Atrazine (ATZ) is an agricultural herbicide. The US Environmental Protection Agency has set the maximum contaminant level at 3 μg/l in potable water, though concentrations can greatly exceed this amount depending on the time of year. Epidemiological studies report associations with developmental health outcomes with potable water exposure. Studies in model organisms identify ATZ as a neurotoxicant and endocrine disrupting chemical. The zebrafish model system was used to test the hypothesis that developmental ATZ exposure has immediate health consequences as well as in the subsequent generation. It was first hypothesized that developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in larvae. In the exposed generation, targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals. The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group. These findings suggest that developmental ATZ exposure generates metabolite profiles similar to mammals leading to behavioral alterations supporting ATZ as a neurodevelopmental toxicant. In the subsequent generation (F1), it was hypothesized that parental ATZ exposure altered protein expression leading to modifications in morphology and behavior in developing progeny. Proteomic analysis identified differential expression associated with neurological development and disease and organ and organismal morphology, specifically the skeletomuscular system. Head length and the ratio of head length to total length was significantly increased in the F1 in the 0.3 and 30 ppb ATZ treated groups. Craniofacial morphology was assessed based on molecular pathway analysis and revealed decreased cartilaginous structure size, decreased surface area and distance between saccular otoliths, and a more posteriorly positioned notochord, indicating delayed ossification. The visual motor response assay showed hyperactivity in the F1 of the 30 ppb treatment group for total distance and time spent moving in the F1 of the 0.3 and 30 ppb treatment groups for all phases. Collectively, these results demonstrate persistent ATZ developmental toxicity in this multigeneration study.

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Atrazine and other pesticides may damage non-target ecosystems, such as wetlands, by destroying vegetation or by disrupting microbial communities and nutrient cycling. The addition of atrazine to a wetland microcosm will disrupt the nitrogen cycle by inhibiting nitrifying and denitrifying bacteria. The inhibition or absence of functional groups of bacteria will limit the ability of the wetland to remove nutrients and herbicides, thus increasing nitrogen levels in non-point source pollution. All tests were conducted in the laboratory using microcosms established from intact plant/sediment core subsamples collected from a natural freshwater wetland. Each microcosm received distilled water, atrazine, or acidified distilled water (pH 2) by groundwater seepage. Microcosms were dosed with 1.5 mg/L and 4.5 mg/L atrazine. Acid treatment of microcosms was done to provide a perturbation reference to the microcosm. Nitrite, nitrate, and ammonia levels were determined, as was dissolved oxygen, pH, and conductivity. The nitrifying bacteria and denitrifying bacteria were enumerated. Data were analyzed with one way analysis of variance (ANOVA) with block and treatment interaction and Tukey's Studentized Range test. Acid treatment of microcosms provided a positive control of microcosm disturbance. Acidification of the sediment disturbed the nitrogen cycle by suppressing ammonia dependent microbial processes. Atrazine (at 1.5 and 4.5 mg/L atrazine) did not significantly affect the number of nitrifying bacteria. The denitrifying or nitrate reducing bacteria were stimulated by exposure to atrazine and nitrate reduction became the dominant microbial process following exposure to atrazine. This shift toward denitrification and nitrate reduction processes caused increased levels of nitrite in the overflow waters. The continued accumulation of atrazine may cause nitrogen losses from an ecosystem which is already nitrogen limiting. Over time, predominant plant species may change, becoming primarily nitrogen fixing species. Changes in plant species would bring about changes in associated microbial populations. Excess nitrogen present in wetland runoff may cause algal blooms in receiving waters and pose health risks in contaminated drinking water. Accumulation of atrazine in wetlands may, ultimately, result in the loss of buffer zones. / Master of Science

L’atrazine, un des herbicides les plus utilisés pour contrôler le développement des plantes adventices dans les cultures, a conduit à la contamination de l’environnement. L’exposition chronique à cet herbicide a conduit à l’émergence de populations microbiennes du sol capables de dégrader l’atrazine et de l’utiliser comme une source d’azote pour leur croissance. Ces populations microbiennes sont responsables de la biodégradation accélérée (BDA) de l’atrazine, un service écosystémique contribuant à diminuer la persistance de cet herbicidedans l’environnement. L’objectif de ce travail était d’étudier les mécanismes génétiques et physiologiques responsables du fonctionnement et de l’amélioration de ce service écosystémique. Nous avons appliqué une démarche expérimentale allant des gènes codant la dégradation à des communautés microbiennes afin d’identifier les processus adaptatifs impliqués dans l’évolution de la fonction de BDA de l’atrazine.Le premier volet a consisté à évaluer l’importance de mutations accumulées dans le gène atzA dans la transformation de l’atrazine en hydroxyatrazine catalysée par AtzA. Le séquençage de gènes atzA de différents isolats bactériens dégradant l’atrazine (Pseudomonas sp. ADP WT, Pseudomonas sp. ADP Ps et différents Chelatobacter heintzii) a montré que la séquence du gène atzA était très conservée. Toutefois quatre mutations non silencieuses ont pu être identifiées (1 chez Pseudomonas sp. ADP MSE et 3 chez Chelatobacterheintzii). La modélisation de la structure de la protéine AtzA a permis de montrer que trois des mutations étaient situées dans des régions importantes (site actif, poche de liaison avec l’atrazine et liaison avec le métalFe2+. [...] Le second volet a consisté à étudier la plasticité de la voie de biodégradation de l’atrazine dans deux conditions opposées : (i) la première visait à évaluer la persistance de la capacité de dégradation en absence de pression de sélection et (ii) la seconde visait à évaluer l’évolution de la capacité de dégradation en présence d’une pression de sélection élevée. Pour conduire ces études, des manipulations d’évolution expérimentale sur Pseudomonas sp. ADP ont été menées. (i) L’exposition à l’acide cyanurique, intermédiaire métabolique de l’atrazine, a conduit à la sélection d’une population nouvellement évoluée capable de croître plus rapidement dans un milieu de culture ne contenant que l’acide cyanurique comme source d’azote. Cette population est caractérisée par une délétion d’une région de 47 kb du plasmide ADP1 contenant les gènes atzABC. Les analyses conduites ont permis de conclure que le gain de compétitivité de la population évoluée résidait dans la perte du fardeau génétique représenté par la région de 47 kb, la capacité de dégradation de l’acide cyanurique restant inchangée. (ii) L’exposition à l’atrazine a conduit à la sélection d’une populationnouvellement évoluée caractérisée par l’insertion du plasmide ADP1 en quasi-totalité sur le chromosome bactérien. [...] Le troisième volet a consisté à développer un outil permettant d’évaluer, à l’échelle d’une communauté microbienne synthétique, l’évolution du potentiel génétique dégradant. Pour ce faire quatre souches dégradantes dont une, Arthrobacter sp. TES6, isolée au cours de cette étude, ont été choisies. [...] Ces travaux montrent que la fonction de biodégradation accélérée de l’atrazine est très versatile et qu’elle est en constante évolution. Il met en évidence que le principal facteur pilotant cette évolution est le niveau d’exposition des populations dégradantes au pesticide. / Atrazine, one of the most used herbicide to control the development of weeds in crop, has led to the contamination of the environment. Repeated exposure to this herbicide resulted in the emergence of microbial populations able to degrade atrazine and to use it as a nitrogen source for its growth. These microbial populations are responsible for accelerated biodegradation of atrazine (BDA), a key ecosystemic service diminishing the persistence of this herbicide in the environment. The aim of this PhD work was to study genetic and physiological mechanisms responsible for functioning and improving of this ecosystemic service. We applied an experimental approach starting from genes to communities degrading atrazine in order to identify processes of adaptation involved in the evolution of accelerated biodegradation function.The first part of the PhD aimed at evaluating the importance of accumulation of single mutations in the atzA gene for the activity of AtzA transforming atrazine to hydroxyatrazine. Sequencing or atzA genes amplified from different atrazine-degrading isolates (Pseudomonas sp. ADP WT, Pseudomonas sp. ADP Ps and differents Chelatobacter heintzii) showed that atzA sequence was conserved. However, four non synonymous mutations were identified (1 for Pseudomonas sp. ADP Ps and 3 for Chelatobacter heintzii). Modeling of AtzA structure showed that three mutations were located in important regions (active site, interaction with atrazine and with the metal Fe2+). [...] The second part aimed at studying the plasticity of the atrazine-degrading pathway in two opposed conditions: (i) one aiming at evaluating the persistence of degrading capability in absence of selection pressure and (ii) a second one aiming at evaluating the evolution of degrading capability under high selection pressure exerted by atrazine. With these aims, experimental evolutions were carried out with Pseudomonas sp. ADP. (i) We showed that cyanuric acid exposure led to the selection of a newly-evolved population characterized by increased growing ability on culture medium containing this substance as nitrogen source. This population is characterized by the deletion of a 47 kb region containing atzABC genes from ADP1. We showed that increased fitness of newly-evolved population was due to the selective loss of the genetic burden represented by the 47 kb region, the cyanuric acid degrading ability remaining unchanged. (ii) Atrazine exposure led to the selection of population characterized by the insertion of ADP1 plasmid in the bacterial chromosome. [...] The third part aimed at developing a tool allowing monitoring the evolution of atrazine-degrading genetic potential at the scale of a synthetic microbial community. To do so four degrading strains among which, one was isolated in this study, were chosen. [...] Altogether, these results showed that the atrazine accelerated biodegradation function is highly versatile and under constant evolution. Furthermore, they highlight that the exposure to atrazine is the key parameter driving the evolution of degrading population

This study assessed the water quality of the Elm Fork Watershed with regards to the herbicide Atrazine. Atrazine is a potential environmental endocrine disruptor and carcinogen. Overall, concentrations were lower than the four-quarter drinking water average of 3 µg/Lthe Maximum Contaminant Level set by the USEPA. However, three creek stations had four-quarter average concentrations greater than 3 µg/L, and virtually all samples exceeded the 0.1 µg/L standard set in Europe [1,2]. Statistically significant differences in concentrations were detected between the 27 sampling stations and areas of high concentrations were identified. However correlations between Atrazine concentrations and land-use and precipitation were not statistically significant. Further analysis with more detailed data should be conducted before any relationships are discarded.

Les pesticides sont des molécules utilisées en grandes quantités en France et dans le monde entier pour la protection des cultures. Lorsque ces substances sont répandues une grande quantit é rejoint les sols. Dans cette thèse, le devenir de pesticides dans le sol est étudié. Pour cela, trois pesticides ont été sélectionnés : l'atrazine, la métamitrone et le fenhexamide ; et une argile de type montmorillonite a été choisie. Les simulations menées lors de cette thèse emploient le formalisme de la Théorie de la Fonctionnelle de la Densité (DFT). La première partie de cette thèse porte sur l'étude statique de l'interaction de l'atrazine avec la pyrophyllite et la montmorillonite. Dans les chapitres suivants, la Dynamique Moléculaire Car-Parrinello en DFT est utilisée. L'étude débute par les pesticides seuls, métamitrone et fenhexamide, puis leurs complexes avec un ou deux cations Na+ et Ca2+. Ensuite, l'adsorption de ces entités, en présence ou non d'eau, sur une surface de montmorillonite, est envisagée à l'aide de calculs périodiques. Dans le second chapitre de ce travail, des résultats issus d'optimisations de géométries en phase gazeuse sont présentés. La troisième partie développe les résultats obtenus pour l'adsorption de la métamitrone et du fenhexamide sur le modèle d'argile choisi. Dans le dernier chapitre, certaines des structures les plus stables de chaque pesticide adsorbé sur la surface ont été solvatées et une étude de la désorption du fenhexamide de la surface de montmorillonite a été réalisée à l'aide de la méthode Umbrella Sampling. Ce travail a permis de quantifier théoriquement la barrière d'énergie libre à franchir pour désorber le fenhexamide de la montmorillonite hydratée. / Pesticides are widely employed molecules for crop protection in France and all over the world. When these substances are spread an important quantity reaches soils. In the present thesis, pesticide fate is under study. For this purpose, three pesticides have been selected: atrazine, metamitron and fenhexamid ; and the most abundant smectite clay type, montmorillonite, has been chosen. The computational simulations conducted in this thesis use the Density Functional Theory (DFT) formalism. The first part of this thesis treats a static study of atrazine interaction with pyrophyllite and montmorillonite. In the following chapters, Car-Parrinello Molecular Dynamics through DFT is used. This study begins with isolated pesticides, metamitron and fenhexamid, then their complexes with one or two Na+ and Ca2+. Finally, the adsorption of these entities, in the presence of water or not, onto a montmorillonite surface, is considered using periodic calculations. Within the second chapter of the present work, results from geometry optimisations in gas phase are presented. The third part develops the obtained results on the adsorption of metamitron and fenhexamid over the chosen clay model. In the last chapter of this thesis, some of the most stable structures of each adsorbed pesticide onto the surface have been solvated and a study of fenhexamid desorption from the montmorillonite surface using Umbrella Sampling has been done. This work allowed to quantify theoretically the free energy barrier to overcome to desorb fenhexamid from the hydrated montmorillonite.