Evaluation of the Sensitivity of Freshwater Organisms Used in Toxicity Tests of Wastewater from Explosives Company

Explosives industries are a source of toxic discharge. The aim of this study was to compare organisms sensitivity (Daphnia similis, Danio rerio, Escherichia coli and Pseudomonas putida) in detecting acute toxicity in wastewater from two explosives, 2,4,6-TNT (TNT) and nitrocellulose. The samples were collected from an explosives company in the Paraiba Valley, So Paulo, Brazil. The effluents from TNT and nitrocellulose production were very toxic for tested organisms. Statistical tests indicated that D. similis and D. rerio were the most sensitive organisms for toxicity detection in effluents from 2,4,6-TNT and nitrocellulose production. The P. putida bacteria was the organism considered the least sensitive in indicating toxicity in effluents from nitrocellulose.

Sensitivity of Eisenia andrei (Annelida, Oligochaeta) to a commercial formulation of abamectin in avoidance tests with artificial substrate and natural soil under tropical conditions

Obtaining ecotoxicological data on pesticides in tropical regions is imperative for performing more realistic risk analysis, and avoidance tests have been proposed as a useful, fast and cost-effective tool. Therefore, the present study aimed to evaluate the avoidance behavior of Eisenia andrei to a formulated product, Vertimec(A (R)) 18 EC (a.i abamectin), in tests performed on a reference tropical artificial soil (TAS), to derive ecotoxicological data on tropical conditions, and a natural soil (NS), simulating crop field conditions. In TAS tests an adaptation of the substrate recommended by OECD and ISO protocols was used, with residues of coconut fiber as a source of organic matter. Concentrations of the pesticide on TAS test ranged from 0 to 7 mg abamectin/kg (dry weight-d.w.). In NS tests, earthworms were exposed to samples of soils sprayed in situ with: 0.9 L of Vertimec(A (R)) 18 EC/ha (RD); twice as much this dosage (2RD); and distilled water (Control), respectively, and to 2RD: control dilutions (12.5, 25, 50, 75%). All tests were performed under 25 +/- A 2A degrees C, to simulate tropical conditions, and a 12hL:12hD photoperiod. The organisms avoided contaminated TAS for an EC50,48h = 3.918 mg/kg soil d.w., LOEC = 1.75 mg/kg soil d.w. and NOEC = 0.85 mg/kg soil d.w. No significant avoidance response occurred for any NS test. Abamectin concentrations in NS were rather lower than EC50, 48h and LOEC determined in TAS tests. The results obtained contribute to overcome a lack of ecotoxicological data on pesticides under tropical conditions, but more tests with different soil invertebrates are needed to improve pesticides risk analysis.

Ecotoxicological evaluation of wastewater from 2.4.6-TNT production

During the manufacture of explosives, large amounts of water are used to remove unwanted by-products generated. This water in turn, ends up in wastewater treatment plants or water bodies. The aim of this study was to evaluate the toxic potential of effluent generated by 2.4.6-Trinitrotoluene (TNT) production, yellow water, red water and mixture of yellow and red water, produced from a plant located in the Paraiba Valley, Sao Paolo state, Brazil. Daphnia similis, Danio rerio, Escherichia coli, Pseudomonas putida and Pseudokircheneriella subcaptata were used as test organisms. Physicochemical parameters such as color, pH, conductivity, total dissolved solids, dissolved oxygen, chemical oxygen demand (COD) and biochemical oxygen demand (BOD) were evaluated. Effluent from 2.4.6-TNT production was extremely toxic to all test organisms. The physicochemical parameters evaluated showed high levels of conductivity (from 41.533 to 42.344 mu S /cm) and chemical oxygen demand (COD of 8471 to 27.364 mg/L) for the effluents analyzed.

Nova metodologia para a previsão da concentração de glifosato e AMPA em amostras de água.

A utilização intensiva e simultânea de agrotóxicos na agricultura ocasiona a exposição múltipla dos organismos a esses compostos, tornando necessária a avaliação da contaminação de misturas, principalmente, no ambiente aquático. Estudos de toxicidade de agrotóxicos são de suma importância para a comunidade como um todo, principalmente quando estes são realizados em águas, haja vista a possível escassez desse recurso. O objetivo deste trabalho foi avaliar a toxicidade aguda dos herbicidas glifosato (Gly) e seu principal metabólito, ácido aminometilfosfônico (AMPA), ao microcrustáceo Daphnia similis.

Influence of Baculovirus anticarsia on the growth rate and survival of some nontarget aquatic organisms.

Recently, microbial pest control agents (MPCAs) have been worldwide used to reduce chemical pesticide use and to diminish the high risk of those compounds in the environment. Among various MPCAs, the nuclear polyhedrosis virus Baculovirus anticarsia is widely used in Brazil in the biological control of the velvet bean caterpillar. Although literature data do not show adverse effects of baculoviruses to nontarget organisms, it is necessary to evaluate their toxicity or patogenicity in order to study th environmental risk of those products and to register the formulations in the Brazilian Environmental Regularory Agency - IBAMA. In the presente work, the influence of a Baculovirus anticarsia formulation was evaluted to measure the consequences in the growth rateof the green algae Selenastrum capricornutum, the duckweed Lemna valdiviana and the microcrustacean Daphnia similis. The survival of the fish Hyphessobrycon scholzei exposed during 28 days was also evaluated. No significative adverse effects (P > 0.05) were observed in the test organisms which were exposed to 1-1000 times the maximum calculated pesticide concentration following a direct application to 15 cm layer of water.

Risk assessment of the herbicide clomazone in the aquatic life.

Clomazone (2-(2-chlorophenyl)methyl-4.4-dimethyl-3-isoxazolidinone) is a post emergence herbicide widely used in rice fields in Rio Grande do Sul (Brazil) with high activity against Gramineae at the recommended application rate(AR).of 700g/ha. The herbicide input into the aquatic ecosystem may occur by aerial application or water drainage. The presence of this chemical in the water may affect non-target organisms leading to impairments in the aquatic food chain. Studies were conducted in this work to evaluate the risk of Clomazone using the estimated mean affective concentration (EC50) for the microalgae Selenastrum capricornutum(96h), the duckweed Lemna valdiviana(96h) and the crustacean Daphnia similis(48h). The EC50 values were 11.2; 31.7 and 13.8 mg/l, respectively. According to the obtained data, and considering a direct input of the herbicide in a 10cm column water, the estimated maximum application rate that doesn't cause acute effects is 5.3 AR for S. capricornutum, 6.5 AR for D. similis and 15.0 AR for L. valdiviana. The estimated maximum application rate that doesn't cause chronic effects is 2.0 AR for D. similis, 1.6 AR for S. capricornutum and 4.5 AR for L. valviana.

Bisphenol A migration from plastic materials: direct insight of ecotoxicity in Daphnia magna

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) whose migration from food packaging is recognized worldwide. However, the real overall food contamination and related consequences are yet largely unknown. Among humans, children’s exposure to BPA has been emphasized because of the immaturity of their biological systems. The main aim of this study was to assess the reproductive impact of BPA leached from commercially available plastic containers used or related to child nutrition, performing ecotoxicological tests using the biomonitoring species Daphnia magna. Acute and chronic tests, as well as single and multigenerational tests were done. Migration of BPA from several baby bottles and other plastic containers evaluated by GC-MS indicated that a broader range of foodstuff may be contaminated when packed in plastics. Ecotoxicological test results performed using defined concentrations of BPA were in agreement with literature, although a precocious maturity of daphnids was detected at 3.0 mg/L. Curiously, an increased reproductive output (neonates per female) was observed when daphnids were bred in the polycarbonate (PC) containers (145.1±4.3 % to 264.7±3.8 %), both in single as in multigenerational tests, in comparison with the negative control group (100.3±1.6 %). A strong correlated dose-dependent ecotoxicological effect was observed, providing evidence that BPA leached from plastic food packaging materials act as functional estrogen in vivo at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non-PC bottles was slightly but not significantly enhanced (92.5±2.0 % to 118.8±1.8 %). Multigenerational tests also revealed magnification of the adverse effects, not only on fecundity but also on mortality, which represents a worrying trend for organisms that are chronically exposed to xenoestrogens for many generations. Two plausible explanations for the observed results could be given: a non-monotonic dose–response relationship or a mixture toxicity effect.

Presença de Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) na Serra da Bodoquena, Estado de Mato Grosso do Sul, Brasil

O veneno das aranhas do gênero Loxosceles causa lesão dermonecrótica e induz hemólise intravascular dependente de complemento, configurando um quadro clínico de intensa gravidade. No Brasil, as espécies L. gaucho L. intermedia e L. laeta, presentes no ambiente antrópico, têm sido apontadas como principais agentes do loxoscelismo. Além destas, existem outras espécies, que por predominarem no ambiente natural, não têm sido avaliadas quanto ao risco à saúde do homem, como é o caso de Loxosceles similis. O desenvolvimento de projeto de pesquisa, na Serra da Bodoquena, para observações ecológicas e identificação de insetos de interesse médico, possibilitou a captura de espécimens de Loxosceles similis na Serra da Bodoquena, Município de Bonito, Estado do Mato Grosso do Sul, Brasil, nas grutas Pitangueiras e do Lago Azul. Os parâmetros para identificação, características ambientais do habitat da espécie, bem como atualização de sua distribuição geográfica são objetos deste trabalho.

FOOD CONCENTRATION AND TEMPERATURE EFFECTS ON LIFE CYCLE CHARACTERISTICS OF TROPICAL CLADOCERA (Daphnia gessneri Herbst, Diaphanosoma sarsi Richard, Moina reticulata (Daday)): I. DEVELOPMENT TIME

The effects of food concentration and temperature on embryonic and postem-bryonic duration of three tropical species, Daphnia gessneri(1.5mm), Diaphanosoma sarsi(1.2mm) and Moina reticulata(0.8mm), were investigated as part of life cycle studies which included growth, body size and reproduction. These are the very first experimental studies undertaken on these species. The long-term growth experiments were performed under controlled laboratory conditions at all combinations of temperature (22"C, 27"C and 32"C) and constant food concentration (0.03, 0.05, 0.10, 0.25, 0.50 and 1.00 mgC/L) of the unicellular green alga Scenedesmus acutus.Animals were examined twice daily throughout their life cycle from the neonate to third adult instar. In all three species, temperature exerted the most powerful influence on embryonic duration but there was also a smaller food effect. In D. gessneri,postembry-onic durations remained more or less the same at food levels 0.25 mgC/L but were influenced by temperature. At food concentrations of 0.1 mgC/L or lower, postembryonic durations became increasingly prolonged, particularly at high temperatures. This threshold concentration is affected by temperature: in D. gessneri,it was 0.1 mgC/L at 22oC and 27oC but higher at 32oC (between 0.25 and 0.50 mgC/L). At the same temperature of 27oC, the food threshold level varied between species: it was higher (0.25 mgC/L) for D. sarsiand lower (0.05 mgC/L) for M. reticulatacompared with D. gessneri(0.1 mgC/L). In both embryonic and postembryonic durations there is a body size effect as the absolute durations were longest in the largest species and shortest in the smallest species In all three species, prolongation of postembryonic duration at combinations of high temperature and lowered food levels was accompanied by increased number of juvenile instars.

Avaliação da toxicidade das águas do rio Cubatão (Norte) - Joinville, SC, com base em ensaios ecotoxicológicos com Daphnia magna

Dissertação de mestrado em Ecologia

Susceptibilidade das bananeiras prata e mysore aos nematóides Radopholus similis e Helicotylenchus multicinctus

Os nematóides Radopholus similis e Helicotylenchus multicinctus são importantes inimigos da bananicultura no Brasil e alhures. Afim de verificar a susceptibilidade dos cultivares Prata e Mysore à infestação pelos mesmos, foram conduzidos dois experimentos de campo no Centro Nacional de Pesquisa de Mandioca e Fruticultura, em Cruz das Almas, BA. Os dois cultivares se revelaram tolerantes aos dois nematóides, sendo a sua reprodução menor nestas bananeiras do que em plantas do cultivar nanicão, referido como testemunha. Prate revelou-se o pior hospedeiro.

Espécies novas de Ischnoptera (Blattellidae, Blattellinae) do Estado do Mato Grosso, Brasil e considerações sobre I. similis

Três espécies novas de Ischnoptera Burmeister, 1838 do Estado do Mato Grosso, Brasil são descritas (I. miuda, I. irregulata, I. pantaneira). Ilustrações da genitália do macho e fêmea são apresentadas. Ischnoptera cristata nom. nov. para I. similis Rocha e Silva, 1984, non I. similis R. S.-Albuquerque & Lopes, 1977.

Shotgun Ecotoxicoproteomics of Daphnia pulex: Biochemical Effects of the Anticancer Drug Tamoxifen.

Among pollutants released into the environment by human activities, residues of pharmaceuticals are an increasing matter of concern because of their potential impact on ecosystems. The aim of this study was to analyze differences of protein expression resulting from acute (2 days) and middle-term (7 days) exposure of aquatic microcrustacean Daphnia pulex to the anticancer drug tamoxifen. Using a liquid chromatography-mass spectrometry shotgun approach, about 4000 proteins could be identified, providing the largest proteomics data set of D. pulex published up to now. Considering both time points and tested concentrations, 189 proteins showed a significant fold change. The identity of regulated proteins suggested a decrease in translation, an increase in protein degradation and changes in carbohydrate and lipid metabolism as the major effects of the drug. Besides these impacted processes, which reflect a general stress response of the organism, some other regulated proteins play a role in Daphnia reproduction. These latter results are in accordance with our previous observations of the impact of tamoxifen on D. pulex reproduction and illustrate the potential of ecotoxicoproteomics to unravel links between xenobiotic effects at the biochemical and organismal levels. Data are available via ProteomeXchange with identifier PXD001257.

Ecotoxicological approaches to assess the long-term effects of four anticancer drugs and metabolites on Daphnia pulex

The aquatic environment is exposed continuously and increasingly to chemical substances such as pharmaceuticals. These medical compounds are released into the environment after having being consumed and body-excreted by patients. Pharmaceutical residues are synthetic molecules that are not always removed by traditional sewage treatment processes and thus escape degradation. Among pharmaceuticals that escape sewage treatment plants (STPs), the anticancer drugs were measured in STP effluents and natural waters. In the aquatic environment, their long-term effects at low concentrations are sparsely known on non-target species. Tamoxifen is an anticancer drug that is widely prescribed worldwide for the prevention and treatment of hormone receptor-positive breast cancers. Two of its metabolites, i.e., endoxifen and 4-hydroxy- tamoxifen (4OHTam), have high pharmacological potency in vivo and such as tamoxifen, they are excreted via faeces by patients. Tamoxifen was measured in STP effluents and natural waters but, to the best of our knowledge, its metabolites concentrations in waters have never been reported. Imatinib is another and recent anticancer compound that targets specific tumour cells. This pharmaceutical is also body excreted and because of its increasing use in cancer treatment, imatinib may reach the natural water. The effects of tamoxifen and imatinib are unknown upon more than one generation of aquatic species. And the effects of 4OHTam, endoxifen have never been studied in ecotoxicology so far. The aims of this thesis were threefold. First, the sensitivity of D. pulex exposed to tamoxifen, 4OHTam, endoxifen or imatinib was assessed using ecotoxicological experiments. Ecotoxicology is the science that considers the toxic effects of natural or synthetic substances, such as pharmaceuticals, on organisms, populations, community and ecosystem. Acute and multigenerational (2-4 generations) tests were performed on daphnids considering several studied endpoints, such as immobilisation, size, reproduction, viability and intrinsic rate of natural increase. Additional prospective assays were designed to evaluate whether 1) low concentrations of tamoxifen and 4OHTam were able to induce toxic effects when used in combination, and 2) daphnids were able to recover when offspring were withdrawn from solutions carrying the pharmaceutical. Second, the stability of tamoxifen, 4OHTam and endoxifen in incubation medium was evaluated in solution exempted from daphnids. Because the nominal concentrations of tamoxifen, 4OHTam and endoxifen did not correspond to the measured, we provide a predictive method to estimate the concentrations of these chemicals during long-term ecotoxicological tests. Finally, changes in protein expressions were analysed in D. pulex exposed 2 or 7 seven days to tamoxifen using ecotoxicoproteomic experiments with a shot-gun approach inducing a peptide fractionation step. Our results show that tamoxifen, 4OHTam and endoxifen induced adverse effects in D. pulex at environmentally relevant concentrations. At very low concentrations, these molecules displayed unusual and teratogenic effects because morphological abnormalities were observed in offspring, such as thick and short antennas, curved spines, premature neonates and aborted eggs. Tamoxifen was the most toxic compound among the test chemicals, followed by 4OHTam, endoxifen and imatinib. Tamoxifen no-observed effect concentrations (NOECs) that were calculated for size, reproduction and intrinsic rate were below or in the range of the concentrations measured in natural waters, i.e., between 0.12 µg/L and 0.67 µg/L. For instance, the tamoxifen NOECs that were calculated for reproduction were between 0.67 and 0.72 µg/L, whereas the NOEC was < 0.15 µg/L when based on morphological abnormalities. The NOECs of 4OHTam were higher but still in the same order of magnitude as tamoxifen environmental concentrations, with a value of 1.48 µg/L. Endoxifen NOEC for the intrinsic rate of natural increase (r) and the reproduction were 0.4 and 4.3 µg/L, respectively. Daphnids that were withdrawn from tamoxifen and 4OHTam were not able to recover. Also, the reproduction of D. pulex was reduced when the treated animals were exposed to the combination of tamoxifen and 4OHTam while no effects were observed when these chemicals were tested individually at the same concentration. Among the anticancer drugs that were tested during this thesis, imatinib was the less toxic molecule towards D. pulex. No effects on size and reproduction were observed within two generations, except for the first whose reproduction decreased at the highest test concentration, i.e., 626 µg/L. Our results also underline the need to use measured or predicted concentrations instead of the nominal during aquatic experiments, particularly when lipophilic molecules are tested. Indeed, notable differences between nominal (i.e., theoretical) and measured concentrations were found with tamoxifen, 4OHTam and endoxifen at all test concentrations. A cost and time sustainable method was proposed to predict the test exposure levels of these chemicals during long-term experiments. This predictive method was efficient particularly for low concentrations, which corresponded to the test concentrations in multigenerational tests. In the ecotoxicoproteomic experiments a total of 3940 proteins were identified and quantified in D. pulex exposed to tamoxifen. These results are currently the largest dataset from D. pulex that is published and the results of proteomic analyses are available for the scientific community. Among these 3940 proteins, 189 were significantly different from controls. After protein annotation, we assumed that treated daphnids with tamoxifen had shifted cost-energy functions, such as reproduction, to maintain their basic metabolism necessary to survive. This metabolic cost hypothesis was supported by the presence of proteins involved in oxidative stress. Biomarkers for early detection of tamoxifen harmful effects on D. pulex were not discovered but the proteins of the vitellogenin-2 family (E9H8K5) and the ryanodine receptor (E9FTU9) are promising potential biomarkers because their expression was already modified after 2 days of treatment. In this thesis, the effects of tamoxifen, 4OHTam and endoxifen on daphnids raise questions about the potential impact of tamoxifen and 4OHTam in other aquatic ecosystems, and therefore, about metabolites in ecotoxicology. Because the NOECs were environmentally relevant, these results suggest that tamoxifen and 4OHTam may be interesting pharmaceuticals to consider in risk assessment. Our findings also emphasize the importance of performing long-term experiments and of considering multi-endpoints instead of the standard reproductive endpoint. Finally, we open the discussion about the importance to measure test exposures or not, during ecotoxicological studies. -- Les milieux aquatiques sont exposés continuellement à un nombre croissant de substances chimiques, notamment les médicaments issus de la médecine vétérinaire et humaine. Chez les patients, les substances administrées sont utilisées par le corps avant d'être éliminées par l'intermédiaire des excrétas dans le système d'eaux usées de la ville. Ces eaux rejoignent ensuite une station de traitement afin d'y éliminer les déchets. Dans le cas des molécules chimiques, il arrive que les processus de traitement d'eaux usées ne soient pas suffisamment efficaces et que ces molécules ne soient pas dégradées. Elles sont alors libérées dans le milieu aquatique avec les effluents de la station d'épuration. Une fois dans l'environnement, ces résidus de médicaments sont susceptibles d'induire des effets sur la faune et la flore aquatique, dont les conséquences à long terme et à faibles concentrations sont peu connues. Les anticancéreux sont une famille de médicaments qui peuvent échapper aux traitements des stations d'épuration et qui sont retrouvées dans le milieu aquatique naturel. Parmi ces substances, le tamoxifen est une molécule utilisée dans le monde entier pour prévenir et traiter les cancers hormonaux dépendant du sein, notamment. Une fois ingéré, le tamoxifen est transformé par le foie en métabolites dont deux d'entre eux, le 4-hydroxy-tamoxifen (4OHTam) et l'endoxifen, possèdent un affinité pour les récepteurs aux estrogènes et une efficacité sur les cellules tumorales supérieure au tamoxifen lui- même. Tout comme la molécule mère, ces métabolites sont principalement éliminés par l'intermédiaire des fèces. Le tamoxifen a déjà été mesuré dans les effluents de stations d'épuration et dans les eaux naturelles, mais aucune valeur n'a été reportée pour ses métabolites jusqu'à présent. Un autre anticancéreux, également éliminé par voie biliaire et susceptible d'atteindre l'environnement, est l'imatinib. Cette récente molécule a révolutionné le traitement et la survie des patients souffrant de leucémie myéloïde chronique et de tumeur stromales gastrointestinales. Les effets du tamoxifen et de l'imatinib sur plusieurs générations d'organismes aquatiques, tels que les microcrustacés Daphnia, sont inconnus et le 4OHTam et l'endoxifen n'ont même jamais été testés en écotoxicologie. Cette thèse s'est articulée autour de trois objectifs principaux. Premièrement, la sensibilité des D. pulex exposés au tamoxifen, 4OHTam, endoxifen et imatinib a été évaluée par l'intermédiaire de tests aigus et de tests sur deux à quatre générations. La mobilité, la taille, la reproduction, la viabilité et la croissance potentielle de la population ont été relevées au cours de ces expériences. Des tests supplémentaires, à but prospectifs, ont également été réalisés afin d'évaluer 1) la capacité de récupération des daphnies, lorsque leurs descendants ont été placés dans un milieu exempté de tamoxifen ou de 4OHTam, 2) les effets chez les daphnies exposées à une solution contenant de faibles concentration de tamoxifen et de 4OHTam mélangés. Le deuxième objectif a été d'évaluer la stabilité du tamoxifen, 4OHTam et endoxifen dilué dans le milieu des daphnies. Après analyses, les concentrations mesurées ne correspondaient pas aux concentrations nominales (c.-à-d., théoriques) et il a été nécessaire de développer une méthode efficace de prédiction des niveaux d'exposition lors de tests de longue durée réalisés avec ces trois molécules. Finalement, des changements dans l'expression des protéines chez des daphnies exposées au tamoxifen ont été investigués par l'intermédiaire d'expériences écotoxicoprotéomiques avec une approche dite de shot-gun avec une étape de fractionnement des protéines. Les résultats obtenus dans cette thèse montrent que le tamoxifen, le 4OHTam et l'endoxifen induisent des effets indésirables chez les daphnies à des niveaux d'exposition proches ou identiques aux concentrations du tamoxifen mesurées dans l'environnement, c'est-à-dire 0.12 et 0.67 µg/L de tamoxifen. Ces molécules ont induit des effets inhabituels tels que la production de : nouveau-nés anormaux, avec des antennes et des queues déformées, des prématurés et des oeufs avortés. Le tamoxifen fut la molécule la plus toxique pour les D. pulex suivie du 4OHTam, de l'endoxifen et enfin de l'imatinib. Lors des expériences sur plusieurs générations, les concentrations n'ayant statistiquement pas d'effet (c.à.d. NOEC en anglais) sur la taille, la reproduction et la croissance intrinsèque de la population étaient du même ordre de grandeur que les concentrations environnementales du tamoxifen. Par exemple, les NOECs du tamoxifen calculées pour la reproduction étaient de 0.67 et 0.72 µg/L, tandis que celle calculée sur la base des anomalies chez les nouveau-nés était < 0.15 µg/L. Les NOECs du 4OHTam se situaient entre 0.16 et 1.48 µg/L et celles de l'endoxifen pour la croissance intrinsèque de la population, ainsi que pour la reproduction, étaient de 0.4 et 4.3 µg/L, respectivement. Dans l'expérience basée sur la récupération des daphnies, la taille et la reproduction ont diminué bien que la descendance fût placée dans un milieu sans substances chimiques. Les daphnies exposées au mélange de tamoxifen et de 4OHTam ont produit moins de nouveau-nés que les contrôles, alors que ces concentrations n'ont pas induit d'effets lorsque testées individuellement. Finalement, l'imatinib n'a pas montré d'effets sur les deux générations testées. Seule la première génération exposée à la plus haute concentration (626 µg/L) a montré une diminution de la reproduction. Les résultats obtenus lors de l'évaluation de la stabilité du tamoxifen, 4OHTam et endoxifen dans le milieu des daphnies ont souligné l'importance d'utiliser des concentrations mesurées ou prédites en écotoxicologie. En effet, des différences notables entre concentrations nominales et mesurées ont été observées à toutes les concentrations et l'hypothèse d'un phénomène d'adsorption sur le verre des récipients a été posée. De ce fait, il a été nécessaire d'élaborer une méthode prédictive efficace et acceptable, en terme de temps et de coûts. Une régression polynomiale basée sur des concentrations mesurées et nominales a permis de prédire avec efficacité les faibles niveaux d'exposition utilisés lors d'expériences écotoxicologiques à long terme, sur plusieurs générations. Suite aux expériences d'écotoxicoprotéomiques, un total de 3940 protéines ont été identifiées et quantifiées chez des daphnies exposées au tamoxifen. Ce nombre est actuellement la plus large série de données publiées et mises à disposition pour la communauté scientifique. Parmi ces protéines, 189 sont significatives et possiblement reliées à des processus de reproduction et de stress. Sur cette base, nous avons émis l'hypothèse que les individus subissant un stress, lié à l'exposition au tamoxifen, ont utilisé leur énergie de base pour favoriser leur survie plutôt que la reproduction. Enfin, la détermination de bio-marqueurs exprimant des dommages précoces des daphnies exposées au tamoxifen n'a pas abouti en tant que telle, mais des protéines prometteuses, telle que la famille de viellogenin-2 (E9H8K5) et le récepteur à la ryanodine (E9FTU9), ont été exprimées après deux jours d'exposition déjà. Ces protéines pourraient faire l'objet d'investigations écotoxicoprotéomiques futures. Les résultats de cette thèse posent certaines questions quant au risque du tamoxifen, du 4OHTam et de l'endoxifen sur la faune et la flore aquatique et plus particulièrement sur les anticancéreux présents dans l'environnement. Les effets toxiques de ces molécules ont été observés à des concentrations environnementales et sur plusieurs générations. La question de considérer les métabolites, et ainsi les pro-médicaments, en écotoxicologie est soulevée, notamment parce que ces molécules peuvent être plus actives et efficaces que la molécule mère. Les expériences chroniques, sur plusieurs générations sont également à favoriser car elles offrent un meilleur reflet de la réalité environnementale que des essais aigus ou d'une génération. L'utilisation de la protéomique permet d'agrandir les connaissances sur les effets des médicaments à un niveau inférieur de l'organisation biologique et ainsi, de mieux comprendre de potentiels mécanismes d'action ou de déterminer de potentiels biomarqueurs. Finalement, il semble important de discuter de l'opportunité de mesurer les concentrations qui sont testées en écotoxicologie afin de ne pas sous-estimer le risque pour la faune et la flore aquatique.