Search

Contamination of aquatic ecosystems is considered as one of the main threats to global freshwater biodiversity. Within the European Water Framework Directive (EU-WFD) a particular attention is dedicated to assess ecological risks of surface water contamination and mitigation of chemical pressures on aquatic ecosystems. In this work, we evaluated ecological risks of surface water contamination for fish populations in four EU-WFD rivers through an integrative approach investigating three Lines of Evidence (chemical contamination, biomarker responses as early warning signals of contamination impacting individuals and ecological analyses as an indicator of fish community disturbances). This work illustrates through 4 case studies the complementary role of biomarkers, chemical and ecological analyses which, used in combination, provide fundamental information to understand impacts of chemical pressures that can affect fish population dynamics. We discuss the limitations of this approach and future improvements needed within the EU-WFD to assess ecological risk of river contamination for fish populations.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA [ADD1_IRSTEA]Systèmes aquatiques soumis à des pressions multiples; International audience; To improve the assessment of aquatic organism responses to environmental stressors, there is an interest in studying epigenetic marks in addition to other validated biomarkers. Indeed, the epigenetic marks may be influenced by the surrounding environment. Non-model invertebrates such as gammarids are sentinel organisms representative of the diversity of natural stream communities. Despite their ecologically relevance, the epigenetic responses have been to date poorly documented in these species. The present study explores the measurement of the global cytosine methylation level in the genome of the freshwater crustacean Gammarus fossarum. In a first step, natural variability of global cytosine methylation level (basal level) was assessed by studying the effect of sex, age and sampling site of organisms. Results showed a significant effect of age and sampling site. In a second step, effects of water temperature and food starvation were studied. For both factors, a hypermethylation was observed after 1 month of exposure. In a third step, gammarids were exposed to a range of environmentally relevant cadmium concentrations (0.05-5 µg/L) in order to assess the effect of a chemical stress. Whatever the cadmium concentration used, a significant hypomethylation was observed after 14 days followed by a trend for hypermethylation after 1 month of exposure. These results are the first ones dealing with the 5Cmethylation status in gammarids. The results constitute potential markers of environmental stresses in relevant sentinel species widely used in ecotoxicological studies.

Waters receiving textile discharges can exhibit genotoxic and mutagenic activity, which has been related to the presence of dyes and aromatic amines as synthesis precursors or byproducts. The aim of this study was to identify dyes and aromatic amines in water samples impacted by textile discharges, and to evaluate the genotoxic responses of these samples using the Salmonella/microsome assay in strains TA98 and YG1041, and the Fpg-modified comet assay in the RTL-W1 fish cell line. The genotoxicity of river samples downstream of the discharge was greater than the upstream samples in both of the Ames tests. The Fpg-modified comet assay detected similar levels of DNA damage in the upstream and downstream samples. Mutagenicity was not detected with TA98, except for the Quilombo River samples, but when YG1041 was used as the tester strain mutagenicity was detected for all sites with a very different profile in upstream sites relative to the other sites. The mutagenic response strongly indicated that aromatic amines or dyes were contributing to the mutagenic activity downstream. The impact of textile discharges was also confirmed by chemical analysis, because the highest concentrations of azo dyes and aromatic amines were detected in the river downstream. This study shows the value of combining assays measuring complementary endpoints to better characterize the mutagenicity of environmental samples, with the advantage that this approach provides an indication of what classes of compounds are responsible for the effect. Environ. Mol. Mutagen. 57:559-571, 2016. © 2016 Wiley Periodicals, Inc.

Les performances atteintes par les deux systemes biologiques etudies – boues activees conventionnelles (BA) et reacteur biologique a biomasse fluidisee (MBBR) – dans le traitement des dix micro— polluants d’origine pharmaceutique quantifies sont similaires et cela grâce au niveau d’epuration identique en nitrification totale.Les abattements quantifies sont variables et atteignent en moyenne pour les sept composes refractaires seulement 24% (carbamazepine, sulfamethoxazole, propranolol, atenolol, diclofenac, econazole, acide salicylique). Les trois autres composes, bien elimines en traitement biologique, atteignent en moyenne 96% (paracetamol, ketoprofene et ibuprofene).Le traitement biologique combine a l’ozonation tertiaire elimine efficacement les composes pharmaceutiques de l’eau des l’utilisation des doses faibles d’ozone (0,45 gO3/g de carbone organique dissous COD). Un abattement moyen de 92% d’efficacite est quantifie sur la moyenne de composes pharmaceutiques dissous, a comparer aux 24% a 45% d’abattement obtenu en traitement bio logique seul. Le passage a une dose d’ozone de 1,30 gO3/gCOD permet d’augmenter de 6% l’abattement moyen des micropolluants. Cette performance n’est pas amelioree par l’utilisation des doses d’ozone plus importantes.Contrairement aux tests d’ecotoxicite normalises (peu sensibles pour discriminer des effets toxiques dans les eaux traitees biologiquement ou chimiquement), les effets mesures sur les embryons du poisson Danio rerio comme la mortalite, les anomalies sur le developpement et la genotoxicite ont montre un effet toxique (nul a faible) pour les effluents biotraites ou ozones. Par rapport au traitement biologique, l’ozonation tertiaire ameliore significativement le potentiel de perturbation endocrinienne des effluents par la reduction complete de l’activite oestrogenique et glucocorticoide des effluents.L’ozonation n’augmente ni ne diminue le niveau de toxicite des effluents deja atteint avec le traitement biologique seul. Il existe un bruit persistant probablement lie a la presence d’autres micro - polluants remanents dans l’eau et non quantifies par la methode d’analyse ciblee. Leur activite peut etre a l’origine du niveau d’effet toxique faible obtenu pour certains biomarqueurs. Ces resultats montrent la necessite d’ameliorer la sensibilite des bioessais dans le but d’optimiser l’evaluation des effets ecotoxicologiques residuels dans les differents types d’effluents.L’obtention des signatures chimiques, au travers d’une approche non ciblee, s’avere un outil puissant et complementaire. Elles revelent la complexite des effluents etudies par le nombre important de signaux obtenus. Les cartographies realisees, par l’analyse en composantes principales, permettent d’avoir une vision globale des effluents et de leur mise en relation respective. Cette approche globale complete efficacement les resultats obtenus par le dosage cible des micropolluants.

International audience; Toxicity of wastewater treatment plant (WWTP) effluents is usually assessed with standardized bioassays, as e.g. the Fish Embryo Toxicity test (FET), but assessment of sub-lethal toxic effects requires to develop more adapted tests. The present work aimed to add the recording of several sub-lethal endpoints in exposed zebrafish embryo-larval stages in order to increase the sensitivity of residual toxicity evaluation of WWTP effluents using a semi-quantitative method (iFET score). This approach was complemented with the genotoxicity assessment on the exposed embryos, a sub-acute hazard particularly relevant to evaluate a potential chronic toxicity risk of low and multi-contaminated environmental matrices. Additionally, endocrine activities of effluents were quantified using human reporter cell lines. This test battery was applied to the assessment of the residual toxicity of five biological treatment effluents, further treated or not using various ozonation treatments intended to improve pharmaceutical compounds removal. Acute toxicity towards zebrafish embryos was very low. However, iFET score approach proposed was able to reveal residual toxicity through the presence of developmental abnormalities in all samples tested. Additionally, a low residual genotoxicity was measured in embryos exposed to two of the WWTP effluents when all excepted one exhibited a residual endocrine activity potential in the ng/L range providing complementary information on the occurrence of endocrine active chemicals and their reduction by different processes. To sum up, such a simplified and ecologically relevant test battery was found sensitive enough to characterize and differentiate various residual effluent’s ecotoxicity at contaminant levels of environmental concern.

International audience; Standardized ecotoxicity bioassays show some limits to assess properly long-term residual toxicity of complex mixture of pollutants often present at low concentration, such as stormwaters. Among invertebrate organisms used for ecotoxicity testing, the microcrustacean Ceriodaphnia dubia (C. dubia) is considered as one of the most sensitive, especially regarding reproduction impairment as a toxicity endpoint. Consequently, the present work explores the interest to perform a multi-generational assay based on the study of the reproduction of C. dubia to assess long-term ecotoxicity of complex mixture, using stormwater samples. With this in mind, a battery of standardized bioassays (Daphnia magna mobility, Pseudokirchneriella subcapitata population growth, Heterocypris incongruens growth and one generation C. dubia reproduction inhibition assays) was performed in parallel to a three generation C. dubia reproduction inhibition assay on 2 stormwater samples. Results highlighted that while all standardized bioassays failed to reveal residual toxicity in the stormwater samples, the C. dubia multi-generational assay exhibited an higher sensitivity than the previous ones. No adverse effect was observed for the first exposed generation, but an increase in mortality and a reproduction disturbance was obtained in the second and third exposed generation depending of the sample. Further experiments are now needed to optimize the exposure protocol of this multi-generational assay.

International audience; Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 ␣sh liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with expo- sure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modi␣ed comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were signi␣cant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was signif- icantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the ␣rst to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity.

International audience; Wastewater treatment plants (WWTPs) are one of the main sources of freshwater pollution eventually resulting in adverse effects in aquatic organisms. Treated effluents can contain many micropollutants at concentrations often below the limit of chemical quantification. On a regulatory basis, WWTP effluents have to be non-toxic to the aquatic environment, wherefore not only chemical abatement but also ecotoxicological evaluation through relevant bioassays is required. Standardized bioassays currently used are often not sensitive enough to reveal a residual toxicity in treated effluents. Therefore, attention must be paid to the development of better-adapted approaches implementing more sensitive organisms and relevant endpoints. In this study, the toxicity of two differently treated effluents (activated sludge treated effluents with and without ozonation) towards the ecologically relevant species Gammarus fossarum was evaluated. Organism fitness traits such as reproduction and sperm DNA integrity were followed in exposed organisms. In complement, enzymatic biomarkers were measured indicating the presence of neurotoxic compounds (acetylcholinesterase activity), the presence of pathogens likely to increase the toxic effects of chemical compounds (phenol-oxidase activity), and the presence of toxic compounds inducing detoxification mechanisms (glutathione-S-transferase activity). Enzymatic activities were not modified, but significant sub-lethal effects were observed in exposed organisms. In both effluents, females showed a retarded molt cycle, a reduced fecundity and fertility, and > 90% of developed embryos exhibited developmental malformations. In addition, a slight but significant genotoxic effect was measured in gammarid sperm. In a whole, no difference in toxicity was found between both effluents. Coupling reproduction impairment and genotoxicity assessment in Gammarus fossarum seems to be a valuable and sensitive tool to reveal residual toxicity in effluents containing a mixture of micropollutants at very low concentrations. Finally, a direct relationship between the observed toxic responses and the quantified micropollutant concentrations could not be evidenced.

International audience; Data regarding the link between DNA integrity of germ cells and the quality of progeny in fish exposed to genotoxicant are scarce although such information is of value to understand genotoxic effects of contaminants in aquatic fauna. This work aimed at studying the consequences of a parental exposure during the breeding season on offspring quality in three-spined stickleback. After in vivo exposure of adult fish to methyl methane sulfonate, a model alkylating compound, a clear increase in DNA damage was observed in erythrocytes of both genders, here used as a biomarker of exposure. MMS exposure significantly affected sperm DNA integrity but neither female fecundity nor fertilization success. In order to understand the contribution of each sex to potential deleterious effects in progeny due to parental exposure, mating of males and females exposed or not to MMS, was carried out. Exposure of both males and females or of males alone led to a significant increase in both mortality during embryo-larval stages and abnormality rate at hatching that appeared to be sensitive stages. Thus, in accordance with recent studies carried out in other freshwater fish species, such development defects in progeny were clearly driven by male genome, known to be devoid of DNA repair capacity in spermatozoa. The next step will be to investigate the link between DNA damage in stickleback sperm and reproductive impairment in natural populations exposed to complex mixture of genotoxicants.

International audience; The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O6-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly estab- lished whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities in fish could be the base for the development of new biomarkers of genotoxicity. Knowing the importance of the germ cell DNA integrity in the reproductive success of aquatic orga- nisms, the DNA repair capacity of such cells deserve to be more studied, as well as DNA repair capacities of established fish cell lines. The limited amount of available data, which shows low/slow DNA repair capacities of fish cell lines compared with mammalian cell lines, concerned mainly the NER system; thus this point merits to be explored more deeply. Additionally, since some of the DNA repair systems appear more efficient in embryo larval stages, it would be of interest to consider embryonic cell lineages more closely.

International audience; A mixture of urban and hospital effluents (50 % v/v) was evaluated for ecotoxicity with an advanced bioassay battery. Mixed effluents were tested before any treatment, af- ter biological treatment alone, and after biological treatment followed by a tertiary ozonation (15 mg O3/L). Laying a high value on the continuance of organisms’ fitness, essential to preserve a healthy receiving ecosystem, the main objective of this study was to combine normalized bioassays with newly developed in vivo and in vitro tests in order to assess alteration of embryo development, growth and reproduction, as well as genotoxic effects in aquatic organisms exposed to complex wastewater effluents. Comparison of the bioassays sensitivity was considered. Contrary to the lack of toxicity observed with normalized ecotoxicity tests, endpoints measured on zebrafish embryos such as developmental abnormalities and genotoxicity demonstrated a residual toxicity in wastewater both after a biological treatment followed or not by a tertiary O3 treatment. However, the ozonation step allowed to alleviate the residual endocrine disrupting potential measure in the bi- ologically treated effluent. This study shows that normalized bioassays are not sensitive enough for the ecotoxicological evaluation of wastewaters and that there is a great need for the development of suitable sensitive bioassays in order to characterize properly the possible residual toxicity of treated effluents.

As a large array of hazardous substances exhibiting genotoxicity are discharged into surface water, this work aimed at assessing the relevance of adding a genotoxicity biomarker in a battery of biomarkers recently developed in the model fish three-spined stickleback (Gasterosteus aculeatus). First the confounding influence of gender, body length, and season (used as a proxy of age and of the fish reproductive status, respectively) on the level of primary DNA damage in erythrocytes was investigated in wild sticklebacks. Then, the genotoxity biomarker was included in a large battery of biomarkers assessing xenobiotic biotransformation, oxidative stress and neurotoxicity, and implemented in five sites. Gender, age and reproductive status did not influence DNA damage level in fish from the reference site. A significant relationship between the level of primary DNA damage and fish length (as a proxy of age also correlated to the season) was highlighted in the contaminated site. Among all biomarkers investigated in the field, the level of DNA damage was one of the four most discriminating biomarkers with EROD, catalase activity and the level of lipid peroxidation representing together 75.40% of the discriminating power in sampled fish. The level of DNA damage was correlated to the EROD activity and to the level of peroxidation, which mainly discriminated fish from sites under urban pressure. Finally, Integrated Biomarker Response indexes (IBRv2), which were calculated with the whole biomarker response dataset exhibited higher values in the Reveillon (9.62), the Scarpe and Rhonelle contaminated sites (5.11 and 4.90) compared with the two reference sites (2.38 and 2.55). The present work highlights that integration of a genotoxicity biomarker in a multiparametric approach is relevant to assess ecotoxicological risk in freshwater aquatic organisms.

International audience; In the perspective of a biomonitoring application for assessing genotoxicity of freshwater ecosystems, the Comet assay has recently been developed on spermatozoa in the amphipod Gammarus fossarum, in order to propose a sensitive and reliable genotoxicity biomarker in an ecologically relevant freshwater species. The appropriate use of a genotoxicity biomarker requires good knowledge of its basal level and its natural variability related to intrinsic biotic and environmental abiotic factors. We propose a procedure for which the lowest biomarker variability related to methodological and intrinsic biotic factors is obtained and a reference value of biomarker basal response taking into account its spatio-temporal changes has been defined. A strong impact of spermatogenesis status and exposure time on the response to genotoxicant pressure was observed. These reports led us to select a standard organism, i.e., the mature male gammarid in precopula. No effect of temperature and conductivity on baseline DNA damage was observed in the laboratory for the tested range (6-24 degrees C and 300/600 mu S cm(-1)). Similarly, no spatio-temporal change relative to season or the physicochemical characteristics of the water was recorded during the field survey. On the basis of these results, a reference level with maximal threshold values has been proposed for the standard gammarid. (C) 2011 Elsevier B.V. All rights reserved.

The discovery of drugs in surface waters is relatively recent. The huge number of molecules (in a city or in hospital) raises the issue of appropriate choice of drugs for priority consideration. In order to study the impact of released drugs on aquatic ecosystems, it is important to consider bioaccumulation as a discriminating parameter. On this basis, among 966 medications prescribed at the Hospices Civils de Lyon, we identified 14 of them, which could represent a critical risk toward aquatics ecosystems, especially during chronic exposure to low concentration. In this group, many therapeutic classes are represented, including widely used drugs such as cardiac tropism and antibiotics. Other compounds, although less consumed in terms of tonnage should be examined, such as the female sex hormones and anti-cancer drugs. Several factors led us to choose mitotane to be studied at first : a very high BCF (7330), no available ecotoxicological data and a very close structural similarity with the DDT insecticide. In order to study the impact of mitotane on an aquatic animal model, we chose an in vitro approach using fish cell lines as a model of the fish internal environment. Therefore, cell culture exposure levels were considered as reflecting the internal mitotane concentrations to which the animal is exposed once the drug is concentrated in its tissues. Two parameters of toxicity were studied on the basis of dose-effect relationships : cytotoxicity (via cell death) and genotoxicity (via DNA primary damages). The threshold cytotoxicity (LC10 at 6 μg/L for the gill cell line RTG-W1 and 18 μg/L for PLHC-1, an hepatocarcinoma originating cell line) are much higher than concentrations that could be observed in the aquatic environment. Thus, using this parameter, the risk for fish exposed at realistic environmental concentration seems very limited. However, with our first results when considering genotoxicity as the toxicity markers much lower active concentrations were found, suggesting a risk of somatic mutations at level close to the plausible environmental reality. Additional studies are presently in progress in order to confirm these first results. The use of cell cultures as a model for the fish internal environment seems promising and appears to be a sensitive tool well adapted to the toxicity screening of many molecules present in the environment., La découverte de la présence de médicaments dans les eaux de surface est relativement récente. Le nombre très élevé de molécules pharmaceutiques (en ville ou a l’hôpital) pose la question de la pertinence des médicaments à étudier en priorité. Dans le but d’étudier l’impact des rejets en médicaments sur les écosystèmes aquatiques, il nous a semble important d’intégrer la bioaccumulation comme facteur discriminant, compte tenu de ses conséquences potentiellement très importantes. Sur 966 molécules pharmaceutiques consommées aux Hospices civils de Lyon, une première liste de 70 molécules prioritaires a été établie, qui a été par la suite réduite a 14 d’entre elles, jugées particulièrement a risque pour les écosystèmes aquatiques. Toutes les classes thérapeutiques sont représentées, notamment des médicaments très utilises comme ceux a tropisme cardiaque et des antibiotiques. D’autres médicaments, moins consommes, sont également a surveiller comme les hormones sexuelles féminines et certains anticancéreux. Notre premier choix d’étude expérimentale s’est porte ensuite sur le mitotane (anticancéreux) en considérant plusieurs paramètres : son facteur de bioconcentration très élevé (FBC de 7 330), la quasi-absence de données écotoxicologiques et, enfin, une très grande proximité structurale avec le DDT. Afin d’étudier la toxicité du mitotane sur un modèle animal représentatif de l’écosystème aquatique, nous avons utilise une approche in vitro sur des lignées cellulaires de poisson. Celles-ci ont été utilisées comme un modèle du milieu intérieur du poisson avec les concentrations d’exposition reflétant les concentrations intra-organisme. Deux paramètres de toxicité ont été suivis : la cytotoxicite (via la mort cellulaire) et la genotoxicite (via la mesure des dommages primaires a l’ADN). Pour le scenario étudié, les valeurs critiques obtenues pour la cytotoxicite (CL10 a 6 μg/L pour la lignée de cellules branchiales RTG-W1 et a 18 μg/L pour la lignée PLHC-1 issues d’un hepatocarcinome) sont supérieures aux concentrations d’exposition intracellulaires calculées. Sur la base de ce paramètre, le risque encouru semble donc limite pour le scenario étudié. Cependant, les premiers résultats obtenus pour la genotoxicite mettent en évidence des concentrations d’effet beaucoup plus faibles, proches d’une réalité environnementale. Des expérimentations complémentaires sont en cours pour confirmer ce dernier résultat. Au final, on peut conclure sur l’intérêt de la prise en compte de la bioaccumulation pour l’évaluation des risques lies aux molécules médicamenteuses. Par ailleurs, l’utilisation des cultures cellulaires comme modèle du milieu intérieur du poisson semble être un outil prometteur, sensible et adapte au screening de ces mêmes molécules.

International audience; This work describes some consequences of paternal germ cell DNA damage on the reproduction success in two fish species. Male brown trout (n = 31) and male Arctic charr (n = 28) were exposed to the model genotoxicant MMS at the end of spermatogenesis to generate a significant DNA damage level in mature spermatozoa (28% and 25% tail DNA in trout and charr sperm, respectively, evaluated through the comet assay). Sperm from each MMS exposed and control fish was then used to fertilize in vitro an aliquot of a single pool of eggs collected from 4 unexposed females for each species. Each batch of fertilized eggs was monitored individually in the hatchery to follow embryonic and larval abnormalities during the fry development. Paternal exposure did not influence fertilization rate or survival rate at hatching in either species. However, MMS paternal treatment resulted in a large array of morphological abnormalities during embryonic and larval development. At the eyed stage, malformations exhibited a 8 fold increase in trout and a 2 fold increase in charr for larvae stemming from MMS treated males as compared with controls. At the end of yolk sac resorption, an increase in the gross morphological abnormality incidence was found in trout larvae originating from MMS exposed males (2.10% vs. 0.93% in control, p < 0.05). When looking more in detail at bony structures after Alizarin red S staining, a 20% incidence of skeletal defects was recorded at the swimming stage. A positive correlation was found between the paternal sperm DNA damage level and the skeletal abnormality incidence of its progeny. During the next 2 months of development, mortality in trout originating from DNA damaged sperm was 3 times higher than in control. After one year, no effect of paternal treatment was found on growth traits (length and weight) but the gross morphological abnormality incidence was still very high in the treated group (27% malformation incidence vs. 0.5% in control). These results demonstrate ecologically relevant consequences of fish spermatozoa DNA damage and stress the value of using this parameter as a biomarker signaling potential long term effects of environmental genotoxins in aquatic systems.

National audience; This study deals with the use of a chronic exposure scenario of zebrafish (Danio rerio) in laboratory conditions to evaluate the genotoxic potential of diuron and azoxystrobin, two pesticides intensively used in vineyard agriculture. Adult male zebrafish were exposed during three weeks in the semi static mode in four 20 L aquaria. Treatment allowed to each aquarium was: negative control (untreated), positive control (methyl methane sulphonate 1 µM), diuron 4.3 nM and azoxystrobin 1.2 nM. Once per week, genotoxicity was assessed (6 fish/treatment) by the use of two complementary biomarkers: the primary DNA damages evaluated in somatic (liver) and germ (spermatozoa) cells by the alkaline version of the Comet assay and the micronucleus formation assessed in erythrocytes. Very low basal DNA damages were obtained with both biomarkers in negative control during the three consecutive weeks and a significant genotoxic response was obtained in 1 µM MMS exposed fish, both in liver and germ cells with the Comet assay and in erythrocytes with the micronucleus test, respectively starting after one and three weeks. With this chronic exposure scenario, both diuron and azoxystrobine revealed a genotoxic potential at realistic environmental concentrations and a significant response was obtained in all cell types investigated and with both biomarkers used, mainly after 7 or 14 days, thus stressing the interest of long-term exposure scenario. Further studies will be undertaken in order to evaluate whether DNA damage observed in spermatozoa of fish exposed to environmental concentration of pesticides could lead to subsequent reproductive disorders.

International audience; The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and common acceptance of FET, it is still performed in multiwell plates and requires laborious and time-consuming manual manipulation of specimens and solutions. This work describes the design and validation of a microfluidic Lab-on-a-Chip technology for automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The innovative device supports rapid loading and immobilization of large numbers of zebrafish embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery. Furthermore, we also present development of a customized mechatronic automation interface that includes a high-resolution USB microscope, LED cold light illumination, and miniaturized 3D printed pumping manifolds that were integrated to enable time-resolved in situ analysis of developing fish embryos. To investigate the applicability of the microfluidic FET (μFET) in toxicity testing, copper sulfate, phenol, ethanol, caffeine, nicotine, and dimethyl sulfoxide were tested as model chemical stressors. Results obtained on a chip-based system were compared with static protocols performed in microtiter plates. This work provides evidence that FET analysis performed under microperfusion opens a brand new alternative for inexpensive automation in aquatic ecotoxicology.

International audience; Tamoxifen, a drug used to treat cancer, is regularly found in hydrosystems at concentrations of several hundred ng L-1. To characterize its ecotoxicity, we implemented a battery of bioassays on organisms belonging to 3 different trophic levels: Pseudokirchneriella subcapitata, Chlorella vulgaris and Chlamydo- monas reinhardtii, for primary producers, Daphnia magna (immobilization, grazing and reproduction) for primary consumers, and Danio rerio for secondary consumers (embryotoxicity test). In view of the results obtained and the ecotoxicity values of tamoxifen available in the literature, we established a PNEC (Predictive No Effect Concentration) equal to 81 ng L-1 for continental water. This PNEC allowed us to cal- culate Risk Quotients (RQ) for 4 continental hydrosystems in 4 different countries in which measures of tamoxifen had already been performed on surface waters. In two of the situations studied, RQs were higher than 1, reaching a maximum of 2.6. These results show the need to deepen the characterization of ecotoxicological risks linked to the discharge of tamoxifen in surface waters. In addition, we propose applying this approach to other drug residues detected in the environment.

International audience; This work aims to explore in the field the relationship between the integrity of sperm DNA and the quality of offspring as a possible cause of the decline of a feral fish population through reproduction impairment. Mature nase (Chondrostoma nasus) were caught during the breeding season in three locations (A–C) of the Rhône River basin and gametes collected by stripping. Sampling locations were chosen according to the following gradient of contamination due to human activities on the watershed: A ≤B < C. Samples of a pool of collected oocytes were fertilized with the sperm of individual males and then incubated individually back in the lab to study embryo-larval development as well as using sperm samples to assess DNA integrity. Genetic analysis clearly showed the absence of a difference in genetic structure between the three studied C. nasus populations from the Rhône basin. Sperm DNA integrity was significantly lower in males from station C compared to other ones while sperm biochemical characteristics and fertilization rate remained almost unchanged whatever the station. Mortality and abnormality rates measured at both hatching and at the end of yolk sac resorption stages followed the same trend as the sperm DNA damage, demonstrating an impact of river water quality on nase fitness through a loss of sperm DNA integrity. Since the level of both abnormalities and mortality measured in offspring of fish caught in the most contaminated area reached high values up to 15% and 80%, respectively, the hypothesis that the observed nase decline in Rhône River stemming through selection forces can be put forward.

International audience; The genotoxic risk associated with deoxynivalenol (DON), a prevalent trichothecene mycotoxin which contaminates cereal-based products has not yet been deeply explored. In this work, the alkaline version of the Comet assay was used to evaluate DNA damage stemming from DON exposure in both dividing and differentiated Caco-2 cells, an epithelial intestinal cell line. To avoid false positive results, cytotoxic and apoptotic thresholds were firstly established using the MTS and neutral red assays and the Hoestch staining method, respectively. Dividing cells were found to be more sensitive to DON than differentiated cells and the lowest IC10 (0.5 mu M) obtained for dividing cells exposed for 72 It was used as the highest working concentration in the genotoxicity study. Both differentiated and dividing cells responded with a dose-dependant relationship to DON in terms of DNA damage in the 0.01-0.5 mu M range. These results demonstrated the existence of a genotoxic potential for DON at low concentrations compatible with actual exposure situations and calls for additional studies to determine the functional consequences which could be taken into account for the risk assessment of this food contaminant

International audience; The exploitation of oil sands has raised major environmental concerns, particularly regarding the presence of high concentration in contaminants such as polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) in oil sands process-affected water (OSPW). The purpose of this study was, first to evaluate the genotoxic impact of OSPW-related compounds such as NAs and PAHs in a salmonid species and secondly to assess if OSPW exposure leads to genotoxicity. For this purpose, rainbow trout hepatocytes were exposed in vitro to environmentally relevant concentrations of synthetic NAs, naphtalene, benzo(a)pyrene, and extracts of synthetic OSPW (generated by a laboratory bitumen extraction) and of oil sands leaching water (OSLW, mimicking leaching of oil sands in river water). Primary DNA damage was assessed by the formamidopyrimidine-DNA glycolyase (Fpg)-modified comet assay. Genotoxicity was observed in hepatocytes exposed to several NAs, mixture of them, OSPW and OSLW extracts. The chemical structure of NAs influences the genotoxicity potential: among the NAs tested, the most cyclic NA was the most genotoxic. It also appears that genotoxicity was more marked for OSPW than for OSLW. Because exposure to OSPW led to oxidative DNA damage, while after exposure to several NAs, these types of DNA damage were limited, the NAs tested in this study could not be qualified as the only major contaminants responsible for OSPW genotoxicity. Notwithstanding, it should be noteworthy that exposure to NAs resulted in genotoxic impact at concentrations lower than those documented by literature for fresh OSPW. Further research is needed to explore the relationships between the chemical structure of NAs and their genotoxicity in the light of the distribution of NAs in fresh OSPW samples as well as in surface waters. (C) 2014 Elsevier B.V. All rights reserved.

A high-performance liquid chromatographic method for the determination of the mycotoxin ergovaline in goat's milk is described here. Ergotamine was used as an internal standard. For a sample size of 5.0 ml, the cleanup method included precipitation of milk protein with acetone. Then, ergovaline was extracted twice with chloroform and purified by elution on an Ergosil ® column. HPLC separation of the extract was accomplished on a C 18 column: an isocratic elution, using acetonitrile–ammonium carbonate, was performed, and the analyte was detected by fluorimetry. The method was found to be linear between 0.7 and 8 ng ml −1 , a mean recovery rate of 99.8% was obtained, and the described assay appeared both repeatable and reproducible. The limit of detection and the limit of quantitation of ergovaline in milk were 0.2 ng ml −1 and 0.7 ng ml −1 , respectively. In order to apply the proposed method, four lactating goats were administered the toxin intravenously at a dose of 32 mg kg −1 body weight. The concentrations of the drug in plasma and milk were then determined at standardized intervals. Ergovaline (unequivocally identified by LC–MS–MS) could not be detected in the milk beyond eight hours post-dosing. Therefore, in goats, milk does not appear to be a major excretion route for the unmetabolized toxin.

A method based on high-performance thin-layer chromatography with UV detection, is described for the rapid determination of the mycotoxin lolitrem B in perennial ryegrass infected with the endophytic fungus Acremonium lolii . The method, which requires a clean-up of the crude extract on C 18 Sep-Pak prior to chromatographic analysis, has been validated according to the criteria established by the Journal of Chromatography B . Its preliminary application to samples of seven perennial ryegrass cultivars infected with A. lolii and cultivated in France, revealed the presence of lolitrem B at concentrations ranging between 0.1 and 0.94 μg/g dry mass. These results are of epidemiological importance for studying the “ryegrass staggers”, a neurotoxic syndrome which is induced in herbivorous animal species mainly by lolitrem B.

International audience; A better knowledge of DNA repair capacities in permanent fish cell lines would contribute to establish their interest in genotoxicity testing for environmental risk assessment studies including the effects of an increase in solar UV radiations on aquatic organisms. NER (Nucleotide Excision Repair) and PER (Photore- activation Repair) are the two repair pathways of choice for UV-induced photo-lesions. In the present paper, these repair processes were characterized in the two rainbow trout cell lines, RTGill-W1 and RTL-W1 (liver), by means of a T4-modified comet assay which allowed to follow the cyclobutane pyrimidine dimers repair kinetics specifically. Both repair processes have been found in the cell lines, PER repairing much faster UV lesions than NER, and NER being slightly more efficient in the gill cell line than in the liver one.

Many surface waters have been reported tobe contaminated whereas the most criticalsources remain to be identified. The waste-water treatment plant (WWTP) effluents dis-charged in surface waters are one of the possi-ble sources. Indeed, these effluents often con-tain well-known contaminants such as heavymetals, pesticides and polycyclic aromatichydrocarbons, and emerging contaminantssuch as pharmaceuticals, personal care prod-ucts, hormones and flame retardants.

International audience; Due to the importance of young-of-the-year (YOY) perch in the peri-alpine regions where they are consumed, the microcystin (MC) contamination of YOY perch was analysed both in field (Lake Bourget, France) and experimentally using force-feeding protocols with pure MCs. In-situ, schools of YOY perch present in the epilimnion of the lake were never found in direct contact with the P. rubescens blooms that were present in the metalimnion. However, MCs were detected in the muscles and liver of the fish and were thus assumed to reach YOY perch through dietary routes, particularly via the consumption of MC-containing Daphnia. Force-feeding experiment demonstrates the existence of MC detoxification/ excretion processes and suggests that in situ, YOY perch could partly detoxify and excrete ingested MCs, thereby limiting the potential negative effects on perch populations under bloom conditions. However, because of chronic exposure these processes could not allow for the complete elimination of MCs. In both experimental and in situ studies, no histological change was observed in YOY perch, indicating that MC concentrations that occurred in Lake Bourget in 2009 were too low to cause histological damage prone to induce mortality. However, Deoxyribonucleic acid (DNA) damages were observed for both the high and low experimental MC doses, suggesting that similar effects could occur in situ and potentially result in perch population disturbance during cyanobacterial blooms. Our results indicate the presence of MCs in wild perch, the consumption of this species coming from Lake Bourget is not contested but more analyses are needed to quantify the risk.

International audience; Many xenobiotics released in the aquatic environment exhibit a genotoxic potential toward organisms. Long term exposure to such compounds is expected to lead to multigenerational reproductive defects, further influencing the recruitment rate and hence, the population dynamics. Paternal exposure to geno- toxicants was previously shown to increase abnormal development in the progeny of mammalian or aquatic species. The aim of this study was to evaluate the relationship between DNA damage in sperm of the fish three-spined stickleback and progeny developmental defects. Spermatozoa were exposed ex vivo to an alkylating agent (methyl methanesulfonate) before in vitro fertilization and DNA damage was assessed by the alkaline comet assay. A significant relationship between abnormal development and sperm DNA damage was underlined. This study illustrates the interest to use germ cell DNA damage after ex vivo exposure to evaluate the impact of genotoxic compounds on progeny fitness in aquatic organisms.

International audience; Amongst DNA-repair processes, base-excision repair (BER) is the major mechanism for removal of DNA-base lesions caused by environmental genotoxicants. BER has been proven to exist in fish but has not been investigated in fish cell-lines, although these constitute increasingly important tools in eco-toxicological assessment. The present study aims at highlighting BER capacity of RTL-W1 and RTG-W1, two trout cell lines used in eco-genotoxicity studies. This is realized by following the kinetics of strand-break repair after a short exposure to model genotoxicants-leading predominantly to BER-specific lesions-by means of the standard alkaline and Fpg-modified comet assays. Results show that both cell lines efficiently repair single-strand breaks and base-alkylation damages within 4h and 24h, respectively. Then, the study shows that after minor modifications of the protocol, the cell extract-based BERc assay can be used to evaluate the base-incision capacity of the cell lines and its variation after exposure of the cells to a model inhibitor of BER (3-aminobenzamide) and to environmental contaminants such as cadmium and tributyltin. This work provides a basis for the further development of DNA-repair activity in fish cell-lines as a new biomarker of genotoxicity. Copyright 2013 Elsevier B.V. All rights reserved.

International audience; In a context of growing awareness of aquatic pollution impacts, there is an increasing need to develop methods for hazard and risk assessment of pollutants. For this purpose, in vitro models such as fish cell lines warrant to be evaluated as possible alternative to in vivo fish testing, and new toxicity endpoints such as genotoxicity deserve to be considered. This study assesses the interest of the formamido pyrimidine glycosylase (Fpg)-modified comet assay applied to three fish cell lines (RTL-W1, RTG-W1, and PLHC-1) regarding the sensitivity of the system for measuring genotoxicity of various classes of pollutants. Cytochrome P450-dependent EROD activity has also been measured to evaluate the importance of the biotransformation capacity of the cell lines in genotoxicity assessment. For all cell lines and chemicals tested, a concentration dependent genotoxic effect was observed with a 10- to 1000-fold increased sensitivity when using the Fpg-protocol compared to the standard comet assay. Such a modified assay led in particular to improve the detection threshold of oxidative and alkylating DNA damages following exposure at environmentally relevant contaminant concentrations and could partly compensate for the lower sensitivity of cell lines versus whole organism testing often cited as a limit of in vitro testing.

International audience; In the present study, juvenile whitefish weighing 2 g were exposed by force-feeding to two ecologically relevant doses (0.05 and 0.5 mu g per fish) of microcystin-LR (MC-LR). Then over 96 h the MC uptake in fish liver and muscle was measured, as the activity of the detoxification enzyme glutathione S-transferase (GST) in the liver, and the genotoxicity impact on red blood cells. Results show that (1) the MC-LR equivalent concentrations increased for both doses and in both organs of whitefish with approximately threefold lower concentrations for the low dose compared to the high dose in both organs and threefold lower concentrations in the muscle compared to the liver for each dose (2) the liver GST activity increased during the first 48 h of exposure with fivefold higher GST activity for the highest dose at 48 h compared to control and (3) MC-LR leads to deoxyribonucleic acid strand breaks that were detected by the comet assay and shown to be partially repaired. This work demonstrates that European whitefish could be impacted by cyanobacteria toxins due to rapid microcystin uptake, especially in the context of chronic contamination, which can occur during long bloom episodes.

International audience; The dredged sediments of polluted seaports now raise complex management problems since it is no longer possible to discharge them into the sea. This results in the need to manage them on land, raising other types of technical, economic and environmental problems. Regarding the technical and economic dimensions, traditional waste treatment methods have proved to be poorly adapted, due to very high costs and low absorbable volumes. In this context, filling quarries in coastal areas with treated sediments could represent an interesting alternative for these materials. Nevertheless, for the environmental dimension, it is necessary to demonstrate that this possibility is harmless to inland ecosystems. Consequently, a specific ecotoxicological risk assessment methodology has been formulated and tested on three sediments taken from seaboards of France, in view to providing an operational and usable tool for the prior validation of any operation to fill quarries with treated seaport sediments. This method incorporates the formulation of a global conceptual model of the scenario studied and the definition of protocols for each of its steps: the characterisation of exposures (based on a simulation of sediment deposit), the characterisation of effects (via the study of sediments ecotoxicity), and the final ecotoxicological risk assessment performed as a calculation of a risk quotient. It includes the implementation in parallel of two types of complementary approach: the "substances" approach derived from the European methodology for assessing new substances placed on the market, and the "matrix" approach which is similar to methods developed in France to assess ecological risks in other domains (waste management, polluted site management, ...). The application of this dual approach to the three sediments tested led to conclude with reliability that the project to deposit sediments "1" and "2" presented a low risk for the peripheral aquatic ecosystems while sediment "3" presented a high risk.

International audience; The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment. (C) 2011 Elsevier Ltd. All rights reserved.

International audience; Germ cells perform a unique and critical biological function: they pass down DNA that will be used for the development of the next generation. Thus there is an increasing need to understand how the adult exposure to genotoxicants could show negative impact on the offspring of aquatic organisms. Hence this work addresses the question of the consequences of germ cell DNA damage resulting from parental exposure on reproduction quality in the freshwater crustacean Gammarus fossarum, a high ecologically relevant species. Initially, the sensitivity response of mature oocytes and spermatozoa to two model genotoxicants, MMS and K2Cr2O7 was compared by implementing the Comet assay after the exposure of these gammarids in the laboratory and after the exposure of caged organisms in the field. Spermatozoa appeared significantly more susceptible than the oocytes to genotoxicants whatever were the exposure conditions. Secondly, a significant correlation between the level of damage to the sperm DNA of exposed parents and the abnormality rate in embryos that had developed in non-contaminated water were demonstrated. Interestingly, this relationship bridges the biomarker response measured in germ cells at molecular level and its consequences at individual level for the subsequent generation. Moreover, reproduction defects were observed for a level of DNA damage exceeding a minimal threshold, which could have significant consequences for the population dynamics of this high ecologically relevant species.

International audience; European flounder responses to the chemical stress were assessed by a comparative approach on four estuaries displaying contrasted patterns of contamination. The contamination typology of the estuaries was investigated by individual measurements of contaminants in fish. Molecular and physiological responses were studied by gene expression, genotoxicity, neurotoxicity and growth rate. Fishes in contaminated estuaries were characterized by high levels of bioaccumulated contaminants, slow energetic metabolism and reduced growth rate, in contrast to the fish responses in the reference site. A seasonal effect was highlighted for contaminated flounder populations, with high PCB levels, high genotoxicity and elevated detoxification rate in summer compared with winter.

International audience; Many xenobiotics and newly developed substances released in the aquatic environment have been found genotoxic for living organisms. There is interest in developing biomarkers of genotoxicity in different phyla and the need to increase our understanding of the impact of genotoxic insult on invertebrates, particularly on crustaceans. Freshwater invertebrates and particularly amphipods are highly relevant species ecologically. However, genotoxic responses of such species are rarely studied, whereas understanding these responses is becoming an urgent concern. The aim of this study was to develop and optimize the Comet assay in the freshwater invertebrate Gammarus fossarum by use of different cell-types: haemocytes, oocytes and spermatozoa. In a first step, the Comet assay was performed on these three cell types after exposure to the model genotoxicant methyl methanesulfonate (MMS) in vitro and in vivo. Results showed a clear doseresponse relationship for all tissues, a low variability and a high sensitivity of the response, demonstrating the effectiveness of the Comet assay to detect genotoxic insult in amphipods. In a second step, to explore the potential of this technique for use in ecotoxicological studies with amphipods, these organisms were exposed to five known or suspected genotoxic compounds. The results demonstrated the possibility to use the freshwater amphipod G. fossarum in environmental genotoxicity studies with the Comet assay.

International audience

The present study deals with the genotoxicity assessement of vineyard pesticides in fish exposed in the field or in mesocosm conditions. Primary DNA damage was quantified as strand-breaks using the single cell gel electrophoresis assay (Comet assay) applied to fish erythrocytes. In a first experiment, a significant genotoxic effect was observed following an upstream-downstream gradient in early life stages of brown trout (Salmo trutta fario) exposed during a three consecutive year survey in the Morcille river contaminated by a mixture of vineyard pesticides. The high response in terms of DNA damage reported in the present study could argue for a high sensitivity of fish early life stage and/or a high level of exposure to genotoxic compounds in the Morcille river. This stresses the interest in using trout larvae incubated in sediment bed to assess genotoxic compounds in the field. In a second experiment, adult European topminnow (Phoxinus phoxinus) were exposed in water running artificial channels to a mixture of diuron and azoxystrobin, two of the main pesticides detected in the Morcille watershed. As compared with the unexposed channel, a 3- to 5-fold increase in the DNA level damage was obtained in fish exposed to chronic environmental pesticide concentrations (1-2 µg.L-1 for diuron and 0.5-1 µg.L-1 for axoxystrobin). A single 6 hour pulse of acute pesticide exposure (14 µg/L of diuron and 7 µg/L of azoxystrobin) was applied to simulate transitory elevated chemical concentrations in the river following storm conditions and did not increase genotoxicity. After a one month recovery period, DNA damage level in exposed fish erythrocytes recovered to unexposed level, suggesting possible involvement of both repair mechanisms and cellular turnover in this transient response. This work highlights that vineyard treatment by pesticides and in particular diuron and azoxystrobin can represent a genotoxic threat to fish from contaminated watershed rivers.

International audience; This study aims to assess the genotoxic potential of nivalenol (NIV) and fusarenon X (FusX), produced by various Fusarium on cereals. Toxins were applied in time and dose-dependent experiments to the human enterocyte-like Caco-2 cell-line, both in dividing (undifferentiated) and in 10–12 days post-confluent cells (differentiated). Genotoxicity was evaluated through the alkaline Comet assay in a concentration range defined for each toxin as below the cytotoxicity threshold IC10, determined by the MTS and the neutral red assays, to prevent false positive results because of DNA damage stemming from necrosis. Thus, genotoxicity was explored in the sub-cytotoxic 0–0.5 μM and 0–0.05 μM ranges respectively for NIV and FusX as the latter was found about 10-fold more cytotoxic than NIV. For both toxins, a 3 h exposure did not cause any DNA damage, unlike after 24 and 72 h exposure in post confluent Caco-2 cells where DNA damage was significantly observed with a dose-dependent relationship. In dividing cells, only FusX increases DNA strand breaks in the 0.01–0.05 μM range after 72 h. These results demonstrated the existence of a genotoxic potential for NIV and FusX at low exposure levels and could contribute to the risk assessment process of these toxins that are of growing concern

International audience; his paper describes a new method of purification of the Lolitrem B, a tremorgenic mycotoxin produced in planta by the endophytic fungus Neotyphodium lolii. The method is based on the large-scale isolation of the toxin by countercurrent chromatography (CCC). The lolitrem B content in endophyted ryegrass seed, 11 mug/g or 11 ppm, is extracted by stirring finely ground seeds with ethanol for 3 h at room temperature. The concentrated crude extract contains about 0.6 mg/g or 600 ppm of lolitrem B. It is then submitted to CCC purification with a biphasic four-solvent liquid system. A 160-fold enrichment was obtained in one step producing a raffinate containing 10% or 100 mg/g of the toxin. Further purifications were then performed by thin layer and low pressure liquid chromatography. Twenty-eight micrograms of lolitrem B with a 96% purity grade were obtained from 8 kg of seeds (yield 32%).

Summary • Variability in the fungal endophytes of 83 natural populations of Lolium perenne (perennial ryegrass) from Europe was assessed. • One plant per population was used for endophyte isolation and mycotoxin analysis. Variability in three isozymes, colony morphology and growth rate on potato dextrose agar (PDA), and synthesis of ergovaline, lolitrem B and peramine was recorded. • Three species were found among 94 strains isolated: Neotyphodium lolii, Neotyphodium sp. (LpTG-2) and Gliocladium-like. The most frequent species was N. lolii, which showed high variability. In 12 populations, a single plant harboured two different endophytes. One-third of the isolates of N. lolii did not produce ergovaline whereas a few isolates did not produce lolitrem B. Ergovaline and lolitrem B-deficient strains, but not the few peramine-deficient isolates, had characteristic morphologies on PDA. No isolate was deficient for both ergovaline and lolitrem B synthesis. • Selection of ergovaline and lolitrem-deficient strains based only on the morphology of the isolates in culture may be possible.

International audience; The toxicokinetics of ergovaline (an ergopeptine mycotoxin present in some grasses infected with endophytic fungus of the genus Neotyphodium) were studied after intravenous administration of a single dose of 15 $\mu$g/kg bwt in four gelding horses. Plasma ergovaline concentrations were measured by high performance liquid chromatography, and the kinetic data were described by a three-compartment model. The elimination half-life and the total clearance of ergovaline were found to be 56.83 $\pm$ 13.48 min and 0.020 $\pm$ 0.004 L/min$\cdot$kg, respectively. According to the toxicological data previously reported in the horse, and in spite of the very low dose administered, clinical signs were observed, including excessive coolness of the ears and the nose, excessive sweating and prostration.; Étude toxicocinétique de l'ergovaline chez le cheval après injection intraveineuse. Les paramètres toxicocinétiques de l'ergovaline (un alcaloïde ergopeptidique présent dans les graminées porteuses d'un champignon endophyte du genre Neotyphodium) ont été déterminés après l'injection intraveineuse d'une dose de 15 $\mu$g/kg chez quatre chevaux hongres. Les concentrations plasmatiques d'ergovaline ont été mesurées par chromatographie liquide haute performance et les données cinétiques décrites selon un modèle à trois compartiments. La demi-vie d'élimination et la clairance totale de l'ergovaline ont été respectivement de 56,83 $\pm$ 13,48 min et de 0,020 $\pm$ 0,004 L/min$\cdot$kg. Malgré la très faible dose administrée, des signes cliniques compatibles avec les données toxicologiques rapportées dans l'espèce équine ont été observés, à savoir hypothermie des oreilles et du nez, hypersudation et prostration.

High performance thin layer chromatography (HPTLC) is used to rapidly determine the presence and amount of ergovalin in seeds of Graminaceae infested with Neotyphodium Coenophialum. Quinine is used as an internal standard. The mycotoxin is solubilized under acid conditions and extracted with chloroform under alkaline conditions. Samples are sprayed on silica gel plates and detected by fluorescence with λ1 (excitation) set at 318 nm and λ2 (emission) > 400 nm. The method developed is specific for endophyte-infested seeds, linear from 3 to 12 μg/g and is both repeatable and reproducible. The limit of detection is 0.5 μg/g and the limit of determination (quantification) is 3.0 μg/g. This technique was applied to several batches of seeds obtained from pastures that may or may not be infested with the fungus. Results were compared with results obtained using a standard High Performance Liquid Chromatography (HPLC) method.

A rapid high-performance liquid chromatographic method for the determination of the mycotoxin ergovaline in ovine plasma is described here. Ergotamine was used as an internal standard. A simple extraction procedure with diethyloxide was carried out, before chromatography on a C8 column, with the excitation and emission wavelengths fixed at 250 and 420 nm respectively, on a fluorimetric detector. The method, which was found to be linear between 3.5 and 15 ng/ml, had good specificity, precision and accuracy. The limit of quantification and the limit of detection were 3.5 and 1.2 ng/ml, respectively. A preliminary application of the described assay to a plasma kinetic study, after intravenous administration of a single dose of ergovaline (17 micrograms/kg body mass) to four sheep, showed a very rapid decrease of the plasma ergovaline levels. The terminal half-life and the total clearance of the mycotoxin were found to be 23.6 min and 0.020 l/min kg-1 body mass, respectively.