Toxicity and teratogenesis in zebrafish embryos (Danio rerio)

The present thesis gives an overview about the potentials zebrafish embryos can be used for in the area of ecotoxicology. The first chapter summarizes the outcome of the ZFET (Zebrafish Embryo Toxicity Test) OECD validation study, an international attempt for the standardization and development of an embryo toxicity test as an (animal) alternative test to the acute (adult) fish test which is a mandatory component of chemical registration worldwide. The overall reproducibility of the ZFET within all participating laboratories was acceptable and the comparison of the fish embryo LC50 data calculated in the validation study with available acute adult fish LC50 data revealed good correlation. Hence, the fish embryo test is a reliable alternative method which is able to replace conventional acute (adult) fish toxicity testing. The following chapters deal with more specific questions on toxicity and teratogenicity. Recently, zebrafish embryos have been shown to be a useful model for the detection of direct acting teratogens. Therefore, chapter II investigated the capability of zebrafish embryos for bioactivation (via CYP P450) of various proteratogenic substances. Apart from one substance, which mainly produced lethal effects, all proteratogens were teratogenic in zebrafish embryos. The test compounds revealed characteristic patterns of fingerprint endpoints. Chapter III investigated detailed both toxic and teratogenic effects of coumarin and warfarin, which are intensively metabolized in animals and humans. Both chemicals produced teratogenic and lethal effects in zebrafish embryos. The comparison of the ratios between the embryo effect concentrations and human therapeutic plasma concentrations revealed a distinct teratogenic potential of warfarin, as well as the equivocal status of coumarin. Since zebrafish embryos are able to (bio)activate proteratogens and also show a promising correlation to humans, in chapter IV very specific teratogenic endpoints were assessed. A dithiocarbamate pesticide (disulfiram) and a hydrazide (acetic acid hydrazide) were selected for the assessment of cartilage and bone teratogenesis in the head of six day old zebrafish larvae. Cartilages of the neurocranium proved to be more stable than cartilages of the pharyngeal skeleton, whereas bones proved more susceptible than cartilages. In chapter V, cartilage and bone malformations after exposure to additional hydrazides and hydrazines were investigated. Despite of the different order of acute toxicity, the dose-dependent malformation of cartilages and decrease of ossification were comparable between all test substances. Chapter VI deals with oxygen consumption of sediments and embryos. The zebrafish embryo test is a widely used bioassay for the testing of effluents and sediments, e.g. in Germany it is now mandatory for effluent testing. In this context, oxygen depletion of sediments and effluents is very important and may be a confounding factor in the interpretation of apparent toxicity. Zebrafish embryos can withstand a broad range of oxygen concentrations, but concentrations lower than 0.88 mg/L are 100 % lethal. In the sediment contact test with zebrafish embryos, native sediments rapidly developed strongly hypoxic oxygen conditions. Chapter VII then summarizes embryotoxic and teratogenic effects of a sediment contact assay with specific sediments from locations in the Tiete River Basin (Brazil), providing a comprehensive and realistic insight into the bioavailable hazard potential of these sediments. High embryo toxicity could be found in samples in the vicinity of the megacity Sao Paulo, but also downstream. Results confirm that most toxicity is due to the discharges of the metropolitan area of Sao Paulo. Along all findings, the overall results indicate that zebrafish embryos are a useful alternative method for traditional toxicity and teratogenicity testing. Regarding chemical testing, the OECD validation study and further work proved that embryos tests are neither better nor worse than conventional testing with adult fish, the correlation including all data available at this time being excellent (r² > 0.9). Furthermore, zebrafish embryos proved to be a suitable model for the determination of teratogenic effects and a potential alternative method for traditional teratogenicity testing including mammalian species.