Natural substances and high pressure processing to preserve sea bass

Esta tese teve como objectivo estudar estratégias de conservação de pescado fresco, recorrendo ao uso de extractos e óleos essenciais de plantas e do processamento por alta pressão (HPP), usando filetes de robalo como um caso de estudo modelo. Relativamente aos extractos e óleos essenciais, avaliaram-se as suas propriedades antibacterianas e antioxidantes. Os extractos aquosos quente de poejo e de orégão e o óleo essencial de cravinho apresentaram a maior actividade antioxidante. Os óleos essenciais foram mais eficientes do que os extractos para inibir o crescimento das estirpes bacterianas testadas, tendo-se observado os menores valores de concentração mínima inibitória nos óleos essenciais de orégão, citronela, alho e orégão Espanhol. De seguida, estudou-se o efeito dos óleos essenciais de orégão Espanhol e de limão na conservação de filetes de robalo fresco tendo em conta critérios microbiológicos, químicos, físicos e sensoriais. A aplicação do óleo essencial de orégão Espanhol aumentou o tempo de vida útil dos filetes sob o ponto de vista bacteriano, mas não em termos sensoriais. A combinação dos óleos essenciais de orégão Espanhol e de limão melhorou o efeito antioxidante e reduziu a intensidade do odor e a sua eficácia em relação às Enterobacteriaceae, comparando com o tratamento com óleo essencial de orégão Espanhol per se. No sentido de reduzir o odor conferido pelos óleos essenciais realizou-se um estudo de conservação para avaliar o efeito de películas com óleos essenciais (citronela, alho e tomilho) em filetes de robalo, recorrendo a uma teste de desafio bacteriano. As películas sem óleos essenciais aumentaram o tempo de vida útil sob o ponto de vista bacteriano, mas este efeito não foi observado com a incorporação dos óleos essenciais nas películas. Em relação ao HPP, testaram-se diversas condições (nível de pressão, tempo de pressurização e taxa de pressurização) e avaliaram-se os efeitos na actividade enzimática, na qualidade global e na conservação de filetes de robalo fresco. Em geral, o aumento do nível de pressão e do tempo de pressurização diminuiu a actividade da fosfatase ácida e das enzimas proteolíticas, a carga bacteriana e a capacidade de retenção de água, enquanto que os filetes ficaram mais brancos. O HPP revelou potencial para o desenvolvimento de novos produtos: mais brancos, não translúcidos, mais firmes e com maior tempo de frescura e estabilidade microbiológica. Em conclusão, os óleos essenciais e o HPP têm potencial para conservar pescado fresco, devido aos seus efeitos na qualidade bacteriana. Ainda assim, mais esforços devem ser feitos no sentido de reduzir a transferência de odor dos óleos essenciais para o pescado e os efeitos do HPP no aspecto do pescado e na oxidação lipídica.

Impact of natural and/or chemical stressors on the freeze-tolerant and euryhaline enchytraeid, Enchytraeus albidus

Rapid climatic changes are taking place in Arctic, subarctic and cold temperate regions, where predictions point to an increase in freeze-thaw events, changes in precipitation, evaporation and salinity patterns. Climate change may therefore result in large impacts in ecosystem functioning and dynamics, especially in the presence of contaminants due to intense anthropogenic activities. Even though multiple stress approaches have received increasing interest in the last decades, the number of such studies is limited. In particular, knowledge on the effect of freezethaw events and salinity fluctuations on ecotoxicology of soil invertebrates is lacking, especially important when considering supralittoral species. Therefore, the aim of this thesis was to investigate the effects of low temperature and salinity fluctuations, singly and in combination with contaminants, in the freeze-tolerant and euryhaline enchytraeid Enchytraeus albidus. The assessment of population level endpoints (survival and reproduction), along with physiological and biochemical parameters such as levels of cryoprotectants, ice/water content, oxidative stress biomarkers, cellular energy allocation, and tissue concentration of chemicals (when applied), provided new and valuable knowledge on the effects of selected physical and chemical stressors in E. albidus, and allowed the understanding of adjustments in the primary response mechanisms that enable worms to maintain homeostasis and survival in harsh environments such as polar and temperate-cold regions. The presence of moderate levels of salinity significantly increased freeze-tolerance (mainly evaluated as survival, cryoprotection and ice fraction) and reproduction of E. albidus. Moreover, it contributed to the readjustments of cryoprotectant levels, restoration of antioxidant levels and changed singnificantly the effect and uptake of chemicals (copper cadmium, carbendazim and 4-nonylphenol). Temperature fluctuations (simulated as daily freeze-thaw cycles, between -2ºC and -4ºC) caused substancial negative effect on survival of worms previsouly exposed to non-lethal concentrations of 4-nonylphenol, as compared with constant freezing (-4ºC) and control temperature (2ºC). The decrease in cryoprotectants, increase in energy consumption and the highest concentration of 4-nonylphenol in the tissues have highlighted the high energy requirements and level of toxicity experienced by worms exposed to the combined effect of contaminants and freezing-thawing events. The findings reported on this thesis demonstrate that natural (physical) and chemical stressors, singly or in combination, may alter the dynamics of E. albidus, affecting not only their survival and reproduction (and consequent presence/distribution) but also their physiological and biochemical adaptations. These alterations may lead to severe consequences for the functioning of the ecosystems along the Arctic, subarctic and cold temperate regions, where they play an important role for decomposition of dead organic matter. This thesis provides a scientific basis for improving the setting of safety factors for natural soil ecosystems, and to underline the integration of similar investigations in ecotoxicology, and eventually in risk assessment of contaminants.

Origin, seasonality and chemical characteristics of rainwater in Aveiro

Este trabalho teve como objectivo caracterizar quimicamente a água da chuva recolhida na cidade de Aveiro, localizada a sudoeste da Europa, no período de Setembro de 2008 a Setembro de 2009. Para matrizes diluídas como a da água da chuva, as metodologias analíticas a utilizar para se conseguir uma rigorosa caracterização química são de grande importância e ainda não estão uniformizadas. Assim, para caracterizar a fracção orgânica, primeiramente foram comparadas duas metodologias de filtração (0.22 e 0.45 μm) e foram estudados dois procedimentos de preservação da água da chuva (refrigeração e congelação), utilizando a espectroscopia de fluorescência molecular. Além disso, foram comparados dois procedimentos de isolamento e extracção da matéria orgânica dissolvida (DOM) da água da chuva, baseados na sorção nos sorbentes DAX-8 e C-18, utilizando as espectroscopias de ultravioleta-visível e fluorescência molecular. Relativamente aos resultados das metodologias de filtração e preservação, é recomendada a filtração por 0.45 μm, assim como, as amostras de água da chuva deverão ser mantidas no escuro a 4ºC, no máximo até 4 dias, até às análises espectroscópicas. Relativamente à metodologia de isolamento e extracção da DOM, os resultados mostraram que o procedimento de isolamento baseado na C-18 extraiu a DOM que é representativa da matriz global, enquanto que o procedimento da DAX-8 extraiu preferencialmente a fracção do tipo húmico. Como no presente trabalho pretendíamos caracterizar a fracção do tipo húmico da DOM da água da chuva, foi escolhida a metodologia de isolamento e extracção baseada na sorção no sorvente DAX-8. Previamente ao isolamento e extracção da DOM da água da chuva, toda a fracção orgânica das amostras de água da chuva foi caracterizada pelas técnicas de ultravioleta-visível e de fluorescência molecular. As amostras mostraram características semelhantes às de outras águas naturais, e a água da chuva do Verão e Outono apresentou maior conteúdo da matéria orgânica dissolvida cromofórica que a do Inverno e Primavera. Posteriormente, a fracção do tipo húmico de algumas amostras de água da chuva, isolada e extraída pelo procedimento baseado na DAX-8, foi caracterizada utilizando as técnicas espectroscópicas de ultravioleta-visível, fluorescência molecular e ressonância magnética nuclear de protão. Todos os extractos continham uma mistura complexa de compostos hidroxilados e ácidos carboxílicos, com uma predominância da componente alifática e um baixo conteúdo da componente aromática. A fracção inorgânica da água da chuva foi caracterizada determinando a concentração das seguintes espécies iónicas: H+, NH4 +, Cl-, NO3 -, SO4 2-. Os resultados foram comparados com os obtidos na chuva colectada no mesmo local entre 1986-1989 e mostraram que de todos os iões determinados a concentração de NO3 - foi a única que aumentou (cerca do dobro) em 20 anos, tendo sido atribuído ao aumento de veículos e emissões industriais na área de amostragem. Durante o período de amostragem cerca de 80% da precipitação esteve associada a massas de ar oceânicas, enquanto a restante esteve relacionada com massas que tiveram uma influência antropogénica e terrestre. De um modo geral, para as fracções orgânica e inorgânica da água da chuva analisadas, o conteúdo químico foi menor para as amostras que estiveram associadas a massas de ar marítimas do que para as amostras que tiveram contribuições terrestres e antropogénicas.

Single and joint effects of natural and chemical stressors to the terrestrial isopod Porcellionides pruinosus

Environmental contamination and climate changes constitute two of the most serious problems affecting soil ecosystems in agricultural fields. Agriculture is nowadays a highly optimized process that strongly relies on the application of multiple pesticides to reduce losses and increase yield production. Although constituting, per se, a serious problem to soil biota, pesticide mixtures can assume an even higher relevance in a context of unfavourable environmental conditions. Surprisingly, frameworks currently established for environmental risk assessments keep not considering environmental stressors, such as temperature, soil moisture or UV radiation, as factors liable to influence the susceptibility of organisms to pesticides, or pesticide mixtures, which is raising increasing apprehension regarding their adequacy to actually estimate the risks posed by these compounds to the environment. Albeit the higher attention received on the last few years, the influence of environmental stressors on the behaviour and toxicity of chemical mixtures remains still poorly understood. Aiming to contribute for this discussion, the main goal of the present thesis was to evaluate the single and joint effects of natural stressors and pesticides to the terrestrial isopod Porcellionides pruinosus. The first approach consisted on evaluating the effects of several abiotic factors (temperature, soil moisture and UV radiation) on the performance of P. pruinosus using several endpoints: survival, feeding parameters, locomotor activity and avoidance behaviour. Results showed that these stressors might indeed affect P. pruinosus at relevant environmental conditions, thus suggesting the relevance of their consideration in ecotoxicological assays. At next, a multiple biomarker approach was used to have a closer insight into the pathways of damage of UV radiation and a broad spectrum of processes showed to be involved (i.e. oxidative stress, neurotoxicity, energy). Furthermore, UV effects showed to vary with the environment medium and growth-stage. A similar biomarker approach was employed to assess the single and joint effects of the pesticides chlorpyrifos and mancozeb to P. pruinosus. Energy-related biomarkers showed to be the most differentiating parameters since age-classes seemed to respond differently to contamination stress and to have different metabolic costs associated. Finally, the influence of temperature and soil moisture on the toxicity of pesticide mixtures was evaluated using survival and feeding parameters as endpoints. Pesticide-induced mortality was found to be oppositely affected by temperature, either in single or mixture treatments. Whereas chlorpyrifos acute toxicity was raised under higher temperatures the toxicity of mancozeb was more prominent at lower temperatures. By the opposite, soil moisture showed no effects on the pesticide-induced mortality of isopods. Contrary to survival, both temperature and soil moisture showed to interact with pesticides to influence isopods’ feeding parameters. Nonetheless, was however the most common pattern. In brief, findings reported on this thesis demonstrated why the negligence of natural stressors, or multiple stressors in general, is not a good solution for risk assessment frameworks.

Photodegradation of sertraline by solar radiation and influencing factors

Photodegradation is considered to be one of the most important processes of elimination of pharmaceutical drugs from natural water matrices. The high consumption and discharge of these substances, in particular antidepressants, to the aquatic environment supports the need to study degradation processes. This dissertation aimed at studying the direct and indirect photodegradation of sertraline, an antidepressant known for its persistence in the environment, and the evaluation of the influence of environmentally relevant factors in its photodegradation. The photodegradation experiments were developed under simulated solar light and the irradiation times converted to summer sunny days (SSD), an equivalent time in natural environmental conditions. The direct photodegradation was evaluated in solutions of sertraline prepared in ultrapure water and the indirect photodegradation was studied through the addition of photosensitizers (humic substances, Fe(III), nitrates and oxygen). Further irradiation studies were perfomed in aqueous samples collected from two wastewater treatment plants, Vouga river and Ria de Aveiro. The samples were chemically characterized (dissolved organic carbon, nitrates and nitrites and iron determination and UV/Vis spectroscopy). The quantification of sertraline was done by HPLC-UV and photoproducts from direct photodegradation were identified by electrospray mass spectrometry. An observed direct photodegradation rate of sertraline of 0.0062 h-1 was determined, corresponding to a half-life time of 111 h (equivalent to 29 SSD). A significant influence of photosensitizers was observed, the best results being achieved in irradiations of sertraline with humic acids, obtaining a half-life time of 12 h. This was attributed to the hydrophobicity of this substance and higher absortivity in the UV/Vis wavelength, which promote processes of indirect photodegradation. The degradation of sertraline in natural samples was also enhanced comparatively to the direct photodegradation, achieving half-life times between 10 and 25h; the best results were achieved in samples from the primary treatment of a wastewater treatment plant and Ria de Aveiro, with half-life times of 10 and 16 h, respectively. A total of six photoproducts formed during the direct photodegradation of sertraline were identified, three of which were not yet identified in the literature. The main factors contributing to the degradation of sertraline were analysed but this was not fully accomplished, requiring further studies of the composition of the natural matrices and the combined influence of distinct photosensitizers during the irradiation. Nevertheless, it was concluded that the photodegradation of sertraline is greatly influenced by indirect photodegradation processes, promoted by the presence of photosensitizers.