Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. There are numerous studies reporting developmental cardiac toxicity in multiple fish species due to PAH exposure. However, there are relatively few instances where the effects of acute PAH exposure in adult fish have been characterized. Furthermore, the majority of experiments comparing PAH toxicity with exposure route in adult fish focus on CYP1A gene expression or enzyme activity, while there is a lack of information about the possible pathophysiological effects. Therefore, the overall objective of this thesis was to characterize the sublethal effects of benzo[a]pyrene (BaP), a prototypical PAH, on adult zebrafish (Danio rerio) cardiorespiratory function and fitness following acute exposure by two different routes. In the first experiment, adult zebrafish were intraperitoneally (i.p.) injected twice (one injection/24 hr) with increasing concentrations of BaP (0.1, 10, and 1000 μg/kg) and compared to corresponding dimethylsulfoxide (DMSO) controls. In a second set of experiments, adult zebrafish were aqueously exposed to BaP (static, renewal at 24 hr; 16.2 and 162 μg/L) and compared to DMSO controls. Following 48 hr exposure, one group of fish (n=10/treatment group) were subjected to swimming performance tests to assess critical swimming speed (Ucrit), oxygen consumption rate (MO2), cost of transport (COT), standard metabolic rate (SMR), active metabolic rate (AMR), and factorial aerobic scope (F-AS). Another group of fish (n=12/treatment group) were subjected to echocardiography following 48 hr BaP exposure to evaluate cardiac function. Following echocardiography analysis, samples were collected for parent compound (BaP) body burden and CYP1A mRNA induction analysis. 48 hr BaP injection resulted in significant sublethal effects on adult zebrafish cardiorespiratory function. Oxygen consumption (MO2) was increased at three swimming speeds in injected BaP groups compared to control. In contrast, aqueously BaP-exposed fish showed increased MO2 only at the single lowest swim speed. COT was also similarly increased for both exposure routes. SMR was elevated with both exposure routes, while AMR remained unchanged. This resulted in a significant decrease in F-AS for all treatment groups compared to corresponding controls with both exposure routes. Cardiac function was significantly affected by both routes of BaP exposure. Ventricular heart rate was significantly decreased in BaP-exposed fish, both injected and aqueously-exposed. However, stroke volume was decreased only in fish aqueously exposed to BaP, which resulted in significantly reduced cardiac output with that exposure route. In contrast, the ratio of atrial to ventricular heart rate (AV ratio) was increased only in fish i.p. injected with BaP, indicating the possibility of cardiac arrhythmias occurring. Analysis of BaP body burdens in fish tissue allowed for identification of an overlapping dose group between exposure routes, through which comparisons of cardiotoxicity were then made. This comparison revealed slight differences in cardiotoxicity between exposure routes. BaP-injected fish suffered from more severe bradycardia than aqueously exposed fish. Furthermore, cytochrome P4501A (CYP1A) mRNA levels in liver and heart tissue showed more significant increases in injected fish, while skeletal muscle CYP1A was increased only following aqueous exposure. In conclusion, acute BaP exposure caused metabolic alterations and impaired cardiorespiratory function in adult zebrafish regardless of exposure route. Interestingly, the primary mechanism behind these effects appeared to differ slightly with exposure route. These results suggest that acute BaP exposure may have negative effects on adult fish survivability in the environment. Overall, this work provides valuable insight into the pathophysiogical consequences of acute PAH exposure in adult stage fish.

1 Abstract The goal of the thesis is to determine acute toxicity indices of seven alcohols (2-ethoxyethanol, 1-propanol, 3-methyl-1-butanol, 3-methyl-2-butanol, 3-hexanol, 2-methyl-3-hexanol, 1-nonanol) by two different alternative methods. Another goal is to create QSAR models to predict the acute toxicities of alcohols which belong into model limits. First of the methods used is based on the movement inhibition of lower animals Tubifex tubifex. The method provides the acute toxicity indices EC50. The second method uses the cell line of mouse fibroblasts called Balb/c 3T3 and it provides the acute toxicity indices IC50. The IC50 were recalculated to the LD50 values that correspond to the oral administration of alcohols to a rat. It has been found that 1-nonanol is included into the category of alcohols classified as acute oral toxic according to the EU CLP system. Other six alcohols are non-classified as acute oral toxic. Subsequently, accurate QSAR models has been created in order to predict the acute toxicity of other alcohols using Tubifex tubifex specie or Balb/c 3T3 cell line. The models are suitable for saturated, branched, ethoxy- and cyclic alcohols with the log POW value from −0.74 to 3.07. It was necessary to exclude the acute toxicity indices of 1-nonanol from these models as some solubility...

The larval and pupal stages of Chironomus riparius are benthic dwellers which ingest sediment, and are commonly prey. In an environment with elevated selenium concentrations, they can accumulate it and transfer it to higher trophic level organisms upon being ingested. This study was conducted to: 1. Develop a method for performing acute toxicity tests using Chironomus riparius. 2. Establish relationships between dry mass and selenium content, and between dry mass and selenium body burden. 3. Determine whether bioaccumulation or bioconcentration contributed more to the selenium content and body burden of individual larvae. 4. Determine selenium concentrations which were acutely and chronically toxic to C. riparius, and whether previous exposure changed the acutely toxic concentration. 5. Evaluate adequacy of national surface water selenium criterion with respect to these data. In acute toxicity tests, 1 larva per 4 ml test solution was an acceptable organism density, and test solutions were successfully renewed by siphoning and refilling the test chamber. Regression showed that larval selenium content was linearly dependent on dry mass within instar, and body burden was negatively exponentially dependent on dry mass. Predicted body burdens were linearly dependent on dissolved selenium concentration. This indicated that bioconcentration was more important than bioaccumulation in determining body burdens. C. riparius readily acclimated after prolonged exposure to dissolved selenium, but became slightly more sensitive to selenium after prolonged exposure to substrate-adsorbed selenium. The national water quality selenium criteria may not protect against biomagnification in all ecosystems, and should be based on some environmental chemical factors. / Master of Science

The toxicity of ammonia has become an issue in recent years, especially in the Chesapeake Bay watershed. Currently, the EPA has set a chronic criterion of 0.035 mg/L (unionized ammonia). The chronic criterion is a four-day average concentration that cannot be exceeded more than once every three years. However, a lack of data exists on the toxicity of ammonia to saltwater organisms. Prior to this research, the chronic criterion was based on two saltwater and four freshwater organisms. This research was conducted to add additional data so more appropriate criteria may be set. Two saltwater fish, the Summer Flounder (Paralichthys dentatus), and the Atlantic Silverside (Menidia menidia) and one invertebrate, the Quahog Clam (Mercenaria mercenaria) were tested for acute toxicity to ammonia. The acute tests performed on the Summer Flounder (2 months old) were 48 and 96-hour tests performed in both synthetic seawater and natural seawater. Seven-day chronic tests were also performed on the Summer Flounder using synthetic seawater. Both 48 and 96-hour acute tests were performed on the Atlantic Silverside (10-14 days old.) using natural seawater. Both 48 and 96-hour acute tests were performed on the Quahog Clam (5mm shell height) using synthetic seawater. The 48-hour LC50s for the Summer Flounder in synthetic and natural seawater were 1.22 mg/L and 1.09 mg/L, respectively. The 96-hour LC50s for the Summer Flounder in synthetic and natural seawater were 1.07 mg/L and 0.889 mg/L, respectively. The 7-day chronic NOEC was 1.37 mg/L. The 48 and 96-hour LC50s for Atlantic Silverside in natural seawater were 1.52 mg/L and 1.18 mg/L, respectively. The 48 and 96-hour LC50s for Quahog Clam in synthetic seawater were 216 mg/L and 36.6 mg/L, respectively. The acute-chronic ratio for the Summer Flounder was 2.27. The refined chronic criterion was 0.081 mg/L based on this research and the research of others. Summer Flounder were more sensitive to ammonia in natural seawater than synthetic seawater. However, Atlantic Silverside showed no difference in sensitivity. / Master of Science

The toxicological screening of the numerous chemicals that we are exposed to requires significant cost and the use of animals. Accordingly, more efficient methods for the evaluation of toxicity are required to reduce cost and the number of animals used. Computational strategies have the potential to reduce both the cost and the use of animal testing in toxicity screening. The ultimate goal of this thesis is to develop computational models for the prediction of toxicological endpoints that can serve as an alternative to animal testing. In Paper I, an attempt was made to construct a global quantitative structure-activity relationship (QSAR)model for the acute toxicity endpoint (LD50 values) using the Munro database that represents a broad chemical landscape. Such a model could be used for acute toxicity screening of chemicals of diverse structures. Paper II focuses on the use of acute toxicity data to support the prediction of chronic toxicity. The results of this study suggest that for related chemicals having acute toxicities within a similar range, their lowest observed effect levels (LOELs) can be used in read-across strategies to fill gaps in chronic toxicity data. In Paper III a k-nearest neighbor (k-NN) classification model was developed to predict human ether-a-go-go related gene (hERG)-derived toxicity. The results suggest that the model has potential for use in identifying compounds with hERG-liabilities, e.g. in drug development.

A poluição marinha por petróleo e seus derivados é uma preocupação crescente de caráter global. O litoral norte do estado de São Paulo é hoje caracterizado como a região mais altamente impactada por esses contaminantes de toda a costa Sul e Sudeste do país. Dentre as substâncias que compõem esses produtos estão os Hidrocarbonetos Aromáticos Policíclicos (HPAs), como o fenantreno, que apresentam potencial de alterar o sistema endócrino de peixes, gerando desajustes hormonais de alto risco para o processo reprodutivo dos animais expostos. Com base nestes fatos, o presente trabalho buscou verificar efeitos do fenantreno como desregulador endócrino em juvenis de E. marginatus. Essa espécie se caracteriza pelo hermafroditismo protogínico, de modo que os juvenis costumam apresentar altas concentrações de esteroides gonadais, o que gera preocupação quanto os efeitos reprodutivos decorrentes de alterações no padrão de produção de esteroides, especialmente em uma espécie que já vem sofrendo declínios populacionais por conta da sobrepesca. A CL50 encontrada para juvenis de E. marginatus foi 1,51 mg⁄L. Em seguida, foi realizado um bioensaio subletal (96h) com quatro grupos experimentais (n=5) em duplicata: grupo controle; duas concentrações distintas de fenantreno (0,1 mg⁄L e 1 mg⁄L) e grupo exposto ao veículo de diluição (0,004% de etanol). Foram dosadas as concentrações de 17β-estradiol (E2), testosterona (T) e 11-cetotestosterona (11-KT) plasmáticos por ELISA. Gônadas, fígado e baço foram processados para análise histológica. Foi conduzido bioensaio in vitro com incubação de fragmentos gonadais sob ação de fenantreno (50μM) ou etanol. Os níveis dos mesmos esteroides também foram dosados por ELISA. A exposição in vivo ao fenantreno resultou em aumento na área dos hepatócitos, bem como no número de centro melanomacrofágicos e hemossiderose no baço. O etanol induziu efeitos similares no baço. Os níveis de E2 e T não se alteraram no plasma ou no meio de incubação in vitro. O fenantreno reduziu a concentração de 11-KT in vitro e in vivo. No plasma, o etanol também causou esse decréscimo. Considerando a importância do 11-KT no desenvolvimento dos ovários em teleósteos, o fenantreno parece causar desregulação da esteroidogênese em juvenis de E. marginatus, possivelmente gerando disfunções durante a inversão sexual / Marine pollution by crude oil and its residual products is a growing global concern. The Northern coast of São Paulo is characterized as the highly impacted area by these contaminants through Brazil\'s South and Southeast coast. Polycyclic aromatic hydrocarbons (PAH), such as phenanthrene, are the main crude oil components and have a significant toxic potential to biota, acting as endocrine disruptors (ED) and negatively impacting reproduction. This study aimed to investigate the effects of phenanthrene as ED on gonadal steroidogenesis in E. marginatus juveniles. As a hermaphrodite protogynous species, E. marginatus juveniles maintain high levels of plasma steroids as substrates for sex inversion, which causes concern about reproductive effects of disruptions in steroid production patterns, especially in an overfished species. LD50 to E. marginatus exposed to phenanthrene was established in 1.51 mg⁄L. An in vivo sublethal exposure (96 h) to phenanthrene was carried out at two concentrations (0.1mg⁄L and 1 mg⁄L); exposure to the vehicle (ethanol) was also performed. Plasma levels of 17β-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT) were measured by ELISA. Gonads, liver and spleen were processed for histological analysis. In an in vitro bioassay, gonad fragments were incubated with phenanthrene (50μM) or ethanol. Steroid levels in the culture media were also measured by ELISA. The in vivo exposure to phenanthrene triggered an increase of the area of the hepatocytes, increased number of melanomacrophagic centers and hemosiderosis in the spleen. Ethanol also induced similar effects on spleen. E2 and T levels did not change neither in plasma nor in vitro media. Phenanthrene sharply reduced 11-KT levels in vitro and in vivo. In plasma, ethanol also decreased 11-KT levels. Considering the importance of 11-KT in fish developing ovaries, phenanthrene seems to disrupt steroidogenesis in juvenile grouper, possibly being able to cause dysfunctions during sex change

A produção de frango de corte é acompanhada por uma geração elevada de resíduos. Assim, o reuso da cama de aviário como adubo tem se mostrado como uma alternativa viável para a destinação adequada deste resíduo. Entretanto o emprego de fármacos utilizados na produção de aves de corte pode atingir o ambiente e gerar efeitos indesejados tanto na microbiota do solo quanto nos organismos aquáticos de ambientes lacustres próximos aos galpões de produção. O objetivo deste trabalho foi quantificar os fármacos norfloxacina (NOR), enrofloxacina (ENR) e ciprofloxacina (CIP) nas matrizes cama de aviário, solos, águas superficiais, subsuperficiais e sedimentos e avaliar o impacto desta contaminação na microbiota do solo e organismos aquáticos. As coletas foram realizadas em seis granjas de produção de frango de corte em municípios na região de Piracicaba/SP. As extrações e posterior determinação dos antimicrobianos nas amostras de cama de aviário e solo foram realizadas pela técnica de cromatografia em fase líquida com detector de arranjo de diodos/ultravioleta (HPLC-DAD/UV) e em água superficial, subsuperficial e sedimento foram realizadas por extração em fase sólida acoplada à cromatografia líquida e espectrometria de massas em tandem (on-line-SPE-LC-MS/MS). A atividade microbiana dos solos foi determinada pelo método da radiorespirometria utilizando 14C-glicose. A estrutura e abundância da comunidade bacteriana foram determinadas pelas técnicas da Reação em Cadeia da Polimerase - Eletroforese em Gel de Gradiente Desnaturante (PCR/DGGE) e da PCR em tempo real (qPCR). Os testes de toxicidade aguda foram realizados com os organismos aquáticos Daphnia magna e Danio rerio. Foram encontradas concentrações entre 1,20 e 8,31 mg kg-1 de fluoroquinolonas (FQs) em cama de aviário, havendo o acúmulo desses fármacos com seu tempo de uso (até 135 dias). As concentrações de FQs nos solos (10,2 a 325 ?g kg-1) foram acima dos níveis tolerados (100 ?g kg-1). Não houve efeitos diretos na atividade microbiana, entretanto houve redução na abundância e modificação na estrutura da comunidade bacteriana em determinadas granjas. Nas amostras de água os resultados foram inferiores ao limite de quantificação (20 ng L-1). Em sedimentos foram encontrados valores entre 22,63 e 76,54 ng kg-1 de FQs. Os resultados de toxicidade aguda para D. magna (CE50- 48 h) foram: 92,49 mg L-1 e 73,04 mg L-1 para ENR e NOR, respectivamente, e para Danio rerio a CL50 (96 h) foi 190,9 mg L-1 para ENR. Observou-se lixiviação de FQs em solos até 4 m de profundidade. Conclui-se que houve contaminação do solo afetando a comunidade bacteriana. Não houve contaminação de águas superficiais e subsuperficiais, sendo que ENR e NOR somente causam toxicidade aguda em organismos aquáticos em concentrações muito acima das encontradas em campo. Esses resultados sugerem medidas mitigadoras de contaminação, como a exposição prévia da cama de aviário à luz solar e a modernização dos sistemas de bebedouro dos galpões, uma vez que a água descartada foi um importante veículo das FQs para a cama de aviário e entorno da granja / The broiler production generates a lot of waste. So the reuse of poultry litter as a fertilizer has proven to be a viable alternative for the appropriate destination of these residues. However the employment of medicines used in the broilers production can reach the environment and cause negative effects in soil microbiota and on aquatic organisms from water bodies close to the production sheds. The objective of this study was to quantify norfloxacin (NOR), enrofloxacin (ENR) and ciprofloxacin (CIP) in poultry litter, soil, surface and subsurface water, sediment and evaluate the impact of these contamination on soil microbiota and aquatic organisms. Samples were collected in six farms in Piracicaba/SP region. Analyses of antibiotics in soil samples and poultry litter were performed by high performance liquid chromatography-diode array/ultraviolet detector (HPLC-DAD/UV) and in surface, subsurface water and sediment were performed by an online solid phase extraction-liquid chromatography tandem mass spectrometry method (on-line SPE-LC-MS/MS). The microbial soils activities were determined by radiorespirometry using 14C-glucose. The diversity and abundance of bacterial communities were determined by Polymerase Chain Reaction/Denaturing Gradient Gel Electrophoresis (PCR/DGGE) and real-time PCR (qPCR). Acute toxicity tests were performed to aquatic organisms Daphnia magna and Danio rerio. It was found concentrations between 1.20 and 8.31 mg kg-1 of fluoroquinolones (FQs) in poultry litter and these antibiotics accumulated in time (up to 135 days). In soils, concentrations of FQs were between 10.2 and 325 ?g kg-1, these values were higher than the allowable levels (100 ?g kg-1). There were no direct effects on soil microbial activity, however there were reduction in abundance and diversity of bacterial community in some farms. In water samples the results were lower than the limit of quantification (20 ng L-1). In sediment, values of FQs were between 22.63 and 76.54 ng kg-1. Acute toxicity for D. magna (CE50-48 h) were 92.49 mg L-1 and 73.04 mg L-1 for ENR and NOR, respectively, and for Danio rerio LC50 (96h) was 190.9 mg L-1 for ENR. It was observed FQs leaching in soil up to 4 m depth. It was concluded that soil contamination impacts bacterial community. There was no contamination in superficias and subsurface waters. ENR and NOR only cause acute toxicity to aquatic organisms in higher concentrations than those found in the environment. These results suggest mitigation measures for contamination, as previous exposure of poultry litter to sunlight and the modernization of water systems, once the wastewater was an important vehicle for FQs to poultry litter and environment

Entre as principais dificuldades que os sistemas de tratamento de esgotos domésticos compostos por lagoas de estabilização enfrentam para atender as exigências impostas pela Resolução CONAMA 357/2005 podem se destacar as concentrações elevadas de nitrogênio amoniacal, fósforo e coliformes fecais e totais. Estes últimos, quando os sistemas utilizam corpos receptores classe 2 ou 3 geram a necessidade de desinfecção. Dentre as diversas tecnologias disponíveis para a desinfecção dos esgotos, a cloração é bastante atrativa pelo custo reduzido, em relação às outras tecnologias, bem como pela elevada experiência que as companhias e municipalidades possuem neste tipo de sistema, advinda dos sistemas de tratamento de água. Dentre os sistemas de cloração, o uso de hipoclorito de sódio torna-se interessante pela segurança, simplicidade das instalações e de operação. A desvantagem do uso deste tipo de sistema se dá na possibilidade da formação de subprodutos tóxicos, dentro os quais pode-se destacar os trihalometanos (THMs) e os ácidos haloacéticos (AHAs), porém esta formação pode ser teoricamente reduzida pela presença de elevadas concentrações de nitrogênio amoniacal nos efluentes destes sistemas, por sua reação preferencial com o cloro, na formação de cloraminas. Outras possibilidades de redução na formação é reduzir a concentração de precursores, reduzir a dosagem de cloro e utilizar método de descloração dos efluentes após a desinfecção. Este trabalho, ambientado no desenvolvimento das pesquisas do Edital 4 do PROSAB/FINEP (Programa Nacional de Pesquisa em Saneamento Básico), desenvolveu testes de desinfecção em efluentes de lagoas anaeróbias e facultativas utilizando como desinfetante hipoclorito de sódio, em dosagens de 8,0 mgCl/L e 12,0 mgCl/L. Desenvolveu também testes de desinfecção com efluentes anaeróbios e facultativos pós-tratados por meio de coagulação/floculação/sedimentação e coagulação/floculação/flotação com ar dissolvido, com dosagens de 2,0 mgCl/L e 4,0 mgCl/L. Para todos os testes de desinfecção foram realizados também testes de descloração das amostras. Desta forma foi possível estudar, dentro de uma faixa limitada, as possibilidades de reduzir a formação de subprodutos tóxicos na desinfecção de efluentes de lagoas de estabilização com hipoclorito de sódio. Nas amostras foram determinados por meio de cromatografia gasosa as concentrações dos THMs e AHAs. Por meio das metodologias do Standard Methods 20th Edition foram realizadas as análises físico-químicas dos parâmetros pH, temperatura, DQO, DBO, Ntotal, N-NH3, NTK, cloro livre, cloraminas, coliformes e outros microrganismos indicadores. A formação de THMs se mostrou bastante pequena, em média abaixo dos 20,0 µg/L em todos os testes, aparentemente devido às elevadas concentrações de N-NH3, da ordem de 30,0 mg/L. O mesmo não ocorreu com os AHAs. Em muitos testes a concentração encontrada de AHAs totais superou o padrão de 80,0 µg/L preconizados pela USEPA para água potável, e aqui adotado como padrão comparativo. A descloração reduziu em média 52% a concentração de AHAs nos testes, colocando quase todas as amostras dentro do padrão de 80,0 µg/L. Os bioensaios mostraram que a presença de cloro livre nas amostras eleva a toxicidade aguda em Daphnia similis provocada pelos efluentes e que a cloração seguida de descloração produz efluentes com toxicidade inferior à toxicidade dos efluentes apenas clorados e toxicidade também inferior àquela dos efluentes sem cloração. / The main difficulties that the stabilization ponds for sewage treatment systems face to meet the requirements imposed by CONAMA Resolution 357/2005 can highlight the high concentrations of ammonia nitrogen, phosphorus and fecal and total coliform. When the systems use receivers water bodies Class 2 or 3 generate the need for disinfection. Among the different technologies available for the disinfection of sewage, the chlorination is very attractive at reduced cost compared to other technologies, as well as the high experience that companies and municipalities have in this type of system, because the water treatment systems. About the chlorination technologies, the use of sodium hypochlorite becomes interesting for security, simplicity of installation and operation. The disadvantage of using this type of system is given the possibility of formation of toxic by-products, within which you can highlight the trihalomethanes (THMs) and haloacetic acids (AHAs), but this formation can theoretically be reduced by the presence of high concentrations of ammoniacal nitrogen in effluents of these systems, for its preferential reaction with the chlorine in the formation of cloraminas. Other possibilities for reducing the training is to reduce the concentration of precursors (organic matter), reduce the dosage of chlorine and use dechlorination method after disinfection. This work, developed in PROSAB / FINEP (National Program for Research on Sanitation) research announcement number 4, developed tests for disinfection of anaerobic and facultative ponds effluents using sodium hypochlorite as a disinfectant, in concentrations of 8.0 mgCl/L and 12.0 mgCl/L. Were also developed tests for disinfection with anaerobic and facultative ponds effluents post-treated by coagulation / flocculation / sedimentation and coagulation / flocculation / flotation with dissolved air, with concentrations of 2.0 mgCl/L and 4.0 mgCl/L. For all of disinfection tests were also conducted dechlorination tests. This made it possible to study the possibilities of reducing the formation of toxic byproducts of the stabilization ponds effluents disinfection with sodium hypochlorite. The THMs and AHAS concentrations were determined by gas chromatography. Through the methods of Standard Methods 20th Edition analyses were carried out physical-chemical parameters of pH, temperature, COD, BOD, total nitrogen, ammoniacal nitrogen, total Kjeldhal nitrogen, free chlorine, chloramines, coliforms and other indicator microorganisms. The formation of THMs was very low, on average below the 20.0 µg/L in all tests, apparently due to high concentrations of NH3-N, the order of 30.0 mg/L. The same did not occur with AHAs. In many tests found concentrations of AHAs exceeded the standard of 80.0 µg/L recommended by the USEPA for drinking water. Dechlorination reduced 52% of AHAs concentrations, in average. Bioassays showed that the presence of free chlorine in the samples increase the acute toxicity in Daphnia similes. Chlorination followed by dechlorination produces effluents with less toxicity than the toxicity of chlorinated and effluent without chlorination.

Os parabenos são conservantes utilizados em alimentos, fármacos, cosméticos e produtos de cuidado pessoal. Pertencem à família de ésteres do ácido p-hidróxibenzoico, distinguindo-se entre si pelo comprimento e ramificação da cadeia lateral alquílica. Nesse contexto, o objetivo principal dessa tese foi avaliar os efeitos dos dois parabenos mais frequentemente utilizados (metilparabeno e propilparabeno) sobre diferentes espécies de ambientes aquáticos. Para isso, inicialmente, foram realizados testes de toxicidade de curta duração com cinco espécies de organismos de diferentes grupos taxonômicos e funcionais, sendo quatro delas de regiões tropicais (Ceriodaphnia silvestrii, Chironomus xanthus, Macrothrix flabelligera e Hydra viridissima) e uma pertencente às regiões temperadas (Hydra attenuata). Os resultados obtidos nesses testes foram comparados com os resultados de literatura aberta, mostraram que Ceriodaphnia silvestrii (MeP CE50=7,56 mgL e PrP, CE50=2,90 mgL) e Macrothrix flabelligera apresentaram-se mais sensíveis em comparação aos organismos padrão de clima temperado. Num segundo momento, foram realizados testes de toxicidade com sete espécies de fungos hifomicetos aquáticos (Articulospora tetracladia Ingold, Flagellospora curta Webster, Heliscus lugdunensis Sacc. & Thérry, Lemonniera aquatica Wildeman, Lemonniera pseudofloscula Dyko et al., Tetracladium apienses R.C. Sinclair & Eicker, Tetracladium marchalianum Wild.) expostas ao conservante propilparabeno, por possuir maior ação antifúngica. Lemonniera aquatica apresentou maior sensibilidade ao propilparabeno, sendo o mais sensível de todos os fungos testados, bem como dos demais organismos aquáticos reportados na literatura. Além de confirmarem que os parabenos representam risco ecológico potencial para os ambientes aquáticos, os resultados obtidos permitiram concluir que há diferenças de sensibilidade entre as espécies tropicais e temperadas e, por isso, as espécies nativas são mais indicadas para se avaliar os efeitos tóxicos dos parabenos nos ambientes. Dada a presença e persistência dos compostos estudados por entrada contínua no ambiente, a magnitude das concentrações determinadas (ng L-1 e μg L-1), a carência de informações relativas à toxicidade e ao risco que representam para os ecossistemas tropicais, incluindo os do Brasil, sugere-se a implantação pelas agências reguladoras de medidas mais restritivas para o uso desses conservantes, visando uma melhor proteção da biota aquática. / Parabens are preservatives used in foods, pharmaceuticals, cosmetics and personal care products. They belong to the family of esters of p-hydroxybenzoic acid, distinguishing each other by the length and branching of the alkyl side chain. The main objective of this thesis was to evaluate the effects of the two most commonly used parabens, methylparaben and propylparaben, on aquatic species. For this purpose, short-term toxicity tests were initially carried out on five species of organisms from different taxonomic and functional groups: four of them from tropical regions, Ceriodaphnia silvestrii, Macrothrix flabelligera, Chironomus xanthus and Hydra viridissima, and one belonging to temperate regions, Hydra attenuata. The results obtained in these tests were compared with the results of the open literature, showing that Ceriodaphnia silvestrii (MeP, EC50 = 7.56 mg L-1 and PrP, EC50 = 2.90 mg L-1) and Macrothrix flabelligera (MeP, EC50 = 8.04 mg L-1 and PrP, EC50 = 3.85 mg L-1) were more sensitive compared to the standard organisms of temperate climate. Secondly, toxicity tests were performed with seven species of aquatic hyphomycetes fungi and only with the preservative propylparaben, because they have a higher antifungal action: Articulospora tetracladia Ingold, Flagellospora curta Webster, Heliscus lugdunensis Sacc. & Thérry, Lemonniera aquatica Wildeman, Lemonniera pseudofloscula Dyko et al., Tetracladium apienses R.C. Sinclair & Eicker, Tetracladium marchalianum Wild. Lemonniera aquatica showed greater sensitivity to propylparaben (EC50 = 1.63 mg L-1), being more sensitive than all fungi, as well as other aquatic organisms. Thus, the results obtained in the present thesis indicate that there is a difference in sensitivity between tropical and temperate species when exposed to parabens, with tropical native species being more sensitive to the effects of these compounds. Given the presence and persistence of the compounds studied by continuous entry into the environment, the magnitude of the concentrations determined (ng L-1 and μg L-1), the lack of toxicity and risk information for tropical ecosystems, including Brazil, it is suggested that regulatory agencies implement more restrictive measures for the use of these preservatives, aiming at a better protection of the aquatic biota.

As neolignanas, bem como as lignanas, apresentam diversas atividades biológicas comprovadas, sendo a licarina A uma neolignana diidrobenzofurânica, detentora de atividade leishmanicida. A partir dessa atividade e do interesse na produção de fármacos eficazes no tratamento dessa doença, a licarina A foi selecionada para estudo de metabolismo in vitro a partir de reações biomiméticas utililizando [Fe(TPP)Cl] e Mn(Salen) como catalisadores; sob ação de oxidantes (PhIO e mCPBA) em quatro solventes de polaridades distintas (AcOEt, MeOH, ACN, DCM), na proporção de 1:30:30 (catalisador:substrato:oxidante). Inicialmente, as reações realizadas foram submetidas às análises por CG-EM comprovando um produto de oxidação majoritário m/z 342, denominado metabólito A. Os solventes de menor polaridade (DCM e AcOEt), bem como o catalisador de Jacobsen, foram mais eficientes na geração desse metabólito. O aumento do tempo reacional bem como o aumento da concentração do oxidante não favoreceu a produção de metabólitos. Posteriormente, as análises dessas reações também foram realizadas por CLUE-DAD-EM/EM e sete metabólitos puderam ser identificados a partir do auxílio do estudo de fragmentação da licarina A bem como a partir de análises de RMN e EM dos metabólitos isolados por CLAE em escala semipreparativa. As análises de RMN dessas frações permitiram a determinação estrutural de um metabólito de m/z 343 [M+H]+ (metabólito 3) constituído de uma epoxidação nos carbonos C-7\'/C-8\' situados na cadeia lateral da licarina A. Sendo assim, quatro dos sete metabólitos (2, 3, 6 e 7) formados apresentaram m/z 343 [M+H]+ e espectros de massas tandem muito semelhantes, correspondendo aos possíveis diasteroisômeros do metabólito epoxidado, visto que a licarina A apresenta dois centros quirais. Outros três metabólitos foram produzidos e exibiram íons de m/z 361 [M+H]+, m/z 315 [M+H]+ e m/z 341 [M+H]+, correspondentes respectivamente a um diol vicinal em C-7\'/C-8\', um aldeído benzóico e um outro aldeído gerado a partir da oxidação da metila terminal em C-9\'. Foi realizado ainda o estudo de metabolismo in vitro com microssomas hepáticos de ratos, sendo que esse estudo foi capaz de reproduzir as reações biomiméticas a partir da produção do mesmo metabólito (2) (apresentando mesmo TR e mesmo espectro de UV). Em relação à toxicidade e aos possíveis danos que a licarina A poderia acarretar, um estudo de toxicidade aguda foi realizado sendo esse capaz de auxiliar na classificação da licarina A, sendo a mesma tóxica em doses > 300mg/kg e 2000mg/kg (categoria 4). Análises dos parâmetros bioquímicos realizadas foram capazes de determinar possível toxicidade hepática, a partir das alterações enzimáticas (AST, ALT, LDH, ALP) ocorridas. Contudo, análise microscópica de cortes dos tecidos (fígado, coração e rins) não determinaram nenhum comprometimento tecidual que pudesse evidenciar toxicidade. / Neolignans and lignans show several biological activities. Licarin A is a dihydrobenzofuranic neolignan, which exhibits leishmanicidal activity. For this reason, licarina A was selected to carry out the studies of in vitro metabolism. So biomimetic reactions were done by the use of [Fe(TPP)Cl] and Mn (Salen) catalysts, different oxidants (PhIO and mCPBA), as well solvents with different polarities (EtOAc, MeOH, ACN, and DCM), and the ratio of 1:30:30 (catalyst:substrate:oxidant). Initially, the reactions were analyzed by GC-MS and showed one major oxidation product of m/z 342 (metabolite A). The less polar solvents (DCM and EtOAc) and Jacobsen catalyst (Mn (Salen)) were more efficient for the production of metabolite A. The reaction time and concentration were increased, but they did not represent a higher production of metabolites. Subsequently these reactions were also analyzed by UPLC-DADMS/ MS and seven metabolites could be identified based on the fragmentation pattern proposed for licarin A, as well as from NMR and MS data of the metabolites isolated by HPLC. A metabolite of m/z 343 [M+H]+ (metabolite 3) was identified by NMR and MS/MS data and it consists of epoxidation in the carbons of C-7\'/C-8\' from licarin A. Therefore, four of the seven metabolites (2, 3, 6 and 7) exhibited m/z 343 [M+H]+ in the MS spectra and the MS/MS showed high similarity with the epoxided metabolite (metabolite 3). So these metabolites could be diasteroisomers of the metabolite 3. Three other metabolites were produced and exhibited ions at m/z 361 [M+H]+, m/z 315 [M+H]+ and m/z 341 [M+H]+, corresponding respectively to a vicinal diol in C-7\'/C-8\', benzylic aldehyde and other aldehyde generated from the oxidation of the terminal methyl at C-9\'. The study of in vitro metabolism by rat liver microsomes was also carried out, and this study was able to reproduce the biomimetic reactions by production of the same metabolite (2). The acute toxicity and potential damage of licarin A were evaluated and the results indicated toxicity for this compound at doses >300mg/kg and 2000mg/kg (category 4). Analysis of biochemical parameters were able to determine possible liver toxicity caused by enzymatic changes (AST, ALT, LDH, ALP). However, microscopic analysis of tissues sections (liver, heart and kidneys) did not show any tissue damage that could indicate toxicity.