Variation of content of lipid classes, sterols and fatty acids in gonads and digestive glands of Scrobicularia plana in relation to environment pollution levels.

Lipids are central for energy metabolism and their fate in bivalves is closely linked to environmental conditions and gametogenic cycle. In order to assess the pollution impact on lipid metabolism of bivalves, storage and structure lipids from samples of Scrobicularia plana were studied. These samples were collected during sexual maturity both from estuaries considered contaminated (Goyen and Blavet) and from a reference site (Bay of St Brieuc) for comparison. Lipids were extracted from the gonads and the digestive glands and further separated by column chromatography. Fatty acids and sterols were then analyzed by gas chromatography-mass spectrometry. Correlations were shown between dioxin-like compounds (Eq-TCDD) and triacyglycerol levels (TAG). In the same way, glycolipids and contamination by polycyclic aromatic hydrocarbons (PAH) and pollutants with estrogenic activity seem to be closely related. In a second time, lipid indices (ratio between storage and structure lipids) were evaluated. Whereas these indices are often used in fish to assess habitat quality with regards to differential anthropogenic pressure, the ratio TAG/sterols was not here significantly influenced by the site of origin of S. plana. Intersite fluctuations of the ratio TAG/phospholipids also remained very limited. This could be explained by the limited contamination level in studied sites but also by a contrasted response from organisms in different taxa (bivalves vs. fish). Environmental pollution is not the only factor able to induce changes in lipid classes. The trophic wealth seemed to be different between the reference site and contaminated estuaries, the total organic carbon content being higher in muddy estuarine sediments.

Integrated assessment of estuarine sediment quality based on a multi-biomarker approach in the bivalve Scrobicularia plana.

The bivalve Scrobicularia plana has been proposed as a sentinel species for the assessment of estuarine sediment quality. The aim of this study was to test the responsiveness of a set of biomarkers in bivalves originating from a moderately contaminated site (Loire estuary, France) and a comparatively cleaner site (Bay of Bourgneuf, France) used as reference. Temporal fluctuations were examined in animals collected on eight occasions from March 2008 to October 2009 for the determination of biochemical (MTs, GST, AChE), physiological (energy reserves as glycogen and lipids, condition, hepato-somatic and gonado-somatic indices), and behavioural biomarkers (burrowing). Metals in clams were examined with the aim of understanding the variations in MT concentrations. The current findings indicate that among biochemical markers MTs must be used in a precautionary fashion, and comparison with a reference site remains indispensable together with an appreciation of any salinity effects. Clearer responses were obtained for GST activity which contributes to defence against organic compounds. However, any such defence was not sufficient to ensure total protection since a number of impairments were observed at the individual level (burrowing behaviour, condition index, gonado-somatic index). The integrated biomarker response (IBR) illustrated higher levels of stress in clams collected from the Loire estuary compared to those gathered from the reference site at most of the sampling periods. The interpretation of responses involved in the cascade of energetic events (from available food, digestive enzyme activities, energy reserves to condition and reproductive status) is complex but did reveal disturbances in the Loire estuary which is far from being the most contaminated estuary in France and over the world. However, no links can be established between effects at individual and supra-individual levels in organisms in the Loire estuary, even in the case of a small oil spill which occurred at the beginning of this study.

Physiological status and intersex in the endobenthic bivalve Scrobicularia plana from thirteen estuaries in northwest France.

The bivalve Scrobicularia plana, an important species for the structure and functioning of estuarine and coastal mudflats, was studied in thirteen sites from NW France differing by their degree of contamination to document the presence of reproduction impairments (intersex, sex ratio, gonadosomatic indices) in relation to the condition revealed by using hepatosomatic and condition indices. In agreement with recent studies in other European estuaries, intersex was revealed in all the studied estuaries, including sites the chemical and ecological status of which is considered "good" according to the criteria of the European Water Framework Directive. The presence of endocrine disrupting chemicals (EDCs) could result in such disturbances. Our results re-inforce the concern linked to the subtle effects of EDCs, which are active at very low doses, often in the absence of any major sign of toxicity. However at this stage, no clear link may be established between intersex and population effects.

Intersite variations of a battery of biomarkers at different levels of biological organisation in the estuarine endobenthic worm Nereis diversicolor (Polychaeta, Nereididae).

The ragworm Nereis diversicolor has been proposed as a sentinel species for the assessment of estuarine sediment quality. The aim of this study was to test the responsiveness of the worms using a battery of biomarkers in specimens from a moderately contaminated site (Loire estuary, Fr.) and a comparatively cleaner site (Bay of Bourgneuf, Fr.) as a reference site. Ragworms were collected on 7 occasions from April 2008 to October 2009 for the determination of biochemical (GST, AChE, digestive enzymes), physiological (energy reserves (glycogen, lipids and proteins), relationship between length and weight), and behavioural (feeding and burrowing) biomarkers. The biomarker responses were tentatively interpreted in terms of the concept of cascading events potentially responsible for local depletion/extinction of populations submitted to chemical stress. Impairments of AChE and amylase activities, feeding rate, energy reserve concentrations (glycogen and lipids), and the relationship between length and weight showed up differences between the reference site and the Loire estuary despite the latter being far from the most contaminated estuary in France or internationally. However, no links could be established in the Loire estuary ragworms between effects at infra-individual and individual levels, nor with worm population density, even though a small oil spill had occurred in the Loire estuary only one month before the beginning of sampling.

Assessment of the influence of confounding factors (weight, salinity) on the response of biomarkers in the estuarine polychaete Nereis diversicolor.

The influences of salinity and body size on biochemical (activities of glutathione-S-transferase, lactate dehydrogenase (LDH), acetylcholinesterase and digestive enzymes amylase and CMCase), physiological (feeding and egestion rates, energy reserves) and behavioural (burrowing speed) biomarkers were examined in the infaunal polychaete Nereis diversicolor. Only a few biomarkers were affected, including increased egestion rate and activities of CMCase and LDH at higher salinity, and higher egestion rate in larger worms. These findings reinforce the status of N. diversicolor as a robust sentinel species for estuaries which are environments that are particularly productive but also particularly at risk.

Influence of anthropogenic stress on fitness and behaviour of a key-species of estuarine ecosystems, the ragworm Nereis diversicolor.

Fitness, (biometric measurements, reproduction) and behaviour that are ecologically relevant biomarkers in assessing the quality of estuarine sediments were studied by comparing the responses of the polychaete worm Nereis diversicolor--a key species in estuaries--along a pollution gradient. Intersite differences were shown for all the measured parameters: size-weight relationships, energy reserves as glycogen and lipids, sexual maturation patterns, total number of oocytes per female, total and relative fecundity, burrowing behaviour. The physiological and behavioural status of N. diversicolor was consistently disturbed in the larger, most contaminated estuaries (Loire and Seine, Fr.) compared to reference sites (Bay of Bourgneuf, Goyen estuary, Fr.). Many classes of potentially toxic chemicals present in these estuaries most likely contribute to these impairments but food availability may act as a confounding factor, interfering with the potential impact of contaminants.

Biological indices, energy reserves, steroid hormones and sexual maturity in the infaunal bivalve Scrobicularia plana from three sites differing by their level of contamination.

The aim of this work was to investigate in situ biometric (condition index, hepato-somatic index, gonado-somatic index), biochemical (glycogen, lipids, sexual steroids) and histological (sex, sexual maturation stage) variables in the infaunal bivalve Scrobicularia plana. First, the reproductive cycle of S. plana was assessed by collecting bivalves from a reference site (the bay of Bourgneuf, Fr) in April, May, July, September and November 2005 and in January and March 2006. Then, S. plana were collected at three key periods of their sexual cycle (March 2006, beginning of gametogenesis; June 2006, spawning; and September 2006, spent) in three sites differing by their level of contamination (bay of Bourgneuf, reference site; Loire and Seine estuaries; Fr, impacted sites). The reproductive cycle of S. plana was well defined with a clear spawning period between May and July, sexual repose from November to January. Development of the gonad began in January and ended in September. Sex-ratio was determined during spawning and the influence of sex on biochemical variables was examined. Progesterone, 17beta-estradiol and testosterone levels in the gonad of S. plana were close to those reported in other bivalves. This study is the first to demonstrate in situ influence of site, sex and sexual maturity on energy reserves, and sexual steroids in S. plana. Even if interpretation of results is complex due to interferences between natural and anthropogenic factors, S. plana is a suitable species for estuarine studies and a better understanding of its reproduction will permit to assess impacts of environmental pollutants.

Metallothionein turnover, cytosolic distribution and the uptake of Cd by the green mussel Perna viridis.

We examined the relationship between Cd kinetics (uptake from solution and diet, and efflux), metallothionein turnover, and changes in the cytosolic distribution of accumulated Cd between protein fractions in the green mussel Perna viridis. We pre-exposed the mussels to 5, 20, 50 and 200 microg l(-1) of Cd for 1 week and determined the biokinetics of Cd uptake and efflux in the mussels. The dietary assimilation efficiency of Cd increased by 2 times following exposure to 20-200 microg l(-1) Cd, but the dissolved uptake rate was unchanged by pre-exposure to any Cd concentrations. The efflux rate of Cd was also similar among control and Cd pre-exposed mussels. The cytosolic distribution of Cd in the mussels that had been exposed to dissolved Cd, showed that besides metallothionein (7000 - 20,000 Da), high molecular weight proteins (>20,000 Da) were important for Cd binding and depuration. In general, the Cd pre-exposed mussels had higher metallothionein turnover with a higher metallothionein synthesis rate, but similar metallothionein breakdown rates as the control mussels. Metallothionein synthesis rate was correlated to the dietary assimilation of Cd, whereas metallothionein breakdown and Cd efflux rate were independent of each other. This study provides important new information for the role of metallothionein turnover on Cd kinetics in an aquatic invertebrate.

Bioaccumulation of metals in sediment elutriates and their effects on growth, condition index, and metallothionein contents in oyster larvae.

The bioavailability of Cd, Cu, Zn, and Pb from two metal-contaminated sediments (Bidassoa and Dunkerque) was studied using Crassostrea gigas larvae exposed to sediment elutriates. The metal contents within the sediments, the larvae and larval growth, the condition index, and the induction of metallothionein in the larvae were measured. The larval growth and condition index were only affected after exposure to the highest elutriates concentration (5 to 25%) from the most contaminated sediment (Dunkerque). Bioaccumulation of all metals was observed in larvae exposed to Dunkerque elutriatre; only Cu bioaccumulation was observed in the Bidassoa elutriate. The results from larvae exposed to both sediment elutriates show a strong correlation between bioaccumulated metal considered individually or in combination and the metallothionein level in larvae presenting no detrimental effect. On the other hand, in the case of larvae exposed to the highest Dunkerque elutriate concentration and showing the highest metal body burden, we observed a drop in the metallothionein level. These results indicate that metallothionein is a more sensitive indicator of heavy metal pollution than physiological endpoints taken into account in bioassays and could be proposed as an early biomarker of metal exposure in larvae. However, care must be taken with "fault control" due to the toxicological effect on larvae metabolism in the case of substantial contaminant exposure.

Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers.

The literature on metallothioneins (MT) and metallothionein-like proteins (MTLP) in aquatic invertebrates is large and increasing, and yet inconsistencies and confusion remain, not least over the physiological role of MT and their use as biomarkers in environmental monitoring programmes. We have collated published information on MT in three important groups of aquatic invertebrates-the molluscs, crustaceans and annelid worms, and attempted to seek explanations for some of the apparent inconsistencies present in the dataset. MTs can be induced by the essential metals Cu and Zn and the non-essential metals Cd, Ag and Hg in both vertebrates and invertebrates, but their induction is variable. Such variation is intraspecific and interspecific, and is down to a variety of reasons environmental and physiological explored here. Against this background of variability MTs do appear to play roles both in the routine metabolic handling of essential Cu and Zn, but also in the detoxification of excess amounts intracellularly of these metals and of non-essential Cd, Ag and Hg. Different isoforms of MT probably play different physiological roles, and the dependence on MT in detoxification processes varies environmentally and between zoological groups. MTs can be used as biomarkers if used wisely in well-designed environmental monitoring programmes.

Mobility and potential toxicity of sediment-bound metals in a tidal estuary.

Sediment toxicity in the Gironde Estuary, France, a site contaminated by such trace metals as Cd, Cu, and Zn, was examined monthly from March to October 1997, using concurrently geochemical procedures to assess the mobility of contaminants and ecotoxicity tests with invertebrate larvae (the oyster Crassostrea gigas, the sea urchin Paracentrotus lividus, and the copepod Tigriopus brevicornis). Higher mobility of Cd than of Cu and Zn was shown by desorption tests. Both the gross concentrations of Cu and Zn in the sediment and the mobility of these metals showed a temporal evolution, and changes in the toxicity of the sediment to invertebrate larvae generally were observed concomitantly. Little damage was registered in the spring, and greater damage was found in the summer and fall. Positive correlations were shown between mortality in copepods or abnormalities in oyster larvae with the quantities of metals remobilized from sediment at pH 4. On the contrary, no clear temporal trend was shown for total PAH levels in sediment. These correlations and previous toxicological data suggest that sediment-bound metals, particularly Cu and Zn, possibly play a role in biological responses.

Tolerance to metals and assessment of energy reserves in the polychaete Nereis diversicolor in clean and contaminated estuaries.

Estuaries are subject to anthropogenic activities. Because the intrasedimentary worm Nereis diversicolor has ecological characteristics and bioindicator abilities, its use was pertinent in investigating the concepts and cost of tolerance to heavy metals (Cd, Cu, Zn). In this context, two approaches were carried out, performing toxicity tests and estimating energy reserves (glycogen and lipids), in populations originating from a contaminated site (Seine estuary) compared with those from a clean site (Authie estuary). Mean lethal times (LT(50)s) of organisms exposed to zinc from the Seine estuary were higher than those from the Authie estuary, but not of organisms exposed to Cd or Cu. The influence of animal weight and salinity on the sensitivity of worms also was studied. The biggest worms were more tolerant to zinc than the smallest ones, and worms survived longer at a reduced salinity (15 per thousand). Concentrations of glycogen and lipids in each sampling season were higher in specimens from the Authie estuary than in worms from the Seine estuary. No influence of salinity on glycogen and lipid levels was observed. Glycogen concentrations were not influenced by the weight of specimens, whereas lipid concentrations were significantly and positively correlated with weight. In conclusion, worms from the Seine estuary exhibited tolerance to Zn, and the depletion of energy reserves observed in this population could be interpreted as a cost of tolerance.

Influence of ploidy and metal-metal interactions on the accumulation of Ag, Cd, and Cu in oysters Crassostrea gigas thunberg.

The present study was designed to compare the response to contaminants in diploid with triploid specimens of the oyster Crassostrea gigas. The reproduction investment in bivalve molluscs has priority on somatic growth. Thus, genetic sterilization by triploidy induction enables the energy flux to be directed toward somatic growth and glucide storage. Bioaccumulation was examined for Ag (10 microg/L), Cd (10 microg/L), and Cu (30 microg/L) to determine if the response to metals follows similar patterns in diploid (2n) and triploid (3n) groups. The effect of ploidy was also evaluated as a function of dry weight of soft tissue and condition index. Moreover, the reciprocal influence of these metals on their incorporation was studied. The results showed that the major factor governing the degree of metal bioaccumulation by oysters is the nature of the metal introduced in the experimental medium. Thus, the uptake of Cd is proportionally more important than in the case of Ag and even more in Cu. For Cu-treated samples, the influence of ploidy on weight and metal body burden (and Cu concentration) was not significant, whereas for Ag and Cd, significant differences according to genetic type were evidenced by higher tissue weight and lower concentrations in triploid than diploid specimens. Metal-metal interactions study especially showed a reciprocal antagonism between Ag and Cu.

Accumulation and soluble binding of cadmium, copper, and zinc in the polychaete Hediste diversicolor from coastal sites with different trace metal bioavailabilities.

Bioaccumulation of cadmium, copper, and zinc was examined in common ragworms Hediste diversicolor from control (Bay of Somme, Blackwater) and metal-rich (Seine estuary, Boulogne harbor, Restronguet Creek) sites in France and the United Kingdom. The degree of exposure in the field was assessed by considering both total concentrations in superficial sediment and the quantities of metals which may be released in vitro at different pH levels. Among the three contaminated sites, release of the three metals was not detectable in Boulogne harbor, in correlation with limited enhancement of the metal concentrations in the common ragworms from this site. Even at those sites where zinc could be released in vitro from the sediment, zinc concentrations were not enhanced in common ragworms, in agreement with previous findings indicating that the body content of this metal is regulated in H. diversicolor. At all the studied sites, bioaccumulated zinc was mainly in cytosolic form. The distribution of cadmium and copper varied according to the origin of the common rag-worms, the insoluble fraction increasing with the degree of contamination (cadmium in the Restronguet Creek, copper in the Seine estuary, and even more in Restronguet Creek). In the cytosolic fraction, metals were partly linked to cytosolic heat-stable thiolic compounds (CHSTC) with molecular masses (5-6 kDa and about 12 kDa) consistent with metallothionein-like proteins (MTLP). Metal-binding to MTLP varied with the degree of contamination and with the metal studied. In contrast to many invertebrates, the presence of metal-binding CHSTC (MM about 2 kDa) other than MTLP seems to be a peculiar feature of H. diversicolor.

Use of metallothionein in gills from oysters (Crassostrea gigas) as a biomarker: seasonal and intersite fluctuations.

The use of oyster gills for the analytical determination of metallothionein (MT) concentration as a biomarker of metal exposure was investigated. Temporal variations in MT and metal concentrations (which can interfere with inter-site differences) were examined over a 7 month period (from spring to autumn) in Japanese oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site (Gironde estuary, France) as well as in individuals translocated from the clean to the contaminated area. The ratio between the annual average of MT concentrations in specimens from the clean and the metal-rich sites was 1.3. During the last 3 months of the experiment, significant differences were no longer registered between transplants and residents from the Gironde estuary. Metals concentrations in oyster gills differed consistently between the clean and the metal-rich sites (annual average ratios of 1.5, 2.7 and 9.8, respectively, for zinc, copper and cadmium) and a fast increase in metal concentrations (over a few months) was observed in transplants, mainly for cadmium. MT and soluble metal concentrations were found to be positively and significantly correlated over the period of the study. This relationship is a positive argument for a possible use of gill MT concentration as a biomarker of metal pollution in contrast to previous findings on the digestive gland, there being a smaller amount of seasonal variability in the weight of oyster gills.

Partitioning of accumulated trace metals in the talitrid amphipod crustacean Orchestia gammarellus: a cautionary tale on the use of metallothionein-like proteins as biomarkers.

The induction of metallothionein-like proteins (MTLP) as biomarkers of trace metal pollution has been investigated in the talitrid amphipod Orchestia gammarellus, an established biomonitor of trace metal availabilities in coastal areas. Sensitivity to metal exposure, MTLP and copper and zinc concentrations have been examined in amphipods from a metal-contaminated site (Dulas Bay, Wales) and two clean sites (Millport, Scotland and Bradwell, England). Groups of 10 amphipods from Dulas Bay and Millport were exposed separately to one of a concentration series (1, 3.16, 10, 31.6, 100 microM) of Cu or Zn for 17 days at 10 degrees C. Specimens from Bradwell were exposed under similar conditions but the experiment was extended to 25 days with intermediate samplings at 5, 10 and 17 days and additional groups were exposed to a mixture of Cu+Zn (7:10 molar ratio). In addition, specimens from Millport were exposed to 1 or 31.6 microM Cd for 17 days. Amphipods from Dulas Bay, which had been chronically exposed to metals in their natural environment had not acquired any tolerance to Cu and Zn since the lowest LC 50s were registered in these samples. Whatever the origin of the amphipods experimentally exposed to metals and whatever the dose of exposure, both Cu and Zn remained approximately equally distributed between cytosolic and insoluble fractions in the amphipods, suggesting that mechanisms of metal storage were identical over the whole range of conditions. Concentrations of MTLP were higher in O. gammarellus from Dulas Bay than in those from Millport analysed directly after collection, although laboratory exposures to dissolved Cd, Cu or Zn have failed to demonstrate differences in the induction of MTLP between amphipods from the clean or contaminated sites. A potential role for metallothionein-like proteins as biomarkers is thus unlikely although it remains plausible that turnover of these proteins does increase in response to increased metal challenges, enabling MTLP to play a role in metal detoxification.

Kinetics of metal elimination in oysters from a contaminated estuary.

In oysters Crassostrea gigas translocated from a metal-enriched estuary (Gironde, France) to a comparatively clean site, the Bay of Bourgneuf (France), Cd, Cu and Zn concentrations were determined monthly in the whole soft tissues, or in different fractions (cytosolic or insoluble) of gills and digestive glands. In all cases, the concentrations of all of the three metals decreased logarithmically and half-lives were always shortest for Cd (86-251 days). After 4 months, the Cd concentration had become not significantly different from the threshold considered safe for human consumption (1 mg kg(-1) wet wt.). In the digestive gland, half-lives were similar in cytosolic and insoluble fractions. In contrast, in the gills, elimination patterns differed markedly between these fractions. The long half-lives calculated for divalent metals in the insoluble fraction of the gills (1505 and 3010 days for Zn and Cu, respectively) is possibly due to a fossilization of metals in intracellular membrane-bound inclusions as shown previously in Ostrea edulis. It is interesting to underline that elimination is fastest for cytosolic metals compared to the insoluble fraction.

Comparison of metallothionein concentrations and tissue distribution of trace metals in crabs (Pachygrapsus marmoratus) from a metal-rich estuary, in and out of the reproductive season.

Crabs, Pachygrapsus marmoratus, were sampled in June 1997 and February 1998 from two sites (at the mouth and 25 km upstream) in the metal-rich Gironde estuary, France. Gills and hepatopancreas were analysed for metal (Cd, Cu, Zn) and metallothionein (MT) contents, in order to examine the influence of both biological and environmental factors on the physico-chemical forms of detoxified metal storage in the crabs. The concentrations of MT and both cytosolic and insoluble metals were not greatly different between males and females, and the influence of organ weights was also minimal. Intersite differences were observed, probably resulting from the gradient of salinity in the estuary, which interacts with both the chemical speciation and bioavailability of metals, and the general protein metabolism of the crabs. Seasonal changes were also important, probably in interaction with the moult and reproductive cycles. In February, concentrations of insoluble metals were generally higher than in June, in both organs, suggesting that essential metals, particularly Zn, are stored during winter then remobilised during the breeding season. The natural variability in the concentrations of MT often concealed any relationship with accumulated metal concentrations. Thus MT in crabs cannot be considered as a useful biomarker of metal pollution.

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site.

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.

The respiratory performance and survival of the bivalve Macoma balthica (L.) at the southern limit of its distribution area: a translocation experiment.

The hypothesis was tested that animals near their extreme Southern limits, due to high temperatures, have a high respiration rate, whereby they reach an extreme low weight-index and ultimately disappear. At estuarine stations the respiration rate of Macoma balthica (L.) (Baltic clam) did not show interseasonal changes, indicating seasonal acclimation, but within the season the respiration increased with increasing temperature, indicating the absence of short-term acclimation. In clams translocated from the Netherlands towards the Bidasoa estuary, 200 km South of their Southern distribution limit, their respiration rate was higher and weight-index lower than in specimens living in Dutch estuaries. Irrespective of an effect of the temperature, clams exposed in experiments to water from Bidasoa showed a higher respiration than clams exposed to water from the other stations. Moreover, at non-estuarine stations with a low food content, the clams showed reversed acclimation, i.e., the respiration rates in winter were much lower than summer rates, most probably a strategy to conserve energy by means of a depressed metabolism. A weight index of 5 mg DW/cm(3) and glycogen content of 2% DW are suggested as the minimal values below which the metabolic energy balance of Baltic clams becomes more negative and the clam population disappears. It was concluded that factors other than temperature influenced the respiration and weight-index of clams, and hence their presence or absence, e.g., food concentration, innate seasonal cycles, and possible pollutants in the water.