The bioavailability of sediment bound contaminants in lentic (Hamilton Harbour, Lake Ontario) and lotic (Detroit River connecting Lakes St. Clair and Erie) environments were assessed by a battery of multi-trophic tests using laboratory grown organisms. Hamilton Harbour is a hyper-eutrophic and highly contaminated environment due to extensive urban and industrial growth, while the Detroit River has been implicated as a major source of contaminants to Lake Erie. An array of sites across Hamilton Harbour and the Detroit River were selected, including the mouth of the Rouge River as well as the Trenton Channel—the contaminated western arm of the Detroit River. Multi-trophic acute assays were conducted using Daphnia magna, Hyalella azteca, Diporeia hoyi, and Lumbriculus variegatus. While the tests were consistent in determining the most toxic hot spots, variability existed in the sensitivities of test organisms to discriminate among less contaminated sites. The most toxic sediment in the Detroit River was at the mouth of the Rouge River, while the site in Windermere Basin in Hamilton harbour was found to be deleterious. The results indicated that the toxicity in a lotic ecosystem such as the Detroit River was caused by both the bottom sediments and the mobile seston component which contributed to the water-borne toxicity. Conversely, lentic and undisturbed ecosystems such as Hamilton Harbour contain much of their toxic component in the bottom sediments and not in the overlying water column. The multi-trophic battery of test approach adopted in our study appears to be effective in detecting and discriminating differential sensitivities of sediment bound contaminants in both lentic and lotic ecosystems. This battery of tests approach needs continued modification, development, and improvement to keep the assays up-to-date, sensitive, cost effective and adaptable in discriminating complex mixtures of sediment bound contaminants found in natural ecosystems.