Investigating the impact of remediation efforts on Hedistediversicolor in the Seine estuary using multiple levels of biological organization.

The Seine Estuary is historically one of the most contaminated estuaries in Europe. In 2002 the estuary underwent major redevelopment which emphasized environmental remediation to measure biological effects. The present study investigated the health status of the sentinel species Hediste diversicolor in the Seine estuary by comparing data from a sampling campaign immediately after the Seine underwent reconstruction and 10 years afterward. Both studies implemented multiple levels of biological organization ranging from enzymatic biomarkers to population density. Integrative modeling was used to establish a holistic status assessment for H. diversicolor in the Seine. H. diversicolor populations in the Seine estuary were impacted in both campaigns suggesting no significant improvements over the 10-year period. This interpretation however can be improved by increasing the number of reference sites used to establish the baseline natural variation, integrating additional environmental parameters into the model and measuring additional biological endpoints, such as reproductive status.

Assessment of carbamazepine acute toxicity in the cockle Cerastoderma edule through chemical, physiological and biochemical tools

Abstract The cockle Cerastoderma edule was exposed to four concentrations (5, 10, 20 and 70 g L-1) of carbamazepine (CBZ). This anticonvulsant was found to alter the mussel behavior of by reducing its clearance rate (CR). Analysis of CBZ accumulation in tissues of C. edule was carried out using HPLC-UV after 48 or 96 hours of exposure. In addition, an overproduction of H2O2 by the bivalves was detected following exposure to CBZ but nitrite levels remained unchanged. Moreover, superoxide dismutase and catalase activities showed a significant increase in relation to their contact with CBZ. The activity of the biotransformation enzyme gluthatione-S-transferase did not change during exposure. Malondialdehyde (MDA) levels indicating cellular damage, increased when bivalves were exposed to 20 and 70 g l-1 of carbamazepine for 96 h CBZ. The results also indicate that acetylcholinesterase activity (AChE) was inhibited in all CBZ concentrations during the 48 h exposure period. However, during the 96 h exposure period, AChE was only inhibited at the highest concentration. Further studies are needed now for more exploration of the toxicity of CBZ since it could be bioaccumulable throughout the food web and may affect non-target organisms.

Resumo O berbigão Cerastoderma edule foi exposto a quatro concentrações (5, 10, 20 e 70 g L-1) de carbamazepina (CBZ). Este anticonvulsivante alterou o comportamento do mexilhão, reduzindo sua taxa de depuração (CR). A análise do acúmulo de CBZ nos tecidos de C. edule foi realizada por HPLC-UV após 48 ou 96 horas de exposição. Além disso, uma superprodução de H2O2 pelos bivalves foi detectada após a exposição à CBZ, mas os níveis de nitrito permaneceram inalterados. Além disso, as atividades de superóxido dismutase e catalase apresentaram aumento significativo em relação ao contato com CBZ. A atividade da enzima de biotransformação glutationa-S-transferase não se alterou durante a exposição. Os níveis de malondialdeído (MDA), indicando dano celular, aumentaram quando os bivalves foram expostos a 20 e 70 g l-1 de carbamazepina por 96 h CBZ. Os resultados também indicam que a atividade da acetilcolinesterase (AChE) foi inibida em todas as concentrações de CBZ durante o período de exposição de 48 horas. No entanto, durante o período de exposição de 96 horas, a AChE foi inibida apenas na concentração mais alta. Mais estudos são necessários agora para uma maior exploração da toxicidade da CBZ, uma vez que pode ser bioacumulável em toda a cadeia alimentar e pode afetar organismos não alvo.

Assessment of carbamazepine acute toxicity in the cockle Cerastoderma edule through chemical, physiological and biochemical tools / Avaliação da toxicidade aguda da carbamazepina no berbigão Cerastoderma edule por meio de ferramentas químicas, fisiológicas e bioquímicas

The cockle Cerastoderma edule was exposed to four concentrations (5, 10, 20 and 70 µg L-1) of carbamazepine (CBZ). This anticonvulsant was found to alter the mussel behavior of by reducing its clearance rate (CR). Analysis of CBZ accumulation in tissues of C. edule was carried out using HPLC-UV after 48 or 96 hours of exposure. In addition, an overproduction of H2O2 by the bivalves was detected following exposure to CBZ but nitrite levels remained unchanged. Moreover, superoxide dismutase and catalase activities showed a significant increase in relation to their contact with CBZ. The activity of the biotransformation enzyme gluthatione-S-transferase did not change during exposure. Malondialdehyde (MDA) levels indicating cellular damage, increased when bivalves were exposed to 20 and 70 µg l-1 of carbamazepine for 96 h CBZ. The results also indicate that acetylcholinesterase activity (AChE) was inhibited in all CBZ concentrations during the 48 h exposure period. However, during the 96 h exposure period, AChE was only inhibited at the highest concentration. Further studies are needed now for more exploration of the toxicity of CBZ since it could be bioaccumulable throughout the food web and may affect non-target organisms.

O berbigão Cerastoderma edule foi exposto a quatro concentrações (5, 10, 20 e 70 µg L-1) de carbamazepina (CBZ). Este anticonvulsivante alterou o comportamento do mexilhão, reduzindo sua taxa de depuração (CR). A análise do acúmulo de CBZ nos tecidos de C. edule foi realizada por HPLC-UV após 48 ou 96 horas de exposição. Além disso, uma superprodução de H2O2 pelos bivalves foi detectada após a exposição à CBZ, mas os níveis de nitrito permaneceram inalterados. Além disso, as atividades de superóxido dismutase e catalase apresentaram aumento significativo em relação ao contato com CBZ. A atividade da enzima de biotransformação glutationa-S-transferase não se alterou durante a exposição. Os níveis de malondialdeído (MDA), indicando dano celular, aumentaram quando os bivalves foram expostos a 20 e 70 µg l-1 de carbamazepina por 96 h CBZ. Os resultados também indicam que a atividade da acetilcolinesterase (AChE) foi inibida em todas as concentrações de CBZ durante o período de exposição de 48 horas. No entanto, durante o período de exposição de 96 horas, a AChE foi inibida apenas na concentração mais alta. Mais estudos são necessários agora para uma maior exploração da toxicidade da CBZ, uma vez que pode ser bioacumulável em toda a cadeia alimentar e pode afetar organismos não alvo.

Assessment of carbamazepine acute toxicity in the cockle Cerastoderma edule through chemical, physiological and biochemical tools.

The cockle Cerastoderma edule was exposed to four concentrations (5, 10, 20 and 70 µg L-1) of carbamazepine (CBZ). This anticonvulsant was found to alter the mussel behavior of by reducing its clearance rate (CR). Analysis of CBZ accumulation in tissues of C. edule was carried out using HPLC-UV after 48 or 96 hours of exposure. In addition, an overproduction of H2O2 by the bivalves was detected following exposure to CBZ but nitrite levels remained unchanged. Moreover, superoxide dismutase and catalase activities showed a significant increase in relation to their contact with CBZ. The activity of the biotransformation enzyme gluthatione-S-transferase did not change during exposure. Malondialdehyde (MDA) levels indicating cellular damage, increased when bivalves were exposed to 20 and 70 µg l-1 of carbamazepine for 96 h CBZ. The results also indicate that acetylcholinesterase activity (AChE) was inhibited in all CBZ concentrations during the 48 h exposure period. However, during the 96 h exposure period, AChE was only inhibited at the highest concentration. Further studies are needed now for more exploration of the toxicity of CBZ since it could be bioaccumulable throughout the food web and may affect non-target organisms.

A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor.

Identifying environmental damage due to anthropogenic activities is a focal point for scientists and policy makers like those involved in the European Water Framework Directive (WFD). Many of these approaches focus on ecological endpoints for assessing environmental perturbations, which lead to policies emphasizing mitigation rather than prevention. Biomarkers provide early-warning indicators of stress but it is necessary to distinguish their natural variations from those induced by chemical stress. The global aim of this study was to establish a baseline assessment criterion (BAC) using historical data in a reference site to define toxicity thresholds. We have developed a multiple polynomial regression model (MPR) accounting the influence of salinity, temperature and size of individual on energetic reserves (glycogen and lipids) in the marine polychaete Hediste diversicolor. The model identified a complex, orthogonal relationship between confounding factors and glycogen and a linear relationship between lipids and size of individuals.

Thyroid endocrine status of wild European eels (Anguilla anguilla) in the Loire (France). Relationships with organic contaminant body burdens.

In teleost fish, thyroid function is involved in various critical physiological processes. Given the complexity of the hypothalamo-pituitary-thyroid (HPT) axis, a large number of genes and proteins can be the potential target of endocrine-disrupting compounds (EDCs). The aim of this study was to evaluate, in yellow and silver European eels (Anguilla anguilla), potential effects of EDCs on thyroid status by analyzing the associations between EDC body burdens and thyroid hormones (THs). In yellow individuals, greater free T3/T4 ratios (FT3/FT4) and lower plasma FT4 levels were associated with greater concentrations of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), as highlighted by significant correlations with many congeners. Few positive relationships with alkylphenols were noticed. In contrast, silver eels usually exhibited less significant correlations between THs and contaminant loads. Expression of a series of genes involved in the HPT axis was also investigated in the silver individuals. Concerning mRNA expression in silver females, some main correlations were noticed: thyroid-stimulating hormone (TSHß) gene expression was significantly correlated to numerous PCBs, and hepatic mRNA levels of deiodinase 2 (Deio 2) were negatively correlated to 2-hydroxyfluorene (2-OHF) and 2-naphtol (2-OHNa). Thyroid receptor (TRα and TRß) mRNA levels exhibited weak negative correlations with some PBDEs in silver females and males. Hepatic vitellogenin (Vtg) mRNA levels were detected in all silver males but at lower levels than in silver females. In males, Vtg mRNA levels were positively associated to FT4/TT4. In silver females, strong positive correlations were found between congeners of PCBs, PBDEs and PFAS suggesting potential estrogenic effects. Overall, the observed results indicate that several organic contaminants, mainly dl-, ndl-PCBs and PBDEs, could be associated with changes in thyroid homeostasis in these fish, via direct or indirect interactions with peripheral deiodination, metabolism of T4 and mechanisms involved in TSHß, Deio 2 and Vtg gene transcription.

Occurrence of POPs and other persistent organic contaminants in the European eel (Anguilla anguilla) from the Loire estuary, France.

The chemical contamination of the Loire estuary by three classes of persistent organic pollutants (POPs): the polychlorinated biphenyls (PCBs), the polybrominated diphenyl ethers (PBDEs) and the perfluorinated and polyfluorinated alkyl substances (PFAS), and three families of organic contaminants, the alkylphenols (APs), the polycyclic aromatic hydrocarbon metabolites (OH-PAHs) and the bisphenol A (BPA) were investigated in the muscles and bile of European eel (Anguilla anguilla). Yellow eels (n=30) were caught in three different points along the estuary to highlight variations between sites and sources of contaminations. Silver eels (n=15) were also studied to compare contaminant impregnation between different life stages of the species. Average concentrations in the muscles of the eel ranged between: 857 and 4358 ng/gLW for the PCBs, 26 and 46 ng/gLW for the PBDEs, 130 and 1293 ng/gLW for the PFAS; and in bile: 31 and 286 µg/g protein for the APs, 9 and 26 µg/g protein for the OH-PAHs and ND-1213 µg/g protein for the BPA. Among PCBs, PCB 153 (40% contribution to the sum of PCBs) was predominant in all eel muscles. PBDE 47 (60%) was the most predominant PBDE congeners, while perfluorooctanesulfonic acid (85%) was the most widely detected PFAS. For APs, 4p-nonylphenol (91%) was the most abundant and for the OH-PAHs, it was 1OH-Pyrene (63%). All the eels exceeded the environmental quality standards (EQS) for biota for the PBDEs and about 75% were higher than the EQS specific to PFOS. Finally, 20% of the analyzed eels presented TEQ concentrations above the maximum limits for lipid-rich species. These results supplied new data on the occurrence, levels, and patterns of 53 organic chemicals in the eels from the Loire estuary and they highlighted the need of further investigations focused notably on the potential effects of these chemicals on this species and their analysis in the water and sediments of the estuary.

Dioxin-like, non-dioxin like PCB and PCDD/F contamination in European eel (Anguilla anguilla) from the Loire estuarine continuum: spatial and biological variabilities.

To characterize the eel contamination by dioxin-like (dl) and non dioxin-like (ndl) polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), sixty-two eels from the Loire estuary (France) were analyzed. PCB contamination significantly increased from glass eel stage (3.71 ± 1.85 and 15.2 ± 4.2 ng g(-1) dw) to other life stages (for yellow eels: 62.8 ± 34.4 and 382 ± 182 ng g(-1) dw; for silver eels: 93.7 ± 56.3 and 463 ± 245 ng g(-1) dw respectively for dl and ndl-PCBs). An inter-site variability based on PCB levels and profiles was observed among the three studied sites. For glass eels, the profile was mainly characterized by less chlorinated PCBs contrary to the other eels, displaying a different bioaccumulation pathway. Overall, the contamination level in the eels from this estuary was shown to be low for PCDD/Fs and intermediate for dl and ndl-PCBs, compared to other international/national areas. However, more than 60% of the studied silver eels displayed higher values for PCDD/F and dl-PCB WHO2005 TEQ than the EU permissible level of 10 pg g(-1) ww. This statement suggests a potential exposure to PCBs through eel consumption, especially with silver eels, and also points out apparent contamination that could eventually affect the reproductive success of the species.

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.

One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha).

A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.

One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha).

A 12-month active biomonitoring study was performed in 2008-2009 on a northern French river system using the freshwater mussel Dreissena polymorpha as a sentinel species. Allochtonous mussels originating from a reference site (Commercy) were caged at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle River basin. The main objective of the study was to characterize the influence of biotic (sex, food availability) and abiotic (temperature, chemicals) factors on the reproductive and energy reserve (glycogen, lipids) cycles of exposed mussels. Both cycles were markedly disturbed at the Bouy and Sept-Saulx sites where the lowest chlorophyll a levels were recorded during the study. At these sites, mussels obviously faced a negative energy balance, as confirmed by the impairment of their physiological state and byssal attachment. At other exposure sites, reproductive and energy reserves cycles were less impacted but were still dependent on the nutritional state of mussels. The latter appeared as a significant natural confounding factor in ecotoxicological survey performed in low polluted areas.

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.

Oxidative stress responses in bivalves (Scrobicularia plana, Cerastoderma edule) from the Oued Souss estuary (Morocco).

The aim of this work was to study the responses of oxidative-stress biomarkers (catalase: CAT, thiobarbituric acid reactive substances: TBARS) in bivalves (Scrobicularia plana and Cerastoderma edule) from the Oued Souss estuary (Morocco). This estuary, subjected to large amounts of sewage discharges and industrial effluents, do not receive since November 2002 any waste outlets due to their connection to a wastewater purification plant. High CAT and TBARS values were recorded in both species collected before the implantation of wastewater treatment. These results confirm that these bivalves were submitted to various contaminants generating oxidative stress and membrane lipid peroxidation. The diminution of these two biomarkers following the setting up of the purification plant indicates probably a reduction in contaminants in the Oued Souss estuary. We recommend the use of CAT and TBARS as oxidative-stress biomarkers in S. plana and C. edule.

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.

Linking steroid hormone levels to sexual maturity index and energy reserves in Nereis diversicolor from clean and polluted estuaries.

The objective of this work was to compare seasonal variations of reproduction physiology of the ragworm Nereis diversicolor --a key species in estuarine ecosystems--originating from a clean (Authie) and multi-polluted (Seine) estuaries. A particular attention was carried out in female worms, on relationships between sexual maturity stages, energy reserves (glycogen and lipids) and steroid hormone levels (progesterone, 17beta-estradiol, and testosterone). Sexual maturity index (SMI), energy reserves and steroid hormones are clearly influenced by season in worms from both sites. Depleted steroid hormone levels were depicted in specimens exhibiting high sexual maturity stage and energy reserves. Intersite analysis has revealed all over the sampling period:--a sexual precocity in worms from Seine,--glycogen concentrations generally higher in worms from Authie,--no clear tendency for lipids,--no differences in steroid hormone levels. Sexual precocity and lower glycogen levels in Seine could be explained by a specific strategy above all devoted to reproduction in these worms. Chemical stress could be a possible explanation of these observations.

Marine water quality assessment using transplanted oyster larvae.

Active bio-monitoring in terms of biomarkers was attempted using Crassostrea gigas larvae produced in the laboratory and transplanted using special containers to two sites at the entrance (A) and inner part (P) of the harbour of Arcachon (French Atlantic Coast). The larvae were kept in the medium for 48 h. Their physiological status and their biomarker levels : acetylcholinesterase AChE, catalase CAT and glutathione S-transferase GST activities were determined together with metallothionein MT and Thiobarbituric Acid Reactive Substances TBARS concentrations. Copper and PAH (polycyclic aromatic hydrocarbon) concentrations were determined in the exposed larvae and in the sediments collected under the containers. Cadmium, lead and zinc could be also analyzed in the sediments. Toxicity tests demonstrate that the larvae are in better physiological conditions in A compared to P. Larvae transplanted in the inner harbour (P) present relatively high GST activity (869.1+/-39.3 nmol min(-1)mg protein(-1)), TBARS (2.74+/-0.19 nmol mg protein(-1)), compared to those exposed at the harbour entrance (A). Copper measured in the sediments (65+/-1 mg kg(-1) d.w.) collected under the cages at P is higher than at A. Larvae placed in A present higher total PAH concentrations compared to the inner part. The data tend to reveal a lower copper and higher PAH contamination in A than in P. Therefore larvae, developing in the natural medium, show different responses according to their immersion sites. These responses, obtained within 48 h, may be related to the chemical contamination of the environment and may be used for seawater quality assessment in future studies.

Tolerance and biomarkers as useful tools for assessing environmental quality in the Oued Souss estuary (Bay of Agadir, Morocco).

The aim of this study was to assess aquatic environmental quality of Oued Souss (Agadir, Morocco). This estuary has been subjected for a long time to large amounts of sewage discharges and industrial effluents. Since November 2002, no waste outlets have been discharged in this site due to their connection to a wastewater purification plant. Firstly, we have compared metal tolerance of the annelid polychaete (Nereis diversicolor) originating from Oued Souss and a relatively clean site (Oualidia, Morocco). Secondly, we have evaluated with a multi-marker approach (acetylcholinesterase [AChE], glutathione-S-transferases [GSTs], catalase, thiobarbituric acid reactive substances [TBARs]) responses of worms to the pollution gradient. Results have shown that worms from Oued Souss have acquired tolerance to copper and zinc due to a long-term sub-lethal metal exposure and this metal tolerance was maintained in spite of the end of wastewater discharges in this site. Higher catalase, GSTs and TBARs values have been observed in worms from Oued Souss sampled before implantation of wastewater treatment. The multi-marker approach confirms that these worms have been submitted to various contaminants. In contrast, high inhibition in AChE activities measured in worms from Oued Souss could be explained by the continuous agricultural influence of nearest areas. The level of contamination was probably maintained since biomarker values were generally higher in worms from Oued Souss when compared to Oualidia.