A study was conducted to identify and determine the relative importance of the acutely toxic fractions of wastewater from oil sands extraction using a bioassay directed toxicity identification evaluation (TIE), to use these data to construct a toxic balance, and to determine whether the same fractions were acutely toxic to Microtox, Daphnia, and rainbow trout. Samples of extraction wastewater were obtained from the Mildred Lake tailings pond in Alberta during summer 1991 and 1992. The samples were centrifuged to remove suspended solids and the toxicity of the supernatant was evaluated. Seven manipulations, each of which was designed to remove a different class of compounds, was performed on the supernatant samples. A complete TIE was performed using the Microtox bacterial bioassay. All acute toxicity of the surface tailings pond water to Microtox was removed by precipitation of organic acids or by removal of nonpolar organics. These results suggest that the main toxic fraction was a surfactant. In the interstitial water of the fine tails, volatiles accounted for 15% of the acute toxicity. However, all the acute toxicity of interstitial water was removed by precipitation of organic acids or by removal of nonpolar organics. Organic acids accounted for all the acute toxicity of tailings pond water to Daphnia and rainbow trout. Differences in relative sensitivity of test organisms to the toxic fraction demonstrate the importance of using more than one test organism to evaluate acute toxicity. Daphnia was 0.4 times less sensitive and rainbow trout 3 times more sensitive than Microtox. 20 refs., 6 tabs