Biomonitoring requirements are continually being added to NPDES permits. As a result, many municipal wastewater treatment facilities have been identified as having effluent acute toxicity. To solve this problem, the Environmental Protection Agency (EPA) has developed a Toxicity Identification Evaluation (TIE) protocol. This protocol lists a set of simple procedures which are used to separate whole effluent samples into different fractions containing different classes of similar compounds. Toxic fractions are further separated and concentrated using various techniques to assist in the possible identification of certain classes of suspected toxic compounds. Once sample fractionation and toxicity testing have been thoroughly investigated, chemical-specific analyses are conducted to tentatively identify toxic constituents. The C18 (Octadecyl) Solid Phase Extraction (SPE) Column is used in the TIE protocol to separate and concentrate moderately polar to nonpolar organic compounds from toxic effluent samples. An increasing gradient of methanol (MeOH) in water is used to elute the column; the objective is to separate retained compounds into eight different fractions based on their polarity. The objective of conducting this research is to determine if the C18 SPE Column elution procedure is a viable technique for the identification and characterization of toxic effluents. This study showed that the C18 column was able to remove compounds causing acute toxicity from samples collected at the Cross Creek Wastewater Treatment Plant (WWTP) in Fayetteville, NC. The 80 to 85 percent MeOH/H20 fractions contained the most toxicity. However, laboratory tests of the procedure using known target compounds indicated that several different MeOH/H20 fractions contained each individual compound thus showing deficiencies in resolution. Moreover, the target compounds that were selected differed widely in polarity (as indicated by the compounds octanol/water partitioning coefficient), yet this did not cause a wide separation of these compounds into specific MeOH/H2O fractions. This research showed the C18 SPE column is capable of retaining relatively non-polar compounds as indicated by the target compound evaluation. These compounds were effectively eluted from the CIS SPE column with MeOH/H20, but separation was not well defined. In the situation of toxic wastewaters, where numerous nonpolar organic compounds may possibly be present in WWTP's discharge, the C18 SPE column provides little information regarding the identity of the non-polar organics causing toxicity in a toxic MeOH/H20 fraction.