In recent years consumption of drugs and thus the quantity of their residues in natural ecosystems is increasing. It is necessary to deal with their possible negative effects on the environment. This thesis is focused on the ecotoxicological evaluation of pharmaceuticals. Diclofenac and ibuprofen from the group of non-steroidal anti-inflammatory substances, penicillin G and ampicillin from the group of antibiotics were tested. Mainly tests on aquatic organisms were used to evaluate of ecotoxicity, because the tested pharmaceuticals are predominantly occurred in the aquatic environment. Daphnia magna, Thamnocephalus platyurus, Artemia salina, Sinapis alba, Lemna minor, and Brachionus calyciflorus were used as testing organisms. Values of LC50, EC50 and IC50 were determinated and the ecotoxicity of pharmaceuticals was compared.

This diploma thesis deals with a use of algal test in ecotoxicology. A freshwater algae, Desmodesmus subspicatus, was used to asses the ecotoxicity of selected chemical substances. A spectrophotometry method, of VIS spectrum, was used in evaluation of the EC50. A correlation, between a number of algal cells and the wavelength absorbance at 683 nm, has been determined for this purpose. Following chemicals were tested, 2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid, (RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid and N-(4-hydro-xyphenyl)acetamide. An EC50 value was determined for all these substances and also their ecotoxicity has been evaluated.

Products of daily use, which are mainly cosmetics and personal care products could significantly affect the environment. Recently, in terms of environmental protection, organic compounds responsible for the pleasant smell of these products - synthetic fragrances or musk compounds - are often mentioned. This diploma thesis aims to assess the ecotoxicity of several new substances from the group linear (alicyclic) musk compounds. From this group following fragrance ingrediens were tested: lilial, linalool, arocete and aroflorone. Since the majority of their residues enter the aquatic ecosystem, aquatic freshwater crustacean Daphnia magna and Thamnocephalus platyurus and aquatic plant Lemna minor were selected as test organisms. Individual ecotoxicity of above mentioned substances was evaluated as well as toxicity of binary mixture of arofloron and linalool on organisms Daphnia magna and Thamnocephalus platyurus. Based on the test results the ecotoxicological values LC50, EC50 and IC50 for the tested substances were evaluated and their ecotoxicity was compared. Lilial showed the highest ecotoxicity. Value of 24h EC50 for organism Daphnia magna was 2.13 mg/l and value of 24h LC50 for the organism Thamnocephalus platyurus was 10.86 mg/l.

This diploma thesis evaluates the influence of contaminated matrices introduced into the ecosystem in terms of ecotoxicity. It is focused mainly on matrix generated by anthropogenic activities, especially waste of various origin - industrial, energetical, biodegradable and more. For experimental purposes sewage sludge as the bulk waste with the ever-increasing production was selected. Sewage sludge was tested at its most common use - application on the agricultural land. This application is limited by legislative through the concentration limits of hazardous elements. For this reason, the potential ecotoxicity of sludge was evaluated. Ecotoxicological evaluation was performed using the contact bioassays. As the test organisms Eisenia foetida, Folsomia candida, Heterocypris incongruens a plant Lactuca sativa were selected. Sludge from wastewater treatment plants Brno - Modřice, Valtice, Mikulov and Lednice was tested. Samples of sewage sludge showed no ecotoxicity, while respecting the application amount established by the regulation. On the contrary, it can be concluded that soils enriched by sewage sludge show positive effect on soil biota.

III-V materials such as indium arsenide (InAs) and gallium arsenide (GaAs) are increasingly used in electronic and photovoltaic devices. The extensive application of these materials may lead to release of III-V ionic species during semiconductor manufacturing or disposal of decommissioned devices into the environment. Although arsenic is recognized as an important contaminant due to its high toxicity, there is a lack of information about the toxic effects of indium and gallium ions. In this study, acute toxicity of As(III), As(V), In(III) and Ga(III) species was evaluated using two microbial assays testing for methanogenic activity and 02 uptake, as well as two bioassays targeting aquatic organisms, including the marine bacterium Aliivibrio fischeri (bioluminescence inhibition) and the crustacean Daphnia magna (mortality). The most noteworthy finding was that the toxicity is mostly impacted by the element tested. Secondarily, the toxicity of these species also depended on the bioassay target. In(III) and Ga(III) were not or only mildly toxic in the experiments. D. magna was the most sensitive organism for In(III) and Ga(III) with 50% lethal concentrations of 0.5 and 3.4 mM, respectively. On the other hand, As(III) and As(V) caused clear inhibitory effects, particularly in the methanogenic toxicity bioassay. The 50% inhibitory concentrations of both arsenic species towards methanogens were about 0.02 mM, which is lower than the regulated maximum allowable daily effluent discharge concentration (2.09 mg/L or 0.03 mM) for facilities manufacturing electronic components in the US. Overall, the results indicate that the ecotoxicity of In (III) and Ga(III) is much lower than that of the As species tested. This finding is important in filling the knowledge gap regarding the ecotoxicology of In and Ga.

Nanoparticles (NPs) have unique electronic, optical and chemical properties due to the extreme small size. Engineered nanoparticles (ENPs) are intentionally produced for desired applications, with specific properties related to shape, size, surface properties and chemistry. Nano-sized silica (SiO2), alumina (Al2O3) and ceria (CeO2) are three important ENPs with large production and wide applications. One of the principal uses of these ENPs is in chemical and mechanical planarization (CMP), a key process applied to polish wafers when fabricating integrated circuits in semiconductor manufacturing, in which SiO2, Al2O3 and CeO2 NPs are used as abrasive particles in CMP slurries. CMP generates large amounts of waste effluents containing high levels of ENPs. Some ENPs have been proven to be able to cause toxicity to microorganisms and higher life forms, including humans. Therefore, there are concerns about the potential risks that ENPs may pose to the natural environment and human health. In addition, III-V materials like indium arsenide (InAs) and gallium arsenide (GaAs) are increasingly used in electronic and photovoltaic devices. Besides ENPs, the waste streams from III-V manufacturing also contain dissolved and particulate materials removed from III-V films during CMP. Arsenic is one of the most notorious contaminants that has been widely studied, while only very limited ecotoxicity information is available for gallium and indium. Finally, since ENPs have high surface area, it is very likely they will interact with the soluble species (such as arsenic ions) in CMP wastewater. Therefore, it is of great importance to understand whether the interactions between these materials could alter their fate and toxicity. The objective of this work is to investigate the potential environmental and health risks from the ENPs and III-V materials used in semiconductor manufacturing. To this end, the physical, chemical and toxicological characterization of ENPs used in CMP was performed (Chapter 3). Furthermore, the fate and transport of the most used ENP, SiO2, in porous media was studied (Chapter 4). In addition, acute toxicity of As(III), As(V), In(III) and Ga(III) species was evaluated using different bioassays (Chapter 5). Finally, the cytotoxicity of ENPs used in CMP slurries to human lung bronchial epithelial cells was evaluated using an impedance based real time cell analysis (RTCA) assay (Chapter 6). In Chapter 3, four model slurries containing ENPs including colloidal silica (c-SiO2), fumed silica (f-SiO2) cerium oxide (CeO2) and aluminum oxide (Al2O3) were characterized for their physical, chemical and toxicological properties. Ecotoxicity of these slurries to the marine bacterium, Aliivibrio fischeri, was evaluated by measuring its bioluminescence activity as a function of the ENP concentration dosed. The results showed that f-SiO2 and CeO2 were not toxic at concentrations up to 700 and 1000 mg/L, respectively. On the other hand, c-SiO2 and Al2O3 were inhibitory only at very high concentrations (>600 mg/L). At about 1300 mg/L, c-SiO2 and Al2O3 led to 37.6% and 28.4% decrease of cell activity after 30 min exposure, respectively. The inhibitory effect from c-SiO2 was related to additives in the slurry. In summary, the results indicate that these slurries are not likely to cause acute toxicity at environmentally relevant concentrations. The potential risks from ENPs are dependent on their fate and transport in the environment. In Chapter 4, the transport and abatement of SiO2 NPs was studied through laboratory scale column experiments. Synthetic fluorescent core-shell SiO2 NPs (83 nm) were used to facilitate NP traceability. Three widely used filtering materials, i.e., sand, anthracite and granular activated carbon (GAC), were used as porous media. Sand showed very poor capacity for the filtration of SiO2 NPs due to its limited surface area and high concentration of negative surface charge. In addition, the stability and transport of SiO2 NP was strongly dependent on the ionic strength of the solution. High ionic strength led to NP agglomeration and facilitated SiO2 NP retention, while low ionic strength resulted in release of captured NPs from the sand bed. Compared to sand, anthracite and GAC showed higher efficiency for SiO2 NP capture. The superior capacity of GAC was primarily due to its porous structure and high surface area. A process model was developed to simulate NP capture in the packed bed columns and determine fundamental attraction parameters. This model provided an excellent fit to the experimental data. Taken together the results obtained indicate that GAC is an interesting material for SiO2 NPs filtration. With the increasing usage of III-V materials, there are concerns about the ecological threats posed by III-V ions released during semiconductor manufacturing and from disposal of decommissioned electronic devices. In Chapter 5, the acute toxicity of As(III), As(V), In(III) and Ga(III) species was evaluated using different bioassays, including three microbial assays, testing for methanogenic activity, O2 uptake and bioluminescence inhibition of marine bacterium A. fischeri. Acute toxicity to the freshwater crustacean Daphnia magna was also tested. The results showed that In(III) and Ga(III) were generally not toxic or only mildly toxic in all assays, while both As(III) and As(V) showed strong inhibitory effects on different microbial activities (methanogenic and bioluminescence). The toxicity of these ions was strongly dependent on the bioassay target. For In(III) and Ga(III), D. magna was the most sensitive organism with 50% lethal concentrations (LC50) of 57.4 and 237.0 mg/L, respectively. On the other hand, As(III) and As(V) were particularly toxic to methanogens. The 50% inhibitory concentrations (IC50) of both species were about 1.5mg/L. Mixed aerobic heterotrophic culture was highly resistant to all four ions and O2 uptake by the aerobes was not affected in the tested concentrations. Overall, the results indicate that the ecotoxicity of In(III) and Ga(III) is much lower than that of the As species. This finding is important in filling the knowledge gap regarding the ecotoxicology of In and Ga. Besides ecotoxicity, ENPs and III-V materials in CMP effluents could also pose a threat to human health. In Chapter 6, the cytotoxicity of CMP slurries to human bronchial epithelial cells (16HBE14o-) was assessed using a novel impedance based real time cell analyzer (RTCA). Cell death and detachment was observed in assays supplied with high concentrations of c-SiO2 and f-SiO2 NPs (≥250 mg/L). On the other hand, CeO2 and Al2O3 slurries were not inhibitory at concentrations up to 1250 mg/L. In addition, since CMP wastewater generated during the planarization of III-V films contains a mixture of ENPs and soluble III-V species, it is important to understand whether the interactions between these materials could alter their fate and toxicity. As(III) toxicity to human lung cells in the presence and absence of CeO2 NPs was evaluated using the RTCA assay. Exposure to As(III) (0.5 mg/L) for 48 h resulted in 81.3% inhibition of cell viability and proliferation, while cell inhibition decreased to only 13.0% when As(III) was dosed together with sub-toxic levels of CeO2 NPs (250 mg/L). This detoxification effect was mainly due to As(III) adsorption onto CeO2 NPs. When the NPs were added, the soluble arsenic concentration was reduced significantly from 0.5 mg/L to 0.03 mg/L. This work demonstrates that adsorption of As(III) on CeO2 NPs can lower As(III) concentration in the solution and reduce its bioavailability and subsequently result in As(III) detoxification. In conclusion, this dissertation indicates that the ENPs (SiO2, CeO2 and Al2O3) used in semiconductor industry are not expected to cause acute toxicity to the natural environment and human health under environmentally relevant concentration (<1 mg/L). Among the soluble III-V species, In(III) and Ga(III) showed no or mild acute inhibitory effects in different bioassays even at comparatively high concentration. However arsenic species are highly toxic to various important microbial populations in the environment and human cells. The results showed that arsenic could induce toxic effects under current discharge limit set for semiconductor industry. Finally, we demonstrated that the adsorption of As(III) on CeO2 NPs can lower the concentration of soluble As(III) and subsequently resulted in As(III) detoxification.

Nas últimas três décadas, o crescimento na demanda por biocombustíveis promoveu a expansão das culturas de cana-de-açúcar no Brasil e, consequentemente, o aumento no consumo de pesticidas. Diversas espécies de anfíbios estão sujeitas à exposição a pesticidas por ocorrerem dentro e ao redor de canaviais. Entretanto, mesmo avaliações de risco preliminares são inviabilizadas pela raridade de estudos ecotoxicológicos com espécies de anfíbios nativas. Esta dissertação propôs testar experimentalmente as hipóteses de que (1) os principais herbicidas utilizados na cultura de cana-de-açúcar no Brasil, embora desenvolvidos especificamente para o controle de ervas daninhas, são capazes de causar mortalidade em larvas de anfíbios (2) esta mortalidade se manifesta mesmo quando larvas de anfíbios são expostas a concentrações ambientalmente relevantes destes herbicidas (3) além de efeitos letais, a exposição de larvas de anfíbios a concentrações ambientalmente relevantes dos principais herbicidas utilizados na cultura da cana-de-açúcar no Brasil causa efeitos subletais em termos de crescimento, desenvolvimento, comportamento, imunocompetência, e atividade enzimática, e estes efeitos subletais podem ser importantes em influenciar o rendimento dos indivíduos. Foram utilizadas como modelo biológico larvas do leiuperídeo Physalaemus cuvieri e dos hilídeos Hypsiboas faber e Hypsiboas pardalis. As três espécies têm áreas de distribuição parcialmente coincidentes com a área de cultivo da cana-de-açúcar, sendo que as duas primeiras sabidamente ocorrem dentro de canaviais. Estas espécies foram expostas aos herbicidas glifosato, ametrina, 2,4-D, metribuzim e acetocloro, na forma de seus ingredientes ativos. Somados, estes herbicidas correspondem a ~2/3 do volume de herbicidas utilizados em importantes regiões produtoras de cana-de-açúcar; por sua vez, herbicidas constituem a maior parte dos pesticidas usados na cultura. Os experimentos de exposição aguda com P. cuvieri e H. pardalis demonstraram que todos os herbicidas testados são tóxicos a larvas de anfíbios, embora alguns deles apenas em concentrações bastante elevadas. Houve uma hierarquia relativamente clara na letalidade dos herbicidas testados (glifosato < metribuzim < ametrina < acetocloro). Uma comparação com os dados disponíveis da toxicidade dos mesmos herbicidas para outras espécies de anfíbios, e para outros animais aquáticos, sugere que as espécies testadas nesta dissertação tendem a ser relativamente robustas, e que, ao contrário de uma hipótese amplamente difundida, anfíbios não aparentam ser particularmente sensíveis a contaminantes. Os experimentos de exposição crônica procuraram simular cenários realistas de exposição ao manipular as doses mínimas (H. faber) e máximas (H. faber, H. pardalis) de aplicação recomendadas pelos fabricantes para o controle de ervas daninhas na cultura de cana-de-açúcar. Conforme esperado, um prolongamento da exposição induziu mortalidade em concentrações muito inferiores àquelas necessárias para causar efeito em exposição aguda. Glifosato, ametrina e acetocloro em doses ambientalmente relevantes induziram mortalidade significativa de larvas de anfíbios, ao menos para H. pardalis. Glifosato, 2,4-D e metribuzim retardaram significativamente o desenvolvimento de H. faber, enquanto que ametrina, testada apenas para H. pardalis, levou a uma diminuição no ganho de massa e no estágio de desenvolvimento final atingido pelas larvas. A atividade de AChE foi inibida pelo metribuzim em H. faber e estimulada pelo glifosato, ametrina e 2,4-D em H. pardalis; por sua vez, a atividade de GST, inalterada pela exposição a herbicidas em H. faber, foi estimulada pela ametrina e pelo acetocloro em H. pardalis. Nenhum composto induziu alterações significativas no perfil leucocitário. Herbicidas também tiveram efeitos comportamentais: a taxa de atividade de H. pardalis foi significativamente reduzida sob exposição à ametrina e ao acetocloro. Esta dissertação sugere portanto que a exposição a doses ambientalmente relevantes dos principais herbicidas utilizados em cultura de cana-de-açúcar no Brasil podem causar efeitos letais e subletais em larvas de espécies nativas de anfíbios. Alguns destes efeitos subletais, como uma diminuição nas taxas de atividade, de crescimento e de desenvolvimento, podem ter importantes consequências para o sucesso dos indivíduos por prolongar o período larval e assim aumentar o risco de dessecação em corpos d´água temporários e semi-permanentes. Estudos futuros devem buscar realismo adicional rumo a uma análise de risco da contaminação ambiental por herbicidas por meio de experimentos manipulando não apenas ingredientes ativos, mas também formulações comerciais, bem como interações dos contaminantes entre si e com outros estressores ambientais, calibrados por concentrações medidas em campo. / In the last three decades, the increasing demand for biofuels in Brazil favored the expansion of sugarcane and, as a consequence, an increase in the consumption of pesticides. Several species of amphibians are subject to pesticide exposure for occuring in or around sugarcane fields. However, a preliminary risk assessment of pesticide exposure is not possible due to the absolute scarcity of ecotoxicological studies with native amphibian species. This thesis proposed to test the hypotheses that (1) the main herbicides used in sugarcane crops in Brazil, although designed for weed control, cause mortality of amphibian larvae (2) this mortality manifests even when amphiban larvae are exposed to environmentally relevant herbicide concentrations (3) in addition to any lethal effects, exposure of amphibian larvae to environmentally relevant herbicide concentrations causes sublethal effects in terms of growth, development, behavior, immunocompetence, and enzimatic activity, and these effects may be important in influencing individual performance. We used as model system the leiuperid frog Physalaemus cuvieri and the hylid frogs Hypsiboas faber and Hypsiboas pardalis. The distributional ranges of these species partially overlap with the region of cultivation of sugarcane in the country, and the first two species are known to colonize waterbodies in or around sugarcane fields. These three species were exposed to glyphosate, ametryn, 2,4-D, metribuzin and acetochlor, presented as active ingredients. These herbicides account to ~2/3 of the volume of herbicides employed in an important sugarcane producing region in the country; herbicides correspond to the majority of pesticides used in this crop. Experiments of acute exposure of P. cuvieri and H. pardalis showed that all herbicides are toxic to amphibian larvae, albeit some of them only in very high doses. There was a relatively consistent ranking in lethality among the compounds (glyphosate < metribuzin < ametryn < acetochlor). A literature review suggests that the species tested tend to be relatively robust when compared to other amphibians and other freshwater animals, and that, contrary to a broadly disseminated hypothesis, amphibians do not seem to be particularly sensitive to contaminants. Experiments of chronic exposure aimed at simulating realistic scenarios of exposure by manipulating minimum (H. faber) and maximum application doses (H. faber, H. pardalis), as recommended by manufacturers for weed control in sugarcane fields. As expected, prolonged exposure induced mortality at much lower concentrations than those manipulated in the experiments of acute exposure. Environmentally relevant concentrations of glyphosate, ametryn and acetochlor induced significant mortality in larval amphibians, at least for H. pardalis. Glyphosate, 2,4-D and metribuzin significantly slowed development in H. faber, whereas ametryn slowed development and growth in H. pardalis. Herbicides also had behavioral effects: activity rates of H. pardalis were significantly reduced under exposure to ametryn and acetochlor. AChE activity was inhibited by metribuzin in H. faber but stimulated by glyphosate, ametryn and 2,4-D in H. pardalis; in turn, GST activity, which was not influenced by exposure to herbicides in H. faber, was stimulated by ametryn and acetochlor in H. pardalis. No herbicide induced significant changes in leukocyte profiles. In conclusion, this thesis suggests that exposure to environmentally relevant doses of the main herbicides employed in sugarcane fields in Brazil can cause lethal and sublethal effects on native species of larval amphibians. Some of these sublethal effects, including the decrease in activity, growth and developmental rates, may have important consequences for individual performance for extending the larval period and as such increasing the risk of dessication in temporary and semi-permanent ponds. Future studies should seek additional realism towards a risk analysis of the environmental contamination by herbicides through experiments manipulating not only active ingredients but also commercial formulations, as well as interactions among contaminants and other environmental stressors, calibrated by field-measured conditions.

Os éteres de difenilas polibromados (PBDEs) são largamente usados como retardantes de chama e têm sido detectados em diversas amostras biológicas, devido às suas características físico-químicas e bioacumuladoras, as quais fazem com que estes compostos tenham uma elevada persistência no meio ambiente. A exposição a estes compostos pode ser nociva ao homem e aos organismos vivos, uma vez que muitos estudos têm relatado que este composto possui atividade citotóxica. Desta forma, no presente estudo, investigamos o mecanismo de ação dos BDE-47, BDE-99, BDE-154 e BDE-209 sobre o processo autofágico de células HepG2, pois além de ser um processo de reciclagem de moléculas intracelular, a autofagia atua ainda na citoproteção celular. Foram realizados ensaios de imunocitoquímica para detecção de alterações no processo autofágico, no qual foi avaliado, sob microscopia de fluorescência, o padrão de distribuição das proteínas LC3-I e LC3-II; paralelamente, foi realizada a técnica de western blotting para a quantificação de três proteínas associadas ao processo de autofagia - LC3, Beclin-1 e p62. Em nossas análises de imunocitoquímica pode-se observar que os quatro PBDEs em estudo foram capazes desencadear o processo de autofagia, sendo que a magnitude do efeito tóxico foi dependente do tempo de exposição (24 e 48 horas), da concentração utilizada (0,1; 1; 5; 25 ?mol/L) e variável de acordo com o congênere em questão, sendo os BDE-47 e BDE-99 os que apresentaram maior potencial de ativação da autofagia. Além disso, esta indução de autofagia ainda pode ser detectada nos ensaios de western blotting, pois os PBDEs alteraram os níveis de LC3-II, Beclin-1 e p62, havendo um padrão de toxicidade semelhante ao visualizado nos ensaios de imunoistoquímica. Adicionalmente, devido ao fato dos PBDEs poderem se acumular no meio ambiente, principalmente em organismos aquáticos, uma análise dos efeitos destes compostos sobre um modelo ecotoxicológico foi realizada. O zebrafish (D. rerio) foi utilizado como organismoteste, a fim de avaliar a toxicidade destes contaminantes ambientais sobre seus estágios embrionários e larvais. Foram analisados endpoints de letalidade, subletalidade e teratogenicidade após a exposição aos PBDEs em estudo. Sendo que os compostos BDE-47, BDE-99 e BDE-209 (na presença de luz) apresentaram alterações em pelo menos um dos indicadores avaliados, sendo que o parâmetro mais afetado foi a inflação das bexigas natatórias, indicando uma possível teratogenicidade causada por estes compostos. Por outro lado, os BDE-154 e BDE- 209 (na ausência de luz) não causaram malformações no organismo-teste. Diante dos resultados obtidos, pode-se concluir que estes compostos podem causar alterações na homeostase celular desencadeando o processo autofágico, sendo que se absorvidos por organismos, podem afetar o desenvolvimento de espécies como o zebrafish. / Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and they have been detected in several biological samples due to their physicochemical and bioaccumulating characteristics, which make these compounds to have a high persistence in the environment. The exposure to these compounds can be harmful to man and other living organisms, since many studies have reported that this compound has cytotoxic activity. Thus, in the present study, we investigated the mechanism of action of the BDE-47, the BDE-99, the BDE-154 and the BDE-209 on the autophagic process of HepG2 cells, because beyond being an intracellular molecule recycling process, autophagy acts also in the cell cytoprotection. Immunocytochemistry assays were performed to detect changes in the autophagic process, in which the distribution pattern of the LC3-I and LC3-II proteins was evaluated under fluorescence microscopy. In parallel, the western blotting technique was used to quantify three proteins associated with the autophagy process - LC3, Beclin and p62. In our analyzes of immunocytochemistry it can be observed that the four PBDEs in the study were able to trigger the autophagy process, and the magnitude of the toxic effect was dependent on the exposure time (24 and 48 hours), on the used concentration (0.1; 1; 5; 25 ?mol/L), and it varies according to the congener. The BDE-47 and BDE-99 are the ones with the highest potential for autophagy activation. In addition, this autophagy induction can still be detected in the western blotting assays, because the PBDEs altered the levels of LC3-II, Beclin-1 and p62, with a similar pattern of toxicity as seen in the immunohistochemical assays. In addition, due to the fact that PBDEs may accumulate in the environment, mainly in aquatic organisms, an analysis of the effects of these compounds on an ecotoxicological model was performed. The Zebrafish (D. rerio) was used as a test organism in order to evaluate the toxicity of these environmental contaminants on their embryonic and larval stages. In this study, lethality, sublethality and teratogenicity endpoints were analyzed after exposure to the PBDEs. The BDE-47, BDE-99 and BDE-209 compounds (in the presence of light) presented alterations in at least one of the indicators evaluated, and the most affected parameter was the swim bladder inflation, indicating a possible teratogenicity caused by these compounds. On the other hand, the BDE-154 and the BDE-209 (in the absence of light) did not cause malformations in the test organism. Considering the results, it can be concluded that these compounds are able to cause changes in cellular homeostasis triggering the autophagic process, and whether absorbed by organisms, they can affect the development of species such as zebrafish.

Neste trabalho foi feito o desenvolvimento e validação de métodos para a determinação do hormônio 17\'alfa\'-metiltestosterona em amostras de água e de sedimentos de uma piscicultura situada no município de Socorro-SP. O local de estudo foi escolhido porque no município de Socorro há um grande número de pisciculturas e também pelo fato da cidade fazer parte do Circuito das Águas. Toda a água utilizada no processo de reversão sexual das tilápias do Nilo (Oreochromis niloticus) é descartada no Ribeirão dos Machados, um dos afluentes do rio Mogi-Guaçu, sem o devido tratamento. Foi desenvolvido o método analítico para a extração do hormônio das amostras de água, empregando a extração em fase sólida com adsorvente C18 (octadesilsilano). Também foi desenvolvido o método para a extração do hormônio das amostras dos sedimentos, empregando o equipamento de ultra-som. Foram feitos estudos de recuperação para as amostras de água e sedimentos em três níveis de fortificação. Empregou-se 300 mL da água de água de nascente (branco), e água do rio Santa Maria do Leme para os estudos de recuperação. Empregou-se 10 g de sedimentos (branco) coletados no rio Santa Maria do Leme, em São Carlos. Estas amostras foram fortificadas com o hormônio estudado. A técnica analítica empregada para a determinação do hormônio foi a cromatografia líquida de alta eficiência com detector UV-Vis e coluna RP-18. Para a determinação do hormônio foi utilizada a fase móvel acetonitrila/água 35:65 v/v. O volume injetado foi de 20 \'mü\'L, o fluxo de 1,0 mL/min e comprimento de onda de 245 nm. Os resultados indicaram que os métodos desenvolvidos e validados se mostraram lineares, sensíveis, seletivos, precisos e exatos nas condições estabelecidas, com limites de detecção e de quantificação adequados para a análise do hormônio. A aplicação desses métodos só foi possível pela adição de padrão, que possibilitou a identificação e quantificação do hormônio nas amostras de água e de sedimentos. Foram também feitos testes de ecotoxicidade do hormônio empregando-se os organismos Daphnia similis e Ceriodaphnia silvestrii para verificar a reprodução das espécies. Foram feitos testes para verificar a reversão sexual com a Ceriodaphnia silvestrii. Foi utilizado o programa computacional TOXSTAT 3.3 para a aplicação dos testes estatísticos com a Daphnia similis, pois os testes com a espécie Ceriodaphnia silvestrii não apresentou o número de 15 neonatos por organismo, não sendo possível a aplicação do referido teste. Verificou-se que não ocorreu reversão sexual nos testes com a Ceriodaphnia silvestrii nas concentrações estudadas (1,0 e 50,0 \'mü\'g/L). O estudo de avaliação e identificação de substâncias emergentes deve ser incorporado nos programas de controle e gestão de recursos hídricos, porque é importante verificar se o hormônio é responsável por algum tipo de toxicidade. / In this work the development and validation of methods for the determination of 17\'alfa\'-methyltestosterone hormone in water and sediments samples was achieved. The samples studied were extracted from pisciculture sites in the city of Socorro, Sao Paulo State. This place was chosen because it has a great number of pisciculture sites and is also localized near important tourist resources, known as the \'Circuito das Águas\'. One of the main concerns is the fact that water volumes used in the process of sexual reversion of the Nile Tilapia (Oreochromis niloticus), are discarded without previous treatment in the Machado river, a tributary of Mogi- Guaçu river, the main water resource in this area. Therefore, there are evident potential threats and care must be taken to analyze the potential contamination of water resources in the neighborhood. Two analytical methods were developed to extract hormone from water and sediments samples: a) using solid phase extraction with sorbente C18; b) using ultrasonic facilities. The recovering of the water and sediments samples was made in three levels of fortification. To perform comparisons, standards were built using 300 mL water samples collected in safe places and samples of fortication in the Santa Maria do Leme river, in Sao Carlos, and 10 g of sediments collected in the Santa Maria do Leme river, in Sao Carlos. These samples were contaminated with hormone under carefully controlled conditions. Hormone level determinations were carried out using HPLC with UVVis detector and column RP-18. The hormone determinations were made using the mobile phase acetonitrile/water 35:65 v/v. The experiments were carried out under the following conditions: a) the injected volume was 20 \'mü\'L; b) the flow speed was 1,0 mL/min ; c) a wavelength of 245 nm was used. The results obtained shows efficiency of the methods developed and validated in this as the correlations observed were linear, sensible, selective and accurate under the established conditions. Therefore, the experimental conditions used for detention and quantification were adequate for hormone analysis. Is worth to note that the identification and quantification of the hormone in the samples of water and sediments, is possibly only by making comparison with the standard discussed above. The organisms Daphnia similis and Ceriodaphnia silvestrii were also used to verify the ecotoxicity of the hormone, using reproduction of these species as the parameter to be monitored. Therefore, tests were made to verify the sexual reversion of Ceriodaphnia silvestrii. In these investigations, the computational program TOXSTAT 3.3 was used to perform the statistical tests. Nevertheless, only the studies performed with the Daphnia similis had statistical means, as the tests with the Ceriodaphnia silvestrii species did not present statistical significance. Therefore, it was verified that Ceriodaphnia silvestrii in the conditions studied here (concentrations of 1,0 and 50,0 \'mü\'g/L) did not present evidences of sexual reversion. As a main conclusion, the present study demonstrates the need of incorporating the evaluation and identification of emergent substances in the protocols used for the control and management of water resources. Among the concerns, special care must be taking to verify correlations between hormone and toxicity.

Boreal regions account for a significant portion of Canada's landmass and economic resources (e.g., forestry, mining, and oil and gas). The inadvertent release of pollutants from industrial activities within these regions stress the need for realistic ecological risk assessments, which can be attained through the use of standardized soil toxicity test methods. Despite the geophysical and economic significance of boreal regions, standardized soil toxicity test methods applicable to these regions are lacking. To help alleviate this gap in ecotoxicology, the objectives of this thesis included the development and application of a new suite of boreal species for the assessment of contaminants in boreal soils. Specifically, research was directed towards the development of a new oribatid mite test, using Oppia nitens (C.L. Koch). Oribatid mites represent the most abundant and diverse microarthropod species in forest soils, significantly contributing to nutrient cycling and soil formation processes; however, these organisms are consistently under-represented in soil ecotoxicological assessments. The applicability and limitations of the use of O. nitens were demonstrated such that lethal and sublethal endpoints could be derived from the assessment of soils. The performance of O. nitens across numerous soils varied such that adult survival was unaffected by soil characteristics, however, reproduction was limited by soil organic matter content. An evaluation of the sensitivity of O. nitens, using a model polycyclic aromatic hydrocarbon was also conducted in a standard test soil. Phenanthrene is a common contaminant in soils, and accumulates within organic-rich horizons, within which oribatid mites live. Therefore, the study was designed to examine the toxicity and bioaccumulation potential of phenanthrene to O. nitens, with a comparison of effect to other soil invertebrate species, as presented in the literature. Oppia nitens was susceptible to phenanthrene at levels comparable to other soil invertebrate species, and the bioaccumulation of the phenanthrene increased with increasing exposure concentration, although steady-state was not achieved during the four-week exposure duration. The accumulation was biphasic in nature, likely a result of initial cuticular sorption processes, followed by the contribution of other processes (e.g., dietary uptake). The elimination of the phenanthrene was not complete, in that at the end of the elimination phase, tissue residues were similar to that observed in the initial gradual accumulation (e.g., by cuticular processes). However, the resultant bioaccumulation factors (BAFs) were relatively low, indicative of limited trophic transfer and biomagnification for this species. Following the development of an O. nitens assay, a further study was conducted to compare the sensitivity of O. nitens to other boreal soil invertebrate species, as well as to standard test species. Soil toxicity tests were designed, using field-collected reference and contaminated (petroleum hydrocarbon- and salt-impacted) soils, using an expanded suite of boreal species (plants: Pinus banksiana, Picea glauca, Picea mariana, Populus tremuloides, Calamagrostis canadensis and Solidago canadensis; earthworms: Dendrodrilus rubidus; and springtails: Folsomia nivalis and Proisotoma minuta). The sensitivity of the boreal species was then compared to that of currently published standard soil toxicity test species (agronomic plants: Elymus lanceolatus and Trifolium pratense; earthworms: Eisenia andrei; and springtails: Folsomia candida). Estimated species sensitivity distributions (ESSDs) were generated to determine whether the boreal and standard test battery of species exhibited differences in their overall sensitivity to the contaminated soil. With regards to the petroleum-hydrocarbon impacted soil, the suite of boreal species was more sensitive relative to the suite of standard test species; however, upon exposure to the salt-impacted soils, no differences in sensitivity were evident between the boreal and standard suite of test species. In both soils, the performance of O. nitens was similar to that observed by the collembolan species. The evaluation of boreal species and soils also took into consideration the use of distinct soil horizons. The layering of horizons was feasible from the initial collection to reassembly for testing in the laboratory, and plant growth was unaffected by this design. However, soil invertebrates demonstrated a preference or avoidance tendency for one horizon over another, and as a result, the assessment of individual horizons was recommended for all future testing. The compilation of research presented herein provides the basis for the standardization of ecologically-relevant test species and methods for the assessment of contaminated soils in boreal regions.