Phosphorothioate insecticides such as parathion (0,0, diethyl O-pnitrophenyl phosphorothioate) must undergo metabolic activation to form oxygen analogs in order to exert their toxicity. The specific isoforms of cytochrome P450 involved in this oxidative desulphuration reaction were examined in rat liver microsomes and a panel of sixteen human liver microsomes. In the rat, parathion (20μM and 200μM) was activated to paraoxon with an apparent Km of 10.4±0.25μM (n=3), the metabolic rates were 241±17 and 256±18 pmol/min/mg protein, respectively. p-Nitrophenol was also formed, at 235±15 and 220±23 pmol/min/mg protein. Human liver microsomes activated parathion (20μM and 200μM) with an apparent Km of 9μM-16μM (n=3), the metabolic rates were 23.3-199.3 and 18.7-310.3 pmol/min/mg protein (n=16). p-Nitrophenol was also formed, at 321.1- 769.2 and 406.2-778.3 pmol/min/mg protein. The activation of parathion (200μM) by human liver microsomes was positively correlated with nifedipine oxidation, indicating the involvement of CYP3A. Correlations were not significant with ethoxyresorufin-0-dealkylation, pentoxyresorufin-0-dealkylation, pnitrophenol hydroxylation, paraoxon hydrolysis or phenylvalerate hydrolysis. Paraoxon formation from parathion by human liver microsomes was markedly inhibited by the CYP3A inhibitors ketoconazole, quercetin and naringenin (apparent Ki=21μM). Metyrapone and a-naphthflavone had some inhibitory effect. The inhibitors were generally less effective towards parathion metabolism by rat liver microsomes. Experiments with EDTA indicated that A-esterase was not functionally important at low levels of paraoxon. Human P450s 3A4 and 3A5 expressed microsomes were the most efficient at biotransforming parathion to paraoxon, although P450s 1A1,2B6 and 2C8 also catalysed the reaction. The present study has shown marked interindividual variation in the metabolism of parathion, which may influence toxicity following exposure to this or other phosphorothioates. Co-administration of inhibitors or inducers of the enzymes involved may affect the fate of parathion and thus enhance or reduce its toxicity.

Neste trabalho, foi realizada uma triagem com os fungos marinhos Aspergillus sydowii CBMAI 934, Aspergillus sydowii CBMAI 935, Aspergillus sydowii 1241, Penicillium decaturense CBMAI 1234, Penicillium raistrickii CBMAI 931, Penicillium raistrickii CBMAI 1235 e Trichoderma sp. CBMAI 932 com o objetivo de avaliar o potencial enzimático destes micro-organismos frente à biodegradação dos pesticidas organofosforados clorpirifós, metil paration e profenofós. Os fungos selecionados para as reações em meio líquido de malte 2%, que melhor adaptaram-se na presença do pesticida clorpirifós, foram as cepas de A. sydowii CBMAI 935 e Trichoderma sp. CBMAI 932, na presença de metil paration foram A. sydowii CBMAI 935 e P. decaturense CBMAI 1234 e na presença de profenofós foram os fungos A. sydowii CBMAI 935 e P. raistrickii CBMAI 931. Foram realizadas curvas analíticas com o objetivo de estimar a extensão da biodegradação dos pesticidas clorpirifós, metil paration, profenofós, e seus respectivos produtos de hidrólise, os derivados fenólicos 3,5,6-tricloro-2-piridinol, 4-nitrofenol e 4-bromo-2-clorofenol, respectivamente. As reações de biodegradação em meio líquido de malte 2% foram avaliadas com 10, 20 e 30 dias de reação com concentração inicial dos pesticidas organofosforados de 50 ppm. O pesticida, profenofós, apresentou uma biodegradação completa com o fungo P.raistrickii CBMAI 931 com 30 dias de reação, e 70% com o fungo A. sydowii CBMAI 935, para o mesmo período. Os fungos também apresentaram potencial para a degradação (ou conjugação) do metabólito, 4-bromo-2-clorofenol. Na presença dos fungos A. sydowii CBMAI 935 e Trichoderma sp. CBMAI 932, clorpirifós apresentou a menor degradação entre os pesticidas avaliados, com uma taxa de degradação de 63 e 72%, respectivamente, em 30 dias de reação. Os fungos selecionados não foram capazes de promover uma degradação apreciável do 3,5,6-tricloro-2-piridinol. Na presença dos fungos A. sydowii CBMAI 935 e P. decaturense CBMAI 1234, o metil paration apresentou uma degradação de 100% em 20 dias de reação. O 4-nitrofenol apresentou degradação (ou conjugação) de aproximadamente 50 e 40% na presença dos fungos A. sydowii CBMAI 935 e P. decaturense CBMAI 1234, respectivamente. Finalmente, os estudos apresentados comprovam a eficiência dos fungos de ambiente marinhos na biodegradação de pesticidas organofosforados. / In this work was performed a screening with the marine fungi Aspergillus sydowii CBMAI 934, Aspergillus sydowii CBMAI 935, Aspergillus sydowii 1241, Penicillium decaturense CBMAI 1234, Penicillium raistrickii CBMAI 931, Penicillium raistrickii CBMAI 1235 e Trichoderma sp. CBMAI 932 to evaluate the enzymatic potential of these microorganisms in the presence of organophosphate pesticides chlorpyrifos, methyl parathion and profenofos. The selected fungi for the reactions in liquid medium of malt 2%, which demonstrated better adaptation in presence of the pesticide chlorpyrifos, were the strains of A.sydowii CBMAI 935 and Trichoderma sp. CBMAI 932, in the presence of methyl parathion were A.sydowii CBMAI 935 and P. decaturense CBMAI 1234 and, in presence of profenofos were the fungi A. sydowii CBMAI 935 and P. raistrickii CBMAI 931. Analytical curves were performed in order to estimate the extent of biodegradation of the pesticide chlorpyrifos, methyl parathion, profenofos, and their hydrolysis products, 3,5,6-trichloro-2-pyridinol, 4-nitrophenol, 4-bromo 2-chlorophenol, respectively. Biodegradation reactions in liquid medium of malt 2% were evaluated with 10, 20 and 30 days with an initial concentration of 50 ppm of organophosphate pesticides. Profenofos, presented a complete biodegradation (100%) with the fungus P.raistrickii CBMAI 931, in 30 days of reaction, and 70% of biodegradation in the presence of the fungus A. sydowii CBMAI 935, at the same period. The fungi also showed a potential for degradation (or conjugation) of 4-bromo-2-chlorophenol. In the presence of the fungus A. sydowii CBMAI 935 and Trichoderma sp. CBMAI 932, chlorpyrifos exhibited the lowest degradation among the evaluated pesticides, presenting a degradation rate of 63 and 72%, respectively, at 30 days of reaction. The selected fungi were not capable of promoting an appreciable degradation of 3,5,6-trichloro-2-pyridinol. In the presence of A. sydowii CBMAI 935 and P. decaturense CBMAI 1234, methyl parathion showed a degradation of 100%, at 20 days of reaction. The 4-nitrophenol showed a degradation (or conjugation) of approximately 50 and 40% in the presence of the fungus A. sydowii CBMAI 935 and P. decaturense CBMAI 1234, respectively. Finally, the presented studies demonstrated the efficiency of the marine fungi in biodegradation of organophosphate pesticides.

Neste trabalho, foi realizada uma triagem com os fungos marinhos Aspergillus sydowii CBMAI 934, Aspergillus sydowii CBMAI 935, Aspergillus sydowii 1241, Penicillium decaturense CBMAI 1234, Penicillium raistrickii CBMAI 931, Penicillium raistrickii CBMAI 1235 e Trichoderma sp. CBMAI 932 com o objetivo de avaliar o potencial enzimático destes micro-organismos frente à biodegradação dos pesticidas organofosforados clorpirifós, metil paration e profenofós. Os fungos selecionados para as reações em meio líquido de malte 2%, que melhor adaptaram-se na presença do pesticida clorpirifós, foram as cepas de A. sydowii CBMAI 935 e Trichoderma sp. CBMAI 932, na presença de metil paration foram A. sydowii CBMAI 935 e P. decaturense CBMAI 1234 e na presença de profenofós foram os fungos A. sydowii CBMAI 935 e P. raistrickii CBMAI 931. Foram realizadas curvas analíticas com o objetivo de estimar a extensão da biodegradação dos pesticidas clorpirifós, metil paration, profenofós, e seus respectivos produtos de hidrólise, os derivados fenólicos 3,5,6-tricloro-2-piridinol, 4-nitrofenol e 4-bromo-2-clorofenol, respectivamente. As reações de biodegradação em meio líquido de malte 2% foram avaliadas com 10, 20 e 30 dias de reação com concentração inicial dos pesticidas organofosforados de 50 ppm. O pesticida, profenofós, apresentou uma biodegradação completa com o fungo P.raistrickii CBMAI 931 com 30 dias de reação, e 70% com o fungo A. sydowii CBMAI 935, para o mesmo período. Os fungos também apresentaram potencial para a degradação (ou conjugação) do metabólito, 4-bromo-2-clorofenol. Na presença dos fungos A. sydowii CBMAI 935 e Trichoderma sp. CBMAI 932, clorpirifós apresentou a menor degradação entre os pesticidas avaliados, com uma taxa de degradação de 63 e 72%, respectivamente, em 30 dias de reação. Os fungos selecionados não foram capazes de promover uma degradação apreciável do 3,5,6-tricloro-2-piridinol. Na presença dos fungos A. sydowii CBMAI 935 e P. decaturense CBMAI 1234, o metil paration apresentou uma degradação de 100% em 20 dias de reação. O 4-nitrofenol apresentou degradação (ou conjugação) de aproximadamente 50 e 40% na presença dos fungos A. sydowii CBMAI 935 e P. decaturense CBMAI 1234, respectivamente. Finalmente, os estudos apresentados comprovam a eficiência dos fungos de ambiente marinhos na biodegradação de pesticidas organofosforados. / In this work was performed a screening with the marine fungi Aspergillus sydowii CBMAI 934, Aspergillus sydowii CBMAI 935, Aspergillus sydowii 1241, Penicillium decaturense CBMAI 1234, Penicillium raistrickii CBMAI 931, Penicillium raistrickii CBMAI 1235 e Trichoderma sp. CBMAI 932 to evaluate the enzymatic potential of these microorganisms in the presence of organophosphate pesticides chlorpyrifos, methyl parathion and profenofos. The selected fungi for the reactions in liquid medium of malt 2%, which demonstrated better adaptation in presence of the pesticide chlorpyrifos, were the strains of A.sydowii CBMAI 935 and Trichoderma sp. CBMAI 932, in the presence of methyl parathion were A.sydowii CBMAI 935 and P. decaturense CBMAI 1234 and, in presence of profenofos were the fungi A. sydowii CBMAI 935 and P. raistrickii CBMAI 931. Analytical curves were performed in order to estimate the extent of biodegradation of the pesticide chlorpyrifos, methyl parathion, profenofos, and their hydrolysis products, 3,5,6-trichloro-2-pyridinol, 4-nitrophenol, 4-bromo 2-chlorophenol, respectively. Biodegradation reactions in liquid medium of malt 2% were evaluated with 10, 20 and 30 days with an initial concentration of 50 ppm of organophosphate pesticides. Profenofos, presented a complete biodegradation (100%) with the fungus P.raistrickii CBMAI 931, in 30 days of reaction, and 70% of biodegradation in the presence of the fungus A. sydowii CBMAI 935, at the same period. The fungi also showed a potential for degradation (or conjugation) of 4-bromo-2-chlorophenol. In the presence of the fungus A. sydowii CBMAI 935 and Trichoderma sp. CBMAI 932, chlorpyrifos exhibited the lowest degradation among the evaluated pesticides, presenting a degradation rate of 63 and 72%, respectively, at 30 days of reaction. The selected fungi were not capable of promoting an appreciable degradation of 3,5,6-trichloro-2-pyridinol. In the presence of A. sydowii CBMAI 935 and P. decaturense CBMAI 1234, methyl parathion showed a degradation of 100%, at 20 days of reaction. The 4-nitrophenol showed a degradation (or conjugation) of approximately 50 and 40% in the presence of the fungus A. sydowii CBMAI 935 and P. decaturense CBMAI 1234, respectively. Finally, the presented studies demonstrated the efficiency of the marine fungi in biodegradation of organophosphate pesticides.

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Made available in DSpace on 2016-08-19T12:56:28Z (GMT). No. of bitstreams: 1 Dissertacao_FernandaGabrielleSoaresSilva.pdf: 1148440 bytes, checksum: 5fda93ea5a22fe5304a59aa6f8b2f3b0 (MD5) Previous issue date: 2016-06-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The quality control of soil and groundwater has become an increasing need because human activities have led to contamination of ecosystems and affected human health. Agriculture, for instance, has left ecosystems waste organophosphate insecticides (OFs), highly toxic. In order to make an analysis of the impacts of these compounds in agricultural areas, it is required to have reference values taken in undisturbed areas, presenting the same type of soil and groundwater. In this regard, the ideal is that these compounds are not present in these ecosystems. In this paper, an electrochemical biosensor based on the acetylcholinesterase enzyme, immobilized by crosslinking with glutaraldehyde in the presence of estuarine macroalgae, was used to analyze levels of OFs pesticides in soil and groundwater samples collected in areas without human interference, throughout the state of Maranhão, Brazil. Biosensors applied presented, after optimization, sensitivity with detection limit between 0,054 and 1,44 μg/L, reproducibility with variation coefficient, CVs, internal measure between 8.3 and 9% and stability, showing minor variation in responses after being stored for 3 months), which made them suitable to be used in the organophosphates monitoring in the environmental matrices. Were also analyzed some physico-chemical parameters of quality of waters and soils, it has been verified that these are within the standards of the Brazilian legislation. The monitoring of the presence of levels, using the biosensor described herein, enabled to discard contamination hypothesis by these pollutants, nevertheless it is known that such compounds can have high mobility in environmental compartments. / O controle da qualidade de solos e águas subterrâneas tem-se tornado uma necessidade cada vez maior, pois as atividades humanas têm levado à contaminação de ecossistemas e afetado a saúde humana. A agricultura, por exemplo, tem deixado nos ecossistemas resíduos de inseticidas organofosforados (OFs), altamente tóxicos. Para se fazer uma análise dos impactos provocados por esses compostos em áreas agrícolas, é necessário que se tenha valores de referência de qualidade tomados em áreas não antropizadas, que apresentem mesma tipologia de solos e águas subterrâneas. Nesse particular, o ideal é que estes compostos não estejam presentes nesses ecossistemas. No presente trabalho, um biossensor eletroquímico à base da enzima acetilcolinesterase, imobilizada por ligação cruzada com glutaraldeído em presença de uma macroalga estuarina, foi utilizado para analisar pesticidas OFs em amostras de solo e de águas subterrâneas coletadas em áreas sem interferência humana, em todo o Estado do Maranhão, Brasil. Os biossensores empregados apresentaram, após otimização, sensibilidade com limite de detecção entre 0,054 e 1,44 μg/L, reprodutibilidade com coeficientes de variação, CVs, intramedidas entre 8,3 e 9% e estabilidade, apresentando mínimas variações nas respostas após armazenados por 3 meses, o que os tornou adequados para serem utilizados no monitoramento de organofosforados nas matrizes ambientais. Também foram analisados alguns parâmetros físico-químicos de qualidade de águas e solos, tendo sido verificado que estes encontram-se dentro dos padrões da legislação brasileira. A monitoração da presença de OFs, empregando o biossensor aqui descrito, possibilitou descartar a hipótese de contaminação por esses poluentes, não obstante se saiba que tais compostos podem ter elevada mobilidade nos compartimentos ambientais.

[ES] La presente Tesis Doctoral tiene dos objetivos claramente diferenciados, siendo el primero de ellos la realización de un estudio de optimización de la síntesis de nanoestructuras de óxido de wolframio (WO3) mediante el uso de un diseño de experimentos, mientras que el segundo de ellos es el uso de estas nanoestructuras en la degradación fotoelectrocatalítica de 4 pesticidas organofosforados de diferentes subfamilias (diazinon, fosmet, clorfenvinfos y fenamifos). El uso del óxido de wolframio como fotocatalizador en el proceso fotoelectrocatalítico (FEC) despierta un gran interés, ya que se trata de un semiconductor con gran fotoestabilidad en electrolitos acuosos ácidos, excelente conductividad eléctrica, tiene la capacidad de absorber la parte azul del espectro visible además de la luz ultravioleta, y el borde superior de la banda de valencia es mayor que el potencial de oxidación de H2O/O2. Todo ello hace que el WO3 sea capaz de fotooxidar eficazmente una amplia gama de compuestos orgánicos. Las nanoestructuras estudiadas en la Tesis Doctoral se sintetizaron mediante anodizado electroquímico, ya que se trata de una técnica sencilla que permite un control de sus parámetros de manera fácil y efectiva, permitiendo obtener las nanoestructuras directamente sobre el propio sustrato metálico. Además, la necesidad de controlar y eliminar los contaminantes emergentes en el medio ambiente se ha vuelto cada vez más crucial durante las últimas décadas. Así, en esta Tesis se han degradado 4 pesticidas tóxicos y persistentes en el medioambiente mediante la técnica de fotoelectrocatálisis (FEC) utilizando las nanoestructuras de WO3. En esta técnica, los fenómenos electrolíticos y fotocatalíticos actúan juntos para mineralizar el contaminante orgánico. Además, la FEC está atrayendo la atención de los investigadores por su capacidad para degradar contaminantes orgánicos y transformarlos en compuestos inocuos con condiciones de trabajo no extremas. Por tanto, en el diseño de experimentos realizado en la Tesis Doctoral se modificaron 3 variables con tres niveles cada una, por tanto se escogió un diseño 33. Las variables que se modificaron fueron el electrolito utilizado durante el anodizado, la temperatura y atmosfera en el proceso de post-anodizado (tratamiento térmico), obteniendo de esta manera nanoestructuras con diferentes propiedades tanto estructurales como fotoelectroquímicas. Los resultados obtenidos mostraron que las nanoestructuras que presentan mejores propiedades morfológicas y fotoelectroquímicas, y con una estructura cristalina más adecuada fueron las obtenidas con el ácido metanosulfónico (CH4O3S) como electrolito y calentadas en el proceso de post-anodizado a 600 ¿C y en atmósfera de aire. Con estas nanoestructuras optimizadas, se realizó el proceso de degradación de los 4 pesticidas seleccionados mediante fotoelectrocatálisis. En este proceso, se partió de una concentración inicial de 20 ppm en todos los pesticidas, haciéndose un seguimiento del pesticida mediante UV-Visible y cromatografía líquida de ultra alto rendimiento acoplada a la espectrometría de masas (UHPLC-MS/Q-TOF). Tras 24 horas de ensayo se consiguió degradar el diazinón hasta 2 ppm (consiguiendo un 90% de degradación), el clorfenvinfos se degradó hasta 1 ppm (consiguiendo un 95% de degradación) y el fosmet y fenamifos se degradaron al 100%. Para cada uno de los pesticidas se ha propuesto una ruta de degradación según los compuestos intermedios identificados mediante el UHPLC-MS/Q-TOF, dando como resultado final moléculas más pequeñas y más inocuas para los seres humanos y para el medioambiente. / [CA] La present tesi doctoral té dos objectius clarament diferenciats, sent el primer la realització d'un estudi d'optimització de la síntesi de nanoestructures d'òxid de wolframi (WO3) mitjançant l'ús d'un disseny d'experiments, mentre que el segon és l'ús d'aquestes nanoestructures en la degradació fotoelectrocatalítica de quatre pesticides organofosforats de diferents subfamílies (diazinon, fosmet, clorfenvinfòs i fenamifòs). L'ús de l'òxid de wolframi com a fotocatalitzador en el procés fotoelectrocatalític (FEC) desperta un gran interès, ja que es tracta d'un semiconductor amb gran fotoestabilitat en electròlits aquosos àcids; amb una excel·lent conductivitat elèctrica; té la capacitat d'absorbir la part blava de l'espectre visible, a més de la llum ultraviolada, i la vora superior de la banda de valència és major que el potencial d'oxidació d'H2O/O2. Tot això fa que el WO3 siga capaç de fotooxidar eficaçment una àmplia gamma de compostos orgànics. Les nanoestructures estudiades en la tesi doctoral es van sintetitzar mitjançant anodització electroquímica, ja que es tracta d'una tècnica senzilla que permet un control dels seus paràmetres de manera fàcil i efectiva, i permet obtenir les nanoestructures directament sobre el mateix substrat metàl·lic. A més, la necessitat de controlar i eliminar els contaminants emergents en el medi ambient s'ha tornat cada vegada més crucial durant les últimes dècades. Així, en aquesta tesi s'han degradat quatre pesticides tòxics i persistents en el medi ambient mitjançant la tècnica de la fotoelectrocatàlisi (FEC) utilitzant les nanoestructures de WO3. En aquesta tècnica, els fenòmens electrolítics i fotocatalítics actuen junts per a mineralitzar el contaminant orgànic. La FEC està atraient l'atenció del personal investigador per la seua capacitat per a degradar contaminants orgànics i transformar-los en compostos innocus amb condicions de treball no extremes. Per tant, en el disseny d'experiments realitzat en la tesi doctoral es van modificar tres variables amb tres nivells cadascuna, per tant, es va triar un disseny 33. Les variables que es van modificar van ser: l'electròlit utilitzat durant l'anodització, la temperatura i l'atmosfera en el procés de postanodització (tractament tèrmic), i es van obtenir d'aquesta manera nanoestructures amb diferents propietats, tant estructurals com fotoelectroquímiques. Els resultats obtinguts van mostrar que les nanoestructures que presenten millors propietats morfològiques i fotoelectroquímiques, i amb una estructura cristal·lina més adequada, van ser les obtingudes amb l'àcid metanosulfònic (CH4O3S) com a electròlit, i calfades en el procés de postanodització a 600 ¿C i en atmosfera d'aire. Amb aquestes nanoestructures optimitzades, es va realitzar el procés de degradació dels quatre pesticides seleccionats mitjançant fotoelectrocatàlisi. En aquest procés, es va partir d'una concentració inicial de 20 ppm en tots els pesticides, i es va fer un seguiment del pesticida mitjançant UV visible i cromatografia líquida d'ultraalt rendiment acoblada a l'espectrometria de masses (UHPLC-MS/Q-TOF). Després de 24 hores d'assaig, es va aconseguir degradar el diazinon fins a 2 ppm (es va assolir un 90% de degradació), el clorfenvinfòs es va degradar fins a 1 ppm (es va assolir un 95% de degradació) i el fosmet i el fenamifòs es van degradar al 100%. Per a cadascun dels pesticides s'ha proposat una ruta de degradació segons els compostos intermedis identificats mitjançant l'UHPLC-MS/Q-TOF, que dona com a resultat final molècules més xicotetes i més innòcues per als éssers humans i per al medi ambient. / [EN] This Doctoral Thesis has two clearly differentiated objectives. The first objective is to carry out an optimization study of the synthesis of tungsten oxide nanostructures (WO3) using a design of experiments. The second objective is to use of these nanostructures in the photoelectrocatalytic degradation of 4 organophosphate pesticides of different subfamilies (diazinon, phosmet, chlorfenvinphos and fenamiphos). The use of tungsten oxide as a photocatalyst in the photoelectrocatalytic (PEC) process arouses great interest, since it is a semiconductor with great photostability in acidic aqueous electrolytes, excellent electrical conductivity, it has the ability to absorb the blue part of the visible spectrum in addition to ultraviolet light, and the upper edge of the valence band is greater than the oxidation potential of H2O / O2. All this makes WO3 capable of efficiently photo-oxidizing a wide range of organic compounds. The nanostructures studied in the Doctoral Thesis were synthesized using electrochemical anodization, since it is a simple technique that permits the control their parameters easily and effectively, allowing the nanostructures to be obtained directly on the metal substrate itself. Furthermore, the need to control and eliminate emerging pollutants in the environment has become increasingly crucial over the past decades. Thus, in this Thesis, 4 toxic and persistent pesticides in the environment have been degraded by the photoelectrocatalysis (PEC) technique using the WO3 nanostructures. In this technique, the electrolytic and photocatalytic phenomena act together to mineralize the organic contaminant. PEC is attracting the attention of researchers for its ability to degrade organic pollutants and transform them into harmless compounds under non-extreme working conditions. Therefore, in the design of experiments carried out in the Doctoral Thesis, 3 variables were modified with three levels each one, therefore a 33 design was chosen. The variables that were modified were the electrolyte used during the anodization, the temperature and the atmosphere in the post-anodization process (annealing treatment), thus obtaining nanostructures with different structural and photoelectrochemical properties. The results obtained showed that the nanostructures with the best morphological and photoelectrochemical properties, and with adequate crystalline structure werethose obtained with methanesulfonic acid (CH4O3S) as electrolyte and annealed in the post-anodization process at 600 ¿C and in an air atmosphere. With these optimized nanostructures, the degradation process of the 4 selected pesticides was carried out by photoelectrocatalysis. This process started from an initial concentration of 20 ppm in all pesticides, using UV-Visible and Ultra-High Performance Liquid Chromatography coupled to Mass Spectrophotometry (UHPLCMS / Q-TOF) to monitoring the process. After 24 hours of experiment, the diazinon was degraded to 2 ppm (achieving 90% degradation), chlorfenvinphos was degraded to 1 ppm (achieving 95% degradation) and phosmet and fenamiphos were 100% degraded. For each of the pesticides, a degradation route has been proposed according to the intermediate compounds identified by UHPLC-MS/Q-TOF, resulting in smaller and more innocuous molecules for humans and the environment. / Agradezco a la Generalitat Valenciana y al Fondo Social Europeo por la ayuda predoctoral recibida para la realización de la presente Tesis Doctoral (ACIF 159- 2018) así como para la realización de una estancia predoctoral en la Universidad de Lisboa. También quiero agradecer al Ministerio de Economía, Industria y Competitividad, por la concesión de los proyectos CTQ2016-79203-R (2016) y PID2019-105844RB-I00 (2019) en los cuales he podido participar durante la Tesis Doctoral. / Roselló Márquez, G. (2021). Eliminación de pesticidas organofosforados mediante fotoelectrocatálisis con fotoánodos de WO3 [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/174712

Thesis (PhD (Botany and Zoology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The use of organophosphate pesticides is still an integral part of commercial farming activities and these substances have been implicated as a major source of environmental contamination in South Africa. Evidence exists that many non target animals in and around agricultural areas are at risk of being affected due to the mobile nature of pesticides and the intermittent nature of pesticide application. The extent to which non-target animals are affected by exposure to two organophosphates (azinphos-methyl and chlorpyrifos) was investigated through monitoring selected biomarker responses and life cycle effects under laboratory conditions in two selected test species. A representative species from both the aquatic and terrestrial environment was used as these two compartments of the environment are inevitably linked due to the mobility of pesticides from the area of application to surrounding areas. The earthworm Eisenia fetida was used as test organism in the terrestrial environment while the fish Oreochromis mossambicus served as representative of the aquatic environment. Juvenile life stages of both species were subjected to standard acute toxicity tests which showed that for both species, juvenile life stages were more sensitive to both pesticides than adults. It was also illustrated that azinphos-methyl is more toxic than chlorpyrifos to both species. Both test species were also subjected to an intermittent exposure regime in order to assess the effects of repeated pesticide application on biomarker, life-cycle and behaviour responses. The results indicated that for similar exposure regimes, azinphos-methyl was more toxic to E. fetida than chlorpyrifos and detrimentally affected all endpoints investigated. The present study suggests that exposure concentration may have a more pronounced effect in inducing a toxic response than exposure interval, irrespective of the pesticide used. In addition to this, E. fetida was unable to avoid the presence of these pesticides in soil, even at concentrations as high as 50% of the LC50 value, indicating that the presence of pesticides in the soil pose a realistic threat to earthworms and other soil dwelling organisms. Biomarker responses, morphological effects and feeding behaviour was assessed for O. mossambicus and similar to the terrestrial toxicity experiments, there was evidence to suggest that in the case of an intermittent exposure scenario, azinphos-methyl was more hazardous than chlorpyrifos to this species. For the majority of endpoints that were investigated, it appeared that exposure interval played a more important role in inducing an effect than exposure concentration. At a shorter exposure interval, the majority of endpoints showed no difference between higher and lower exposure concentrations, while at a longer exposure interval the effects of exposure concentration became evident. In addition, feeding behaviour was affected by pesticide exposure in a dose-dependent manner. The present study yielded important results that improve the understanding of biological impacts of pesticide pollution on the environment. This can aid in optimising farming practices such as pesticide application not only in terms of eradicating the pest organisms, but also in terms of mitigating the environmental effects associated with large-scale pesticide use, thereby ensuring sustained biodiversity in these areas. / AFRIKAANSE OPSOMMING: Die gebruik van organofosfaat plaagdoders is ‘n integrale deel van kommersiële landbou aktiwiteite maar hierdie middels is ook ‘n prominente bron van omgewingsbesoedeling in Suid-Afrika. Daar is bewys dat verskeie nie-teiken diere in en om landbouareas geaffekteer word weens die nie-statiese aard van plaagdoders in die omgewing, sowel as die herhalende aard van plaagdodertoediening. Die graad waartoe nie-teiken diere geaffekteer word deur die plaagdoders azinphos-metiel en chlorpyrifos is ondersoek deur die monitering van verskeie biomerkerresponse en lewenssiklus-effekte in geselekteerde toetsspesies binne ‘n beheerde laboratoriumomgewing. ‘n Verteenwoordigende spesie van beide die akwatiese en die terrestriële omgewing is gebruik aangesien hierdie twee dele van die omgewing onlosmaaklik verbind is weens die beweging van plaagdoders vanaf die area van toediening na omringende areas. Die erdwurm Eisenia fetida is gekies as toetsorganisme vir die terrestriële omgewing en die varswatervis Oreochromis mossambicus het gedien as verteenwoordigende spesie vir die akwatiese omgewing. Onvolwasse diere van beide spesies is onderwerp aan standaard akute toksisiteitstoetse en daar is gevind dat, vir beide spesies, onvolwasse diere meer sensitief vir die betrokke plaagdoders is as volwasse diere. Dit is ook gevind dat azinphosmetiel giftiger is as chlorpyrifos vir beide spesies. Beide toetsspesies is ook onderwerp aan ‘n chroniese blootstellingsregime om die effek van herhaalde plaagdodertoediening op biomerker-, lewenssiklus- en gedragsresponse te ondersoek. Die resultate van die herhaalde blootstelling het aangedui dat vir soortgelyke blootstellingsregimes, azinphos-metiel giftiger is as chlorpyrifos vir E. fetida en dat beide middels alle eindpunte wat ondersoek is, nadelig affekteer. Die huidige studie toon ook bewyse dat blootstellingskonsentrasie ‘n meer prominente effek as blootstellingsinterval kan hê in die teweegbringing van ‘n toksiese respons. Verder was E. fetida nie in staat om die teenwoordigheid van die plaagdoders in grond te vermy nie, self nie by konsentrasies so hoog as 50% van die LC50 waarde nie. Laasgenoemde resultaat dui dus aan dat die aanwesigheid van plaagdoders in die grondomgewing ‘n realisitese bedreiging inhou vir erdwurms en ander grondorganismes. Soortgelyk aan die terrestriële toksisiteitseksperimente, was daar getuienis vir die verhoogde toksisiteit van azinphos-metiel relatief tot chlorpyrifos vir O. mossambicus. Dit blyk dat blootstellingsinterval ‘n meer prominente rol as blootstellingskonsentrasie speel in die teweegbringing van effekte vir die meerderheid van die eindpunte wat ondersoek is. In die geval van ‘n korter blootstellingsinterval het die meerderheid van eindpunte wat ondersoek is geen verskille getoon tussen ‘n hoër en ‘n laer konsentrasie nie, terwyl met ‘n langer blootstellingsinterval daar ‘n aanduiding was dat blootstellingskonsentrasie ‘n meer prominente rol gespeel het. Verder is gevind dat voedingsgedrag in O. mossambicus geaffekteer is op ‘n konsentrasie verwante manier. Die huidige studie toon resultate wat ‘n belangrike bydrae kan lewer tot die begrip van die biologiese impakte van organofosfaat plaagdoders op die omgewing. Die resultate kan gebruik word vir die optimisering van boerderypraktyke soos plaagdodertoediening, sodat laasgenoemde effektief is vir die beheer van pes-organismes, maar ook die impakte van grootskaalse plaagdodertoediening kan minimaliseer en sodoende die biodiversiteit binne hierdie areas sal beskerm.

Contexte : L’organisme en développement est très sensible à son environnement. Plusieurs études épidémiologiques ont suggéré la toxicité développementale d’une dizaine de polluants suite à des expositions prénatales. L’objectif général de cette thèse est d’évaluer les conséquences des expositions à des polluants chimiques (consommation de produits de la mer comme vecteur de contamination, solvants organiques et insecticides organophosphorés) pendant la grossesse sur le développement de l’enfant à 2 ans en se focalisant sur le développement neuro-comportemental, le développement respiratoire et immunitaire et le développement staturo-pondéral. Matériel et méthodes : Ce travail s’est appuyé sur la cohorte mère-enfant PELAGIE, mise en place en Bretagne entre 2002 et 2006. Près de 3500 femmes ont été incluses en début de grossesse et environ 1500 couples mère-enfant ont été suivis à 2 ans. Les expositions ont été évaluées en début de grossesse à partir de questionnaires, de matrices emplois-expositions ou de dosage de biomarqueurs d’exposition. Les indicateurs de santé ont été mesurés lors du suivi à 2 ans par questionnaire permettant de recueillir des éléments sur le comportement, les manifestations respiratoires à type de sifflement, les maladies allergiques et la croissance entre la naissance et 2 ans. Résultats : L’exposition professionnelle prénatale aux solvants organiques était associée à des déficits de l’attention/hyperactivité et des niveaux d’agressivité plus importants chez l’enfant à 2 ans. Cette exposition n’était pas associée avec les manifestations respiratoires et allergiques. Nous avons aussi montré que la consommation maternelle de coquillages et crustacés pendant la grossesse était associée à une augmentation du risque d’allergie alimentaire chez l’enfant. Enfin, l’exposition prénatale aux insecticides organophosphorés a été associée à une diminution de la vitesse de croissance en taille à 2 ans. Conclusion : Ce travail renforce l’hypothèse de l’implication de l’exposition prénatale aux polluants chimiques de l’environnement, y compris à faible dose, dans l’origine développementale de la santé et des maladies. / Background: The organism is very sensitive to environment during its developmental period. Number of epidemiological studies has suggested the developmental toxicity of about ten chemical pollutants after prenatal exposure. The general objective of the thesis is to explore the effect of prenatal exposure to certain chemical pollutants (organic solvents, organophosphates pesticides and maternal consumption of fish and shellfish (vectors of pollutants)) on child development at age 2. Methods: This thesis is based on the PELAGIE mother-child cohort that was set up in 2002, in Brittany (FRANCE). In total, 3421 women were included in this cohort at the beginning of pregnancy and 1500 mother- child pairs were followed when the child was 2 years old. Exposures were evaluated at the beginning of pregnancy from questionnaires, job-exposure matrices or measurement of biomarkers of exposure. Health indicators were measured at the 2 years follow-up. At follow-up, questionnaires allowed to obtain information on child behaviour, respiratory manifestations like wheezing, allergies (eczema and food allergy) and growth between birth and the age of two. Results: Prenatal occupational exposure to solvents was associated with higher level of hyperactivity and attention deficit at age 2. This exposure was not associated neither with respiratory nor with allergic manifestations. We also observed that maternal shellfish consumption during pregnancy was associated with a higher risk of food allergy in preschoolers. Finally, higher prenatal exposure to organophosphate pesticides was associated with a decrease height growth velocity at age 2. Conclusion: The present thesis based on the PELAGIE study supported for the hypothesis that prenatal exposure to environmental chemicals may be implicated in the developmental origin of health and diseases.

Pesticides are among the most pervasive environmental contaminants and they are an important potential risk for human health. Agricultural workers are constantly exposed to pesticide spray, drift and residues in the soil and foliage. Many agricultural pesticides are readily absorbed by the body, through contact with the skin, the respiratory track, the eyes, and the gastrointestinal system. Multiple studies have reported a strong association between pesticide exposure and various health outcomes including cancer. Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects and neurological diseases. The focus of the present translational study is to examine the relationship between human exposure to the organophosphate pesticide azinphos methyl (AZM) and oxidative stress by measuring biomarkers of oxidative stress in biological fluids (i.e., urine, serum) and peripheral blood lymphocytes (PBLs) of agricultural workers. The findings from these field studies will be validated in vitro by examining cultures of human lymphocytes treated with AZM for similar biomarkers of oxidative stress. Since the collection of PBLs from study participants is highly invasive and not suitable for studies involving younger subjects, we also examined buccal cells for biomarkers of oxidative stress (i.e., DNA damage) as a more universal source of human tissue to assess oxidative stress in pesticide exposed individuals. We demonstrated in this study that AZM induces oxidative stress and causes DNA damage in human tissues. Agricultural workers who had been exposed to AZM showed elevated serum levels of lipid peroxides, increased urinary levels of 8-OH-dG, and lymphocytes from these individuals showed increased DNA damage and associated changes in oxidative DNA repair enzymes. Biomarkers of oxidative stress were also elevated in human lymphocytes treated with physiologically relevant concentrations of AZM. In cultures of human lymphocytes, AZM caused a concentration-dependent loss of viability and associated increases in ROS and a reduction in intracellular GSH. We also demonstrated that viable leukocytes from the oral cavity can be readily obtained from humans and these buccal cells can be used to assess DNA damage following exposure to occupational and environmental genotoxicants. We also noted that oral leukocytes are especially sensitive to cryopreservation with DMSO and thus, these cells must be cryoprotected with 5% DMSO to preserve the viability of these cells for subsequent biochemical studies. In summary, these in vivo and in vitro studies demonstrated that AZM induces oxidative stress in a dose-dependent matter and that oral lymphocytes are a good source of human tissue for assessing DNA damage and possibly other biochemical changes. The possible health implications of the variations in these biomarkers of oxidative stress and DNA damage are undetermined. Yet the findings from these studies have provided a strong foundation for determining the mechanism by which pesticide induce oxidative stress, to explore the putative relationship between pesticide-induced oxidative stress and disease (e.g. cancer, neurodegenerative disorders) and determine whether tissue damage in humans is brought about by direct or by indirect action of organophosphate pesticides. / Graduation date: 2010