Differences in chemical contaminants bioaccumulation and ecotoxicology biomarkers in Mytilus edulis and Mytilus galloprovincialis and their hybrids.

The Mytilus mussels are spread all over the world and many related species coexist in several areas and can produce hybrid offspring. Mussels have been used for decades in national and international programs to monitor chemical contamination in the environment. Differences in bioaccumulation and biotransformation abilities between species and their hybrids should be evaluated to assess the comparability of the results obtained within the international biomonitoring programs. The objective of this study was to characterize bioaccumulation abilities and biomarker responses in Mytilus edulis, Mytilus galloprovincialis and their hybrids via an in situ transplantation experimentation on their progenies. Four mussel groups (M. edulis, M. galloprovincialis and two hybrids batches) issued from genetically characterized parents were transplanted for one year in Charente Maritime (France) to ensure their exposure to identical sources of contamination. The bioaccumulation of several families of contaminants (trace metals, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, polychlorinated biphenyls), the response of several biomarkers (DNA strand breaks level, lysosomal membrane stability, metallothionein content, acetylcholine esterase activity) and some physiological parameters (growth, mortality, gonadal development), were analyzed. Differences were observed between species, however they were contaminant-specific. Variations in contaminants levels were observed between progenies, with higher levels of Cu, PBDE, PCB in M. edulis, and higher levels of Cd, Hg, Zn in M galloprovincialis. This study demonstrated that variations in contaminant bioaccumulation and different biomarker responses exist between Mytilus species in the field. Data on species or the presence of hybrid individuals (or introgression) is an important additional parameter to add to biomonitoring programs databases.

Realistic environmental exposure to microplastics does not induce biological effects in the Pacific oyster Crassostrea gigas.

The aim of the present study was to evaluate the presence and potential toxic effects of plastic fragments (<400 µm) of polyethylene and polypropylene on the Pacific oyster Crassostrea gigas. Oysters were exposed to environmentally relevant concentrations (0, 0.008, 10, 100 µg of particles/L) during 10 days, followed by a depuration period of 10 days in clean seawater. Effects of microplastics were evaluated on the clearance rate of organisms, tissue alteration, antioxidant defense, immune alteration and DNA damage. Detection and quantification of microplastics in oyster's tissues (digestive gland, gills and other tissues) and biodeposits using infrared microscopy were also conducted. Microplastics were detected in oyster's biodeposits following exposure to all tested concentrations: 0.003, 0.006 and 0.05 particles/mg of biodeposits in oysters exposed to 0.008, 10 and 100 µg of particles/L, respectively. No significant modulation of biological markers was measured in organisms exposed to microplastics in environmentally relevant conditions.

The influence of natural dissolved organic matter on herbicide toxicity to marine microalgae is species-dependent.

Microalgae, which are the foundation of aquatic food webs, may be the indirect target of herbicides used for agricultural and urban applications. Microalgae also interact with other compounds from their environment, such as natural dissolved organic matter (DOM), which can itself interact with herbicides. This study aimed to evaluate the influence of natural DOM on the toxicity of three herbicides (diuron, irgarol and S-metolachlor), singly and in ternary mixtures, to two marine microalgae, Chaetoceros calcitrans and Tetraselmis suecica, in monospecific, non-axenic cultures. Effects on growth, photosynthetic efficiency (Ф'M) and relative lipid content were evaluated. The chemical environment (herbicide and nutrient concentrations, dissolved organic carbon and DOM optical properties) was also monitored to assess any changes during the experiments. The results show that, without DOM, the highest irgarol concentration (I0.5: 0.5 mg.L-1) and the strongest mixture (M2: irgarol 0.5 µg.L-1 + diuron 0.5 µg.L-1 + S-metolachlor 5.0 µg.L-1) significantly decreased all parameters for both species. Similar impacts were induced by I0.5 and M2 in C. calcitrans (around -56% for growth, -50% for relative lipid content and -28% for Ф'M), but a significantly higher toxicity of M2 was observed in T. suecica (-56% and -62% with I0.5 and M2 for growth, respectively), suggesting a possible interaction between molecules. With DOM added to the culture media, a significant inhibition of these three parameters was also observed with I0.5 and M2 for both species. Furthermore, DOM modulated herbicide toxicity, which was decreased for C. calcitrans (-51% growth at I0.5 and M2) and increased for T. suecica (-64% and -75% growth at I0.5 and M2, respectively). In addition to the direct and/or indirect (via their associated bacteria) use of molecules present in natural DOM, the characterization of the chemical environment showed that the toxic effects observed on microalgae were accompanied by modifications of DOM composition and the quantity of dissolved organic carbon excreted and/or secreted by microorganisms. This toxicity modulation in presence of DOM could be explained by (i) the modification of herbicide bioavailability, (ii) a difference in cell wall composition between the two species, and/or (iii) a higher detoxification capacity of C. calcitrans by the use of molecules contained in DOM. This study therefore demonstrated, for the first time, the major modulating role of natural DOM on the toxicity of herbicides to marine microalgae.

Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 µg L-1 Cu2+) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 µg L-1, while significant genotoxic effects were detected at 1 µg L-1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such a correlation was diminished following copper exposure. The methylation level of some specific gene regions (HoxA1, Hox2, Engrailed2 and Notochord) displayed changes upon copper exposure. Such changes were gene and exon-specific and no obvious global trends could be identified. Our study suggests that the embryotoxic effects of copper in oysters could involve homeotic gene expression impairment possibly by changing DNA methylation levels.

Parental diuron-exposure alters offspring transcriptome and fitness in Pacific oyster Crassostrea gigas.

One of the primary challenges in ecotoxicology is to contribute to the assessment of the ecological status of ecosystems. In this study, we used Pacific oyster Crassostrea gigas to explore the effects of a parental exposure to diuron, a herbicide frequently detected in marine coastal environments. The present toxicogenomic study provides evidence that exposure of oyster genitors to diuron during gametogenesis results in changes in offspring, namely, transcriptomic profile alterations, increased global DNA methylation levels and reduced growth and survival within the first year of life. Importantly, we highlighted the limitations to identify particular genes or gene expression signatures that could serve as biomarkers for parental herbicide-exposure and further for multigenerational and transgenerational effects of specific chemical stressors. By analyzing samples from two independent experiments, we demonstrated that, due to complex confounding effects with both tested solvent vehicles, diuron non-specifically affected the offspring transcriptome. These original results question the potential development of predictive genomic tools for detecting specific indirect impacts of contaminants in environmental risk assessments. However, our results indicate that chronic environmental exposure to diuron over several generations may have significant long term impacts on oyster populations with adverse health outcomes.

Comparative embryotoxicity and genotoxicity of the herbicide diuron and its metabolites in early life stages of Crassostrea gigas: Implication of reactive oxygen species production.

Herbicides are one of the major classes of pollutants contaminating coastal waters over the world. Among them, diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a phenylurea herbicide frequently detected in oyster-producing area, known to be toxic for this important exploited non-target species. With the aim to investigate the mechanisms by which diuron displays its toxicity in oyster, the implication of both biotransformation and oxygen reactive species (ROS) production was studied considering embryotoxicity and genotoxicity as endpoints. Comparative embryotoxicity and genotoxicity of diuron and its main metabolites (DCPMU, DCPU and 3,4-DCA) were thus studied on oyster larvae by the embryo-larval bioassay on D larvae and the comet assay on trochophore larvae, respectively. Exposures were also performed in presence and absence of known ROS scavenger compounds - ascorbic acid and N-acetylcysteine, to evaluate the involvement of oxyradicals in the toxic responses. In the case of diuron, the production of ROS on exposed oyster larvae was also measured using 2',7'-dichlorodihydrofluorescein diacetate as a probe for flow cytometric analysis. The results we obtained showed the embryotoxicity and genotoxicity of diuron and its metabolites in early life stages of the Pacific oyster. For concentrations ranging from 0.05 to 0.5µgL(-1), diuron appeared significantly more embryotoxic than DCPMU and DCPU (p<0.001). Embryotoxicity decreased with diuron metabolism as follows: diuron≥DCPMU=DCPU, highlighting that biotransformation can constitute a true detoxication pathways in oyster larvae by decreasing the toxicity of the parent compound. In the opposite, no difference was observed between diuron and its metabolites concerning larval development when considering a lower and more environmentally realistic range of concentrations (0.002-0.050µgL(-1)). 3,4-DCA was the only compound that did not show any sign of embryotoxicity, even at concentrations up to 5µgL(-1). Concerning genotoxicity, no significant difference was observed between diuron and all of its metabolites including 3, 4 DCA with damages detected from the concentration of 0.05µgL(-1). As for diuron, the toxicity of the metabolites seems to be mediated in some part by ROS production as clearly demonstrated by the decrease in genotoxicity and developmental abnormalities in the presence of the oxidant scavenger, ascorbic acid.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas.

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

Multigenerational exposure of the microalga Tetraselmis suecica to diuron leads to spontaneous long-term strain adaptation.

To investigate the ability of microalgae to develop stable, long-term resistance to herbicides, the marine microalga Tetraselmis suecica was exposed to the herbicide diuron (5 µg/L) for a 43-generation exposure period followed by a 12-generation depuration phase. During the first 25 generations, diuron-exposed cultures showed doubling times ranging from 1.95 to 2.6 days, which was 2 to 2.5-fold longer than control cultures. Between generations 25 and 38, during diuron exposure, two out of the three exposed cultures exhibited a spontaneous drop in doubling time. These results provided evidence of culture adaptation to diuron. To assess persistence of the diuron adaptation observed on growth performance, one of the adapted cultures (D3) was maintained for 12 months in unexposed conditions and then tested by a second, short-term exposure to diuron 5 µg/L, in parallel with a control culture (C1) for six generations. Flow cytometry analyses were used to monitor cell density, viability, morphology, relative chlorophyll content and intracellular reactive oxygen species (ROS) level. Under these conditions, diuron induced a strong increase of doubling time in exposed-C1 cultures (2.5-fold longer than unexposed-C1 cultures), but no significant increase occurred in exposed D3-cultures compared with unexposed D3- and unexposed C1-cultures, showing the persistence of adaptation in the previously-exposed strain D3. Intracellular ROS level showed the same trend. Significant differences were observed between these strains, with weaker effects of diuron on strain D3 compared with strain C1: forward scatter (FSC), representing relative cell size, decreased in exposed cultures (67.8% and 95% of the controls for C1 and D3, respectively), whereas FL3 as relative chlorophyll content increased in exposed cultures (115.6% and 108.6% of the controls for C1 and D3, respectively). Results of second exposure to diuron revealed that the adaptation of strain D3 had persisted after 12 months of depuration, as no growth impairment was observed. This study demonstrates the possible appearance of stable diuron resistance in microalgae in cases of strong, multigenerational chronic exposure to this herbicide in polluted environments.