Advancing evaluation and applicability of zebrafish embryo behavior assays through sophisticated mathematical methods and innovative technology for on-line monitoring and assessment

The assessment of environmental risks of chemicals is a major challenge. This has been acknowledged by the introduction of legislative frameworks that aim to minimize negative impacts of chemicals in the environment. The assessment of European water systems is regulated by the European Water Framework Directive, which was implemented in order to achieve a good ecological and chemical status for all European water bodies. Under this framework the assessment of water quality is based on the monitoring of 45 Priority Substances. It is known that these substances only reflect a small fraction of the overall composition and potential toxic impact of environmental pollutants present in water systems. Hence, it was strongly recommended to enhance monitoring studies by including biological tools. The implementation of effect-based methods in regulatory frameworks for the management of water quality will greatly enhance the predictive power of monitoring strategies. Here, it is important that sensitive methods are used that represent different trophic levels. With regard to the 3R principle, tests based on adult vertebrates need to be replaced or refined by sensitive alternative methods. The fish embryo toxicity test (FET) with zebrafish embryos has been approved by the OECD for the testing of chemicals and has been proven to be a good alternative to the acute fish toxicity test (AFT) that utilizes adult fish. The FET is frequently applied in ecotoxicological risk assessment and has already replaced the AFT in whole effluent testing in Germany. Recently, the emergence of automated tracking technology made tests based on zebrafish embryo behavior available to a broader research audience, including ecotoxicological research. Fish early life stage behavior has been proven to be a sensitive and ecologically relevant endpoint for the assessment of toxicity of single chemicals and environmental samples. Furthermore, the method holds the potential to be applied in high-throughput automated testing strategies, and hence should be assessed in regard of its implementation in large-scale monitoring and environmental risk assessment strategies. Apart from this, it is important that the mechanistic understanding of toxicity in in vivo models advances in order to link molecular effects that can be readily assessed in in vitro bioassays to higher functions and adverse outcomes at the organismal or population level. This concept has been applied in the adverse outcome pathway (AOP) framework, which can potentially lead to the acceptance of mechanistic assays in risk assessment studies and will therefore contribute to the replacement of animal experiments. In addition to the continuous contamination of the environment with low levels of toxic substances, accidental or deliberate contamination of water resources with contaminants at acute toxic concentrations pose a risk to environmental and human health. For instance, the well documented Sandoz incident in Basel in 1986 polluted the river Rhine with pesticides to an extent that resulted in fish mortality and further severe damage to the river biotope. This accident demonstrates the need for efficient and sensitive on-line monitoring strategies for water resources. In addition to conventional sensor technology the implementation of biological early warning systems (BEWS) has been demonstrated to be a sensitive addition to real-time monitoring systems. The aim of the present thesis was to (1) assess whether hatched zebrafish embryos react with changes in behavior to acute exposure to different model toxicants, (2) expand the area of application for zebrafish embryo behavioral studies in ecotoxicological risk assessment and to the field of real-time monitoring of water quality, (3) develop sophisticated methods for the evaluation of behavioral data, (4) advance the mechanistic understanding of the developmental and behavioral toxicity of a complex environmental sample, represented by the water soluble fraction of crude oil, and finally (5) develop appropriate system for the on-line exposure of zebrafish embryos to samples from a water stream. Two studies evaluated the behavioral responses of hatched zebrafish embryos after the acute manual exposure to a set of model compounds. The results showed that behavior represents a sensitive indicator for the presence of neurotoxic and narcotic compounds. The introduction of a novel parameter for the specific detection of the initial escape response of zebrafish embryos in reaction to the toxic compounds proved to be a sensitive additional evaluation parameter for the evaluation of zebrafish embryo behavioral data. In combination with the novel evaluation method for data from the light/dark transition test that is based on the calculation of the mean peak activity area, these studies demonstrated that there exists a potential to develop sophisticated mathematical methods that aid in the evaluation and understanding of zebrafish embryo behavioral responses. Additionally, the results demonstrated that acute peak contamination leads to stress-induced behavioral responses in fish early life stages, which indicates that acute exposure scenarios should be considered in studies aimed at evaluating the behavioral toxicity of chemicals. Finally, the evaluation of potential fields of application found that utilizing zebrafish embryos as an on-line water quality monitoring tool constitutes a system that is sensitive and at the same time considerably smaller compared to existing BEWS. The major advantage of this small-scaled system is that it allows to include a control group into the biosensor which would reduce the rate of false positive alarms generated by the sensor. Whole transcriptome gene expression analysis of zebrafish embryos exposed to a complex sample of crude oil components found that several biological pathways were affected that had been previously described in similar experiments with other fish species. Affected pathways included phase I and II metabolism, cardiovascular development, ocular development, energy metabolism and antioxidant responses. Furthermore, the analysis revealed that the circadian clock was affected at the gene expression level, which has not been previously described for crude oil toxicity. It can be assumed that most of the detected dysregulation of genes connected to the affected biological functions can be linked to the initial binding of certain PAHs to the aryl hydrocarbon receptor. Additionally, the study found that the embryo’s swimming activity was significantly affected by exposure to a concentration in the range of the LC10 for the tested samples. The gene expression analysis identified impaired ocular function, strain on energy metabolism, and the shift of the circadian rhythm to be likely underlying molecular causes for the effects on zebrafish behavioral responses. The study enhanced the understanding of the mechanistic toxicity of crude oil in developing fish and will advance the predictive power of effect-based methods in ecotoxicological risk assessment. Finally, this thesis introduced two novel systems that allow for (1) the automated exchange of exposure medium in multiwell plate based bioassays by a custom-built pipetting robot and (2) the exposure of organisms under flow-through conditions on a miniaturized scale in a novel flow-through well plate. The application of the pipetting robot in bioassays was successfully validated by experiments with zebrafish embryos exposed to permethrin and cadmium. The results of the exposure experiments highlighted the importance of controlling exposure concentrations of lipophilic substances in microwell plate-based bioassays. The higher medium exchange frequency performed by the pipetting robot greatly reduced variation and prevented the steep decrease in exposure concentrations for permethrin that was observed for the static and semi-static exposure regimes. This thesis demonstrated that the developed systems have the potential to be implemented in automated high-throughput testing strategies in environmental risk assessment and in on-line monitoring strategies as part of a biological early warning system. Overall, the work described here greatly enhances usability, reliability and applicability of zebrafish embryo behavior as a sensitive biomarker for exposure to waterborne contaminants