (1996) Chang et al. (1996) Krassoi (1995) Krassoi et al. (1996)

Remarks Rearing Rearing Bioassay Bioassay Rearing Bioassay Bioassay Bioassay Bioassay Bioassay Bioassay Bioassay Bioassay Bioassay


45

conditions, whether in the field or in the laboratory (ASTM, 1980, 1989; Krassoi et al., 1996). The best results are usually obtained during the regular spawning season by using adults from areas where bivalves are cultivated (see Section 2.2). On the other hand, when bioassays need to be conducted out of season, ripe adults may be obtained by conditioning techniques (see Section 2.3.1). Most commercial hatcheries are capable of supplying mature oysters, Crassostrea spp., which are the ones used most extensively in bioassays (see Table l ) , to laboratories lacking the facilities for conditioning bivalves. A word of caution is in order, however, because the adults from commercial hatcheries are sometimes overripe, i.e. they have been conditioned for too long without spawning. “. . . Oysters resorbing their unspawned products . . . result in poor bioassay material” (Woelke, 1972, p. 31), with abnormality levels near, or above 20% in the controls, thus generating results of questionable validity (see Section 3.1.3.2). As noted by Eertman et al. (1993, p. 38) in the case of C. gigas embryos and larvae, “despite the oysters being conditioned for spawning, the autumn and winter periods seem less suitable for performing toxicity tests” as larval abnormalities reached 40% in October and March. Moreover, many hatcheries clean out their facilities in autumn and winter and are therefore unable to provide spawners during this time. If the spawners are subjected to unfavourable environmental conditions, this will affect the quality of the gametes and influence the results of the test (Widdows, 1993; see Section 2.2.2). In Mytilus rrossulus and Mizuhopecten yessoensis, exposure of the adults to sublethal doses of heavy metals (Cu and Zn) prevents the formation of gametes capable of normal embryonic development (Karaseva and Medvedeva, 1993). On the other hand, adaptive mechanisms such as the formation of metalloproteins have also been found, particularly in the case of cadmium (see Webb, 1979), and these metalloproteins are later found in the larvae upon their exposure to the contaminant (e.g. Pavicic et at., 1984, 1994a,b; Roesijadi et al., 1996, 1997; Ringwood and Brouwer, 1995). Similarly, Hoare et al. (1995a,b) found that Mytilus edulis embryos obtained from spawners originating from a site polluted by heavy metals had higher tolerance to copper than those spawned by adults from a non-polluted area; tolerance appeared to be “maternally determined”. There have been attempts to cryopreserve bivalve larvae in order to dispense with the need to obtain mature adults, but little progress has been made since the first experiments by Lannan (1971; see also Renard, 1991). It has usually not been possible to obtain adequate percentages of normal D-larvae (80% or higher) from cryopreserved gametes. Nevertheless, McFadzen (1992), Cleary et al. (1993) and McFadzen and Cleary (1994) have performed bioassays with veligers of C. gigas cryopreserved 24 hours

46


M. N . L. SEAMAN

after fertilization, and of Tapes philippinarum 48 hours old. Contrary to the authors’ contention, it is not at all certain that 48-hour cryopreserved larvae are more sensitive as test organisms than the embryonic stages favoured by most other workers. More recently, Chao et al. (1997) have proposed the use of C. gigas and Meretrix lusoria cryopreserved late embryos and early larvae in aquaculture management programmes on the basis of survival rates from 62 to 84%. Future progress may yet facilitate the development of routine bioassays with cryopreserved material, making it possible, for example, to exchange genetically homogeneous material between laboratories. At present, however, cryopreservation techniques are not sufficientlyreliable and widespread for routine employment. We agree with Widdows (1993, p. 153), that the use of cryopreserved D-larvae “still remains to be fully evaluated”. 3.1.3. Spawning and fertilization Spawners must be used within 24 hours after being obtained from the field or the hatchery; they need to be carefully cleaned of fouling organisms and scrubbed in seawater. Two-year-old animals are preferable in the case of oysters, in order to have a fair chance of obtaining both males and females (see Section 2.2.1). 3.1.3.1. Induction of spawning. Gametes may be obtained either by stimulating the animals to spawn, or by “stripping” the gonad. The former method, recommended by Woelke (1972), has been adopted by ASTM (1989) in Crassostrea gigas, C. virginica, Mytifus spp. and Mercenaria mercenaria. Mature adults are placed in 1 litre beakers with filtered seawater and stimulated to spawn by varying the temperature. According to ASTM (1989, Table 2, p. 341) the temperature may be raised by 5°C to 10°C above the temperature of conditioning, but it is not supposed to exceed 20°C for mussels. On the other hand, bivalves may be subjected in the field to temperatures which fluctuate from more than 30°C (at low tide and strong solar irradiation) to 20°C at high tide. His and Beiras (1995) and His et al. (1997a) have found that 30 minute periods at temperatures alternating between 18°C and 28°C serve very well to induce spawning in both C. gigas and Mytilus galloprovincialis. Dixon and Prosser (1987) induced M . edufis to spawn at 30”C, and C. Bittkau (personal communication) obtained healthy gametes of the same species from the Baltic in winter (ambient temperature 5°C) after exposing them to fluctuations between 18°C and 28°C immediately after field sampling. An additional spawning stimulus may be provided by adding a suspension of sperm in filtered seawater to the beakers containing the mature adults (obtained by stripping the gonads of a male). To avoid


47

undesired fertilizations in such a case, it is also possible to use a suspension of previously frozen sperm which has been brought to ambient temperature. Ringwood (1992b) used heat-killed sperm to stimulate spawning in Zsognomon californicum. 3.1.3.2. Stripping the gonad. With regard to the production of gametes by stripping the gonad, Allen et al. (1988, p. 7) states, “during our research, we investigated the conventional wisdom that stripped eggs of Pacific oysters are inferior to naturally spawned ones. We found nothing to support that belief”. Alliegro and Wright (1983) stress that stripping avoids the often tedious induction of spawning by other methods, which may take hours. According to Thain (1991), there is no important difference between the use of larvae obtained by stripping or by natural spawning in the conduction of ecotoxicological bioassays. Most other authors, however, disagree (Woelke, 1961, 1966; Loosanoff and Davis, 1963; Loosanoff, 1965b, 1969; Wilson, 1981; ASTM, 1989; Widdows, 1993). Although stripping of mature females can provide some uninjured ripe eggs that can be fertilized and develop normally, among the eggs obtained by induced spawning there are always considerably fewer abnormal ones than among eggs obtained by stripping (Loosanoff and Davis, 1963). As early as 1961, Woelke stated that “this practice [stripping] was dropped” because of “the frequency with which unsatisfactory results were achieved, apparently due to immature eggs” (Woelke, 1961, p. 115). Moreover, in Mercenaria mercenaria it is not possible to fertilize stripped eggs until they have been treated by a weak ammonia solution “to cause the germinal vesicle to break and the eggs become physically prepared for fertilization” (Loosanoff, 1969, p. 21). In Mytilus edulis “attempts to obtain mature (fertilizable) eggs by removing the ovary of apparently ripe females have met with unsatisfactory results, because it is only during their stay in the ovary, and during and following the act of spawning, that the ova become physiologically mature” (Longo and Anderson, 1969a, p. 73). In C. gigas, spawned oocytes are considerably less susceptible to polyspermy (fertilization of one egg by more than one sperm) than oocytes mechanically removed from the ovary (Stephano and Gould, 1988; Konar and Stephenson, 1995). In short, we agree with ASTM, that “use of eggs stripped from female bivalves is not recommended because it often results in an excess of poorly developed and malformed embryos” (ASTM, 1989, p. 343). 3.1.3.3. Number of parents. Opinions differ with regard to the number of parents whose spawn should be used in bioassays. Thain (1991) states that, for Crassostrea gigas, after stripping the gonads, “three batches of eggs are pooled and two batches of sperm are also pooled”. Johnson (1988), in Mytilus edulis, and Krassoi et al. (1996) in Chlamys

48


asperrima mixed the gametes of three males and three females; Beaumont et al. (1987) also preferred to use gametes of several spawners, both in M. edulis and Pecten maximus, “in order to avoid working with the very restricted genetic variation inherent in full or half sibs” (p. 300), as did Spangenberg and Cherr (1996) in M. californianus and Mercenaria mercenaria, and Laughlin et al. (1988, 1989) in M. mercenaria. Cherr et al. (1990) conducted separate fertilizations of several females of Mytilus californianus with sperm of two or three males, and Roberts (1987), in Crassostrea virginica and Mercenaria mercenaria, fertilized the eggs of several females with the sperm of one or several males. Finally, Ringwood (1991, 1992a,b) mixed the gametes of several males and females in Zsognomon californicum. ASTM (1980) recommended using gametes from two individuals of each sex in order to obtain a genetic mix of embryos. In contrast, ASTM (1989) recommends in order to verify differences in sensitivity between parental couples “ideally, the test should be conducted by subjecting progeny from each of at least three individual male-female pairings to each of the one or more control treatments” (p. 242). Woelke (1972) and Chapman et al. (1992) in Crassostrea gigas, and Granmo et al. (1988, 1989) in Mytilus edulis, recommended the use of a single male and a single female in a given bioassay; the same approach was chosen by Butler et al. (1990) in M. edulis: “eggs and sperm from two individuals were selected to give a single pairing, to minimize genetic variability in the test embryos” (p. 214). 3.1.3.4. Elimination of impurities in the spawn. Loosanoff and Davis (1963) recommended sieving the gametes to eliminate tissue fragments emitted by the bivalves along with the gametes. Butler et al. (1990) passed the gametes through a 1OOpm mesh, and Thain (1991) through 90pm. ASTM (1989) recommends a mesh of 75pm for the eggs and 37 pm for the sperm, the fertilized eggs being retained on a sieve of 37 pm for rinsing and counting. Krassoi et al. (1996) retained the eggs on a sieve of 45 pm, after passing them through a mesh of 100 pm. Granmo (1972), on the other hand, rinsed the eggs three times, allowing them to decant between rinsings, prior to fertilization. Spangenberg and Cherr (1996) simply rinsed the eggs in 0.45 pm filtered seawater after fertilization, and Ringwood (1992a,b) eliminated tissue debris by rinsing and gentle centrifugation. His and Beiras (1995) and His et al. (1997a) obtained excellent results by passing the eggs and sperm through sieves of 100 p m and 32 pm respectively, before proceeding to fertilize, without further rinsing or sieving after fertilization. 3.1.3.5. Counting the eggs. After spawning, and sieving or rinsing, the first step in a bioassay is to count the eggs. The method proposed for


49

this purpose by Loosanoff and Davis (1963) has been adopted by most of the workers in the field (e.g. Woelke, 1972). It consists in placing the fertilized eggs in a graduated cylinder of 1 litre volume filled with filtered seawater and gently mixing the water column with a “plunger constructed by drilling holes in a disc of acrylic plastic or fibreglass of suitable diameter and attaching it to a PVC or acrylic rod of suitable length” (ASTM, 1989, p. 343; see Figure 7 in Woelke, 1972). Several samples are taken by pipette and counted under the microscope in a haematocytometer or well slide. A sample (usually 100 p1, or less) should consist of about 50 eggs, and, as the number of eggs in the cylinder is initially unknown, it may be necessary to adjust the volume of the samples accordingly, or to take a large sample from the graduated cylinder and dilute it according to need. The total number of eggs is computed by extrapolating the average number of eggs in the samples to the volume of the cylinder. 3.1.3.6. Fertilization. The sperm suspension should be obtained at approximately the same time as the eggs are collected: “Fresh actively-moving sperm are used to assure a normal fertilization of eggs” (Loosanoff and Davis, 1963, p. 35). To obtain a concentrated suspension, males should be made to spawn in the smallest possible volume of filtered seawater; some authors propose to count the sperm with a haematocytometer (Cherr et al., 1990; Ringwood, 1992b; Konar and Stephenson, 1995; Krassoi et al., 1996), particle counter (Thain, 1991), or by “turbidimeter readings previously calibrated with a hematocytometer” (Eyster and Morse, 1984, p. 642). ASTM (1989) admits that “precise sperm counts are unnecessary after one gains a little experience” (p. 343), even though optimum fertilization depends on the ratio of eggs to sperm (Section 2.3.3.1). This differs between species (and between authors), and it is also strongly influenced by gamete quality (potency of the sperm and receptiveness of the eggs, which are difficult to appraise). Most authors add a few millilitres of dense, freshly spawned sperm suspension and verify fertilization success under the microscope; 5 to 10 sperm (24 at most) should be visibly attached to each egg membrane (Konar and Stephenson, 1995; His and Beiras, 1995; His et al., 1997a). Because the biological quality of the gametes deteriorates rapidly after spawning (Section 2.3.3.2), fertilizations should preferably be undertaken within 30 minutes. After eggs and sperm have been united in one container, the actual fertilization takes place within minutes and the polar bodies are expelled within half an hour (Allen et al., 1988; see also Section 2.2.3). As a rule, fertilization of the gametes for bioassays should be undertaken as soon as the first females in the batch have spawned.

E. HIS, R. BEIRAS AND M. N.

L. SEAMAN

3.2. Bioassay procedures In assays with bivalve embryos and larvae, we may distinguish between acute toxicity tests, when response is recorded after short-term exposure (a few days or hours), and chronic toxicity tests, when the exposure period covers the majority of the larval life span (several weeks). The former tests frequently record embryogenesis success or larval mortality as the response, whereas the latter record sub-lethal responses, usually larval growth. Both kinds of tests are complementary. Acute toxicity tests are easier to standardize and provide a rapid assessment, whereas chronic toxicity tests are more sensitive and better approximate the environmental conditions. The execution of longer-term sub-lethal tests requires perfect capabilities in larval rearing - particularly with regard to the culture of food algae - as well as the availability of relatively sophisticated equipment, including a unit for the cultivation of algae. Sub-lethal tests cannot, therefore, be performed as a routine method in most marine biological laboratories, yet they remain an integral part of more detailed studies on particular chemicals or environmental hot spots. The first step in all bioassays - whether they are acute tests with embryos, chronic exposure studies lasting several days with larvae, or investigations on pediveligers and toxic effects on metamorphosis - is the production of gametes, embryos and D-larvae of excellent quality and the elimination of all artefacts resulting from any type of deficiency in the biological material. We have already reviewed the methods to obtain mature adults, induce spawning, and perform in vitro fertilization in Section 2.3. The principal causes of failure in bioassays with Chlamys asperrima have been summarized by Krassoi et al. (1996, Table 7). These include loss of broodstock, spawning of broodstock in holding tanks, poor spawning, poor fertilization success, low percentage of normal larval development (loo >loo >loo >loo

>loo

Martin ef al.. 1981; Cu,Zn and Ni sulphates; abnormal larvae excluded Wathng, 1981; *calculated by

160* 130* 13

1100 >loo >1w >loo >loo >loo >loo >loo

>1W >100

His and Robert, 1981; abnormal larvae

5.3

119

349

758

611

326

4538

excluded

larvae excluded Coghanese, 1982; Ag nitrate; abnormal larvae excluded

218 5-6.5 W13.5 5 4 . 5

>so 44

1&100

24 b, 24°C 24 d-', 0.2 pm FSW

13

42-48 h, 26°C 15-17 ml-', ASW 44.48 h, 26psu. 20-22d-', 1 pm FSW uv sterilized 48h, 20. 25, 30°C 22.5, 27.5psu, 1 pm FSW

5.6

5.8

103

310

20°C 11.4

24.2 35.3 32.2

15.1 18.7 18.3

206 325 230

25'C 12.6 30°C 10.2

1&20

extrapolation.

His and Robert, 1982; sulphate; abnormal

10

SPP.)

C. gigas

Okubo and Okubo. 1%2; Cu and Zn sulphates Brereton d al., 1973: most larvae abnormal from Zn 150. Glickstem, 1978; abnormal larvae excluded Se decreases Hg toxicity; *nitrate. Cardwell ef ul., 1979a; all sulphate; 'fully shelled regardless of shape.

5w1000

1180 2450 3800 7500* 10300

Robert and His, 1985; abnormal larvae excluded Zhadan ef al., 1992; approximate ranges; sulphates; abnormal larvae excluded Beuas and His,1994; abnormal larvae excluded 16000 Calabrese ef al., 1973; *arsenite MacInnes and Calabrese. 1978; abnormal larvae excluded MacInnes and Catabrese. 1979; 220 at 2% 27.5 psu; abnormal larvae excluded

48h, 25psu. 60ml-, FSW sterilized

C. iredalei

fertilized egg

M. edulis

fertilized egg

1?-17"C

M. edulis

fertilized egg

M. edulrr


fertilized egg (2 h) fertilized egg (0.5-2 h) fertilized egg

48 b, 17°C. 2 4 2 8 ml-l,1 pm FSW uv sterilized 48 h, 19°C. 26psu, 151&', 5 pm FSW uv sterilized 2 d. W C , 35 psu, 30 I&'


fertilized egg

M.galloprovincialk

fertilized egg

M. mercenaria

fertilized egg

M. galloprovmcialis

S.soltdissimo

I. califomicum

fertllued egg (1h) fertilized egg (2 h) fertilized egg (4 h) fertilized egg (15min) sperm + egg (1 h later)

I. califomicum

fertilized egg

M. lateralis

S. plano I. californicum

(M Chlamys asperrima Chlamys aspemma

fertilized egg (>I h) fertilized egg (30 min)

10-32 5.8

14

32-100 3.2-10* 5.8

24

3.5

891

476

13'7

us*

4426 4743**

145

4055

Hrs-Brenko el al., 1977; abnormal larvae excluded Pavicic. 1980: Z n sulohate. *60h at 16°C; "60h at 1 6 k abnormal larvae excluded Pavicic er al... 1994a:. abnormal larvae excluded Beiras and Hi$ 1995a; abnormal larvae excluded Calabrese and Nelson, 1974

10 4.8

21

166

310

780

Eyster and Morse, 19W, abnormal larvae excluded

6.4-9.5 c. 16

48 h, 21°C. 10, 3Opsu, 75 I&'

18,17

48 h, ZOS'C, 24psu. 25 I&', 0.45 pm FSW 48 h, 2 4 T, 34psu (24psu), 2 ml-', 0.22 wm FSW 2 h. temnerature not eiven. 34psd 200 I&', 0.8 FSW 48 h, temperature not given, 0.45 pm 34psu, 17 d-', FSW 48 h, 18°C 33psu, 30ml-', 1pm FSW uv sterilized 48h, 18'C, 33psu, U)ml-', 5 sm FSW uv sterilized

10-20

&

320loo0 175

250-500

48 h, 20"C, 37-38psu 48 h, 20°C 37-38 psu, 3 W m1-l. marselv FSW 48 h, 20°C, ml-', 0:2 pm FSW 42-48 h, 26"C, 15-17 m1-I. ASW 48 h, 20"C, 3Opsu, 30 ml-l, 0.22 pm FSW uv sterilized

Ramachandran er al., 1997; Cu sulphate; abnormal larvae excluded 3 2 W >loo00 Okubo and Okuho. 1962; Cu,Zn and too00 Cr sulphate; *acetate. 1200 >3000 4469 Martin el al., 1981; Cu,Zn and Ni sulphate; abnormal larvae excluded 30000 Morgan er al., 1986. 459

81

Momson and Petrocelli, 1990. sulphate. Ruiz el 111.. 1995~:nitrate; abnormal larvae excluded, control embryogenesis success loo

>loo

>lW

85 1100 75 >lo0

mortality

>lW >loo

C cucullata

3d,75pm 13d, 254 pm 24 h

60 >1M) 2447

>loo

c.gigas

1d,Mpm 211 pm 3 1 0 ~

C. virginica

2d

C. virginica

2d

C virginicn

48 h, 70 mn

0. edulis

1-3 d

12 d. 25°C. 10-12 I&', mix algal food, 1pm FSW 48h, 17.5.22.5,27.Jpsu, 20 d-'mix , algal food, 1pmFSW 8 d. 26°C. 27-28 psu, 20 I&', monoalgal food, 1 pm carbon FSW uv sterilized 4.2 h, 15°C0.8 ID-',no food

Cr

Referenceandnotes

Watling, 1978

200

60 >loo

monoalgal food. 0.8pm FSW 48 h, 24°C 24 I&', no food, 0.2 pm FSW

Cd

Boyden er d., 1975; sulpbate

mortality

3d,80pm 13d, 239 pm

5 d, 26°C. 28psu. 8 DIP,

Pb

>500

mortality

C margaritam

C gigas

Ni

mortality mortality (tissue degeneration)

m

mortality

12.0

Watling, 1982

80

>loo

>lo0

His and Robert, 1981 1994 & Beiras and €I

33

115 25.0

32.8

1200

Calabrese el a/., 1977a

>90 60-90

mortality

Macnes and Calabrese, 1979: *3060 at 1 7 . 5 salinity ~ ~ ~

&!XI*

mortality

mortality (no response touching with needle)

>86

3.3

227

Wikfors and Ukeles 1982

Connor, 1972

M. edulis

3d

15 d, 15% 32psu. 5 ID-'.mix algal food, 0.2 pm FSW

M. edulis

150 pm

10d, 1SoCc, 32psu, l o r n - ' , monoalgal food

M.galloprovincialis 2 d. 76 um 141 pm 225 pm 258 p m

48 h, 20"C, 24 m1-l. no food, 0.2 rn FSW

mortality (tissue degeneration) mortality

M. rnercenaria

2d

b 1 0 d. 25°C. 10-12 ID-',mix algal food, 1 pm FSW

I. californicurn

3d 10 d 24 d 36 d

48 h. 24'C. 34psu ( 2 4 ~ s ~ ) . mortality 1ID-],no fbod,'O.ZZ pm FSW

P mnxirnus

3d

C gigas

2 d, 50-70 pm

C. gigas

5d,88pm 16 d, 260 pm

15 d, 15°C. 32 psu. 5 ml-'. mix mortality (inactive algal food, 0.2 pm FSW oesophageal cilia) growth (length) 5 d + 5 d recovery, 26"C, 5 ID-',mix algal food, 5 pro FSW uv sterilized 7 d , 2 5 T , 2.5 d-' growth (length) (young), 5 d, 23°C 1.5 ID-' (old), mix algal food, 5 pm FSW growth (length) 4 d, 22-23", 2.5 ID-' (young) 1.5ml-l (old), mix algal food, 5 pm FSW

C. gigus

6 d, 135 pm

C cucullnra

51 164 322 383 14.7 32.4

Stromgren and Nielsen. 1991; sulphate; silicon-coated culture jars Beiras and His,1995a

16.4

195

Calahrese e t a / . , 1977a

5700

Ringwood, 1990.results at 24psu salinity in brackets

100

Beaumont er 01.. 1987 Brereton er a/., 1973; sulphate

-150

>1M)

75 120

3d,@Jw 13d, 239 pm

35 85

45

40

85

1W

3 d, 75 pm 13 d, 254 pm

40 85

50

45 120

egg 24 h

C. gigas

egg 24 h fertilized egg

7 d, 26°C 28psu. 6 ml-', growth (height) monoalgal food, 0.8 pm FSW mix growth (height) 12 d, 24% 32psu. 8 d-', algal food, 0.2 pm FSW 10d, 24°C 20,25,30psu, growth (height) 8 mix algal food, 0.2 pm FSW

I&',

Watling, 1978

1W7.00 80

C. gigas

C gigas

10

50 75

16d,309~ C margariracee

Beaumont er al., 1981

2W500

mortality (inactive oesophageal cilia) mortality

95

85

Watling, 1982

-12 12-24

His and Robert, 1981

613 -64

His and Robert, 1982: sulphate Robert and His, 1985

250

~~

~

Exposure wnditions (time, temperature, salinity, density, food, seawater)

Initial age/size

Test species

LC, or ECx (pg metal ion I-') End-point

Hg

c. gigar

ld,60&m

10 d, 24°C 8 I&', mix algal food 0.2 pm FSW

growth (height)

7.1

C virginica

2d

growth (length)

11.8

C. virginica

48 h, 70 ~ L I J

, 12 d, 25°C 10-12 d-'mix algal food, 1f l FSW 8 d, 26'C. 27-28psu. 20 ml-', monoalgal food, 1 pm carbon FSW uv sterilized

growth (length)

0.edulis

179,181pm

%h

3d

M. edulis

150 m

15 d, 15% 32psu. 5 d-l,mix growth (height) algal food 0.2 ~ L IFSW J 10d, 15°C32psu. lOml-', growth (length) monoalgal food

M.mercenaria

2d

b 1 0 d, 25°C. 10-12 d-'mix , algal food, 1pm FSW

growth (length)

21.4

fl maximur

3d

15 d. 15°C 3 2 ~ s5~d.-'. mix growth (height) algal food, 0.2 pm FSW

1. californicum

3d.85~1~

28 d, 24°C 34psu. l O d - ' , mix algal food, 0.45 pm FSW 4 d. temperature not given, 34 psu. 10 d-', mix algal food, 0.45 pm F'SW

growth (dry weight) (tissue dry weight)

72 h. 2C-22pC. 34psu. Mml-', 1~ L I FSW J

Ca uptake

5 d, 21°C. 1ml-', mix algal

settlement

21 d, 250-320 eyed

~LIJ,

food. 2-5 sterilized

~ L I FSW J uv

Cd

Cr

Referenceandnotes

30.2

47.1

Calabrese ei al., 1977a

1078 >27

Wikfors and Ukeles 1982

Stromgreu and Nielsen, 1991: sulphate; siliwnsoated culture jan 7.4

C. gigas

Pb

Walne. 1970

growth (height)

9d, 118pm

Ni

Beaumont et al., 1981

10d, 20°C. 8 d-'mix . algal food, 0.2 pm FSW

M. laiernlir

Zn

>86

M. galloprovincialis 2d,75fim

3d

Cu

Beiras and His 1994

growth (length)

M. edulis

I. californicum

Ag

Beiras and His 1995a 42.2

16.9

232

20

Ringwood, 1992a

-20 -100

>20

growth

Ringwood, 1992b

(dry weight) 26.5

18.5

176 500'*

Ho and Zuhkoff, 1982 Boyden et al., 1975; sulphate; ' 5 d continuous exposure; **5 d exposure C5 d rewvery in clean FSW; at Z n 125 and 250 no marked reduction in settler number but 2 d delayed settlement

C. gigas

16 d

C. gigas

19 d

c. gigas

2290 pm, eyed

C. gigas

2280. eyed

C. virginica

2230, eyed

C virginica

eyed

6 d exposure f 3 d recovery, 22-u"C 1.5 ml-'. mix algal food, 5 pm FSW 20 d

3445

settlement*

1&20*

idem

4 d, 24T, 11 n f ' , no food, 0.2 pm FSW, eplnephrine lO-'mol. added d 2 4 d, 37 psu, ASW, epinephrine mol. added at different tunes

metamorphosis* metamorphosis

c. 1wo

c. loo0

4 d, 25 psu, ASW, mol. epinephrine added at different times 20 I&', flowing 0.5 p m FSW

metamorphosis

c. 500

c. 5M)

settlement (4 h) +spat mortality (4-7d)*

534 ppm**

ASW, artificial seawater; FSW, filtered seawater.

2&25

3c-35

3244

1&20 1&20*

1c-20

Watling, 1983; *fixation to bard substrate 1&20

Watling, 1983 (only approximate figures given); '1 d delayed settlement Beiras and Hi$1994 *adulr shell formation G a n g ef ul., 1996: nominal concentrations; estimated actual concentrations 2 orders of magnitude lower for cu

Phelps and Mihursky, 1986: *fan-shaped non-empty shells; **Cuconcentration in the setting surface micro-organism laver; nitrate

94


n = 16); nickel, 683 (2425, n = 4); lead, 968 (2847, n = 5); cadmium, 2219 (23312, n = 18); arsenic 3913 (25073, n = 2); chromium, 4564 (23663, n = 6); and manganese, 23 000 (*9899, n = 2). In the case of copper toxicity the estimation of the EC5,, is much lower and more precise if the EC5o values extrapolated from Watling's (1981) data are considered as outliers and disregarded: 24 2 28 (n = 24). According to their embryotoxicity, metals can be classified into three groups. Dissolved mercury, silver and copper are toxic at the 10ppb (i.e. lo-*) level, zinc, nickel and lead are usually toxic at levels from 100 to 1000ppb to and cadmium, arsenic and chromium often show embryotoxicity to level only. Other bivalves at concentrations above the 1 ppm elements occasionally tested and found virtually non-toxic are selenium (EC50 > 10ppm), iron (ECSO> 10 ppm), strontium (ECS0> 50 ppm), and molybdenum (EC50 > 50 ppm) (Okubo and Okubo, 1962; Glickstein, 1978; Martin et al., 1981; Morgan et al., 1986; Spangenberg and Cherr, 1996). Data on aluminium toxicity (not included in Table 9) to bivalve embryos are scarce and contradictory. Calabrese et al. (1973) found no effects on embryogenesis at concentrations up to 7500 pg A1 1-', while recently Pagano et al. (1996) found toxic effects between 54 and 162 pg 1-', and Wilson and Hyne (1997) report ECSovalues of about 200 pg 1-l. In all three cases pH was within the limits of tolerance for bivalve embryos and larvae (27, Calabrese and Davis, 1966,Wilson and Hyne, 1997). However, Calabrese et al. (1973) used aluminium chloride, whereas Pagano et al. (1996) and Wilson and Hyne (1997) used aluminium sulphate; the differences in toxicity may therefore be a result of differences in speciation (see Section 4.3) of the salts in seawater. 4.1.1.2. Larval mortality and growth. The lethal and effective concentrations shown in Table 10 are even more difficult to compare than those for embryos (see Table 9) because of two additional variables, age of the larvae and duration of the experiment. As discussed below, larval tolerance increases with size in terms of survival (Watling, 1978;Beiras and His, 1994, 1995a) and growth (Watling, 1982). Also, detectable effective concentrations impairing larval growth depend on the duration of the experiment, and in the studies reviewed here this ranged from 4 to 28 days. In a number of cases, larval growth is a more sensitive response to toxicants than inhibition of embryogenesis (e.g. Beiras and His, 1994, 1995a). Cadmium, for instance, has been shown to inhibit larval growth at significantly lower concentrations than those which inhibit embryogenesis (compare Tables 9 and 10). Irrespective of its sensitivity, however, a bioassay using growth as the biological endpoint is neither simple nor quick thus, it is not suited for routine screening tests. Nevertheless, effective pollutant concentrations at which larval growth is significantly reduced are still worth studying because of their ecological relevance.


95

Ho and Zubkoff (1982) studied the effective concentrations of some heavy metals which reduce calcium uptake in bivalve larvae, and found these concentrations to be similar to those affecting growth (see Table 10). Therefore, calcification of the organic matrix of the shell may be one of the processes impaired by heavy metals and can therefore be used as a measure of sub-lethal toxicity to bivalve larvae. 4.1.1.3. Settlement. Data on the effects of heavy metals on settlement and metamorphosis in competent pediveligers are scarce (Table lo), but they do indicate that the sensitivity of this ecologically relevant stage is similar to that of embryos and larvae. Bioassays with settlement as the endpoint might be particularly relevant for testing the toxicity of surfaceassociated toxicants, such as those that adsorb readily to sediment particles. A shortcoming for the development of settlement bioassays is the limited knowledge of the physico-chemical factors that naturally promote attachment to the substratum and morphogenic change in bivalves. Some advances have been made with the use of epinephrine, a potent inductor of metamorphosis in bivalve larvae, allowing massive and synchronous settlement in the controls (see Section 3.2.4.5). On the other hand, heavy metals do not necessarily inhibit larval settlement. Sub-lethal concentrations of copper are known to stimulate settlement in oyster larvae (Prytherch, 1934, in C. virginica; Nell and Holliday, 1986, in Saccostrea commercialis; P. Salkeld, Plymouth Marine Laboratory, personal communication, in C. gigas). 4.1.1.4. Respiration. Heavy metals are known to increase respiration in all developmental stages of bivalves, including eggs and larvae. Oxygen consumption increased by 1.2 times to 2.7 in Spisula solidissima larvae reared at 50 pg Ag 1-' (Thurberg et al., 1975) and by about 2 times in unfertilized eggs of Mytilus edulis exposed to 30 000 pg Cu 1-' (Akberali et al., 1985). Mercury also increased oxygen consumption of 5-day-old C. virginica larvae after 24-hour exposure to 1-100 pg 1-' (Cunningham, 1972, cited by Cunningham, 1979). However, zinc (100 pg 1-') and cadmium (2000 pg 1-l) have been reported to cause slight (1620%) decreases in respiration in mussel embryos (Pavicic, 1980). The sensitivity of this response is not substantially different from the test of embryogenesis inhibition, whereas the latter is much more simple, rapid and accurate to measure. Moreover, these tests have yielded contradictory results (generally an increase, but also some cases of decrease in respiration) which are difficult to interpret. Therefore respiration does not seem to be a suitable response for embryo and larval bioassays. 4.1.1.5. Genotoxicity. Mercury has been shown to be genotoxic, as exposure to 30 pg HgC121-1 doubles the mean number of sister-cromatid exchanges (SCE, a sensitive indicator of chromosomal damage), in fertilized mussel eggs (Brunetti et al., 1986). Nitrilotriacetate (a com-

96

E. HIS, R.

BEIRAS AND M. N. L. SEAMAN

ponent of some phosphate-free detergents), which is suspected of having mutagenic effects, did not increase SCE at the concentration tested (5mg1-'), nor did it interact with the genotoxicity of mercury chloride (Brunetti et al., 1986). TBT-oxide (up to 1pgl-') was not found to be genotoxic to 12-hour-old mussel larvae either, based on the results of SCE and chromosomal aberrations (Dixon and Prosser, 1986). Fractions extracted with an organic solvent from oil-polluted sediments have also been assayed for genotoxicity in bivalve larvae (Wolfe et at., 1995), but the results are difficult to interpret, because there was significant SCE induction by the reference sediment as well. 4.1.2. Biocides

The available data on biocide concentrations causing 50% reduction in normal embryogenesis, larval survival, growth and settlement are summarized in Table 11. Chemicals are listed in tentative order of decreasing toxicity, based on the highest toxicity values reported. In order to focus on the most relevant environmental pollutants, we are limiting this review to the most toxic biocides for bivalve larvae, i.e. those with ECS0values of less than 10 ppm (10 000 pg 1-') in at least one experiment. Substances of lesser toxicity, for which all published studies give an ECS0higher than lOppm, are not considered here regardless of the fact that they may be highly toxic to organisms other than bivalve larvae. Tributyl-tin (TBT) is the most toxic chemical assayed to date with bivalve embryos and larvae, with an EC50 below the unit ppb level in several instances. This is not surprising, considering that oysters and other molluscs are target species for this active component of antifouling paints. Halogens (chlorine, bromine) and halogen-produced oxidants (such as monochloramine, chloroform, bromamines, bromide, bromate, halomethanes, etc.) are also extremely toxic. Accurate measurement of EC50 values is difficult owing to the high volatility of these substances, and experimental designs should employ flow-through systems. Identification of individual halogen compounds is usually not attempted, but Stewart et at. (1979) found high toxicity of bromate and comparatively low toxicity of chloroform and bromoform. Data on ozone-produced oxidants are shown in Table 11 for comparison. Again, bivalve larvae are target species of these compounds, used in energy plants to prevent their cooling-water pipes clogging. Cyanide (CN-) is highly toxic to all aerobic organisms, and early developmental stages of bivalves are no exception, with ECsovalues in the 10-100 pg 1-' range (Okubo and Okubo, 1962; Pavicic and Pihlar, 1982; Pablo et al., 1997).


97

Intensive agriculture increasingly relies on the use of phyto-sanitary chemical products for pest control. In 1992 the world consumption of these products was approximately 1.4 million tons (Andral, 1996). In coastal areas these products are leached from the soil by precipitation and drained into the sea. The toxicity of a great number of insecticides, herbicides and other biocides has been assayed with early developmental stages of bivalves, but the most important contribution still remains the pioneering study by Davis and Hidu (1969a). More recently, His and Seaman (1993) extended these studies of lethal and sub-lethal toxicity in embryos and larvae to 12 commercially available pesticides. Some organochlorinated pesticides such as trichlorocarbanilide (TCC), 2,3dichloro-l,4-naphthoquinone(Phygon), dichlorodiphenyltrichloroethane (DDT), pentachlorophenol (PCP), dehydroabyetilamine compounds (Delrad, Rosin Amine D), and cyanide can be toxic at concentrations below 100 pg 1-l. Toxicity of chlorophenols increases with the number of chlorine substituents, from an ECsO in the order of 20000pg1-' for 4-chlorophenol (4-CP, one chlorine atom) (Krassoi et al., 1997), to values around 1000 for tetrachlorophenol (TCP, four chlorine atoms) (Davis and Hidu, 1969a), and 27-55 pg 1-' for pentachlorophenol (PCP) (Woelke, 1972). Another group of persistent chlorinated insecticides (dieldrin, endrin, lindane, methoxychlor, toxaphene, aldrin) and copepodicides (dichlorvos) can be toxic at 100-500ppb. Dichlorvos, a highly toxic pesticide used in salmon farming for the control of ectoparasitic copepods, is particularly relevant in marine toxicology, because it is deliberately introduced into the marine environment in appreciable quantities. Crustacean larvae are ten times more sensitive to Dichlorvos than molluscs (LCs0: 5-50 pg 1-' for lobster larvae, according to McHenery et al., 1991). Organo-phosphorated pesticides are generally less toxic, with EC50values in the range of 500-1000 pg 1-' (Parathion, Fenitrothion, Guthion, etc.), or higher (Malathion). Herbicides (e.g. 2,4-D, atrazine-simazine, Neburon, Diuron, etc.) are usually toxic to bivalve embryos and larvae only at concentrations above the ppm level (e.g. Robert ef al., 1986), with the exception of Dinoterbe, a highly toxic phenolic herbicide used in maize culture, which has an ECsOof 50-100 pg 1-'. As in the case of metals, pesticides affect embryonal development more than larval survival. Growth reduction may take place at concentrations below those inhibiting embryogenesis, although this is not always the case (see Table 11). The sensitivity of larval growth is particularly dramatic in the case of TBT, which reduces the rate of growth at concentrations in the order of 1ng 1-l (His et al., 1983; Lapota et al., 1993). In those cases in which there are sufficient data for comparison, the various bivalve species (oysters, mussels and clams)

Table 11 Toxicity of pesticides, biocides and miscellaneous chemicals to early developmental stages of bivalves. LCso: toxicant concentration causing 50% mortality; otherwise ECSo:toxicant concentration causing 50% reduction in the end-point. Chemicals are tentatively ranked in decreasing toxicity order. Except for TBT,data on other toxicants are mostly from the classic work by Davis and Hidu (1969a); these authors refer to Loosanoff and Davis (1963) for details on experimental procedures. Davis and Hidu (1969a) and His and Seaman (1993) used acetone as a carrier when appropriate. Pesticide TBT

Test sp.

Initial age/&

Exposure conditions

End-voint

Notes

C. gigas

30 min fertilized egg

24h, F W 24% ZEpsu, 0.8 pm

embryogenesis

14 d fertilized egg

24°C. 12-14psu, no food, 1pn mortality' FSW, flow-through system (48, .% h) Bow-through system mortahty (48 h)

M. lateralis C. virginica

M. arenaria

97-117 pm 308 pn 100% eyed

27-28°C. 15-16psu. diluted sewage, no food*, flow-through system

3648 h

17-20-C 13psu, 1wm FSW, flow-through system

18 d

Davis and Hidu, 1969a

ZpentachJornphenyl acetate

R. Brown, cited by Dimick and Breese, 1965

'Na pentachloropheuate

Roosenhurg et a!., 1980b

*no movement and disturbed

>25

300, 64 -300, -300 27,46** two separate experiments

internal organization *author mentions monochloramine, bromamine. bromide, halomethanes and bromate

Roberts 1980

38 mortality (96 h) 500 (16 h) disturbed internal W 5 0 0 (24 h) lo0

Dowcide G

M. rnercenarin

fertilized egg 2d

48h 10 d. 24°C. mix algal food

embryogenesis mortality

m

egg egg 1st polar body (25 min) 2ceU (1h) 64cell or more (4 h) blastula (8 h) trocophore (20 h) veliger (32 h) fertilized egg 2d

48h 10 d, 24°C. mix algal food

embryogenesis mortality growth

3820 2500 1W2500

2300 (1300:) Stewart ef al., 1967 20 700 (24 5 0 0 3 Armstrong and 5300 (5200') Millemann, 1974

7000 8300 16 000 19 000

2Aooo Davis and Hidu, 1969a

Fenvalerate

C. virginica

not given

48 h, not given

emhryogenesis

>loo0

P. W. Borthwick, cited by

Neburon

M. mercenan'a

fertilized egg 2d

48 h 10 d, 24°C. mix algal food


So00

Tagatz and hey. 1981

*values for I-naphthol, Sevin hydrolytic product

Exposure conditions

End-point

C. virginica

fertilized egg 2d

48h 12 d. 24"C, mix algal food


Davis and Hidu. 1%9a

C. virginica M. mercenaria

fertilized egg fertilized egg 2d

48 h 48h 10 d, 24"C, mix algal food

embryogenesis embryogenesis mortality growth

Davis and Hidu, 1%9a Davis and Hidu, 1%9a

embryogenesis

Okubo and Okubo, 1%2

Test sp.

Pesticide Malathion

Rhodamine B (stain)

M. edulis

T m i c acid

M. edulis

Initial age/size

20°C ==25psu,20-30 ml-', FSW uv sterilized

C gigas KKIO,

c

embryogenesis embryogenesis (48 h*)

3200-10000 210 000

Okuho and Okubo, 1962 Cardwell er 01.. 1979a

embryogenesis

3200-10 000 3200-10 000

Okubo and Okubo, 1962

M. edulis

Dicapthon

Phosphamidon Monuron

M. mercenarin

C. gigas M. mercenaria

Reference

fertilized egg 2d

48 h

10 d, 24°C mix algal food


3340 5740 >2000

fertilized egg

20% ~ 2 5 p s u 20-30 . ml-', FSW uv sterilized

embryogenesis

4lmo-mm

48h 10 d, 24'C. mix algal food


fertilized egg 2d

Notes

*fully shelled, even misshapen or undersized

Davis and Hidu, 1%

Woelke, 1972

(48h*)

*fully shelled, even misshapen or undersized

Davis and Hidu. 1%9a

growth

Fenuron

M. mercenaria

fertilized egg 2d

48h 10 d, 24°C mix algal food


Davis and Hidu, 1%9a

Carbofuran

C. gigas

fertilized egg (30 min)

9d. 24% Bpsu, 3 O d P (egg), 6 d - l (larva), mix algal food, 0.2 p m FSW

mortality growth

His and Seaman, 1993

Bromoxynil

c gigas


9 d, 24°C 28psu, 30 I&' (egg), E d - ' (larva), m h algal food, 0.2 pm FSW

mortality growth

7000 5000-10000


Metaldehide

C. gigas


9 d, 24% 28psu, 30 m1-l (egg), 8 d P (larva), mix algal food, 0.2 pm FSW

mortality growth

7400 5000-10 000*

His and Seaman. 1993

Carbetamide

c. gigas


9 d, 24°C 28psu. 30 ml" (larva), mix (egg), 6 d-' algal food, 0.2 FSW

mortality growth

93w


210000

Bold: 95% CI provided. FSW, filtered seawater.

*6 d

*6 d

107


Table 12 Interspecific comparison of sensitivity of bivalve embryos to Sevin insecticide. Median effective concentrations (EC,,) inhibiting normal embryogenesisare given.

Species

EGO(mg 1-7 ~~

Crassostrea gigas C. virginica Mytilus edulis


2.2 1.8-3.7 2.04.0

1.6-3.2 2.3 2.5-4.0

Reference ~

~~

Stewart et al., 1967 Woelke, 1972 Davis and Hidu, 1969b Dimick and Breese, 1965 Stewart et al., 1967 Davis and Hidu, 1969b

show similar sensitivity to biocides (e.g. Sevin, Table 12); this corresponds to the similarity in their sensitivity to metals. The effects of biocides on larval settlement are poorly documented. DDT did not greatly affect settlement in C. gigas (Loosanoff, 1954), but DDT and other insecticides did affect settlement in Ostrea edulis (Waugh et al., 1952). Cardwell et al. (1979b), working with C. gigas embryos at 20°C, reported a 48-hour EC50ammonia of 15 mg NH3-N1-’. Gormly et al. (1996) assayed the toxicity of microbial pest-control agents (Bacillus thuringiensis, B. alvei, Metharyzium anisopliae) and of a virus of the gipsy moth to D-larvae of Mulinia lateralis, finding strong effects of B. thuringiensis upon this non-target species. 4.1.3. Detergents and oil Data on the toxicity of detergents and oil to bivalve embryos and larvae are given in Table 13. Detergents are usually a mixture containing at least one surfactant and one builder. The surfactant is the active component that reduces the surface tension of the water and dissolves the organic molecules of a stain. The first synthetic detergents included non-linear surfactants, which were poorly degradable. Owing to environmental concerns, these so-called “hard” detergents have largely been replaced by “soft” detergents, which use readily degradable compounds with a linear alkyl group, such as the linear alkylate sulphonates (LAS) as surfactants. The builder of a detergent contributes to the cleaning action by sequestering cations that would otherwise interfere in the action of the surfactant. Polyphosphates used to be the typical cation-sequestering agents in detergents, but concerns about their role in water eutrophication have prompted their replacement. Nitrilotriacetate (NTA) was one of the alternative builders, but its innocuity to mammals has been under discussion (Stoker and Seager, 1976), and zeolites (sodium aluminium

Table 13 Toxicity of detergents, oil and detergent-oil mixtures to early developmental stages of bivalves. LC5,,: toxicant concentration causing 50% mortality; otherwise ECS0:toxicant concentration causing 50% reduction in the end-point. Several detergents were assayed as mixtures: in these cases, only the active components are mentioned and their percentages in the assayed mixture provided. Concentrations are expressed as p,g I-’ of active cornponenth except when otherwise stated. Detergent!Oil

Test species

Cationic surfactants para diisobutyl C.virginicn phenoxyethoxy ethyl dimethyl benzyl ammonium chloride monohydrate 98.8%

lauryl pyridinium chloride

Initial agelsize

Exposure wnditions

EC& or L C ,

Reference

490

Hidu, 1965

abnormal larvae excluded

Hidu, 1965


fertilized egg

24°C 2C-30 I&’, 15 pm FSW uv sterilized

embryogenesis

2d

24°C 1C-15 d-l,mix algal food, 15 pm FSW uv sterilized

mortality (12 d) growth (12 d) (length) embryogenesis mortality (10 d) growtb (10 d) (length) embryogenesis mortality (12 d) growth (12 d) (length) embryogenesis mortalilty (10 d) growth (10 d) (length)

8.5 100-250 -250

M.mercenarin

idem

idem

C virginiur

idem

idem

M. rnercenaria

End-point

>So0 100-250 1270

>So0

100-250

90

100-250

50-100

idem

idem

Id

not given

mortality (6 h) growth (1week) settlement (6 h expo.)

1ooO* 50” 1ooO***

26°C 13 UP, FSW uv sterilized

embryogenesis (48 h)

25-250

Linear anionic

Notes

SUrfaclanlS

h e a r akylate sulphonate (LAS) dodecylbenzene (12 C) (anionic biodegradable surfactant)

0.edulis

Linear alkylate sulphonate’ 60.8% (LAS) (anionic biodegradable surfactant)

C virginica

8-10 d

fertilized e g

‘referred to as “lethal concentration”; “slightly superior” for I-d-old C. gigar larva “conc. mentioned as “seriously affecting” growth for 0. edulis and C gigas ***cOnc mentioned as “significantly reducing” settling and metamorphosis Calabrese and Davis, *described as “compo&e of a 1%7 number of wmmercially available product$ typical of the LAS presently (1%5) being marketed” Renzoni, 1973b

48h linear alkylate sulphonate ( + A S ) (12 C) (anionic hiodeeradable surfactant)

M. edufir

LAS (12 C)

M. edulis

egg

324 pm larvae

26°C 1&12 ml-l, mix algal food, FSW uv sterilized ZOT, 10 I&', food not given, paper FSW. uv sterilized

22% 20%, 1 1 0 ml-', monoalgal diet, 20 Fm FSW

mortality (10 d) 500-1000 growth (10 d) (length) 250-500 fertilization (2&24 h) embryogenesis (% h) mortalitv (10 d) growth (10 d) iength mortality* (24 h) (48 h) (72 h)

(96h)

C. gigas LAS 10 C 11 c 12 c 13 C 14 C short chain blend medium chain blend long chain blend LAS degradation products: DTW lienosulvhonic acid orher pioducts sodium dodecyl sulphate 85% C. gigas

Granm6, 1972

>m >4M) 7500 4500

34cQ

Hansen er of., 1997 *no ciliary movement **length increase and C content abnormal swimming at 35 ppb

38M) 1400 8206500

growth** (9 d) settling (7 d) 10000) fertilized egg

(SDSI* SDS

-300 -50

20°C 29% 2&30

I&',

unfiltered SW C gigas Protorhacu staminea Tresus capax

>iom

embryogenesis (48 h) 840*sd0.09 (normalshell development) 910*sd0.14 mortality (48 h) embryogenesis (48 h) 930 (1ooO) 450 (870)

Cardwell er af.,

197%

Cardwell er ul., 1979a

*14% 10 C, 72% 12 C,14% 14 C *means of 20 batches

fully shelled (total)

Trous nurralfi SDS

M. larerutis

2 h fertilized egg

21°C 75 ml-'

M. lateralis

embryogenesis (48 h) 8200 (10%) 5800 (30%)

SDS

48 h D-larva

22-25°C %28%, 5-8ml-', 0.22 pm FSW uv sterilized

mortality* (48 h)

SDS

Chlamys ospem'ma fertilized egg ('1 h) Chfamys arpewima fertllized egg

SDS

(30 min)

18°C 33%, 30 ml-', 1pm embryogenesis (48 h) FSW uv sterilized 18°C 33%, 3Oml-', 5 pm embryogenesis (48 h) FSW uv sterilized

6300

845

1wO

Momson and Petrocelli, 1990 G o d y er al., 19% *loss of respiratory circulation and ciliary motion, disfiguration and eventual decomposition Krassoi er af., 1996 abn. lar. exc.; LOEC 6-56, NOEC 500 Krassoi er of., 1997 abn. lar. exc.: LOEC 950, NOEC 530

DetergenUOil

Test species

Non-linear anionic surfactanrs alkyl naphthalene sulphonate’ C. virginica

M. memotarin

Initial age/size

Exposure conditions

fertilized egg

24‘C 2&30ml-’, 15 pm FSW uv sterilized

2d

24°C 10-15 de1. mix algal food, 15 pm FSW uv sterilized

idem

idem

End-point

embryogenesis (48h) (abn. lar. exc.)

mortality (12 d) growth (12 d) (length) embryogenesis (48 h) (abn. l a . exc.)

ECm or LC, (I% 1-7 1630

Reference

Notes

Hidu, 1965

abnormal larvae excluded; *(ANS+polyphosphate+fatty alcohol sulphates; percentages unknown; w n c expressed as gross product)

Hidu, 1%5


Hidu, 1965


Hidu, 1965


>2500

looo-2500 5830

mortality (10 d) 210 000 growth (10 d) (length) 5wO-10000 alkyl benzene sulphonate 54.8%

C virginica

M.mercenaria triethanolamine dodeeyl benzene sulphonate 609.

alkyl sulphate 26.&28.9%, free fatty alwhol 1.&2.5%

tetrapropylene benzene sulphonate

idem idem

idem idem

C virginica

idem

idem

M.mercenarin

idem

idem

C virginica

idem

idem

M. mercenaria

idem

idem

Id

not given

0. edulir

b10 d

embryogenesis mortality (12 d) growth (12 d) (length) embryogenesis mortality (1Od) growth (10 d) (length) embryogenesis mortality (12 d) growth (12 d) (length) embryogenesis mortality (10 d) growth (10 d) (length) embryogenesis mortality (12 d) growth (12 d) (length) embryogenesis mortality (10 d) growth (10 d) (length) mortality (6 h) growth (1 week) settlement (6 h expo.)

270 500-1000

25&500

940 >2500 1ooO-2500 390 1wO-2500 -1000 1030

25W5000 -2500 370 2500-5000 2500-5000 470 5000-10000 5000-10000 2000‘ 50.’ l000***

Renzoni. 1973b

‘referred to as “lethal wncentration”; “slightly superior” for I-d-old C.gigas larva **cone mentioned as “seriously affecting” growth for 0.edulis and C. gigas ***wnc mentioned as “sigm5cantly reducing” settling and metamorphosis

Non-ionic su@ctrrnts iso-octyl phenoxy polyethoxy C virginica ethanol

fertilized egg 2d

24°C. 2C-30 d - l , 15 pm FSW uv sterilized 24"C, 1C-15 tn-', mix algal food, 15 pm FSW uv sterilized

M. mercenaria alkyl polyether alcohol tallow alcohol decaethyleneglycolether (non-ionic biodegradable surfactant) Builden sodium nitrilotriacetate W.4)

Oil dispersants Chevron

E-314 Hollchem Aquaclean Houghtosol Polyclens Janslov Spillex Seasweep Polywmplex W1439 Emsol Slik Gamlen Dasic

idem

idem

idem idem egg

idem idem 72 h, 18°C. 33%.

C virginicrr

M. mercenaria M. edulis

M. galloprovincialis fertilized egg

20% 35%. 40 d-'

860

embryogenesis mortality (12 d) growth (12 d) (length) embryogenesis mortality (10 d) growth (10 d) (length) embryogenesis mortality (12 d) growth (12 d) (length) embryogenesis mortality (10 d) growth (10 d) (length) fertilization (22 h) embryogenesis (46 h) embryogenesis (72 h)

770 1000-2500 2500-5000 1600 1000-2500 1000-2500 1750 2500-5000 2500-5000 100-500 100-500 >loo0

embryogenesis (40 h)

>20 ooO*

(30 min)

C gigas

C. gig?

0.cdulrr C gigas 0. edulis

0. edulrr

c, gigm

0.edulrr

C gigas

fertilized egg

developing egg 1 week developing egg 1 week

1 week developing egg 1 week developing egg

20T, a5%, 2&3l d-' embryogenesis' FSW uv sterilized (4 h)

23°C 23°C

23°C

embryogenesis (24 h) growth (2 d) embryogenesis (24 h) growth (2 d)

growth (2 d) embryogenesis (24 h) erowth (2 d) kmbryogenesis (24 h)

Hidu, 1965


1000-2500 1000-2500

Hidu, 1965 abnormal larvae excluded

Granmo and J@rgensen, 1975

Brunetti et 01.. 1989 'increasing embryogenesis success with increasing NTA mnc.

0.1-1

Woelke. 1972

1-10 1&100 la1000 400 c. 1 m lW**

embryogenesis (48 h) 1000 (Ven) 430 (No. 2 ) (vital staining) mortality (10 d) (vital staining)

1600 (Ven) 530 (No.2)

loo0 (Ven) 570 (No. 2) 440 (450) (No. 6) 450 (410) (No. 2) mortality (4 d) (larval 1700 (d 1) density in the water 1700 (d 2) column) 200 (d 3) 1wO (d 4)

Woelke, Woelke, Woelke, Woelke,

1972 1972 1972 1972

Byme and Calder, WSF 12 h gyratory shaking (200 rpm)t 24 h equilibrium+glass 1911 wool filtration '100% mortality at d 6 Renzoni, 1973a *also with a 4 1 mixture of oil and dispersant (Corexit 8666). **oiI+dispersant, oysters only Conc. mentioned are those used to make up experimental solutions (30min agitation); actual oil wnc. accommodated in SW not measured Byme and Calder. WSF: 12 h gyratory shaking (2CQrpm)+24h 1977 equilibrium+glass wool filtration

length growth (10 d)

relined oil (No. 2 and No. 6 fuel oil)

C. gigas

fertilized egg

relined oil (No. 2 fuel oil)

C virginicu

-120 pm

embryogenesis (4s h)

22.5% 21%. mix algal food. ASW

CardweU era[., 1979a

water-accommodated fraction

Sigler and Leibovitz, 1982

WSF k100 oil:water 20 h magnetic stirring, resulting 8.65 ppm fuel oil

refined oil (No. 2 fuel oil)

refined oil (No. 2 fuel oil, No. 6 fuel oil) refined oil (diesel oil) nude oil (Venezuelan)

M. lateralis

M. edulis

48 h D-larva

22-25"C, 2&28%, 5 4 I&'. 0.22 pm FSW uv sterilized

crude oil (Algeria, Libya, Iraq, Kuwait, Indonesia)

4: Irq pediveliger > adult (Figure 14). Among the various possible toxicity tests with bivalve larvae the embryogenesis bioassay will usually be the method of choice for both pure chemical toxicity tests and routine environmental monitoring. Metamorphosis success (i.e. settlement of pediveligers) and measurement of chronic effects on larval growth may be similar in sensitivity to the criterion of embryogenesis success, but they require rearing the larvae for weeks, entailing considerable effort and cost (see Section 3.2.3) and rendering these bioassays impractical for routine investigations. They may well be preferable in studies of particular hot spots and for risk assessment of new chemical products likely to enter the marine environment. Tests with gametes (spermiotoxicity, unfertilized eggs) may be simple from a methodological point of view, but are not commonly employed, and their sensitivity is lower than that of the embryogenesis test. 6.2. Assessment of the toxicity of various contaminants

Any discussion on ecotoxicological bioassays must make a careful distinction between toxicological investigations with identified compounds on one hand, and routine monitoring techniques with environmental samples on the other. The former mostly concern basic research on specific toxicants (e.g. new industrial compounds) and on methodological aspects (e.g. comparison of bioassay techniques); the latter focus on general

133


I

1

SUB-LETHAL EFFECTS

LETHAL EFFECTS Lc50

EC50

larva growth embryogenesis D-larva metamorphosis

adult (Connor, 1972)

1

ybyrd:eliger

estuaries :P

I

I 0.01

+

I

I

0.1

1

&:-:a Iw w p J 21 8 PfJl 0

,c

A

A

v

10

v

100

1

1000

v

10 000

Hg (P9. I-’)

Figure 14 Sub-lethal (0)and lethal (+) effects of mercury upon the oyster. Larval growth and embryogenesis are the most sensitive responses, but the latter is more easily and rapidly assessed.Data from Beiras and His (1994) except for the adult. Usual mercury concentrations in estuarine waters are also shown.

pollution monitoring and abatement. With regard to both, we need to keep in mind that biological systems are extremely diverse and complex, and that no single method or species could ever be adequate to measure pollution “as such”. A battery of bioassays may often be required for a fairly adequate assessment of a particular pollution phenomenon. B a k e r et al. (1990), for instance, advocate the use of the oyster embryo bioassay in samples determined as toxic by the Microtox bioassay, which is supposedly more sensitive, but less reliable. The most toxic compound in the marine environment bioassayed to date with bivalve embryos and larvae is TBT, the toxicity of which is generally one or two orders of magnitude greater than that of any other compound bioassayed to date. The further ranking of pollutants is heavy metals (especially mercury, silver and copper), chlorine and derived oxidants, organics and pesticides (especially organo-chlorine), detergents, petroleum products and, depending on their composition, industrial1 and urban effluents and sediments (Figure 15). Apart from a considerable amount of variation between the results of different authors, the toxicity of many compounds is affected by chemical and physical conditions and interactions of a complex nature, often defying precise analysis. ‘These include bioassay methodology, chemical speciation and complexation, synergistic and antagonistic effects, and seasonality in the bioassay organisms (see also Sections 4.2 and 4.3). As Figure 15 shows, the range of reported effective concentrations spans at least two orders of magnitude, even in the case of precisely defined

134

E. HIS, R. EElRAS AND M. N. L. SEAMAN

1 PPt 0

y

In

1PPm

1 PPb

Materials Figure 15 Toxicitv of various materials to bivalve embryos and larvae in the publiiations reviewed here (range of median effective concentrations;EC50).TBT tributyl-tin; HM: heavy metals; C1: halogens and halogen-produced oxidants; C1-Pest: organochlorinated pesticides; Pest: other pesticides; Det: surfactants and other components of detergents; Petr: petroleum products; Effl: industrial and urban effluents; Sed: sediments.

compounds, depending on biological and chemical species, larval stages, experimental regimes and criteria of toxicity. On the other hand, scientists are often asked to provide clear and simple data as a basis for political decision making. This underscores the continuing need for standardization. In the case of many substances (contaminated sediments in particular), there are considerable problems with regard to the methodology of sampling and the handling of samples; standardization of these methods is urgently needed, but it will not be easy to achieve. Correspondingly,the use of bioassays for the purpose of enacting and monitoring pollution abatement measures may yield contradictory results for some time yet. 6.3. Bivalve embryo and larval bioassay methodology

In the case of using bivalve embryos and larvae for the purpose of pollution assessment, decades of experience as well as recent improvements and simplifications in methodology do render standardization achievable (e.g. ASTM, 1980, 1989; Thain, 1991; Krassoi et al., 1996; His et al., 1997a). With respect to routine investigations of environmental quality, the embryogenesis bioassay is generally regarded as the method of


135

choice, even though differences of opinion may exist concerning certain details of methodology. The following aspects should be considered in future standardization efforts.

6.3.1. Bivalve species The various species commonly used in embryotoxicity bioassays (Crassostrea gigas, C. virginica, Mytilus edulis, M. galloprovincialis, Mercenaria mercenaria, Mulinia lateralis) appear to have similar sensitivity to environmental contaminants (see Section 4.2.1 and Table 12). The choice of species to be used in a bioassay therefore depends mostly on practical considerations. In studies concerning a shellfish-culture area, for instance, the species of choice will be the one being cultivated on site. Profound knowledge of its biology and maintenance in the laboratory is essential to prevent artefacts in the bioassay. Other aspects include availability (e.g. use of a species during its spawning season rather than using conditioned broodstock), sensitivity, commercial interest and ecological relevance. With regard to more sophisticated basic research (e.g. chronic and persistent toxicity, physiological and genetic adaptation to pollution) the American coot clam Mulinia lateralis offers interesting perspectives. Its small size and the brevity of its life cycle make it relatively easy to cultivate several generations per year in adequately equipped laboratories, although these advantages have not been exploited fully in toxicological research to date. Doubtless this is largely because of its limited natural distribution (the Atlantic coast of North America) and the present-day difficulties and necessary precautions required for importing and holding exotics in the laboratory. Possibly, species with similar Characteristics may yet be found in Europe and other parts of the world.

6.3.2. Broodstock availability and gamete production Ripe broodstock of Crassostrea and/or Mytilus is generally available almost year-round because conditioning techniques extend the seasonal availability. Even though it may be considered controversial by solme of the researchers in the field, we propose that “stripping” be abandoned as a method of obtaining gametes for embryotoxicity bioassays. Most authors who have worked with bivalve larvae will undoubtedly concur, because this method results in unacceptably high levels of mortality and abnormality in the controls, introducing gamete quality as an undesirable factor of variation in the test. When inducing spawning by thermal stimulation of the broodstock, rigid temperature limits are useless; adult bivalves should be stimulated to

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spawn by temperature variations of about lWC, for example 5°C above and below the ambient water temperature at the time of sampling. 6.3.3. Incubation water At many marine laboratories the quality of the water is inadequate for rearing bivalve larvae, even though it may be sufficient for holding the adults of various species. In such cases water should be obtained 1 or 2 days before the experiment from areas known to be unpolluted. Filtration of the water at 0.2 pm is generally recommended. Alternatively, artificial seawater is adequate, and it also eliminates any possibility of contaminants in natural seawater (for example dissolved organic matter); the cost is negligible when only small volumes are used (see below).

6.3.4. Toxicant exposure and observation of the response Fertilization should be achieved as soon as possible after the first females spawn, in order to avoid deterioration of gamete quality. Toxicant exposure should begin as soon as fertilization of the gametes has been achieved because this enhances the sensitivity of the test; there is usually no particular reason for delay. Similarly, the bioassay should be terminated as soon as embryogenesis is achieved (24 hours in most bivalve species, 48 hours in Mytifus).There is no particular reason to continue a test of embryogenesis beyond the time required for embryogenesis, as prolonged incubation introduces the risk of mortalities from causes other than the toxicant under study (e.g. bacterial proliferation resulting from decomposition of undeveloped eggs and dead embryos). For the sake of accuracy and precision, further manipulation of the biological material (sieving, sub-sampling, etc.) should be kept to a minimum once a bioassay has been initiated. Therefore, small volume incubations in suitable vessels permitting a direct assessment of the material under the microscope (e.g. transparent 30 ml vials, and 3 ml microwells) are particularly suitable. 6.3.5. Assessment of embryogenesis success Many workers have in the past employed definitions of larval normality which do not reflect the appearance of larvae from the natural environment; by definition, however, the criteria of normality should conform to the natural situation. A strict and accurate definition of larval abnormality (see Section 3.2.2.5, Figure 7) has yet to become generally accepted. At the same time, a valid acceptable level of abnormalities in the controls needs to be agreed upon; we suggest that embryogenesis bioassays are invalid if


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the level of larval abnormality in the controls is higher than 20%. The percentage of abnormal controls must always be stated when publishing an investigation. 6.3.6. Statistical evaluation of bioassays In embryo-larval bioassays end points are often measured as proportions. Angular transformation is generally sufficient to overcome the difficulties of binomially distributed variables, but this must not be taken for gr.anted and alternative methods are available (see Section 3.4.1). Besides, very little attention is often paid to the question of sample size required for precise estimation of the biological responses and powerful detection of differences among them (see Section 3.4.3). The assessment of environmental samples by means of bivalve Iiioassays has relied too often on arbitrary scales (e.g. Woelke, 1966), or at best, calculation of “percent net risk” (PNR; equation 2, Section 3.2.2.5) The PNR is highly influenced by the control response, and comparison of PNR values calculated with controls that exhibit highly different responses is unacceptable. As mentioned above, we consider that the PNR values have limited value when the level of larval abnormality in controls is :.20%. Alternatively, a more systematic approach based on ANOVA-multiple range tests is recommended (see Section 3.4.2). 6.4. Perspectives in future research on bivalve larval bioassays

Despite the fact that bivalve embryogenesis and larval bioassays are presently approaching the status of routine methods, there is still ample room for improvement of the techniques. Research in the immediate future should focus on the following aspects:

1. The definition of a “standard” organism that is easy to cultivate in the laboratory (e.g. Mulinia lateralis) as a reference species, particularly if a genetically homogeneous laboratory strain is created. This would help in the standardization of toxicological investigations and routine monitoring studies, as well as in the inter-calibration between different laboratories, rendering all types of studies directly comparable. 2. Cryopreservation of fertilized or unfertilized gametes and the evaluation of gamete quality immediately after spawning. In conjunction with (1) above, this would eliminate the effects of genetic, seasonal and inter-annual variability, and facilitate the performance of bioassays at all times of the year and at laboratories lacking sophisticated conclitioning and larval-rearing equipment.

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3. Development of simple methods for performing long-term bioassays on larval performance (e.g. growth, swimming behaviour, feeding behaviour) to assess sub-lethal effects of toxicity. One central element is the development of an adequate, commercially available and standardized artificial diet (e.g. freeze-dried algae), to liberate long-term tests from the slavery of algal production. In this respect, toxicological research again shares a common interest with aquaculture (commercial hatcheries and nurseries). 4. Automatization of the assessment responses to toxicant exposure. Small incubation volumes in standardized incubation recipients, as well as technological progress, may transform this aspect of marine ecotoxicological research in the near future. Automatic image analysis systems may soon permit the evaluation of larval abnormalities in acute toxicity tests and larval growth in chronic toxicity studies with a minimum of human intervention and with a maximum of comparability.

6.5. Concluding remarks

Bivalve embryo and larval bioassays have shown that in various cases where environmental concerns have led to the replacement of toxic substances by new compounds (e.g. linear instead of non-linear surfactants), the replacement may be more hazardous to the environment than the original compound. This may also be the case when a remedy is used to combat an environmental threat (e.g. the greater toxicity of oil dispersants in comparison to the oil itself). This not only raises questions with regard to environmental regulation and pollution abatement actions but also emphasizes the necessity of assessing the biological effects of new compounds before they are mass produced and before they are introduced into the natural environment in appreciable quantities. Finally, after all of this is said and done, we should keep in mind that there is one decisive aspect preferable to each and every refinement of toxicity assessment, environmental monitoring methodology, pollution abatement and bioassay techniques, and that is to introduce fewer contaminants, and in lesser quantities, into the natural environment.

ACKNOWLEDGEMENTS We thank M. L'Excellent and A. Radenac (central library of IFREMER, Nantes) and S. Robinson (library of Plymouth Marine Laboratory) for


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their invaluable help in obtaining rare specimens of literature, and C. Cantin (IFREMER, Arcachon) for his important technical assistance with respect to many of the illustrations.

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Population Structure and Dynamics of Walleye Pollock. Theragra chalcogramma K . M. Bailey'. T. J. Quinn 11'. F! Bentzen3 and W. S. Grant4 'Resource Assessment and Conservation Engineering Division. Alaska Fisheries Science Center, 7600 Sand Point Way NE. Seattle W A 98115 Email:[email protected]. Phone: 206.526.4243 . Fax: 206-526-6723 2Juneau Center, School of Fisheries and Ocean Sciences, University of Alaska Fairbanks, 1120 Glacier Highway, Juneau A K 99801-8677 3MarineMolecular Biology Laboratory. School of Fisheries. University of Washington. Seattle W A 98195 4ConservationBiology Division. Northwest Fisheries Science Center, 2725 Montlake Blvd., Seattle W A 98112 1. Introduction ......................................................................... 2. Background: The Fishery. Life History and Ecosystem Interactions 2.1. The fishery for walleye pollock 2.2. Life history 2.3. Predator-prey interactions and ecosystem considerations 3 Population Ecology 3.1. Macroecology 3.2. Population dynamics 3.3. Recruitment 4. Population Structure 4.1. Methods for estimating stock structure ...................................... 4.2. Phenotypic population structure 4.3. Genetic population structure 4.4. Metapopulationstructure ..................................................... 4.5. Populationstructuring mechanisms 5. Management Implications Acknowledgements References

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The population biology of walleye pollock. Theragra chalcogramma, is described including its life history. population dynamiq genetic structure

ADVANCBS IN MARINE BIOLOGY VOL. 37 ISBN &12-(126137-5

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and metapopulation structure. Walleye pollock is an important species in the ecosystems of the subarctic PaciJic Ocean, and is one of the world’s largest fisheries. The population dynamics of pollock is driven by recruitment, which is associated with environmental variability. Management of pollock stocks is based on harvests from large geographic regions. However, lumping stocks within these regions may be adverse to conservation and management goals. Historical genetic studies of pollock have produced some conflicting results and comprehensive genetic studies are needed. A summary view of genetic structure in walleye pollock to date suggests a pattern of geographic stock structure, with varying levels of gene flow between major regions. Phenotypic differences between stocks, elemental composition of otoliths and parasite studies indicate restricted mixing of juveniles and adults. Genetic differences appear between broad regions, but resolution between adjacent stocks, especially within the eastern Bering Sea, is currently lacking. Recent studies indicate genetic differentiation among pollock in the Gulf of Alaska and Bering Sea, possibly resulting from reduced gene flow owing to larval retention mechanisms or strong natal homing. The global population of pollock does not fit into a strict metapopulation framework, but some neighbouring populations may be considered as metapopulations. Whether there is either density-driven migration of strong recruitment cohorts, or population sinks, is controversial and more information is needed. Stock mixing problems can be best addressed by means of high resolution genetic techniques in conjunction with tagging and the use of natural environmental markers. 1. INTRODUCTION

Walleye pollock, Theragra chalcogramma (see cover picture) is a dominant groundfish in many ecosystems across the North Pacific Ocean and plays an important role in the dynamics of higher trophic levels (National Research Council, 1996). For example, about 70% of the groundfish biomass in the eastern Bering Sea consists of pollock (Wespestad, 1993). Pollock is the target of one of the world’s largest fisheries, with annual harvests ranging from 4 to 7 million metric tons (mt) in the North Pacific over the past decade. This species represents about 5 % of the world’s harvest of fishes. In US waters, catches have been in the order of 1.5 million mt, about 40% of US fisheries, with an ex-vessel value of 200 to 400 million dollars and a post-primary processing value of 600 to 900 million dollars from 1992-96 (Kinoshita et al., 1998). It has been suggested that declines in abundance of pollock are associated with declines in the abundances of other groups of animals, such as marine mammals and birds in the North Pacific (Springer, 1992; Merrick et al., 1997).

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Many issues in the management of pollock have not been resolved. These include the delineation of population boundaries and the dynamics of pollock populations, discoveries of new populations, and the effects of fishing on migratory individuals in areas of population mixing. Fishery resource management is based on the concept of sustainable stocks. The idea behind this concept is the identification of groups of fish whose demographics (population statistics) and abundances are largely independent of other groups. Ideally, each group produces a surplus of fish that when harvested does not threaten the ability of the stock to persist, nor does its harvest affect the abundances of fish in other stocks. The identification of these idealized units, however, is often controversial because the motivations of fishery managers may be variously influenced by biological, practical or political considerations (Carvalho and Hauser, 1994). In the case of pollock in US waters, populations in the Gulf of Alaska are presently managed independently of populations in the Bering Sea, as are populations in the eastern and western Bering Sea and in the Aleutian Basin. However, demographically independent sub-populations may exist within some of these areas, for example, there are spawning aggregations within the Gulf of Alaska in Shelikof Strait, Prince William Sound, and the regions around the Shumigan Islands (Figure 1). A further complication is that inshore fisheries in the US are currently managed by state agencies, whereas offshore fisheries are under federal or international jurisdiction. In addition to the identification of population boundaries, little is known about the amount of mixing of fish from different populations on summer feeding grounds, or of the sources of fish for the recolonization of depleted areas. In fisheries, attempts to define local populations date back t o the conceptual developments of Schmidt, Heincke and Hjort in the early 1900s (reviewed by Sinclair, 1988). Information on the spatial structure of populations, their intermixing and the ecology and life-history dynamics of individual populations is vital to formulating a sound policy for the harvest management of walleye pollock, as well as for other species. The identification of population boundaries and the measurement of migration between populations are especially important in pollock because high levels of harvest may rapidly deplete individual segments of a population. Such information can potentially be used to address several questions. If a population is depleted by overharvesting, will it recover quickly by replenishment from neighbouring populations through migraticon of juveniles or adults or through transport of eggs and larvae? How much migration is required for the recovery of a depleted population on a time scale that is of interest to the fishing industry? How can levels of migration between populations be measured? We know that pollock populations in previously glaciated areas have been transient over a scale of tens of

Figure 1 The distribution range (:::) of walleye pollock and major spawning locations ( 0 ) .


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thousands of years, but how stable are populations on shorter time scales? What is the effect of intense harvests on natural cycles of population variability? The role of spatial factors in population dynamics, and especially the importance of habitat subdivision and spatially distributed dynamics is relatively unknown for most animals (Kareiva, 1990). However, a growing body of theory and empirical observations exists in the fields of landscape ecology, macroecology, population dynamics, population genetics and metapopulation dynamics that can be used to address the above questions and to understand the dynamics of walleye pollock populations and guide future research. Individuals are not scattered uniformly within the distributional range of a species, and the theories in these disciplines provide a framework for understanding the effects of population subdivision and demography on the harvest management of walleye pollock. Terminology is somewhat different in ecology and population gen etics. Population sub-units are sometimes referred to as populations, local populations, sub-populations, stocks, or sub-stocks. In this review, we define a population as a group of individuals in an area that is distinguishable from other groups in other areas. Populations may have different demographic trajectories; that is, they may have different age structures that result from different birth, recruitment, death and dispersal rates. Populations may also be reproductively isolated from one another to some degree, and may show genetic differentiation if they have been isolated for a long time. The term stock has been variously used in the literature to designate collections of fish. These collections have variously been defined as fish occurring in a particular locality, for example, a current system or within political boundaries, or merely as fish harvested by a partiicular method. The term stock is sometimes used in place of population and may or may not imply genetic discreteness. Indeed, fisheries scientists and managers usually use the term stock for a population component for which assessment information can be determined and effective management regulations can be developed. We use the word here in a more general sense indicating a group of fish. In this chapter, we review the natural history of walleye pollock with particular reference to population dynamics, including what is known about early life-history stages, demography, and population structure. We attempt to integrate and revise current knowledge linking dynamics and population structure of pollock (Bailey el al., 1997, 1998) as a framework for future research. We have included studies on North Pacific and B,ering Sea oceanography, and on larval life history patterns as they influence population structure and dynamics, as well as studies of the popullation genetics of pollock based on recently developed molecular methods. The scope of the review is to concentrate on pollock stocks most familiar to us,

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those of the northeastern Pacific Ocean, but we refer to other stocks, such as those around the Japan Archipelago, when appropriate. Pollock has a relatively short history as a harvested species and has not been studied as thoroughly as other demersal species, for example Atlantic cod, Gudus morhua, which has a rich literature on population and catch histories, and population structure. Thus, comparisons with cod are frequently made. 2. BACKGROUND: THE FISHERY, LIFE HISTORY AND ECOSYSTEM INTERACTIONS

In this section, background information is presented on the fishery and life history of walleye pollock as it applies to population structure and dynamics. The role of pollock as a predator and as a prey in its ecosystem is also reviewed. 2.1. The Fishery for Walleye Pollock

In the waters around Japan, a fishery for pollock existed as early as the eighteenth century, although it remained at a low level until the 1970s (Saito, 1972; Tsugi, 1989). In the northeastern Pacific Ocean, commercial catches of pollock are recorded as early as 1954, but it was the development of at-sea processing of surimi (see below) in the early 1960s that led to a large-scale directed fishery. The fishery in the eastern Bering Sea rapidly expanded to a peak catch of 1.9 million mt in 1972. Bilateral agreements between nations followed soon afterwards and then the passage of the 1976 Magnuson Fisheries Conservation Management Act (MFCMA) extended the US fishery management jurisdiction to 200 miles offshore, and led to regulated harvest levels. In the early history of eastern Bering Sea and Gulf of Alaska fisheries, harvests were made by foreign nations (mainly Japan, USSR, Poland and Korea), but in the 1980s a domestic US fishery developed. Concentrations of pollock were discovered in the 1970s in the central Bering Sea in an area outside the US and USSR exclusive economic zones (EEZs), the so-called “Donut Hole”. Fish in this area were outside national jurisdiction and harvests were unregulated until 1988, when international treaties and cooperative efforts curtailed fishing (Wespestad, 1993). In the early period of development of pollock fisheries in the northeast Pacific, the major gear used was bottom trawls, but targeted pollock fisheries have used mainly large midwater trawls deployed close to the bottom (Megrey, 1989). In Funka Bay, Japan, walleye pollock are caught with bottom gillnets (Kendall and Nakatani, 1992), and in Nemuro Strait, Japan, fish are caught with longlines and gillnets (Yoshida, 1989).


185

Pollock is utilized in a varied array of products. In 1996, about 51% of pollock landed off Alaska was used to make surimi, a minced fish product that is further modifed into artifical crab, scallop, or shrimp meat, fish sausage (kameboko) and other products. Another 20% of the landed Alaska catch was used to make fillets, and the remainder was used for roe, other minced fish, whole fish, fish meal and other products. About 8% of the catch was discarded because fish were too small or pollock wals an undesirable bycatch. The overall biomass and catch of pollock on the US side of the North Pacific Ocean is relatively stable, but some populations have experienced declines and fisheries closures in recent years, including stocks in F’uget Sound, Shelikof Strait, the Donut Hole and near Bogoslof Island. More recently, there are reports that the western Pacific stocks are in a state of decline. Eastern Bering Sea continental shelf populations have berm at healthy levels in the past, although there is concern about the sustainability of present harvest levels. Pollock harvests were especially high from the mid-1980s through about 1992 resulting from relatively strong recruitment, high abundance levels and unrestricted high seas fisheries. Wespestad (1996) lists 12 geographically distinct (although not necessarily genetically distinct) stock groupings and their catch trends (Table 1). Biomass and catch trends for the major stock groupings indicate generally declining levels since the late-1980s in the major fishing grounds (Figure 2a and b). The greatest relative declines in biomass and catches for pollock have been away from the centire of pollock’s distribution. For example, in the Gulf of Alaska catches peaked at 307 thousand mt in 1984 and have declined to 55 thousand mt in ’1996. In this region catches are regulated by quotas that reflect biomass levels. At the extreme southern end of its range in the eastern North Pacific, pollock in southern Puget Sound may almost be extinct (Palsson et ul., 1996). Likewise for the southern end of its range in the western North Pacific around northern Japan, recent catches are reduced by three to four times from their maximal values in the 1970s (H. Yoshida, personal communication, 1994). 2.2. Life History

Walleye pollock is one of seventeen gadiform species represented by four families in the northeastern Pacific Ocean and is one of five species iin the family Gadidae (Dunn and Matarese, 1987). There is one other member of the Therugru genus which is 7: jinmurchicus, a small fish infrequently found in the Barents Sea (Pethon, 1989). Most pollock populations spawn at predictable times, in the late winter

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and early spring, in the same locations year after year, usually in sea valleys, canyons, or indentations in the outer margin of the continental shelf. They are also known to spawn in fjords or deep-water bays (such as Puget Sound) and in some deep-water locations over the Aleutian Basin. Since observations began about 15 years ago the bulk of spawning of the Shelikof Strait population has occurred in a 2-week period in April, within a few kilometres of Cape Kekurnoi. In the Bogoslof Island region in the Aleutian Basin, pollock spawn from early to mid-March, and over the southeastern Bering Sea shelf, most fish spawn from April to mid-May. In Funka Bay, Japan, spawning occurs in January and February (Kendall and Nakatani, 1992). Some populations of pollock apparently spawn at a particular time of year following a spawning migration to a specific location; presumably the eggs and larvae can then reach nursery areas that are favourable for survival (Kim, 1987; Kendall and Nakatani, 1992). This presumed adaptive strategy may vary among populations. For example, in Funka Bay fish spawn near the entrance of the bay in an area where eggs and larvae drift into the bay and in Shelikof Strait fish spawn in an area where larvae are retained on the shelf and are transported into coastal nurseries (Kendall and Nakatani, 1992). Pollock have complex pairing and mating behaviour during spawning (Sakurai, 1982; Baird and Olla, 1991). Female pollock spawn numerous batches of eggs over a relatively short time (Sakurai, 1982; Hinckley, 1987). Eggs are spawned at depth, and in most areas remain deep in the water column (100 to 400m) (Kendall et al., 1994). However, they are shallower in Funka Bay and over the Bering Sea shelf. The deep distribution of eggs presents a problem for interpreting spawning distributions from historical ichthyoplankton surveys that usually sample only to depths of 100 to 200 m. For example, in the Aleutian Basin, most eggs are found at 300 to 400 m (Dell'Archiprete, 1992). Eggs take from 7 to 30 days from fertilization to hatch, depending on ambient temperature. After hatching, the larvae are located in the upper portion of the water column (generally from 20 to 60 m depth); larvae undertake limited die1 migrations (Kendall et al., 1994). Larvae grow relatively slowly (about 0.10 to 0.20 mm.d-l; Nishimura and Yamada, 1984; Kendall et al., 1987; Bailey et al., 1996a). Larvae metamorphose to juveniles at about 18 mm and undergo associated life-history changes (Bailey, 1989; Grover, 1990;Merati and Brodeur, 1996; Brodeur, 1998). Young-of-year juveniles grow about 1mm.d-l, reaching 80 to 100 mm by 6 months and 120 to 140 mm by the end of their first year. Juveniles mature sexually at about age 4 and a length of 40 to 45cm. Pollock can live as long as 20 years and attain a maximum length of 75 cm (Wespestad, 1993).


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Table I Geographical distribution of walleye pollock stocks, modified from Wespestad (1996).

Stock

Stock characteristics

North American

Southeast Alaska-Canada Westem-Central Gulf of Alaska (GOA) Eastern Bering Sea (EBS) Aleutian Basin (AB) Aleutian Islands (AI)

Small stock, minor fisheries Variable stock, 50-200 thousand t catch Large stock, 1-2 mt catch Variable stock (0.1-1.4 mt catch) Small stock, minor fisheries

Asian

Northwest Bering Sea Western Bering Sea East Kamchatka West Kamchatka North Sea of Okhotsk Sakhalin Kurd Islands Japan Sea Japan Pacific

Mix of US and Russian fish, 0.5-1 mt catch Medium stock, 0.5-1 mt catch Small-medium stock, 100-300 thousand t catch Large stock, near 1 mt catch Medium stock, 0.5-1 mt catch Small stock, 65 thousand t average catch Small-medium stock Heavily fished Moderate catch to 0.5 mt

2.3. Predator-Prey Interactions and Ecosystem Considerations

Early-stage larvae feed mainly on copepod nauplii (Nakatani, 1988; Canino et al., 1991), the success of this behaviour is related to survival (Nakatani, 1988; Bailey et al., 1995; Paul et al., 1997). Between larval and juvenile life, pollock become crepuscular feeders (Merati and Brodeur, 1996). Juveniles prey mostly on euphausiids, decapod larvae and copepods (Grover, 1990; Brodeur, 1998). As adults, pollock feed mainly on euphausiids, small fishes, copepods and amphipods but are capable of eating all manner of smaller marine organisms. Cannibalism is a particularly important aspect of adult pollock feeding in the eastern Bering Sea (Dwyer et al., 1987; Livingston, 1989, 1993). In some years, during the autumn and winter, up to 80% of the mean stomach contents of an adult pollock may be composed of age-0 juvenile pollock. Cannibalism is also prevalent in other regions, but is noticeably less so in the Aleutian Basin and Gulf of Alaska. Pollock eggs and larvae are preyed upon by a wide assortment of animals, including euphausiids and amphipods. Small invertebrate predators may consume from 4 to 17% of the total number of eggs present in the water column (Bailey et al., 1993; Brodeur and Merati, 1993). The impact of predation by small fishes on eggs and larvae is not well known

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16 14 12 E

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2 0 1984

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1879

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lQs5

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Figure 2 Trends in major stock groupings of walleye pollock (a) catch trends for Asian and North American stocks, (b) abundance trends for major stocks (after Wespestad, 1996).

(Brodeur et al., 1991; Brodeur and Merati, 1993). Egg cannibalism occurs but is a fairly small component of the total egg mortality, with consumpto 3% of the total egg production tion estimates ranging from 4% (Brodeur et al., 1991). Pollock are prey to many groundfishes and are a critical prey component of the ecosystem both in the Gulf of Alaska and eastern Bering


189

Sea (Springer, 1992; Livingston, 1993). Marine mammals and seabirds depend on strong year classes of pollock in the eastern Bering Sea. Juvenile pollock, primarily age 0 and age 1, are the main prey of northern fur seals (about 80% of stomach content is pollock), and feeding on pollock varies with the recruitment level of juveniles (Sinclair et al., :1996). Swartzman and Haar (1983) proposed an interaction between the fishery harvests, cannibalism and fur seal feeding that includes the fishery removing older cannibalistic fish, thus reducing mortality of young pollock and making them more available to fur seals.

3. POPULATION ECOLOGY Geographic range considerations and aspects of macroecology (Brown, 1995) are an important population characteristic of species. Species with broad niches may become both widespread and locally abundant (Brown, 1984), and large ranges, abundance and invasion ability are linked characteristics within a species (Lawton et al., 1994). Species with extraordinary invasion abilities are generally those best adapted for marginal habitats (MacArthur and Wilson, 1967). As described below, these concepts are especially relevant to pollock population biology., Our treatment of population dynamics covers stock assessment, harvest management, and recruitment. Stock assessment is the tool used to detect changes in abundance in the population, estimate natural and fishing mortality and make harvest recommendations. Since this study is key to understanding the pollock population, this section is covered in detail. Recruitment and mortality are the main factors causing changes in abundance in the population.

3.1. Macroecology

Walleye pollock has a broad geographical niche. Pollock is comrrionly associated with the outer shelf and slope regions of coastal waters, but they are capable of utilizing a wide variety of habitats including nearshore eelgrass beds (Sogard and Olla, 1993), large estuaries like Puget Sound, coastal embayments, and open ocean basins such as the Aleutian Basin of the Bering Sea. Adults are often described as semi-demersal, although in some areas they are strictly pelagic (Bakkala, 1993). In some regions pollock is even considered a benthic fish (Tsuji, 1989). As noted previously, pollock commonly feed on a wide assortment of prey from pelagic copepods to epibenthic organisms and pelagic and demersal fishes.

K. M. BAILEY ETAL.

190 Larvae Posehatchage (days) 0

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Figure 3 The spatial patchiness of walleye pollock eggs and larvae as indicated by Lloyd's index of patchiness (after Stabeno et al., 1996a).

Pollock are distributed from Puget Sound to the northern Bering Sea and across the North Pacific Ocean and are most abundant in the eastern Bering Sea and the Sea of Okhotsk. The local abundance of pollock is usually high and they often dominate regional groundfish communities. Given its ecological plasticity, broad range and high levels of abundance, pollock appears to be a classical generalist species capable of invading and adapting to marginal habitats. Spatial patchiness of the early life stages varies with size (and age) (Stabeno et al., 1996). Based on Lloyd's index of patchiness (the ratio of mean crowding of a population to its mean density; Lloyd, 1967), patchiness increases from the egg stage to newly hatched larvae, and tends to decrease through the late larval stage (Figure 3). By the early juvenile stage, patchiness increases again as fish begin to school. Three types of larval pollock patches were described in the above study: those created by the interaction of larvae with time-dependent currents, those associated with eddies, and those associated with geographic structure, such as islands. In the laboratory, the formation and maintenance of larval pollock aggregations has been related to prey patches (Davis and Olla, 1995), and as well, in field studies the distribution of pollock larvae often coincides with that of their prey (Nakatani and Maeda, 1983). The geographic distribution of pollock varies with ontogeny. The distribution patterns of several year classes in the eastern Bering Sea were tracked to examine ontogenetic dynamics in distribution of different year

191

POPULATION STRUCTURE AND DYNAMICS OF WALLEYE POLLOCK -170'00'

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Figure 4 Relative distribution patterns of the 1982 year class of walleye pollock in the eastern Bering Sea: (a) larvae in June from ichthyoplankton surveys, (b) age-0 juveniles in autumn from midwater trawl surveys, (c) age-1 in summer from bottom trawl surveys, (d) age-3 in summer from bottom trawl surveys. 0 shows area of highest abundance. --- shows approximate region surveyed.

classes (Bailey et al., in press). For example, the 1982 year class was .found predominantly in the outer-shelf region of the southeastern Bering Sea as larvae (Figure 4a); as age-0 juveniles they had moved northward and inshore (Figure 4b). As age-1 fish, they had distributed themselves farther northward and also a large portion of the population was found shoreward (Figure 4c). As age-3 fish in summer, a portion of the 1982 year class returned to the southern outer-shelf region, but a large number of fish remained in the northeastern outer shelf (Figure 4d). Similar patterns were observed with other year classes. Overall, these distribution patterns indicate generally northward movements of age-0 and age-I1 fish.

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However, distributions vary considerably between years and sometimes these age groups move shoreward. By a g e 3 it appears that pre-spawning fish move southward again. Hydrographic fronts, temperature, prey availability and depth influence the distribution of adults and juveniles (Bailey, 1989; Swartzman et al., 1994; Brodeur et al., 1997). The interacting effects of temperature, light and prey levels on the vertical migratory behaviour of pollock have been well-studied in the laboratory (Olla et al., 1996; Sogard and Olla, 1996a and b). In the sea, the geographical distribution of pollock is limited by low bottom temperatures, from 0°C to 2”C, as shown by distribution of commercial catches (Figure 5 ) , and catches of juveniles and adults in research surveys (Francis and Bailey, 1983; Wyllie-Echeverria and Wooster, 1998). However, when bottom temperatures are very low, concentrations of pollock may reside in warmer water above the cold pool (Swartzman et al., 1994), but juveniles are sometimes found in the cold pool (Francis and Bailey, 1983; Wyllie-Echeverria and Wooster, 1998). Kihara and Uda (1969) and Maeda (1972) believed that pollock in the Bering Sea are associated with the Alaska Stream “extension water mass” (temperature 3 4 ° C and salinity 32 to 34 ppt). Detailed studies have not been conducted at high temperatures, but the range of pollock appears to be limited by temperatures of 10°C to 12°C. Changes in spatial distribution, such as patchiness, geographic distribution and ontogenetic changes in vertical distribution play a key role in ecological interactions. Patchiness of larvae may influence the aggregation of predators, a potential density-dependent regulating factor. The mechanisms that cause larval patchiness, such as eddies, may increase retention of larvae in favourable nursery areas. In daytime, juvenile pollock aggregate in dense concentrations near the bottom and are more diffuse and shallow at night (Brodeur and Wilson, 1996). Aggregating behaviour of juvenile pollock has been shown from laboratory studies to have energetic implications that may influence growth rates (Ryer and Olla, 1997). The geographic distribution of juveniles may affect cannibalism as a result of horizontal overlapping of juveniles and adults, and thus be a factor in recruitment (Francis and Bailey, 1983). The vertical overlap of juveniles and adults, influenced by water column structure and prey availability, is also an important factor in cannibalism (Bailey, 1989). Characterization of pollock as a colonizing species lends some support to suggestions that the rapid increase in pollock occurring in the late 1960s was related to the harvesting of Pacific ocean perch (Somerton, 1978) and Pacific herring, thus reducing competition and initiating an “ecological release” (MacArthur and Wilson, 1967). Pollock could be considered a classic r-selected species with opportunistic rapid growth, early maturity and high fecundity that is able to rapidly occupy a niche opening.

193


60N

55N

50N

60N

55N

50N

Figure 5 Monthly catch rates by Japanese fishing vessels in 1"X 1" statistical squares during June 1976 and June 1978. Shaded area is the boreal bottom water mass (sometimes known as the cold pool, less than 2°C and 31-32 ppt). 0 represent catches >lo00 mt and 0 are catches between 16999 mt).

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In the Gulf of Alaska, an increase of pollock in the mid-1980s coincided roughly with a regime shift occurring in 1977 to 1978 (Hollowed and Wooster, 1995) and possibly was associated with good conditions for juveniles of the 1976 to 1978 year classes. Recently, however, pollock stocks in the Gulf of Alaska declined markedly until 1995 with a small increase since then. Curiously, the age of first maturity has increased (Megrey, 1988) in spite of declining density. Since the mid-1980s juvenile survival has been relatively poor; from 1980 to 1985 age 0-2 mortality was lower than the 1980 to 1991 mean value for five of six year classes. By contrast, from 1986 to 1991, five of six year classes had higher than the mean long-term mortality (from data in Bailey et al., 1996~).These data tend to indicate trends of increasing predation pressure on juveniles or eroding environmental conditions for juveniles and adults. Range expansions may be limited by physical impediments, such as temperature, salinity and substratum availability, and by biological factors including the presence of competitors and predators. There is also historical structure in the environment, such as changes in the occupation of niches owing to disease and environmental events. Some populations may expand their range as they become more abundant, although others do not show this trend but show increases in local density. For example, species with highly specialized niches may not expand readily compared with species that have more generalized requirements. Ontogenetic changes in the relationship of range expansiodcontraction and abundance may also exist; for example, Schneider et al. (1997) found that the range of juvenile Atlantic cod did not contract with decreasing abundance, whereas the opposite is true for adult cod (Swain and Wade, 1993; Atkinson et al., 1997). The relationship of distributional range and population abundance has not been formally examined for most pollock stocks, as historical records over a long time span and over the whole range of pollock are not currently available. However, based on the historical declines of abundance in populations outside the major fishery area, while the central area of pollock biomass has remained relatively stable, pollock can be characterized as a species with a main central range and numerous fringe populations; the overall range of the population may not shrink with population decreases unless local fringe populations become extinct. Within local populations there may be a positive range and abundance association, similar to that found for Atlantic cod. Tsugi (1989) asserted that during times of increasing commercial catch levels (and therefore abundance) pollock expand into adjacent waters. Likewise, Stepanenko (1997) reports range expansions of pollock in the Bering Sea related to increasing abundance and warming temperatures. However, range con-


195

tractions with declining abundance may not occur for juvenile pollock due to their dispersal potential. Instead, the range extent may be controlled by transport and other environmental factors. Wyllie-Echeverria (1995) related invasions of juvenile pollock into the Chukchi Sea with warm ocean conditions. As described below, density-driven migrsations into new or previously colonized habitats have been proposed, but definitive data supporting these movements are lacking. 3.2. Population Dynamics

Most knowledge about walleye pollock on a stock-scale comes from stock assessment information and modelling. This information is used to monitor the status of the stocks and to evaluate the effects of fisheries upon them. Detailed history of the pollock fisheries and stock assessment procedures are found in Bakkala et al. (1987), Quinn and Collie (1990), Marasco and Aron (1991), Hollowed et ai. (1997) and Wespestad et al. (1997). Analysis of the population dynamics of pollock involves using fisheries survey data and catch-at-age population models that make some assumptions about stock structure and natural mortality to estimate trends in fishing mortality, recruitment and population abundance. There are two types of survey used in stock assessment of pollock: a hydroacoustic (sonar) survey assesses the midwater component o f the population and a bottom trawl survey assesses the near bottom (within 3 m off bottom) component. Surveys provide a consistent sampling of fish from year to year, give information on abundance in areas not commercially fished, and contribute information on small, pre-recruit fish that are not caught by the commercial fishery. National Marine Fisheries Service (NMFS) survey data used in stock assessment date back to the 1970s. Hydroacoustic surveys occur annually in the Bogoslof area and in Shelikof Strait. The eastern Bering Sea is surveyed with hydroacoustics every 3 years. In hydroacoustic assessment, electrical energy from a transmitter is converted into acoustic energy by an underwater tramducer. The energy reflected from fish is converted back into electrical energy and the signal is processed. Generally, NMFS survey transect lines are run 5 to 10 nautical miles apart. Midwater trawls are used to collect samples for species identification and length and age composition. Typically, for NMFS bottom trawl surveys stations are located 20 nautical miles apart. The gear is a US east coast-type otter trawl with a 31.4 m long footrope, equipped with a small mesh liner (3.2 cm str,etched mesh). The catch from each tow is sorted by species, weighed and counted. Otoliths are collected for age and growth information. Total biomass is

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estimated using an area-swept method (length of the net opening multiplied by the distance the net is towed). The density of fish from all survey stations is averaged and extrapolated to the surveyed area to provide a total biomass estimate. In the Bering Sea, bottom trawl surveys are annual, while in the Gulf of Alaska, they are triennial. Stock assessment from the commercial fishery is based on catch-at-age data. Standard virtual population analysis (VPA, also known as cohort analysis) (Pope, 1972) was originally used. VPA is a back-calculation procedure where catch-at-age data from the fishery is used in Baranov catch equations (Ricker, 1975) along with assumptions of natural mortality and terminal fishing mortality to estimate to numbers-at-age, exploitation rates and biomass levels over time (Quinn and Deriso, 1999). A basic assumption of VPA methods is that the catch-at-age data are measured without error. More recently, statistical catch-at-age models, summarized in Quinn and Deriso (1999) have been utilized. In the statistical age-structured (SAM) and stock synthesis models, the catch-at-age in any given year is assumed to be measured with an amount of error. The stock assessment model contains parameters for recruitment, fishing and natural mortality, selectivity of the gear, and possible stock-recruitment relationships. These stock assessment methods are also fitted to the catch, survey and other information to estimate parameters. In the more recent forms of the analysis hundreds of parameters are estimated by the model, mostly dealing with year-to-year and age-specific deviations in coefficients such as selectivity and catchability. The main advantage of the current stock assessment models is their ability to integrate the various sources of information into a single framework. For the purpose of stock assessment and management, walleye pollock in the US EEZ is divided into four stocks: eastern Bering Sea (EBS), Aleutian Islands (AI), Bogoslof Island-Aleutian Basin (AB) (Figure 6), and Gulf of Alaska (GOA), (Wespestad et al., 1997; Hollowed et al., 1997). Catches in the eastern Bering Sea have been far higher than those in other regions, except during a period in the late 1980s when Aleutian Basin (Donut hole) catches were very high. Different assessments are made for these stocks because of differences in spawning time, weight-at-age, fecundity and other characteristics. Furthermore there are seasonal allocations in some fisheries. There are assessment problems related to stock structure for each of the stocks. The stock structure of the Bering Sea as a whole is poorly understood for assessment purposes (Wespestad et al., 1997). On the Russian side, the western Bering Sea shelf has a unique walleye pollock stock of lesser abundance than the eastern Bering Sea. In the northern Bering Sea (mainly in Russia), there is thought to be intermingling of


197

3000000

2000000

.-c% f- : m c

1000000

i

.

0 1984 1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996

’

Year . _

---

Figure 6 Time trends of catches of walleye pollock in waters off the coast of

Alaska. eastern and western Bering Sea pollock. With current large catches coming out of this area, there is concern about impacts on the eastern Bering Sea population. For current management considerations, the Aleutian Basin (or Donut Hole or Central Bering Sea) is thought to contain fish originating both in the east and the west (Wespestad et al., 1997). These fish can be either a separate stock or stocks with some intermingling with the shelf populations or a spillover of immigrants from strong cohorts (such as 1978,1982 and 1984) from the shelves. Some component of the Aleutian Basin “stock” travels to the vicinity of Bogoslof Island each year. Large fislheries in the Aleutian Basin and Bogoslof region in the late 1980s, led to substantial reductions in these populations. Recent surveys have shown little biomass in the Bogoslof or the Aleutian Basin proper. One exception was a relatively large hydroacoustic survey biomass of 1.1million mt in the Bogoslof region in 1995, which was confirmed by a replicate survey. It is suspected that some rebuilding of this population occurred as a result of a strong 1989 year class. The Aleutian Islands region, a broad area stretching from 170W to 170”E, contains a small pollock population (Wespestad et al., 1997). Trawl surveys taken at roughly three-year intervals (1980,1983,1986,1991,1994,

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1997) covers this area during the summer. The winter-roe fishery has been moving farther to the west and possibly catches Bogoslof fish as well as Aleutian Islands fish. Wespestad et al. (1997) suggest that much of the 1994 Aleutian Islands catch was Bogoslof fish. The proximity of eastern Bering Sea fish to the Aleutian Islands region and the variable movement of eastern Bering Sea fish create further problems for the Aleutian Islands assessment, in that data in this region contain contributions from both the Aleutian BasidBogoslof and eastern Bering Sea stocks. The Gulf of Alaska pollock population is subdivided into WesterdCentral and Eastern Gulf stocks (Hollowed et al., 1997). Little information is available about the latter so recommendations are inferred from the WesternKentral Gulf based on relative biomass distributions in the two areas. The most recent assessment has shown a large increase in biomass owing to strong appearances of the 1988/89 and 1994 year classes in the WesterdCentral Gulf. The 1989 year class did not appear there until 1994 but has been steady since then, raising the possibility of migration from the eastern Bering Sea (which had an exceptionally strong 1989year class), among other possible reasons. Alternatively, a portion of the strong adjacent 1988 year class has been mis-aged as these fish have become older than 6 years of age. Recommended catches are partitioned among the Shumagin, Chirikof and Kodiak areas in relation to relative biomass in the most recent bottom trawl survey. These distributions are variable over time. Another stock issue relates to a population of pollock discovered in Prince William Sound (PWS). It is not known whether these fish are accounted for in the regular bottom trawl survey as a part of the Westendcentral or Eastern stock or whether they are a separate stock component altogether. A stock/spatial issue emerges in these assessments relative to marine mammal and seabird populations. In the eastern Bering Sea, a Catcher Vessel Operational Area (CVOA) was installed in 1992 to provide catcher vessels with a better opportunity to harvest pollock (compared to larger factory trawlers and motherships). This action has tended to increase fishing effort in areas adjacent to endangered Steller sea lion and seabird populations. At the current time, there is little scientific evidence about the effects of concentrated fishing in time or space on pollock, marine mammal or seabird populations. Nevertheless, concern has been expressed and further management actions may be taken to reduce any risk. A multiple-area age-structured model was developed to provide abundance and exploitation information for the entire Bering Sea (Quinn and Wespestad, unpublished reports to the International Pollock Workshops 1992, 1994). The model is based on a back-calculation procedure similar to cohort analysis. It has three regions (western Bering Sea, Aleutian Basin, and eastern Bering Sea) with interchange between the western

.

199


-

1.5E + 07

-EBS

1.OE + 07

A

--

I

m m

0

5.OE

I O.OE

Basin Elomass

-8-Bogoslof Stmey Biomass Model Biomas

5

C

+ 06

0

EBSSurvey Biomass WBS Mod~l Biomass Japanese ( F U E

I

I

+ 00 1978

1980

1982

ISM

1-

1988

1990

1992

Figure 7 Biomass estimates from a multi-area, cohort analysis model (Quinn and

Wespestad, unpublished data). Some of the data series used in fitting the model are also shown: biomass estimates from the Bogoslof hydroacoustic survey (expected to be 77% of the modelled Basin biomass shown), biomass estimates from the eastern Bering Sea triennial trawVhydroacoustic survey, and Japanese CPUE data from trawlers operating in the Basin (adjusted by the model's estimated catchability for this gear).

Bering Sea and Aleutian Basin and the eastern Bering Sea and Aleutian Basin. Biomass in Bogoslof is assumed to be a constant proportion P E of the Aleutian Basin biomass and is also interpreted as the proportion of Aleutian Basin fish which originated in the eastern Bering Sea. Migration is defined in terms of the proportion of Aleutian Basin fish at age a which moved from the shelf to the Aleutian Basin at the end of the previous year. This proportion is estimated within the model as a linear function starting at the value 1 at age 5 and ending at the value 0 at age 1:l. The rationale for this function is that Aleutian Basin fish are older than shelf fish and do not appear in the Aleutian Basin before age 5 and that there does not seem to be much recruitment of older fish to the Aleutian Basin. The model integrates catch, (catch per unit effort) and survey information from all three regions. Estimates of biomass (ages 3-t) shown in Figure 7 show a maximum Aleutian Basin population of about 7 million mt in 1986 declining to under 1million mt by 1992. In contrast the eastern Bering Sea population was at least twice as large in 1986 and its relative decline was much smaller than that for the Aleutian Basin. The western Bering Sea population was about one-half the size of the Aleutian Basin population in 1986 but became larger than the Aleutian Basin population in 1990,

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K. M. BAILEY ET AL.

because its biomass has been fairly stable. The great decline in the Aleutian Basin population is attributed to a much larger exploitation rate than those on parts of the shelf. Biomass estimates from the model are quite sensitive to choices for model parameters and several combinations of parameters explain the data comparably. Thus, the single-area assessment models are considered superior at the present time, pending better information on stock structure, movement and natural mortality. Natural mortality for eastern Bering Sea pollock is traditionally assumed to be 0.45 at age 2 and 0.3 at older ages. In the Bering Sea multi-area model, natural mortality of 0.9 at age 1, 0.45 at age 2 and 0.2 at other ages was assumed. A lower natural mortality is needed in the multi-area model because part of natural mortality in the traditional model is essentially the loss of fish from the shelf to the Basin. For the Gulf of Alaska, the traditional choice for natural mortality is 0.3 for ages 2 and older. A multi-species predation model has been developed for Gulf of Alaska pollock to investigate this assumption (Hollowed et at., 1997). Using consumption rates of pollock by arrowtooth flounder, Pacific halibut and Steller sea lions, the authors extended the use of the stock synthesis model to include predation mortality. Their results showed that estimated natural mortality ranged from 0.63 to 0.99 at age 1, to 0.25 to 0.49 at ages 3 and older. However, their results were based on limited data about consumption rates and were viewed as preliminary. Nevertheless, their results show that natural mortality is probably not constant by age or by year. Furthermore, changes in natural mortality are confounded with changes in recruitment, so better understanding of multi-species interactions would improve understanding of pollock population dynamics. In particular, current high abundance of flatfishes in the Gulf of Alaska and eastern Bering Sea may be influencing biomass and recruitment of pollock. Fishing mortality is well understood for the eastern Bering Sea and Gulf of Alaska pollock populations because catch and biomass are both measured directly. Catches are kept low via conservative procedures used by the North Pacific Fishery Management Council, wherein scientists make recommendations about conservative harvest levels and managers recommend allowable catches under these levels. For Gulf of Alaska pollock, fishing mortalities for fully recruited fish has generally been less than 0.25 (Hollowed et al., 1997), meaning that average exploitation rates have been of the order of 10 to 15% or less. For eastern Bering Sea pollock, average exploitation rates have ranged between 7 and 22%, below the maximum sustainable yield (MSY) level of 30% (Wespestad et al., 1997). Furthermore, these rates are lower than those found in several other fisheries on gadids (Wespestad et al., 1997).

201

POPULATION STRUCTURE AND DYNAMICS OF WALLEYE POLLOCK 4.5

,

1

2.5

4

2

3.5

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c

- 3

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2.5

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8

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6

.-a

1 .-P c

B

1.5

v)

1

0.5

0.5 0

0

1967

1972

1977

1982

1987

Year Class

Figure 8 Year class abundance as age-2 recruits (bars) compared to spawning biomass (line) of walleye pollock in the Gulf of Alaska, showing the dramatic effects of year class strength on population biomass.

3.3. Recruitment For all of the major pollock groupings stock fluctuations are strongly influenced by intermittent recruitment of strong year classes. For example, Figure 8 shows the ‘‘logged’’ impact of a series of strong year classes on stock abundance in the Gulf of Alaska, as well as the drop in abundance related to subsequent recruitment of relatively poor year classes. In the eastern Bering Sea the 1978 year class comprised 67% of the pollock population in 1981 and 53% of the population in 1982 (Figure 9). Many regions share the same strong year classes, for example 1978 was a strong year class in the Gulf of Alaska, Aleutian Basin, eastern Bering Sea, western Bering Sea and Sea of Okhotsk. Likewise 1982, 1984 and I989 were strong across the Bering Sea although not necessarily in the Gulf of Alaska. Strong year classes in the Gulf of Alaska including 1976, 1977, 1979 and 1988 did not appear strong in the Bering Sea. Therefore, although some strong year classes are shared, there is not a consistent association of strong year classes among the Bering Sea and Gulf of Alaska populations that would clearly indicate density-dependent dispersal between these geographic regions or large-scale conditions favourable

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K. M. BAILEY ETAL.

35 30

25

1

p 20

.-

P

.=a 2 8 a

15

10

5 0 1964

1969

1974

1979

1984

1989

Year class

Figure 9 Year class abundance as age-2 recruits (bars) compared to spawning biomass (line) of walleye pollock in the eastern Bering Sea.

to recruitment in all areas. Within the Bering Sea, there appears to be an association of strong year classes among the different regions. The occurrence of similar strong year classes across the Bering Sea has been cited as evidence of panmixia within the Bering Basin (Dawson, 1994). However, Francis and Bailey (1983) showed some evidence for shifts in dominance of year classes between north and south portions of the eastern Bering Sea shelf. At the extreme ends of the range of pollock (e.g. Puget Sound), the year classes of good recruitment (1972 to 1975) were quite different from those in the Gulf of Alaska and Bering Sea. Age-specific life tables for walleye pollock in the western Gulf of Alaska for the 1980 to 1991 year classes were compiled to perform exploratory key factor analyses (Bailey et al., 1996~).Early larval mortality was significantly correlated with generational mortality (-In(Recruits/Eggs)), but patterns in juvenile mortality were also similar to generational mortality, and in some years were clearly dominant in determining the fate of a cohort (Figure 10). Density-dependent mortality was indicated only for the late larval to early juvenile stage. Time trends in juvenile mortality were associated with the increasing abundance of arrowtooth flounder, a major predator. These authors proposed that pollock recruitment levels could be influenced at any life stage, but

203


PerallK

1

Juvenile k k

! =Larval

Year Class Figure 10 Results of key factor analysis, comparing annual values of generational mortality ( K ) , larval mortality (kz + k3) and juvenile mortality (k5 + k6) (from Bailey et al., 1996~).

depends partly on a sufficient supply of individuals from earlier stages of development. Forecasts of future abundance and biomass in the eastern Bering, Sea and Gulf of Alaska are currently made by a linear prediction of recruitment from relative estimates of age-1 abundance from the bottom trawl survey and hydroacoustic survey respectively. Recently, these fisheries have been supported by only a few strong year classes, which has made forecasts of total abundance more dependent on the forecasts of recruitment. This approach is advantageous in that a direct measurement of the year class at age 1 is used and the linear relationship has lbeen strong based on R2 values. Disadvantages include the presumably large measurement errors in age-1 abundance from a bottom trawl survey and the likelihood of inter-annual variability in the linear relationship. These problems stem from a finding that pollock at age 1 in the Bering Sea are only partially vulnerable to bottom trawl gear, because the survey is conducted in deep water, although many young pollock typically reside higher in the water column (Smith, 1981). In the Gulf of Alaska, the survey is conducted to assess abundance of the spawning stock, and only a portion of the age-1 stock may be assessed if their distribution does not overlap completely with spawners. The major factors that affect recruitment to a fish population

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20 4

+ Observed Expected -Standard

1978

h

16

14

OY 0

I

1

2

3

4

5

6

7

8

9

10

Spawning Biomass ( a t )

Figure 11 Ricker spawner-recruit relationship for pollock in the eastern Bering Sea without and with environmental data. The predicted recruitment using environmental data is shown as a smoothed line. The strong 1978 year class is noted (after Fair 1994).

are egg production effects, biological effects on early life survival, environmentaYoceanographic effects on early life survival (Heath, 1992) and overlapping of juvenile fish with their predators. The first factor relates to maturity, fecundity and spawning characteristics. Spawning biomass can be used as a proxy for egg production as long as fecundity is roughly proportional to fish weight. Biological effects on early life history are generally referred to as density-dependent effects and include such mechanisms as cannibalism, density-dependent predation, and within-species competition for food and/or space. Such effects can be explored by fitting spawner-recruit relationships. Since adult pollock in the eastern Bering Sea are known to be cannibalistic (Dwyer et al., 1987; Livingston, 1993), there should be a dome-shaped or Ricker-like spawner-recruit relationship if the effect is strong (Quinn and Deriso, 1999). Early analyses showed that a Ricker relationship can be fitted to eastern Bering Sea pollock spawner-recruit data (Quinn and Collie, 1990; Wespestad, 1995; Quinn and Niebauer, 1995), although the spread of data around the modelled curve is broad. Figure 11 shows a fit of the Ricker relationship for estimates of eastern Bering Sea pollock recruitment and spawning biomass obtained from catch-age analysis using data through 1994 (after Fair, 1994). The Ricker fit is statistically significant, suggesting that density dependence is present.


205

The most recent stock assessment (Wespestad et al., 1997), however, did not show a strong Ricker relationship with data up to 1997. Part of the problem is a result of using only part of the time series (data starting in 1978 since there is some question of the accuracy of prior data), but some of the discrepancy is owing to recent strong year classes in 1989 and 1996 being spawned from somewhat large spawning biomasses. Perhaps recent compression of the age composition of the pollock population has made cannibalism less of a factor in recent pollock dynamics. In the Gulf of Alaska, no clear spawner-recruit relationship emerges, and it is of interest that cannibalism is not a major factor there. The effects of density dependence on pollock dynamics were modelled by Wespestad and Quinn (1996). They performed deterministic, retrospective simulations of the eastern Bering Sea population under different levels of density dependence and fishing mortality. Their results showed that fishing has little effect on pollock recruitment, but when it did have an effect it tended to increase recruitment. The reason for this phenomenon in the simulations is that very high levels of pollock spawning biomass would result in decreased recruitment because of increased mortality from cannibalism. Environmental and oceanographic effects on recruitment are also well studied. Larval mortality has been loosely linked to temperature (Bailey et al., 1996c), and storms (Bailey and Macklin, 1994). Larval growth and survival has also been associated with prey levels for early feeding lairvae (Haldorson et al., 1989; Canino et al., 1991; Bailey et al., 1995; Paul et al., 1997). Better environmental conditions may thus be conducive to better development and growth, and perhaps greater ability to avoid predation. But mismatches between the emergence of larval pollock and their food (Brase, 1996) and episodes of catastrophic mortality (Bailey et al., 1995) have also been shown. Since the bulk of spawning in some stocks, such as Shelikof Strait, takes place over a short 2-week period, an episode of high larval mortality can have a significant effect on the survival of an annual cohort. Correlation studies indicate that environmental factors appear to significantly affect eastern Bering Sea pollock recruitment (Quinn and Niebauer, 1995). Seven environmental variables were used in the analyses: air temperature, bottom temperature, sea surface temperature, ice colver, wind, the Southern Oscillation Index and the Pacific North American pressure index. High pollock recruitment coincided with above average air temperatures and reduced ice cover when pollock are about age 1, suggesting that oceanographic conditions associated with warmer temperatures in the Bering Sea during the early life history are conducive to higher recruitment (Quinn and Niebauer, 1995). Ohtani and Azurriaya (1995) found similar results with temperature during the first winter and

206

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fecundity closely related to recruitment. These results are in accord with the current understanding of the physical processes of the eastern Bering Sea (Niebauer and Day, 1989). As these processes are atmospherically driven, air temperature is likely to be a proxy variable representing those physical processes, which follows empirically that high correlations occur among these environmental variables. Francis and Bailey (1983) proposed a complex recruitment process where the interaction between temperature over the shelf and spawning location puts eggs and larvae in different current regimes, affecting larval transport patterns, and the overlap of cannibalistic adults and juveniles. Consistent relationships between environmental variables and pollock recruitment occur when using monthly, quarterly or annual environmental breakdowns. The most consistent relationships occur with annual averages of environmental data, suggesting that an integration of the effects of environmental variables occurs in determining pollock recruitment. Figure 11 shows predicted recruitments from fitting a generalized Ricker spawner-recruit model including environmental data, showing that both biological and environmental factors appear to be important. Similar studies with environmental and early life history variables have been conducted in the Gulf of Alaska and other regions as well. Megrey et al. (1995, 1996) showed that pollock recruitment in the Gulf was correlated with precipitation (as an indicator of eddies), an index of atmospheric sea-level pressure gradient, and wind mixing energy. Balykin (1996) correlated recruitment indices of pollock in the western Bering Sea to adult stock abundance and temperature during the first year of life, and Vasil’kov and Glebova (1984) associated recruitment variations of pollock of western Kamchatka to thermal variability during egg and larval stages. 4. POPULATION STRUCTURE

On short time scales, differences between populations may arise because of environmental influences on larvae and juveniles. Elemental composition of hard parts, parasite load, morphology or meristics may differ between areas because of differences in temperature, elemental composition of sea water, food and growth. Many of these differences result from plastic responses to environmental variability and may reflect only short-term population structure that is not related to the level of genetic differentiation among populations. Genetic differentiation is determined by rates of mutation, random genetic drift, natural selection and gene flow between populations, and is influenced as well by demographic structure (such as the type of connections among populations, population size,

POPULATION STRUCTURE AND DYNAMICS OF WALLEYE

POLLOCK

207

extinction rates, etc.). Although mutation is the ultimate source of genetic diversity, recurrent mutation by itself is not a strong force in creating differences between populations. When the number of breeding individuals in a population is small, random drift may lead to genetic changes because of the incomplete representation of parental genes in offspring. Recent studies of marine organisms indicate that population sizes may be highly variable for many species because of high larval mortality and because variability in ocean currents and food availability leads to highly variable recruitment (Hedgecock, 1994a and b). When the reproductive variance among families is large, even species with large census population sizes may have small effective population sizes. Random drift in populations of these species may therefore be important in creating differences between populations. Natural selection may lead to different results depending upon the kind of selection operating. For example, selection for local adaptation in low gene flow species may produce a mosaic of genetically distinct populations in coarse-grain environments, but the same mode of selection may produce genetic homogeneity in high gene flow species which experience the environment as finely grained. The genetic variants detected by most molecular techniques appear to be quasi-neutral relative to levels of natural selection and are therefore useful for estimating rates of gene flow between populations under some circumstances (see Waples, 1998). Gene flow tends to reduce genetic differences between populations and to counter random drift by homogenizing allelic frequencies between populal.ions and by increasing the effective size of local populations. An important consideration in the use of genetic methods for stock identification for management is that only a small amount of gene flow is needed to produce genetic similarity among populations that nevertheless may be demographically distinct (Waples, 1998). Many of the early models of genetic population structure incorporated migration between partially isolated sub-populations, but assumed that sub-population sizes were constant (e.g. Wright, 1931; Kimura and Weiss, 1964). These models have been used to a large extent to interpret the results of empirical studies and to estimate levels of gene flow between populations from molecular data. The importance of local extinctions and recolonizations, in addition to gene flow, random drift and selection, in influencing population structure has been recognized in the last few years and has been incorporated recently into population genetic models (Hanski and Gilpin, 1991). In this view, a species consists of a collection of sub-populations (a metapopulation), which are tied together by gene flow, but which respond to local environmental variability independently of other sub-populations. High rates of local extinction and recolonization may lead to an accelerated loss of genetic variability relative to a group of

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stable populations and may tend to genetically homogenize the subpopulations (Gilpin, 1991; Grant and Leslie, 1993). 4.1. Methods for Estimating Stock Structure

Several methods have been used to infer population structure. Nongenetic methods measure traits that reflect environmental influences during the lifetime of a fish and are often useful for identifymg short-term population structure. For example, tagging provides evidence of migration from one locality to another, while the analysis of parasites and trace element composition in calcified parts of a fish may provide evidence of separate areas of origin for adults on feeding grounds. Methods that measure phenotypic variables, such as meristics, morphology or behaviour, provide population information that reflects both environmental and genetic influences. Even when purely genetic information is available, understanding morphological and life-history variability is essential for comprehending the mechanisms shaping population structure (Grant et al., 1999). Several molecular genetic methods have been applied to delineating the genetic boundaries of fish populations. Since the 1970s, the electrophoretic analysis of enzymatic proteins has been widely used to infer population structure. This method detects Mendelian variants (allozymes) at a single gene locus and provides genotypic and allelic data to infer breeding structure within populations and to measure the amount of genetic differentiation between populations (see Ryman and Utter, 1987). Analysis of allozymes has been useful for studying levels of divergence on a scale of hundreds to a few million years. However, this method is limited in its ability to infer short-term population structure in species of marine fishes with high levels of gene flow. Small amounts of gene flow tend to produce genetic homogeneity among populations, even though the populations may be demographically independent. The direct analysis of DNA often reveals greater levels of genetic variability within populations than the analysis of the encoded proteins. One class of DNA that has been used to study fish populations occurs as a circular loop in mitochondria and carries a unique set of genes not found in nuclear DNA. Mitochondria1 DNA (mtDNA) lacks recombination during reproduction and is inherited from the maternal parent in fishes, so that gene lineages may be inferred from RFLP (restriction fragment length polymorphism) analysis with restriction enzymes, or from sequences of fragments that have been amplified with the polymerase chain reaction (PCR). In some cases, analysis of mtDNA may be more powerful than the analysis of allozymes because the smaller female effective


209

population size and haploid condition is expected to lead to greater amounts of random drift, and hence to larger differences between populations. The analysis of VNTRs (variable number of tandem repeats) shows promise for distinguishing populations. One class of VNTR genes, microsatellites, consists of repeated 1-4 nucleotide motifs with high rates of length mutation. Microsatellite sequences may be the most powerful Mendelian population markers yet developed for detecting short-term population events (Jarne and Logoda, 1996). Microsatellites typically exhibit much higher levels of variability than do other genetic marlkers, such as allozymes and mtDNA. In marine fishes, polymorphic microsatellite loci typically have 10 or more alleles and may have heterozygosities of 50 to 95% (Wright and Bentzen, 1994; Bentzen et al., 1996; Garcia de Le6n et al., 1997). These high levels of polymorphism, which reflect high mutation rates, may confer a greater ability to resolve some aspects of population structure than do the levels of polymorphism revealed by the analysis of allozymes or mtDNA (Bentzen et al., 1996; Ruzzante e,t al., 1996 a and b). Mutation rates at microsatellite loci appear to be three to five orders of magnitude greater than those for non-repetitive DlNA. Mutations are usually a result of replication slippage errors that cause size polymorphisms, rather than point mutations resulting in single base pair changes. The large numbers of low-frequency alleles at a locus, however, point to the necessity of using large sample sizes for population analysis (Ruzzante, 1998). Furthermore, microsatellite variants may be less prone to selection than are allozymes, which increases their value for estimating gene flow rates (Wright and Bentzen, 1994). The identification of demographically independent management units is often complicated by the use of different phenotypic and genetic met hods that individually may be capable of measuring only limited aspects of population structure. For example, vertebral number, which is generally elevated in fish developing at high latitudes in cold water, may indicate environmental conditions during early life history stages, but may not reflect adult migration or gene flow patterns. As a specific example, meristics in Atlantic cod vary significantly among populations on the Newfoundland and Labrador shelves in the northwestern Atlantic (Lear and Wells, 1984; Pepin and Carr, 1993; Templeman, 1981). The results of an analysis of mtDNA variability, however, failed to find genetic differences between these populations (Pepin and Carr, 1993). Yet the analysis of other kinds of DNA, such as mini- (Galvin et al., 1995) and microsatellite loci (Bentzen et al., 1996), and anonymous nuclear loci (Pogson et al. 1995), showed significant frequency differences between samples that were collected over distances as small as several hundred kilometres. The analysis of parasite distributions, elemental composition

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K. M. BAILEY ETAL.

and morphology may also indicate the existence of stock structure that may or may not correspond to patterns of genetic differentiation between populations. The present-day population structure of a fish may reflect mechanisms and events operating on several different temporal and spatial scales. Some events, such as Pleistocene coastal glaciation, may have led to large-scale geographic differences in morphology, life history or demography that may be reflected in temporally stable genetic differences between populations. Other events, such as annual or decadal shifts in temperatures and current systems, food availability, predation and harvesting may lead to changes in geographic distributions and demography. Some of these population events may produce genetic variability between groups of fish that may be detectable with genetic methods. For example, populations of Atlantic haddock (Melanogrammus aeglefinus) are characterized by a large amount of variability in annual recruitment and by a recent decrease in population size in the northwestern Atlantic most likely resulting from overharvesting (Clark et al., 1982). An analysis of mtDNA variability in archived scales collected in 1975 and 1985 and in fresh samples collected in 1995 revealed significant temporal variability in haplotype frequencies in populations on Georges Bank in the northwest Atlantic (Purcell et al., 1996). These results were interpreted to indicate that fish from genetically distinct populations episodically contribute to the Georges Bank population. Measurable genetic differences have been found on even smaller temporal and spatial scales. Ruzzante et al. (1996b) found frequency differences at microsatellite loci between larval cohorts of Atlantic cod in a retentive gyre of warm mixed water off Nova Scotia. The low level of differentiation, however, indicates that the samples were drawn from different components of a single population and not from different populations. 4.2. Phenotypic Population Structure

Phenotypic characteristics of pollock, as reflected in meristic and morphometric variability, both within small geographic regions and across much broader areas indicate population structure (Table 2). For example, Koyachi and Hashimoto (1977) and Hashimoto and Koyachi (1977) used differences in allometrics, vertebral, gill raker and fin ray counts to distinguish 11 to 12 groups of pollock across its range (Sea of Japan: western Hokkaido, two populations in northwestern Honshu, and Pormorskaya; Pacific coast of Japan: southern Hokkaido, northern Honshu, southern Kuril; southwestern and northern Sea of Okhotsk: Kamchatka Peninsula, eastern Bering Sea, Gulf of Alaska and west coast of Canada).

Table 2 Summary studies of pollock stock structure using phenotypic characteristics.

Author

Method

Area

Results

Ogata (1959)

meristic - vertebral counts

Sea of Japan and Pacific Ocean side of Japan

Iwata and Hamai (1972)

meristic - vertebral counts

Sea of Japan, Okhotsk Sea and Pacific Ocean near Hokkaido

Hashimoto and Koyachi (1969)

northern Japan

Janusz (1994) Temnykh (1991)

morphometrics - body length and other morphological features meristics and morphometrics morphometrics

Sea of Japan has 3 different stocks. Sea of Japan differs from the Pacific Ocean side 8 “local forms” 2 groups in the Sea of Japan 3 groups in the Okhotsk Sea 3 groups in the Pacific Ocean 3 groups discriminated

Temnykh (1994)

morphometrics

western Bering Sea and eastern Kamchatka

Ishida (1954)

morphometrics - otoliths

northern Sea of Japan, Okhotsk Sea and northern Pacific Ocean coast of Japan

Shaw and McFarlane (1986) morphometrics length-at-age

Sea of Okhotsk Sea of Okhotsk

British Columbia - Dixon Entrance, Strait of Georgia

3 stocks distinguished Southern Kurils population distinguished from northern Sea of Okhotsk western Bering and eastern Kamchatka stocks distinguished otolith size is larger in Sea of Japan pollock than Okhotsk Sea. Otoliths are similar between Sea of Japan and Pacific Ocean pollock 2 stocks discriminated - Strait of Georgia pollock are smaller. Little interaction between pollock north and south of Queen Charlotte Sound

Author

Method

Area

Results

Thompson (1981)

morphornetrics length-at-age

Saunders et al. (1989)

morphornetrics, life history

British Columbia - Dixon Entrance, Strait of Georgia and Queen Charlotte Sound British Columbia

L p d e et al. (1986)

morphometrics length-at-age

eastern Bering Sea and Bering Sea basin

Hinckley (1987)

spawning time and location; morphornetrics length-at-age; fecundity

Aleutian Basin and eastern Bering Sea shelf and slope

Mulligan et al. (1989)

spawning time and location

eastern Bering Sea and Aleutian Basin

Serobaba (1977)

morphometrics and rneristics

Dawson (1994)

morphometrics

northern, western, eastern and southern Bering Sea Bering Sea

3 separate stocks - each area contains its own distinct stock. Little mixing occurred between them separate stocks in Strait of Georgia, Hecate StraWDixon Entrance, Queen Charlotte Sound and western Vancouver Island northeastern slope and Aleutian Basin represent 1 stock distinct from other regions of the eastern Bering Sea 3 spawning stocks in the eastern Bering Sea - basin, northeastern slope and eastern shelf and slope 3 spawning areas separated in space and time: eastern Bering Sea southeastern Shelf, eastern Bering Sea northwestern shelf, Aleutian Basin different stocks occupy each region 3 stocks - eastern Bering Sea sheK Aleutian Basin, and Aleutian Islands

Janusz et al. (1989)

meristics and morphometrics

Nitta and Sasaki (1990)

morphometrics

Donut Hole and eastern Bering Sea shelf Donut Hole, eastern Bering Sea, near Japan

Gong et al. (1990)

meristics

Asian and Bering Sea

Wilimovsky et al. (1967)

meristics - fin ray and vertebral counts and morphometrics

entire Pacific Ocean

Koyachi and Hashimoto (1977)

meristics - fin ray, gill raker and vertebral counts

entire Pacific Ocean

2 stocks distinguished in Donut Hole and eastern Bering Sea characteristics distinguish 3 stocks, with about 90% classification accuracy Asian stock and Bering Sea stocks distinguished but stocks within these regions not distinguished morphometric - no strong evidence for discrete stocks meristic - no differences between Bering Sea and Puget Sound pollock. No differences between Gulf of Alaska and northern British Columbia pollock 12 sub-populations, including the Bering Sea and Gulf of Alaska

K. M. BAILEY ETAL.

214

55t

/ .....

40f;p 3!

4

5

6

8

7

9

10

Age

Figure 12 Mean length-at-age for male pollock in different regions of the Bering Sea, 1978 to 1983. Age is in years (after Lynde et al., 1986).

Within much smaller geographic regions there are also distinct groups. For example, around the islands of Japan and Sea of Okhotsk, Iwata and Hamai (1972) identified eight groups, and in the Sea of Okhotsk three stocks were distinguished (Janusz, 1994). Within the eastern Bering Sea including the eastern Aleutian Basin, three to five stocks have been distinguished using morphometrics and life history characters (Hinckley, 1987; Dawson, 1994). Lynde et al. (1986) showed differences in length-atage among fish collected in summer on the southeastern Bering Sea shelf and fish collected in the Aleutian Basin or northeastern Bering Sea slope region (Figure 12). Fish collected in the southeastern slope were intermediate in growth characteristics, possibly indicating mixing, and fish from the northeastern shelf were relatively small when young and large when older, perhaps resulting from ontogenetic migrations of Aleutian Basin fish. Within a fairly small region around Hokkaido Island of Japan, lwata and Hamai (1972) found eight local forms based on vertebral counts. Naturally acquired tags such as elemental composition of otoliths and parasite characteristics indicate restricted mixing among pollock juveniles and adults of different sub-populations (Table 3). For example, the chemical “fingerprint” of otoliths near the nucleus (deposited during early larval life) can be utilized to assign fish to their capture location as juveniles with 70 to 85% accuracy over broad regions of the eastern Bering Sea (Mulligan et al., 1989), indicating limited movement and

Table 3 Summary of studies of pollock stock structure using acquired characteristics.

Author Nakano et al. (1991) Mulligan et al. (1989)

Severin et al. (1995)

Method

Area

otolith chemistry: adults, whole otolith homogenates otolith chemistry: juveniles, inner early life otolith increments

eastern Bering Sea, western Bering Sea, Donut Hole eastern Bering Sea, (southeastern shelf, northwestern shelf, Aleutian Basin) eastern Bering Sea (Bristol Bay), Gulf of Alaska British Columbia - Strait of Georgia, west side of Vancouver Island, Queen Charlotte Sound and Dixon Entrance Sea of Okhotsk, Kommander Islands., Kamchatka Peninsula

Arthur (1983)

otolith chemistry: juveniles, outer otolith increments parasites

Avdeev and Avdeev (1989)

parasites

Miscellaneous Authors, see Figures 14 and 15

tagging studies

western and eastern Bering Sea, Japan

Results differences in 3 areas, little mixing differences in 3 areas, some mixing distinguish 5 areas, some mixing 3 stocks in this area: Strait of Georgia, Vancouver Island and Queen Charlotte Sound/ Dixon Entrance seven distinct groups within the northeastern Sea of Okhotsk, distinct groups in Kommander Islands, eastern Kamchatka and Shirshov Ridge broad movements, homing migrations to spawning site

216

K.

M. BAILEY H A L .

65 N

60 N

55 N

I

I

165 E

/

I

1

I

I

I

1

170 E

175 E

180

175W

17DW

165 W

I

Figure 13 Movement of walleye pollock tagged by Japanese scientists in the Bering Sea (from Dawson 1994). The dashed line represents the US-Russia conventional boundary. The area in the centre of the figure designated by a solid line is the area outside the exclusive economic zone of either country, known as the “donut hole”. Note: most tagging and recoveries occurred during the summer/autumn feeding season.

mixing of fish from different geographic regions. However, results of otolith composition studies should be viewed with some caution because of potential artefacts caused by sample preparation (Proctor and Thresher, 1998). Using parasite frequencies, adult pollock caught on the south side of Vancouver Island can be distinguished from those on the west side with about 75% accuracy (Arthur, 1983). In the Sea of Okhotsk, several different populations of pollock were distinguished based on parasite frequencies (Avdeev and Avdeev, 1989). By contrast, mark-recapture studies where pollock were tagged in summer indicate broad movement of individuals across areas of the Bering Sea (Figure 13). However, critical studies of spawning populations are rare. Tagging results thus far indicate potential for dispersal during the summer feeding period. Historical tagging studies do not indicate whether dispersal patterns in the Bering Sea are part of ordered seasonal migrations, or whether individual tag

217

POPULATION STRUCTURE AND DYNAMICS OF WALLEYE POLLOCK -136"OO'

-138"OO'

-140"OO'

-142"OO'

-144"OO'

-146"OO'

-148"OO'

48"OO'

46"OO'

44"OO'

42"OO'

-136"OO'

-138"OO'

-14O"OO'

-142"OO'

-144"OO'

-146"OO'

-148"OO'

Figure 14 Recapture distribution for an experiment in which 666 pollock were tagged and released (A) in the northern Sea of Japan on 17 April, 1968. Loca.tions and dates of recaptures are shown by ( 0 )(redrawn after Tsugi, 1989).

returns represent fish moving within large schools as migrating populations or as sole immigrants mixing with local populations. However, tagging studies around Japan support a model of dispersed feeding migrations and homing migrations to specific spawning areas (Figure 14; Tsuji, 1989).

4.3. Genetic Population Structure

Different molecular methods are variously suited to detecting population structure on different temporal and spatial scales, and the results of the various genetic studies of pollock must be interpreted in this light (Table 4). On a scale of thousands of years, allozyme studies have detected genetic subdivision across the North Pacific for several marine fishes that apparently was the result of geographic isolation by coastal glaciation during Pleistocene ice ages. During the last ice age, which peaked about 18000 years ago, much of the coastline of south central Alaska and the Kamchatka Peninsula was covered with ice and much of the present-day Bering Sea was dry land (CLIMAP, 1976). Allozyme divergence between

Table 4 Summary of stock structure studies on walleye pollock using biochemical genetics characteristics. Author

Method

Area

Mulligan et al. (1992)

mtDNA RFLP

eastern Bering Sea basin, Aleutian Islands and Gulf of Alaska

Shields and Gust (1995)

mtDNA sequencing

across Bering Sea and Gulf of Alaska

Grant and Utter (1980)

allozyme

southeastern Bering Sea and Gulf of Alaska

Johnson (1977)

allozyme

Iwata (1973)

allozyme

Iwata (1975a and b)

allozyme

Efremov et al. (1989)

allozyme

eastern Bering Sea and Gulf of Alaska northern Sea of Japan and north Pacific coast of Japan northern Sea of Japan and eastern Bering Sea northern Sea of Okhotsk

Powers, unpublished data, see text

mtDNA RFLP and DNA microsatellite

Bering Sea and Gulf of Alaska

Results Aleutian Islands and Donut HoleBogoslof, Gulf and Donut HoleBogoslof have informative differences, but sample sizes are small minor differences between eastern and western Bering Sea minor genetic differences between the two areas. No differences within the areas no significant differences found no differences found significant differences found between the two areas allozyme variability suggesting that aconitase could be genetic marker eastern and western Bering Sea distinguished using mtDNA, Gulf of Alaska and eastern Bering Sea stocks have informative differences using microsatellite DNA

POPULATION STRUCTURE AND DYNAMICS

OF WALLEYE POLLOCK

219

western and eastern North Pacific populations of Pacific herring (Crrant and Utter, 1984), Pacific cod (Grant et al., 1987), and chum salmon (Seeb et al., in review) appear to have originated from this glaciation. FsT (an index of population differentiation, the variance of allele frequencies among populations) values among populations of these species across the North Pacific are 0.15 or larger (Table 5 ) , however, the boundaries of the eastern and western groups differ among species. The genetic demarcations between eastern and western races of Pacific herring and chum salmon are around the Alaska Peninsula, but those for Pacific cod and pollock are in the western North Pacific Ocean or western Bering Sea. An east-west subdivision among pollock populations was detected at a siingle allozyme locus (Sod) in the combined studies of Iwata (1973,1975a arid b) and Grant and Utter (1980) and appears to be located on the Asian side of the Bering Sea or in the Okhotsk Sea. The precise location of the boundary is uncertain, however, because of the paucity of samples in these areas. Genetic population structure arising on shorter time scales may als80be apparent in the distributions of allozyme frequencies. This variation is difficult to measure, however, because the magnitude of sample error is often the same as the level of genetic differentiation between populations of high gene-flow species (Waples, 1998). Within the eastern North Pacific race of pollock, no significant allele-frequency differences were found between samples collected within the southeastern Bering Sea or within the Gulf of Alaska (Grant and Utter, 1980). However, a small, but significant, amount of allele-frequency heterogeneity was detected between populations in the southeastern Bering Sea and the Gulf of Alaska. FsT among samples within each region was 0.021, and is typical of values for several other marine fishes (Table 5; Waples, 1998) with apparently high equilibrium levels of gene flow between populations. Although the analysis of mtDNA is expected to reveal a greater amount of genetic population structure because of its maternal inheritance, little genetic structure has been found between populations of pollock in some studies of mtDNA variability. Mulligan et al. (1992) found 65 RFLP haplotypes in 168 fish from four localities: 1) the Gulf of Aliiska, 2) the Donut Hole in the middle of the Bering Sea, 3) Bogoslof Island in the southeastern Bering Sea, and 4) Adak Island in the Aleutian Archipelago. Two haplotypes occurred in 36% of the individuals and 51 haplotypes were represented by a single fish. Monte Carlo chi-square tests and cluster analysis of sequence divergence showed that the sample from Adak Island was distinct from the other three samples, which were. not distinct from each other after correction for multiple tests (Figure 15). Haplotypic diversity was 0.918, and FST among samples was 0.019 (Table 6), which is similar to that estimated from allozyme frequencies.

Table 5 Estimates of allozyme Hs (the mean sub-population heterozygosity over all populations), and population differentiation FsT (the variance of allele frequencies among populations (From Seeb et al., in preparation)).

No. of samples

Mean sample size

No. of loci

HS

HT

FS T

Walleye pollock

14

44.4

28

0.045

0.046

0.021

Yellowfin sole

3 16

93.3 68.9

31 31

0.089 0.051

0.090 0.053

0.004 0.043

Pacific herring Western Pacific

21 11

95.7 101.5

40 40

0.083 0.073

0.159 0.012

Eastern Pacific

10

89.3

40

0.098

0.007

Pacific ocean Perch

27

53.1

25

0.069

0.023

Pacific cod

11

71.2

41

0.025

9

75.2

41

5 9

95.6 97.8

19 10

Species

Atlantic cod

0.032

0.189 0.031

0.071 0.021 0.014

Geographical range

Reference

1

Southeastern Bering Sea, Gulf of Alaska same Japan, Bering Sea, Gulf of Alaska North Pacific Japan to eastern Bering Sea Gulf of Alaska to California Eastern Bering Sea to Washington Westem-eastern North Pacific Bering Sea to Gulf of Alaska North Atlantic

7

(recalculated)

8

2 3

4

5 6

1. Calculated from data in Grant and Utter (1980); 2. Seeb, unpublished manuscript; 3. Grant et al. (1983); 4. Grant and Utter (1984); 5. Seeb and Gunderson (1988); 6. Grant et al. (1987); 7. Mork er al. (1985); 8. Pogson et al. (1995).

221


r

Gulf of Alaska

4

Donut Hole

Bogoslof Island

I

I

0.6

A

d

1

0.45

a

I

0.3

k Island

1

0.15

1

0.0

Divergence

Figure 15 UPGMA clustering of genetic distances among walleye pollock stocks in the eastern Bering Sea and Shelikof Strait (after Mulligan et al., :1992).

Nucleotide diversity (n-) was 0.75. This relatively low level of divergence among haplotypes is typical of species of marine fishes that have had recent population origins (Grant and Bowen, 1998). A second study of mtDNA also points to low levels of genetic differentiation among pollock populations. Shields and Gust (1995) examined nucleotide sequence variability in a 76 base pair (bp) spacer region and in a 250 bp segment of the control region of mtDNA in 162 pollock from 32 localities. However, the seasonal timing of sample collection is unknown and samples may have been collected when fish from different spawning groups mix. Samples were grouped into six regions for analysis: 1) western Bering Sea, 2) northwestern Bering Sea, 3) the Donut Hole, 4) Aleutian Island chain, 5) southeastern Bering Sea, and 6) Gulf of Alaska. Data for the two segments were analysed separately since not all fish were successfully sequenced for both mt DNA segments. Twenty spacer-region haplotypes were found in 110 fish. A total of 83 fish (75%) had the same haplotype, and 17 of the 20 haplotypes each occurred in a single fish. A Monte Carlo chi-square test of haplotypic frequencies in the six regions was non-significant. Seventeen controlregion haplotypes were found in 140 fish, of which 114 had the same haplotype. Eleven of the 17 haplotypes each occurred in a single fish. A Monte Carlo test of the 11 most frequent haplotypes among the six

Table 6 Estimates of mtDNA haplotypic (h) and nucleotide (n,*) diversities and population differentiation, F,, among populations of demersal fishes. (From Seeb et al., in preparation.) h Species Walleye pollock Atlantic haddock Atlantic cod Greenland halibut Red drum Black drum Red snapper

No. of localities

Mean size

No. of haplotypes

4 5 5 4 7

42.0 10.8 26.6 25.8 40.0 63.0 37.5 46.8

65 21 22 10 22 99 37 68

11 8 9

TI,*

(%)

Mean

Pooled

F,,

Mean

Pooled

Reference

0.900 0.888 0.769 0.335 0.692 0.954

0.918 0.867 0.789 0.330

0.019

0.69

0.75 0.61

0.025 0.003 0.003 0.002 0.011 0.0

1 2 2 3

0.25 0.59

0.951 0.780 0.750

~

4 0.58 0.48 0.50

5

6 6 ~

Data from: 1. Mulligan et al. (1992); 2. Zwanenburg et al. (1992); 3. Pepin and Carr (1993); 4. Morgan et al. (1997); 5. Gold et al. (1993); 6. Gold et al. (1994).


223

regions also failed to detect significant heterogeneity. However, an a posteriori test of regions 1 and 2 versus regions 4 and 5 showed a significant difference for combined spacer- and control-region haplo types. The finding of very low levels of population differentiation is similar to the results of Mulligan et al. (1992), except for the lack of differentiation of Aleutian Island samples. In contrast, another study of mtDNA variability appears to have detected high levels of variability among populations in the Bering Sea and Gulf of Alaska. Quattro and Powers (unpublished data, reported in Macklin, S. A. 1999, Bering Sea FOCI Final Report, Environmental Research Laboratory Special Report, Pacific Marine Environmental Laboratory, Seattle, WA (in press); see also Bailey et al., 1997) sequenced two 300 bp segments each in coding regions of cytochrome-b and ATPase and found three haplotypes that were subsequently screened with PCRRFLP methods in 262 pollock from nine regions. These regions included 1) the Gulf of Alaska, 2) southeastern and 3) northeastern Bering Sea, 4) southeastern and 5 ) northeastern Aleutians, 6), Aleutian Basin, 7) western Bering Sea, 8) east Kamchatka Peninsula and 9) Sea of Japan. Regions 1, 3 and 5 were tested for temporal variability between samples collected in 1988 and 1991 or 1994, and none of these comparisons was signilicant. These data were pooled by region and the nine regions were then tested for frequency heterogeneity. The amount of differentiation among these regions was considerable; an analysis of molecular variance (AN'OVA, equivalent to FsT) showed that 14.5% of the total variability resulted from differences among samples. A cluster analysis of sequence divergences between samples indicated that the greatest amount of differentiation was between Gulf of Alaska/eastern Bering Sea populations and western Bering Sea/Sea of Japan populations. This major subdivision is consistent with allozyme data but not with some studies of mtDNA. Bering Sea populations and those in the Gulf of Alaska also appear to be differentiated from each other, but to a lesser extent. The analysis of microsatellite loci in pollock has apparently dei ected differences among genetically discrete groups of fish on finer temporal and spatial scales than has the analysis of allozyme loci or mtDNA. Pollock primers for two microsatellite loci found in Atlantic cod (Gmo-2, Gmo-145) were used by Villa and Powers (unpublished data, reported in: Macklin, S. A. 1999, Bering Sea FOCI Final Report, Environmental Research Laboratory Special Report, Pacific Marine Environmental Laboratory, Seattle, WA (in press); see also Bailey et al., 1997) to arialyse pollock from six localities that had been collected from 1990 to 1996. These included 1) western Bering Sea off Kamchatka Peninsula, 2) southeastern Bering Sea, 3) Pribilof Islands and 4) Shelikof Strait in the Gulf of Alaska. Eleven alleles were detected for Gmo-2 and 20 alleles

224

K. M. BAILEY ETAL.

were detected for Gmo-145. ANOVAs for both loci showed significant differences among the four regions, and paired a posteriori comparisons using 'lhkey's test showed significant differences between all pairs of samples except between regions 2 and 3. However, the time scale over which the samples were taken and the lack of temporal sampling make it difficult to interpret these results. Furthermore, in the case of Gmo-145 a null allele was responsible for the major east-west differences. The presence of a null allele indicates that the nucleotide sequences in the flanking regions of microsatellite markers were not identical between these populations, resulting in no PCR product for those individuals bearing the null allele. Cluster analyses of two genetic distances indicated that the sample from Shelikof Strait was more closely related to samples from the western Bering Sea than they were to samples in the southeastern Bering Sea. These results differ to some degree with the analysis of rntDNA and with results from allozymes. In a study of allozyme, mtDNA and microsatellite variability in three samples collected off Bogoslof Island, Shelikof Strait and in Prince William Sound, Seeb et al. (unpublished data) found significant differences among various combinations of samples for each method. Eleven of 31 allozyme loci were polymorphic (common allele frequency 0.095 or less) and the average heterozygosity among samples was 0.08. The sum of chi-square values over these 11 polymorphic loci showed a significant difference between the Gulf of Alaska samples and the Bogoslof sample, but no significant difference was found between the two Gulf of Alaska samples. Monte Carlo comparisons of frequencies of 68 composite PCR-RFLP haplotypes for the mtDNA genes cytochrome b, cytochrome oxidase and NADH Dehydrogenase 5/6 also revealed significant differences between the Bogoslof Island sample and the two samples from the Gulf of Alaska, but not between the two Gulf of Alaska samples. The results for the microsatellite loci were less certain because of the apparent presence of null alleles at two of the loci, Gmo-9 and Gmo-132, as revealed through inheritance studies. However, significant differences between Prince William Sound and Bogoslof Island ( P = 0.038) were detected for the Gmo-1 locus, which appeared to give better results than the other microsatellite loci. Together, these results reflect other studies in showing little differentiation among Gulf of Alaska populations and moderate amounts of differentiation between Gulf of Alaska and southeastern Bering Sea populations. These genetic studies permit a few tentative conclusions about the temporal and spatial patterns of genetic structure in pollock. As with several other fishes in the North Pacific, the imprint of isolation by Pleistocene glaciation appears to be visible in allozyme differences between Asian and North American populations and in mtDNA dif-


225

ferences in one of two studies where the extent of sampling permits this comparison. Microsatellite data, however, failed to reflect this major east-west subdivision. These data appear to indicate that Gulf of Alaska populations are more closely related to western Bering Sea populations than they are to geographically nearby southeastern Bering Sea populations. This is similar to microsatellite data for Pacific herring, which also failed to detect strong east-west differences that appeared in allozyme (Grant and Utter, 1984) and mtDNA data (P. Bentzen, unpublished data). The reason for the apparent genetic similarities between east-west populations across the North Pacific of both pollock and Pacific herring could be convergence in the microsatellite length alleles and not longdistance gene flow. Within the North Pacific races, small genetic differences have been detected between the Gulf of Alaska and the southeastern Bering Sea for pollock in a study of allozymes, in three studies of mtDNA variability, and in a study of microsatellite variability. The Alaska Peninsula and the Aleutian Archipelago are obvious barriers to gene flow between these two areas. Genetic differences between localities have been detected within these areas in some studies, but no clear picture of geographically stable populations has so far emerged. Several reasons may contribute to the lack of clear population definition. One reason is that for some molecular techniques, such as allozymes and mtDNA, only a small amount of gene flow between populations is required to produce the appearance of genetic homogeneity (Waples, 1998). The genetic structure detected by these methods is the result of random drift, which is small in large populations, and of gene flow, which is potentially large in marine species. Microsatellite loci, on the other hand, are often characterized by elevated mutation rates, which may produce population-specific frequency profiles that depict fine-scale population structure. Because of the nature of length mutatialns at microsatellite loci, however, allelic convergence is common SCI that same-length alleles in two fish may not be identical by descent, an assumption that is important for inferring genetic relatedness between populations from molecular data. More studies on the temporal/spatial stability of these markers in populations are also needed. Another reason for the lack of genetic definition may be that the samples for some studies were collected out of the spawning season. If the homing of pollock to specific spawning localities leads to genetically discrete populations, samples collected in mixed-stock (feeding) areas maly not resolve the genetic structure of breeding populations. Another poss4ibility is that the genetic differences detected in some of the studies reviewed here may result from fine-scale structure related to short-term isadation of population segments by physical oceanographic features.

226

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4.4. Metapopulation Structure Metapopulations are broadly defined as a collection of partially isolated populations that are connected by migration and gene flow (Harrison and Taylor, 1997). The metapopulation concept can provide a link between landscape ecology, population dynamics and population genetics. Metapopulation dynamics may be important for marine fishes because episodes of climatic and oceanic change may influence local population abundances. A metapopulation approach requires that: 1) local populations have spatial structure where local populations are separated by unsuitable habitat, and 2) migration has an effect on local dynamics (Hanksi and Simberloff, 1997). Pollock dispersed from the Atlantic through the Arctic Ocean and into the Pacific with the submergence of the Bering Strait during the Pliocene (Svetovidov, 1948). Some populations have apparently colonized available habitats relatively recently. For example, Prince William Sound, Puget Sound and other fjords and bays were either ice covered, or in the process of being formed during the last period of heavy glaciation about 10 to 18 thousand years ago. At this time, sea levels could have been as much as 150 m lower than present-day levels, and the major basins would have been substantially more isolated with strong geographical barriers between populations. New local populations are in the process of being discovered, such as the Aleutian Basin population found in the 1970s and the Shumgin Island population discovered in the early 1990s. Whether these are newly established populations or whether they existed previously at low levels without being recognized is unknown. Local pollock populations also appear to be susceptible to extinction. A recent example is in south Puget Sound, where a healthy charter boat recreational fishery existed in the 1970s to 1980s. Since about 1985 pollock are rarely caught in south Puget Sound by either recreational fishers or scientists, and for all practical purposes are considered to be extinct in this part of their range (Palsson et al., 1996). It is not always clear whether application of the metapopulation concept is appropriate for marine fish populations because of the issues of non-continuous distributions separated by uninhabitable areas and the extent of exchange of individuals between populations. Genetically distinct populations would indicate little exchange of individuals between sub-populations on an evolutionary scale, much less on an ecological scale. However, even if there is movement between sub-populations, life-history differences in spawning time or mating behaviour may prevent gene flow. In the case of pollock, local spawning populations are patchily distributed, but during the summer feeding period they may mix and are not clearly separated by uninhabitable areas, although there appear to be


227

less populated habitats between major aggregations. The concept of metapopulations was derived from land animals where the landscape is discontinuous, whereas the ocean is clearly different. Furthermore, the distribution of fish at different stages changes with ontogeny, arid the exchange of individuals among populations may occur at differeint life stages. Movement of individuals among walleye local pollock populations on an ecological time scale remains a subject of controversy. In spite of these issues, the framework of metapopulation dynamics may have utility for assessing population dynamics of neighbouring local populations where there is a large potential for exchange, for examiple in the Bering Sea. It is likely that some groups of local pollock populations have characteristics that may fit into a metapopulation framework. However, within the metapopulation concept there may be different types of connections between local populations including: mainland-aland, source-sink, non-equilibrium and stepping stone models. A strawman conceptual model of population structure for walleye pollock is presented in Figure 16. This hypothetical view shows some populations (e.g. Puget Sound) that are relatively isolated from other distant populations, and groups with varying degrees of population exchange ranging from a lot (within the eastern Bering Sea) to a little (across the Bering Sea). However, there are correspondences between stock groupings, diffeirentiation and the potential for mixing among different current systems. Most pollock stock members appear to complete their life cycle within specific current systems (Figure 17). Another way of conceptualizing pollock population structure is through MacCall’s (1990) geographic basin model. In this model, a population is analogous to liquid in a basin, where the volume of the basin represents abundance, the depth is the density, and the extent of shoreline is the range. Population growth rate is set by in sits growth (birth and death rates) and dispersal (which is determined by viscosity and habitat suitability). Characterizing pollock in particular, local stocks may be represented as shallow-basin populations (tending to increase range as the population grows), with low viscosity (high mobility). Across its range pollock may be viewed as a multi-basin array (multi-stock) connected by sills of varying heights (limiting dispersal between basins to varying degrees). Whether there are source-sink relationships among stocks of pollock is controversial. The existence of commercial concentrations of pollock in the Aleutian Basin is commonly considered to represent density-driven outflow of fish from the eastern Bering Sea continental shelf. Furthermore, as surplus fish they could be harvested severely without affecting their source population (Wespestad, 1993). Unfortunately, hypotheses about Aleutian Basin fish originate from sparse scientific observations. The

228

K. M. BAILEY ETAL.

Figure 16 Hypothetical model of the population structure of walleye pollock. Filled circles represent spawning populations scaled to approximate abundance. Hatched regions represent the proposed distributions of several major populations (Okhotsk, east Bering, west Bering, Shelikof) during the feeding season, indicating overlap. Lines show some of the potential connections among populations scaled to relative gene flow through migration or larval transport.

pollock in the Aleutian Basin are largely composed of fish older than 5 years. Juveniles are believed to be rare in the Aleutian Basin (Mulligan et at., 1989), and only recently have they been found along the northeastern side of the basin (Tang et al., 1996). Spawning occurs in the central basin and in the southeastern part of the basin as well (Hinckley, 1987; Sasaki, 1988; Mulligan et al., 1989). These offshore spawning aggregations are separated from continental shelf spawning groups by as much as 900 km. Pollock in the basin region also have different length-at-age and fecundity characteristics compared with shelf fish (Hinckley, 1987; Dawson, 1994), and these differences indicate the existence of different spawning groups. Eggs and larvae from the southeastern part may drift with prevailing currents onto the outer continental shelf and slope of the eastern Bering Sea (Bailey et at., 1997). In contrast to the idea of separate spawning populations, Shuntov (1992) and Dawson (1994) proposed that large

Figure 17 Major currents in the North Pacific Ocean.

230

K. M. BAILEY ETAL.

numbers of pollock found in the Aleutian Basin make seasonal migrations from the eastern and western Bering Sea shelves to graze there. However, grazing conditions over the basin region cannot be especially good, because size-at-age and fecundity of fish caught there are lower than over the shelf regions (Hinckley, 1987), and stomach contents indicate poor feeding (Dwyer, 1984; Yoshida, 1994). Density-driven migrations have important implications for harvesting strategies, demographic structure and genetic structure of the population. However, most of the support for the source-sink concept in the Aleutian Basin comes from fragmented or anecdotal observations. We believe that scientific data collected within a solid theoretical framework is needed. Density-dependent movement of the 1989 year class from the Bering Sea into the Gulf of Alaska also merits further examination. The 1989 year class was believed to be weak in the Gulf of Alaska from observations of juvenile abundance, so its increasing abundance in the Gulf in subsequent years has been interpreted as evidence of outflow of the strong 1989 year class from the Bering Sea. Alternatively, larvae and juveniles may have .been transported out of the normal area of juvenile surveys in the Gulf, followed by homing of adults. A similar situation has been observed in haddock (Frank, 1992). A portion of the strong 1988 year class in the Gulf may also be misclassified to another year class owing to imprecision in assigning fish ages as they get older and their otoliths become more difficult to interpret. 4.5. Population Structuring Mechanisms

In marine fishes, the importance of larval dispersal on gene flow and population structure has been shown by several studies (Waples, 1987; Waples and Rosenblatt, 1987; Doherty et al., 1995; Shulman and Bermingham, 1995; also see Avise, 1994). However, high dispersal potential may not always translate into high gene flow and genetic homogeneity (Palumbi, 1995). For example physical impediments, such as fronts and eddies (Iles and Sinclair, 1982), may limit dispersal when early life stages are not passively drifting but are actively and purposefully swimming, and when later stages have natal homing behaviour. Pollock have been the subject of studies of larval transport in relation to oceanographic conditions in the Gulf of Alaska since 1981 (Kim and Kendall, 1989). Pollock eggs and larvae are found in concentrations that can be tracked and monitored (Bailey et al., 1996c) and their distributions can be predicted reasonably well from modelled ocean currents (Hermann et al., 1996). Pollock larvae are often associated with oceanographic features such as eddies (Vastano et al., 1992; Schumacher et at., 1993;


231

Bailey et al., 1997). In the following section we discuss the rode of variability in larval transport and retention by features, such as eddies, in structuring the walleye pollock population, and we explore the possible linkages between them. Other mechanisms of population structuring in pollock are discussed with the main emphasis on populations in the Bering Sea and Gulf of Alaska. Considerable knowledge of ocean currents and their variability is required to understand patterns of gene flow in pollock populations. In the Gulf of Alaska, the major spawning of pollock is in Shelikof !$trait, a deep sea valley (>250 m) penetrating the continental shelf between the Alaska Peninsula and Kodiak Island. Flow in this region is dominated by the Alaska Coastal Current, which is one of the most vigorous coastal currents in the world with speeds of 25 to lOOcm.s-' (Stabeno et al., 1996b; Figure 18). The Alaska Coastal Current bifurcates east of Sutwik Island, with one branch continuing along the shallow continental shelf (-100 m) along the Alaska Peninsula. This branch has relatively weak flow, with speeds of about 10cm.s-'. The other branch flows seaward through the sea valley with a portion that joins the Alaska Stream (with speeds of 50 to 100cm.s-') and the remainder recirculates onto the shallow shelf. Frontal features, meanders and eddies are prominent in the Shelikof sea valley (Vastano er al., 1992; Napp et al., 1996). Horizontal density gradients and vertical shear in the flow result in baroclinic instabilities which generate eddies in the region. The location of eddy formation coincides with the area of pollock spawning (Schumacher and Kendall, 1995) and in the springtime spawning period three to four eddies form per month (Bograd et al., 1994), some of which may remain stationary for weeks (Schumacher et al., 1993). About 70 satellite-tracked drifters drogued at 40 m to simulate larval drift have been released in the Shelikof sea valley. The drifters show the pattern of bifurcation of the Alaskan Coastal Current and the variability in the fate of larvae during the 50 days or so when they are planlktonic (Figure 19). Residence time of the drifters on the shelf ranges from 35 to 122 days, with a mean of 55 days. Drifters can exit the sea valley as early as 15 days and enter the Alaska Stream where they are quickly transported westward. In Shelikof Strait, pollock show a mean downstream progression of their centre of abundance over time (Figure 20). However, these patterns vary greatly between years. Larval transport patterns show reasonably good correspondence with satellite-tracked drifter patterns. In years when larvae are transported onto the shallow shelf west of the sea valley, larvae drift downstream at speeds of 4 to 6 cm.s-' (Hinckley et al., 1991). Although passive physical transport seems important, larvae are able to

-175O00'

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-17PoO'

- 16S000'

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-155000'

-165"00'

-1W00'

-155"00'

-150"00'

Figure 18 Currents in the Gulf of Alaska and Bering Sea. Hatched areas are known pollock spawning locations.

233


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170-w

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166~1 1 6 4 ~ 1i

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Figure 19 Composite of representative satellite-tracked drifter trajectories in the Gulf of Alaska. Drifters were drogued at 40m,released near larval pollock aggregations, and tracked for 50 days, approximating the duration of larval drift. Dashed line is the 1OOOm isobath. Solid dark lines are drifter paths and circles mark position at 10 day intervals (after Bailey et al., 1997).

control their vertical movement and thus may have an active influence in their distribution (Olla et al., 1996), especially for older larvae and juveniles. Eddies in the Shelikof Sea Valley can retain larvae for several weeks in the region. For example, in 1990 an eddy was found with an aggregation of larvae. Satellite-tracked drifters remained in the eddy for about 22 days, until the eddy moved westward, interacted with sh.allow topography of the shelf and disintegrated (Bailey et al., 1995). In some years with strong and frequent storm activity, wind-driven transport increases and flushes most larvae from the sea valley into the Alaskan Stream. An example of this was in 1991 when winds were extremely high, with three storm events during the late April early May period, and drifter trajectories indicated that most water vigorously flowed out of the sea valley (Bailey et al., 1995). Under these conditions, primary production and microzooplankton levels were depressed, 1 arval feeding and nutritional conditions were poor and larval mortality was high (Bailey et al., 1995). In general, pollock larvae experiencing

234

K. M. BAILEY ETAL.

0 March 29-April

13

&lApril 15-April 29

@ @

April 30-May 15 May 16-June 2

Figure 20 Centroids of larval pollock distribution tracked by 2-week periods, averaged over 1987 to 1992 (after S. Picquelle, unpubl. data).

conditions associated with these high transport conditions will have low survival. In the Bering Sea, a cyclonic gyre dominates circulation, with the Kamchatka Current flowing southward along the western side, and the Bering Slope Current northward along the eastern side of the central Aleutian Basin (Figure 17). Alaska Stream water can enter the Aleutian Basin through any of the deep passes through the Aleutian Islands, but predominately through Amchitka, Amutka and Buldiur Passes and Near Strait. The transport through the passes varies on time scales of months. Over the main area of our interest, the southeast basin and the shelves, the major flows are the Bering Slope Current (BSC) and Aleutian North Slope Flow (ANSF) (Figure 18). The Aleutian North Slope Flow goes eastward along the north slope of the Aleutian Islands and connects the flow through the passes with the Bering Slope Current. Instabilities in the Bering Slope Current can result in onshelf flow from the basin onto the shelf. The shelf is divided into three regions, the inner shelf (depth

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