Pharmaka werden nach ihrer Einnahme bzw. Verabreichung über verschiedene Pfade in die Umwelt eingetragen. Obwohl Arzneimittel zu den toxikologisch best-untersuchten und -charakterisierten Stoffen gehören, ist ihre Wirkung auf die Umwelt und die darin lebenden Organismen weit weniger gut untersucht. Wenn in der Literatur Daten zur Ökotoxizität vorhanden sind, so beziehen sich diese meist nur auf die Wirkung von Einzelstoffen. In der Umwelt sind die Organismen jedoch gegenüber Mischungen exponiert. Aufgrund der geschilderten Problematik wurden eine Reihe von Arzneimitteln unterschiedlicher Indikationsgruppen einzeln und in Kombination mit dem Embryotest mit dem Zebrabärbling (Danio rerio, DarT) untersucht. Dieses Testsystem wurde durch Schulte & Nagel (1994) als Alternativmethode zum akuten Fischtest nach OECD 203 entwickelt und bietet den Vorteil neben letalen auch eine Reihe von subletalen Endpunkten erfassen zu können. Es handelt sich zudem nach dem deutschen Tierschutzgesetz nicht um einen Tierversuch. Die generelle Vergleichbarkeit der ermittelten Werte mit Daten aus akuten Fischtests nach OECD 203 sowie die Anwendbarkeit für verschiedenste Fragestellungen konnten in einer Reihe von Studien gezeigt werden (Nagel, 2002). Für die hier vorgestellten Untersuchungen wurden zunächst 32 Pharmaka und drei Pflanzenschutzmittel als Einzelstoffe mit dem DarT untersucht. Basierend auf den Ergebnissen der Einzelstofftests wurden Mischungen sowohl aus Substanzen mit ähnlichen als auch unähnlichen Wirkmechanismen getestet. Es zeigte sich, dass unabhängig vom Wirkmechanismus die Mischungstoxizität durch das Konzept der Konzentrationsadditivität gut vorhergesagt wurde, während das Konzept der Unabhängigen Wirkung die Mischungstoxizität unterschätzte. Ebenfalls konnte gezeigt werden, dass die Kombination der Stoffe auf Basis der NOEC, die im DarT anhand der Herzschlagfrequenz nach 48 Stunden ermittelt wird, zu deutlichen Mischungseffekten führt.

L'uranium est un radioélément naturel, généralement retrouvé à l'état de traces, mais dont la concentration peut être significativement augmentée à proximité de certaines installations du cycle du combustible nucléaire ou de zones d'agriculture intensive. En raison de son association dans les minerais avec différents éléments traces métalliques tels que le cadmium, l'U est la plupart du temps présent en mélange avec d'autres contaminants dans l'environnement. L'étude de leurs effets combinés est indispensable afin de mieux appréhender le risque engendré par les contaminations métalliques multiples pour les écosystèmes. L'évaluation de la toxicité des mélanges et l'identification des interactions synergiques ou antagonistes sont généralement réalisées sur la base de modèles d'additivité de référence intégrés à des approches descriptives, purement statistiques et sans réelles considérations biologiques. Plus récemment, des modèles mécanistes ont été proposés en alternative afin de mieux rendre compte de la dynamique des processus biologiques et des mécanismes de toxicité des contaminants. Ceux-ci n'ont cependant été mis en pratique que dans un nombre limité de cas d'étude de la toxicité des mélanges. L'objectif de ce projet de thèse a été d'étudier les effets combinés chroniques de l'U et du Cd sur les traits de vie du nématode Caenorhabditis elegans, en utilisant en parallèle une approche descriptive (MixTox) et une approche mécaniste (DEBtox). Pour cela, une exposition des nématodes à différentes concentrations d'U et de Cd, seuls ou en mélange, a été réalisée durant onze jours. Une interaction antagoniste importante entre l'U et le Cd a été identifiée pour les critères d'effet de croissance et de reproduction, à partir des deux approches. L'étude du transfert de l'U et du Cd du milieu vers la nourriture ainsi que de leur bioaccumulation par C. elegans nous a permis de montrer que la coprésence d'U diminuait la fraction de Cd disponible pour l'exposition des nématodes. Afin de déceler la présence d'une éventuelle interaction au niveau des organismes, durant les phases toxicocinétique ou toxicodynamique, les données d'effet ont été réanalysées à partir des concentrations d'U et de Cd dans la nourriture, supposées plus proches des concentrations disponibles pour C. elegans. Des effets combinés globalement additifs, sans interaction notable, ont été mis en évidence pour l'U et le Cd même si des conclusions légèrement contrastées ont été obtenues à partir des approches descriptives et mécanistes. La présente étude permet d'illustrer la complexité de l'étude des effets des mélanges et de l'identification des interactions entre les substances. Malgré quelques difficultés d'application, l'approche mécaniste DEBtox semble particulièrement prometteuse pour décrire la toxicité de mélanges au cours du temps et tester d'éventuels mécanismes d'interaction. À terme, le perfectionnement des outils d'analyse des effets combinés des contaminants devrait permettre une meilleure prise en compte de la problématique des mélanges dans les démarches d'évaluation des risques. / Uranium is a natural radioactive trace element for which elevated concentrations can be found in the vicinity of some nuclear fuel cycle facilities or of intensive farming areas. Due its co-occurrence with different trace metals, such as cadmium, in geological ores, U is generally found associated with other contaminants in the environment. The study of their combined effects on ecosystems is of interest to better characterize such multi-metallic polluted sites. The mixture toxicity assessment and the identification of synergistic or antagonistic interaction are generally performed on the basis of additive reference models integrated to descriptive and purely statistical approaches with no real biological basis. Recently, mechanistic models were proposed to better account for the dynamics of biological and toxicological processes. However, such models have only been put into practice in a few number of mixture toxicity case studies. The aim of this PhD project was to assess the chronic U/Cd combined toxicity on the life history traits of the nematode Caenorhabditis elegans using both a descriptive (MixTox) and a mechanistic (DEBtox) approach. To do so, nematodes were exposed during eleven days to different U and Cd concentrations, alone or in mixture. A strong antagonistic interaction between U and Cd was identified for length increase and brood size endpoints on the basis of both approaches. From the study of the U and Cd media-to-food transfer and of the U and Cd bioaccumulation by C. elegans, we showed that the co-presence of U reduced the available Cd fraction for nematodes. To identify a possible interaction at organism level, occurring during the toxicokinetic or toxicodynamic steps, data were re-analyzed on the basis of U/Cd concentrations in food, assumed to be more closely related to available concentrations for C. elegans. Overall additive effects, without interaction, were identified between U and Cd, even if slightly contrasted conclusions were obtained on the basis of the descriptive and mechanistic approaches. The present study underlines the complexity of studying mixture toxicity and identifying chemical interactions. Despite some application problems, the mechanistic approach DEBtox is particularly promising to describe the toxicity of chemical mixtures over time and to test hypothetical interaction mechanisms. In the future, the improvement of tools to analyze the combined toxicity of contaminants would allow to better address the issue of mixtures in ecotoxicological risk assessment processes.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Resolução CONAMA N 430/2011 exige a utilização de dois bioensaios (dois níveis tróficos) para avaliação ecotoxicológica de efluentes, mas a seleção ao acaso de bioensaios pode permitir lançamentos tóxicos. A sensibilidade dos bioindicadores irá depender da substância tóxica avaliada. Assim, baterias de bioensaios sensíveis devem ser estabelecidas às classes de contaminantes. Na literatura não há estudos que indiquem uma bateria de bioensaios ecotoxicológicos sensíveis para avaliação de efluentes contendo principalmente metais. Esse trabalho teve como objetivo selecionar uma bateria de bioensaios ecotoxicológicos que conjuntamente detectem toxicidade ao maior número de metais isolados e em misturas e que sejam realizados no menor tempo indicado pelas normas de padronização. Foram avaliadas as sensibilidades de seis bioensaios, incluindo três níveis tróficos (produtores, algas: Pseudokirchneriella subcapitata e Chlorella vulgaris; consumidores primários, cladóceros: Daphnia similis e Ceriodaphnia dubia; consumidores secundários, peixes: Poecilia reticulata e Danio rerio), a 10 espécies metálicas individuais (Ag+, Cd2+, Cu+, Cu2+, Cr3+, Cr6+, Pb2+, Ni2+, Zn2+ e Hg2+) e a efluentes reais (siderúrgicos) e simulados em laboratoriais (baseado nos limites máximos permitidos para descarte). Os bioensaios com peixes foram os menos sensíveis, D. rerio não detectou toxicidade em nenhum dos efluentes testados. P. subcapitata foi um bom bioindicador de toxicidade de Cr3+ e D. similis foi o organismo mais sensível a Hg2+. O uso combinado do bioensaio crônico de 72h com C. vulgaris e do bioensaio agudo de 48h com C. dubia garantiu a detecção das menores concentrações dos metais tanto individualmente quanto em efluentes reais e simulados. Apesar de P. subcapitata ser um bom bioindicador da toxicidade de Cr3+, a interação dos metais em misturas tornou C. vulgaris igualmente sensível. Da mesma forma, apesar de D. similis ter sido mais sensível ao Hg2+, o efeito da toxicidade dos efluentes com maiores teores de Hg2+ foi detectado por C. dubia / CONAMA Resolution No. 430/2011 requires the use of at least two bioassays (two trophic levels) for ecotoxicological evaluation of effluents. The use of bioassay batteries is necessary to evaluate toxic effects at various biological levels. The selection of bioassays without prior testing and determination of the most sensitive/suitable groups for each impact may allow the discharge of effluents that pose a threat to the environment. In the present study we tested and selected a battery of sensitive ecotoxicological bioassays for detecting toxic effects of metals. The sensitivities of six organisms were evaluated (algae Pseudokirchneriella supcapitata and Chlorella vulgaris, Cladocera Daphnia similis and Ceriodaphnia dubia, and fish Poecilia reticulata and Danio rerio) after exposure to 10 individual metal species deemed toxic to the aquatic environment (Ag+, Cd2+, Cu+, Cu2+, Cr3+, Cr6+, Pb2+, Ni2+, Zn2+, and Hg2+) and to real (steel-mill) and laboratory simulated effluents (based on maximum allowable cocentration of metal in effluent for disposal). In the bioassays, fish were the least sensitive; D. rerio showed no sensitivity to any of the effluents tested. P. subcapitata was a good bioindicator of Cr3+ toxicity, and D. similis was the most sensitive organism to Hg2+; but the toxic effect of effluents with higher levels of Hg2+ was better detected by C. dubia. Although P. subcapitata be a good bioindicator of toxicity of Cr3+, the interaction of metals in mixtures became equally sensitive C. vulgaris. The most sensitive battery of bioassays to detect low concentrations of dissolved metals in effluents was the 72-h chronic test with C. vulgaris and the 48-h acute test with C. dubia. The identification of these responses is important for countries that are starting to ecotoxicological assessment of their effluents, as well as Brazil, and serves as a reference for the environmental agencies in the management and control of effluents. The limits of acceptability of toxicity should be based on batteries of tests with sensitive organisms

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Resolução CONAMA N 430/2011 exige a utilização de dois bioensaios (dois níveis tróficos) para avaliação ecotoxicológica de efluentes, mas a seleção ao acaso de bioensaios pode permitir lançamentos tóxicos. A sensibilidade dos bioindicadores irá depender da substância tóxica avaliada. Assim, baterias de bioensaios sensíveis devem ser estabelecidas às classes de contaminantes. Na literatura não há estudos que indiquem uma bateria de bioensaios ecotoxicológicos sensíveis para avaliação de efluentes contendo principalmente metais. Esse trabalho teve como objetivo selecionar uma bateria de bioensaios ecotoxicológicos que conjuntamente detectem toxicidade ao maior número de metais isolados e em misturas e que sejam realizados no menor tempo indicado pelas normas de padronização. Foram avaliadas as sensibilidades de seis bioensaios, incluindo três níveis tróficos (produtores, algas: Pseudokirchneriella subcapitata e Chlorella vulgaris; consumidores primários, cladóceros: Daphnia similis e Ceriodaphnia dubia; consumidores secundários, peixes: Poecilia reticulata e Danio rerio), a 10 espécies metálicas individuais (Ag+, Cd2+, Cu+, Cu2+, Cr3+, Cr6+, Pb2+, Ni2+, Zn2+ e Hg2+) e a efluentes reais (siderúrgicos) e simulados em laboratoriais (baseado nos limites máximos permitidos para descarte). Os bioensaios com peixes foram os menos sensíveis, D. rerio não detectou toxicidade em nenhum dos efluentes testados. P. subcapitata foi um bom bioindicador de toxicidade de Cr3+ e D. similis foi o organismo mais sensível a Hg2+. O uso combinado do bioensaio crônico de 72h com C. vulgaris e do bioensaio agudo de 48h com C. dubia garantiu a detecção das menores concentrações dos metais tanto individualmente quanto em efluentes reais e simulados. Apesar de P. subcapitata ser um bom bioindicador da toxicidade de Cr3+, a interação dos metais em misturas tornou C. vulgaris igualmente sensível. Da mesma forma, apesar de D. similis ter sido mais sensível ao Hg2+, o efeito da toxicidade dos efluentes com maiores teores de Hg2+ foi detectado por C. dubia / CONAMA Resolution No. 430/2011 requires the use of at least two bioassays (two trophic levels) for ecotoxicological evaluation of effluents. The use of bioassay batteries is necessary to evaluate toxic effects at various biological levels. The selection of bioassays without prior testing and determination of the most sensitive/suitable groups for each impact may allow the discharge of effluents that pose a threat to the environment. In the present study we tested and selected a battery of sensitive ecotoxicological bioassays for detecting toxic effects of metals. The sensitivities of six organisms were evaluated (algae Pseudokirchneriella supcapitata and Chlorella vulgaris, Cladocera Daphnia similis and Ceriodaphnia dubia, and fish Poecilia reticulata and Danio rerio) after exposure to 10 individual metal species deemed toxic to the aquatic environment (Ag+, Cd2+, Cu+, Cu2+, Cr3+, Cr6+, Pb2+, Ni2+, Zn2+, and Hg2+) and to real (steel-mill) and laboratory simulated effluents (based on maximum allowable cocentration of metal in effluent for disposal). In the bioassays, fish were the least sensitive; D. rerio showed no sensitivity to any of the effluents tested. P. subcapitata was a good bioindicator of Cr3+ toxicity, and D. similis was the most sensitive organism to Hg2+; but the toxic effect of effluents with higher levels of Hg2+ was better detected by C. dubia. Although P. subcapitata be a good bioindicator of toxicity of Cr3+, the interaction of metals in mixtures became equally sensitive C. vulgaris. The most sensitive battery of bioassays to detect low concentrations of dissolved metals in effluents was the 72-h chronic test with C. vulgaris and the 48-h acute test with C. dubia. The identification of these responses is important for countries that are starting to ecotoxicological assessment of their effluents, as well as Brazil, and serves as a reference for the environmental agencies in the management and control of effluents. The limits of acceptability of toxicity should be based on batteries of tests with sensitive organisms

A literature review of metal mixture interaction analyses identified that there was not a consistent method to determine the impact of metal mixtures on an aquatic organism. The review also revealed that a majority of the research on mixtures made use of water concentrations only. Therefore research was conducted to determine the relationship between exposure, bioaccumulation and chronic effects of the four elements As, Co, Cr and Mn individually. Mechanistically based saturation models of bioaccumulation and toxicity were determined for the benthic invertebrate Hyalella azteca, from which lethal water concentrations and body concentrations were also determined. These models were then combined with those previously done for the metals Cd, Cu, Ni, Pb, Tl and Zn to model the impact of 10 metal mixtures on bioaccumulation in short term (1-week) exposures and on bioaccumulation and toxicity in chronic (4-week) exposures at “equi-toxic” concentrations. Interactions between the metals were identified in which; Cd, Co and Ni bioaccumulations were significantly inhibited, Tl and Zn bioaccumulations were marginally inhibited, there was no impact on Cr, Cu or Mn bioaccumulation, and both As and Pb bioaccumulation were enhanced by some mixtures of metals. It was determined that strict competitive inhibition may be a plausible mechanism of interaction affecting Co, Cd and Ni bioaccumulation but not for any of the other metals. However, it is possible that other interactions such as non-competitive or anti-competitive inhibition may have been responsible. A metal effects addition model (MEAM) was developed for Hyalella azteca based on both the bioaccumulation (body concentrations) to effects and the exposure (water concentration) to effects relationships developed from the single metal only studies The MEAM was used to predict the impact of metal mixture exposures on mortality. Toxicity was under-estimated when based on measured water or body concentrations, however, its best prediction was based on body concentrations. The MEAM, when based on measured body concentrations, takes bioavailability into account, which is important since the chemical characteristics of water can greatly alter the bioavailability and therefore toxicity of metals. The MEAM was compared to the traditional Concentration Addition Model (CAM), which calculates toxic units based on water concentrations and LC50s or body concentrations and LBC50s. The CAM overestimated toxicity, but had its best prediction when based on water concentrations. Over all, the best fit to observed mortality was the prediction by the MEAM, based on body concentrations. The measurement of bioaccumulated metals and the use of the MEAM could be important in field site assessments since it takes into account changes in bioavailability due to different site water chemistries whereas the traditional CAM based on water concentration does not.

A literature review of metal mixture interaction analyses identified that there was not a consistent method to determine the impact of metal mixtures on an aquatic organism. The review also revealed that a majority of the research on mixtures made use of water concentrations only. Therefore research was conducted to determine the relationship between exposure, bioaccumulation and chronic effects of the four elements As, Co, Cr and Mn individually. Mechanistically based saturation models of bioaccumulation and toxicity were determined for the benthic invertebrate Hyalella azteca, from which lethal water concentrations and body concentrations were also determined. These models were then combined with those previously done for the metals Cd, Cu, Ni, Pb, Tl and Zn to model the impact of 10 metal mixtures on bioaccumulation in short term (1-week) exposures and on bioaccumulation and toxicity in chronic (4-week) exposures at “equi-toxic” concentrations. Interactions between the metals were identified in which; Cd, Co and Ni bioaccumulations were significantly inhibited, Tl and Zn bioaccumulations were marginally inhibited, there was no impact on Cr, Cu or Mn bioaccumulation, and both As and Pb bioaccumulation were enhanced by some mixtures of metals. It was determined that strict competitive inhibition may be a plausible mechanism of interaction affecting Co, Cd and Ni bioaccumulation but not for any of the other metals. However, it is possible that other interactions such as non-competitive or anti-competitive inhibition may have been responsible. A metal effects addition model (MEAM) was developed for Hyalella azteca based on both the bioaccumulation (body concentrations) to effects and the exposure (water concentration) to effects relationships developed from the single metal only studies The MEAM was used to predict the impact of metal mixture exposures on mortality. Toxicity was under-estimated when based on measured water or body concentrations, however, its best prediction was based on body concentrations. The MEAM, when based on measured body concentrations, takes bioavailability into account, which is important since the chemical characteristics of water can greatly alter the bioavailability and therefore toxicity of metals. The MEAM was compared to the traditional Concentration Addition Model (CAM), which calculates toxic units based on water concentrations and LC50s or body concentrations and LBC50s. The CAM overestimated toxicity, but had its best prediction when based on water concentrations. Over all, the best fit to observed mortality was the prediction by the MEAM, based on body concentrations. The measurement of bioaccumulated metals and the use of the MEAM could be important in field site assessments since it takes into account changes in bioavailability due to different site water chemistries whereas the traditional CAM based on water concentration does not.

Pharmaka werden nach ihrer Einnahme bzw. Verabreichung über verschiedene Pfade in die Umwelt eingetragen. Obwohl Arzneimittel zu den toxikologisch best-untersuchten und -charakterisierten Stoffen gehören, ist ihre Wirkung auf die Umwelt und die darin lebenden Organismen weit weniger gut untersucht. Wenn in der Literatur Daten zur Ökotoxizität vorhanden sind, so beziehen sich diese meist nur auf die Wirkung von Einzelstoffen. In der Umwelt sind die Organismen jedoch gegenüber Mischungen exponiert. Aufgrund der geschilderten Problematik wurden eine Reihe von Arzneimitteln unterschiedlicher Indikationsgruppen einzeln und in Kombination mit dem Embryotest mit dem Zebrabärbling (Danio rerio, DarT) untersucht. Dieses Testsystem wurde durch Schulte & Nagel (1994) als Alternativmethode zum akuten Fischtest nach OECD 203 entwickelt und bietet den Vorteil neben letalen auch eine Reihe von subletalen Endpunkten erfassen zu können. Es handelt sich zudem nach dem deutschen Tierschutzgesetz nicht um einen Tierversuch. Die generelle Vergleichbarkeit der ermittelten Werte mit Daten aus akuten Fischtests nach OECD 203 sowie die Anwendbarkeit für verschiedenste Fragestellungen konnten in einer Reihe von Studien gezeigt werden (Nagel, 2002). Für die hier vorgestellten Untersuchungen wurden zunächst 32 Pharmaka und drei Pflanzenschutzmittel als Einzelstoffe mit dem DarT untersucht. Basierend auf den Ergebnissen der Einzelstofftests wurden Mischungen sowohl aus Substanzen mit ähnlichen als auch unähnlichen Wirkmechanismen getestet. Es zeigte sich, dass unabhängig vom Wirkmechanismus die Mischungstoxizität durch das Konzept der Konzentrationsadditivität gut vorhergesagt wurde, während das Konzept der Unabhängigen Wirkung die Mischungstoxizität unterschätzte. Ebenfalls konnte gezeigt werden, dass die Kombination der Stoffe auf Basis der NOEC, die im DarT anhand der Herzschlagfrequenz nach 48 Stunden ermittelt wird, zu deutlichen Mischungseffekten führt.