Development of an integrative approach for the characterization of ecological risk on marine transition ecosystems: the Sado Estuary as a case study

Estuaries and other transitional waters are complex ecosystems critically important as nursery and shelter areas for organisms. Also, humans depend on estuaries for multiple socio-economical activities such as urbanism, tourism, heavy industry, (taking advantage of shipping), fisheries and aquaculture, the development of which led to strong historical pressures, with emphasis on pollution. The degradation of estuarine environmental quality implies ecologic, economic and social prejudice, hence the importance of evaluating environmental quality through the identification of stressors and impacts. The Sado Estuary (SW Portugal) holds the characteristics of industrialized estuaries, which results in multiple adverse impacts. Still, it has recently been considered moderately contaminated. In fact, many studies were conducted in the past few years, albeit scattered due to the absence of true biomonitoring programmes. As such, there is a need to integrate the information, in order to obtain a holistic perspective of the area able to assist management and decision-making. As such, a geographical information system (GIS) was created based on sediment contamination and biomarker data collected from a decade-long time-series of publications. Four impacted and a reference areas were identified, characterized by distinct sediment contamination patterns related to different hot spots and diffuse sources of toxicants. The potential risk of sediment-bound toxicants was determined by contrasting the levels of pollutants with available sediment quality guidelines, followed by their integration through the Sediment Quality guideline Quotient (SQG-Q). The SQG-Q estimates per toxicant or class was then subjected to georreferencing and statistical analyses between the five distinct areas and seasons. Biomarker responses were integrated through the Biomarkers Consistency Indice and georreferenced as well through GIS. Overall, in spite of the multiple biological traits surveyed, the biomarker data (from several organisms) are accordant with sediment contamination. The most impacted areas were the shipyard area and adjacent industrial belt, followed by urban and agricultural grounds. It is evident that the estuary, although globally moderately impacted, is very heterogeneous and affected by a cocktail of contaminants, especially metals and polycyclic aromatic hydrocarbon. Although elements (like copper, zinc and even arsenic) may originate from the geology of the hydrographic basin of the Sado River, the majority of the remaining contaminants results from human activities. The present work revealed that the estuary should be divided into distinct biogeographic units, in order to implement effective measures to safeguard environmental quality.

Characterization of the volatile fraction emitted by phloems of four pinus species by solid-phase microextraction and gas chromatography–mass spectrometry

Pine forests constitute some of the most important renewable resources supplying timber, paper and chemical industries, among other functions. Characterization of the volatiles emitted by different Pinus species has proven to be an important tool to decode the process of host tree selection by herbivore insects, some of which cause serious economic damage to pines. Variations in the relative composition of the bouquet of semiochemicals are responsible for the outcome of different biological processes, such as mate finding, egg-laying site recognition and host selection. The volatiles present in phloem samples of four pine species, P. halepensis, P. sylvestris, P. pinaster and P. pinea, were identified and characterized with the aim of finding possible host-plant attractants for native pests, such as the bark beetle Tomicus piniperda. The volatile compounds emitted by phloem samples of pines were extracted by headspace solid-phase micro extraction, using a 2 cm 50/30 mm divinylbenzene/carboxen/polydimethylsiloxane table flex solid-phase microextraction fiber and its contents analyzed by high-resolution gas chromatography, using flame ionization and a non polar and chiral column phases. The components of the volatile fraction emitted by the phloem samples were identified by mass spectrometry using time-of-flight and quadrupole mass analyzers. The estimated relative composition was used to perform a discriminant analysis among pine species, by means of cluster and principal component analysis. It can be concluded that it is possible to discriminate pine species based on the monoterpenes emissions of phloem samples.

Comparative effects of sediments contaminated by carcinogenic and non-carcinogenic PAHs in Dicentrarchus labrax: a semi-quantitative histopathological approach

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

Analysis of the interaction of polycyclic aromatic compounds in a model organism: integration of genotoxic and histopathological effects

Due to their toxicity, especially their carcinogenic potential, polycyclic aromatic hydrocarbons (PAHs) became priority pollutants in biomonitoring programmes and environmental policy, such as the European Water Framework Directive. The model substances tested in this study, namely benzo[b]fluoranthene (B[b]F), considered potentially carcinogenic to humans and an effector carcinogenic PAH to wildlife, and phenanthrene (Phe), deemed a non-carcinogenic PAH, are common PAHs in coastal waters, owning distinct properties reflected in different, albeit overlapping, mechanisms of toxicity. Still, as for similar PAHs, their interaction effects remain largely unknown. In order to study the genotoxic effects of caused by the interaction of carcinogenic and non-carcinogenic PAHs, and their relation to histopathological alterations, juvenile sea basses, Dicentrarchus labrax, a highly ecologically- and economically-relevant marine fish, were injected with different doses (5 and 10 μg.g-1 fish ww) of the two PAHs, isolated or in mixture, and incubated for 48 h. Individuals injected with B[b]F and the PAH mixture exhibited higher clastogenic/aneugenic effects and DNA strand breakage in blood cells, determined through the erythrocytic nuclear abnormalities (ENA) and Comet assays, respectively. Also, hepatic histopathological alterations were found in all animals, especially those injected with B[b]F and the PAH mixture, relating especially to inflammation. Still, Phe also exhibited genotoxic effects in sea bass, especially in higher doses, revealing a very significant acute effect that was accordant with the Microtox test performed undergone in parallel. Overall, sea bass was sensitive to B[b]F (a higher molecular weight PAH), likely due to efficient bioactivation of the pollutant (yielding genotoxic metabolites and reactive oxygen species), when compared to Phe, the latter revealing a more significant acute effect. The results indicate no significant additive effect between the substances, under the current experimental conditions. The present study highlights the importance of understanding PAH interactions in aquatic organisms, since they are usually present in the aquatic environment in complex mixtures.

Toxicity of carcinogenic and non-carcinogenic Polycyclic Aromatic Hydrocarbons and their mixtures to aquatic organisms under ecologically-relevant scenarios

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous priority pollutants that tend to be trapped in aquatic sediments due to their high hydrophobicity. Nonetheless, the differential toxicological effects and mechanisms between the various classes of PAHs and their mixtures, as they invariably occur in the environment, are scarcely known, especially under ecologically-relevant scenarios. This thesis aimed at establishing a bridge between the study of mechanistic pathways and environmental monitoring of carcinogenic and non-carcinogenic PAHs, by introducing ecological-relevance in the research with model PAHs. A first bioassay conducted in situ with the mussel Mytilus edulis demonstrated that, dredging operations in harbours increase PAH bioavailability, eliciting genotoxicity, and showed that established environmental guidelines underestimate risk. Subsequent ex situ bioassays were performed with the carcinogenic benzo[b]fluoranthene (B[b]F) and non-carcinogenic phenantrene (Phe), selected following preceding results, and revealed that low-moderate concentrations of these PAHs in spiked sediments induce genotoxic effects to the clam Ruditapes decussatus, therefore contradicting the general notion that bivalves are less sensitive to PAHs than vertebrates due to inefficient bioactivation. Also, it was demonstrated that passive samplers permit inferring on PAH bioavailability but not on bioaccumulation or toxic effects. On the other hand, sea basses (Dicentrarchus labrax), yielded a complex pattern of effects and responses, relatively to genotoxicity, oxidative stress and production of specific metabolites, especially when exposed to mixtures of the PAHs which led to additive, if not synergistic, effects. It was shown that Phe may elicit significant genotoxicity especially in presence of B[b]F, even though the low, albeit realistic, exposure concentrations diluted dose- and time-independent relationships. The present work demonstrated that environmental quality guidelines underestimate the effects of PAHs in realistic scenarios and showed that the significant genotoxic and histopathological effects caused by mixed PAHs may not be reflected by oxidative stress- or CYP-related biomarkers. Besides important findings on the metabolism of PAH mixtures, the work calls for the need to re-evaluate the criteria for assessing risk and for the disclosure of more efficient indicators of toxicological hazard.