Immunotoxicological effects of perfluorooctanesulfonic acid on European seabass are reduced by polyethylene microplastics.

Marine environments receive plastic waste, where it suffers a transformation process into smaller particles. Among them, microplastics (MPs; <5 mm) are ingested by aquatic organisms leading to negative effects on animal welfare. The interactions between MPs, contaminants and organisms are poorly understood. To clarify this issue, European seabass (Dicentrarchus labrax L.) were fed with diets supplemented with 0 (control), polyethylene (PE) MPs (100 mg/kg diet), perfluorooctanesulfonic acid (PFOS, 4.83 µg/kg diet) or PFOS adsorbed to MPs (MPs-PFOS; final concentrations of 4.83 µg and 100 mg of PFOS and MP per kg of feed, respectively). Samples of skin mucus, serum, head-kidney (HK), liver, muscle, brain and intestine were obtained. PFOS levels were high in the liver of fish fed with the PFOS-diet, and markedly reduced when adsorbed to MPs. Compared to the control groups, liver EROD activity did not show any significant changes, whereas brain and muscle cholinesterase activities were decreased in all the groups. The histological and morphometrical study on liver and intestine showed significant alterations in fish fed with the experimental diets. At functional level, all the experimental diets affected the humoral (peroxidase, IgM, protease and bactericidal activities) as well as cellular (phagocytosis, respiratory burst and peroxidase) activities of HK leukocytes, being more marked those effects caused by the PFOS diet. Besides, treatments produced inflammation and oxidative stress as evidenced at gene level. Principal component analysis demonstrated that seabass fed with MPs-PFOS showed more similar effects to MPs alone than to PFOS. Overall, seabass fed with MPs-PFOS diet showed similar or lower toxicological alterations than those fed with MPs or PFOS alone demonstrating the lack of additive effects or even protection against PFOS toxicity.

Sub-lethal effects of waterborne copper in early developmental stages of rainbow trout (Oncorhynchus mykiss).

The aim of this work was to study the impact of copper during a sub-chronic exposure to environmental concentrations in the early life stages of rainbow trout (Oncorhynchus mykiss). Eyed-stage embryos of rainbow trout, at 265 °D, were exposed in semi-static conditions to sub-lethal concentrations of CuSO4 up to the larval stage (528 °D) under laboratory-controlled conditions. During 3 weeks, they were exposed to the environmentally-realistic concentration of 2 µg/L Cu and to a 10-fold higher concentration, 20 µg/L Cu. Several biological (survival, hatching success, malformation, growth) and behavioral (swimming activity) and molecular endpoints (genotoxicity and gene transcription) were studied. Exposure to 20 µg/L Cu had an inhibitory effect on hatching and increased half-hatched embryos (25%). At the end of the exposure, no significant differences were observed in growth of the larvae exposed to the highest Cu concentration. However, larvae exposed to 2 µg/L Cu exhibited increased growth in comparison with non-exposed larvae. The percentage of malformed larvae was significantly higher for both copper conditions, with skeletal malformations being the most observed. Expression of several genes was evaluated in whole larvae using quantitative real-time PCR. Genes involved in detoxification (gst, mt1 and mt2) and in cell cycle arrest (p53) were significantly repressed in both copper conditions when compared to control. In addition, potential genotoxic effects on larvae were investigated by the comet assay on blood cells, but this test did not demonstrate any significant DNA damage on larvae exposed to copper. This study confirms the adverse effects of copper on early life stages of rainbow trout even at the lowest environmentally relevant tested concentration.

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

Fish 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.

A comprehensive study of the toxicity of natural multi-contaminated sediments: New insights brought by the use of a combined approach using the medaka embryo-larval assay and physico-chemical analyses.

Sediment compartment is a long term sink for pollutants and a secondary source of contamination for aquatic species. The abiotic factors controlling the bioavailability and thus the toxicity of complex mixtures of pollutants accumulated in sediments are poorly documented. To highlight the different factors influencing sediment toxicity, we identified and analyzed the physico-chemical properties, micro-pollutant contents, and toxicity level of six contrasted sediments in the Lot-Garonne continuum. Sediment toxicity was evaluated using the recently described Japanese medaka (Oryzias latipes) embryo-larval assay with direct exposure to whole sediment (MELAc). Multiple toxicity endpoints including embryotoxicity, developmental defects and DNA damage were analyzed in exposed embryos. Chemical analyses revealed significant variations in the nature and contamination profile of sediments, mainly impacted by metallic trace elements and, unexpectedly, polycyclic aromatic hydrocarbons. Exposure to sediments induced different toxic impacts on medaka early life stages when compared with the reference site. Principal component analysis showed that the toxic responses following exposure to sediments from the Lot River and its tributary were associated with micro-pollutant contamination: biometric measurements, hatching success, genotoxicity, craniofacial deformities and yolk sac malabsorption were specifically correlated to metallic and organic contaminants. Conversely, the main biological responses following exposure to the Garonne River sediments were more likely related to their physico-chemical properties than to their contamination level. Time to hatch, cardiovascular injuries and spinal deformities were correlated to organic matter content, fine particles and dissolved oxygen levels. These results emphasize the necessity of combining physico-chemical analysis of sediment with toxicity assessment to accurately evaluate the environmental risks associated with sediment contamination.

Using an Integrated Approach to Assess the Sediment Quality of an Mediterranean Lagoon, the Bizerte Lagoon (Tunisia).

The present study investigates the quality of surface sediments from the Bizerte lagoon (North Tunisia) using an integrated approach including chemical contaminant analysis, bioassays and sediment quality guidelines (SQGs). Sediment samples were collected at 9 sites and analyzed for eight heavy metals (Hg, Cd, Cr, Cu, Pb, Zn, Ni, Fe and Mn). PAHs, PCBs, OCPs were measured previously in the same sediment samples. Our results indicated that the highest concentrations of metals were found near urban areas due to the municipial and industrial wastewater discharges. Sediment pollution assessment was carried out using geoaccumulation index and enrichment factor, which indicate a widespread pollution by Cd, Pb, Ni and Zn in the studied sediments. For bioassays, aqueous and organic extracts were used to assess toxicity and genotoxicity in sediments by using Microtox(®) and SOS Chromotest, respectively. Toxicity levels were compared to metallic and organic pollutants contents. Our results highlight differences in the pattern of responses between the different assays and show no correlation with all the studied contaminants, emphasizing the influence of other contaminants not analyzed in the present study. Based on SQGs, the results of toxicity assessment indicated that adverse effects caused by Ni and Zn would be expected frequently. Nickel was found to have the highest predicted acute toxicity, followed by Zn, Pb, Cd, Cu and Cr. There was no significant relationship between sediment toxicity calculated from heavy metal concentrations (SQG approach) and those measured with bioassays. These findings support the use of integrated approachs for evaluating the environmental risks of sediments.

Toxicity evaluation of water-accommodated fraction of heavy and light oils on the rainbow trout fish cell line RTL-W1.

Fish are currently used models for the toxicity assessment of chemicals, including polycyclic aromatic hydrocarbons (PAHs). Alternative methods including fish cell lines are currently used to provide fast and reliable results on the toxic properties of chemicals while respecting ethical concerns about animal testing. The Rainbow trout liver cell line RTLW1 was used to analyze the effects of two water-accommodated fractions from two crude oils: Arabian Light crude oil (LO) and refined oil from Erika (HO). Several toxicity endpoints were assessed in this study, including cytotoxicity, EROD activity, DNA damage (comet and micronucleus assays), and ROS production. RTL-W1 cells were exposed for 24 h at two or three dilutions of WAF at 1000 µg/L (0.1% (1 µg/L), 1% (10 µg/L), and 10% (100 µg/L)) for cytotoxicity and EROD activity and 1% and 10% for ROS production and genotoxicity). Exposure of RTL-W1 cells to LO WAF induced a significant increase of EROD activity and ROS production and altered DNA integrity as revealed by both the comet assay and the micronucleus test for 10 µg/L of LO. On the other hand, HO WAF exhibited limited toxic effects except for an EROD induction for 1% WAF dilution. These results confirmed the usefulness of RTL-W1 cells for in vitro toxicological assessment of chemical mixtures.

Water-accommodated fractions of heavy and light oils impact DNA integrity, embryonic development, and immune system of Japanese medaka at early life stages.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants generally found in complex mixtures. PAHs are known to cause pleiotropic effects on living organisms, including developmental defects, mutagenicity, carcinogenicity and immunotoxicity, and endocrine disruptions. The main goal of this study is to evaluate the toxicity of water-accommodated fractions (WAFs) of oils in two life stages of the Japanese medaka, larvae and juveniles. The deleterious effects of an acute exposure of 48 h to two WAFs from Arabian light crude oil (LO) and refined oil from Erika (HO) were analyzed in both stages. Relevant endpoints, including ethoxy resorufin-O-deethylase (EROD) activity, DNA damage (Comet assay), photomotor response, and sensitivity to nervous necrosis virus (NNV) infection, were investigated. Larvae exposed to both oil WAFs displayed a significant induction of EROD activity, DNA damage, and developmental anomalies, but no behavioral changes. Deleterious effects were significantly increased following exposure to 1 and 10 µg/L of LO WAFs and 10 µg/L of HO WAFs. Larval infection to NNV induced fish mortality and sharply reduced reaction to light stimulation. Co-exposure to WAFs and NNV increased the mortality rate, suggesting an impact of WAFs on fish defense capacities. WAF toxicity on juveniles was only observed following the NNV challenge, with a higher sensitivity to HO WAFs than to LO WAFs. This study highlighted that environmentally realistic exposure to oil WAFs containing different compositions and concentrations of oil generated high adverse effects, especially in the larval stage. This kind of multi-marker approach is particularly relevant to characterize the toxicity fingerprint of environmental mixtures of hydrocarbons and PAHs.

New insights into the impact of leachates from in-field collected plastics on aquatic invertebrates and vertebrates.

The impact of leachates from micronized beached plastics of the Mediterranean Sea and Atlantic Ocean on coastal marine ecosystems was investigated by using a multidisciplinary approach. Chemical analysis and ecotoxicological tests on phylogenetically distant species were performed on leachates from the following plastic categories: bottles, pellets, hard plastic (HP) containers, fishing nets (FN) and rapido trawling rubber (RTR). The bacteria Alivibrio fischeri, the nauplii of the crustaceans Amphibalanus amphitrite and Acartia tonsa, the rotifer Brachionus plicatilis, the embryos of the sea urchin Paracentrotus lividus, the ephyrae of the jellyfish Aurelia sp. and the larvae of the medaka Oryzias latipes were exposed to different concentrations of leachates to evaluate lethal and sub-lethal effects. Thirty-one additives were identified in the plastic leachates; benzophenone, benzyl butyl phthalate and ethylparaben were present in all leachates. Ecotoxicity of leachates varied among plastic categories and areas, being RTR, HP and FN more toxic than plastic bottles and pellets to several marine invertebrates. The ecotoxicological results based on 13 endpoints were elaborated within a quantitative weight of evidence (WOE) model, providing a synthetic hazard index for each data typology, before their integrations in an environmental risk index. The WOE assigned a moderate and slight hazard to organisms exposed to leachates of FN and HP collected in the Mediterranean Sea respectively, and a moderate hazard to leachates of HP from the Atlantic Ocean. No hazard was found for pellet, bottles and RTR. These findings suggest that an integrated approach based on WOE on a large set of bioassays is recommended to get a more reliable assessment of the ecotoxicity of beached-plastic leachates. In addition, the additives leached from FN and HP should be further investigated to reduce high concentrations and additive types that could impact marine ecosystem health.

Locomotion behavior of juveniles of the freshwater pearl mussel Margaritifera margaritifera: A new non-invasive tool for the evaluation of stress effects.

The lack of knowledge about the sensitivity of the endangered freshwater pearl mussel (FWPM) Margaritifera margaritifera to environmental pollution and the rapid decline of its populations in Europe, have led to the need of developing non-destructive experimental protocols in order to assess the impact of such pollution. This species has a complex life cycle and the early life stages are considered the most sensitive. This study deals with the development of a methodology for the assessment of juvenile mussels' locomotor behavior using an automated video tracking system. Different parameters were determined such as the duration of the video recording and light exposure as a stimulus during the experiment. Locomotion behavior pattern of juveniles was assessed in control condition and also following exposure to sodium chloride as a positive control in order to validate the experimental protocol developed in this study. Results showed that juveniles locomotion behavior was stimulated under light exposure. Moreover, exposure to sublethal concentrations of sodium chloride (0.8 and 1.2 g/L) for 24 h was found to decrease juveniles' locomotion by almost three-times, thus validating our experimental methodology. This study allowed to provide a new tool for the assessment of stress condition impacts on the juveniles of the endangered FWPM, highlighting the interest of such non-destructive biomarker of health for protected species. Consequently, this will help in the improvement of our knowledge on M. margaritifera sensitivity to environmental pollution.

Comparison of the accumulation and effects of copper pyrithione and copper sulphate on rainbow trout larvae.

Copper pyrithione (CuPT) is used as a co-biocide in new antifouling paints but its toxicity remains little known. To compare the toxicity of copper-based compounds, rainbow trout (Oncorhynchus mykiss) larvae were exposed for 8-day to CuPT and CuSO4 at equivalent copper concentrations. CuPT exposure led to the greatest accumulation of Cu in larvae. Exposure to 10 µg.L-1 CuPT induced 99% larval mortality but only 4% for CuSO4-exposed larvae. The larval development and growth were affected by CuPT (from 0.5 µg.L-1 Cu) but not by CuSO4. Lipid peroxidation was not induced by either contaminant. The expression of genes involved in oxidative stress defence, detoxification and copper transport was induced in larvae exposed to CuSO4 and CuPT but at higher concentrations for CuPT. This study highlights the marked toxicity of CuPT for early life stages of fish and raises the question of the possible environmental risks of this antifouling compound.

Plastic debris, persistent organic pollutants and their toxicity impacts in coastal areas in Central Chile.

The chemical components of plastic wastes have made their disposal a major economic, social, and environmental problem worldwide. This study evaluated the acute toxicity and genotoxicity of marine plastic debris on the beaches of Concepción Bay, Central Chile, taken during three periods (spring, summer, and winter). An integrated approach was used, including chemical and toxicological data, using the Microtox® test with Vibrio fischeri and SOS chromotest with Escherichia coli and concentrations of polychlorinated biphenyls (PCBs), Organochlorine Pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs). The results presented here exclusively include the novel data obtained from the winter campaign, revealing high concentrations of PBDEs (238 ± 521 ng g-1). In addition, the genotoxicity and acute toxicity tests were sensitive for most of the samples studied. This investigation is the first attempt to analyse the toxicity of plastic debris in coastal areas along the Chilean coast.

Spatial distribution of anthropogenic particles and microplastics in a meso-tidal lagoon (Arcachon Bay, France): A multi-compartment approach.

Assessment of microplastic (MP) contamination is still needed to evaluate this threat correctly and tackle this issue. Here, MP contamination was assessed for a meso-tidal lagoon of the Atlantic coast (Arcachon Bay, France). Sea surface, water column, intertidal sediments and wild oysters were sampled. Five different stations were studied to assess the spatial distribution of the contamination. Two were outside of the bay and three were inside the bay (from the inlet to the back). A distinction was made between all anthropogenic particles (AP, i.e. visually sorted) and MP (i.e. plastic polymer confirmed by ATR-FTIR spectroscopy). The length of particles recovered in this study ranged between 17 µm and 5 mm. Concentration and composition in sea surface and water column samples showed spatial variations while sediment and oyster samples did not. At outside stations, the sea surface and the water column presented a blended composition regarding shapes and polymers and low to high concentrations (e.g. 0.16 ± 0.08 MP.m-3 and 561.7 ± 68.5 MP.m-3, respectively for sea surface and water column), which can be due to coastal processes and nearby input sources. The inlet station displayed a well-marked pattern only at the sea surface. High AP and MP concentrations were recorded, and fragments along with polyethylene overwhelmed (respectively 76.0 % and 73.2 %). Higher surface currents could explain this pattern. At the bay back, AP and MP concentrations were lower and fibers were mainly recorded. Weaker hydrodynamics in this area was suspected to drive this contamination profile. Overall, fragments and buoyant particles were mainly detected at the sea surface while fibers and negatively buoyant particles prevailed in other compartments. Most of the studied samples presented an important contribution of fiber-shaped particles (from 31.5 % to 94.2 %). Finally, contamination was ubiquitous as AP and MP were found at all stations in all sample types.

A protocol for lixiviation of micronized plastics for aquatic toxicity testing.

Plastics contain various types and amounts of additives that can leach into the water column when entering aquatic ecosystems. Some leached plastic additives are hazardous to marine biota at environmentally relevant concentrations. Disparate methodological approaches have been adopted for toxicity testing of plastic leachates, making comparison difficult. Here we propose a protocol to standardize the methodology to obtain leachates from microplastics (MPs) for aquatic toxicity testing. Literature reviewing and toxicity tests using marine model organisms and different types of MPs were conducted to define the main methodological aspects of the protocol. Acute exposure to leachates from the studied plastics caused negative effects on the early life stages of sea urchins and marine bacteria. We provide recommendations of key factors influencing lixiviation of MPs , such as particle size (<250 µm), solid-to-liquid ratio (1-10 g/L), mixing conditions (1-60 rpm), and lixiviation time (72 h). The proposed methodology was successful to determine the toxicity of leachates from different micronized plastics on marine biota. Our recommendations balance sensitivity, feasibility and environmental relevance, and their use would help ensure comparability amongst studies for a better assessment of the toxicity of plastic leachates on aquatic biota.

Metal contamination and heat stress impair swimming behavior and acetylcholinesterase activity in embryo-larval stages of the Mediterranean mussel, Mytilus galloprovincialis.

Behavioral parameters are increasingly considered sensitive and early bioindicators of toxicity in aquatic organisms. A video-tracking tool was specifically developed to monitor the swimming behaviour of D-larvae of the Mediterranean mussel, Mytilus galloprovincialis, in controlled laboratory conditions. Both maximum and average swimming speeds and trajectories were recorded. We then investigated the impact of copper and silver with or without a moderate rise of temperature on swimming behavior and acetylcholinesterase (AChE) activity of mussel D-larvae and the possible mechanistic link between both biological responses. Our results showed that copper and/or silver exposure, as well as temperature increase, disrupts the swimming behavior of mussel larvae which could compromise their dispersal and survival. In addition, the combined effect of temperature and metals significantly (p < 0.05) increased AChE activity in mussel larvae. Pearson's correlation analysis was performed and results showed that the AChE activity is positively correlated with maximum speeds (r = 0.71, p < 0.01). This study demonstrates the value of behavioral analyzes of aquatic invertebrates as a sensitive and integrate marker of the effects of stressors.

Intergenerational effects of environmentally-aged microplastics on the Crassostrea gigas.

This study focused on the impacts of aged aquaculture microplastics (MPs) on oysters (Crassostrea gigas). Adult oysters were exposed for two months to a cocktail of MPs representative of the contamination of the Pertuis Charentais area (Bay of Biscay, France) and issuing from oyster framing material. The MPs mixture included 28% of polyethylene, 40% of polypropylene and 32% of PVC (polyvinyl chloride). During the exposure, tissues were sampled for various analyzes (MP quantification, toxicity biomarkers). Although no effect on the growth of adult oysters was noted, the mortality rate of bivalves exposed to MPs (0.1 and 10 mg. L-1 MP) increased significantly (respectively 13.3 and 23.3% of mortalities cumulative). On the one hand, the responses of biomarkers revealed impacts on oxidative stress, lipid peroxidation and environmental stress. At 56 days of exposure, significant increases were noted for Glutathione S-Transferase (GST, 10 mg. L-1 MP), Malondialdehyde (MDA, 10 mg. L-1 MP) and Laccase (LAC, 0.1 and 10 mg. L-1 MP). No variations were observed for Superoxyde Dismutase (SOD). Besides, ingestion of MPs in oyster tissues and the presence in biodeposits was highlighted. In addition, in vitro fertilisations were performed to characterize MPs effects on the offspring. Swimming behavior, development and growth of D-larvae were analysed at 24-, 48- and 72-h after fertilisation. D-larvae, from exposed parents, demonstrated reduced locomotor activity. Developmental abnormalities and arrest as well as growth retardation were also noted. This study highlighted direct and intergenerational effects of MPs from aged plastic materials on Pacific oysters.

Impacts of chemical stress, season, and climate change on the flounder population of the highly anthropised Seine estuary (France).

The main objective of this study was to improve our knowledge on the responses of fish populations to multistress (diffuse pollution and warming waters) in estuaries. Adult flounders were caught in two estuaries in the Eastern English Channel: the heavily polluted Seine estuary vs the moderately contaminated Canche estuary. Fish samplings were conducted in January just before the reproduction period, and in July when gonads were at rest. The overall rise in coastal winter water temperatures detected over the Channel impairs the flounder's phenology of reproduction in the two estuaries, inducing a delay of maturation process and probably also spawning. The higher liver histopathology index in Seine vs Canche could be the consequence of the fish exposition to a complex cocktail of contaminants in a strongly industrialized estuary. Higher levels of neurotoxicity, gill lipid peroxidation, and liver EROD activity were observed in Seine vs Canche. Furthermore, a possible impairment in mitochondrial metabolism was suggested in the Seine flounder population. We confirmed in this study the potential role of two membrane lipids (sphingomyelin and phosphatidylserine) in the resistance towards oxidative stress in Seine and Canche. Finally, we suggest that the Seine flounder population (and possibly the connected Eastern English Channel flounder populations over the French Coast) could be seriously impacted in the future by multistress: higher winter temperatures and chemical contamination.

Environmental microplastics disrupt swimming activity in acute exposure in Danio rerio larvae and reduce growth and reproduction success in chronic exposure in D. rerio and Oryzias melastigma.

Microplastics (MPs), widely present in aquatic ecosystems, can be ingested by numerous organisms, but their toxicity remains poorly understood. Toxicity of environmental MPs from 2 beaches located on the Guadeloupe archipelago, Marie Galante (MG) and Petit-Bourg (PB) located near the North Atlantic gyre, was evaluated. A first experiment consisted in exposing early life stages of zebrafish (Danio rerio) to MPs at 1 or 10 mg/L. The exposure of early life stages to particles in water induced no toxic effects except a decrease in larval swimming activity for both MPs exposures (MG or PB). Then, a second experiment was performed as a chronic feeding exposure over 4 months, using a freshwater fish species, zebrafish, and a marine fish species, marine medaka (Oryzias melastigma). Fish were fed with food supplemented with environmentally relevant concentrations (1% wet weight of MPs in food) of environmental MPs from both sites. Chronic feeding exposure led to growth alterations in both species exposed to either MG or PB MPs but were more pronounced in marine medaka. Ethoxyresorufin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities were only altered for marine medaka. Reproductive outputs were modified following PB exposure with a 70 and 42% decrease for zebrafish and marine medaka, respectively. Offspring of both species (F1 generation) were reared to evaluate toxicity following parental exposure on unexposed larvae. For zebrafish offspring, it revealed premature mortality after parental MG exposure and parental PB exposure produced behavioural disruptions with hyperactivity of F1 unexposed larvae. This was not observed in marine medaka offspring. This study highlights the ecotoxicological consequences of short and long-term exposures to environmental microplastics relevant to coastal marine areas, which represent essential habitats for a wide range of aquatic organisms.

Natural distribution of pure and hybrid Mytilus sp. along the south Mediterranean and North-east Atlantic coasts and sensitivity of D-larvae stages to temperature increases and metal pollution.

The distribution of the Mediterranean mussel Mytilus galloprovincialis extends more and more northwards in the Atlantic. Crossings are frequently observed with the blue mussel Mytilus edulis along the French and English coasts. The aim of this study is firstlyto identify the co-presence of M. galloprovincialis, M. edulis, and their hybrids in different sites of the Mediterranean and Atlantic coasts, and to provide insights for the thermal tolerance and toxicant susceptibility of Mytilus edulis, Mytilus galloprovincialis and their hybrids. Mussels were collected from the shore at 20 sampling sitesin Europe and Tunisia and identified using Me 15/16 primers targeting the adhesive protein gene sequence. Samples were screened for the presence of Mytilus edulis, Mytilus galloprovincialis, and hybrids alleles using PCR. To get more information on hybrids sensitivities to temperature and metals, freshly fertilized eggs of the two species and their hybrids were reared at four temperatures 18, 20, 22, and 24 °C and exposed to concentrations of Cu, Ag, and a mixture of both metals. Arrests of development and malformations were recorded after 48 h of exposure. The genotypic identification of the two species on 20 sites of the Mediterranean and Atlantic coasts carried out during this study confirms the presence of pure and hybrid species of mussel. Our results highlighted that hybrid larvae from a female of M. galloprovincialis are significantly more tolerant to temperature increases than pure larvae of M. galloprovincialis and pure and hybrid larvae of M. edulis. No significant interspecies-differences of sensitivity were noted for metal exposure alone. However, a co-exposure of larvae to both metal and high temperature highlighted the higher tolerance of hybrid larvae from a female of M. galloprovincialis to both stresses. The overall results could allow the prediction of the future evolution of mussel populations facing environmental changes.

Toxicity and risk assessment of six widely used pesticides on embryo-larval development of the Pacific oyster, Crassostrea gigas.

This study aims to assess the toxic effects and the potential risk of widely used agricultural pesticides on the development (malformations and developmental arrest), growth and swimming activity of oyster D-larvae (Crassostrea gigas). Freshly fertilized oyster embryos were exposed for 24 h at 24 °C to different concentrations (0, 0.01, 0.1, 1 and 10 µg.L-1) of six different pesticides: Glyphosate and its commercial solution (Roundup), Isoproturon, Nicosulfuron, Chlortoluron and Boscalid. The six pesticides tested induced a significant increase in larval malformations and developmental arrests. All pesticides except Glyphosate and Isoproturon affected larval growth. Roundup, Nicosulfuron, Chlortoluron and Boscalid also affected the swimming behaviour of the D-larvae, with a significant decrease recorded in their maximum swimming speed. Comparison of the LOEC (Lowest-Observed-Effect Concentration) of each compound led to the following toxicity classification: Boscalid > Chlortoluron = Nicosulfuron > Glyphosate > Roundup > Isoproturon, with respectively LOEC of 0.0028; 0.015; 0.017; 0.11; 0.3 and 0.78 µg.L-1. By comparison of the maximum concentrations in the Pertuis Charentais (South West, France) and LOEC of each pesticide, the following risk scale was obtained: Chlortoluron > Boscalid > Glyphosate > Roundup > Nicosulfuron > Isoproturon. Our results revealed that Chlortoluron, Boscalid and to a lesser extent Glyphosate represent a potential threat to early life stages of oyster living in the Pertuis Charentais marine area.

Comparative developmental toxicity of conventional oils and diluted bitumen on early life stages of the rainbow trout (Oncorhynchus mykiss).

Petroleum hydrocarbons are widely used and transported, increasing the risks of spills to the environment. Although conventional oils are the most commonly produced, the production of unconventional oils (i.e. diluted bitumen or dilbit) is increasing. In this study, we compared the effects of conventional oils (Arabian Light and Lloydminster) and dilbits (Bluesky and Clearwater) on early life stages of a salmonid. To this end, aqueous fractions (WAF: water accommodated fraction) of these oils were extracted using mountain spring water. Rainbow trout (Oncorhynchus mykiss) larvae were exposed to 10 and 50% dilutions of these WAFs from hatching (340 DD; degree days) until yolk sac resorption (541 DD). Exposure to WAFs increased skeletal malformations (both dilbits) and hemorrhage (both conventional oils and Bluesky) and decreased head growth (Arabian Light). In addition, increases in EROD activity and DNA damage were measured for all oils and an increase in cyp1a gene expression was measured for Arabian Light, Bluesky and Clearwater. The PAH and C10C50 concentrations were positively correlated to total larval EROD activity, whereas concentrations of total hydrocarbons, VOCs, PAHs, and C10C50 were positively correlated to cyp1a expression. Total hydrocarbon, VOC, and C10C50 concentrations were also negatively correlated to larval growth. This study supports that petroleum hydrocarbons are toxic to early developmental stages of rainbow trout and show that their degree and spectrum of toxicity depends on their chemical composition.