Before treated wastewater can be discharged to the environment, utilities are required to perform a direct toxicity assessment (DTA), which usually requires that live aquatic animals, such as fish, swim in the clean discharge water for 4-10 days while their growth and activity are observed and measured. This preparatory research investigated the potential of in vitro cell culture assays for replacing whole animal tests. It was concluded that there are no legal or regulatory barriers, that in vitro cell culture has higher social and ethical acceptance than in vivo animal tests, and that three organisms (bacteria, algae and fish) could provide the cells needed to conduct seven toxicity tests that are good candidates for future development as alternatives to existing DTA methods.