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Evidence is growing that agrochemicals are playing a role in the decline of amphibians in California. An area of concern is the Sierra Nevada Mountains, where insecticides used in the Central Valley are aerially transported to amphibian habitats. I examined the effects of two of these insecticides, endosulfan and chlorpyrifos, in two experiments on anuran larvae. For the first experiment I exposed Sierra Mountain yellow-legged frog (Rana sierrae) larvae starting at Gosner stage 25 to each insecticide for 63 d to determine median lethal concentrations (LC50) and sublethal effects on growth and development. In the second experiment Sierran treefrog (Pseudacris regilla) larvae were exposed to chlorpyrifos and endosulfan individually and in combination from Gosner stage 25 through metamorphosis to evaluate the interaction between these insecticides. In the first experiment the endosulfan LC50 was 19.8 µg/L (95% confidence interval, 15.3-52.2 µg/L) and the chlorpyrifos LC50 was 595 µg/L (95% confidence interval could not be determined). Endosulfan concentrations greater than 8 µg/L reduced growth but had no effect on time to metamorphosis. No larvae exposed to chlorpyrifos reached metamorphosis. All larvae exposed to greater than 737 µg/L died before the end of the experiment. Growth was reduced above 325 µg/L and cholinesterase was depressed at 737 µg/L compared to controls. In the second experiment the interactive effects of the insecticides depended on concentration and exposure duration. Chlorpyrifos alone did not affect survival or body size after 30 d, even at concentrations greater than the previously reported LC50. Survival and body size decreased with increasing endosulfan concentrations. In combination, 137 µg/L chlorpyrifos inhibited the negative effects of endosulfan on growth and survival and the positive effects of endosulfan on cholinesterase.

Incidence of endocrine disruption in wild fish species has been documented globally and is well characterised in the UK, where the occurrence of intersex in roach (Rutilus rutilus) is widespread. Although this has been associated with concentrations of steroid oestrogens, research indicates that anti-androgenic chemicals may also play a role in inducing these effects. Anti-androgenic activity is commonly detected in wastewater treatment works effluents and some receiving waters, but the chemicals responsible remain largely uncharacterised. This thesis aimed to identify environmental anti-androgens in UK and South Australian catchments and to produce environmentally relevant exposures to assess their potential impacts on sexual disruption in fish, alone and in combination with steroid oestrogens. By using hydrological modelling techniques, pharmaceuticals with an anti-androgenic mode of action were predicted to occur in the ng/L concentration range in UK and South Australian wastewater treatment works effluents and river catchments. This work included analysis of future trends in environmental concentrations of the pharmaceuticals and the steroid oestrogens in these catchments. Modest increases in concentrations by 2050 were predicted in the absence of mitigation, which could increase in the risk posed to fish health by the steroid oestrogens in the future. The effects of the predicted concentrations of two pharmaceuticals, bicalutamide and cyproterone acetate, were then assessed in fathead minnows (Pimephales promelas) and Japanese medaka (Oryzias latipes) based on the UK modelling for the present day. These concentrations did not contribute to endpoints characteristic of sexual disruption, alone or in combination with steroid oestrogens. However, the results did support an environmental role for the steroid oestrogens in intersex induction. Concurrently, effect directed analysis identified some highly potent anti-androgens, such as triclosan and pyrene, in wastewater treatment works effluents from the UK. However, they are likely to make a minor contribution to overall anti-androgenic activity due to their low concentrations. Consequently, more work is required to identify the causes of this activity in the environment and its implications for wild fish health.

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The presence of human pharmaceuticals in the environment has raised concerns regarding their potential adverse effects on non-target aquatic organisms. Pharmaceuticals are designed to target specific molecular pathways in humans in order to produce known pharmacological and physiological responses, before toxicological effects are seen. The “Read-Across Hypothesis” stipulates that pharmaceuticals can produce similar biological effects in fish, as in humans, if the molecular target is conserved, and the internal (blood plasma) concentrations are similar. The read-across hypothesis was tested using ibuprofen, a non-steroidal anti-inflammatory drug, and the model fish test species, the fathead minnow (Pimephales promelas), to determine if ibuprofen can cause similar target-mediated effects in teleost fish and humans, at comparable blood plasma concentrations. Fathead minnows were exposed, using continuous flow-through systems, for ≤96 hours to a range of ibuprofen water concentrations (100, 270, 370 and 500 µg/L) to determine if plasma concentrations similar to human therapeutic plasma concentrations (HTPCs, or Cmax) could be established in fish blood plasma. The mode of action of ibuprofen was used to identify relevant endpoints (i.e. cyclooxygenase (COX) enzyme) in order to examine target-mediated effects following drug exposure. The water and plasma ibuprofen concentrations were determined using LC-MS/MS. The measured ibuprofen plasma concentrations in individual fish were linked to target-mediated effects on COX gene expression, COX enzyme activity and prostaglandin E2 (PGE2) synthesis (products of COX activity), which were quantified using molecular (QPCR) and biochemical (colourimetric and enzyme immunoassay) assays, and linked with the Cmax of ibuprofen. It was demonstrated that in fish with a mean ibuprofen plasma concentration 1.8-fold below the Cmax, PGE2 concentrations (the most robust endpoint) was significantly inhibited following ibuprofen exposure. However, in fish exposed to an ibuprofen concentration closer to (2 to 3-fold above) environmentally relevant water concentrations (i.e. 9 µg/L), when the mean plasma concentration was 224-fold below the Cmax, fish did not respond to ibuprofen exposure. This study provides qualitative and quantitative evidence for the applicability of the “read-across hypothesis”, and highlights its potential utility for prioritising pharmaceuticals for environmental risk assessment.

Levando-se em consideração a grande demanda por água e por fim a poluição emergente nos dias de hoje, o gerenciamento dos corpos hídricos deve ser algo constante, com o propósito preventivo e corretivo, sendo o objetivo deste trabalho a análise das condições ecotoxicológicas e limnológicas dos reservatórios Guarapiranga, Billings e Paiva Castro, na Região Metropolitana de São Paulo, que é abastecida por tais reservatórios. Foram realizadas duas coletas, sedo uma em Maio de 2011, na estação seca, e outra em Janeiro de 2012, na estação chuvosa. Em cada reservatório foram analisados cinco pontos próximos à captação de água pela SABESP. A água e sedimento foram avaliados quanto à toxicidade aguda e crônica através de bioensaios com os cladóceros Daphnia similis e Ceriodaphnia dubia para a água e D. similis e Chironomus xanthus para o sedimento. O tratamento dos dados ocorreu através dos testes de Dunnett\'s ou Steel\'s Many-one Hank e Kruskal-Wallis (fecundidade / sobrevivência) e Fisher Exact Test (mortalidade). Os parâmetros limnológicos analisados na água foram: pH, temperatura, condutividade elétrica, material em suspensão (orgânico e inorgânico), sólidos totais, oxigênio dissolvido, DQO, DBO, nitrogênio total, nitrato, nitrito, amônio, fósforo total, ortofosfato e clorofila a. Os parâmetros físico-químicos do sedimento analisados foram: pH, temperatura, granulometria, teor de Matéria Orgânica e metais (Cd, Cu, Ni, Zn, Pb e Mn). O grau de associação entre as variáveis limnológicas da água, sedimento, e testes ecotoxicológicos, foi avaliado em teste não-paramétrico, através do Coeficiente de Correlação de Spearman. As mesmas variáveis também foram analisadas através da Análise dos Componentes Principais (ACP). O Índice de Estado Trófico mostrou serem os reservatórios Guarapiranga e Billings os mais eutrofizados dentre os corpos hídricos estudados. Conforme os parâmetros analisados na água, alguns parâmetros de certos pontos amostrais estiveram fora dos Padrões estabelecidos pela Resolução CONAMA nº 357/05. Houve influência das chuvas em relação aos parâmetros limnológicos da água, mas o mesmo não foi evidenciado em relação aos resultados dos testes ecotoxicológicos. No geral os dados sugerem ser o sedimento dos reservatórios com maior potencial tóxico do que a água. Os dados ainda sugerem um gradiente de toxicidade, começando pelo reservatório Billings, Guarapiranga e por último Paiva Castro, sendo este o menos tóxico. Houve uma possível correlação entre os metais encontrados no sedimento dos reservatórios mais eutrofizados e a toxicidade obtida nos testes ecotoxicológicos. Os dados sugerem que maiores esforços preventivos e corretivos devem ser alocados para os reservatórios mais eutrofizados e com maiores ocorrências de toxicidade, como é o caso de Guarapiranga e Billings, enquanto que no terceiro reservatório, Paiva Castro, o monitoramento e esforços preventivos serão satisfatórios, mantendo os padrões de qualidade de suas águas em relação aos parâmetros analisados neste trabalho. / Considering the great water demand and the increase pollution in the present-day, the management of the hydric bodies must be something constant, with the preventive and corrective purpose, being the objective of this work to analyze the ecotoxicological and limnological conditions of Guarapiranga, Billings and Paiva Castro reservoirs, in the Metropolitan Region of São Paulo, that\'s supplied by these reservoirs. It was made 2 collections, one in May 2011, in the dry season, and the other in January 2012, in the wet season. In each reservoir it was analyzed 5 points next to the water captation station by the SABESP. The water and sediment were analyzed as for acute and chronic toxicity through bioEssays with the cladocerans Daphnia similis and Ceriodaphnia dubia to the water and D. similis and Chironomus Xanthus to the sediment. The data treatment occurred through the Dunnett\'s or Steel\'s Many-one Hank and Kruskal-Wallis (fecundity/ survival) and Fisher Exact Test (mortality). The limnological water parameters analyzed were: pH, temperature, electrical conductivity, suspended solids (organic and inorganic), total solids, dissolved oxygen, COD, BOD, total nitrogen, nitrate, nitrite, ammonium, total phosphorus, orthophosphate and chlorophyll a. The physical-chemical analyzed parameters in sediment were: pH, temperature, granulometry, organic matter, and metals (Cd, Cu, Ni, Zn, Pb and Mn). The association level between the limnological variables in water, sediment, and ecotoxicological tests, was available in non-parametric tests, through the Spearman correlation coefficient. The same variables were analyzed through the Principal Component Analysis. The Trophic State Index show the Guarapiranga and Billings reservoir as the most eutrophicated between the hydric bodies studied. According to the analyzed parameters in the water, some parameters of certain points were out of the standards established by CONAMA Resolution nº 357/05. There was influence from the rain in relation to the limnological parameters from water, but the same standard was not evidenced in relation to the results from the ecotoxicological tests. In general, the data suggest being the reservoir sediments with more toxic potential than the water. The data still suggest a toxicity gradient, beginning in the Billings and Guarapiranga reservoir, and for last Paiva Castro, being this one the less toxic. There was one possible correlation between the metal found in the more eutrophicated reservoir sediment and the toxicity obtained in the ecotoxicological tests. The data suggest that greater preventive and corrective efforts must be allocated to the more eutrophicated reservoirs with more toxicity occurrences, as the case of Guarapiranga and Billings, while in the third reservoir, Paiva Castro, the monitoring and preventive efforts will be satisfactory, keeping the water quality standard in relation to the parameters analyzed in this work.

Brown algae are an important group of organisms inhabiting coastal ecosystems worldwide. Because of their sessile nature, they are exposed to natural abiotic stresses such as high and low irradiances, desiccation, thermal fluctuations and mechanical stress, as well as anthropogenic-derived stresses such as chemical pollution. While the impacts of metal pollution affect brown algae on a local scale, there is growing concern on the potential interactions between pollutants and abiotic pressures resulting from global climate change. The main objective of this study was to determine the nature of the interactions (synergistic, additive or antagonistic) of different concentrations of copper in combination with increased temperatures in controlled laboratory experiments using the model brown alga Ectocarpus siliculosus as a proxy for brown seaweeds, which are globally important primary producers and bioengineers of near-shore waters. The responses in E. siliculosus were evaluated at different levels of biological organisation. At the whole organism level Cu or temperature affected growth but no interactions occurred. Antagonistic interactions occurred between stressors in the photosynthetic efficiency response (measured as chlorophyll a fluorescence), being less affected by Cu at higher temperatures. The bioaccumulation of Cu ions showed and antagonistic response to temperature as less Cu ions were accumulated at elevated temperature. The concentrations of H2O2 and lipid peroxides (TBARS), which are indicators of oxidative stress, were synergistically affected by interactions of stressors. In contrast, the concentrations of antioxidants ascorbate and glutathione reflected both additive and antagonistic interactions respectively. This also occurred in the activity of antioxidant 8 enzymes (superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase) and the expression of related genes. Finally, the results of the biochemical and physiological tests were integrated with the whole transcriptome response to temperature and Cu stress. These results showed that interactions between temperatures and Cu stress could be highly complex, but also lead to the discovery of potential stress markers such as light harvesting complex proteins and several transporters. This research provides new insights into the responses of brown macroalgae to metal and thermal stress. Those responses indicate that synergistic or antagonistic interactions can occur at different levels of organisation, being the regulation of antioxidant metabolism, photosynthetic physiology and related gene expression, the most important mechanisms involved. This information will aid to understand potential effects of climate change on the toxicity of metals for macroalgae in estuaries and coasts affected by pollution.

O mexilhão marrom Perna perna (Linnaeus, 1758) auxilia no monitoramento de compostos químicos em ecossistemas marinhos. No entanto, os mecanismos moleculares de detoxificação e resposta ao estresse são desconhecidos. Elucidar esses mecanismos é crucial para entender os efeitos tóxicos dos poluentes químicos e desenvolver biomarcadores para avaliar a qualidade ambiental dos ecossistemas marinhos. No presente estudo, indivíduos da espécie P. perna foram expostos a antraceno (ANT) e os RNAs mensageiros (mRNA) das brânquias foram sequenciados com a plataforma Illumina. A análise química do tecido mole dos animais identificou concentrações de ANT 268 a 715 vezes mais alta no grupo exposto comparado ao grupo controle, demonstrando que a exposição foi realizada com sucesso. O sequenciamento do transcritoma do P. perna gerou 273.152.390 pares de reads, resultando na montagem de 231.728 contigs com tamanho médio de 720 pb e N50 de 1.083 pb, os quais 66.563 contigs (28,7%) pode ser anotado utilizando banco de dados como GenBank, Pfam, Gene Ontology e KEGG. Os resultados obtidos a partir da anotação funcional sugerem que as brânquias tenham papel na biotransformação de xenobióticos, resposta antioxidante, sinalização, resposta imunológica inata, e osmorregulação. Foi possível identificar genes de biotransformação de fase I, II e III, incluindo CYPs e GSTs. Transcritos similares a CYPs e GSTs estavam sendo expressos no grupo exposto, porém nenhum deles foram classificados como diferencialmente expressos. Contudo, muitos genes hipotéticos foram diferencialmente expressos, o que sugere que P. perna utilize mecanismos desconhecidos de biotransformação para lidar com a contaminação de ANT. Genes de sistema imune inato foram regulados tanto positivamente quanto negativamente, assim como observado para Perna viridis exposto a benzo(a)pireno, sugerindo que ANT promove alterações da capacidade de resposta do sistema imune inato do P. perna. / The brown mussel Perna perna (Linnaeus, 1758) helps the monitoring of chemical compounds in marine ecosystems. However its molecular mechanisms of detoxification and stress response remain unclear. Elucidating these mechanisms is crucial to understand the toxic effects of chemical pollutants and to develop biomarkers to assess marine ecosystems. In this study, P. perna individuals were exposed to anthracene (ANT) and its mRNA complement was sampled sequenced with Illumina technology. Chemical analysis of the soft tissue identified ANT concentrations 268 - 715 fold higher in the exposed group compared to controls, demonstrating that the exposure procedure was successfully accomplished. Transcriptome sequencing of P. perna generated 273.152.390 paired reads that were assembled in 231.728 contigs of average length 720 bp and N50 1.083 bp , which 66.563 contigs (28,7%) could be annotated using GenBank genes, Pfam domains, Gene Ontology (GO) terms and KEGG pathways. The results obtained from functional annotation suggest gills play a role in xenobiotics biotransformation, antioxidant response, signal transduction, innate immune response, and osmoregulation. It was possible to identify transcripts similar to genes related with biotransformation reactions of phases I, II and III, including CYPs and GSTs. Transcripts similar to CYPs and GSTs isoforms were highly expressed in the group exposed to ANT, however no CYP, GST, or even other genes related with biotransformation reactions were classified as differentially expressed. On the other hand, several hypothetical genes were differentially expressed, which suggests that P. perna uses unknown mechanisms of biotransformation to deal with ANT stress contamination. Immune related-genes were both up and down-regulated, as was also observed for Perna viridis exposed to benzo(a)pyrene, suggesting that ANT promotes alteration in the immune response of P. perna.

Orientador: Carmem Silvia Fontanetti Christofoletti / Resumo: O Brasil é o maior produtor mundial de cana-de-açúcar. O fenilpirazol fipronil está dentre os inseticidas mais utilizados na cultura de cana-de-açúcar. Sua molécula é extremamente ativa e um potente desregulador do sistema nervoso central dos insetos. Devido a sua elevada toxicidade, moléculas como o ethiprole estão sendo empregadas na agricultura em busca de uma alternativa mais segura. Produzido a partir da modificação da molécula do fipronil, poucos estudos têm demonstrado que o ethiprole surgiu no cenário agrário como uma alterativa mais segura que o fipronil aos organismos não alvos em geral. O uso de bioindicadores permite o estudo dos possíveis riscos de inseticidas. Entre estes, plantas e organismos aquáticos são excelentes para avaliar efeitos de agrotóxicos no ambiente. No presente estudo, foi avaliada a ação de diferentes concentrações dos inseticidas Regent® e Curbix® em organismos não alvos, por meio de ferramentas macroscópicas em Allium cepa e Lactuca sativa e microscópicas em Oreochromis niloticus. Os testes de germinação de sementes e alongamento radicular em A. cepa e L. sativa foram utilizados para a avaliação dos efeitos fitotóxicos no desenvolvimento inicial das plantas. As alterações histopatológicas no fígado de O. niloticus, a quantificação de peroxidação lipídica (TBARS), localização in situ das proteínas de choque térmico (HSP70) e de fragmentação do DNA (TUNEL) foram empregadas para verificar o potencial tóxico dos inseticidas a nível celular. Os re... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Brazil is the world’s largest sugar cane producer. Phenylpyrazole fipronil is included among the most commonly used insecticides in sugarcane cultivation. Its molecule is extremely active and a powerful deregulator of the central nervous system of insects. Given its high toxicity, molecules such as ethiprole are being employed in agriculture to find a safer alternative. Developed by modifying the Fipronil molecule, few studies have demonstrated whether ethiprole appeared as a safer alternative to Fipronil to non-target organisms in general. The use of bioindicators allows the study of possible risks of insecticides. Among them, plants and aquatic organisms are excellent for assessing the effects of pesticides on the environment. In the present study, it was assessed the effects of different concentrations of Regent® and Curbix® insecticides on non-target organisms, employing macroscopic tools in Allium cepa and Lactuca sativa and microscopic ones in Oreochromis niloticus. Seed germination and root elongation tests on A. cepa and L. sativa were used to evaluate phytotoxic effects on initial plant development. Histopathological alterations in the liver of O. niloticus, quantification of lipid peroxidation (TBARS), in situ locations of thermal shock proteins (HSP70), and DNA fragmentation (TUNEL) were used to verify the toxic potential of insecticides at the cellular level. The results of phytotoxic analyses on A. cepa showed that Curbix® induced a reduction in germination perce... (Complete abstract click electronic access below) / Doutor

The introduction of pharmaceuticals and personal care products (PPCPs) to aquatic systems has impacted development and reproductive health of fish in many regions of the world. This study investigated western mosquitofish in the bayou systems of Harris County, Texas for evidence of morphological and reproductive abnormalities. Mosquitofish were sampled above and below WWTPs on five streams during May and August 2010, and specimens were dissected and analyzed for reproductive (egg/embryo weight, embryo/egg number and embryos staging), morphological (liver and gonad weight, body length, gonadosomatic index and hepatosomatic index) and histological indicator variables. In addition, water samples were analyzed for concentrations of PPCPs. Spatial and temporal variation was observed among all indicator variables, however no consistent differences were found above versus below WWTP discharges. Histopathology showed no evidence of lesions or presence of intersex individuals. Chemical analysis revealed a variety of pharmaceuticals and anthropogenic chemicals present in the Houston area waterways, however all were at concentrations lower than those known to cause impacts to fishes. These results suggest that the current concentrations of chemicals being discharged from WWTPs into headwater reaches of streams in the suburban area of west Houston are below levels that impact the physiology of male and female mosquitofish.