Identification of the isoflavonoid genistein in bleached kraft mill effluent

Plants synthesize many phytochemicals, including flavonoids, which may be present in the heartwood of trees used in the pulp and paper industry. Extracts were prepared from wood pulp and mill effluent collected from a bleached kraft mill in Ontario, Canada, and these extracts were subfractionated by LH-20 gel filtration chromatography and analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and LC-ESI-tandem mass spectrometry (LC-ESI-MS-MS). Initial LC-MS analysis in negative ion mode was conducted by monitoring ions corresponding to the deprotonated molecular ions of a range of flavonoid compounds. The presence of chromatographic peaks generated by selected ion monitoring (SIM) in samples of both mill effluent and wood pulp encouraged further analysis. Of the compounds highlighted by LC-ESI MS-SIM analysis, the isoflavonoid genistein was positively identified by LC-ESI-MS-MS. Genistein was quantified at a concentration of 30.0 mug/kg in air-dried wood pulp and concentrations of 13.1 mug/L and 10.5 mug/L in untreated and treated (final) effluent, respectively. Genistein is a known endocrine disrupter substance and,therefore, could contribute to the alterations in sex steroid levels and reduced reproductive capacity observed in fish captured near the discharges of pulp mills.