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.

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.

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.

Influence of field and experimental exposure of mussels (Mytilus sp.) to nickel and vanadium on metallothionein concentration.

In mussels impacted by the V- and Ni-rich oil dispersed in the environment after the << Erika >> wreck, a significant and positive correlation was observed between metallothionein (MT) and V concentrations whereas no correlation was observed between MT and Ni concentrations. Mussels were exposed separately to V and Ni in the laboratory. The highest concentrations tested (> or =2000 ng V per individual; > or =50 ng Ni per individual) were responsible for V and Ni bioaccumulation. In the case of V, the metal was preferentially stored in the insoluble fraction of the soft tissues but with increasing body burden, the part of soluble V increased from 19 to 41%. Metallothionein was induced in mussels exposed to V but not in those exposed to Ni. In addition, in mussels originating from a shellfish farming area which showed abnormally high levels of V, depuration showed concomitant decreases of V and MT. It is the first report in the literature of a functional link between MT and V. When interpreting surveys of metal pollution based upon the use of MT, one must be aware that V may be responsible for MT synthesis, explaining likely high levels of this biomarker in the absence of metals known as MT inducers.

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.

Immunological and biochemical responses in Mya arenaria (Mollusca Bivalvia) exposed in vivo to estradiol-17beta.

Soft-shell clams Mya arenaria were injected with 10, 20 or 40 nmol of estradiol 17beta (E2). We observed a significant inhibiting effect of E2 on phagocytic activity of hemocytes from clams exposed to 10 and 20 nmol. A dose-response increase of the glycogen phosphorylase in the gonad tended to show a remobilisation of glycogen reserves involved in vitellogenesis although the exposure time must have been too short to observe a decrease in glycogen reserves or an increase in RNA concentration. Both results corroborate those of other studies about estrogen involvement in controlling immune capacity and energy metabolism related to vitellogenesis in bivalves. We can assume that immune parameters should now be taken into consideration in assessing endocrine disruption in bivalves. Nevertheless further studies are needed to understand the controlling pathways of E2 with a special regard on its interactions with other effectors involved in bivalve immunity and reproduction as well.

Estradiol-17beta and testosterone concentrations in male and female Mya arenaria (Mollusca bivalvia) during the reproductive cycle.

Perturbation of the reproductive cycle as well as vitellin-like protein synthesis have already been reported in Mya arenaria sampled in contaminated areas of the St. Lawrence maritime estuary (Québec, Canada). To assess the potential role of endocrine disruptors in modulating the reproductive cycle in clams, the role of sex steroids has to be clarified. We determined the levels of estradiol-17beta and testosterone during the reproductive cycle in female and male M. arenaria. Both steroids were measured by ELISA in the gonads and no differences in steroid profiles appeared between sexes. Estrogen levels varied between 150 and 400 pg g(-1) wet weight over gametogenesis and were near 10 times higher than testosterone levels. Results showed transient increases of both steroids at the onset of vitellogenesis in females and during the spawning stage in both sexes. These findings indicate that these hormones could have a role as endogenous modulators of gametogenesis. Further studies are, however, needed to describe the pathway of steroid synthesis in clam gonad and elucidate steroid involvement in controlling gametogenesis and as well as their relationship with neurohormones since these latter are required to promote sexual maturation.

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.

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.

Estimates of trace metal bioavailability to humans ingesting contaminated oysters.

Oysters, as very popular food items in France, are subject to rules concerning the maximum acceptable contents of trace metals in foods. The food standards for the quantities of metals permitted are always based on total metal concentrations, and not on the metal concentrations that are potentially bioavailable to the consumer (man). In order to estimate the percentages of accumulated trace metals (i.e. Ag, Cd, Cu and Zn) that are potentially bioavailable during consumption, we have used a simple chemical digestion simulation on the insoluble fraction of oysters. These quantities have been added to the soluble fraction, assuming that metals in this fraction are completely bioavailable. Our experiments were conducted on oysters Crassostrea gigas sampled from five sites on the French Atlantic coast and on oysters Ostrea edulis sampled from Restronguet Creek in the United Kingdom. These sites are characterised by various degrees of metal contamination. This study has allowed us to gain a better estimate of the real concentrations of metals bioavailable to the consumer. Only a part of the total metal present is bioavailable: 36-68% for silver, 44-75% for cadmium, 26-80% for copper and 50-80% for zinc. These new estimates have the potential to contribute to any re-evaluation of the food standards for metals.

Variation of metallothioneins in gills of the clam Ruditapes decussatus from the Gulf of Gabès (Tunisia).

Cadmium (Cd) and metallothionein (MT) concentrations were determined in the subcellular fractions of the gills of the clam Ruditapes decussatus. Clams were collected monthly during the period of sexual development (from June to August 2001) from two different sites situated in the Gulf of Gabès (Tunisia). These sites differ by their degree of metal contamination. Differences in condition index (CI) were observed in June and August between clams from both sites. In clams from both sites, cadmium was approximately equally distributed between the soluble (S1) and insoluble (C1) fractions. MT levels varied according to month but not sex. Site and body mass of clams seem to be important factors to explain the variation of MT levels compared to cadmium.

Multimarker approach in transplanted mussels for evaluating water quality in Charentes, France, coast areas exposed to different anthropogenic conditions.

An active biomonitoring experiment was performed using mussels collected at a clean site, Fier d'Ars, and transplanted to two locations, outside the harbor of La Rochelle and in the Baie de L'Aiguillon along the coast of Charentes (French Atlantic coast) beginning in April for several months. Mussels were collected in June and October. The cadmium, copper, and zinc concentrations of all resident and transplanted mussel samples and the polycyclic aromatic hydrocarbon and polychlorinated biphenyl concentrations in some mussel samples and in the sediment samples were determined. Mussel response was evaluated for several biochemical biomarkers: concentrations of metallothionein, activities of glutathione S-transferase and acetylcholinesterase (AChE) and levels of thiobarbituric reactive substance (TBARS). The physiological status of the animals was assessed using the condition index. A principal component analysis performed with the chemical and biochemical results of the evaluations of the resident and transplanted mussels collected in June allowed them to be separated into three groups: resident mussels from la Rochelle with high metal and TBARS levels, resident mussels from Baie de L'Aiguillon with a very high condition index, and resident mussels from Fier d'Ars and transplanted mussels at La Rochelle and Baie de L'Aiguillon with low TBARS and AChE activities. Strong seasonal variation from June to October of all parameters was noted. Mussels transplanted to La Rochelle appeared to be the most "polluted" in their pollutant concentrations and biochemical responses; moreover, the La Rochelle site had the highest concentration of organics in sediments of all the sites. The choice of Fier d'Ars as a reference site may be questionable because some of the biomarker responses of the mussels were higher than expected there, although these pollutants in mussels and sediment were present at the lowest concentrations measured.

Delayed gametogenesis and progesterone levels in soft-shell clams (Mya arenaria) in relation to in situ contamination to organotins and heavy metals in the St. Lawrence River (Canada).

There is a growing awareness that contaminants in the aquatic environment may alter steroid hormone levels and affect the reproductive success of the invertebrates. To verify if heavy metals and organotins affect sexual maturation in Mya arenaria, individuals were collected from July to November 1998, at different sites along the South coast of the St. Lawrence maritime estuary. Near the Rimouski harbour, clams showed high levels of tributyltin (TBT), DBT in the gonad, along with a lower gonado-somatic index [GSI=gonad wet weight (g)/body wet weight without shell (g)x100], low progesterone levels and a delay in sexual maturation when compared to the reference site. Sites that had intermediate levels of contaminants exhibited intermediate responses of hormones and sexual maturation stages. It is therefore suggested that TBT, DBT are endocrine disruptors in clams. Further studies will however be necessary to investigate in more details how contaminants as TBT can affect the steroid hormones production in the gonads of M. arenaria.

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.

Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabès (Tunisia).

Metallothioneins (MTs), malonedialdehyde (MDA), and glycogen concentrations were determined in order to assess the health status of Ruditapes decussatus exposed to in situ contamination, showing the effects of time, site, and metal contamination on these three biomarkers. Metallothionein was positively correlated with Cd and Zn. If introduced in a multiple model, the factors site and time were shown to decrease significantly MT concentrations. MDA was inversely correlated with size of the animals and was affected by the factor time. Glycogen was inversely correlated with zinc and was affected by both site and time.This study constitutes a field-based validation of a multiparametric approach using specific and nonspecific biomarkers.

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.

Seasonal gonad progesterone pattern in the soft-shell clam Mya arenaria.

Steroidogenesis, which plays a major role in the reproductive cycle of vertebrates, is still for the most part, unknown in invertebrates. The aim of this study was to examine the link between progesterone and the reproductive cycle in Mya arenaria. The soft-shell clams, Mya arenaria were collected in Anse à l'Orignal (Parc Provincial du Bic, Québec, Canada) from July to November 1998. Histological data have shown that female gonads of M. arenaria were in the spawning stage in August and September, while the male gonads were in the ripe stage. This period of active gametogenesis was associated with a depletion of lipid reserves. These lipids could be used as a source of energy and as a substrate for steroidogenesis. Liquid Chromatography coupled to Mass Spectroscopy (LC-MS) and quantified by Enzyme Linked ImmunoSorbent Assay (ELISA) determined progesterone. Progesterone levels in the gonad were increased during the ripe stage in the male and during the spawning stage in the female. These results indicate, for the first time, that progesterone, as in vertebrates, may play a role in the reproductive cycle of M. arenaria.