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Recently, research has contributed to better knowledge on the occurrence of pesticides in coastal water by identifying frequently detected substances, their concentration range and their acute and chronic toxicity for organisms. Pesticide pollution is of particular concern in France due to important agricultural activities and presence of several exoreic catchment areas that vehicle pesticides up to coastal waters, impacting non-target marine species. Several ecotoxicology questions remain to be addressed concerning the long-term effects of chronic pesticide exposure and the mechanisms involved in adaptation to chemical stress. In the present study, we brought new insights on the genetic and epigenetic effects of the herbicide diuron in oyster genitors. During gametogenesis, we exposed Crassostrea gigas to environmentally realistic herbicide concentrations (0.2-0.3 μg L -1 during two 7-day periods at half-course and end of gametogenesis). Diuron exposure was shown to decrease global DNA methylation and total methyltransferase activity in whole oyster tissue; this is consistent with the previous observation of a significant decrease in DNMT1 gene expression. Diuron effect seemed to be tissue-specific; hypermethylation was detected in the digestive gland, whereas diuron exposure had no effect on gill and gonad tissue. The genotoxicity of diuron was confirmed by the detection of one adduct in gonad DNA. By using in vitro approaches and human DNMT1 (DNMT1 has not been purified yet in bivalves), the presence of DNA lesions (adduct, 8-oxodGuo) was shown to interfere with DNMT1 activity, indicating a complex interaction between DNA damage and DNA methylation. Based on our results, we propose mechanisms to explain the effect of diuron exposure on DNA methylation, a widespread epigenetic mark.

Genitors of the Pacific oyster Crassostrea gigas were submitted during gametogenesis to a short pulse exposure to the herbicide diuron at a realistic environmental concentration. Histological analysis showed no effect of diuron on gametogenesis course, sex ratio and reproductive effort. A non-significant increase in testosterone and progesterone levels was observed in genitors exposed to the herbicide. At cell level, diuron exposure was shown to modulate the phagocytic activity of circulating hemocytes. The results of a transcriptional analysis showed that diuron affected the expression of genes belonging to functions known to play a major role during oyster gametogenesis such as gene transcription regulation, DNA replication and repair, DNA methylation and cytokinesis. Taking into account the results we previously obtained on the same genitors, this study showed a negative effect of diuron on oyster reproduction by inducing both structural and functional modifications of the DNA.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be present at high levels as mixtures in polluted aquatic environments. Many PAHs are potent mutagens and several are well-known carcinogens. Despite numerous studies on individual compounds, little is known about the toxicity of PAHs mixtures that are encountered in environmental situations. In the present work, zebrafish were continuously fed from 5 days post-fertilisation to 14 months post-fertilisation (mpf) with a diet spiked with fractions of either pyrolytic (PY), petrogenic light oil (LO), or petrogenic heavy oil (HO) origin at three concentrations. A decrease in survival was identified after 3 mpf in fish fed with the highest concentration of HO or LO, but not for PY. All PAH fractions caused preneoplastic and neoplastic disorders in long-term-exposed animals. Target tissues were almost exclusively of epithelial origin, with the bile duct epithelium being the most susceptible to chronic exposure to all PAH fractions, and with germ cells being the second most responsive cells. Significantly higher incidences of neoplasms were observed with increasing PAH concentration and exposure duration. The most severe carcinogenic effects were induced by dietary exposure to HO compared to exposure to LO or PY (45, 30 and 7 %, respectively, after 9 to 10 months of exposure to an intermediate concentration of PAHs). In contrast, earliest carcinogenic effects were detected as soon as 3 mpf after exposure to LO, including the lowest concentration, or to PY. PAH bioactivation and genotoxicity in blood was assessed by ethoxyresorufin-O-deethylase activity quantification and comet and micronuclei assays, respectively, but none of these were positive. Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenotoxic events that impair survival and physiology of exposed fish.

The Seine Estuary is well known to be widely contaminated by organic pollutants and especially by polycyclic aromatic hydrocarbons (PAHs). Fish are known to metabolize PAHs, leading to different toxic effects at both cellular and sub-cellular levels. In this work, we studied the relationships between the 7-ethoxyresorufin-O-deethylase (EROD) activity in the liver, the level of DNA strand breaks in blood cells and the concentration of PAH metabolites in the bile of the sentinel flatfish species Limanda limanda. Muscle and liver samples were analysed for parent PAH levels. Female and male dabs of two size classes (juveniles and adults) were collected by trawling in two sites with different degrees of pollution during March and September 2005 and 2006. Significant effects of sex, age, site and season were demonstrated on EROD activity and the level of strand breaks. Parent PAH concentrations in dabs did not allow discriminating of the two sampling sites. However, for PAH metabolites, significant differences were observed with sites and seasons. Dabs collected at the mouth of the estuary appeared to be the most impacted when looking at the results obtained with the three selected markers. The significant correlations observed between the level of PAH metabolites and the level of DNA lesions showed the importance of a combined analysis of chemical and biochemical markers to correctly assess the contribution of chemical contamination to the toxic effects measured in situ in fish.

In this study, laboratory experiments were carried out in order to come to a better understanding of the fate of polycyclic aromatic hydrocarbons (PAHs) in the marine environment and especially on their bioaccumulation, biotransformation and genotoxic effects in fish. Juveniles of turbot (Scophthalmus maximus) were exposed to PAHs through different routes via (1) a mixture of dissolved PAHs, (2) a PAH-polluted sediment and (3) an oil fuel elutriate. Fish were exposed 4 days followed by a 6-day depuration period. In each experiment, PAH concentrations in the seawater of the tanks were analysed regularly by gas chromatography coupled with mass spectrometry. Muscle and liver samples were also analysed for parent PAH levels and PAH bioconcentration factors were calculated. Biotransformation was evaluated by measuring the levels of PAH metabolites in fish bile. Genotoxicity was assessed by the alkaline comet assay. Regardless of exposure route, the parent PAH concentrations in the liver and muscle showed a peak level 1 day after the beginning of the exposure, followed by a decrease up to the background level towards the end of the experiment, except for the exposure to dissolved PAHs for which levels were relatively low throughout the study. As a consequence, no bioaccumulation was observed in fish tissues at the end of the experiment. In contrast, regardless of exposure routes, a rapid production of biliary metabolites was observed throughout the whole exposure experiment. This was especially true for 1-hydroxypyrene, the major metabolite of pyrene. After 6 days of recovery in clean water, a significant decrease in the total metabolite concentrations occurred in bile. Fish exposed through either route displayed a significant increase in DNA strand breaks after 4 days of exposure, and significant correlations were observed between the level of biliary PAH metabolites and the level of DNA lesions in fish erythrocytes. Overall results indicate that exposure to either a mixture of dissolved PAHs, a PAH-contaminated sediment or a dispersed oil fuel elutriate leads to biotransformation and increase in DNA damage in fish. The quantification of PAH metabolites in bile and DNA damage in erythrocytes appear to be suitable for environmental monitoring of marine pollution either in the case of accidental oil spills or sediment contamination.