Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-05-17T18:17:01Z No. of bitstreams: 1 DissRVM.pdf: 3835144 bytes, checksum: b2d0ed5ccb40ffab36e4731ad8d6c688 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-22T17:46:53Z (GMT) No. of bitstreams: 1 DissRVM.pdf: 3835144 bytes, checksum: b2d0ed5ccb40ffab36e4731ad8d6c688 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-22T17:47:00Z (GMT) No. of bitstreams: 1 DissRVM.pdf: 3835144 bytes, checksum: b2d0ed5ccb40ffab36e4731ad8d6c688 (MD5) / Made available in DSpace on 2017-05-22T17:51:18Z (GMT). No. of bitstreams: 1 DissRVM.pdf: 3835144 bytes, checksum: b2d0ed5ccb40ffab36e4731ad8d6c688 (MD5) Previous issue date: 2016-06-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The morphological variability in the Selenastraceae family has been a barrier to a good establishment of well-defined taxonomic groups. Several studies with molecular compounds have already demonstrated its importance in the elucidation of this relation. In this scenario, the present study used a matrix-assisted laser desorption ionization timeof- flight mass-spectrometry (MALDI-TOF MS) technique as a tool to differentiation of the microalgae of this family at the species and strains levels. A group of 18 strains, belong to in 12 different species, of freshwater microalgae was selected. Cells and dissolved organic matter (DOM) were analyzed in the middle exponential growth phase. The analyzes were performed in two different mass ranges: 400 to 2,000 Da and 2,000 to 20,000 Da. Each strain that yielded unique spectra with a good resolution of peaks and reproducibility was selected for clusters analyzes. These spectra were used to make a dissimilarity analysis that showed the capability of differentiation of the strains and species. The strains of the genera Monoraphidium were not all grouped, possibly because it is a polyphyletic group. The praticity and quickness of this technique for data acquisition, allied with the low cost of the analysis, are factors that favor its application in taxonomic studies. / A baixa variedade morfológica presente na família Selenastraceae tem sido uma barreira para o estabelecimento de grupos taxonômicos bem definidos. Estudos realizados com diversos marcadores moleculares já demonstraram a importância dos compostos produzidos por esses organismos na elucidação dessas relações. Nesse cenário, o presente estudo buscou avaliar a aplicação da espectrometria de massa por MALDI-TOF como ferramenta para a discriminação de cepas e espécies dessa família. Foram selecionadas 18 cepas, classificadas em 12 espécies diferentes, de microalgas de água doce. As células e a matéria orgânica dissolvida (MOD) analisadas foram amostradas de cultivos no meio da fase exponencial de crescimento. As análises foram feitas em duas extensões de massa: massas baixas (400 – 2000 Da) e massas altas (2 – 20 kDa). Para cada tipo de análise foram selecionadas as cepas que renderam espectros com boa resolução de picos. Esses espectros foram então utilizados em análises de dissimilaridade para a elaboração de dendrogramas, evidenciando a capacidade da técnica para a distinção de cepas e espécies. Apesar das cepas do gênero Monoraphidium não ficarem todas agrupadas, o que possivelmente ocorre devido ao fato do grupo ser polifilético. A grande rapidez e praticidade desta técnica para a obtenção de dados, aliado ao baixo custo das análises, são fatores que favorecem a sua aplicação neste tipo de estudo.

Microalgas e cianobactérias têm sido amplamente recomendadas para biomonitoração de metais pesados e outros poluentes, sendo considerados indicadores sensíveis às alterações ambientais e utilizados como organismos testes na regulamentação dos níveis de metal. Estes micro-organismos fotossintetizantes são produtores primários da base da cadeia alimentar aquática e são os primeiros a serem afetados pela poluição por metais pesados. O cobre é um metal normalmente considerado como nutriente essencial para a vida aquática mas pode ser tóxico para algumas espécies. Portanto, neste estudo foram avaliados o efeito tóxico e a bioacumulação de cobre (II) em quatro espécies de micro-organismos fotoautotróficos componentes do fitoplâncton dulcícola, duas cianobactérias filamentosas (Anabaena sp. e Oscillatoria sp) e duas microalgas da classe das clorofíceas (Monorraphidium sp. e Scenedesmus sp.). O meio de cultivo utilizado nos ensaios foi o ASM-1 com e sem a presença de cobre (0,6 mg/L a 12 mg Cu2+/L) onde, o efeito tóxico do metal foi monitorado por contagem celular para as microalgas e por peso seco para as cianobactérias. A bioacumulação do metal foi avaliada da mesma forma para todos os micro-organismos, através de coletas de amostras no decorrer do experimento e determinação da concentração de cobre em solução por espectrometria de absorção atômica com chama. Os resultados obtidos mostram que o efeito tóxico do metal é diretamente proporcional à concentração inicial para os micro-organismos estudados, mas que o cobre (II) foi mais tóxico para as cianobactérias que para as microalgas verdes. A bioacumulação teve uma relação direta com o efeito tóxico do metal sobre os micro-organismos. Os resultados obtidos permitem sugerir que cobre (II) tem efeito negativo no fitoplâncton, inibindo o crescimento e alterando parâmetros metabólicos como a fotossíntese. A bioacumulação do metal pode comprometer os níveis tróficos da cadeia alimentar, afetando seu transporte para seres superiores / Microalgal and cyanobacterial cells are widely used in the biomonitoring of metal contaminated areas as well as other pollutant agents, being considered sensitive microorganisms in relation to environmental changes and also used as test-organisms in the regulation of low levels of metals. These photosynthesizing microorganisms are primary producers in the aquatic food chain, and are the first ones to be affected by heavy metal pollution. Copper is a metal usually considered as an essential nutrient for aquatic forms of life, however toxic for some species. In this way, in the present study, the toxic effect of copper ion and its bioaccumulation by photoautotrophic microbes from freshwater phytoplankton: two filamentous cyanobacteria (Anabaena sp. and Oscillatoria sp) and two chlorophyte microalgae Monorraphidium sp. and Scenedesmus sp.). The culture medium used in the tests was the ASM-a medium in absence and contaminated with copper ion (0,6 mg/L to 12 mg Cu2+/L). The toxic effect of copper was monitored through cell count (microalgal cells) and dry weight measurements (cyanobacterial cells), and copper bioaccumulation was evaluated for all the species tested, through determination of the residual metal concentration in solution, by flame atomic absorption spectrometry. Results obtained indicated that the toxic effect of the metal was directly proportional to the initial concentration of the ion for all the species tested; however, copper ion was more toxic to cyanobacterial than to microalgal cells. The bioaccumulation of copper was directly related to the toxic effect of the ion over the microorganisms. Results obtained suggest that copper ion has a negative effect on the phytoplankton, inhibiting cell growth and also affecting metabolic parameters such as photosynthesis. The bioaccumulation of the metal can markedly affect the trophic levels in the food chain, thus affecting its transport to higher species

Microalgas e cianobactérias têm sido amplamente recomendadas para biomonitoração de metais pesados e outros poluentes, sendo considerados indicadores sensíveis às alterações ambientais e utilizados como organismos testes na regulamentação dos níveis de metal. Estes micro-organismos fotossintetizantes são produtores primários da base da cadeia alimentar aquática e são os primeiros a serem afetados pela poluição por metais pesados. O cobre é um metal normalmente considerado como nutriente essencial para a vida aquática mas pode ser tóxico para algumas espécies. Portanto, neste estudo foram avaliados o efeito tóxico e a bioacumulação de cobre (II) em quatro espécies de micro-organismos fotoautotróficos componentes do fitoplâncton dulcícola, duas cianobactérias filamentosas (Anabaena sp. e Oscillatoria sp) e duas microalgas da classe das clorofíceas (Monorraphidium sp. e Scenedesmus sp.). O meio de cultivo utilizado nos ensaios foi o ASM-1 com e sem a presença de cobre (0,6 mg/L a 12 mg Cu2+/L) onde, o efeito tóxico do metal foi monitorado por contagem celular para as microalgas e por peso seco para as cianobactérias. A bioacumulação do metal foi avaliada da mesma forma para todos os micro-organismos, através de coletas de amostras no decorrer do experimento e determinação da concentração de cobre em solução por espectrometria de absorção atômica com chama. Os resultados obtidos mostram que o efeito tóxico do metal é diretamente proporcional à concentração inicial para os micro-organismos estudados, mas que o cobre (II) foi mais tóxico para as cianobactérias que para as microalgas verdes. A bioacumulação teve uma relação direta com o efeito tóxico do metal sobre os micro-organismos. Os resultados obtidos permitem sugerir que cobre (II) tem efeito negativo no fitoplâncton, inibindo o crescimento e alterando parâmetros metabólicos como a fotossíntese. A bioacumulação do metal pode comprometer os níveis tróficos da cadeia alimentar, afetando seu transporte para seres superiores / Microalgal and cyanobacterial cells are widely used in the biomonitoring of metal contaminated areas as well as other pollutant agents, being considered sensitive microorganisms in relation to environmental changes and also used as test-organisms in the regulation of low levels of metals. These photosynthesizing microorganisms are primary producers in the aquatic food chain, and are the first ones to be affected by heavy metal pollution. Copper is a metal usually considered as an essential nutrient for aquatic forms of life, however toxic for some species. In this way, in the present study, the toxic effect of copper ion and its bioaccumulation by photoautotrophic microbes from freshwater phytoplankton: two filamentous cyanobacteria (Anabaena sp. and Oscillatoria sp) and two chlorophyte microalgae Monorraphidium sp. and Scenedesmus sp.). The culture medium used in the tests was the ASM-a medium in absence and contaminated with copper ion (0,6 mg/L to 12 mg Cu2+/L). The toxic effect of copper was monitored through cell count (microalgal cells) and dry weight measurements (cyanobacterial cells), and copper bioaccumulation was evaluated for all the species tested, through determination of the residual metal concentration in solution, by flame atomic absorption spectrometry. Results obtained indicated that the toxic effect of the metal was directly proportional to the initial concentration of the ion for all the species tested; however, copper ion was more toxic to cyanobacterial than to microalgal cells. The bioaccumulation of copper was directly related to the toxic effect of the ion over the microorganisms. Results obtained suggest that copper ion has a negative effect on the phytoplankton, inhibiting cell growth and also affecting metabolic parameters such as photosynthesis. The bioaccumulation of the metal can markedly affect the trophic levels in the food chain, thus affecting its transport to higher species

Microalga-based technologies are introduced to provide a new generation of biofuels. The cultivation of microalgae in wastewater as the growth medium may offer the dual benefit of wastewater treatment and sustainable biofuel production. The potential of microalgal biomass to be converted into biodiesel depends on the accumulation of lipids, mainly triacylglycerols. However, the presence of toxic contaminants in municipal or industrial wastewater could negatively affect the biomass growth and modify the pattern of lipid accumulation in algae cells. This research aims to evaluate the potential of cultivating microalgae in wastewater for the removal of selected contaminants and the production of biodiesel. The hypothesis underlying this work was that growing microalgae in the presence of contaminants, typically found in wastewater, may induce a stress response that potentially increases the lipid productivity and enhances the biofuel yield. This research explored the potential of microalgae to be used for the removal of wastewater pollutants while accumulating high concentrations of neutral lipid that can be converted into biodiesel. Physiological and transcriptional responses of two genera of green algae were observed in the presence of stresses induced by toxic metals in raw hydraulic fracturing flowback water and pharmaceuticals found in municipal wastewater. Two green algae, Dunaliella salina and Chlorella vulgaris, were selected based on the availability of genetic information, current trends in the alga industry, and specific growth requirement in hypersaline flowback water and municipal wastewater. The first part of this research focuses on the possibility of using the salt-tolerant green microalga, D. salina, for the removal of toxic metals from hydraulic fracturing flowback water. In the second part of this research, the freshwater green microalga, C. vulgaris, was exposed to a suite of pharmaceuticals, commonly found in municipal wastewater, and the effects on the biomass growth and lipid accumulation were evaluated. In Chapter 3, aqueous geochemical characteristics of hydraulic fracturing flowback water were determined. Metagenomic analysis was also used to characterize the microbial community structure of the water samples. This technique included DNA extraction, PCR amplification of genes for 16S and 18S ribosomal RNA, and pyrosequencing. Although the two flowback water samples were shown to have water property measures in the range of reported values in literature, they were significantly different from each other regarding water properties such as total dissolved solids and total organic carbon. They also had distinct microbial community structures. It was shown the flowback water samples contained mainly halophilic, anaerobic, and thermophilic species with a pattern that was expected to be seen in natural gas shale reservoirs. Chapter 4 covers the characterization of the effects of contaminants (toxic metals) in hydraulic fracturing flowback water on the biomass growth and cellular lipid accumulation in Dunaliella salina. Metals were analyzed using ICP-MS. Biomass accumulation was determined by spectrophotometry and gravimetric methods. Lipid accumulation and composition were determined by GC-MS after conversion into fatty acid methyl esters. D. salina was successfully cultivated in saline hydraulic fracturing flowback water, and algal biomass growth was associated with a significant reduction of several toxic metals in the samples. D. salina, grown in flowback water, accumulated high levels of cellular lipids. Also, the estimated biodiesel properties were comparable with other algal biodiesel and complied with the US and EU biodiesel standards. The last chapter of part one presents the effects of a collection of metals commonly found in hydraulic fracturing flowback water and other industrial wastewaters on growth and lipid accumulation of D. salina. The impacts of the nitrogen concentration in the growth medium were also observed. The Nile red assay (fluorescence measurement after Nile red staining) was used to assess lipid accumulation. A range of toxic effects on the algal growth was observed. However, the effects were lower at higher nitrogen level that may indicate to an improved toxicity resistance when nutrient stress is minimal. Even with a high dose-dependent toxicity on growth, some toxic metals increased lipid accumulation. Chapter 6 describes the microplate-based toxicity test conducted to characterize the effect of a range of emerging contaminants (pharmaceuticals) commonly found in municipal wastewater on the biomass growth and cellular lipid accumulation measured by Nile red staining assay. Similar to what was observed for metals in Chapter 5, some pharmaceuticals increased lipid accumulation, even with a high dose-dependent toxicity on algal growth. Most tested pharmaceuticals at municipal wastewater levels (low) did not have a significant effect on the algal growth and lipid accumulation. Toxicity data (EC50 of growth) were well in agreement with what was reported in literature and antibiotics were the most toxic compounds among the different pharmaceutical classes. Chapter 7 presents a further characterization of the effects of a few selected compound from the previous chapter. Changes in the algal lipid composition upon exposure to the selected compounds were determined by qualitative and quantitative analysis of the extracted lipids using GC-MS. FT-IR spectroscopy was used to detect changes in the cellular constituents. The transcriptional responses of the key genes involved in the lipid metabolism of C. vulgaris were also investigated upon exposure to tetracycline using reverse-transcription real-time PCR and the results were interpreted based on the measured levels of cellular lipids and other metabolites. Lipid accumulation was changed upon exposure of algae to pharmaceuticals; however, the FAMEs profile did not show a significant variation. FT-IR spectroscopy highlighted the subtle changes in the cellular basis, and FT-IR peak associated with lipids was strongly correlated with total FAMEs quantified by GC-MS (Pearson correlation coefficient = 0.89). Gene expression analysis showed a general down-regulation of genes involved in the lipid biosynthesis. Results from this research are expecting to help develop and optimize innovative algal-based technologies combining the benefits of wastewater treatment, sustainable biofuel production, and mitigation of carbon dioxide emissions. / Civil Engineering

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A aplicabilidade de um método selecionado de medição indireta de vitelogenina (Vtg) em plasma sanguíneo de peixe, baseado na quantificação de fosfato álcali-lábil (alkali-labile phosphate-ALP) para acessar estrogenicidade em água, foi investigada na presente tese. O método foi originalmente desenvolvido para a espécie de peixe Carassius carassius (Carpa cruciana) e aplicado pela primeira vez na espécie Oreochromis niloticus (Tilápia do Nilo) no presente estudo. Com o objetivo de acessar a sensibilidade do método, em uma primeira etapa da investigação foram realizados estudos laboratoriais com soluções estoques de 17-ethinylestradiol (EE2), 17-estradiol (E2), e estrona (E1). Os efeitos destes hormônios foram investigados com base tanto na concentração quanto na carga, utilizando-se para tanto, unidades experimentais com volumes distintos (2 L e 130 L). Após a validação do método de ALP, a estrogenicidade foi avaliada nas seguintes águas contaminadas: (i) afluente e efluente de uma grande estação de tratamento de esgotos convencional (ETE) e de uma estação descentralizada de tratamento de esgoto de pequeno porte (Ecossistema Engenheirado-DEE); (ii) água superficial (SW) e água subterrânea (GW) coletadas em uma área de brejo contaminada com gasolina; (iii) água de uma lagoa urbana (LRF) da cidade do Rio de Janeiro, com alta densidade populacional e descarte clandestino de esgoto. Na segunda etapa foram analisados em microalgas os efeitos (outros que não disrupção endócrina) causados pelos hormônios EE2, E2 e E1. Os hormônios foram testados individualmente e em misturas, em culturas individuais e combinada (S+) das espécies de microalgas unicelulares P. subcapitata e D. subspicatus. Com base nos níveis de ALP para a espécie de peixe e no EC50 para as espécies de algas, os resultados mostraram que o EE2 e o E2 causaram disrupção endócrina superior e foram mais tóxicos do que o E1 para peixes e microalgas respectivamente. Quando em misturas (E+) de concentrações equivalentes (EE2:E2:E1), os estrogênios resultaram em efeito aditivo para as espécies O. niloticus e P. subcapitata, e menos que aditivo para D. subspicatus e cultivo misto de algas (S+). Culturas contendo ambas as espécies de algas (S+) por um longo período de exposição (96 h) resultaram na atenuação dos efeitos tóxicos causados pela exposição, tanto individual (EE2, E2 ou E1), quanto na mistura (E+) dos estrogênios, medidos em termos de EC50 (T0h 0,07; 0,09; 0,18; e 0,06 g mL-1; e T96h 1,29; 1,87; 5,58; e 4,61 g mL-1, respectivamente). O DEE apresentou uma maior eficiência na remoção dos disrutores endócrinos do que a ETE convencional. Foi detectada estrogenicidade em amostras da LRF, e de água SW e GW em área brejosa contaminada com gasolina. Os resultados dos ensaios sugerem que as interações (efeitos aditivos ou menos que aditivo) causadas pela mistura dos estrogênios assim como, as interações entre as espécies de algas afetaram o resultado final dos ensaios ecotoxicológicos. Um fator raramente abordado em estudos ecotoxicológicos que foi destacado na presente tese refere-se à importância de considerar não somente a concentração e a dosagem, mas também a carga aplicada e o volume das unidades experimentais. Devido à boa sensibilidade do O. niloticus quando exposto às concentrações relativamente baixas dos estrogênios, a combinação do método de ALP com os biomarcadores auxiliares (particularmente MN) pode ser um protocolo adequado para a detecção de estogenicidade e genotoxicidade respectivamente em diferentes ambiente aquáticos contaminados, como parte de um programa de monitoramento ambiental / The applicability of one selected method for indirect measurement of vitellogenin (Vtg) in fish plasma based on the quantification of alkali-labile phosphates (ALP method) to assess estrogenicity in water was investigated. The ALP method applied in this investigation was originally developed with Carassius carassius (Crucian carps). This thesis describes the first attempt of using this method with Oreochromis niloticus (Nile tilapia). In a first part of the investigation, laboratory studies were conducted with water spiked with 17-ethinylestradiol (EE2), 17-estradiol (E2), and estrone (E1) in order to assess the method sensitivity. The effects of these estrogens were investigated on the basis of both load and concentration, using experimental units with two different volumes (2 L and 130 L). After validation of the method, the estrogenicity of the following contaminated waters was assessed: (i) affluent and effluent of one large conventional municipal wastewater treatment plant (WWTP) and one small decentralized wastewater treatment plant (Decentralized Engineered Ecosystem-DEE); (ii) surface water (SW) and groundwater (GW) obtained from a gasoline-contaminated marshland; (iii) samples from a urban lagoon (LRF) located in Rio de Janeiro city with high density population and clandestine sewage discharge. An additional goal of the thesis was to assess the effect (other than endocrine disruption) caused by EE2, E2 and E1 to microalgae. Assays with single and mixed estrogens and single and combined cultures (S+) of the green microalgae P. subcapitata and D. subspicatus were carried out. The results have shown that EE2 and E2 were more estrogenic and toxic than E1 to the fishes and to the microalgae respectively. Mixed solutions of estrogens (E+) in equal proportions (EE2:E2:E1) resulted in additive effect on O. niloticus and P. subcapitata and less-than-additive effect on D. subspicatus and S+ measured as ALP (for fish) and EC50 (for microalgae). Combined cultivation of both algae species and longer exposure time (96 h) resulted in attenuation of the toxic effects caused by single (EE2, E2 or E1) and mixed (E+) estrogens according to EC50 (T0h 0.07, 0.09, 0.18, and 0.06 g mL-1; and T96h 1.29, 1.87, 5.58, and 4.61 g mL-1, respectively). The decentralized engineered ecosystem was more efficient than the conventional WWTP regarding estrogenicity removal from the final effluent. Estrogenicity was detected in some samples of the LRF and the SW and GW of the gasoline-contaminated marshland. Therefore, the investigations suggested that interactions (additive and less-than additive effect) take place when different estrogens are present in the water environment and interactions also occur between algae species, which affect the final toxicity. Additionally, the study highlighted the importance of taking into account not only concentration and dose regime but also the mass load and therefore, the volume used in the experimental units, which are rarely addressed in ecotoxicity assays. Considering the good sensitivity of O. niloticus exposed to relatively low concentrations of estrogens, the combination of the ALP method with auxiliary biomarkers (particularly MN) can be a suitable protocol for estrogenicity and genotoxicity detection in different contaminated waters as part of water environmental monitoring programs

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A aplicabilidade de um método selecionado de medição indireta de vitelogenina (Vtg) em plasma sanguíneo de peixe, baseado na quantificação de fosfato álcali-lábil (alkali-labile phosphate-ALP) para acessar estrogenicidade em água, foi investigada na presente tese. O método foi originalmente desenvolvido para a espécie de peixe Carassius carassius (Carpa cruciana) e aplicado pela primeira vez na espécie Oreochromis niloticus (Tilápia do Nilo) no presente estudo. Com o objetivo de acessar a sensibilidade do método, em uma primeira etapa da investigação foram realizados estudos laboratoriais com soluções estoques de 17-ethinylestradiol (EE2), 17-estradiol (E2), e estrona (E1). Os efeitos destes hormônios foram investigados com base tanto na concentração quanto na carga, utilizando-se para tanto, unidades experimentais com volumes distintos (2 L e 130 L). Após a validação do método de ALP, a estrogenicidade foi avaliada nas seguintes águas contaminadas: (i) afluente e efluente de uma grande estação de tratamento de esgotos convencional (ETE) e de uma estação descentralizada de tratamento de esgoto de pequeno porte (Ecossistema Engenheirado-DEE); (ii) água superficial (SW) e água subterrânea (GW) coletadas em uma área de brejo contaminada com gasolina; (iii) água de uma lagoa urbana (LRF) da cidade do Rio de Janeiro, com alta densidade populacional e descarte clandestino de esgoto. Na segunda etapa foram analisados em microalgas os efeitos (outros que não disrupção endócrina) causados pelos hormônios EE2, E2 e E1. Os hormônios foram testados individualmente e em misturas, em culturas individuais e combinada (S+) das espécies de microalgas unicelulares P. subcapitata e D. subspicatus. Com base nos níveis de ALP para a espécie de peixe e no EC50 para as espécies de algas, os resultados mostraram que o EE2 e o E2 causaram disrupção endócrina superior e foram mais tóxicos do que o E1 para peixes e microalgas respectivamente. Quando em misturas (E+) de concentrações equivalentes (EE2:E2:E1), os estrogênios resultaram em efeito aditivo para as espécies O. niloticus e P. subcapitata, e menos que aditivo para D. subspicatus e cultivo misto de algas (S+). Culturas contendo ambas as espécies de algas (S+) por um longo período de exposição (96 h) resultaram na atenuação dos efeitos tóxicos causados pela exposição, tanto individual (EE2, E2 ou E1), quanto na mistura (E+) dos estrogênios, medidos em termos de EC50 (T0h 0,07; 0,09; 0,18; e 0,06 g mL-1; e T96h 1,29; 1,87; 5,58; e 4,61 g mL-1, respectivamente). O DEE apresentou uma maior eficiência na remoção dos disrutores endócrinos do que a ETE convencional. Foi detectada estrogenicidade em amostras da LRF, e de água SW e GW em área brejosa contaminada com gasolina. Os resultados dos ensaios sugerem que as interações (efeitos aditivos ou menos que aditivo) causadas pela mistura dos estrogênios assim como, as interações entre as espécies de algas afetaram o resultado final dos ensaios ecotoxicológicos. Um fator raramente abordado em estudos ecotoxicológicos que foi destacado na presente tese refere-se à importância de considerar não somente a concentração e a dosagem, mas também a carga aplicada e o volume das unidades experimentais. Devido à boa sensibilidade do O. niloticus quando exposto às concentrações relativamente baixas dos estrogênios, a combinação do método de ALP com os biomarcadores auxiliares (particularmente MN) pode ser um protocolo adequado para a detecção de estogenicidade e genotoxicidade respectivamente em diferentes ambiente aquáticos contaminados, como parte de um programa de monitoramento ambiental / The applicability of one selected method for indirect measurement of vitellogenin (Vtg) in fish plasma based on the quantification of alkali-labile phosphates (ALP method) to assess estrogenicity in water was investigated. The ALP method applied in this investigation was originally developed with Carassius carassius (Crucian carps). This thesis describes the first attempt of using this method with Oreochromis niloticus (Nile tilapia). In a first part of the investigation, laboratory studies were conducted with water spiked with 17-ethinylestradiol (EE2), 17-estradiol (E2), and estrone (E1) in order to assess the method sensitivity. The effects of these estrogens were investigated on the basis of both load and concentration, using experimental units with two different volumes (2 L and 130 L). After validation of the method, the estrogenicity of the following contaminated waters was assessed: (i) affluent and effluent of one large conventional municipal wastewater treatment plant (WWTP) and one small decentralized wastewater treatment plant (Decentralized Engineered Ecosystem-DEE); (ii) surface water (SW) and groundwater (GW) obtained from a gasoline-contaminated marshland; (iii) samples from a urban lagoon (LRF) located in Rio de Janeiro city with high density population and clandestine sewage discharge. An additional goal of the thesis was to assess the effect (other than endocrine disruption) caused by EE2, E2 and E1 to microalgae. Assays with single and mixed estrogens and single and combined cultures (S+) of the green microalgae P. subcapitata and D. subspicatus were carried out. The results have shown that EE2 and E2 were more estrogenic and toxic than E1 to the fishes and to the microalgae respectively. Mixed solutions of estrogens (E+) in equal proportions (EE2:E2:E1) resulted in additive effect on O. niloticus and P. subcapitata and less-than-additive effect on D. subspicatus and S+ measured as ALP (for fish) and EC50 (for microalgae). Combined cultivation of both algae species and longer exposure time (96 h) resulted in attenuation of the toxic effects caused by single (EE2, E2 or E1) and mixed (E+) estrogens according to EC50 (T0h 0.07, 0.09, 0.18, and 0.06 g mL-1; and T96h 1.29, 1.87, 5.58, and 4.61 g mL-1, respectively). The decentralized engineered ecosystem was more efficient than the conventional WWTP regarding estrogenicity removal from the final effluent. Estrogenicity was detected in some samples of the LRF and the SW and GW of the gasoline-contaminated marshland. Therefore, the investigations suggested that interactions (additive and less-than additive effect) take place when different estrogens are present in the water environment and interactions also occur between algae species, which affect the final toxicity. Additionally, the study highlighted the importance of taking into account not only concentration and dose regime but also the mass load and therefore, the volume used in the experimental units, which are rarely addressed in ecotoxicity assays. Considering the good sensitivity of O. niloticus exposed to relatively low concentrations of estrogens, the combination of the ALP method with auxiliary biomarkers (particularly MN) can be a suitable protocol for estrogenicity and genotoxicity detection in different contaminated waters as part of water environmental monitoring programs