Validation of the REA bioassay to detect estrogenic activity in the water cycle

► The REA assay was validated as a qualitative screening assay for estrogenic activity in water. ► The procedure was sensitive and specific, not influenced by androgens or progesterones. ► The LOQ of the procedure is below 1 ng EEQ/L, sufficient for water threshold levels. ► Some WWTP effluents showed a clear estrogenic response above LOQ. ► No significant amounts of conjugated estrogens were present in these water samples.