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Abstract

The present study was conducted as part of the research project "Tox-Box: securing drops of life - an enhanced health-related approach for risk assessment of drinking water in Germany”, funded by the Federal Ministry of Education and Research (02WRS1282G). The main fields of research within this project were endocrine disruption, genotoxicity, germ cell toxicity and neurotoxicity. The central task was to provide a compilation of methods to gain information about emerging pollutants to rank these in the health-related indicator value (HRIV) supported by the Federal Environmental Agency. The thesis, embedded in the field of neurotoxicity, aims at developing and validating a test battery for neurotoxicity, based on zebrafish embryo within scope of the 3R principle. The test systems comprised: (1) an extended embryo toxicity test (FET) to assess the general toxicity of substances and sediment extracts as well as to discriminate between neurotoxic and general physiological effects, (2) the acetylcholinesterase activity, (3) the lateral line system, (4) the olfactory epithelium and (5) the retina of zebrafish embryos. The methods were evaluated by screening test with various substances, namely amidotrizoic acid, caffeine, cypermethrin, 2,4-dichlorophenol, 2,4-dinitrotoluene, dichlorvos, 4-nonylphenol, paraoxon-methyl, perfluorooctanoic acid and perfluorooctane sulfonic acid. The extended embryo toxicity test proved useful to detect physiopathological effects and led to the determination of the EC10 value, which was used in subsequent neurotoxicity test to exclude side effects by physiological influences. The acetylcholinesterase test detects both, inhibition (dichlorvos / paraoxon) and activation (cypermethrin) and can be used as an in vivo and in vitro assay, as well as for pure substances and sediment extracts. A few substances were positive in one method exclusively, either in vivo or in vitro. Three out of nine substances triggered the test system at all, but, far below the EC10, constituting a high sensitivity and high specificity. The neuromast assay proved to be more sensitive than most comparable studies. Observing anterior and posterior neuromasts separately, led to a reduction of false-negative results. Additionally, 80 % of the substances showed effects at the EC10, which documents a high sensitivity, but limited specificity. Furthermore, being affected by endocrine disruptors (nonylphenol, perfluorooctansulfonic acid) as well, this methods also covers effects outside neurotoxicity. Specific olfactory antibodies (Golf , ANO2-, GAP43-Antibody) have been detected in the olfactory system. Anti-Golf was detected in the cilia of ciliary receptor neurons, leading to the use of Golf as a marker in the olfactory epithelium. The combination of wholemount staining and cryosectioning led to a sufficient structure resolution. Since only the positive control zinc sulfate and none of the standard substances had effects in the olfactory epithelium, the use in a test battery is not yet given. For the presentation of histopathological effects in the retina, a classic hematoxylin / eosin staining of paraffin sections has proved useful. However, effects in layer arrangement could be detected for any of the substances tested at the EC10. Therefore, the retina is quite insensitive for neurotoxicity testing in the range of EC10. In summary, the combination of embryo toxicity testing, the acetylcholinesterase assay (in vivo and in vitro) and the neuromast assay, seems suitable for a neurotoxicity test battery.