Among all nanomaterials, which are intentionally manufactured and applied, nanosilver (nAg) is one of the most frequently applied nanomaterials. It is introduced into wastewater treatment plants (WWTPs) due to its use as antimicrobial resource in household and medical care products and hence concern raised regarding its impact on activated sludge organisms and their purification efficiency. Within this thesis, the effects of nAg on single species, simple food chains and communities related to activated sludge were investigated. Among all tested species in this thesis, the gammaproteobacteria R. planticola was the most sensitive organism regarding the tested nAg material, NM-300K. The environmental risk assessment (ERA), based on an assumed predicted environmental concentration derived from modeled concentrations of other types of nAg, revealed no risk for the activated sludge. This result should be interpreted with care, considering the tolerantly chosen safety factor for calculation of the predicted no effect concentration and the assumptions which were made concerning environmental concentrations. Differences in acute toxic effects of nAg on the ciliate P. tetraurelia were observed depending on the type of medium and the exposure pathway (via medium or via bacterial food). More detailed investigations are required to analyze the distribution, availability and uptake of nAg into ciliates in the respective tests. In chronic experiments concentration response was very steep in the range between the effect concentration determined in acute toxicity testing (resulting in 100% mortality) and a tenfold lower concentration (no effect observed) for both exposure pathways. Community experiments with activated sludge exposed to realistic and high concentrations of nAg revealed acute effects on the protozoan community at high nAg concentration using multivariate statistics for data analysis. In contrast, the sludge biotic index was not meaningful for data interpretation, as no differences were observed between the samples of different treatments. For chronic testing, more preliminary work is required to develop a protocol for artificial wastewater which serves the needs of activated sludge organisms over longer time periods and which retains a typical composition of the activated sludge community.

Triclosan (TCS) is a personal care product with many fields of application and is of public interest for several years now. Monitoring studies showed that TCS is a ubiquitous chemical in the aquatic environment. Aquatic organisms are exposed to TCS in a broad range of concentrations, from ng L-1 up to lower μg L-1. TCS has a bactericidal effect for various types of gram-positive and gram negative bacteria. TCS targets a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase (Schweizer, 2001). Therefore the terminal reaction in the fatty acid elongation cycle is inhibited (Levy et al., 1999). Although effects on non-target organisms are reported, the Mode of Action (MoA) of TCS is not well examined for those organisms. The aim of this PhD thesis was to investigate effects of TCS on non-target autotrophic organisms at different levels of biological complexity in the aquatic environment. In this thesis microalgae have been found to be very sensitive to TCS. In some cases even higher sensitivities than in bacteria were observed, which is in accordance with published effect data (Harada et al., 2008; Orvos et al., 2002). Similarly to bacteria, high species sensitivity differences were observed for algae (Franz et al., 2008). In bacteria these sensitivity differences can be ascribed to several resistance mechanisms reported in Schweizer (2001). These findings lead to the question about the reasons for species sensitivity differences in algae. A mesocosm study was performed to detect effects of TCS across levels of biological organization and to investigate the impact of sensitivity differences on complex aquatic communities. For that purpose, structural and functional effects parameters were observed.

In the European Union an environmental risk assessment is required for the registration of new chemicals, biocides, pesticides and pharmaceuticals. In order to avoid the release of potential hazardous substances, various ecotoxicity tests are performed, including acute and chronic fish tests. As a consequence of the new program of the European Union “Registration, Evaluation and Authorisation of Chemicals” (REACH) the number of animal experiments for environmental risk assessment is expected to increase remarkably within the next years. On the other hand there is a strong societal demand for reducing the number of animal tests by using alternative in vitro models. According to EU directives, investigations using non-human vertebrate embryos are considered pain free in vitro methods and are therefore accepted as alternatives to animal experiments. For the acute fish test, the Danio rerio embryo test (DarT) has been established as a replacement method and included in national regulations at least for waste water (German Waste Water Dues Law). However, no alternatives for chronic fish tests are currently available. The overall goal of this thesis was to work towards such a replacement by extending DarT zu Gene-DarT. Toxicants will initially interact at the molecular level with consequences for physiology, fitness and survival. The analysis of gene expression patterns may unravel elements of these molecular events before any phenotypic changes are visible. The hypothesis of this thesis therefore was that chemical-sensitive genes in embryos exposed in a conventional DarT may indicate toxic impact of substances at sub-acute concentrations and thus enhance the sensitivity of the embryo toxicity test. Furthermore, unlike the conventional DarT-endpoints, gene expression analysis will provide insights into mechanistic processes underlying toxicity. The 3,4-dichloroaniline (3,4-DCA), which is used as a reference compound in the DarT, was selected as model chemical in this thesis. In a first step, differentially expressed genes in embryos exposed to 3,4-DCA were identified by microarray technology and RT-PCR techniques. Six dose-dependent significant differentially expressed genes were identified. These genes were involved in biotransformation pathways (cyp1a, ahr2), stress response (nrf2, maft, ho-1) and cell cycle control (fzr1). Differential expression upon 3,4-DCA exposure was detected below the LOEC (lowest observed effect concentration = 6.2 µM) of survival or developmental disorders of the embryo test (0.78 µM and above). For the validation of stage specific sensitivity, genes were also analysed in post-hatched stages. Extension of exposure to post-hatched stages resulted in a differential expression at lower concentrations as for the embryonic stages, indicating an improved sensitivity due to stage-specific sensitivity or exposure time. To confirm the adaptive function of the 3,4-DCA-sensitive genes, embryonic mRNA abundance was experimentally manipulated by knock down and overexpression. By injection of sense (mRNA) or antisense (siRNA) RNA in one-cell-stages of embryos, the transcript levels of genes were transiently enhanced or repressed in embryos exposed to 3,4-DCA. mRNA injection of the genes cyp1a, ho-1 and nrf2 reduced the number of embryos with 3,4-DCA-induced malformations. In contrast, siRNA injections for the same genes led to an increase in the severity and frequency of developmental disorders. The results clearly indicate the adaptive functions of the investigated genes or their corresponding proteins. This study demonstrates that the analysis of chemical-sensitive gene expression shows the potential to increase the sensitivity of conventional toxicity tests. The analysis of gene expression also provides additional mechanistic information for toxic action, e.g. in the presented study, the involvement of Ah-receptor regulated pathways as an adaptive response. Furthermore, the presented data indicate that functional manipulations, using mRNA and siRNA-injection, are suitable to evaluate the role of differentially expressed genes for toxicity.

<p>  </p> <p>Over the past two decades, emerging fungal pathogens have been reported in numerous groups ranging from mammals to key crop species across the globe. To combat the rise of these fungal pathogens in industrial agriculture, fungicides have been developed and subsequently applied en masse. Despite their growing usage, research examining the effects of these anthropogenic contaminants on natural systems is severely limited compared to other major classes of pesticides. Of particular concern are their impacts on aquatic systems, which can be especially at risk due to their proximity to agriculture. Herein I used a tiered approach to assess the impacts of this class of pesticides on aquatic ecosystems using amphibians as model system. In the first chapter, I assessed the acute toxicity of two widely applied fungicides, pyraclostrobin and chlorothalonil, to six different species of amphibians commonly found in the Midwest. My results showed that these fungicides are very acutely toxic to several species of amphibians at levels within the expected environmental concentration (EEC). In the second chapter, I examined the impacts of long-term low-dose exposure of pyraclostrobin on the growth, development, and activity levels of three species of amphibians. Despite the acute toxicity of pyraclostrobin, sublethal effects of the fungicide were rather limited with only minor effect on growth and activity. In the third chapter, I investigated the effects of fungicide exposure on host-parasite interactions using trematodes and American Bullfrog tadpoles. I found that in all treatments pyraclostrobin increased parasite loads from ~3 to 8 times compared to control tadpoles. Additionally, parasite loads were approximately 2 times higher in tadpoles with the continued fungicide exposures compared to those tadpoles that were moved to fresh water following initial fungicide exposure. Finally, my fourth chapter investigated the impact of pyraclostrobin and chlorothalonil application regime on community-level interactions under semi-natural conditions. While chlorothalonil had limited effects on the community, I found that pyraclostrobin was acutely toxic to Gray Treefrogs at environmentally relevant concentrations and these effects were stronger with more frequent applications. Collectively, my research has demonstrated that fungicides can be acutely toxic to amphibians at EECs under laboratory and semi-natural conditions. Additionally, they have the potential to alter disease dynamics by increasing infection risk. Given our increasing usage of fungicides to control emerging fungal pathogens, a greater emphasis on understanding the environmental implications of this management strategy is needed to inform ecological risk assessments. </p>

There is growing concern that current toxicological test methods are too slow and expensive to evaluate the safety of the thousands of chemicals in the Canadian economy. In this thesis, a novel zebrafish embryo test, with integrated behaviour, energy expenditure and gene transcription assays, was used to assess the hazard of a diverse suite of 29 chemicals. I hypothesized that points of departure (PODs) from the integrated test would be protective of the long-term toxic effects of these chemicals. I found that: 1) integrating alternative test methods enhanced the sensitivity of the zebrafish embryo acute toxicity (FET) test, 2) integrated results provided a holistic understanding of potential mechanisms of action and effects, and 3) transcriptional PODs were protective of PODs from traditional long-term and short-term juvenile and adult fish toxicity tests reported in the literature. This integrated zebrafish embryo test is a sensitive, informative and protective chemical hazard screening tool.

No description available.

Algumas atividades antrópicas podem provocar alterações nos recursos naturais, seja ela no rio, no solo ou no mar. Os efeitos destas alterações podem ser observados na biota, na qualidade das águas, na disponibilidade de nutrientes, interferindo em todos os elementos que compõem o ecossistema, influenciando-o em maior ou menor grau. O presente estudo procurou determinar o efeito dos poluentes sulfato de zinco, cloreto de amônia, dodecilsulfato de sódio e benzeno, numa espécie bioindicadora marinha, el larvas de mexilhão (Perna perna), além de acrescentar dados sobre a biologia e analisar quimicamente a presença de hidrocarbonetos nos tecidos dos animais adultos e das larvas. Para tanto foram utilizados testes de toxicidade, estudos sobre a bioenergética (consumo de oxigênio e excreção de amônia), enzimas biomarcadoras e excreção de fósforo e nitrogênio. Com relação à biologia, foram encontrados indivíduos sexualmente maduros a partir de 26,1 e 27 mm de comprimento para fêmeas e machos, respectivamente, e há diferença, ainda que esta não seja estatisticamente significativa, entre ovócitos e larvas submetidos a ação de poluentes. A análise de hidrocarbonetos indicou que existe uma contribuição biogênica e petrogênica no litoral norte do Estado de São Paulo. Para as demais análises verificou-se que, quando comparados aos grupos controles, as larvas foram sensíveis e responderam aos diferentes poluentes, geralmente com inibição da atividade. / Some antropic activities may be the cause fo alterations in natural resources, being it river, soil or sea. These alteration effects may be observed in the biota, water quality, nutrients disponibility, interfering with all elements that are part of a ecosystem, with greater or lesser influence degree. The present study was directed to determine the effect of pollutants, such as zinc sulphate, ammonia chlorate, sodium dodecilsulphate and benzene, acting over larvae of a marine bioindicator, the mussel (Perna perna), besides adding data on biology and chemically analysing hidrocarbon presence in larvae and adult animal tissues. In order to obtain these results, toxicity tests were used and bioenergetic (oxygen consumption and ammonia excretion), biomarkers, phosphorus and nitrogen excretion, were studied. As for biology, individuals sexually mature were found starting with 26,1 and 27 mm length females and males, respectively, and there is difference between larvae and ovocites, although statistically not significant, submitted to pollutants action. The hydrocarbon analysis indicates a biogenic and petrogenic contribution to north coast of São Paulo State. For the other analyses the observed results, when compared to control groups, showed that larvae are sensitive, and responded to different pollutants, generally with activity inhibition.

No description available.

Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.

Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.