The mitochondria are organelles where energy in the form of ATP (Adenosine Triphosphate) is produced. During low oxygen supply (hypoxia) and mitochondrial uncoupling, ATP synthesis is reduced and AMP (Adenosine Monophosphate) accumulates in the cells. This increase in AMP: ATP ratio stimulates the AMP-Activated Kinase (AMPK) pathway, known to improve healthspan and lifespan by increasing mitochondrial biogenesis (making new mitochondria), decreasing oxidative stress and inflammation, and proteotoxicity (by degrading non-functional organelles and proteins). Here, the life/healthspan extending potential of chronic mild intermittent hypoxia (CMIH) and mitochondrial uncoupling using 2,4 -Dinitrophenol (DNP) was investigated in an emerging model organism, an aquatic crustacean, Daphnia magna. First, the effect of CMIH (4mgO2/L) on longevity in four different genotypes of Daphnia magna was investigated. All individuals were kept in similar conditions with controls in normoxia (8mgO2/L). Hypoxia was created by bubbling compressed nitrogen gas through the water twice daily. Survival was assessing through censuses conducted every 3 days and gene expression changes in response to CMIH were assessed by RNA sequencing using Oxford Nanopore Technology. Briefly, RNA was isolated from genotypes after hypoxic treatments and reverse transcribed to cDNA, libraries were multiplexed and sequenced using Oxford Nanopore MinION for 24-48 hours. Lastly, the effect of prolonged exposure to DNP on longevity was evaluated. Daphnia were chronically exposed to either 0 (control), or 0.1, 1, and 5μM of DNP. Genotypes displayed different tolerance to hypoxia and DNP treatments. Contrary to the expectations, CMIH and DNP reduced longevity, but only in genotypes from permanent ponds, while having no effect on the survival of genotypes from intermittent ponds, arguably better adapted to naturally occurring hypoxic conditions. We uncovered 11 candidate genes that were differentially expressed in these genotypes. In particular, genes involved in mTOR, p53, and sirtuin pathways showed patterns of expression consistent with protection against hypoxia. These pathways are known to regulate autophagy, apoptosis, inflammation, and cell cycle. Because our findings elucidate genotype-specific physiological and transcriptomic responses to respiratory perturbations (CMIH and DNP) we may be able to make a step towards the understanding of a model organism’s response to respiratory stress.

The diploma thesis is primarily focused on the standardization of Daphnia magna breeding in laboratory conditions of the Department of Zoology, Fisheries, Hydrobiology and Beekeeping at the Faculty of Agronomy of the Mendel University in Brno. The main aim of the work is to simplify the preparation of medium for breeding Daphnia magna due to time saving. During testing, we were interested in births and mortality of individuals observed during 14-day tests in two media. We focus on chemical and physical properties of the media, namely the pH, temperature and intensity of the light.

The aim of this thesis was to derermine the values of toxic effect of chemical sub-stance chemical on the tested organism Daphnia magna. For testing, these materials were selected: polyaluminiumchloride (PAX 18), its aqueous solution (PAX 19) and potassium dichromate. The thesis is divided into two parts, in the first half I devote to theory of the basic terminology of the issue and the general characterization of the tested organism. In the practical part I focus on the characteristics of the used substances, the course of the tes-ting and the results of the work together with the discussion. Experimental concentrations for PAX 18 were between 1-100 mg.l-1, for PAX 19 10-300 mg.l-1 and for potassium dichromate 40-150 mg.l-1. Testing was perfomed in 3 repeatings with ten individuals + control. 24hEC50 values were: for PAX 18 45,06 mg.l-1, for PAX 19 97,82 mg.l-1 and for potassium dichromate 1,569 mg.l-1. The values of 48hEC50 for PAX 18 byly 22,62 mg.l-1, for PAX 19 37,20 mg.l-1 and for potassium dichromate 0,864 mg.l-1. In all tests, fluctuations in pH and dissolved oxygen content, as well as the number of inhibited individuals, were observed. The results of the observations were plotted and then commented verbally.

Ecotoxicology aims to understand toxic effects of chemicals in the environment. Effects can be observed at different levels of the biological organization, from molecular to ecosystem level. Biomarkers on molecular and biochemical levels are used in ecotoxicology as early warning signals of chemical exposure, possible toxic effects and underlying mechanisms. As methods and technologies improve, more biomarkers are being implemented in ecotoxicological studies, due to the general interest in early detection and thus efficient prevention of environmental risks. However, to be of value in ecotoxicological assessment, a connection between biomarker response and effects at higher levels of biological organization should be established. Also, baseline variability for the biomarker in question as well as response to natural fluctuations of environmental factors should be evaluated. The aim of this thesis was to increase value and understanding of biomarkers in ecotoxicological assessment by (1) linking responses across different levels of biological organization, and (2) gaining better understanding of the relative importance of ecological and physiological factors affecting oxidative biomarkers. Paper I is focused on evolutionary conserved drug targets and the toxicity of pharmaceuticals for non-target organisms. The main conclusion from this study is that pharmaceuticals with conserved drug targets in non-target organisms have a higher toxicity than pharmaceuticals for which drug-targets have not been identified in the species. The effects were evaluated using end points at molecular, biochemical and individual levels. Consistent with the expected higher sensitivity of molecular and biochemical end points, the effects on the low-level biomarkers were observed at lower concentrations than at the individual level.   Paper II is focused on delineating effects of feeding and toxic exposure on oxidative biomarkers commonly used in ecotoxicology. The results are in agreement with the theory of caloric restriction that links enhanced caloric intake to increased pro-oxidative processes in animals. In our experiments with the cladoceran Daphnia magna, we observed positive effects for both antioxidant capacity and oxidized lipids in response to enhanced feeding rates. This have implications for the use of oxidative stress biomarkers in ecotoxicology as many substances have inhibitory effects on feeding rate and thus, changes in oxidative biomarkers can result from the altered feeding rate rather than other toxic mechanisms. Therefore, possible changes in feeding rate should be assessed when conducting exposure experiments or interpreting field data in studies employing oxidative stress biomarkers. However, it was concluded that the ratio between antioxidative capacity and protein content was independent of feeding rate. Thus, this biomarker is suitable for xenobiotic exposure in D. magna. This thesis have contributed to better understanding of molecular and biochemical biomarkers in ecotoxicological studies in regard to the connections between effects at different biological levels and confounding factors in biomarker response. / <p>At the tiem of defence the following papers wer unpublished and had a status as follows:</p><p>Paper 1: Manuscript; Paper 2: Manuscript</p>

Climate change has caused an increase in extreme rain events and flooding in certain regions across the globe. During rain events, water flows over impervious surfaces structures such as roads and sidewalks, picking up contaminants such as metals, fertilizers and other nutrients, and various organics that which may impact organisms in such as streams, river, and lakes. Previous work has found significant differences in survival of organisms that were exposed to contaminated stormwater runoff. This study investigated stormwater chemistry parameters at collection sites on the East Tennessee State University campus. Sites were selected based on the extent of human interaction and traffic in the areas. Additionally, acute toxicity of stormwater samples was investigated through 48-h bioassays with the cladoceran, Daphnia magna. In September and November 2022, water chemistry and toxicity analyses were conducted across multiple rain events and over a six-hour time course of an individual rain event. For each of the events and the time course, chlorophyll levels, specific conductivity, pH, temperature, and dissolved oxygen were measured. No statistical difference between the water chemistry parameters between sampling sites or between rain events were observed. Additionally, no significant differences in 48-h survival of D. magna were detected between sampling locations or during the single event time course study. These data suggest that there were no pollutant surges at the collection sites and that D. manga survival was not affected by the contaminants.

No description available.

No description available.

Made available in DSpace on 2017-07-10T19:24:14Z (GMT). No. of bitstreams: 1 TESE ADRIANA_ JOHANN.pdf: 2610924 bytes, checksum: 3d2882b78e66f6ce454900b7a8b0d486 (MD5) Previous issue date: 2015-07-29 / Fundação Araucária / Environmental impacts have become a reality ever more present in aquatic ecosystems, suffering changes in the constitution of the chemical elements of water, availability and their nutrient dynamics. Therefore, comprehensive methodologies are needed to assess the actual condition of these ecosystems, following their changes. In this sense, the objective of this study was to evaluate the water quality of the basin of the São Francisco Verdadeiro river, by analyzing the physical, chemical, microbiological variables, and acute and chronic toxicity tests with the test organism Daphnia magna, to identify possible natural or anthropogenic interference. Thus, fortnightly collections were carried out in six sampling locations (P1, P2, P3, P4, P5 and P6), on the period from April 2013 to April 2014, totalizing 143 samples, of which 31 water quality variables were evaluated, with subsequent calculation of the Water Quality Index (WQI), and acute and chronic toxicity tests. The results obtained for the physical, chemical and microbiological variables showed that dissolved oxygen (Sites P3, P5 and P6), turbidity and ammonia nitrogen (Site P6), pH, biochemical oxygen demand, total phosphorus, fecal coliform bacteria (Escherichia-coli), cadmium, lead, manganese, nickel and zinc (All locations), presented different values of the limits recommended by CONAMA resolution No 357/2005, for Class II waters. It is noteworthy that such disagreements have occurred at different times. WQI values ranged from good to great quality, despite high values of some variables. For acute tests, the results showed no toxic effect on any of the sites sampled throughout the study period. For acute tests, the results showed no toxic effect on any of the evaluated sites throughout the study period. With regard to chronic tests, toxic effect occurred in 5.59% of the samples for the parameter longevity, observed by the death of Daphnia magna organisms during the test, and 97.90% of the samples for fertility parameter, verified by the reduction in reproduction, in which the samples showed a reduced number of offspring compared to the control (M4 culture medium). Toxic effect events registered for fertility were compared with pH the variables, total phosphorus, BOD, fecal coliforms, cadmium, lead, copper, nickel and zinc, which presented different values of the limits established legislation by CONAMA Nº 357/2005, for water class II, since it was observed that the toxic effect events, often coincided with at least one variable above the limits. Therefore, the results obtained for the physical, chemical and microbiological variables showed that the São Francisco Verdadeiro river is impacted by the use and occupation of the basin; receiving contributions of rural and urban áreas, and chronic toxicity tests indicated inadequate conditions for the protection of aquatic life, showing that they are effective tools for assessing a body of water, can serve as allies of the classic valuation methods. / Os impactos ambientais têm se tornado uma realidade cada vez mais presente nos ecossistemas aquáticos, que sofrem mudanças na constituição dos elementos químicos da água, na disponibilidade e na sua dinâmica de nutrientes. Sendo assim, metodologias abrangentes são necessárias para avaliar o estado real desses ecossistemas, acompanhando suas mudanças Nesse sentido, o objetivo desse trabalho foi avaliar a qualidade da água da bacia do Rio São Francisco Verdadeiro, por meio da análise de variáveis físicas, químicas, microbiológicas e testes de toxicidade agudos e crônicos, com o organismo-teste Daphnia magna, visando identificar possíveis interferências naturais ou antrópicas. Para tanto, foram realizadas coletas quinzenais, em seis locais de amostragem (P1, P2, P3, P4, P5 e P6), no período de abril de 2013 a abril de 2014, totalizando 143 amostras, das quais foram avaliadas 31 variáveis de qualidade de água, com posterior cálculo do Índice de Qualidade da Água (IQA) e os testes de toxicidade agudos e crônicos. Os resultados obtidos para as variáveis físicas, químicas e microbiológicas, mostraram que oxigênio dissolvido (Locais P3, P5 e P6), turbidez e nitrogênio amoniacal (Local P6), pH, demanda bioquímica de oxigênio, fósforo total, coliformes fecais (Escherichia-coli), cádmio, chumbo, manganês, níquel e zinco (Todos os locais) apresentaram valores discordantes dos limites preconizados pela resolução CONAMA No 357/2005, para águas de Classe II. Vale salientar que estas discordâncias ocorreram em períodos distintos. Os valores do IQA variaram de boa a ótima qualidade, apesar dos valores elevados de algumas variáveis. Para os testes agudos, os resultados mostraram que não houve efeito tóxico em nenhum dos locais amostrados em todo o período avaliado. Com relação aos testes crônicos, ocorreu efeito tóxico em 5,59% das amostras, para o parâmetro longevidade, constatado pela morte dos organismos Daphnia magna durante o teste e em 97,90% das amostras, para o parâmetro fecundidade, verificado pela redução na reprodução, em que as amostras apresentaram um número reduzido de filhotes com relação ao controle (Meio de cultivo M4). Os eventos de efeito tóxico registrados para a fecundidade foram comparados com as variáveis pH, fósforo total, DBO, coliformes fecais, cádmio, chumbo, cobre, níquel e zinco, que apresentaram valores discordantes dos limites estabelecidos pela legislação CONAMA No 357/2005 para águas de Classe II, em que observou-se que os eventos de efeito tóxico, em quase todo o período coincidiram com pelo menos uma variável acima dos limites. Dessa maneira, os resultados obtidos para as variáveis físicas, químicas e microbiológicas demonstraram que o rio São Francisco Verdadeiro é impactado pelo uso e ocupação do solo da bacia, recebendo contribuições das áreas rurais e urbanas, e os testes de toxicidade crônicos indicaram condições inadequadas para a proteção da vida aquática, mostrando que são ferramentas eficazes para a avaliação de um corpo de água, podendo servir como aliados dos métodos clássicos de avaliação.

Made available in DSpace on 2017-05-12T14:47:30Z (GMT). No. of bitstreams: 1 TESE ADRIANA_ JOHANN.pdf: 2610924 bytes, checksum: 3d2882b78e66f6ce454900b7a8b0d486 (MD5) Previous issue date: 2015-07-29 / Fundação Araucária / Environmental impacts have become a reality ever more present in aquatic ecosystems, suffering changes in the constitution of the chemical elements of water, availability and their nutrient dynamics. Therefore, comprehensive methodologies are needed to assess the actual condition of these ecosystems, following their changes. In this sense, the objective of this study was to evaluate the water quality of the basin of the São Francisco Verdadeiro river, by analyzing the physical, chemical, microbiological variables, and acute and chronic toxicity tests with the test organism Daphnia magna, to identify possible natural or anthropogenic interference. Thus, fortnightly collections were carried out in six sampling locations (P1, P2, P3, P4, P5 and P6), on the period from April 2013 to April 2014, totalizing 143 samples, of which 31 water quality variables were evaluated, with subsequent calculation of the Water Quality Index (WQI), and acute and chronic toxicity tests. The results obtained for the physical, chemical and microbiological variables showed that dissolved oxygen (Sites P3, P5 and P6), turbidity and ammonia nitrogen (Site P6), pH, biochemical oxygen demand, total phosphorus, fecal coliform bacteria (Escherichia-coli), cadmium, lead, manganese, nickel and zinc (All locations), presented different values of the limits recommended by CONAMA resolution No 357/2005, for Class II waters. It is noteworthy that such disagreements have occurred at different times. WQI values ranged from good to great quality, despite high values of some variables. For acute tests, the results showed no toxic effect on any of the sites sampled throughout the study period. For acute tests, the results showed no toxic effect on any of the evaluated sites throughout the study period. With regard to chronic tests, toxic effect occurred in 5.59% of the samples for the parameter longevity, observed by the death of Daphnia magna organisms during the test, and 97.90% of the samples for fertility parameter, verified by the reduction in reproduction, in which the samples showed a reduced number of offspring compared to the control (M4 culture medium). Toxic effect events registered for fertility were compared with pH the variables, total phosphorus, BOD, fecal coliforms, cadmium, lead, copper, nickel and zinc, which presented different values of the limits established legislation by CONAMA Nº 357/2005, for water class II, since it was observed that the toxic effect events, often coincided with at least one variable above the limits. Therefore, the results obtained for the physical, chemical and microbiological variables showed that the São Francisco Verdadeiro river is impacted by the use and occupation of the basin; receiving contributions of rural and urban áreas, and chronic toxicity tests indicated inadequate conditions for the protection of aquatic life, showing that they are effective tools for assessing a body of water, can serve as allies of the classic valuation methods. / Os impactos ambientais têm se tornado uma realidade cada vez mais presente nos ecossistemas aquáticos, que sofrem mudanças na constituição dos elementos químicos da água, na disponibilidade e na sua dinâmica de nutrientes. Sendo assim, metodologias abrangentes são necessárias para avaliar o estado real desses ecossistemas, acompanhando suas mudanças Nesse sentido, o objetivo desse trabalho foi avaliar a qualidade da água da bacia do Rio São Francisco Verdadeiro, por meio da análise de variáveis físicas, químicas, microbiológicas e testes de toxicidade agudos e crônicos, com o organismo-teste Daphnia magna, visando identificar possíveis interferências naturais ou antrópicas. Para tanto, foram realizadas coletas quinzenais, em seis locais de amostragem (P1, P2, P3, P4, P5 e P6), no período de abril de 2013 a abril de 2014, totalizando 143 amostras, das quais foram avaliadas 31 variáveis de qualidade de água, com posterior cálculo do Índice de Qualidade da Água (IQA) e os testes de toxicidade agudos e crônicos. Os resultados obtidos para as variáveis físicas, químicas e microbiológicas, mostraram que oxigênio dissolvido (Locais P3, P5 e P6), turbidez e nitrogênio amoniacal (Local P6), pH, demanda bioquímica de oxigênio, fósforo total, coliformes fecais (Escherichia-coli), cádmio, chumbo, manganês, níquel e zinco (Todos os locais) apresentaram valores discordantes dos limites preconizados pela resolução CONAMA No 357/2005, para águas de Classe II. Vale salientar que estas discordâncias ocorreram em períodos distintos. Os valores do IQA variaram de boa a ótima qualidade, apesar dos valores elevados de algumas variáveis. Para os testes agudos, os resultados mostraram que não houve efeito tóxico em nenhum dos locais amostrados em todo o período avaliado. Com relação aos testes crônicos, ocorreu efeito tóxico em 5,59% das amostras, para o parâmetro longevidade, constatado pela morte dos organismos Daphnia magna durante o teste e em 97,90% das amostras, para o parâmetro fecundidade, verificado pela redução na reprodução, em que as amostras apresentaram um número reduzido de filhotes com relação ao controle (Meio de cultivo M4). Os eventos de efeito tóxico registrados para a fecundidade foram comparados com as variáveis pH, fósforo total, DBO, coliformes fecais, cádmio, chumbo, cobre, níquel e zinco, que apresentaram valores discordantes dos limites estabelecidos pela legislação CONAMA No 357/2005 para águas de Classe II, em que observou-se que os eventos de efeito tóxico, em quase todo o período coincidiram com pelo menos uma variável acima dos limites. Dessa maneira, os resultados obtidos para as variáveis físicas, químicas e microbiológicas demonstraram que o rio São Francisco Verdadeiro é impactado pelo uso e ocupação do solo da bacia, recebendo contribuições das áreas rurais e urbanas, e os testes de toxicidade crônicos indicaram condições inadequadas para a proteção da vida aquática, mostrando que são ferramentas eficazes para a avaliação de um corpo de água, podendo servir como aliados dos métodos clássicos de avaliação.

Les paramètres comportementaux sont de plus en plus considérés comme étant des indicateurs sensibles et précoces de toxicité chez les organismes aquatiques. Cependant, l’utilisation du comportement natatoire comme critère de toxicité pour l’analyse de risque environnemental ou pour le contrôle de la qualité de l’eau reste pour l’heure encore limitée. En effet, il est actuellement difficile de quantifier la sensibilité des paramètres comportementaux et d’établir un lien entre les seuils d’effets comportementaux et les effets aigus et subaigus classiquement mesurés dans les tests en écotoxicologie. Dans le but d’améliorer notre compréhension des effets comportementaux des polluants sur Daphnia magna, nous avons développé un nouveau système de mesure du comportement multi-cuves baptisé «Multi-DaphTrack ». Douze substances toxiques, couvrant une large gamme de modes d’action différents ont été testées dans ce système. Un test standard d’immobilisation ainsi que des analyses de comportement dans le toximètre DaphToxI®, ont également été effectués pour chaque substance afin de comparer les seuilsd’effets comportementaux avec les paramètres classiques d’immobilisation. Les résultats des expositions aux différentes substances ont démontré que notre nouveau système d’exposition multi-cuves permet de détecter des effets comportementaux (i.e.,augmentation de la vitesse de nage) significatifs et précoces pour l’ensemble des substances testées et ce, à des concentrations proches de la CE10(48 h) du test aigu d’immobilisation dès la première heure d’exposition. Des profils comportementaux différents ont été observés selon les substances testées (i.e., intensité, temps de latence et durée de l’effet) mais ceux-ci ne sont pas spécifiques d’un mode d’action particulier. Les résultats obtenus avec le toximètre DaphToxI®ont révélé des profils d’effet similaires (i.e.,augmentation de la vitesse de nage) bien que ce système soit globalement moins sensible par rapport au système «Multi-DaphTrack». Pour conclure, notre nouveau système d’exposition multi-cuves «Multi-DaphTrack» est un outil plus sensible et précoce que letest standard d’immobilisation pour l’évaluation de la toxicité de substances chimiques. L’utilisation du système «Multi-DaphTrack» pourrait donc être envisagée, après quelques améliorations et validation supplémentaires, pour l’évaluation de la qualité des masses d’eauet des effluents. / Swimming behavior is increasingly reported as a sensitive and early indicator of toxicant stress in aquatic organisms. However, it remains unclear how to quantify the sensitivity of swimming behavioral endpoints and how to compare these effect thresholds with standard ecotoxicological endpoints used in risk assessment. To date, the systematic assessment of the sensitivity of swimming behavioral endpoints in daphnia is limited because of the restrained test capacity of existing behavioral analysis systems. Hence, we developed a new behavioral analysis multi-cellsystemnamed “Multi-DaphTrack” with a high throughput testing capacity in order to enhance our understanding of swimming behavioral effects in Daphnia magnaunder chemical exposure. Twelve compounds covering different modes oftoxic action were selected and tested in this new system andin a single-cell commercialized biomonitor (DaphToxI®) and with the acute standard test.Our new multi-cellexposure system detectedsignificant and early swimming behavioral effects (i.e.,increase of the average speed) for most of the tested compounds and this, from the first hour of exposure at concentrations near the EC10(48h). Contrastedbehavioral profiles were observed for average speed (i.e., intensity, time of effect onset, effect duration), but no distinct behavioral profiles could be drawn from the chemical mode of action. Despite less sensitive,the DaphToxI®gave similar trends (i.e.,rapid peak increase) compared to our “Multi-DaphTrack” system. To conclude, behavior analysis using our “Multi-DaphTrack”systemcould be used as an alternative or complement to the current acute standard test for toxicity assessment of chemicals. With some additional improvements and validations, it also could be used forquality assessment of waterbodiesand sewages.