The lower Genesee River and Rochester Embayment of Lake Ontario are a designated Area of Concern (AOC) under the binational Great Lakes Water Quality Agreement. The “degradation of phytoplankton and zooplankton populations” or plankton Beneficial Use Impairment (BUI) was classified as unknown and in need of further assessment in this AOC because water quality data suggested plankton communities could be effected and community data were either unavailable or indicated impacts. The plankton BUI may now be obsolete because local contaminant sources have been largely eliminated. The present study was conducted between July 2013 and August 2014 to assess the BUI-removal criteria: “AOC plankton bioassays confirm that toxicity in ambient waters (i.e., no growth inhibition) is not significantly higher than comparable non-AOC controls”. Acute and chronic toxicity of waters from 13 sites were quantified seasonally using standardized bioassays with the green alga Pseudokirchneriella subcapitata and water flea Ceriodaphnia dubia to test the hypothesis that toxicity of waters from AOC sites was not higher than that of waters from comparable non-AOC reference sites. Survival and reproduction of C. dubia did not differ significantly between site types, systems, or months. The growth of P. subcapitata did not differ between site types, but differed among systems and months. All results indicate that waters from AOC sites were no more toxic to both plankton species than waters from reference sites. Assuming test species represent natural plankton assemblages, water quality should not negatively affect survival and growth of resident plankton populations in the Rochester Embayment AOC.