In the European Union an environmental risk assessment is required for the registration of new chemicals, biocides, pesticides and pharmaceuticals. In order to avoid the release of potential hazardous substances, various ecotoxicity tests are performed, including acute and chronic fish tests. As a consequence of the new program of the European Union “Registration, Evaluation and Authorisation of Chemicals” (REACH) the number of animal experiments for environmental risk assessment is expected to increase remarkably within the next years. On the other hand there is a strong societal demand for reducing the number of animal tests by using alternative in vitro models. According to EU directives, investigations using non-human vertebrate embryos are considered pain free in vitro methods and are therefore accepted as alternatives to animal experiments. For the acute fish test, the Danio rerio embryo test (DarT) has been established as a replacement method and included in national regulations at least for waste water (German Waste Water Dues Law). However, no alternatives for chronic fish tests are currently available. The overall goal of this thesis was to work towards such a replacement by extending DarT zu Gene-DarT. Toxicants will initially interact at the molecular level with consequences for physiology, fitness and survival. The analysis of gene expression patterns may unravel elements of these molecular events before any phenotypic changes are visible. The hypothesis of this thesis therefore was that chemical-sensitive genes in embryos exposed in a conventional DarT may indicate toxic impact of substances at sub-acute concentrations and thus enhance the sensitivity of the embryo toxicity test. Furthermore, unlike the conventional DarT-endpoints, gene expression analysis will provide insights into mechanistic processes underlying toxicity. The 3,4-dichloroaniline (3,4-DCA), which is used as a reference compound in the DarT, was selected as model chemical in this thesis. In a first step, differentially expressed genes in embryos exposed to 3,4-DCA were identified by microarray technology and RT-PCR techniques. Six dose-dependent significant differentially expressed genes were identified. These genes were involved in biotransformation pathways (cyp1a, ahr2), stress response (nrf2, maft, ho-1) and cell cycle control (fzr1). Differential expression upon 3,4-DCA exposure was detected below the LOEC (lowest observed effect concentration = 6.2 µM) of survival or developmental disorders of the embryo test (0.78 µM and above). For the validation of stage specific sensitivity, genes were also analysed in post-hatched stages. Extension of exposure to post-hatched stages resulted in a differential expression at lower concentrations as for the embryonic stages, indicating an improved sensitivity due to stage-specific sensitivity or exposure time. To confirm the adaptive function of the 3,4-DCA-sensitive genes, embryonic mRNA abundance was experimentally manipulated by knock down and overexpression. By injection of sense (mRNA) or antisense (siRNA) RNA in one-cell-stages of embryos, the transcript levels of genes were transiently enhanced or repressed in embryos exposed to 3,4-DCA. mRNA injection of the genes cyp1a, ho-1 and nrf2 reduced the number of embryos with 3,4-DCA-induced malformations. In contrast, siRNA injections for the same genes led to an increase in the severity and frequency of developmental disorders. The results clearly indicate the adaptive functions of the investigated genes or their corresponding proteins. This study demonstrates that the analysis of chemical-sensitive gene expression shows the potential to increase the sensitivity of conventional toxicity tests. The analysis of gene expression also provides additional mechanistic information for toxic action, e.g. in the presented study, the involvement of Ah-receptor regulated pathways as an adaptive response. Furthermore, the presented data indicate that functional manipulations, using mRNA and siRNA-injection, are suitable to evaluate the role of differentially expressed genes for toxicity.

Um die Effekte von Umweltchemikalien auf Populationen von Fließgewässerorganismen zu untersuchen, sind an der TU Dresden Fließrinnen entwickelt worden. Die Komplexität wurde bewusst reduziert, um die beobachteten Effekte besser kausal analysieren zu können. Nach einer Erweiterung des Nahrungsnetzes um Grazer (Eintagsfliegen-Larven) und der weiteren Optimierung der Methode zur Bestimmung des Aufwuchses (Periphyton) wurden 2 Experimenten mit einem Pflanzenschutzmittel (Fenoxycarb) und einer Industriechemikalie (Bisphenol A) durchgeführt, um die Effekte dieser Chemikalien auf den Aufwuchs, die Eintagsfliegen-Larven Rhithrogena semicolorata und Seratella ignita, sowie Oligochaeten der Art Lumbriculus variegatus zu erfassen. Die Wirkung von Fenoxycarb in 4 Konzentrationen von 0,05 bis 50 µg L-1 wurde über eine Dauer von 101 Tagen untersucht. Auf die physiko-chemischen Parameter, den Aufwuchs und L. variegatus wurden keine Effekte festgestellt. Bei R. semicolorata wurden Effekte auf das Überleben und die Emergenz beobachtet. Für den Endpunkt "Anzahl der toten Larven in der Fließrinne" wurde eine LC50 von 3,3 µg L-1 bestimmt. Die akute Toxizität (48 h LC50) von Fenoxycarb für S. ignita ist größer als 5 mg L-1. Bei Larven von S. ignita wurden 72 Tage nach Applikation Effekte beobachtet werden, die dem Wirkmechanismus von Fenoxycarb entsprechen. In der höchsten Konzentration zeigten ca. 80 % der emergierten Tiere morphologische Abnormalitäten an 4 Segmenten des Abdomens. Die Exposition erfolgte möglicherweise durch an Aufwuchs bzw. Detritus gebundene Substanz oder deren Metabolite, da die Substanz 19 Tage nach Applikation im Wasser nicht mehr nachweisbar war. Im zweiten Experiment wurde Wirkung von Bisphenol A in 3 Konzentrationen von 5 bis 500 µg L-1 (effektiv 0,24 bis 24,1 µg L-1) über eine Dauer von 103 Tagen untersucht. Auf die physiko-chemischen Parameter wurde kein Einfluss festgestellt. Der Aufwuchs in der höchsten Konzentration erreichte nur die Hälfte des Biomasse-Maximums der Kontrolle. Die EC10 beträgt 1,8 und die EC50 21,7 µg L-1 (effektiv). Das Populationswachstum von Lumbriculus variegatus war in den beiden höchsten Konzentrationen im Vergleich zur Kontrolle um ca. 50 % reduziert. Die akute Toxizität (LC50 96 h) für L. variegatus beträgt 5,3 mg L-1. Für das Populationswachstum in den Fließrinnen wurde eine EC10 von 0,11 µg L-1 (effektiv) ermittelt, die um den Faktor 48184 unter der akuten Toxizität liegt. - Anlage: Rohdaten (0,53 MB)- Nutzung: Referat Informationsvermittlung der SLUB"

Triclosan (TCS) is a personal care product with many fields of application and is of public interest for several years now. Monitoring studies showed that TCS is a ubiquitous chemical in the aquatic environment. Aquatic organisms are exposed to TCS in a broad range of concentrations, from ng L-1 up to lower μg L-1. TCS has a bactericidal effect for various types of gram-positive and gram negative bacteria. TCS targets a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase (Schweizer, 2001). Therefore the terminal reaction in the fatty acid elongation cycle is inhibited (Levy et al., 1999). Although effects on non-target organisms are reported, the Mode of Action (MoA) of TCS is not well examined for those organisms. The aim of this PhD thesis was to investigate effects of TCS on non-target autotrophic organisms at different levels of biological complexity in the aquatic environment. In this thesis microalgae have been found to be very sensitive to TCS. In some cases even higher sensitivities than in bacteria were observed, which is in accordance with published effect data (Harada et al., 2008; Orvos et al., 2002). Similarly to bacteria, high species sensitivity differences were observed for algae (Franz et al., 2008). In bacteria these sensitivity differences can be ascribed to several resistance mechanisms reported in Schweizer (2001). These findings lead to the question about the reasons for species sensitivity differences in algae. A mesocosm study was performed to detect effects of TCS across levels of biological organization and to investigate the impact of sensitivity differences on complex aquatic communities. For that purpose, structural and functional effects parameters were observed.

In the European Union an environmental risk assessment is required for the registration of new chemicals, biocides, pesticides and pharmaceuticals. In order to avoid the release of potential hazardous substances, various ecotoxicity tests are performed, including acute and chronic fish tests. As a consequence of the new program of the European Union “Registration, Evaluation and Authorisation of Chemicals” (REACH) the number of animal experiments for environmental risk assessment is expected to increase remarkably within the next years. On the other hand there is a strong societal demand for reducing the number of animal tests by using alternative in vitro models. According to EU directives, investigations using non-human vertebrate embryos are considered pain free in vitro methods and are therefore accepted as alternatives to animal experiments. For the acute fish test, the Danio rerio embryo test (DarT) has been established as a replacement method and included in national regulations at least for waste water (German Waste Water Dues Law). However, no alternatives for chronic fish tests are currently available. The overall goal of this thesis was to work towards such a replacement by extending DarT zu Gene-DarT. Toxicants will initially interact at the molecular level with consequences for physiology, fitness and survival. The analysis of gene expression patterns may unravel elements of these molecular events before any phenotypic changes are visible. The hypothesis of this thesis therefore was that chemical-sensitive genes in embryos exposed in a conventional DarT may indicate toxic impact of substances at sub-acute concentrations and thus enhance the sensitivity of the embryo toxicity test. Furthermore, unlike the conventional DarT-endpoints, gene expression analysis will provide insights into mechanistic processes underlying toxicity. The 3,4-dichloroaniline (3,4-DCA), which is used as a reference compound in the DarT, was selected as model chemical in this thesis. In a first step, differentially expressed genes in embryos exposed to 3,4-DCA were identified by microarray technology and RT-PCR techniques. Six dose-dependent significant differentially expressed genes were identified. These genes were involved in biotransformation pathways (cyp1a, ahr2), stress response (nrf2, maft, ho-1) and cell cycle control (fzr1). Differential expression upon 3,4-DCA exposure was detected below the LOEC (lowest observed effect concentration = 6.2 µM) of survival or developmental disorders of the embryo test (0.78 µM and above). For the validation of stage specific sensitivity, genes were also analysed in post-hatched stages. Extension of exposure to post-hatched stages resulted in a differential expression at lower concentrations as for the embryonic stages, indicating an improved sensitivity due to stage-specific sensitivity or exposure time. To confirm the adaptive function of the 3,4-DCA-sensitive genes, embryonic mRNA abundance was experimentally manipulated by knock down and overexpression. By injection of sense (mRNA) or antisense (siRNA) RNA in one-cell-stages of embryos, the transcript levels of genes were transiently enhanced or repressed in embryos exposed to 3,4-DCA. mRNA injection of the genes cyp1a, ho-1 and nrf2 reduced the number of embryos with 3,4-DCA-induced malformations. In contrast, siRNA injections for the same genes led to an increase in the severity and frequency of developmental disorders. The results clearly indicate the adaptive functions of the investigated genes or their corresponding proteins. This study demonstrates that the analysis of chemical-sensitive gene expression shows the potential to increase the sensitivity of conventional toxicity tests. The analysis of gene expression also provides additional mechanistic information for toxic action, e.g. in the presented study, the involvement of Ah-receptor regulated pathways as an adaptive response. Furthermore, the presented data indicate that functional manipulations, using mRNA and siRNA-injection, are suitable to evaluate the role of differentially expressed genes for toxicity.

Modellierung, Analyse und Bewertung des chemischen Gewässerzustandes in Flussgebieten Der Schwerpunkt der chemischen Belastungen von Oberflächengewässern durch Abwasseremissionen verlagert sich in jüngerer Zeit, durch die Ertüchtigung der Abwasserreinigungsanlagen, von biologisch leicht abbaubaren organischen Substanzen hin zu Mikroverunreinigungen. Die Expositionsanalyse von Gewässersystemen gegen xenobiotische Substanzen mit dem Ziel einer Steuerung der Belastungen rückt immer mehr in den Vordergrund des Interesses (EG 2000, EG 2001). Am Beispiel des nordrhein-westfälischen Rheineinzugsgebiets wird in der vorliegenden Arbeit eine Analyse und Bewertung des chemischen Gewässerzustandes durch georeferenzierte Modellierung von Flussgebieten durchgeführt. Eingesetzt wird das Modellsystem GREAT-ER (Georeferenced Regional Exposure Assessment Tool for European Rivers). Der methodische Teil der Arbeit beschreibt die Kalibrierung des Modellsystems für das Einzugsgebiet des Rheins in Nordrhein-Westfalen. Weiter werden die für die Modellierung notwendigen Eingangsparameter verschiedener beispielhafter Substanzen aus verschiedenen Quellen hergeleitet. In den Anwendungsstudien werden Simulationsergebnisse für die Stoffe Bor, EDTA, HHCB, und Diclofenac sowie Diuron und Ammoniumstickstoff dargestellt und mit Messwerten der Gewässerkonzentrationen verglichen. Die Emissionsmengen für Bor und EDTA aus dem Gebrauch im Haushalt sind gut quantifizierbar. Beide Substanzen verhalten sich in den Gewässern konservativ und konnten deshalb für die Kalibrierung des Modellsystems genutzt werden. HHCB und Diclofenac sind Substanzen, die typischerweise über Haushaltsabwässer in die Gewässer gelangen, aus diesen jedoch gut eliminiert werden. Das Pestizid Diuron gelangt mit dem Oberflächenabfluss von versiegelten Flächen in das Abwasser und die Gewässer. Die Elimination aus den Gewässern ist gering. In der Arbeit wird eine Quantifizierung der Emissionsmengen auf Basis der versiegelten Flächen durchgeführt und damit eine räumliche Zuordnung der Eintragsmengen erreicht. Mit Ammoniumstickstoff wird schließlich die Gewässerexposition einer Substanz berechnet, die auch über diffuse Quellen in die Gewässer gelangt. Grundannahme ist hier, dass die Frachten aus Punktquellen die diffusen Einträge überlagern. In Abhängigkeit von der jeweiligen Substanz und Lage der Messstellen zeigen die Ergebnisse sowohl gute Übereinstimmung als auch stellenweise große Abweichungen zu den gemessenen Substanzkonzentrationen in den Gewässern. Für die auftretenden Abweichungen ergeben sich Erklärungsansätze, aber auch weiterer Untersuchungsbedarf wird deutlich. Die Ergebnisse der Arbeit belegen, dass das mit GREAT-ER entwickelte Werkzeug zur georeferenzierten Modellierung von Substanzkonzentrationen in Gewässern auf dem Gebiet der zeitlichen und räumlichen Analyse von realen Messwerten und im Rahmen eines Immissions- und Belastungsmanagements einsetzbar ist. Es können aus den Umgebungsparametern begründete Hypothesen zu lokalen Substanzkonzentrationen in Gewässern entwickelt werden, deren Informationsgehalt gegenüber Messung und generischer Modellierung höher ist.

Modelling and evaluating the aquatic fate of detergents - Abstract Within this thesis an environmental assessment and evaluation method for analysing aquatic ecotoxicological impacts of household laundry is developed. The methodology allows comparative assessments of different product alternatives, washing habits, and wastewater treatment techniques in order to identify their relevance with respect to waterborne discharges. Elements from both analytical tools Life Cycle Assessment (LCA) and Environmental Risk Assessment of chemicals (ERA) are combined in this methodology. The core consists of the Geography-referenced Regional Exposure Assessment Tool for European Rivers (GREAT-ER), which calculates concentrations of `down-the-drain' chemicals in surface waters due to point releases. In order to simulate the aquatic fate of detergents, a new GREAT-ER emission model is developed, called GREAT-ER product mode, which calculates calculates concentration increases of detergent ingredients in surface waters based on product formulations and assumptions concerning washing habits. Two evaluation methods, the Critical Length (CL) and the Product Risk Ratio (PRRx), are defined for evaluating the results. CL is the sum of mean concentration increases, divided by substance-specific no effect concentrations (NECs), over all river stretches and all ingredients weighted by the lengths of the stretches. PRRx is the (percentual) number of river stretches in a region, in which the x-percentiles of the predicted concentration increases of at least one ingredient exceed a substance-specific NEC. The emission model requires input data that can be derived from the functional unit of an LCA, which allows an assessment of other impact categories by using any existing LCA method. The methodology is applied to a case study which is based on scenarios given in the comprehensive product assessment `Washing and washing agents' (`Produktlinienanalyse', PLA). In order to apply the GREAT-ER product mode, the Rur river basin in Western North-Rhine Westphalia is chosen as study area. The catchment integration includes the development of a simple hydrological model that combines a nonlinear regression analysis with a local refinement procedure. The quality of the integration of the Rur catchment data is analysed by a comparison of monitoring data and predicted concentrations of detergent and cleaning agent ingredients using actual consumption data of the two years 1993 and 2000. The product mode results show that use habits have a larger influence on the results than product formulations. However, the largest influence is caused by varying wastewater treatment techniques. Boron and the surfactants are the most relevant detergent ingredients. Furthermore, using different detergents for white and coloured laundry lowers the predicted emissions significantly. Based on this methodology, sustainable development indicators (SDIs) for describing the aquatic aspects of household laundry are defined. CL is proposed as pressure indicator and PRRx as state indicator for describing aquatic aspects of the sustainability of household laundry in a region. Different regions can be compared by normalising the CL by the region's population and expressing the PRRx as a percentage of stretches in a region. Annually evaluating regional CLs and PRRxs allows the assessment whether a region is moving towards a more sustainable state. Concluding, the new method analyses and evaluates the environmental fate of detergents discharged after use via the wastewater pathway. It provides information not obtainable by other existing methods, which has been made possible due to the focussing on a specific application, for which the method is developed. Its application in the context of sustainable development offers a means to evaluate environmental implications of this important human activity.