Chlorine disinfection, widely used in wastewater reclamation, can form toxic and harmful disinfection by-products (DBPs), some of which are associated with endocrine disruption. In this study, the presence of bromide was found to promote an increase in antiestrogenic activity using a yeast two-hybrid assay in the sewage secondary effluent during chlorine disinfection. Among the dissolved organic matters in the secondary effluent, hydrophobic acids and hydrophilic substance fractions were determined as potential precursors associated with increase in antiestrogenic activity in the secondary effluent induced by bromide. Further antiestrogenic activity evaluation and mass spectrum characterization following the semipreparative liquid chromatography fractionation of a natural organic matter precursor, tyrosine, after chlorination under the presence of bromide revealed, for the first time, that 2-(bromo-4-hydroxyphenyl) acetonitrile (Br-HPAN) and 2-(dibromo-4-hydroxyphenyl) acetonitrile (DBr-HPAN) exhibited antiestrogenic activity. Br-HPAN and DBr-HPAN were the DBPs involved in the increase in antiestrogenic activity in the tyrosine solution. Bromide was shown to induce the formation of Br-HPAN and DBr-HPAN in the secondary effluent during chlorine disinfection. (C) 2014 Elsevier B.V. All rights reserved.