Η παρούσα μελέτη διερευνά την ικανότητα των πολυκυκλικών αρωματικών υδρογονανθράκων να (PAHs), όπως είναι το φαινανθρένιο και το ανθρακένιο, να επάγουν συνθήκες stress (stress syndrome) σε ιστούς (π.χ. βράγχια) του είδους Mytilus galloprovincialis. Σύμφωνα με τα αποτελέσματα αυτής της μελέτης, τα μύδια τα οποία εκτέθηκαν για 7 ημέρες σε PH και/ή AN (τελικής συγκέντρωσης 0,1 mg/L σε κάθε περίπτωση) καθώς και σε μίγμα αυτών των οργανικών ρύπων (τελικής συγκέντρωσης 0,2 mg/L), παρουσίασαν αυξημένη συχνότητα εμφάνισης πυρηνικών ανωμαλιών, όπως είναι οι μικροπυρήνων (MN), και επαγωγή νευρολογικών διαταραχών, πράγμα που υποδεικνύεται από την μέτρηση της δραστικότητας της ακετυλχολινεστεράσης (AChE). Επιπλέον, τα αυξημένα επίπεδα της MDA στα βράγχια των εκτιθέμενων ατόμων καθώς και η μείωση του χρόνου ημιζωής (LT50) των μυδιών που εκτέθηκαν στον αέρα (τεχνική Stress on Stress), αντανακλούν στις οξειδωτικές επιπτώσεις που επάγουν οι πολυκυκλικοί αρωματικοί υδρογονανθράκες στους ιστούς των εκτιθέμενων στον αέρα μυδιών, πράγμα που οδηγεί στην επαύξηση των τοξικών επιδράσεων, σε διαφορετικά επίπεδα της λειτουργίας του οργανισμού. / This study investigates the ability of certain polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PH) and anthracene (AN), to induce stress conditions (stress syndrome) in tissues (e.g. gills) of mussel Mytilus galloprovincialis. According to the results of this study, mussels exposed for 7 days to PH and/or AN (at a final concentration of 0,1mg/L in each case) as well as to a mixture of these organic pollutants (at a final concentration of 0,2 mg/L) showed increased frequencies of nuclear abnormalities, such as the presence of micronuclei (MN), and induction of neurological disorders, as shown by measuring the activity of acetylcholinesterase (AChE). Furthermore, increased levels of MDA in the gills of exposed individuals, and the reduction of the lethal time (LT50) of challenge mussels superimposed in air (Stress on Stress technique), merely reflect PAHs-induced oxidative effects on tissues of challenge mussels, thus leading to the enhancement of toxic effects to different levels of organism function.

Japanese eels (Anguilla japonicas) are snakelike fishes living in waters in the Asian region. In contrast to most fishes which are stenohalines that can only live in waters with a narrow range of salinity, Japanese eels are classified as euryhalines that can habitat in a broad range of salinity. As the lifecycle of Japanese eels consists of stages across fresh and seawater districts, a well-developed osmoregulation mechanism is needed to balance the intra- and extra- cellular osmolarity of the fishes throughout the seawater acclimation process. While fish gills are one of the organs that separating the ambient water and the inner body fluid of the fish, the fish gills of the Japanese eels have been studied as one of the most crucial organs for osmoregulation purposes. Yet, the osmoregulation and survival strategies of Japanese eels under hyperosmotic stress has not been fully elucidated. In chapter 2, this study has performed a transcriptome study on the ex vivo gill filament model of the Japanese eel to profile the molecular responses after a hypertonic treatment of 4 hours or 8 hours. The experiment is aimed to mimic the gill cells exposed to seawater in the seawater acclimation process of Japanese eels. A profile of differential expressed genes (DEGs) has been revealed that 577 DEGs were commonly upregulated and 711 DEGs were commonly downregulated in both 4- and 8-hours hypertonic treatment. Functional analysis and annotation have been processed with these DEGs, including Ingenuity Canonical Pathways analysis and gene ontology. These analyses have revealed that the cellular homeostasis of the gill cells has been disrupted and cell death responses has been induced by osmotic stress. The results have raises a concern that the maintenance of cellular viability and a cell death regulation mechanism are needed for the fishes to survive in the early stage of seawater acclimation. In chapter 3, this chapter demonstrated that gill cells in Japanese eels are susceptible to apoptosis when they are exposed to hyperosmotic treatments in both in vitro gill cell and the ex vivo gill filament model. To maintain the viability of the gills cells, two inhibitors of apoptosis, XIAP, and survivin, were seen to be expressed in gills cells. The expression of XIAP and survivin were upregulated by dexamethasone, which is an agonist mimicking the effect of cortisol on fishes in seawater acclimation. Meanwhile, the expression levels of the apoptosis executor, caspase 3, were downregulated. These data suggested that with the regulation of cortisol express in the fishes, XIAP and survivin are effective apoptosis regulators in the gill cells of Japanese eels. The study has demonstrated the molecular responses of the gills of Japanese eels exposed to hyperosmotic stress at the transcriptional level and post-translational level by using transcriptome studies and protein study respectively. The study has paved cell death regulation to be another the key field to study in understanding the ability of salinity tolerance in euryhalines.

Le Loup ou Bar Dicentrarchus labrax est une espèce euryhaline dont les femelles pondent généralement en eau de mer. Pendant son cycle de vie, des migrations vers les estuaires et les lagunes, peuvent exposer très tôt les jeunes stades à des variations de salinité. Les mécanismes de l'osmorégulation sont bien connus chez les prélarves, les larves et les adultes de D. labrax en eau de mer (EM, 38) et en eau de mer diluée (EMD, 5), cependant les possibilités d'osmorégulation et leurs mécanismes sont inconnus chez les embryons. Le but de cette étude a été d'évaluer la mise en place de la fonction osmorégulatrice chez les embryons de D. labrax.Tout d'abord le développement embryonnaire des différents sites osmorégulateurs a été décrit, en se focalisant sur le tube digestif, en incluant le pharynx et les premières fentes branchiales. La formation de ces structures débute au stade 12 somites (S) et a été décrite jusqu'à l'ouverture de la bouche, 5 jours après l'éclosion.En second lieu, le lieu et la cinétique d'apparition des premières cellules osmorégulatrices, les « ionocytes » ont été recherchés. Ces cellules ont été identifiées au stade 12S sur la membrane de la vésicule vitelline et au niveau des premières fentes branchiales et du tube digestif primitif au stade 14S. La fonctionnalité de ces cellules a été étudiée grâce à des immunomarquages des principales protéines transmembranaires impliqués dans l'osmorégulation [l'ATPase Na+/K+ (NKA), le cotransporteur Na+/K+/2Cl- (NKCC) et le canal à chlore (CFTR)], et avec une étude ultrastructurale. Des ionocytes potentiellement fonctionnels sont présents à partir du stade 25S au niveau de la membrane de la vésicule vitelline et du tube digestif primitif, mais les ionocytes des premières fentes branchiales ne sont pas totalement fonctionnels à l'éclosion. L'existence d'un phénomène de boisson passive qui permettrait la régulation hydrique chez D. labrax est envisagé.Finalement, l'osmorégulation embryonnaire existant en EM et en EMD a été étudiée. Des mesures nanoosmométriques des fluides embryonnaires indiquent une capacité à hyper- et hypo-osmoréguler. Cependant, en EMD, des analyses en qRT-PCR et des immunomarquages de NKA, NKCC et CFTR révèlent que les mécanismes de l'hyper-osmorégulation peuvent limiter les pertes ioniques mais ne sont pas suffisamment efficaces pour permettre une acclimatation totale à l'EMD à ce stade très précoce. / The European sea bass Dicentrarchus labrax is a euryhaline species which usually spawns in seawater. Due to its life cycle that includes migrations to lagoon and estuaries, young stages can be exposed early to salinity variations. Osmoregulatory patterns are well known in prelarvae, larvae and adults D. labrax in seawater (SW, 38) and in dilute seawater (DSW, 5), but the possibility and mechanisms of embryonic osmoregulation are still unknown. The goal of this study was to investigate the occurence of the omoregulatory function in the embryos of D. Labrax.First, the embryonic development of the different osmoregulatory sites was described, focusing on the digestive system including the pharynx and the first gill slits. The formation of these structures is initialized at stage 12 somites (S) and was described throughout the opening of the mouth five days after hatching.Secondarily, the time and the location of the occurrence of the first osmoregulatory cells, the ionocytes were followed. These cells were identified at stage 12S on the yolk sac membrane and at stage 14S in the first gill slits and in the posterior primitive gut. The functionality of these cells was studied, using immunostaining of the main ionic transporters involved in osmoregulation [the Na+/K+ ATPase (NKA), the Na+/K+/2Cl- cotransporter (NKCC) and the chloride channel (CFTR)], and through ultrastructural investigations. Potentially functional ionocytes are present from stage 25S in the yolk sac membrane and in the gut, but gill slits ionocytes are not fully functional at hatching. Passive drinking is suspected to regulate water balance in D. labrax.Finally, the embryonic osmoregulation in SW and DSW was investigated. Nanoosmometry measurements of the embryonic fluids demonstrated some capabilities of hyper- and hypo-osmoregulation. However, in DSW, qRT-PCR and imunostaining of NKA, NKCC and CFTR, reveal that hyper-osmoregulatory mechanisms can only limit ion loss but are not efficient enough to allow a full acclimation at this early life stage.

Animais presentes em regiões de maior contaminação apresentam diferenças fisiológicas, quando comparados com animais de regiões menos contaminadas, provavelmente permitindo a sua sobrevivência em ambientes poluídos. Assim, o objetivo da tese foi caracterizar o transporte de cádmio em brânquias e hepatopâncreas de Ucides cordatus, como também comparar o transporte nestes órgãos, em animais de regiões poluídas (Itanhaém) e não poluídas (Juréia), além de analisar a porcentagem de ácidos graxos na membrana, níveis de metalotioneína (MT), lipoperoxidação (LPO) e mecanismo de acúmulo do metal. Assim, as células de brânquias anteriores (BA), posteriores (BP) e hepatopâncreas (H) foram separadas. Em seguida, realizou-se o transporte de cádmio em BA, BP, e em cada tipo celular do H, com ou sem a utilização de inibidores; o mesmo procedimento foi utilizado para caracterizar o transporte de cádmio em cada região estudada. Também foram analisadas a porcentagem de ácidos graxos, níveis de MT, LPO e mecanismos de detoxificação de cádmio. Observou-se um maior transporte através de canais de cálcio e trocadores Na+/Ca2+ em BP e nas células E, R e F de H, pois são células específicas para as trocas iônicas. Em animais de regiões poluídas, observou-se um maior transporte que ocorreu nos mesmos parâmetros, devido aos tipos celulares específicos envolvidos. Em animais de regiões contaminadas verificou-se maior porcentagem de MUFA em hepatopâncreas, acarretando em menor fluidez na membrana, e maior índice de LPO, devido ao estresse causado pela presença do contaminante no meio. Já em animais de regiões não poluídas, observou-se maior porcentagem de PUFA, com maior fluidez na membrana, e maior quantidade de MT, principalmente no hepatopâncreas, resultando na detecção de pequenas alterações ambientais. Nas brânquias, o cádmio se acumula em organelas intracelulares, enquanto em hepatopâncreas se ligam a MTs do citoplasma celular. Portanto, acredita-se que estes animais apresentaram diferentes mecanismos fisiológicos que permitiram a sua sobrevivência na região poluída / Animals resident in regions polluted by metals must have physiological mechanisms for survival, compared with animals from unpolluted regions. Therefore, the objective of this study was to characterize cadmium transport in gills and hepatopancreas of Ucides cordatus, and to compare this transport in animals from polluted (Itanhaém) and unpolluted (Juréia) regions. In addition, the animals were analyzed for cell membrane fatty acids composition, metallothionein levels (MT), lipid peroxidation (LPO) and mechanisms of accumulation of intracellular Cd. The anterior gills (BA), posterior gills (BP) and hepatopancreas (H) cells were separated. After, the transport of cadmium was realized in BA, BP and in each cellular type of H, with or without various inhibitors present. The same approach was utilized to characterize cadmium transport from crabs of polluted and unpolluted regions: fatty acids composition, MT levels, LPO and cadmium detoxification mechanisms were also analyzed. An increase in the transport of cadmium was observed through calcium channels and Na+/Ca2+ exchangers, in BP and in E, R and F cells of H, probably because they are specific cells for ion transport. In animals from polluted regions, we observed an increase in cell cadmium transport when compared with animals from unpolluted regions. Animals from polluted environments also presented more MUFA in H, leading to lower membrane fluidity, and higher LPO, probably due to the stress caused by pollution. In animals from unpolluted regions, we observed more PUFA in the plasma membrane, resulting in higher membrane fluidity, and more MT levels, mainly in H. In gills, intracellular cadmium accumulation occurred in organelles, whereas in H, Cd binding to MT in cellular cytoplasm was more evident. Therefore, we believe that animals from metal polluted environments present the above-described physiological mechanisms to deal with Cd, allowing their survival in this environment

Le loup ou bar européen effectue une partie de son cycle de vie dans les lagunes et estuaires, des eaux de transitions caractérisées par des fluctuations environnementales plus marquées qu’en milieu marin. Les mécanismes d’acclimatation à la salinité à des températures plus élevées qu’en milieu marin sont encore relativement inexplorés. Dans cette étude, des juvéniles de loup ont été préacclimatés pendant deux semaines à l’eau de mer (EM) à 18 °C (eau tempérée) ou à 24 °C (eau chaude) puis transférés soit dans l’eau douce (ED) soit dans l’EM aux deux températures testées. À 24 °C, les loups parviennent à maintenir leur pression osmotique sanguine relativement constante, quelle que soit la salinité testée. En revanche, la hausse de température affecte significativement différents traits physiologiques liés à l’osmorégulation, la régulation acido- basique, l’excrétion azotée et la production de mucus. Cette étude a notamment montré qu’une acclimatation thermique à 24 °C modifie la structure morphologique de la branchie et induit un déséquilibre de la balance ionique sanguine. Ce déséquilibre concerne particulièrement les ions Na+ et a pour conséquence une diminution du ratio Na+/Cl- plasmatique, pouvant être symptomatique d’une acidose sanguine. Après transfert en douce, une augmentation moins importante de la densité des ionocytes branchiaux et de l’activité de la pompe Na+/K+ ATPase a été observée à 24 °C comparé à 18 °C. De plus, certains transporteurs ioniques jouant un rôle clé dans l’absorption ionique en eau douce n’ont pas été induits au niveau transcriptionnel à 24 °C. Cela suggère une capacité réduite des poissons de passer d’un épithélium branchial hypo-osmorégulateur vers un épithélium hyper-osmorégulateur lors d’un transfert en eau douce à 24 °C vs 18 °C. Au niveau moléculaire, la hausse de température a affecté l’expression de plusieurs transporteurs ioniques branchiaux, notamment en EM. À 24 °C, la surexpression de transporteurs liés au transport de proton H+ pourrait être le signe d’un déséquilibre acido- basique. De plus, l’induction de transporteurs d’ammonium pourrait indiquer un besoin accru d’excréter de l’azote par voie branchiale, potentiellement lié à un métabolisme plus élevé en eau chaude. L’expression des gènes codant pour la protéine chaperonne HSP90 a été fortement affectée par la hausse de température et dans une moindre mesure par la dessalure. Pour finir, l’analyse des mucocytes suggère que la production de mucus pourrait être induite en réponse au stress thermique. Ce type d’étude fonctionnelle sur l’acclimatation à différents régimes de température permet d’apporter des éléments de compréhension pour pouvoir prédire les réponses des téléostéens face aux conséquences du changement global. / The European sea bass Dicentrarchus labrax undertakes seasonal migrations to estuaries and lagoons that are characterized by fluctuations in environmental conditions. It is unclear to what extent salinity acclimation mechanisms are affected at temperatures higher than in the sea, as usually encountered in transitional waters in spring and summer. In this study, juvenile sea bass were pre-acclimated to seawater (SW) at 18 °C (temperate) or 24 °C (warm) for two weeks and then transferred to either fresh water (FW) or SW at the considered temperatures. We have shown that sea bass are able to efficiently maintain blood osmolality at 24 °C at both salinities. However, temperature increase induced significant changes regarding several physiological traits related to osmoregulation, acid-base regulation, ammonia excretion and mucus production. This study showed that thermal acclimation at 24 °C affects gill morphology through gill remodeling and whole-organism ion balance. Plasma Na+ levels seemed to be particularly affected leading to decreased plasma Na+/Cl- ratio in warm conditions, suggesting a blood acidosis. Following FW transfer, the major effects observed were a lower increase in the density of branchial ionocytes and in Na+/K+-ATPase activity at 24 °C compared to 18 °C. Moreover, several key ion transporters involved in ion uptake were not transcriptionally induced following FW transfer at 24 °C. These data suggest a less effective capacity to switch from hypo-to hyper-osmoregulation after FW transfer when fish are exposed to higher temperature. At the molecular level, the temperature increase affected the expression of several branchial transporters, notably in SW. At 24 °C, higher expression of transporters linked to H+ transport might be linked to an acid-base imbalance. Moreover, the induction of ammonia-transporting channels might also indicate increased need for nitrogen excretion, potentially due to enhanced metabolism in warm conditions. Molecular chaperones HSP90 expression was strongly affected by temperature increase and to a lesser extent by salinity decrease. Finally, gill goblet cells analysis suggested that mucus production is increased in response to thermal stress. This study and additional functional studies investigating different temperature regimes provide insights on the effect of increased temperatures on fish responses and may help to predict how teleost will face the consequences of global change.

O impacto dos metais na fisiologia de peixes tem sido considerado preocupante, pois são contaminantes críticos nos ecossistemas aquáticos devido ao seu alto potencial de entrar no organismo, acumular-se e ser transferido na cadeia trófica e muitos estudos têm demonstrado os efeitos sobre várias funções em animais, quando expostos a estes metais. O rio Paraíba do Sul vem apresentando concentrações de alumínio (Al) e manganês (Mn) acima dos valores permitidos pela legislação Brasileira e, além disso, fatores abióticos tais como a acidez da água, podem contribuir para alteração do potencial de toxicidade, podendo acarretar em danos à biota aquática. As respostas desencadeadas pelos animais a estas mudanças ambientais podem levá-los a um estado de estresse, que é altamente energético devido à realocação de substratos inicialmente destinados às atividades de elevada demanda energética, como crescimento e reprodução, em direção às atividades que requerem intensificação para restaurar a homeostase. O presente estudo teve como objetivo avaliar os efeitos dos metais Al e Mn em pH ácido, isoladamente e em combinação, durante 96 horas (teste agudo) nas brânquias e nos substratos metabólicos de fêmeas vitelogênicas de Astyanax bimaculatus e, adicionalmente após um período de 96 horas em água limpa, verificar a habilidade dos animais em se recuperar na ausência destes metais. Os animais foram divididos em 5 grupos experimentais: CTR em pH neutro; pH ácido; Al; Mn; e Mn+Al. Os dados mostram que quando expostos a estes metais isoladamente, a atividade enzimática da bomba Na+/K+ ATPase e a quantidade de células de cloreto foram diminuídas, no entanto quando combinados, estes efeitos não foram observados. O Al isolado e em associação com o Mn promoveu peroxidação de fosfolipídios de membrana. Além disso nos animais expostos aos dois metais juntos, houve um aumento de ácidos graxos monoinsaturados (MUFA) e saturados (STA) acompanhado pela diminuição dos ácidos graxos polinsaturados (PUFA) o que pode ter evitado alterações na atividade de bomba e na quantidade de células de cloreto na combinação destes metais. Considerando-se os substratos metabólicos, a exposição ao Al diminuiu a concentração de proteínas nos ovários e no plasma, glicogênio muscular, além de alterar as porcentagens de MUFA e PUFA no fígado, e PUFA nos ovários. O Al também promoveu lipoperoxidação hepática, e no período de recuperação houve diminuição da concentração de lipídios em vários tecidos analisados. A exposição ao Mn promoveu diminuição das concentrações de proteínas totais em todos os tecidos analisados, seja no período de exposição agudo ou de recuperação, não havendo a recuperação deste substrato no fígado e nos ovários; o Mn promoveu lipoperoxidação nos fosfolipídios das membranas do fígado e, em associação com o Al, pareceu interferir na dinâmica dos PUFA das membranas dos ovários. Várias interações foram observadas entre o Al e Mn, demonstrando casos de antagonismo, potenciação e até mesmo de sinergismo entre estes metais, dependendo do parâmetro fisiológico analisado, evidenciando que a exposição de fêmeas vitelogênicas a estes metais pode ser prejudicial ao processo reprodutivo principalmente devido à ação observada, inclusive nos ovários / The impact of metals in fish physiology has been considered worrying due to their high potential of entering in the organism, to accumulate and be transferred through the food chain and many studies have shown the effects on several functions in animals, when exposed to these metals. The Paraíba do Sul river showed aluminum (Al) and manganese (Mn) concentrations above the values allowed by the Brazilian law and moreover abiotic factors, such as acidic water, can contribute to changing the toxicity potential and can result in damage to aquatic biota. The answers triggered by the animals to these environmental changes can lead to a stress state which has a high energetic cost due to the substrate reallocation, initially designated to activities with high energetic demand, as growth and reproduction, to activities that need to be intensified, to reestablish the homeostasis. The main goals of this study were to evaluate the effects of the metals Al and Mn in acidic pH, alone and in combination, during 96 hours (acute test) in the gills and in metabolic substrates in Astyanax bimaculatus vitelogenic females and, additionally, verify the ability of the animals to recover in the absence of the metals after the 96 hours in clear water. The animals were divided in 5 groups : CTR in neutral pH; acidic pH; Al; Mn; and Mn+Al. The results showed that when the animals were exposed to the isolate metals, there was a decrease in the Na+/K+ ATPase pump activity and in the amount of chloride cells, however when combined, these effects were not observed. Al, isolated or in association with Mn, promoted phospholipids peroxidation in gill membranes. Furthermore, in the animals exposed to both metals together, there were an increase in monounsaturated fatty acids (MUFA) and saturated fatty acids (STA) and decrease in polyunsaturated fatty acids (PUFA) which may have avoided alterations in pump activity and in the amount of chloride cells. In the metabolic substrates, Al exposition decreased plasma and ovaries protein concentration, muscle glycogen, and altered the percentage of MUFA and PUFA in the liver, and PUFA in the ovaries. Al also promoted liver lipoperoxidation, and in the recovery period lipid content decreased in most tissues analyzed. Mn exposition promoted a decrease in total protein content in all tissue analyzed, both in the acute and recovery periods, and there was no recovery of this substrate in liver and ovaries; Mn promoted lipoperoxidation in liver phospolipids and, in association with Al, interfered in the PUFA dynamic in ovaries membranes. Many interactions were observed between Al and Mn, evidencing antagonism, potentiation and also synergism, depending on the physiologic parameter analyzed, showing that the exposition of vitellogenic females to these metals can be harmful to reproductive process due to the observed actions, including in ovaries

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-22T18:15:15Z No. of bitstreams: 1 MayaraCruzRibeiro.pdf: 1477635 bytes, checksum: 3daf1f5d95a16f48c327f3fb3a6b3b89 (MD5) / Made available in DSpace on 2017-06-22T18:15:15Z (GMT). No. of bitstreams: 1 MayaraCruzRibeiro.pdf: 1477635 bytes, checksum: 3daf1f5d95a16f48c327f3fb3a6b3b89 (MD5) Previous issue date: 2014-04-29 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / The diversity of the oscillations in the aquatic environment can be enhanced by the lack of preventive methods in management. The lack of knowledge about the parameters of water quality, such as transparency, alkalinity, hardness, dissolved oxygen, pH, ammonia, nitrite and pollutants cause various problems. The increase in the concentration of ammonia causes toxicity to aquatic organisms as it interacts with the biological parameters and can be intensified by the lack of appropriate management in water. Animals exposed to these contaminants may change in metabolic, immunologic, physiologic, and tissue damage in target organs such as gills and liver processes. The tetra (Bryconops caudomaculatus) is a native species with potential for aquaculture, with great potential for expansion, have high proliferation, short production cycle, accepts artificial food and serves as food for other species. The lack of information and the demand for knowledge about the disturbances caused by these animals increased concentrations of ammonia in the water require more specific studies in these target organs. The objective of this experiment was to evaluate the effects of exposure of un-ionized ammonia (NH3) in juvenile Bryconops caudomaculatus, and morphological and histopathological changes in the gills and liver. 150 juvenile minnows subjected to five treatments in tripicata with concentrations of NH3 (mg L-1) were control 0.003 ± 0.4; 0.15 ± 0.5; 0.30 ± 0.3; 0.50 ± 0.5; 1.00 ± 0.4. After the experimental period, 12 animals from each treatment were collected to obtain the gills and liver. After, target organs passed by fixation, dehydration, and paraffin embedding diafranização processes. The staining technique used was toluidine blue for gills and hematoxylin-eosin for the liver. Analysis of variance was used for parametric data and Kruskal-Wallis test for the descriptive analyzes. Morphological changes were observed in the secondary lamellae in the gills from 0.15 mg L-1 as hyperplasia, hypertrophy, epithelial displacement, lamellar melt, edema, aneurysm rupture lamellar epithelium and increase in width of the lamellae. There was also the presence of liver histopathologies from 0.30 mg L-1 as the capillary congestion, inflammation, pigmentation and endogenous treatment with the highest concentration in hepatocytes showed necrosis. These changes caused by the processes of intoxication impaired vital functions, metabolism, processes of respiration and detoxification of the body, probably in hematopoiesis and osmoregulation. / A diversidade das oscilações no ambiente aquático pode ser intensificada pela falta de metodologias preventivas no manejo. A falta de conhecimento a respeito dos parâmetros de qualidade de água, como transparência, alcalinidade, dureza, oxigênio dissolvido, pH, amônia, nitrito e poluentes, causa vários problemas. A elevação na concentração da amônia causa toxicidade para os organismos aquáticos, pois interage com os parâmetros biológicos e pode ser intensificada pela falta de um manejo adequado na água. Animais expostos a esses contaminantes podem sofrer alterações nos processos metabólicos, imunológicas, fisiológicos, lesões teciduais e nos órgãos-alvo como brânquias e fígado. O lambari (Bryconops caudomaculatus) é uma espécie nativa com potencial para piscicultura, com grandes possibilidades de expansão, tem alta proliferação, ciclo curto de produção, aceita alimento artificial e serve de alimento para outras espécies. A falta de informação e a demanda por conhecimento sobre os distúrbios causados nesses animais pelo aumento das concentrações de amônia na água exigem estudos mais específicos nesses órgãos-alvo. O objetivo deste experimento foi avaliar os efeitos da exposição de amônia não ionizada (NH3) em juvenis de Bryconops caudomaculatus, e as alterações histopatológicas e morfológicas nas brânquias e no fígado. Foram utilizados 150 juvenis de lambaris submetidos a cinco tratamentos, em tripicata, com concentrações de NH3 (mg L-1): controle 0,003 ± 0,4; 0,15 ± 0,5; 0,30 ± 0,3; 0,50 ± 0,5; 1,00 ± 0,4. Após o período experimental foram coletados 12 animais de cada tratamento, para a obtenção das brânquias e do fígado. Após, os órgãos-alvo passaram pelos processos fixação, desidratação, diafranização e inclusão em parafina. A técnica de coloração utilizada foi a Azul de Toluidina para brânquias e a Hematoxilina-Eosina para o fígado. Foi utilizada análise de variância para os dados paramétricos e teste de Kruskall-Wallis para as análises descritivas. Observou-se alterações morfológicas nas lamelas secundárias nas brânquias a partir de 0,15mg L-1, como hiperplasia, hipertrofia, deslocamento epitelial, fusão lamelar, edemas, aneurisma lamelar com ruptura do epitélio e aumento na largura das lamelas. Houve presença também de histopatológias no fígado a partir de 0,30 mg L-1 como congestão nos capilares, processos inflamatórios, pigmentação endógena e no tratamento com maior concentração os hepatócitos apresentaram necrose. Essas alterações causadas pelos processos de intoxicação prejudicaram as funções vitais, metabolização, os processos de respiração e desintoxicação do organismo, hematopoiese e provavelmente na osmorregulação.

The overall objective of my thesis research was to develop methodologies for assessing effects of mining effluents on pristine and sensitive northern rivers. I used a multi-trophic level approach in field studies to evaluate current monitoring methods and to determine whether metal mining activities had affected two otherwise pristine rivers that flow into the South Nahanni River, NWT; a World Heritage Site. Upstream reference conditions in the rivers were compared to sites downstream and further downstream of mines. The endpoints evaluated included concentrations of metals in river water, sediments and liver and flesh of slimy sculpin (Cottus cognatus); benthic algal and macroinvertebrate abundance, richness, diversity, and community composition; and various slimy sculpin measures. Elevated concentrations of copper (p=0.002)and iron (p=0.001) in liver tissue of sculpin from the Flat River were associated with high concentrations of mine-derived iron in river water and copper in sediments that were above national guidelines. In addition, sites downstream of the mine on the Flat River had increased algal abundances (p=0.002) and altered benthic macroinvertebrate communities ((p<0.001) whereas the sites downstream of the mine on Prairie Creek had increased benthic macroinvertebrate taxa richness (p=0.050) and improved sculpin condition (males: p=0.008; females: p=0.001). Biological differences in both rivers were consistent with mild enrichment of the rivers downstream of current and historical mining activity. Although the effects of mining activities on riverine biota in these northern rivers are currently limited, results of this research show that there is potential for effects to occur with proposed growth in mining activities.<p> Laboratory exposures were conducted using slimy sculpin, identified as a sentinel fish species in pristine northern rivers, to identify alternative methods for assessing toxicity of contaminants of concern in mining effluents. Ammonia was selected for the exposures based on effluent characteristics of northern mining effluents. Ammonia is known to be an important toxicant in aquatic environments. Although ammonia toxicity has been well studied in many fish species, effects of chronic exposure of slimy sculpin, a critical biomonitoring species for northern aquatic habitats, are not well known. Slimy sculpin were exposed to six concentrations of un-ionized ammonia relevant to concentrations found in northern mining effluents: control (0 ppm), 0.278 ppm, 0.556 ppm, 0.834 ppm, 1.112 ppm, and 1.668 ppm. An LC50 of 1.529 ppm was calculated from mortality data. Histopathological examination of gills indicated significant tissue damage, measured as lamellar fusion and epithelial lifting, at 0.834, 1.112, and 1.668 ppm. Using gill endpoints, NOEC and LOEC were calculated as 0.556 ppm and 0.834 ppm respectively. An EC50 of 0.775 ppm was determined for lamellar fusion and an EC50 of 0.842 ppm for epithelial lifting. Hemorrhage of gills was present in mortalities which occurred at 1.668 ppm of un-ionized ammonia. A significant decrease in liver somatic index (LSI) was seen in both male and female fish at 0.834 and 1.112 ppm, respectively. Gonadosomatic index (GSI) in female fish significantly increased at 1.668 ppm un-ionized ammonia with an associated significant increase in total whole body testosterone concentrations. GSI in male fish also significantly increased at 1.668 ppm but no differences were seen in testosterone concentrations. No significant differences were seen in gonad histopathological assessments or condition factor. Results from this study indicate that ammonia concentrations commonly reported in northern mine effluents hold potential to affect the health of slimy sculpin including acute, chronic, histological and endocrine endpoints. <p> Results from both the field study and laboratory exposures provide direction for future monitoring programs in pristine northern rivers and emphasize the importance of monitoring tools to detect change in these ecosystems. I recommend that monitoring of northern pristine rivers focus on a multi-trophic monitoring approach including indicators in algal and benthic macroinvertebrate communities due to their responsiveness. Laboratory exposures using slimy sculpin should be considered to obtain toxicological information for northern contaminants of concern. Gill histopathology endpoints may be a more sensitive indicator for detecting effects in slimy sculpin exposed to ammonia than traditional chronic endpoints. I also recommend monitoring of metal burdens in periphyton and benthic invertebrates for assessment of exposure to mine effluent and causal association in areas of low fish abundance.

Rainbow trout were exposed to aluminum at pH 7.25 and 8.25 and four hardnesses (10, 30, 80, and 120 ppm CaCO₃) for 96 hours in a continuous-flow system and mortality and aluminum accumulation in the gills were determined. Temperature, pH, and dissolved oxygen were measured daily for each treatment. Dissolved and total aluminum concentrations and hardness were determined following exposure periods of 48 and 96 hours. Aluminum was most toxic at pH 8.25, and was more toxic at lower than at higher hardnesses. Water hardness provided a significant protective effect against aluminum-induced mortality (p < 0.05), and there were no significant effects for water hardness on gill accumulation at either of pH. At pH 7.25 no mortalities occurred under any conditions. At pH 8.25, the accumulation of aluminum in gill tissues was higher than for pH 7.25 following exposure for 96 hours. In addition, aluminum concentration and exposure time had a significantly cumulative effect on fish mortality (p < 0.05). Possible mechanisms for aluminum toxicity and the accumulation of aluminum in the gills of rainbow trout were attributed to the forms and solubilities of aluminum species at different pH values. Competition between Ca²⁺ and aluminum for binding sites on the gills likely influenced aluminum toxic action. / Graduation date: 1992

The overall objective of my thesis research was to develop methodologies for assessing effects of mining effluents on pristine and sensitive northern rivers. I used a multi-trophic level approach in field studies to evaluate current monitoring methods and to determine whether metal mining activities had affected two otherwise pristine rivers that flow into the South Nahanni River, NWT; a World Heritage Site. Upstream reference conditions in the rivers were compared to sites downstream and further downstream of mines. The endpoints evaluated included concentrations of metals in river water, sediments and liver and flesh of slimy sculpin (Cottus cognatus); benthic algal and macroinvertebrate abundance, richness, diversity, and community composition; and various slimy sculpin measures. Elevated concentrations of copper (p=0.002)and iron (p=0.001) in liver tissue of sculpin from the Flat River were associated with high concentrations of mine-derived iron in river water and copper in sediments that were above national guidelines. In addition, sites downstream of the mine on the Flat River had increased algal abundances (p=0.002) and altered benthic macroinvertebrate communities ((p<0.001) whereas the sites downstream of the mine on Prairie Creek had increased benthic macroinvertebrate taxa richness (p=0.050) and improved sculpin condition (males: p=0.008; females: p=0.001). Biological differences in both rivers were consistent with mild enrichment of the rivers downstream of current and historical mining activity. Although the effects of mining activities on riverine biota in these northern rivers are currently limited, results of this research show that there is potential for effects to occur with proposed growth in mining activities.<p> Laboratory exposures were conducted using slimy sculpin, identified as a sentinel fish species in pristine northern rivers, to identify alternative methods for assessing toxicity of contaminants of concern in mining effluents. Ammonia was selected for the exposures based on effluent characteristics of northern mining effluents. Ammonia is known to be an important toxicant in aquatic environments. Although ammonia toxicity has been well studied in many fish species, effects of chronic exposure of slimy sculpin, a critical biomonitoring species for northern aquatic habitats, are not well known. Slimy sculpin were exposed to six concentrations of un-ionized ammonia relevant to concentrations found in northern mining effluents: control (0 ppm), 0.278 ppm, 0.556 ppm, 0.834 ppm, 1.112 ppm, and 1.668 ppm. An LC50 of 1.529 ppm was calculated from mortality data. Histopathological examination of gills indicated significant tissue damage, measured as lamellar fusion and epithelial lifting, at 0.834, 1.112, and 1.668 ppm. Using gill endpoints, NOEC and LOEC were calculated as 0.556 ppm and 0.834 ppm respectively. An EC50 of 0.775 ppm was determined for lamellar fusion and an EC50 of 0.842 ppm for epithelial lifting. Hemorrhage of gills was present in mortalities which occurred at 1.668 ppm of un-ionized ammonia. A significant decrease in liver somatic index (LSI) was seen in both male and female fish at 0.834 and 1.112 ppm, respectively. Gonadosomatic index (GSI) in female fish significantly increased at 1.668 ppm un-ionized ammonia with an associated significant increase in total whole body testosterone concentrations. GSI in male fish also significantly increased at 1.668 ppm but no differences were seen in testosterone concentrations. No significant differences were seen in gonad histopathological assessments or condition factor. Results from this study indicate that ammonia concentrations commonly reported in northern mine effluents hold potential to affect the health of slimy sculpin including acute, chronic, histological and endocrine endpoints. <p> Results from both the field study and laboratory exposures provide direction for future monitoring programs in pristine northern rivers and emphasize the importance of monitoring tools to detect change in these ecosystems. I recommend that monitoring of northern pristine rivers focus on a multi-trophic monitoring approach including indicators in algal and benthic macroinvertebrate communities due to their responsiveness. Laboratory exposures using slimy sculpin should be considered to obtain toxicological information for northern contaminants of concern. Gill histopathology endpoints may be a more sensitive indicator for detecting effects in slimy sculpin exposed to ammonia than traditional chronic endpoints. I also recommend monitoring of metal burdens in periphyton and benthic invertebrates for assessment of exposure to mine effluent and causal association in areas of low fish abundance.