The need to protect aquatic organisms from the toxic potential of environmental contaminants with minimal use of animal testing has been highlighted by the introduction of a number of regulatory directives. This thesis proposes the potential of 3D cell culture models to ‘bridge the gap’ between the use of existing subcellular and in vivo systems for the testing of compounds for potential persistence, bioaccumulation and toxicity, improving the predictive power of in vitro screening procedures. In this project, a trout hepatocyte spheroid model was optimised and using metabolic, fluorescence, transport and gene expression assays, the utility of this system in such studies as a superior alternative to currently used in vitro models was demonstrated. Hepatocytes cultured as spheroids remained viable for up to 40 days, exhibiting significantly greater functional xenobiotic metabolism and transport, as well as expression of genes, compared to other in vitro systems. These features of spheroids present a profile more closely representative of that of the whole liver. Together with the potential advantages of screening highly persistent compounds that require long term incubation and possible use in the assessment of the toxicity of compounds exhibiting chronic effects, this system successfully enriches current in vitro testing strategies.