Orientadores: Leonardo Fernandes Fraceto, Vera Lucia Scherholz Salgado de Castro

Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia

Resumo: A ecotoxicologia de nanopartículas de dióxido de titânio (nano- TiO2) tem sido amplamente estudada nos últimos anos, mas os resultados obtidos ainda são inconclusivos. Assim, permanecem dúvidas sobre a aplicabilidade dos atuais protocolos ecotoxicológicos para avaliação dos possíveis...

Resumo: A ecotoxicologia de nanopartículas de dióxido de titânio (nano- TiO2) tem sido amplamente estudada nos últimos anos, mas os resultados obtidos ainda são inconclusivos. Assim, permanecem dúvidas sobre a aplicabilidade dos atuais protocolos ecotoxicológicos para avaliação dos possíveis impactos do uso da nanotecnologia. Poucas investigações toxicológicas tem considerado as propriedades fotocatalíticas da substância, que podem aumentar a sua toxicidade para a biota aquática. O objetivo deste trabalho foi avaliar os efeitos em organismos aquáticos expostos a diferentes nano- TiO2, sob diferentes condições de iluminação. A interação de variáveis como concentração, fase cristal (anatase puro - TA , ou uma mistura de anatase e rutilo - TM) e da condição de iluminação ( luz visível ou luz ultravioleta e visível) foram investigadas através da observação de parâmetros de letalidade e de efeitos subletais em peixes juvenis (pacu caranha, Piaractus mesopotamicus), embriões de peixe (zebrafish, Danio rerio) e microcrustáceos (Daphnia similis e Artemia salina). A exposição aguda e prolongada de peixes juvenis não causou mortalidade nem acúmulo de Ti em músculo dos peixes, mas houve efeitos bioquímicos e genéticos, que dependeram da fase cristal e da condição de iluminação empregada. A atividade de fosfatase ácida (FA), bem como os níveis de proteínas carboniladas (PCO) e de metalotioneína foram biomarcadores úteis de exposição aguda ao nano -TiO2. Por outro lado, os resultados mostraram que para a exposição prolongada, a atividade específica da catalase (CAT), glutationa S-transferase (GST), os níveis de PCO e o ensaio cometa foram os biomarcadores mais úteis. O nano-TiO2 também foi considerado praticamente não tóxico sob luz visível para D. similis e A. salina. A exposição ao nano -TiO2, sob luz visível e ultravioleta aumentou a toxicidade dos nano-TiO2 para microcrustáceos. No caso de D. similis, TM foi mais tóxico do que o TA , mostrando valores de CE5048h = 60,16 e 750,55 mg/L, respectivamente. A A. salina foi mais sensível do que D. similis, com CE5048h = 4 mg/L para ambos os produtos. Em concentrações subletais, o nano- TiO2 não apresentou qualquer impacto negativo sobre o crescimento de Daphnia e Artemia. As atividades específicas de CAT, superóxido dismutase e FA foram biomarcadores úteis de exposição ao nano- TiO2 em Daphnia. Para embriões, a exposição ao nano-TiO2 causou eclosão precoce. Sob luz ultravioleta, o nano- TiO2 causou redução no comprimento das larvas. Além disso, um aumento no número de larvas com alteração de equilíbrio e na mortalidade foi observado nos grupos expostos a TM sob luz ultravioleta. As atividades específicas de CAT e GST apresentaram boa resposta em embriões expostos ao nano-TiO2. A determinação da toxicidade do nano- TiO2 depende do organismo, meio de cultura e o tempo de exposição utilizado nos bioensaios. Depende também da fase cristal e das condições de iluminação. Verificou-se que a exposição à radiação ultravioleta a níveis ambientais mínimos aumenta a toxicidade do nano-TiO2. Os resultados indicam a ocorrência de estresse oxidativo em conseqüência da exposição ao nano-TiO2, mas em geral, não houve uma clara relação concentração- resposta ao considerar parâmetros subletais. Isto pode estar relacionado com a instabilidade dos sistemas de exposição, devido a agregação e precipitação das nanopartículas. Entretanto, nossos resultados indicam que a influência de fatores abióticos sobre a ecotoxicidade do nano- TiO2 deve ser explorada em detalhes, a fim de estabelecer modelos experimentais adequados para estudar a sua toxicidade em espécies de relevância ambiental e contribuir para o desenvolvimento sustentável da nanotecnologia

Abstract: The ecotoxicology of titanium dioxide nanoparticles (nano-TiO2) has been extensively studied in recent years but the results are so far confusing. Hence, doubts remain about the applicability of current ecotoxicological protocols to evaluate the possible impacts of nanotechnology. Few...

Abstract: The ecotoxicology of titanium dioxide nanoparticles (nano-TiO2) has been extensively studied in recent years but the results are so far confusing. Hence, doubts remain about the applicability of current ecotoxicological protocols to evaluate the possible impacts of nanotechnology. Few toxicological investigations have considered the photocatalytic properties of the substance, which can increase its toxicity to aquatic biota. The aim of this work was to evaluate the effects on aquatic organisms exposed to different nano-TiO2, under different illumination conditions. The interaction of variables as concentration, crystal phase (pure anatase ¿ TA, or a mixture of anatase and rutile ¿ TM) and illumination condition (visible light or ultraviolet and visible light) were investigated by observing lethal an sublethal parameters in juveniles fishes (pacu caranha, Piaractus mesopotamicus), fish embryos (zebrafish, Danio rerio) and microcrustaceans (Daphnia similis and Artemia salina). The acute and prolonged exposure of juvenile fishes caused no mortality neither Ti accumulation in fish muscle, but showed biochemical and genetic effects, which depends on the crystal phase and the illumination condition employed. The acid phosphatase activity (AP) as well as the metallothionein and protein carbonylation (PCO) levels and the micronucleus test were useful biomarkers of acute exposure of fish to nano-TiO2. On the other side, the findings showed that for prolonged exposure, the specific activity of catalase (CAT), glutathione S-transferase (GST), PCO levels and comet assay were more useful as biomarkers. Nano-TiO2 was also considered pratically non-toxic under visible light to D. similis and A. salina. Exposure to nano-TiO2 under visible and ultraviolet light enhanced the toxicity of nano-TiO2 to microcrustaceans. In the case of D. similis, TM was more toxic than TA, showing values of EC5048h = 60.16 and 750.55 mg/L, respectively. A. salina was more sensitive than D. similis, with EC5048h = 4 mg/L for both products. At sublethal concentrations, the nano-TiO2 did not show any negative impacts on the growth of Daphnia and Artemia. The specific activities of CAT, AP and superoxide dismutase were usefull biomarkers of nano-TiO2 exposure in Daphnia. To embryos the nano-TiO2 exposure caused early hatching. Under ultraviolet light, nano-TiO2 caused reduction in larvae length. Also, an increase in larvae with alteration in equilibrium and larvae mortality was observed in groups exposed to TM under ultraviolet light. The specific activities of CAT and GST showed good answer in embryos exposed to nano-TiO2. Determination of the nano-TiO2 toxicity using bioassays depends on the organism, culture medium, and exposure time employed. It also depends on the crystal phase and the illumination condition. Exposure to ultraviolet light at minimal environmental levels increases the nano-TiO2 toxicity. The results indicate the occurrence of oxidative stress in consequence of nano-TiO2 exposure, but in general there was not a clear concentration-response relationship when considering sublethal parameters. This can be related to the instability of exposure systems, due to nanoparticles aggregation and precipitation. However, our results indicates that the influence of abiotic factors on nano-TiO2 ecotoxicity must be explored in detail, in order to establish experimental models to study their toxicity to environmentally relevant species and contribute to nanotechnology development in a sustainable way. The ecotoxicology of titanium dioxide nanoparticles (nano-TiO2) has been extensively studied in recent years but the results are so far confusing. Hence, doubts remain about the applicability of current ecotoxicological protocols to evaluate the possible impacts of nanotechnology. Few toxicological investigations have considered the photocatalytic properties of the substance, which can increase its toxicity to aquatic biota. The aim of this work was to evaluate the effects on aquatic organisms exposed to different nano-TiO2, under different illumination conditions. The interaction of variables as concentration, crystal phase (pure anatase ¿ TA, or a mixture of anatase and rutile ¿ TM) and illumination condition (visible light or ultraviolet and visible light) were investigated by observing lethal an sublethal parameters in juveniles fishes (pacu caranha, Piaractus mesopotamicus), fish embryos (zebrafish, Danio rerio) and microcrustaceans (Daphnia similis and Artemia salina). The acute and prolonged exposure of juvenile fishes caused no mortality neither Ti accumulation in fish muscle, but showed biochemical and genetic effects, which depends on the crystal phase and the illumination condition employed. The acid phosphatase activity (AP) as well as the metallothionein and protein carbonylation (PCO) levels and the micronucleus test were useful biomarkers of acute exposure of fish to nano-TiO2. On the other side, the findings showed that for prolonged exposure, the specific activity of catalase (CAT), glutathione S-transferase (GST), PCO levels and comet assay were more useful as biomarkers. Nano-TiO2 was also considered pratically non-toxic under visible light to D. similis and A. salina. Exposure to nano-TiO2 under visible and ultraviolet light enhanced the toxicity of nano-TiO2 to microcrustaceans. In the case of D. similis, TM was more toxic than TA, showing values of EC5048h = 60.16 and 750.55 mg/L, respectively. A. salina was more sensitive than D. similis, with EC5048h = 4 mg/L for both products. At sublethal concentrations, the nano-TiO2 did not show any negative impacts on the growth of Daphnia and Artemia. The specific activities of CAT, AP and superoxide dismutase were usefull biomarkers of nano-TiO2 exposure in Daphnia. To embryos the nano-TiO2 exposure caused early hatching. Under ultraviolet light, nano-TiO2 caused reduction in larvae length. Also, an increase in larvae with alteration in equilibrium and larvae mortality was observed in groups exposed to TM under ultraviolet light. The specific activities of CAT and GST showed good answer in embryos exposed to nano-TiO2. Determination of the nano-TiO2 toxicity using bioassays depends on the organism, culture medium, and exposure time employed. It also depends on the crystal phase and the illumination condition. Exposure to ultraviolet light at minimal environmental levels increases the nano-TiO2 toxicity. The results indicate the occurrence of oxidative stress in consequence of nano-TiO2 exposure, but in general there was not a clear concentration-response relationship when considering sublethal parameters. This can be related to the instability of exposure systems, due to nanoparticles aggregation and precipitation. However, our results indicates that the influence of abiotic factors on nano-TiO2 ecotoxicity must be explored in detail, in order to establish experimental models to study their toxicity to environmentally relevant species and contribute to nanotechnology development in a sustainable way