Environmental concentrations of irgarol, diuron and S-metolachlor induce deleterious effects on gametes and embryos of the Pacific oyster, Crassostrea gigas

Irgarol and diuron are the most representative "organic booster biocides" that replace organotin compounds in antifouling paints, and metolachlor is one of the most extensively used chloroacetamide herbicides in agriculture. The toxicity of S-metolachlor, irgarol and diuron was evaluated in Pacific oyster (Crassostrea gigas) gametes or embryos exposed to concentrations of pesticides ranging from 0.1× to 1000×, with 1× corresponding to environmental concentrations of the three studied pesticides in Arcachon Bay (France). Exposures were performed on (1) spermatozoa alone (2) oocytes alone and (3) both spermatozoa and oocytes, and adverse effects on fertilization success and offspring development were recorded. The results showed that the fertilizing capacity of spermatozoa was significantly affected after gamete exposure to pesticide concentrations as low as 1× of irgarol and diuron and 10× of metolachlor. The offspring obtained from pesticide-exposed spermatozoa displayed a dose-dependent increase in developmental abnormalities. In contrast, treating oocytes with pesticide concentrations up to 10× did not alter fertilization rate and offspring quality. However, a significant decline in fertilization success and increase in abnormal D-larvae prevalence were observed at higher concentrations 10× (0.1 μg L(-1)) for S-metolachlor and 100× for irgarol (1.0 μg L(-1)) and diuron (4.0 μg L(-1)). Irgarol, diuron and S-metolachlor also induced a dose-dependent increase in abnormal D-larvae prevalence when freshly fertilized embryos were treated with pesticide concentrations as low as concentration of 1× (0.01 μg L(-1) for irgarol or S-metolachlor, and 0.04 μg L(-1) for diuron). The two bioassays on C. gigas spermatozoa and embryos displayed similar sensitivities to the studied pesticides while oocytes were less sensitive. Diuron, irgarol and S-metolachlor induced spermiotoxicity and embryotoxicity at environmentally relevant concentrations and therefore might be a threat to oyster recruitment in coastal areas facing chronic inputs of pesticides

Comparative responses of sperm cells and embryos of Pacific oyster (Crassostrea gigas) to exposure to metolachlor and its degradation products

International audienceMetolachlor is one of the most intensively used chloroacetanilide herbicides in agriculture. Consequently, it has been frequently detected in coastal waters as well as its major degradation products, metolachlor ethane sulfonic acid (MESA) and metolachlor oxanilic acid (MOA) which are encountered at higher concentrations than metolachlor. Although a few studies of metolachlor toxicity have been conducted on marine organisms, little is known about the environmental toxicity of metolachlor degradation products. In this study, the deleterious effects of metolachlor and its degradation products on spermatozoa and embryos of Crassostrea gigas have been compared using biomarkers of developmental defects, DNA damage and gene transcription levels. After 24h exposure, significant increases in the percentage of abnormal D-larvae and DNA damage were observed from 0.01μgL(-1) for S-metolachlor and 0.1μgL(-1) for MESA and MOA. Results showed that S-metolachlor was more embryotoxic and genotoxic than its degradation products. Oyster sperm was also very sensitive to metolachlor exposure and followed the pattern: metolachlor (0.01μgL(-1))>MOA (0.1μgL(-1))>MESA (1μgL(-1)). Metolachlor and MESA mainly triggered variations in the transcription level of genes encoding proteins involved in oxidative stress responses (mitochondrial superoxide dismutase and catalase). Overall, no significant variation in transcription levels could be detected in C. gigas embryos exposed to MOA. This study demonstrates that metolachlor and its main degradation products have the potential to impact several steps of oyster development and therefore recruitment in coastal areas exposed to chronic inputs of pesticides

Developmental toxicity of PAH mixtures in fish early life stages. Part I: adverse effects in rainbow trout

A new gravel-contact assay using rainbow trout, Oncorhynchus mykiss, embryos was developed to assess the toxicity of polycyclic aromatic hydrocarbons (PAHs) and other hydrophobic compounds. Environmentally realistic exposure conditions were mimicked with a direct exposure of eyed rainbow trout embryos incubated onto chemical-spiked gravels until hatching at 10 °C. Several endpoints were recorded including survival, hatching delay, hatching success, biometry, developmental abnormalities, and DNA damage (comet and micronucleus assays). This bioassay was firstly tested with two model PAHs, fluoranthene and benzo[a]pyrene. Then, the method was applied to compare the toxicity of three PAH complex mixtures characterized by different PAH compositions: a pyrolytic extract from a PAH-contaminated sediment (Seine estuary, France) and two petrogenic extracts from Arabian Light and Erika oils, at two environmental concentrations, 3 and 10 μg g−1 sum of PAHs. The degree and spectrum of toxicity were different according to the extract considered. Acute effects including embryo mortality and decreased hatching success were observed only for Erika oil extract. Arabian Light and pyrolytic extracts induced mainly sublethal effects including reduced larvae size and hemorrhages. Arabian Light and Erika extracts both induced repairable DNA damage as revealed by the comet assay versus the micronucleus assay. The concentration and proportion of methylphenanthrenes and methylanthracenes appeared to drive the toxicity of the three PAH fractions tested, featuring a toxic gradient as follows: pyrolytic < Arabian Light < Erika. The minimal concentration causing developmental defects was as low as 0.7 μg g−1 sum of PAHs, indicating the high sensitivity of the assay and validating its use for toxicity assessment of particle-bound pollutants

Usefulness of RTL-W1 and OLCAB-e3 fish cell lines and multiple endpoint measurements for toxicity evaluation of unknown or complex mixture of chemicals

International audienceFish are currently used for the assessment of chemical toxicity. The REACh regulation and the European directive on the protection of animals used for scientific purposes both recommend the use of methods other than animal testing. In view of this, fish cell lines are increasingly used to provide fast and reliable toxic and ecotoxic data on new chemicals. The sensitivity of the Rainbow trout liver cell line RTL-W1 and Japanese medaka embryos cell line OLCAB-e3 were used with different toxicity endpoints, namely cytotoxicity, EROD activity, ROS production and DNA damage for various classes of pollutants displaying different modes of action but also with complex environmental mixtures. Toxicity tests were coupled with chemical analysis to quantify the chemical concentrations in cell cultures. Differences in sensitivity were found between fish cell lines. MTT reduction assay revealed that OLCAB-e3 cells were more sensitive than RTL-W1 cells. On the contrary, RTL-W1 gave higher response levels for the Fpg-modified comet assay and ROS assay. The OLCAB-e3 cell line did not express EROD activity unlike RTL-W1. This study highlights the capacity of the two different fish cell lines to measure the toxicity of individual toxicants but also environmental mixtures. Then, results obtained here illustrate the interest of using different cell lines and toxicity endpoints to assess the toxicity of complex or unknown mixture of chemicals

Developmental toxicity of PAH mixtures in fish early life stages. Part II: adverse effects in Japanese medaka

In aquatic environments, polycyclic aromatic hydrocarbons (PAHs) mostly occur as complex mixtures, for which risk assessment remains problematic. To better understand the effects of PAH mixture toxicity on fish early life stages, this study compared the developmental toxicity of three PAH complex mixtures. These mixtures were extracted from a PAH-contaminated sediment (Seine estuary, France) and two oils (Arabian Light and Erika). For each fraction, artificial sediment was spiked at three different environmental concentrations roughly equivalent to 0.5, 4, and 10 μg total PAH g−1 dw. Japanese medaka embryos were incubated on these PAH-spiked sediments throughout their development, right up until hatching. Several endpoints were recorded at different developmental stages, including acute endpoints, morphological abnormalities, larvae locomotion, and genotoxicity (comet and micronucleus assays). The three PAH fractions delayed hatching, induced developmental abnormalities, disrupted larvae swimming activity, and damaged DNA at environmental concentrations. Differences in toxicity levels, likely related to differences in PAH proportions, were highlighted between fractions. The Arabian Light and Erika petrogenic fractions, containing a high proportion of alkylated PAHs and low molecular weight PAHs, were more toxic to Japanese medaka early life stages than the pyrolytic fraction. This was not supported by the toxic equivalency approach, which appeared unsuitable for assessing the toxicity of the three PAH fractions to fish early life stages. This study highlights the potential risks posed by environmental mixtures of alkylated and low molecular weight PAHs to early stages of fish development

Combined effects of pollutants and salinity on embryo-larval development of the Pacific oyster, Crassostrea gigas

For several years, low larval recruitment has been observed in Arcachon Bay, in southwest France. Exposure to pollutants could partly account for the reduction of early life stages of the Pacific oyster. This study evaluated the effects of copper and S-metolachlor in combination with salinity on the early life stages of Crassostrea gigas. Embryos were exposed to concentrations of copper (1, 10 and 50 μg L(-1)) or S-metolachlor (10, 100 and 1000 ng L(-1)) and six salinities (18, 21, 24, 27, 30 and 33 u.s.i). Embryotoxicity was measured by considering both the percentage of abnormalities and arrested development in D-shaped larvae. Embryo-larval development was only affected at salinities ≤24 u.s.i, which have never been observed during C. gigas reproduction period in Arcachon Bay. Both contaminants had an effect at environmental concentrations. Our results suggest that copper and metolachlor toxicity was enhanced with decreasing salinity

Environmental concentrations of benz[a]anthracene induce developmental defects and DNA damage and impair photomotor response in Japanese medaka larvae

Benz[a]anthracene (BaA) is a ubiquitous polycyclic aromatic hydrocarbon found in numerous aquatic ecosystems. However, ecotoxicological data in aquatic organisms are scarce. To remedy this lack of data, Japanese medaka (Oryzias latipes) embryos were exposed to BaA and toxic effects were investigated at multiple toxicological endpoints. Japanese medaka embryos were incubated onto BaA-spiked artificial sediment for 9 days at low or moderate environmental concentrations ranging from 0.9 to 12 µg g−1 dw. BaA-exposed embryos exhibited significant tachycardia. BaA exposure was also shown to increase CYP1A activity in the hepato-biliary tissue as well as craniofacial deformities and DNA damage in pro-larvae. The photomotor response of BaA-exposed larvae was reduced in comparison to the control group. According to this set of tests, the lowest tested and observed effect concentration (LOEC) for Japanese medaka early life stages was equivalent to 0.92 µg g−1 dw of BaA. This concentration fall into the range of concentrations frequently encountered in sediments of polluted aquatic ecosystems. Taking into consideration these results, BaA represents a threat for fish early life stages in particular those developing onto or into contaminated sediments