Seismic vulnerability and risk assessment of historic masonry buildings

Seismic risk evaluation of built-up areas involves analysis of the level of earthquake hazard of the region, building vulnerability and exposure. Within this approach that defines seismic risk, building vulnerability assessment assumes great importance, not only because of the obvious physical consequences in the eventual occurrence of a seismic event, but also because it is the one of the few potential aspects in which engineering research can intervene. In fact, rigorous vulnerability assessment of existing buildings and the implementation of appropriate retrofitting solutions can help to reduce the levels of physical damage, loss of life and the economic impact of future seismic events. Vulnerability studies of urban centresshould be developed with the aim of identifying building fragilities and reducing seismic risk. As part of the rehabilitation of the historic city centre of Coimbra, a complete identification and inspection survey of old masonry buildings has been carried out. The main purpose of this research is to discuss vulnerability assessment methodologies, particularly those of the first level, through the proposal and development of a method previously used to determine the level of vulnerability, in the assessment of physical damage and its relationship with seismic intensity.

Development of open models and tools for seismic risk assessment: application to Portugal

The exponential growth of the world population has led to an increase of settlements often located in areas prone to natural disasters, including earthquakes. Consequently, despite the important advances in the field of natural catastrophes modelling and risk mitigation actions, the overall human losses have continued to increase and unprecedented economic losses have been registered. In the research work presented herein, various areas of earthquake engineering and seismology are thoroughly investigated, and a case study application for mainland Portugal is performed. Seismic risk assessment is a critical link in the reduction of casualties and damages due to earthquakes. Recognition of this relation has led to a rapid rise in demand for accurate, reliable and flexible numerical tools and software. In the present work, an open-source platform for seismic hazard and risk assessment is developed. This software is capable of computing the distribution of losses or damage for an earthquake scenario (deterministic event-based) or earthquake losses due to all the possible seismic events that might occur within a region for a given interval of time (probabilistic event-based). This effort has been developed following an open and transparent philosophy and therefore, it is available to any individual or institution. The estimation of the seismic risk depends mainly on three components: seismic hazard, exposure and vulnerability. The latter component assumes special importance, as by intervening with appropriate retrofitting solutions, it may be possible to decrease directly the seismic risk. The employment of analytical methodologies is fundamental in the assessment of structural vulnerability, particularly in regions where post-earthquake building damage might not be available. Several common methodologies are investigated, and conclusions are yielded regarding the method that can provide an optimal balance between accuracy and computational effort. In addition, a simplified approach based on the displacement-based earthquake loss assessment (DBELA) is proposed, which allows for the rapid estimation of fragility curves, considering a wide spectrum of uncertainties. A novel vulnerability model for the reinforced concrete building stock in Portugal is proposed in this work, using statistical information collected from hundreds of real buildings. An analytical approach based on nonlinear time history analysis is adopted and the impact of a set of key parameters investigated, including the damage state criteria and the chosen intensity measure type. A comprehensive review of previous studies that contributed to the understanding of the seismic hazard and risk for Portugal is presented. An existing seismic source model was employed with recently proposed attenuation models to calculate probabilistic seismic hazard throughout the territory. The latter results are combined with information from the 2011 Building Census and the aforementioned vulnerability model to estimate economic loss maps for a return period of 475 years. These losses are disaggregated across the different building typologies and conclusions are yielded regarding the type of construction more vulnerable to seismic activity.

Combined effects of chemical and natural stressors to soil organisms and plants

Nas últimas décadas, a Terra tem experimentado um aquecimento global e mudanças nos padrões de precipitação. Muitos estudos sobre a avaliação de risco de agrotóxicos em organismos não-alvo foram realizados com base em protocolos padronizados, com condições abióticas controladas. Mas, em campo, os organismos são expostos a flutuações de vários fatores ambientais, bem como a poluentes, que podem alterar os limites de tolerância dos organismos aos stressores naturais, bem como alterar a toxicidade ou biodisponibilidade do químico em causa. Considerando isso, o principal objetivo deste trabalho foi o de avaliar de que modo e em que medida os fatores ambientais (temperatura, humidade do solo e radiação UV) podem interagir uns com os outros ou afetar a toxicidade do carbaril para invertebrados do solo e plantas. Para isso, foram utilizadas quatro espécies padrão: Folsomia candida, Eisenia andrei, Triticum aestivum e Brassica rapa, e simulados diferentes cenários climáticos, com vários parâmetros letais e subletais analisados. A exposição combinada foi analisada utilizando, quando possível, a ferramenta MIXTOX, com base no modelo de referência de acção independente (IA) e possíveis desvios, assim como rácios sinergísticos/antagonísticos (a partir de valores de EC50/LC50), quando a dose-resposta de um dos stressores não foi obtida. Todos os fatores de stress aplicados isoladamente causaram efeitos significativos sobre as espécies testadas e sua exposição combinada com carbaril, apresentaram respostas diferenciadas: para as minhocas, a seca e temperaturas elevadas aumentaram os efeitos deletérios do carbaril (sinergismo), enquanto o alagamento e temperaturas baixas diminuíram sua toxicidade (antagonismo). Para os colêmbolos, o modelo IA mostrou ser uma boa ferramenta para prever a toxicidade do carbaril tanto para temperaturas altas como para as baixas. Para as duas espécies de plantas foram encontradas diferenças significativas entre elas: em termos gerais, as interações entre carbaril e os stressores naturais foram observadas, com sinergismo aparecendo como o padrão principal relacionado com a radiação UV, solos secos e temperaturas elevadas, enquanto o padrão principal relacionado com temperaturas baixas e stress de alagamento foi o antagonismo. Quando os efeitos de dois stressores naturais (radiação UV e humidade do solo) em plantas foram avaliados, uma interação significativa foi encontrada: a seca aliviou o efeito deletério da radiação UV em T. aestivum e o alagamento aumentou os seus efeitos, mas para B. rapa a adição de ambos os stresses de água causou um aumento (sinergismo) dos efeitos deletérios da radiação UV para todos os parâmetros avaliados. Portanto é necessário que as diferenças sazonais e latitudinais, bem como as mudanças climáticas globais, sejam integradas na avaliação de risco de contaminantes do solo.

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.

From genes to population: effects of toxicants on Enchytraeus albidus

Nowadays, a systems biology approach is both a challenge as well as believed to be the ideal form of understanding the organisms’ mechanisms of response. Responses at different levels of biological organization should be integrated to better understand the mechanisms, and hence predict the effects of stress agents, usable in broader contexts. The main aim of this thesis was to evaluate the underlying mechanisms of Enchytraeus albidus responses to chemical stressors. Therefore, there was a large investment on the gene library enrichment for this species, as explained ahead. Overall, effects of chemicals from two different groups (metals and pesticides) were assessed at different levels of biological organization: from genes and biochemical biomarkers to population endpoints. Selected chemicals were: 1) the metals cadmium and zinc; 2) the insecticide dimethoate, the herbicide atrazine and the fungicide carbendazim. At the gene and sub-cellular level, the effects of time and dosage were also adressed. Traditional ecotoxicological tests - survival, reproduction and avoidance behavior - indicated that pesticides were more toxic than metals. Avoidance behaviour is extremely important from an ecological point of view, but not recommended to use for risk assessment purposes. The oxidative stress related experiment showed that metals induced significant effects on several antioxidant enzyme activities and substrate levels, as well as oxidative damage on the membrane cells. To increase the potential of our molecular tool to assess transcriptional responses, the existing cDNA library was enriched with metal and pesticide responding genes, using Suppression Subtractive Hybridization (SSH). With the sequencing information obtained, an improved Agilent custom oligonucleotide microarray was developed and an EST database, including all existing molecular data on E. albidus, was made publicly available as an interactive tool to access information. With this microarray tool, most interesting and novel information on the mechanisms of chemical toxicity was obtained, with the identification of common and specific key pathways affected by each compound. The obtained results allowed the identification of mechanisms of action for the tested compounds in E. albidus, some of which are in line with the ones known for mammals, suggesting across species conserved modes of action and underlining the usefulness of this soil invertebrate as a model species. In general, biochemical and molecular responses were influenced by time of exposure and chemical dosage and these allowed to see the evolution of events. Cellular energy allocation results confirmed the gene expression evidences of an increased energetic expenditure, which can partially explain the decrease on the reproductive output, verified at a later stage. Correlations found throughout this thesis between effects at the different levels of biological organization have further improved our knowledge on the toxicity of metals and pesticides in this species.

Seismic vulnerability assessment of slender masonry structures

Slender masonry structures are distributed all over the world and constitute a relevant part of the architectural and cultural heritage of humanity. Their protection against earthquakes is a topic of great concern among the scientific community. This concern mainly arises from the strong damage or complete loss suffered by this group of structures due to catastrophic events and the need and interest to preserve them. Although the great progress in technology, and in the knowledge of seismology and earthquake engineering, the preservation of these brittle and massive structures still represents a major challenge. Based on the research developed in this work it is proposed a methodology for the seismic risk assessment of slender masonry structures. The proposed methodology was applied for the vulnerability assessment of Nepalese Pagoda temples which follow very simple construction procedure and construction detailing in relation to seismic resistance requirements. The work is divided in three main parts. Firstly, particular structural fragilities and building characteristics of the important UNESCO classified Nepalese Pagoda temples which affect their seismic performance and dynamic properties are discussed. In the second part the simplified method proposed for seismic vulnerability assessment of slender masonry structures is presented. Finally, the methodology proposed in this work is applied to study Nepalese Pagoda temples, as well as in the efficiency assessment of seismic performance improvement solution compatible with original cultural and technological value.

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.

Cartografia geoquímica da ilha de Santiago com uma densidade de amostragem média/baixa

A ilha de Santiago é a maior do arquipélago de Cabo Verde, apresenta uma área de 991 km2, com um comprimento e largura máximos de 54,9 km e 29 km, respectivamente, e 1392 m de maior altitude. As condições climáticas e a erosão são alguns dos problemas naturais do arquipélago de Cabo Verde. Além disso, a intervenção humana no ambiente superficial revela-se muitas vezes inadequada e com forte impacto. A construção em solos com aptidão agrícola ou florestal, as actividades industriais, a deposição de materiais sólidos ou líquidos de forma inadequada, as práticas agrícolas incorrectas e intensivas, o uso abusivo de pesticidas e fertilizantes, a rega com água contaminada, a sobreexploração de aquíferos que levam muitas vezes à salinização, etc. têm enorme impacto em termos de contaminação de solos, águas superficiais e subterrâneas nas áreas envolventes. Por conseguinte, o conhecimento da variabilidade geoquímica natural é fundamental para a resolução de questões de índole económica, ambiental e de ordenamento do território, médica, e jurídica. A necessidade de construir uma base de dados de geoquímica georeferenciada que caracterize o ambiente superficial da ilha de Santiago foi a principal motivação para a realização deste estudo. Realizou-se um levantamento geoquímico de 337 amostras de sedimentos de corrente e 249 amostras de solos na ilha de Santiago, tendo sido seguidas as recomendações do Projecto Internacional IGCP 259 não só na fase de amostragem, mas também nas fases seguintes de preparação, análise, tratamento dos dados e elaboração de mapas. Determinaram-se os teores, na fracção < 2 mm, para 36 elementos – 9 elementos maiores (Al, Ca, Fe, K, Mg, Mn, Na, P, Ti) e 27 elementos vestigiais (Ag, As, Au, B, Ba, Bi, Cd, Co, Cr, Cu, Ga, Hg, La, Mo, Ni, Pb, S, Sb, Sc, Se, Sr, Th, Tl, U, V, W, Zn). Efectuou-se ainda a análise textural e estudou-se a composição mineralógica de cerca de 25% das amostras. Analisaram-se, também, 103 amostras de rochas, colhidas nas várias formações da ilha de Santiago, tendo sido determinados os teores de K2O, Na2O, Fe2O3(T), MnO, Sc, Cr, Co, Zn, Ga, As, Br, Rb, Zr, Sb, Cs, Ba, Hf, Ta, W, Th e U e REE, a fim de se fazer uma comparação com os teores destes elementos encontrados nos solos e sedimentos de corrente, averiguando se a sua variação é ou não essencialmente condicionada pela geoquímica da rocha-mãe. Os padrões geoquímicos obtidos através dos mapas de distribuição espacial foram correlacionados com a natureza da rocha mãe, o tipo de solo, e ainda com algumas fontes de contaminação. A interpretação dos resultados foi realizada não só pela observação dos mapas geoquímicos, mas também após análise estatística dos conjuntos de dados obtidos, e apoiada em informação diversa disponível. A utilização da Análise de Componentes Principais permitiu distinguir associações entre elementos químicos, quer de origem geogénica quer antropogénica. Foram ainda elaborados mapas de distribuição espacial de vários índices multielementares de importância ambiental, como o Índice de Acidificação Al/(Ca+Mg+K), Índice de Combi, Índice de Avaliação de Risco Ambiental e Índices de Enriquecimento/Contaminação para vários grupos de elementos considerados como “primary pollutant metals”.

Genotoxic risk of herbicides to Anguilla anguilla L.

No contexto dos contaminantes aquáticos, os herbicidas são considerados como um dos grupos mais perigosos. Uma vez aplicados, estes são facilmente transportados para cursos de água, quer devido a uma pulverização pouco cuidada ou devido a fenómenos de escorrência superficial e/ou subterrânea. A presença destes agroquímicos no ambiente tem vindo a ser associada a efeitos nefastos em organismos não-alvo, como é o caso dos peixes. Contudo, existe ainda uma grande lacuna no que diz respeito à informação científica relacionada com o seu impacto genotóxico. Deste modo, a presente tese foi delineada com o intuito de avaliar o risco genotóxico em peixes de duas formulações de herbicidas: o Roundup®, que tem como princípio activo o glifosato, e o Garlon®, que apresenta o triclopir na base da sua constituição, produtos estes largamente utilizados na limpeza de campos agrícolas, assim como em florestas. Foi ainda planeado desenvolver uma base de conhecimento no que diz respeito aos mecanismos de dano do ADN. Como último objectivo, pretendeu-se contribuir para a mitigação dos efeitos dos agroquímicos no biota aquático, nomeadamente em peixes, fornecendo dados científicos no sentido de melhorar as práticas agrícolas e florestais. Este estudo foi realizado adoptando a enguia europeia (Anguilla anguilla L.) como organismo-teste, e submetendo-a a exposições de curta duração (1 e 3 dias) dos produtos comerciais mencionados, em concentrações consideradas ambientalmente realistas. Para a avaliação da genotoxicidade foram aplicadas duas metodologias: o ensaio do cometa e o teste das anomalias nucleares eritrocíticas (ANE). Enquanto o ensaio do cometa detecta quebras na cadeia do ADN, um dano passível de ser reparado, o aparecimento das ANE revela lesões cromossomais, sinalizando um tipo de dano de difícil reparação. O ensaio do cometa foi ainda melhorado com uma nova etapa que incluiu a incubação com enzimas de reparação (FPG e EndoIII), permitindo perceber a ocorrência de dano oxidativo no ADN. No que diz respeito ao Roundup®, o envolvimento do sistema antioxidante como indicador de um estado próoxidante foi também alvo de estudo. Uma vez que as referidas formulações se apresentam sob a forma de misturas, o potencial genotóxico dos seus princípios activos foi também avaliado individualmente. No caso particular do Roundup®, também foram estudados o seu surfactante (amina polietoxilada; POEA) e o principal metabolito ambiental (ácido aminometilfosfórico; AMPA). Os resultados obtidos mostraram a capacidade do Roundup® em induzir tanto dano no ADN (em células de sangue, guelras e fígado) como dano cromossómico (em células de sangue). A investigação sobre o possível envolvimento do stresse oxidativo demonstrou que o tipo de dano no ADN varia com as concentrações testadas e com a duração da exposição. Deste modo, com o aumento do tempo de exposição, os processos relacionados com o envolvimento de espécies reactivas de oxigénio (ERO) ganharam preponderância como mecanismo de dano no ADN, facto que é corroborado pela activação do sistema antioxidante observado nas guelras, assim como pelo aumento dos sítios sensíveis a FPG em hepatócitos. O glifosato e o POEA foram também considerados genotóxicos. O POEA mostrou induzir uma maior extensão de dano no ADN, tanto comparado com o glifosato como com a mistura comercial. Apesar de ambos os componentes contribuirem para a genotoxicidade da formulação, a soma dos seus efeitos individuais nunca foi observada, apontando para um antagonismo entre eles e indicando que o POEA não aumenta o risco associado ao princípio activo. Deste modo, realça-se a necessidade de regulamentar limiares de segurança para todos os componentes da formulação, recomendando, em particular, a revisão da classificação do risco do POEA (actualmente classificado com “inerte”). Uma vez confirmada a capacidade do principal metabolito do glifosato – AMPA – em exercer dano no ADN assim como dano cromossómico, os produtos da degradação ambiental dos princípios activos assumem-se como um problema silencioso, realçando assim a importância de incluir o AMPA na avaliação do risco relacionado com herbicidas com base no glifosato. A formulação Garlon® e o seu princípio activo triclopir mostraram um claro potencial genotóxico. Adicionalmente, o Garlon® mostrou possuir um potencial genotóxico mais elevado do que o seu princípio activo. No entanto, a capacidade de infligir dano oxidativo no ADN não foi demonstrada para nenhum dos agentes. No que concerne à avaliação da progressão do dano após a remoção da fonte de contaminação, nem os peixes expostos a Roundup® nem os expostos a Garlon® conseguiram restaurar completamente a integridade do seu ADN ao fim de 14 dias. No que concerne ao Roundup®, o uso de enzimas de reparação de lesões específicas do ADN associado ao teste do cometa permitiu detectar um aparecimento tardio de dano oxidativo, indicando deste modo um decaimento progressivo da protecção antioxidante e ainda uma incapacidade de reparar este tipo de dano. O período de pós-exposição correspondente ao Garlon® revelou uma tendência de diminuição dos níveis de dano, apesar de nunca se observar uma completa recuperação. Ainda assim, foi evidente uma intervenção eficiente das enzimas de reparação do ADN, mais concretamente as direccionadas às purinas oxidadas. A avaliação das metodologias adoptadas tornou evidente que o procedimento base do ensaio do cometa, que detecta apenas o dano nãoespecífico no ADN, possui algumas limitações quando comparado com a metodologia que incluiu a incubação com as enzimas de reparação, uma vez que a última mostrou reduzir a possibilidade de ocorrência de resultados falsos negativos. Os dois parâmetros adoptados (ensaio do cometa e teste das ANE) demonstraram possuir aptidões complementares, sendo assim recomendado a sua utilização conjunta com vista a efectuar uma avaliação mais adequada do risco genotóxico. Globalmente, os resultados obtidos forneceram indicações de grande utilidade para as entidades reguladoras, contribuindo ainda para a (re)formulação de medidas de conservação do ambiente aquático. Neste sentido, os dados obtidos apontam para a importância da avaliação de risco dos herbicidas incluir testes de genotoxicidade. A magnitude de risco detectada para ambas as formulações adverte para a necessidade de adopção de medidas restritivas em relação à sua aplicação na proximidade de cursos de água. Como medidas mitigadoras de impactos ambientais, aponta-se o desenvolvimento de formulações que incorporem adjuvantes selecionados com base na sua baixa toxicidade.

Toxicogenomics of natural and anthropogenic effectors in eleutheroembryos

The introduction of chemicals into the environment by human activities may represent a serious risk to environmental and human health. Environmental risk assessment requires the use of efficient and sensitive tools to determine the impact of contaminants on the ecosystems. The use of zebrafish for the toxicity assessment of pharmaceuticals, drugs, and pollutants, is becoming well accepted due to zebrafish unique advantages for the screening of compounds for hazard identification. The aim of the present work is to apply toxicogenomic approaches to identify novel biomarkers and uncovered potential modes of action of classic and emergent contaminants able to disrupt endocrine systems, such as the Retinoic Acid Receptor, Retinoid X Receptor and the Aryl Hydrocarbon Receptor. This study relies on different nuclear and cytosolic protein receptors and other conditional (ligand- or stress- activated) transcriptional factors that are intimately involved in the regulation of defensome genes and in mechanisms of chemical toxicity. The transcriptomic effects of organic compounds, endogenous compounds, and nanoparticles were analysed during the early stages of zebrafish development. Studying the gene expression profiles of exposed and unexposed organisms to pollutants using microarrays allowed the identification of specific gene markers and to establish a "genetic code" for the tested compounds. Changes in gene expression were observed at toxicant concentrations that did not cause morphological effects. Even at low toxicant concentrations, the observed changes in transcript levels were robust for some target genes. Microarray responses of selected genes were further complemented by the real time quantitative polymerase chain reaction (qRT-PCR) methodology. The combination of bio-informatic, toxicological analyses of differential gene expression profiles, and biochemical and phenotypic responses across the treatments allowed the identification of uncovered potential mechanisms of action. In addition, this work provides an integrated set of tools that can be used to aid management-decision making by improving the predictive capability to measure environmental stress of contaminants in freshwater ecosystems. This study also illustrates the potential of zebrafish embryos for the systematic, large-scale analysis of chemical effects on developing vertebrates.

Toxicidade de químicos em mistura: o caso da albufeira do Alqueva

In the environment humans and biota are generally exposed to chemical mixtures rather than individual chemicals. Therefore, when assessing the environmental risk of chemicals, it is important to consider chemical mixtures and their possible interactions. The main objective of this work focused on the environmental risk assessment of pesticides found in the water of the Alqueva reservoir and their binary combinations. In this aquatic ecosystem several pesticides were above of the environmental quality standards. But in addition, there were several sampling points of the reservoir where ecotoxicity was observed despite the presence of these contaminants at low concentrations. Here, a component-based approach was used to assess the effects of the pesticide mixtures. The effects of the binary combinations of four herbicides, atrazine (ATR), terbuthylazine (TER), simazine (SIM) and metolachlor (MET), on the growth rate of the microalgae Pseudokirchneriella subcapitata and the effects of the binary combinations of the s-triazine herbicides ATR and TER and the insecticide chlorpyrifos (CPF) on the swimming behaviour and acetylcholinesterase (AChE) activity of the zebrafish Danio rerio were assessed using the two reference models of concentration addition (CA) and independent action (IA). Moreover, the combined effects of the herbicides (ATR, TER and MET) and the insecticide CPF were also tested on the swimming behaviour and AChE activity of the aquatic midge Chironomus riparius after the cholinesterases characterization. In this risk characterization, the calculated risk quotients for the herbicides ATR, TER, SIM and MET were higher than 1, meaning that these herbicides present a high risk for the Alqueva ecosystem. As expected, the microalgae P. subcapitata was the most sensitive species to the herbicides. However, despite these herbicides pose no or low risk to other aquatic organisms tested in this study, with EC50 values much higher than the concentrations found in this aquatic ecosystem, they are able to increase the toxic effects of CPF when they are tested in binary mixtures. Moreover, the risk quotients of mixtures of these herbicides present simultaneously in three different locations of the reservoir were also higher than 1, so this confirms the fact that these herbicides when present in mixtures, present a greater risk for this ecosystem than the expected considering each single chemical by its own.

Effect assessment of nanoparticles toxicity in the terrestrial compartment

Over 11 million tons of nanomaterials (NMs) have been produced in 2012 and predictions point the increase in production. Despite predictions and extended usage via consumer products and industry, the understanding of the potential impact of these materials on the environment is virtually absent. The main aim of this thesis is to understand how a selected group of nanomaterials (metal based particles) may impact soil invertebrates, with special focus on the mechanisms of response. Since a case-by-case Environmental Risk Assessment (ERA) of all the emerging contaminants (particularly NMs) is impossible, among others due to time and cost reasons, to gain understanding on the mechanism of action and response is very important to reach a common paradigm. Understanding the modes of action provides predictive characters in cross particle extrapolation. Besides, it also provides insight for the production of new and sustainable materials. Overall, the effects of the selected NMs (Copper and Silver, Titanium and Zirconium oxides) and the respective salt forms, were investigated at the gene expression (using high-throughput tools, microarray and qPCR technology), biochemical (using enzymatic assays for analysis of oxidative stress markers) and organism (survival and reproduction as in OECD test guidelines) levels, this using standard soil species (Enchytraeus albidus, Enchytraeus crypticus, Eisenia fetida). Gene expression analysis provided valuable information on the mechanisms affected by each of the NMs. The gene expression profile highlighted a (nano)material signature and the effect of the duration of exposure. The functional analyses integrated with the biochemical and organism data, revealed a good understanding power. The biochemical parameters (oxidative stress related) were distinct across the materials and also influenced by duration of exposure and concentration. The standardized organismal responses differed the least between the various materials. The overall outcome is that, in this context of NMs effect assessment, gene expression and enzymatic assays introduced a very important knowledge gap, which could not had been achieved by the standard organismal effects alone. A reoccurring issue with some metal based NMs is the possible dissolution and subsequent release of ions that then causes toxicity e.g. Cu-NPs or Ag-NPs release Cu2+ or Ag+. The oxidation state of the particles was investigated, although this was not the focus of the thesis. The study of fate, e.g. dissolution of NPs, is also only in its beginning and the appropriate techniques are currently being developed. The results showed a specific nanoparticle effect. The UV exposure with titanium dioxide nanoparticles increased its effect.

Organic contaminants in urban soils: major inputs and potential risks

Urban soil quality may be severely affected by hydrophobic organic contaminants (HOCs), impairing environmental quality and human health. A comprehensive study was conducted in two contrasting Portuguese urban areas (Lisbon and Viseu) in order to assess the levels and potential risks of these contaminants, to identify sources and study their behaviour in soils. The concentrations of HOCs were related to the size of the city, with much higher contamination levels observed in Lisbon urban area. Source apportionment was performed by studying the HOCs profiles, their relationship with potentially toxic elements and general characteristics of soil using multivariate statistical methods. Lisbon seems to be affected by nearby sources (traffic, industry and incineration processes) whereas in Viseu the atmospheric transport may be playing an important role. In a first tier of risk assessment (RA) it was possible to identify polycyclic aromatic hydrocarbons (PAHs) in Lisbon soils as a potential hazard. The levels of PAHs in street dusts were further studied and allowed to clarify that traffic, tire and pavement debris can be an important source of PAHs to urban soils. Street dusts were also identified as being a potential concern regarding human and environmental health, especially if reaching the nearby aquatic bodies. Geostatistical tools were also used and their usefulness in a RA analysis and urban planning was discussed. In order to obtain a more realistic assessment of risks of HOCs to environment and human health it is important to evaluate their available fraction, which is also the most accessible for organisms. Therefore, a review of the processes involved on the availability of PAHs was performed and the outputs produced by the different chemical methods were evaluated. The suitability of chemical methods to predict bioavailability of PAHs in dissimilar naturally contaminated soils has not been demonstrated, being especially difficult for high molecular weight compounds. No clear relationship between chemical and biological availability was found in this work. Yet, in spite of the very high total concentrations found in some Lisbon soils, both the water soluble fraction and the body residues resulting from bioaccumulation assays were generally very low, which may be due to aging phenomena. It was observed that the percentage of soluble fraction of PAHs in soils was found to be different among compounds and mostly regulated by soil properties. Regarding bioaccumulation assays, although no significant relationship was found between soil properties and bioavailability, it was verified that biota-to-soil bioaccumulation factors were sample dependent rather than compound dependent. In conclusion, once the compounds of potential concern are targeted, then performing a chemical screening as a first tier can be a simple and effective approach to start a RA. However, reliable data is still required to improve the existing models for risk characterization.