This paper describes an analytical procedure for free estrogens and their conjugates in domestic wastewater. The procedure demonstrated in this study is innovative in terms of levels of detection and quantification of the following substances: estrone (E1); 17β-estradiol (E2); 17α-ethynylestradiol (EE2); estriol (E3); estrone-3-sulfate (E1-S); β-estradiol 3-sulfate (E2-S); estriol 3-sulfate (E3-S); estrone β-D-glucuronide (E1-G); β-estradiol 17-(β-D)-glucuronide (E2-G); estriol 3-(β-D)-glucuronide (E3-G); β-estradiol 3-sulfate 17-glucuronide (E2-S&G); and estradiol 3,17-disulfate (E2-diS). The detection limits of this method ranged from 0.1 to 1.4 ng/l. The recovery efficiencies of the estrogens in the analysis from influent and effluent of the secondary settling tank in a wastewater treatment plant (WWTP) were higher than 94% for the free estrogens, but were less than 50% for the conjugated estrogens. The field study using this method was conducted at twenty WWTPs in Japan. The median concentrations of the estrogens ranged from ND to as high as >100 ng/l. In the influent and secondary effluent samples, the concentrations of E1, E2 and E3 were the same levels as those previously reported. We found that the conjugated estrogens exist at higher concentrations in the influent and the secondary effluent than in the other studies, and that the concentrations of the conjugated estrogens were higher than those of the free estrogens.

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