Chemical contamination poses a risk to the aquatic environment. Chemicals can affect the biological quality of surface waters, i.e. the composition of aquatic communities. The preservation or restoration of a good ecological status of surface water bodies requires the determination of the chemical status and the assessment of its effects on biological quality. Current regulatory assessments are restricted to single priority chemicals. This approach does not account for the entirety of ecologically relevant chemicals, transformation products, or the joint effects of chemical mixtures that are ubiquitously present in water bodies. Various toxicity tests are used to assess biological effects of environmental chemicals onaquatic organisms. However, acute toxicity tests, such as the fish embryo acute toxicity test (OECD test no. 236), lack sensitivity to detect sub-lethal chemical toxicity, particularly in the realm of chemicals that target the developing nervous system. Therefore, new approaches are required to quantify the effects of chemicals and mixtures on neurotransmission and neurodevelopment. Embryo-larval stages of zebrafish (Danio rerio) represent an excellent model system for the detection of chemical neurotoxicity, particularly during early development which represents one of the most vulnerable life stages to chemical exposure. The functionality of the zebrafish nervous system can be determined using automated behavior assays. However, key variables that influence chemical-dependent behavioral effects are not well characterized. Therefore, this thesis seeks to refine the execution, computational analyses, and applicability domain of an automated light-dark transition zebrafish behavior assay to ultimately delineate distinct behavioral responses following exposure to environmental chemicals and mixtures.