The conservation of biodiversity and endemism in South Africa’s freshwater aquatic ecosystems is a high priority, particularly in the Cape Floristic Region. However, the perception that South Africa lacks suitable fish species for recreational angling, aquaculture and biological control, led to the widespread introduction and use of alien fish species. As a result, formal stocking programs have seen the introduction of five of the world’s top 100 invasive species into South Africa (Dudgeon et al. 2006). According to Dudgeon et al. (2006) freshwater ecosystems are the most endangered ecosystem in the world. The threats to freshwater biodiversity, according to Dudgeon et al. (2006), can be grouped into five categories that interact with one another: overexploitation, water pollution, flow modifications, destruction of habitat and invasion by exotic species. This PhD study took place in the Amatola region of the Eastern Cape Province, South Africa. The Amatola region is a rural area with no large-scale mining or industrial developments, only localised settlements. These developments are mainly situated around impoundments, because of the resources such as water and food that they provide. Thus the dams within the Amatola region should theoretically have no major industrial stressors on them. The three impoundments studied were Binfield Park, Sandile and Wriggleswade Dams. Binfield Park Dam is a 260ha impoundment. It impounds the Tyume River and is used by both subsistence anglers from the local communities and occasionally by recreational bass anglers. Sandile Dam is a 146ha impoundment and is the smallest of the three dams in this study. It impounds the Wolf and Keiskamma Rivers. Wriggleswade Dam is a 1000ha impoundment used extensively by recreational bass and carp anglers, and impounds the Kubusi River. There is a paucity of information regarding the health of the indigenous and alien fish species from the study region, as well as on the parasite diversity of these various fish species. In order to fill the gaps in the information the following hypothesis was proposed. That the necropsy based and histology based fish health assessment can be successfully implemented as tools to assess the effects of heavy metal pollution and alien fish parasites in freshwater fish from selected impoundments in the Amatola region, Eastern Cape Province, South Africa. In order to achieve this hypothesis the main aim of this study will be to use the necropsy- and histology- based fish health assessment to determine the health status of the fish species in these impoundments as well as to understand the potential threat of water pollution and fish parasites. Fish were sampled with the aid of gill nets, fyke nets and by angling from each of the three impoundments over three surveys in July 2011, and March and August 2012. Following capture fish were transported to a field laboratory in aerated containers. At the field laboratory the fish were examined and dissected using the methods recommended by Adams et al. (1993) for a necropsy-based fish health assessment. Gills, livers, kidneys and gonads samples were also collected for histological analysis. Macroscopic and histology-based fish health assessment index was used, as well the analysis of muscle tissue of Micropterus salmoides and surface water and sediment from Binfield Park, Sandile and Wriggleswade Dams. It was shown that, according to the macroscopic fish health assessment index, M. salmoides in Wriggleswade Dam had a higher FHAI score compared to those in Binfield Park and Sandile Dam, there were no significant differences between the FHAI scores. However, the cause of the higher FHAI in the Wriggleswade Dam was because of the external skin damage caused by the presence of the alien parasite Lernaea cyprinacea. The histology-based fish health assessment index, however, showed that M. salmoides from Binfield Park had significantly higher histology Fish Index (IFISH) scores compared to those in Sandile and Wriggleswade Dams. The main contributors to the high IFISH score of Binfield Park were the significantly high Liver Index (IL) and Kidney Index (IK). The increased severity of the alterations observed in the liver and kidney tissue of the Binfield Park M. salmoides samples may have been as a result of the high concentration of mercury found in the muscle tissue of M. salmoides. The water quality and metals detected in the water of Binfield Park, Sandile and Wriggleswade Dams were all below the target water quality guideline values, as well as below those of previous research into the nutrients and presence of metals in these impoundments. The sediment metal analysis showed that the levels of Co, Mn and Ni were above the target guideline levels for Binfield Park, Sandile and Wriggleswade Dams, Cu was above target guidelines levels for Sandile Dam, and uranium was above the target guideline concentrations for Wriggleswade Dam. Binfield Park Dam had significantly high levels of mercury in the muscle tissue of M. salmoides, while Sandile Dam had significantly high levels of zinc in the muscle tissue of M. salmoides. It was shown that M. salmoides from each of the three impoundments are in a healthy state according to the parameters assessed. However, the presence of heavy metals, particularly mercury, uranium and zinc, do indicate the presence of human activities. The indigenous parasites of Anguilla mossambica have been well documented including the gastrointestinal nematode Paraquimperia africana, and the stomach nematode Heliconema africanum. Indigenous parasites such as the swimbladder nematode Anguillicola papernai had no effect on the condition factor of infected and uninfected eels. However, the damage caused by the alien parasites were evident, including the first documented effects of the alien gill monogenean Pseudodactylogyrus anguillae on indigenous wild populations of the longin fin eel A. mossambica from the Eastern Cape, South Africa. Histological observations indicated that an alien gill monogenean caused hyperplasia, increase in mucous cells, rupture of pillar cells as well as telangiectasia. This alien parasite has invaded the Keiskamma and Kei River systems in the Eastern Cape, South Africa. According to the macroscopic fish health assessment index, A. mossambica from Binfield Park, Sandile and Wriggleswade Dams are in a healthy state. However, the histology-based health assessment highlighted that the effects on P. anguillae have a severe negative impact on the health of A. mossambica. Using the macroscopic and histology-based fish health assessment, a comprehensive investigation into the fish health status of Mugil cephalus and Myxus capensis from Binfield Park Dam revealed that human effects and parasites are not the only threats to freshwater fish. Nephrocalcinosis is a non-infectious kidney disease which is characterised by abnormal calcium deposition in the kidneys of humans and some fish species. According to the macroscopic and histology-based fish health assessment, the M. cephalus and M. capensis are not in a healthy condition. The macroscopic and histology-based fish assessment indices are not stressor-specific, and therefore the cause of the poor health state of these two fish species could not be determined. A possible suggestion for the poor health of these two species is the age of the species. Because the two mullet species were stocked into Binfield Park Dam, Ellender et al. (2012) could successfully age them accurately to ten years of age, which is the upper limit of the life span for these species. Macroscopic and histology-based fish heath assessments were conducted on Labeo umbratus from Sandile Dam in order to determine the health of this species. Macroscopic and histology-based fish health assessment indicated that its L. umbratus are in a healthy state. The March 2012 survey specimens had a significantly higher macroscopic FHAI score than those from the July 2011 survey. The increased FHAI score was because of parasite infections, as well as discoloured livers and increased total blood plasma protein levels, which are indicators of nutritional state. However, the presence of the anchor worm parasite Lernaea barnimiana in low numbers had no significant impact on the health of L. umbratus. The effect of the alien anchor worm parasite Lernaea cyprinacea was shown on the transloacted small mouth yellowfish Labeobarbus aeneus. It was also shown that L. aeneus are, according the macroscopic FHAI and the histology-based fish health assessment index, in a healthy state. However, the high scores observed in the macroscopic fish health assessment index were primarily as a result of the presence of the alien parasite L. cyprinacea and its associated affects on the fish host. Because of the significant impact of this alien parasite species on the translocated host species, it can be assumed that this alien parasite species will have a negative effect on the health of indigenous fish species in the Great Kei River. It is clear from the results presented in this study that the necropsy based and histology based fish health assessment can be successfully implemented as tools to assess the effects of heavy metal pollution and alien fish parasites in freshwater fish from selected impoundments in the Amatola region, Eastern Cape Province, South Africa, thus the original hypothesis of this thesis is accepted. Based on work done in this research the gaps in research have been identified. Due to the high levels of mercury indentified in the muscle tissue of M. salmoides from Binfield Park Dam. A human health assessment and edibility should be conducted in order to determine if the fish from Binfield Park Dam is safe for human consumption. In order to conserve South Africa’s Freshwater fish biodiversity, country wide surveys of indigenous fish species must be undertaken so that the health and the parasite diversity can be evaluated. / PhD (Zoology), North-West University, Potchefstroom Campus, 2015

The conservation of biodiversity and endemism in South Africa’s freshwater aquatic ecosystems is a high priority, particularly in the Cape Floristic Region. However, the perception that South Africa lacks suitable fish species for recreational angling, aquaculture and biological control, led to the widespread introduction and use of alien fish species. As a result, formal stocking programs have seen the introduction of five of the world’s top 100 invasive species into South Africa (Dudgeon et al. 2006). According to Dudgeon et al. (2006) freshwater ecosystems are the most endangered ecosystem in the world. The threats to freshwater biodiversity, according to Dudgeon et al. (2006), can be grouped into five categories that interact with one another: overexploitation, water pollution, flow modifications, destruction of habitat and invasion by exotic species. This PhD study took place in the Amatola region of the Eastern Cape Province, South Africa. The Amatola region is a rural area with no large-scale mining or industrial developments, only localised settlements. These developments are mainly situated around impoundments, because of the resources such as water and food that they provide. Thus the dams within the Amatola region should theoretically have no major industrial stressors on them. The three impoundments studied were Binfield Park, Sandile and Wriggleswade Dams. Binfield Park Dam is a 260ha impoundment. It impounds the Tyume River and is used by both subsistence anglers from the local communities and occasionally by recreational bass anglers. Sandile Dam is a 146ha impoundment and is the smallest of the three dams in this study. It impounds the Wolf and Keiskamma Rivers. Wriggleswade Dam is a 1000ha impoundment used extensively by recreational bass and carp anglers, and impounds the Kubusi River. There is a paucity of information regarding the health of the indigenous and alien fish species from the study region, as well as on the parasite diversity of these various fish species. In order to fill the gaps in the information the following hypothesis was proposed. That the necropsy based and histology based fish health assessment can be successfully implemented as tools to assess the effects of heavy metal pollution and alien fish parasites in freshwater fish from selected impoundments in the Amatola region, Eastern Cape Province, South Africa. In order to achieve this hypothesis the main aim of this study will be to use the necropsy- and histology- based fish health assessment to determine the health status of the fish species in these impoundments as well as to understand the potential threat of water pollution and fish parasites. Fish were sampled with the aid of gill nets, fyke nets and by angling from each of the three impoundments over three surveys in July 2011, and March and August 2012. Following capture fish were transported to a field laboratory in aerated containers. At the field laboratory the fish were examined and dissected using the methods recommended by Adams et al. (1993) for a necropsy-based fish health assessment. Gills, livers, kidneys and gonads samples were also collected for histological analysis. Macroscopic and histology-based fish health assessment index was used, as well the analysis of muscle tissue of Micropterus salmoides and surface water and sediment from Binfield Park, Sandile and Wriggleswade Dams. It was shown that, according to the macroscopic fish health assessment index, M. salmoides in Wriggleswade Dam had a higher FHAI score compared to those in Binfield Park and Sandile Dam, there were no significant differences between the FHAI scores. However, the cause of the higher FHAI in the Wriggleswade Dam was because of the external skin damage caused by the presence of the alien parasite Lernaea cyprinacea. The histology-based fish health assessment index, however, showed that M. salmoides from Binfield Park had significantly higher histology Fish Index (IFISH) scores compared to those in Sandile and Wriggleswade Dams. The main contributors to the high IFISH score of Binfield Park were the significantly high Liver Index (IL) and Kidney Index (IK). The increased severity of the alterations observed in the liver and kidney tissue of the Binfield Park M. salmoides samples may have been as a result of the high concentration of mercury found in the muscle tissue of M. salmoides. The water quality and metals detected in the water of Binfield Park, Sandile and Wriggleswade Dams were all below the target water quality guideline values, as well as below those of previous research into the nutrients and presence of metals in these impoundments. The sediment metal analysis showed that the levels of Co, Mn and Ni were above the target guideline levels for Binfield Park, Sandile and Wriggleswade Dams, Cu was above target guidelines levels for Sandile Dam, and uranium was above the target guideline concentrations for Wriggleswade Dam. Binfield Park Dam had significantly high levels of mercury in the muscle tissue of M. salmoides, while Sandile Dam had significantly high levels of zinc in the muscle tissue of M. salmoides. It was shown that M. salmoides from each of the three impoundments are in a healthy state according to the parameters assessed. However, the presence of heavy metals, particularly mercury, uranium and zinc, do indicate the presence of human activities. The indigenous parasites of Anguilla mossambica have been well documented including the gastrointestinal nematode Paraquimperia africana, and the stomach nematode Heliconema africanum. Indigenous parasites such as the swimbladder nematode Anguillicola papernai had no effect on the condition factor of infected and uninfected eels. However, the damage caused by the alien parasites were evident, including the first documented effects of the alien gill monogenean Pseudodactylogyrus anguillae on indigenous wild populations of the longin fin eel A. mossambica from the Eastern Cape, South Africa. Histological observations indicated that an alien gill monogenean caused hyperplasia, increase in mucous cells, rupture of pillar cells as well as telangiectasia. This alien parasite has invaded the Keiskamma and Kei River systems in the Eastern Cape, South Africa. According to the macroscopic fish health assessment index, A. mossambica from Binfield Park, Sandile and Wriggleswade Dams are in a healthy state. However, the histology-based health assessment highlighted that the effects on P. anguillae have a severe negative impact on the health of A. mossambica. Using the macroscopic and histology-based fish health assessment, a comprehensive investigation into the fish health status of Mugil cephalus and Myxus capensis from Binfield Park Dam revealed that human effects and parasites are not the only threats to freshwater fish. Nephrocalcinosis is a non-infectious kidney disease which is characterised by abnormal calcium deposition in the kidneys of humans and some fish species. According to the macroscopic and histology-based fish health assessment, the M. cephalus and M. capensis are not in a healthy condition. The macroscopic and histology-based fish assessment indices are not stressor-specific, and therefore the cause of the poor health state of these two fish species could not be determined. A possible suggestion for the poor health of these two species is the age of the species. Because the two mullet species were stocked into Binfield Park Dam, Ellender et al. (2012) could successfully age them accurately to ten years of age, which is the upper limit of the life span for these species. Macroscopic and histology-based fish heath assessments were conducted on Labeo umbratus from Sandile Dam in order to determine the health of this species. Macroscopic and histology-based fish health assessment indicated that its L. umbratus are in a healthy state. The March 2012 survey specimens had a significantly higher macroscopic FHAI score than those from the July 2011 survey. The increased FHAI score was because of parasite infections, as well as discoloured livers and increased total blood plasma protein levels, which are indicators of nutritional state. However, the presence of the anchor worm parasite Lernaea barnimiana in low numbers had no significant impact on the health of L. umbratus. The effect of the alien anchor worm parasite Lernaea cyprinacea was shown on the transloacted small mouth yellowfish Labeobarbus aeneus. It was also shown that L. aeneus are, according the macroscopic FHAI and the histology-based fish health assessment index, in a healthy state. However, the high scores observed in the macroscopic fish health assessment index were primarily as a result of the presence of the alien parasite L. cyprinacea and its associated affects on the fish host. Because of the significant impact of this alien parasite species on the translocated host species, it can be assumed that this alien parasite species will have a negative effect on the health of indigenous fish species in the Great Kei River. It is clear from the results presented in this study that the necropsy based and histology based fish health assessment can be successfully implemented as tools to assess the effects of heavy metal pollution and alien fish parasites in freshwater fish from selected impoundments in the Amatola region, Eastern Cape Province, South Africa, thus the original hypothesis of this thesis is accepted. Based on work done in this research the gaps in research have been identified. Due to the high levels of mercury indentified in the muscle tissue of M. salmoides from Binfield Park Dam. A human health assessment and edibility should be conducted in order to determine if the fish from Binfield Park Dam is safe for human consumption. In order to conserve South Africa’s Freshwater fish biodiversity, country wide surveys of indigenous fish species must be undertaken so that the health and the parasite diversity can be evaluated. / PhD (Zoology), North-West University, Potchefstroom Campus, 2015

In this thesis the effects of individual and multiple environmental stressors (physical and chemical) are examined in inbred and outbred zebrafish (Danio rerio, Hamilton), a model species used in ecotoxicology and environmental risk assessment (ERA). The central question addressed is, are inbred laboratory animals representative and protective of wild populations? That is, are inbred fish equally or more sensitive to chemicals and other stressors compared with more outbred (wild) fish? A combination of tools and approaches incorporating traditional (eco)toxicology and population genetics have been employed, together with more contemporary molecular genetics and population modelling, to compare and contrast a range of responses in inbred and outbred zebrafish exposed to the endocrine disrupting chemical clotrimazole and/or temperature elevation in the laboratory. The choice of test species was based on our broad understanding of its basic biology, extending from the molecular level to the population level, and its wide use as a model organism in (eco)toxicology. Selection of the test chemical clotrimazole and temperature was based on a shared mode of action, aromatase inhibition, and therefore their ability to block oestrogen production, impair reproduction, promote male development and skew population sex ratios in zebrafish. A cascade of responses were compared in inbred and outbred zebrafish, including changes in the levels of expression of genes for gonadal aromatase and other steroidogenic enzymes, circulating sex steroid hormones, gonadal sex differentiation and development (via gonadal histopathology) and reproductive fitness (female fecundity, paternity and viability of embryos). Amongst the most striking results were directional skews in sex ratio towards males in response to clotrimazole (Chapter 5) and elevated temperature exposure (Chapter 7). Inbred fish were generally more responsive compared to outbreds, which showed evidence of physiological and developmental compensation, resulting in lower male-sex skews and superior fitness in terms of male reproductive success (paternity and viability of embryos). The greater effects observed in inbred fish were attributed to inbreeding×environment interactions and the amplification of inbreeding depression. Although no empirical genetic evidence of this mechanism is presented (loss of heterozygosity at quantitative trait loci and concomitant loss of heterosis and/or the expression of recessive, deleterious alleles in homozygotes), supporting evidence was provided by increased phenotypic variance in some apical endpoints in inbred fish, including specific growth rate and fecundity. This increased variance also has the potential to counteract the higher levels of response observed in inbreds, because the power to detect statistically significant changes in responses is reduced. This trade-off was demonstrated for specific growth rate. Crucially, significant male-sex ratio skews (>80%) were induced at substantially lower clotrimazole exposure concentrations (1.7 µg l-1) in combination with elevated temperature (33°C), compared with exposure concentrations (43.7 µg l-1) generating similar sex ratio skews at the standard test temperature of 28°C. These temperatures represent current and predicted 2100 (elevated) mean temperatures in the zebrafish’s native India and Bangladesh. Although the lowest observed effect concentration was an order of magnitude above the predicted environmental concentration for clotrimazole, it is conceivable that combined environmental exposures to similarly acting chemicals (e.g. other azole compounds used in crop protection, veterinary and human medicine) could produce similar effects to those we observed. The consequent effects of sex ratios skews and reduced fitness (fecundity and embryo viability) on per capita population growth rate (r) and extinction probability were predicted in inbred versus outbred zebrafish populations using stochastic population viability analysis. The results showed that the observed male-skews >80% threaten small zebrafish populations with fewer than 100 breeding adults (<20 adult females). However, small reductions of 2-3% in embryo-juvenile (age 0+) survivorship (including simulated inbreeding depression) were more influential on r and extinction probability than large sex ratio skews and/or reduced female fecundity. The results presented in this thesis support the contention that chemical effects may be exacerbated by other environmental stressors, but also illustrate the importance of considering biological (genetic), as well as physical and chemical interactions in cumulative ERA. Greater sensitivity of inbred versus outbred organisms to the effects of environmental stressors on sexual differentiation and reproductive fitness offers a margin of safety to ERA and the protection of wildlife populations (excluding those that are severely inbred and critically endangered). This is because, as originally stated, laboratory organisms used in ERA are generally more inbred than their wild counterparts. Nevertheless, more attention should be paid to the origin, breeding history and genetics of laboratory strains. This will help to ensure consistency between studies and testing laboratories and provide more confidence in extrapolating the results to wild populations.

La contamination des milieux aquatiques urbains se caractérise aujourd'hui par un mélange de micropolluants d'origines et de types variés. Les conséquences de ce type de contamination, multiples mais faibles et chroniques, sur les écosystèmes aquatiques sont difficiles à évaluer. En effet, il est difficile d'une part de dresser un état des lieux précis de la contamination et d'autre part, de relier cette contamination à la réponse biologique des organismes exposés puisqu'elle n'engendre pas nécessairement des effets visibles et immédiats. Cette étude a pour objectif de développer une méthodologie permettant d'utiliser le périphyton pour évaluer l'impact d'une contamination urbaine typique. Les biofilms sont des communautés aquatiques complexes jouant un rôle primordial dans le fonctionnement des écosystèmes aquatiques. Leur réponse biologique à une perturbation peut s'évaluer au niveau structurel comme au niveau fonctionnel. Une première étape de ce travail a consisté à développer d'une part un test de toxicité aiguë permettant d'évaluer la tolérance de la composante hétérotrophe des biofilms selon le concept PICT, et d'autre part l'utilisation de la technique d'empreinte génétique ARISA pour évaluer des modifications de structure des communautés bactériennes et eucaryotes des biofilms. Les méthodologies développées ont ensuite été testées au laboratoire par des expositions de périphyton à des métaux à des concentrations environnementales. Par la suite, elles ont pu être déployées in situ : d'abord dans un gradient de contamination multi-métallique sur la Seine avec des prélèvements ponctuels de biofilms naturels ainsi qu'une expérience de transposition (amont/aval de l'agglomération parisienne). Enfin, le lien entre la tolérance du périphyton et la contamination métallique globale du milieu a été exploré à l'échelle d'un bassin versant. Les résultats sont prometteurs quant à l'utilisation du périphyton pour évaluer l'impact d'une contamination urbaine / Urban aquatic ecosystems are these days contaminated by mixtures of micropollutants of diverse types and origins. Such contaminations, which are multiple, chronic and non-lethal, raise increasing concern among the scientific community. Indeed, a precise evaluation of exposure levels is difficult to obtain. Moreover, establishing a causal link between contamination and the induced biological response of aquatic organisms is complex as no direct and visible effects can be observed. The aim of this study is to develop a methodology for the use of periphyton as a biomarker of urban pollution. Biofilms are complex aquatic communities and play an important role in aquatic ecosystems. Their response to a disruption can be assessed both in terms of structure or functional modifications. Our first step was to develop both a PICT approach with a new short-term toxicity test to measure the tolerance to metal of the heterotrophic component of biofilms and the use of a fingerprinting technique (ARISA) to reveal structure modifications of both bacterial and eukaryotic communities. The methodologies thus developed were tested in a microcosm study by exposing periphyton to metals at environmental levels. They were then used in field studies: first in a multi-metallic gradient along the Seine river by punctual sampling of natural periphyton and also in a translocation study (upstream/downstream the Paris urban area). Furthermore, the link between biofilm tolerance levels and the global metallic contamination was explored at the watershed scale. It is hoped that the future use of this approach will help to understand the impacts of human activities, especially toxic pressure, on aquatic ecosystems

Sur un site de traitement du bois dont les sols étaient contaminés en Cu, une remédiation par la phytostabilisation aidée a été développée et évaluée. Les objectifs étaient : 1) L’évaluation initiale des risques. 2) La sélection d’amendements efficaces pour immobiliser le Cu et restaurer la croissance des végétaux. 3) La sélection d’espèces végétales tolérantes au Cu. 4) L’établissement in situ et le suivi de parcelles d’amendements et d’espèces potentiellement efficaces pour la phytostabilisation. 5) La caractérisation de mécanismes de tolérance d’une Poaceae, Agrostis capillaris. La contamination des sols de surface augmente la mortalité d’invertébrés terrestres et d’espèces végétales, diminue les biomasses et la diversité des communautés végétales et modifie leur composition. L’exposition des racines du haricot au Cu a été diminuée par l’ajout d’amendements au sol, les plus efficaces étant la matière organique, seule ou combinée avec un composé alcalin ou du fer zérovalent. Dans les communautés végétales établies, des espèces natives tolérantes au Cu, i.e. Agrostis capillaris on été identifiées. Les tests en pot ont confirmé la tolérance de plusieurs espèces et souligné des différences de tolérance inter- et intra-espèces. Après les tests en pots, certaines espèces ont été transplantées in situ ainsi que d’autres espèces identifiées d’après la littérature. Après deux ans de phytostabilisation aidée, l’écotoxicité des sols pour le haricot diminue et l’installation spontanée d’espèces augmente. L’association des arbres avec des ectomycorhizes a permis d’améliorer la croissance de P. nigra, cependant le rôle des mycorhizes et d’autres micro-organismes pour améliorer la phytostabilisation reste à confirmer. Une espèce, A. capillaris, a été utilisée pour identifier des mécanismes de tolérance au Cu dans les racines. Le protèome de deux écotypes, métallicole et non métallicole, a été comparé en conditions d’exposition croissante au Cu, en vue de détecter une synthèse différentielle des protéines solubles et de les séquencer. / At a timber treatment site, there is a major Cu contamination of topsoils. Soil remediation was developed and assessed, especially aided phytostabilisation. Main objectives were: 1) Initial risk assessment; 2) The selection of efficient amendments for increasing Cu stabilization and plant growth; 3) The selection of Cu-tolerant plant species; 4) The in situ appraisal of the best amendments and plant species with a long term monitoring of the plots; 5) The characterisation of the tolerance mechanisms of a Poaceae species, A. capillaris. The topsoil contamination increased the mortality for two terrestrial invertebrates and plant species, decreased the biomass and diversity of plant communities and modified their composition as well. Root exposure to Cu was decreased with the incorporation of amendments, i.e. organic matters, singly or in combination with alkaline materials or zerovalent iron grit. In established plant communities, native Cu tolerant species were recorded. Pot experiments confirmed several plant species as Cu tolerant and highlighted differential tolerance across plant species and populations. Some plant species were transplanted to the site and the soil Cu labile pool was decreased by mixing amendments. After a two-year period of aided phytostabilisation, bean exposure decreased, native plant species naturally disseminated and diversity increased. The association of trees with ectomycorhiza improved the growth of P. nigra. The role of mycorhiza and other micro-organisms in improving phytostabilisation was not clear-cut but is a promising option. One of the tolerant plant species, A. capillaris, was chosen to identify the Cu tolerance mechanisms in roots. The proteome of two populations, one metallicolous and one non metallicolous, was extracted and differential synthesis of soluble proteins compared in order to identify and sequence candidates.

Alquilbenzeno Linear Sulfonado (LAS) é o surfactante aniônico mais utilizado no mundo e é encontrado constantemente em esgoto doméstico e águas residuárias de lavanderia. A remoção de LAS em reatores foi comprovada em estudos anteriores, mas pouco se sabe sobre a toxicidade desse composto em amostras mais complexas para a biota aquática. Neste trabalho, objetivou-se avaliar duas Hipóteses; A: O LAS padrão causa efeitos negativos na biota aquática, e B: O tratamento em reator de leito fluidificado de amostra complexa contendo LAS (água residuária de lavanderia comercial combinada com esgoto doméstico bruto) diminui os efeitos negativos na biota aquática. Ambas as hipóteses foram avaliadas por meio de bioensaios de toxicidade com: Chironomus sancticaroli, Allonais inaequalis e Daphnia magna, analisados a partir da mortalidade dos organismos e análises estatísticas, para determinar os efeitos tóxicos dos LAS padrão e das amostras do reator, comparados ao controle. Para avaliar a Hipótese A foram realizados testes de toxicidade aguda, crônica e crônica de gerações com o LAS padrão. No teste de toxicidade crônica de gerações, com a espécie C. sancticaroli, foram analisados a deformidade bucal, comprimento das larvas, tamanho das asas das fêmeas e fecundidade potencial com o LAS padrão em 3 gerações. Para responder a Hipótese B, foram realizados bioensaios de toxicidade aguda e crônica com os afluentes, efluentes e diluições dos efluentes nas seis fases de operação do reator. C. sancticaroli foi a espécie mais tolerante tanto em teste de toxicidade aguda como crônica com LAS padrão (CL50-96h 25,25 mg.L-1 e CL50-10dias 18,11 mg.L-1), seguido de A. inaequalis (CL50-96h 8,13 mg.L-1 e CL50-10dias 8,56 mg.L-1). A espécie mais sensível ao LAS foi D. magna (CE50-48h 6,11mg.L-1 e CE50-21dias 3,21 mg.L-1). No teste de toxicidade crônica de longa duração, não existiu diferença estatística entre as gerações para o desenvolvimento das larvas do inseto C. sancticaroli, entretanto evidenciaram diferença estatística entre as gerações quanto ao tamanho das asas das fêmeas, indicando que esses organismos podem sofrer alterações fisiológicas quando a exposição ao contaminante é contínua, confirmando a Hipótese A. Os resultados ecotoxicológicos com o reator evidenciaram toxicidade de 100% de todos afluentes e efluentes para A. inaequalis e D. magna. Já C. sancticaroli apresentou menor toxicidade nas primeiras fases de operação (de 20 a 100% de mortalidade). Em geral, foi identificado diferença de toxicidade das fases do reator por meio das diluições. Nas fases menos tóxicas, a diminuição da mortalidade era observada a partir de diluição de 50%; nas fases mais tóxicas, a partir de 20%. É possível concluir que a Fase VIb de operação do reator apresentou diminuição da toxicidade. Entretanto, ao se analisar testes de toxicidade crônica nessa fase, conclui-se que o efluente mesmo depois do tratamento em reator causa efeito negativo nos organismos-teste avaliados, dessa forma é possível refutar a Hipótese B. A partir dos resultados apresentados, é possível concluir que o LAS causa impacto ambiental, principalmente em exposição continua, e que mesmo com remoção em sistema biológico de efluentes reais (água residuária de lavanderia comercial combinada com esgoto doméstico) existe impacto, possivelmente com a interação entre os diversos compostos tóxicos e o LAS. / Linear Alkylbenzene Sulfonate (LAS) is the most commonly utilized anionic surfactant in the world and it is present in domestic sewage and laundry wastewater. Removal of LAS in reactors has been proven in previous studies, but little is known about the toxicity to the aquatic biota. The objectives of this study were tested into two Hypotheses, A: The standard surfactant LAS causes negative effects on aquatic biota, and B: Treatment in complex sample fluidized bed reactor containing LAS (commercial laundry wastewater combined with raw domestic sewage) diminishes the negative effects on aquatic biota. Both Hypotheses were evaluated toxicity bioassays using three test organisms: Chironomus sancticaroli, Allonais inaequalis and Daphnia magna. To evaluate the Hypothesis A, acute, chronic and long-term chronic toxicity tests were performed with standard LAS. In the long-term chronic tests with C. sancticaroli organisms, we analyzed the oral deformity, larval length, female wing size and potential fecundity in contact with the standard LAS in 3 generations. Acute and chronic bioassays with affluents, effluents and effluent dilutions in the VI operating phases of the fluidized bed reactor were proceeded to evaluate the Hypothesis B. The results were analyzed from the mortality of the organisms, and statistical analysis were performed to determine the similarity of the toxic effects of the LAS and reactor samples, compared to the control. The species C. sancticaroli was the most tolerant species in both acute and chronic trials with standard LAS (LC50-96h 25.25 mg.L-1 and LC50- 10 days 18.11 mg.L-1), followed by species of A. inaequalis (LC50-96h 8.13 mg.L-1 and LC50-10 days 8.56 mg.L-1). The species most sensitive to LAS was D. magna (CE50-48h 6.11mg.L-1 and CE50-21dias 3.21mg.L-1). In the long-term chronic test, there is no statistical difference between the generations for the development of C. sancticaroli insect larvae. However, they showed a statistical difference between the generations regarding the size of the female wings, indicating that these organisms may undergo physiological changes when exposure to the contaminant is continuous. From these results it is possible to confirm Hypothesis A. The ecotoxicological results with the reactor showed toxicity of 100% of all tributaries and effluents for A. inaequalis and D. magna. The C. sancticaroli species had low toxicity in the first stages of operation (from 20 to 100% mortality). In general, toxicity difference of the reactor phases was identified by half of the dilution analysis. In the less toxic phases, the decrease in mortality was observed from 50% dilution; in the most toxic phases, from 20%. It is also possible to conclude that Phase VIb showed a decrease in the mortality of the test organisms where a chronic test was performed. However, when analyzing the chronic tests, it can be concluded that the effluent even after the treatment in the reactor causes a negative effect on the test organisms evaluated, so it is possible to refute the Hypothesis B. From the results presented, it is possible to conclude that the LAS causes environmental impact, mainly in continuous exposure, and that even with treatment of actual effluents (commercial laundry wastewater combined with domestic sewage) there is an impact, possibly with the interaction between the various toxic compounds and the LAS.

Conventional ecotoxicological tests (forced exposure) are an important tool when what is sought are the possible acute and chronic effects of environmental pollutants on each individual that is exposed. The disadvantage of this approach lies in the fact that the organisms are kept enclosed within containers of the same concentration for several hours and / or days. The forced exposure test has no ecological relevance when the modeled environment exhibits a contamination gradient and organisms can move along this gradient. In many aquatic ecosystems, it is common to observe a contamination gradient, with concentrations decreasing with distance from the discharge zone, so that organisms do not necessarily exhibit continuous and forced exposure to the contaminant. The objective of this work was to analyze how aquatic pollutants (e.g Triclosan, Bisphenol, Atrazine and Copper) influence the dispersion / habitat selection pattern of two species of fish: Poecilia reticulata and Danio rerio, using a static system (non-forced exposure) with several compartments, forming a gradient of contamination with the compound to be analyzed. All pollutants tested triggered an avoidance response in fish at environmentally relevant concentrations. The concentrations that caused avoidance on the organisms are lower than those that cause sub-lethal effects on aquatic organisms, including fish. We also find in one of our approaches the potential for the formation of a chemical barrier (habitat fragmentation) by the release of pollutants into the water bodies, reducing the migration potential of aquatic organisms. Finally, one of the most important findings is the interaction of the species with each other when exposed to a pollution gradient. In this case, the presence of one species interfered in the distribution of the other (reduction of the migration potential), when both were in the same system. Thus, the non-forced approach demonstrates to be a powerful tool in the evaluation of environmental risk, complementary to the traditional ecotoxicological tests / Os testes ecotoxicológicos convencionais (exposição forçada) são uma ferramenta importante quando o que se busca são os possíveis efeitos agudos e crônicos dos poluentes ambientais sobre cada indvíduo que é exposto. A desvantagem dessa abordagem está no fato de que os organismos são mantidos enclausurados dentro de recipientes com uma mesma concentração por várias horas e/ou dias. O teste de exposição forçada não tem relevância ecológica quando o ambiente modelado apresenta um gradiente de contaminação e os organismos podem se mover ao longo deste gradiente. Em muitos ecossistemas aquáticos, é comum observar um gradiente de contaminação, com as concentrações diminuindo com a distância da zona de descarga, de modo que os organismos não apresentam obrigatoriamente uma exposição contínua e forçada ao contaminante. Desta forma, este trabalho teve como objetivo a análise de como os poluentes aquáticos (e.g. Triclosan, Bisfenol, Atrazina e Cobre) influenciam o padrão de dispersão / seleção de habitat por duas espécies de peixes: Poecilia reticulate e Danio rerio, utilizando um sistema estático (não forçado) com vários compartimentos, formando um gradiente de contaminação com o composto a ser analisado. Todos os poluentes testados dispararam uma resposta de fuga nos peixes em concentrações ambientalmente relevantes. As concentrações que causaram a fuga dos organismos são menores do que aquelas que causam efeitos sub-letais em organismos aquáticos, incluindo peixes. Encontramos também em uma de nossas abordagens o potencial de formação de uma barreira quimica (fragmentação de habitat) pela liberação de poluentes nos corpos hídricos, reduzindo o potencial de migração dos organismos aquáticos. Por fim, um dos achados mais importantes está na interação das espécies entre si quando expostas a um gradiente de poluição. Nesse caso, a presença de uma espécie interferiu na distribuição da outra (redução do potencial de migração), quando ambas se encontravam no mesmo sistema. Sendo assim, a abordagem não forçada demonstra ser uma ferramenta poderosa na avaliação de risco ambiental, complementar aos testes ecotoxicológicos tradicionais

Os organismos chamados de algas apresentam uma grande diversidade de espécies e ocupam uma grande variedade de nichos ecológicos. Fundamentais para a manutenção das condições que permitem a vida no planeta, inclusive porque constituem a base de cadeias tróficas de ecossistemas aquáticos, as algas vêm sofrendo com o descarte contínuo de resíduos resultantes das mais variadas atividades humanas. Por outro lado, as algas, ao serem expostas aos poluentes, podem indicar a presença dos mesmos em seus habitats por meio de seus biomarcadores. Neste sentido, este trabalho preocupou-se em caracterizar as bases moleculares da toxicidade do hidrocarboneto policíclico aromático (HPA) benzo(a)pireno (BaP), presente no petróleo cru e derivado da combustão parcial de matéria orgânica, sobre a macroalga vermelha Gracilaria birdiae, uma Rhodophyta marinha nativa. Para avaliar a agressividade do poluente, a alga foi exposta a diferentes concentrações do HPA em água do mar, tanto em situações de exposição aguda (24 e 96h) quanto crônica (7 e 15 dias). Após estes períodos de exposição, alguns biomarcadores bioquímicos e fisiológicos da alga tiveram seus comportamentos analisados. Foram eles: taxas de crescimento (TC); duas defesas antioxidantes: os níveis do tripeptídeo de baixo peso molecular glutationa (GSH) e a atividade da enzima superóxido dismutase (SOD); os níveis do dímero de glutationa GSSG; a fotossíntese da macroalga; e seu aspecto de pigmentação geral. Foi possível observar que BaP é tóxico para a macroalga G. birdiae. Aumentos da concentração do HPA nos meios de cultura das algas provocaram menores TC. A partir destes dados de TC foi possível determinar uma curva de inibição do crescimento da alga e a 7 respectiva IC50 de BaP para um período de exposição de 15 dias, com valor de 69 ng do HPA para cada mL de água do mar. Após diferentes exposições a BaP, incluindo em IC50, algas expostas a BaP apresentaram níveis reduzidos do antioxidante GSH, o mesmo efeito que fora observado para GSSG (com exceção da exposição de 96h). O desempenho fotossintético, a atividade de SOD e a pigmentação de G. birdiae mostraram ser sistemas menos sensíveis à presença de BaP, e foram prejudicados em concentrações mais elevadas do poluente, em torno de 10 a 20 &#181;g/mL. Nestas situações, a despigmentação severa da macroalga foi acompanhada de decaimentos expressivos de alguns parâmetros fotossintéticos (i.e., rETR, RQE, Ik, &#946;) da alga. Numa outra abordagem, alguns experimentos foram feitos com o intuito de descobrir se a alga era capaz de eliminar e biorremediar BaP presente na água do mar. Aparentemente, a alga não apresenta tal capacidade; porém, uma cinética mais detalhada se faz necessária para a confirmação desta observação. Concluindo, os resultados do trabalho corroboram outros dados da literatura a respeito da toxicidade de BaP sobre sistemas vivos. Ainda, apesar de a sensibilidade de G. birdiae a BaP não ter sido alta, foi possível apontar alguns sistemas bioquímicos da alga capazes de desempenhar papel como biomarcadores de exposição ao poluente estudado, o que pode auxiliar na tomada de medidas para o manejo e a conservação de áreas impactadas / Algae show a great biodiversity and occupy many different ecological niches. Besides being essential for the maintenance that allow life on Earth, algae are on the basis of aquatic food chains and they suffer with continuous residue discharge that comes from different human activities. However, once algae are exposed to pollutants, biomarkers can indicate its presence on the environment. The aim of this work was on the characterization of benzo(a)pyrene (BaP; a polycyclic aromatic hydrocarbon (PAH) derived from crude oil and from incomplete combustion of organic matter) toxicity against the red macroalga Gracilaria birdiae, a marine brazillian Rhodophyta. BaP toxicity in marine water was investigated under different exposure concentrations after acute (24 and 96h) and chronic (7 and 15 days) conditions. At the end of the exposures time, some of the algae`s biochemical and physiological biomarkers were analyzed: growth rate (GR); two antioxidant defenses: the low molecular weight peptide glutathione (GSH) and superoxide dismutase (SOD) enzyme activity; glutathione disulfide (GSSG) levels; the macroalga photosynthetic capacity; and the general colour aspect. Increased BaP concentrations led to a decrease of GR values. From GR data it was possible to obtain an inhibition growth curve and the respective BaP IC50 for a 15-day exposure time period with value of 69 ng/mL. After different BaP exposure conditions, including IC50, exposed algae presented decreased GSH levels, the same effects observed for GSSG (except for 96h exposure). The photosynthetic yield, SOD activity and colour aspect of G. birdiae appeared to be more resistant systems against BaP, and were affected only in higher BaP concentrations (10 to 20 &#181;g/mL). In such situations, G. birdiae lost its red colour, which was accompanied by considerable photosynthetic parameters (i.e., rETR, RQE, Ik, &#946;) decay. In a different approach, some experiments were done in order to discover any BaP clean-up and bioremediation played by G. birdiae. Preliminary results suggests that the alga has low remediation efficiency, although more investigations need to be done to confirm this. In summary, the results presented here show similar tendencies with literature data in respect of BaP toxicity against living organisms. Even though G. birdiae presented reasonable resistance to BaP, it was possible to identify some of the alga`s biochemical systems as BaP exposure biomarkers. This situation can be useful for impacted areas assessment and management.

A avaliação de sedimentos contendo de mistura complexa de substâncias contaminantes é um grande desafio, especialmente em reservatórios localizados próximos a áreas urbanas, onde existe um grande número de fontes de poluição, como é o caso do reservatório Rio Grande. Com o objetivo de proceder uma análise integrada, nós realizamos uma bateria de biotestes usando amostras de sedimento do reservatório Rio Grande e células permanentes da linhagem RTL-W1 e bactérias expostas a extrato acetônico (teste de citotoxicidade, teste de EROD, teste cometa, teste do micronúcleo e teste de flutuação de Ames) e também embriões de Danio rerio (teste de contato com o sedimento). Após a exposição dos embriões, nós retiramos o córion e realizamos medição de metais (ICP-MS) em embriões não coagulados (digestão: H2O2 e HNO3 sob radiação UV). Nós realizamos análises químicas de HPAs alvos no extrato e de dinâmica de metais, esta última avaliada como heterogeneidade temporal e remobilização para a coluna d\'água. O extrato do sedimento se mostrou citotóxico (NR50 = 10,3 mg sedimento equivalente /mL; n=4) e o sedimento se mostrou com elevado potencial tóxico para embriões de peixe (EC50 48h=12 mg sedimento/ mL; n=4). Não foram detectados metais no tecido de embriões não coagulados. Teste de EROD revelou alta atividade enzimática, com EC25 TCDD = 0,82 mg of SEQ/mL, e Bio-TEQ = 1884 pg/g (n=6). A concentração total de HPAs analisados no extrato foi de 763,6 &#181;g/kg; apesar disso, apenas 4.64 % do Bio-TEQ pôde ser explicado pelo PAH-TEQ. O teste de flutuação de Ames e o teste cometa revelaram altos potenciais para mutageno e genotóxicos. Os metais estudados aparentemente não são a causa primária para o efeito agudo observado, assim como os HPAs analisados. Porém, nós encontramos uma alta concentração de metais no sedimento, além de uma heterogeneidade temporal de curto tempo coincidente com a passagem de uma frente fria, e uma possível remobilização de metais para a coluna d\'água, o que levanta a necessidade de um melhor entendimento da dinâmica dos metais em um ambiente aquático como o estudado (tropical e polimítico), além da necessidade de um planejamento cuidadoso de desenhos experimentais de estudos de sedimentos (considerando as escalas temporal e espacial e a massa d\'água como um todo). Análise Integrada por meio de ponderação das evidências indicou a necessidade de ações de manejo para o sedimento do reservatório Rio Grande. No entanto, deve ser realizada a caracterização do risco, no contexto de uma Análise de Risco Ecológica, antes que se decida qual ação tomar, de forma a melhor dirigir o problema. Estudos complementares devem priorizar testes com organismos com exposição crônica a amostras do sedimento do Rio Grande; PCBs e dioxinas devem ser considerados como possíveis indutores de atividade de EROD; análises químicas do sedimento devem ser expandidas e considerar outros compostos orgânicos; técnicas de fracionamento de sedimento devem ser empregadas visando o estabelecimento de causalidade em respeito aos compostos orgânicos / Assessment of complex mixture sediments is a great challenge, especially for reservoir located near urban areas where a multitude of pollution sources is present, such as Rio Grande reservoir. Aiming an integrated assessment, we performed a battery of bioassays on sediment from Rio Grande reservoir, using permanent cell line RTL-W1 and bacteria exposed sediment acetonic extracts (cytotoxicity test, EROD assay, comet assay, micronucleus assay and Ames Fluctuation test) and Danio rerio embryos (whole sediment contact). After exposure, we measured metal (ICP-MS) in dechorionated non-coagulated fish embryos (digestion: H2O2 and HNO3 under UV light radiation). PAH was also measured in extracts and metal dynamics was evaluated as temporal heterogeneity and remobilization to water column. Sediment extract was highly cytotoxic (NR50 = 10.3 mg sediment equivalent /mL; n=4) and whole sediment showed high toxic potential to fish embryos (EC50 48h=12 mg sediment/ mL; n=4). No metal could be detected in the non-coagulated fish eggs, although metals were found in high concentrations with potential to cause negative effect. EROD assay showed dioxin-like activity, with EC25 TCDD = 0.82 mg of SEQ/mL, and Bio-TEQ as high as 1884 pg/g (n=6). Sum of target PAH in sediment extract was 763.6 &#181;g/kg; though 4.64 % of Bio- TEQ could be explained by PAH-TEQ. Ames Fluctuation test and comet assay reveled high potentials for mutageno- and genotoxicity. Studied metals appear not to be the cause of primary acute effect observed toxicity tests, nor seam target PAHs. Yet, we observed high concentration of metal in sediment, short-time temporal heterogeneity of metals coupled with front cold passage and possible remobilization into the water column, what raises the need for better understanding of metal dynamics in such aquatic environment (tropical polymithic) and careful planning for sediment sampling designs (considering temporal and spatial scales and the whole water column above the sediment). Integrated assessment through Weight-of-Evidence (WOE) approach indicates the need of management actions regarding sediment from Rio Grande reservoir. However, characterization of the risk in within an Ecological Risk Assessment (ERA) framework should precede action, in order to better address the problem. Further studies should prioritize chronic exposure of organisms to sediment samples from Rio Grande; PCBs and dioxins should be considered as possible EROD activity inducer; chemical analysis should be expanded and consider other organic chemicals; sediment fractionation technique should also be applied in order to provide insights on causality from organic compounds.

O Estado de São Paulo, devido a seu alto grau de industrialização, grande densidade demográfica e técnicas de produção agrícola, tem grande arte de seus mananciais impactados. As avaliações da qualidade da água são de vital importância para o manejo adequado e recuperação desses sistemas, todavia os programas atuais de monitoramento são incompletos no sentido de não permitirem uma avaliação global dos efeitos dos contaminantes sobre a biota aquática. Esses programas enfocam preferencialmente a coluna d\'água, porém é o sedimento o compartimento preferencial de armazenamento e transformações de contaminantes e nutrientes. Para que, associados a outros estudos e métodos de análise, possam ser desenvolvidas as bases metodológicas para a definição de critérios de qualidade de sedimentos, foram realizados bioensaios crônicos com amostras de sedimento de seis represas do sistema do Rio Tietê (Pedro Beicht, Rasgão, Billings, Barra Bonita, Promissão e Bariri), nos períodos de outubro e novembro de 2000 e de 2001, bem como testes para a determinação da faixa de sensibilidade de Chironomus xanthus às substâncias dicromato de potássio - K2Cr2O7 (4,82 ~ 9,41 mg/L), sulfato de zinco - ZnSO4 (1,46 ~ 13,47 mg/L), cloreto de cádmio - CdCl2 (0,34 ~ 1,11 mg/L), sulfato de cobre - CuSO4(1,13 ~ 1,77 mg/L), cloreto de potássio - KCl (2,91 ~ 3,77 g/L) e atrazina (1,43 ~ 2,72 mg/L). Os resultados dos testes com amostras ambientais indicaram maior toxicidade nos reservatórios de Rasgão e Billings e em menor grau em Barra Bonita e Bariri. / The são Paulo State, due to its high levels of industrialization, great demographic density and agricultural techniques, has most of its water resources compromised. The water quality assessments are fundamental to the appropriate management and recover of these systems, however the existing monitoring programs are incomplete and do not allow the assessment of all the effects that the contaminants can carry out in the aquatic biota. The main approach of these programs is the water column, but the sediment is the preferential compartment of contaminants and nutrient storing and alteration. Associated to other studies and analyses, this work intends to help to develop the methodological basis to define the sediment quality criteria by doing chronic bioassays with sediment samples of six reservoir of the Tietê river system (Pedro Beicht, Rasgão, Billings, Barra Bonita, Promissão e Bariri) , as well as determining the Chironomus xanthus sensibility to the substances K2Cr207 (4,82 ~ 9,41 mg/L), ZnSO4 (1,46 ~ 13,47 mg/L), CdCl2 (0,34 ~ 1,11 mg/L), CuSO4 (1,13 ~ 1,77 mg/L), KCl (2,91 ~ 3,77 g/L) and atrazine (1,43 ~ 2,72 mg/L). The environmental bioassays showed that the highest toxicity was found in Rasgão and Billings reservoirs while Barra Bonita and Bariri showed minor levels of toxic effects.