Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Due to the proximity of crop and fish culture areas, some agrichemicals that could be harmful for fish could enter into fishponds by different ways, such as by leaching through rain. The contamination of the aquatic environment by pesticides is a problem of global importance because of its toxic effects on non-target organisms. These products may affect toxicological endpoints in fish. Among the effects that pesticides can cause fish is the formation of reactive oxygen species (ROS) and changes in antioxidant enzymatic and nonenzyme, which may show a situation of oxidative stress. The formation of ROS may be associated with different pathological processes in fish exposed to pollutants such as pesticides. Several biochemical and physiological responses occur when a pesticide enters the body, resulting in the adaptation of the organism to the contaminant or if the body can not efficiently metabolize these products, they may induce toxicity. Rhamdia quelen (Teleostei) were exposed to sub-lethal concentrations of methyl parathion (MP), a glyphosate based herbicide (Gly), and tebuconazole (Teb). The liver of R. quelen exposed to MP and Teb showed enhanced levels of thiobarbituric acid reactive substances (TBARS), compared to the control fish (56% and 59%, respectively). In contrast, Gly did not alter the TBARS generation. The protein carbonyl content increased only in fish exposed to Teb. Fish exposed to the three agrichemicals showed a significant decrease of catalase activity (52%, 48%, and 67%, respectively) and increased glutathione-S-transferase (57%, 46%, and 160%, respectively) activity. Fish exposed to MP, Gly, and Teb showed higher reduced glutathione (151%, 472%, and 130%, respectively, when compared to the control levels) and ascorbic acid concentrations (121%,102%, and 184%, respectively),while the non-protein thiol content increased only in R. quelen exposed to tebuconazole. Fish exposed to MP and Teb showed several pathological changes in the liver, including hepatocyte degeneration and bile stagnation. The present work reports for the first time the toxicity of the pesticide MP and the fungicide Teb in R. quelen, and as in other works, suggests the relatively lower liver toxicity of Gly for fish. The data presented herein demonstrate that sublethal concentrations of MP and Teb cause changes in oxidative stress parameters as well as hepatic cell injuries in R. quelen, and that these parameters have the potential to be developed as bioindicators of exposure to these agrichemicals. / A contaminação do ambiente aquático por agrotóxicos é um problema de importância mundial, devido ao seu efeito tóxico em organismos não-alvo. Estes produtos podem afetar parâmetros toxicológicos em peixes. Entre os efeitos que os agrotóxicos podem causar em peixes está a formação de espécies reativas de oxigênio (EROs) e alterações em antioxidantes enzimáticos e não-enzimáticos, o que pode evidenciar uma situação de estresse oxidativo. Várias respostas bioquímicas e fisiológicas ocorrem quando um pesticida entra no organismo, resultando na adaptação do organismo ao contaminante ou se o organismo não consegue metabolizar com eficiência estes produtos, os mesmos podem induzir toxicidade. Alevinos de Rhamdia quelen foram expostos a concentrações subletais de parationato metílico (MP), um herbicida a base de glifosato (GLI) e tebuconazole (TEB). O fígado de R. quelen exposto a MP e TEB mostrou um maior nível de substancias reativas do ácido tiobarbitúrico (TBARS), superior nos peixes no grupo controle (56% e 59%, respectivamente). Em contraste, GLI não alterou a produção de TBARS. Ocorreu aumento da carbonilação pretéica apenas nos peixes expostos a TEB. Os peixes expostos aos três agrotóxicos mostraram uma diminuição significativa da atividade da catalase (52%, 48%,e 67%, respectivamente) e aumento da atividade da glutationa-S-transferase (57%, 46% e 160%, respectivamente). Os peixes expostos a MP, GLI e TEB apresentaram um aumento de glutationa reduzida (151%, 472% e 130%, respectivamente, quando comparado com os níveis do grupo controle) e concentrações de ácido ascórbico (121%, 102% e 184%, respectivamente), enquanto que o conteúdo de tióis-não-proteicos aumentou apenas em R. quelen expostos a tebuconazole. Os peixes expostos a MP e TEB mostraram diversas alterações histopatológicas no fígado, incluindo a degeneração dos hepatócitos e estagnação biliar. O presente trabalho relata pela primeira vez a toxicidade dos pesticidas MP e do fungicida TEB em R. quelen, e sugere que a toxicidade de GLI é relativamente menor para os peixes. Os dados apresentados demonstram que concentrações sub-letais de MP, TEB podem causar alterações nos parâmetros de estresse oxidativo, bem como lesões de células hepáticas em R. quelen e que estes parâmetros têm o potencial de serem desenvolvidos como biomarcadores de exposição a esses agrotóxicos.

Effects of developmental exposure to chlorpyrifos (CPS) or methyl parathion (MPS) on visuospatial, adaptive fear response, and passive avoidance memory and the signaling mechanisms responsible for these neurocognitive changes were investigated. Using an incremental low dose regimen, rat pups were orally gavaged daily with either corn oil (vehicle), CPS, or MPS from postnatal day 1 (PND1) -PND21. Cholinesterase activity was significantly inhibited with the highest dosages of CPS and MPS for up to 19 days after the last dosages. OP exposure impaired working and reference memory in males whereas in the females, enhancement occurred following CPS exposure. In addition, the adaptive fear response and passive avoidance retention memory was impaired in males whereas differential changes occurred in females. Accordingly, the behavioral deficits observed in males were persistent whereas the enhancement in females was transient. Males were more sensitive to OPs than females in that the medium and high dosages of CPS and MPS produced greater effects in females whereas all dosages of both compounds produced effects in males. Training in the radial arm maze significantly increased protein kinase C gamma (PKC ) expression and activity in the hippocampal membrane fraction of control rats whereas exposure to OPs exhibited a significant decrease in PKC and PKC immunoreactivity in both untrained and trained rats. However, MPS exposed females exhibited a significant increase in PKC expression in the cytosolic fraction but this was not related to improved memory. Reduction of membrane PKC expression and activity and cytosolic PKC expression and activity seemed to be related to visuospatial learning and memory deficits in exposed males but not exposed females. Brain-derived neurotrophic factor (BDNF) gene expression in the hippocampus was significantly increased (60%) in trained control males as compared to untrained control males. In contrast, trained and untrained females exhibited similar levels of BDNF gene expression. However, exposure of both sexes to either CPS or MPS significantly reduced the expression of BDNF in trained rats. In summary, these data indicate that OP exposure induced gender-specific changes in working memory formation and altered PKC isozyme levels/activity and BDNF expression.

This thesis describes the characterization of SsoPox, a lactonase with promiscuous phosphotriesterase activity from the hyperthermophilic archaeon, Sulfolobus solfataricus, and the potential of the phosphotriesterase from Brevundimonas diminuta (PTEBd) to function as an organophosphate sensor. Arg-223 and Tyr-99 of SsoPox are not essential for lactonase activity, however substitution of a phenylalanine in place of Tyr-97 abolished lactonase activity while reducing paraoxonase activity by 20-fold. Substrate specificity of SsoPox can be modulated through the partial blockage of the hydrophobic binding tunnel adjacent to the active site. The specificity constant for N-(3-oxo-decanoyl)-L-homoserine lactone decreased 37-fold when a phenylalanine was introduced in place of Leu-226. PTEBd was expressed and purified from Pseudomonas putida and, like SsoPox, can be immobilized to Disruptor paper. The immobilized enzyme can be used to detect five organophosphates at concentrations as low as 50 μM. Incubation of PTEBd-immobilized sensors at different temperatures proved that the enzyme is stable for at least 40 days at 23.5 degrees Celsius without any detectable change in activity.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Organophosphorus compounds such as methyl-parathion are used in the various stages of production to control pests both in agricultural activity as in aquaculture. The mechanism of action of this type of compound is the inhibition of the enzyme acetylcholinesterase. The zebrafish (Danio rerio) has been increasingly used as an experimental model in varied fields such as development, genetics and pharmacological research. The methyl-parathion has been characterized as endocrine disruptor of the hypothalamic-pituitary-interrenal axis (HHI). An experiment was carried out of 96 hours of exposure of adult fish to the substance tested, at the concentration of 5.2 mg/L. Was evaluated the whole-body cortisol level in order to measure the endocrine response to a stressful event. Were also investigated the gene expression of glucocorticoid receptor (GR), steroidogenic acute regulatory protein (StAR) and heat shock protein 70 (HSP 70). Fish exposed that have undergone a stressor event demonstrated low levels of cortisol. In addition, the fish stressed and exposed to agro-chemical showed a decreased expression of the StAR, HSP 70 and GR genes. The data indicate that exposure to methyl-parathion causes a decrease in the ability to respond appropriately to a stressor. Fish that have an inability to produce a satisfactory answer by the HHI axis are not able to make the necessary metabolic and ion adjustments for recovery the homeostasis, getting vulnerable to stress caused by aquaculture practices or environmental changes. / Compostos organofosforados como o parationato metílico são utilizados nas diversas etapas de produção para controlar pragas tanto na atividade agrícola como na aquicultura. O mecanismo de ação desse tipo de composto é a inibição da enzima acetilcolinesterase. O peixe-zebra (Danio rerio) vem sendo cada vez mais usado como modelo experimental em variados campos, como desenvolvimento, genética e pesquisa farmacológica. O parationato metílico já foi caracterizado como interruptor endócrino do eixo hipotálamo-hipófise-interrenal (HHI). Foi realizado um experimento de 96 horas de exposição de peixes adultos à substância testada, na concentração de 5,2 mg/L. Foi avaliado o nível de cortisol de corpo inteiro, visando medir a resposta endócrina a um evento estressor. Também foram investigadas a expressão dos genes do receptor de glicocorticoide (GR), da proteína regulatória de esteroidogenese aguda (StAR) e a proteína do choque térmico 70 (HSP 70). Os peixes expostos que foram submetidos a um evento estressor demonstraram baixos níveis de cortisol de corpo inteiro. Além disso, os peixes estressados e expostos ao agroquímico apresentaram uma diminuição da expressão dos genes GR, StAR e HSP 70. Os dados indicam que a exposição ao parationato metílico provoca uma diminuição da capacidade de responder adequadamente a um evento estressor. Peixes que possuem uma incapacidade em produzir uma resposta satisfatória do eixo HHI, não são capazes de realizar os ajustes iônicos e metabólicos necessários à recuperação da homeostase, ficando vulneráveis ao estresse causado pelas práticas aquícolas ou por alterações ambientais.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-05-02T17:14:06Z No. of bitstreams: 2 Dissertação - Josiane Moreira Cardoso - 2017.pdf: 2032261 bytes, checksum: 74966b401eaefa19e8806d5ea51fd25b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-03T11:49:31Z (GMT) No. of bitstreams: 2 Dissertação - Josiane Moreira Cardoso - 2017.pdf: 2032261 bytes, checksum: 74966b401eaefa19e8806d5ea51fd25b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-03T11:49:31Z (GMT). No. of bitstreams: 2 Dissertação - Josiane Moreira Cardoso - 2017.pdf: 2032261 bytes, checksum: 74966b401eaefa19e8806d5ea51fd25b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-07 / Outro / In this work, experiments were performed to study the potential of coconut fiber (Cocos nucifera L.) as adsorbent for removing the pesticide parathion methyl from water. The analytical methodology used to quantify the pesticide in the adsorption studies was based on solid phase extraction (SPE) and gas chromatography with electron capture detection (GC/ECD). The method were validated and showed good analytical features in terms of selectivity, linearity, accuracy (recovery: 100.4%), precision (coefficient of variation: 9.63%;), limit of detection (0.003 mg L-1 ) and quantification (0.010 mg L-1 ). The matrix effect in pesticide quantification was evaluated and the results showed an enhanced chromatographic response for the parathion methyl in all the concentrations evaluated due to interference of endogenous coconut fiber compounds present in the solution after adsorption tests, therefore, the quantification was made using matrix-standard calibration solutions to compensate for matrix-induced effects and to obtain more accurate results. The adsorbent characterization (FTIR, MEV, surface area and pHpcz) showed that coconut fiber surface is composed of very irregular particles, with many cavities (pore diameter: 3.48 nm) and external surface area of 3.64 m2 g -1 . The presence of several functional groups: carboxylate, hydroxyl, carboxyl, among others was observed. The pH at point of zero charge (pHpcz) for the adsorbent was 5.85. The adsorption capacity of the coconut fiber was evaluated under different parameters: adsorbent treatment, adsorbent dosage, contact time and concentration of the pesticide. The adsorbent treatment with water, HCl or NaOH did not affect the adsorption capacity (75.6% of removal was obtained, independently of the treatment). The increasing in the adsorbent dosage of 5 to 20 g L-1 caused an increasing in the adsorption capacity from 25.0% to 76.6%. The adsorption equilibrium process was reached after 90 minutes of contact, with 86.1% of removal. The kinetic study showed that the Avrami kinetic model was the best fit for the experimental data. The adsorption capacity improved as function of the pesticide concentration increasing. The experimental data were fitted to Langmuir, Freundlich and Sips isotherm models and the best fit was obtained with Freundlich and Sips models. The maximum adsorption capacity (Qmax) were 39.8547 mg g-1 . Additional tests showed that the adsorption capacity was slightly affected when using real samples (natural water) in the adsorption experiments (82.24% removal) and presented excellent adsorption capacity when used in an alternative column adsorption procedure (85.73% removal). The results indicate that the coconut fiber has a great potential to be used as adsorbent material for the treatment of water contaminated with the pesticide methyl parathion. / Neste trabalho foi estudada a utilização da fibra de coco (mesocarpo do fruto de Cocos nucifera L.) como adsorvente para a remoção do agrotóxico parationa metílica de meio aquoso. A metodologia analítica empregada para quantificar o agrotóxico nos estudos de adsorção foi baseada nas técnicas de extração em fase sólida (SPE) e cromatografia gasosa com detecção por captura de elétrons (GC/ECD). O método foi validado e apresentou boa performance analítica em termos de seletividade, linearidade, exatidão (recuperação: 100,4%), precisão (coeficiente de variação: 9,63%), limites de detecção (0,003 mg L-1 ) e quantificação (0,010 mg L-1 ). Foi avaliado o efeito de matriz na quantificação do agrotóxico, e os resultados mostraram que ocorreu aumento na resposta cromatográfica para a parationa metílica em todas as concentração avaliadas devido à interferência de compostos endógenos da fibra de coco, presentes na solução após os ensaios de adsorção, desse modo, a quantificação foi realizada utilizando padrões analíticos preparados em extrato branco da matriz para compensar os efeitos de aumento na resposta cromatográfica induzido pela matriz e assim obter resultados quantitativos mais exatos. A caracterização do adsorvente (FTIR, MEV, área superficial e pHpcz) mostrou que a superfície da fibra de coco é constituída de partículas bastante irregulares e com muitas cavidades (diâmetro do poro: 3,48 nm), apresentando área superficial externa de 3,64 m2 g -1 . Foi observada a presença dos grupos funcionais: carboxilato, hidroxila, carboxila, entre outros. O pH no ponto de carga zero (pHpcz) para o adsorvente foi 5,85. A capacidade de adsorção da fibra de coco foi avaliada sob diferentes parâmetros: tratamento do adsorvente, dose de adsorvente, tempo de contato e concentração do agrotóxico. O tratamento do adsorvente com água, HCl ou NaOH não afetou a capacidade de adsorção, obtendo-se cerca de 75,6% de remoção, independentemente do tratamento. O aumento na dose de adsorvente de 5 para 20 g L-1 fez com que a capacidade de adsorção aumentasse de 25,0% para 76,6%. O equilíbrio do processo de adsorção foi alcançado após 90 minutos de contato, com 86,1% de remoção. O estudo cinético do processo mostrou que o modelo cinético de Avrami foi o que melhor ajustou aos dados experimentais. A capacidade de adsorção melhorou em função do aumento na concentração da parationa metílica no meio aquoso. Os dados experimentais foram ajustados às isotermas de Langmuir, Freundlich e Sips, sendo que os melhores ajustes foram observados para os modelos de Freundlich e Sips. A capacidade máxima de adsorção (Qmáx) foi 39,8547 mg g-1 . Ensaios adicionais mostraram que a capacidade de adsorção da fibra de coco foi pouco afetada quando foi utilizada amostra real (água natural) nos ensaios de adsorção (82,24% de remoção) e apresentou excelente capacidade de adsorção quando empregado em procedimento alternativo de adsorção em coluna (85,73% de remoção). Os resultados obtidos indicam que a fibra de coco pode ser utilizada como material adsorvente no tratamento de meio aquoso contaminado com o agrotóxico parationa metílica.

The increased use of pesticides has resulted in a corresponding increase in concern for the effect they may have on the health of humans and other non-target organisms. The two main areas of concern are the toxicological effects that mixtures of pesticides may have as well as the endocrine disrupting effects. Although the individual pesticides may be present at concentrations below the levels deemed to be detrimental to health, it has been argued that their combined effect may still result in elevated health risks. Another important aspect of pesticide risk assessment requires a consideration of the breakdown products of pesticides and their effect on human health. There has been very little research into the effects of degradation products and this issue should be addressed as these could potentially pose a higher risk than their parent compounds. One of the most important bio-markers available for use is the ubiquitous enzyme acetylcholinesterase (AChE). This enzyme is responsible for one of the most important functions in the body; namely nerve impulse transmission, upon which all life depends. The inhibition of this enzyme indicates toxicity and as a subsequence, a threat to the organism’s well-being. Bioassays have also recently been developed to test chemicals for endocrine disrupting effects. These tests rely on a dose response equivalent to that of the most potent well known estrogen 17-β estradiol. Any chemical that has a measurable response is deemed to display endocrine disrupting effects. This first aim of this study was to investigate the toxicological and endocrine disrupting effects of three organophosphorus pesticides; aldicarb, parathion and demeton-S-methyl, in addition to two breakdown products; aminophenol and p-nitrophenol. Two carbamate pesticides; carbaryl and carbofuran were also analysed. The toxicological effects of mixtures of the parent pesticide compounds were tested to assess if any antagonistic, additive or synergistic effects were observed. This data was then used in conjunction with an artificial neural network to assess if individual pesticides could be distinguished from mixtures of pesticides. A final objective was to sample various Eastern Cape water sources, utilising the enzymatic assay to determine the presence of any of these pesticides in these samples. There were several conclusions drawn from this study. AChE was successfully used as an assay to test the toxicity of the pesticides under investigation, based on their inhibition of this enzyme. An important factor for consideration throughout the study was the need to establish basal and monitor AChE activity (i.e. the need to monitor AChE activity in the absence of any pesticide). This ensured accurate comparison of the results obtained. It was found that demeton-S-methyl was the most potent of these pesticides followed by carbaryl, parathion, aldicarb and finally carbofuran, and that carbofuran could potentiate AChE. The results indicated that pesticide mixtures generally exhibited an additive inhibitory effect on AChE, although at some concentrations of pesticides, synergistic and antagonistic effects were noted. From the data using mixtures of pesticides, a feed forward neural network was created that was successfully able to distinguish individual pesticides from mixtures within its training parameters. None of the pesticides tested displayed endocrine disrupting properties in the Yeast Estrogen Screen (YES), T47D-KBluc and MDA-kb2 bio-assays. Other studies reported mixed results in this regard and thus no final conclusions could be drawn. The Blaauwkrantz River, Kariega River, Sundays River, Swartkops River and Kowie River were all tested for pesticides and although positive results were recorded, conventional methods indicated that there were no pesticides in the rivers. There were, however, trace metals present which are known to inhibit AChE, thus causing a false positive result. These results indicated that AChE can be used as a high throughput initial pre-screening tool, but that it cannot serve as a substitute for more accurate conventional testing methods.