The research focused on investigation of two important elements of the toxicity reduction evaluation (TRE) protocol proposed by EPA: (1) toxicity identification evaluation (TIE) and (2) toxicity source evaluation (TSE). The specific objectives were to: challenge the TIE protocol with target compounds to determine whether the toxicity could be properly classified; apply the TIE and TSE protocols to two case studies (High Point, NC and Fayetteville, NC) where pass-through toxicity was highly variable; and investigate the potential for return activated sludge to desorb components that may cause acute toxicity. The refractory toxicity assessment (RTA) protocol was modified toidentify chronic toxicity as the end-point rather than acute toxicity. Results indicated that the TIE Phase I protocol can provide proper direction for further narrowing of potential toxicants but more data are needed on mixtures of target compounds to show reliability.