Amélioration de l'évaluation écotoxicologique des effluents de stations d'épuration : proposition d'une batterie de tests centrée sur les effets à long terme et des paramètres de forme de l'organisme

Cette thèse a visé l’optimisation de l’évaluation de l’écotoxicité d’effluents traités en station d’épuration dans le cadre du projet Européen TRIUMPH “TReatIng Urban Micropollutants and PHarmaceuticals in waste waters”. Le but du projet était de proposer de nouveaux traitements des eaux usées combinant oxydation biologique et chimique par l’ozone pour optimiser l’abattement des micropolluants et de la toxicité résiduelle des effluents de station d’épuration. Les effluents traités étudiés dans cette thèse provenaient d’une station d’épuration pilote traitant un mélange constitué à part égale d’eaux usées urbaine et hospitalière. La première étape du travail a consisté à identifier les bioessais les plus pertinents et plus sensibles au sein d’une batterie de tests in vitro et in vivo, normalisés et non-normalisés, ciblant des paramètres de la fitness des organismes et respectant la Directive 2010/63/EU relative à la protection des animaux utilisés à des fins scientifiques. La toxicité d’un effluent traité par boues activées et par ozonation tertiaire a été évaluée avec le test d’inhibition de la reproduction de Brachionus calyciflorus, le test d’inhibition de la croissance de Pseudokirchneriella subcapitata et d’Heterocypris incongruens. La mortalité a été évaluée avec le Fish Embryo Toxicity test (FET) et des critères sub-létaux (9 anomalies du développement) ont été ajoutés pour proposer un nouveau paramètre appelé iFET score. La génotoxicité a été évaluée sur la lignée cellulaire de poisson RTL-W1, sur Salmonella typhimurium (Ames test) et sur les cellules d’embryons de Danio rerio. Le potentiel de perturbation endocrinienne a été quantifié avec les lignées cellulaires MELN/MDA-kb2. A l’issue de cette première phase, le développement embryonnaire de poisson zèbre (iFET score) ainsi que l’évaluation de la génotoxicité sur cellules embryonnaire de poisson zèbre et sur lignée cellulaire RTL-W1 se sont révélés les plus sensibles pour caractériser une toxicité résiduelle des deux effluents traités conventionnellement. Pour la suite du travail, ces critères ont donc été retenus afin d’évaluer la toxicité résiduelle de plusieurs effluents résultant de traitements innovants répartis en quatre campagnes. Les résultats montrent que les mesures sur embryons de poisson (iFET score et génotoxicité mesurée sur les cellules embryonnaires) sont les plus informatives et discriminantes pour évaluer finement la toxicité résiduelle d’effluent de station d’épuration. Enfin, une exposition de Gammarus fossarum a été réalisée pendant 14 jours directement sur le site pilote au cours de la dernière campagne et plusieurs paramètres reliés à la fitness des organismes (cycle de mue, reproduction et génotoxicité sur les spermatozoïdes) ont été mesurés. Cette dernière expérimentation a démontré l’intérêt d’exposition in vivo à long terme dans le choix d’une batterie de tests en vue d’optimiser la mesure d’une toxicité résiduelle d’effluents de station d’épuration caractérisés par une contamination chimique très complexe et à bas bruit.This thesis deals with the optimization of ecotoxicity evaluation of treated effluents and was part of the European TRIUMPH “TReatIng Urban Micropollutants and PHarmaceuticals in waste waters” project. The aim of the project was to propose innovative ozone-based, cost-efficient wastewater treatments improving micropollutant abatement and reducing effluent toxicity when compared to conventional wastewater treatments. The differently treated effluents studied throughout the thesis came from a pilot wastewater treatment plant, receiving a mixture of 50% urban and 50% hospital wastewaters.The first step of the work was to identify the most pertinent and sensitive bioassays from a battery of standardized and non-standardized in vitro and in vivo bioassays targeting endpoints involved in organism fitness, and respecting the Directive 2010/63/EU on the protection of animals used for scientific purposes. Toxicity of conventional activated sludge treated effluent and of effluent treated by subsequent tertiary ozonation was assessed using reproduction inhibition test with Brachionus calyciflorus, growth inhibition assay with Pseudokirchneriella subcapitata and with Heterocypris incongruens. Mortality was evaluated with Fish Embryo Toxicity test (FET), and sub-lethal criteria (9 developmental abnormalities) were additionally recorded to propose a new parameter named iFET score. Genotoxicity was evaluated on the fish RTL-W1 cell line, on Salmonella typhimurium (Ames test) and on Danio rerio embryo cells. Endocrine disrupting potential was measured with MELN/MDA-kb2 cell lines. Zebrafish embryo development (iFET score) and genotoxicity evaluation on zebrafish embryo cells as well as on RTL-W1 cell line were demonstrated as the most sensitive tests to reveal a residual toxicity in conventionally treated effluents. Thus, they were retained in a second step to evaluate the toxicity of effluents stemming from several innovative treatments structured into four campaigns. Results showed that iFET score approach and subsequent genotoxicity evaluation were the most informative and discriminant methods to properly assess effluent residual toxicity. Finally, a 14 day-in situ exposure of Gammarus fossarum was realized on the pilot site during the last campaign, evaluating fitness relevant endpoints (molting cycle, reproduction and genotoxicity on sperm). This experiment confirmed that toxicity evaluation of treated wastewaters should integrate long-term in vivo exposure in order to take into account their highly complex micropollutant composition

Tamoxifen ecotoxicity and resulting risks for aquatic ecosystems

International audienceTamoxifen, 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

Proposal to optimize ecotoxicological evaluation of wastewater treated by conventional biological and ozonation processes

International audienceA 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

Élimination des composés pharmaceutiques en station d’épuration par traitements biologiques et ozonation tertiaire

National audienceThe choice to operate the CAS and MBBR systems with full nitrification, results in the same level of performance on pharmaceutical compounds removal for both biological systems (despite the differences on process conditions : sludge concentration, hydraulic contact time…).The biological treatment of 10 pharmaceuticals with CAS or MBBR reactor succeeded in 24% of removal of the 7 refractory compounds measured (carbamazepine, sulfamethoxazole, propranolol, atenolol, diclofenac, econazole, salicylic acid), and an average of 45% including also paracetamol, ketoprofen and ibuprofen with an important variability on performances (> 20%). The combined system CAS biolog ical treatment plus tertiary ozonation at low ozone dosage (0.45 gO3/gDOC) improves the average removal up to 92% with only one compound -econaz ole- moderately eliminated (70%). It has been observed that increasing the ozone from the low to the intermediate dosage of 1.30 gO3/gDOC leads to an average removal improvement of around 6% whereas a more important TOD (2.30 gO3/gDOC) did not led to a better performance.Contrary to the lack of toxicity observed with normalized ecotoxicity tests, endpoints measured on zebrafish embryos (FET bioassay) such as mortality, developmental abnormalities and genotoxicity demonstrated a residual toxicity (null/weak) both, in wastewater after a biological treatment followed or not by a tertiary ozonation treatment. However, the tertiary ozonation resulted in an improvement of the residual endocrine disrupting potential measured in the biologically-treated effluents.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 differently treated effluents.The knowledge of the sample chemical fingerprints, by a comprehensive non-target approach based on the comparison of biochemical signatures, is a promising growing strategy to identify and prioritize the emerging contaminants and to contribute to the study of the treatment performance.Les performances atteintes par les deux systèmes biologiques étudiés – boues activées conventionnelles (BA) et réacteur biologique à biomasse fluidisée (MBBR) – dans le traitement des dix micro— polluants d’origine pharmaceutique quantifiés sont similaires et cela grâce au niveau d’épuration identique en nitrification totale.Les abattements quantifiés sont variables et atteignent en moyenne pour les sept composés réfractaires seulement 24% (carbamazépine, sulfaméthoxazole, propranolol, aténolol, diclofénac, éconazole, acide salicylique). Les trois autres composés, bien éliminés en traitement biologique, atteignent en moyenne 96% (paracétamol, kétoprofène et ibuprofène).Le traitement biologique combiné à l’ozonation tertiaire élimine efficacement les composés pharmaceutiques de l’eau dès l’utilisation des doses faibles d’ozone (0,45 gO3/g de carbone organique dissous COD). Un abattement moyen de 92% d’efficacité est quantifié sur la moyenne de composés pharmaceutiques dissous, à comparer aux 24% à 45% d’abattement obtenu en traitement bio logique seul. Le passage à une dose d’ozone de 1,30 gO3/gCOD permet d’augmenter de 6% l’abattement moyen des micropolluants. Cette performance n’est pas améliorée par l’utilisation des doses d’ozone plus importantes.Contrairement aux tests d’écotoxicité normalisés (peu sensibles pour discriminer des effets toxiques dans les eaux traitées biologiquement ou chimiquement), les effets mesurés sur les embryons du poisson Danio rerio comme la mortalité, les anomalies sur le développement et la génotoxicité ont montré un effet toxique (nul à faible) pour les effluents biotraités ou ozonés. Par rapport au traitement biologique, l’ozonation tertiaire améliore significativement le potentiel de perturbation endocrinienne des effluents par la réduction complète de l’activité oestrogénique et glucocorticoïde des effluents.L’ozonation n’augmente ni ne diminue le niveau de toxicité des effluents déjà atteint avec le traitement biologique seul. Il existe un bruit persistant probablement lié à la présence d’autres micro - polluants rémanents dans l’eau et non quantifiés par la méthode d’analyse ciblée. Leur activité peut être à l’origine du niveau d’effet toxique faible obtenu pour certains biomarqueurs. Ces résultats montrent la nécessité d’améliorer la sensibilité des bioessais dans le but d’optimiser l’évaluation des effets écotoxicologiques résiduels dans les différents types d’effluents.L’obtention des signatures chimiques, au travers d’une approche non ciblée, s’avère un outil puissant et complémentaire. Elles révèlent la complexité des effluents étudiés par le nombre important de signaux obtenus. Les cartographies réalisées, par l’analyse en composantes principales, permettent d’avoir une vision globale des effluents et de leur mise en relation respective. Cette approche globale complète efficacement les résultats obtenus par le dosage ciblé des micropolluants

Gammarus fossarum as a sensitive tool to reveal residual toxicity of treated wastewater effluents

International audienc

A posteriori assessment of ecotoxicological risks linked to building a hospital

International audienceHospital wastewater (HWW) contain a large number of chemical pollutants such as disinfectants, surfactants, and pharmaceutical residues. A part of these pollutants is not eliminated by traditional urban wastewater treatment plants (WWTP), leading to a risk for the aquatic ecosystems receiving these effluents. In order to assess this risk, we formulated a specific methodology based on the ecotoxicological characterisation of the hospital wastewater using a battery of three chronic bioassays (Pseudokirchneriella subcapitata, Heterocypris incongruens and Brachionus calyciflorus). We used it for the posteriori risk assessment of a hospital recently built in south-east France, and we studied the evolution of this risk during two years. We also used it to assess the decrease of the ecotoxicological risk after treatment of the effluent in a specific line of the local WWTP. Lastly, we compared these results with the risk assessment made before the building of the hospital in the context of a priori risk assessment. The results obtained showed an important evolution of the risk overtime, according to the hospital activities and the river flows, and a real decrease of the risk after treatment in the dedicated line. They also showed that the a priori assessment of ecotoxicological risks, made previously, was overstated, mainly because of the application of the precautionary principle

Assessment of long term ecotoxicity of urban stormwaters using a multigenerational bioassay on Ceriodaphnia dubia: A preliminary study

International audienceStandardized 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

Assessment of Ozone or Not-Treated Wastewater Ecotoxicity Using Mechanism-Based and Zebrafish Embryo Bioassays

International audienceToxicity 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

Microhabitat use, population densities, and size distributions of sulfur cave-dwelling Poecilia mexicana

The Cueva del Azufre in Tabasco, Mexico, is a nutrient-rich cave and its inhabitants need to cope with high levels of dissolved hydrogen sulfide and extreme hypoxia. One of the successful colonizers of this cave is the poeciliid fish Poecilia mexicana, which has received considerable attention as a model organism to examine evolutionary adaptations to extreme environmental conditions. Nonetheless, basic ecological data on the endemic cave molly population are still missing; here we aim to provide data on population densities, size class compositions and use of different microhabitats. We found high overall densities in the cave and highest densities at the middle part of the cave with more than 200 individuals per square meter. These sites have lower H2S concentrations compared to the inner parts where most large sulfide sources are located, but they are annually exposed to a religious harvesting ceremony of local Zoque people called La Pesca. We found a marked shift in size/age compositions towards an overabundance of smaller, juvenile fish at those sites. We discuss these findings in relation to several environmental gradients within the cave (i.e., differences in toxicity and lighting conditions), but we also tentatively argue that the annual fish harvest during a religious ceremony (La Pesca) locally diminishes competition (and possibly, cannibalism by large adults), which is followed by a phase of overcompensation of fish densities