An acute toxicity identification evaluation following U.S. EPA guidelines was performed with a municipal wastewater to identify effluent components responsible for lethality of larval fathead minnows (Pimephales promelas) and Ceriodaphnia dubia. Ammonia toxicity, also present in the effluent, was not the object of this study. The study was designed to characterize effluent toxicity not due to ammonia. To minimize ammonia toxicity interferences, all Phase I testing was performed at pH's where ammonia toxicity would be negligible. Phase I toxicity characterization results indicated surfactants as the class of compounds causing acute non-ammonia toxicity for both test species. A distinct toxicant characteristic, specifically sublation at alkaline pH, was employed to track suspect surfactant loadings in the collection system. Concurrently, effluent surfactant residue testing determined nonionic surfactants were at adequate concentrations and were sufficiently toxic to cause the measured adverse effects. Influent surfactant toxicity was determined to be much less than in the final effluent indicating the treatment process was enhancing surfactant toxicity. Nonionic surfactants known to behave in this manner are nonylphenol ethoxylates. Degradation of nonylphenol ethoxylates to the more toxic degradate nonylphenol (NP) during wastewater treatment explained the increase in toxicity. Major loadings of NPE, particularly NPE9, were determined through both analytical measurements and toxicity tracking to be a regional hospital laundry facility and a tannery. Substitution of NPE-based detergents to less toxic citrus-based products resulted in dramatic reduction of municipal effluent NPE concentrations and removal of non-ammonia acute toxicity.