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Research has confirmed that elevated synthetic estrogen in surface waters can lead to intersex characteristics in aquatic vertebrates. Unmetabolized antibiotics, hormones from animal wastes, including humans, and discarded pharmaceuticals are some ways synthetic estrogen enter aquatic ecosystems through the release of contaminated effluents. In this investigation, the Agilent 1260 Infinity HPLC was used to detect the possible presence of 17-α-ethindyl estradiol in effluents released from the Chattanooga Sewer Treatment Plant. Results were analyzed by comparing HPLC chromatograms from effluent and spiked samples. We detected a possible peak of synthetic estrogen in the effluent samples with a retention rate of 6 minutes detected at 280 nm. This method could be used to determine if synthetic estrogen is present, within the mdl, in the Tennessee River at various distances downstream from the Chattanooga Sewer Treatment Plant and to compare these levels with those documented as being able to feminize male fish.