In recent years, there has been increasing interest in utilizing fish cell culture in vitro assays, as alternatives to whole fish assays, for assessing the toxicity of aquatic pollutants. The fish cell line RTL-W1, derived from rainbow trout (Oncorhynchus mykiss) liver, was shown to retain several tissue specific features and to respond to chemical insults similarly to fish in vivo. In our study, we investigated the toxicity of two metals silver and cadmium and two pesticides azoxystrobin and paraquat using a cytotoxicity assay that measures simultaneously cell metabolic activity and cell membrane integrity. Moreover, we developed a novel 'high-content imaging' approach to evaluate if mitochondria's surface area is a sensitive and specific indicator of mitochondria toxicity. Initially, the cytotoxicity assay was used to determine the chemicals' effective concentrations (EC