A toxic fraction identification protocol (TFIP) was developed to identify the fractions(s) of a complex wastewater effluent contributing to toxicity. The TFIP is based on sequential physical/chemical fractionations of the effluent with associated toxicity testing using the aquatic invertebrate test organism Daphnia. Fractionation treatments used in the TFIP include filtration, aeration, activated carbon, and cation and anion exchange treatments. Before and after each treatment, acute toxicity tests are conducted to determine the efficacy of the treatments in removing toxicity. Selected chemical analyses are used along with toxicity test results to identify the toxic fraction(s) in the wastewater. The TFIP can determine whether or not the toxicity is associated with filterable solids, volatile and/or biodegradable organics, carbon adsorbable compounds, cationic or anionic inorganic constituents, and charged organic compounds. The TFIP was developed using six industrial effluents possessing a diversity of chemical constituents. It was validated using a nontoxic industrial effluent spiked with hexavalent chromium and naphthenic acids.