Development of Method for Identification of Major Substances Inducing Estrogenic Activity Contained in Sewage and River Waters

Toxicity Identification and Evaluation (TIE) procedure for sewage and river waters was developed to identify the major substances inducing the estrogenic activity through application to various sewage samples (i.e., influent and effluent of sewage treatment plant and returned sludge from final settling tank) and river waters. The TIE procedure was composed of instrumental analysis and chemical fractionation technique combined with bioassay using recombinant yeast. The samples collected from a municipal sewage treatment plant and Tama River receiving a huge treated wastewater were firstly filtrated, and organic compounds in the dissolved and suspended phases were extracted and subjected to the instrumental analysis and the bioassay. At the same time, the parts of the extracts were subjected to fractionation procedures. In the instrumental analysis, natural estrogens and their conjugates, synthetic estrogen and nonylphenol and its metabolites were measured by LC-MS/MS and HPLC. The estrogenic activities measured by the bioassay were almost comparable with those calculated from the concentrations and relative potencies of the target substances for all samples ranging from river waters to sludge samples. Results from the fractionation technique followed by the bioassay, significant estrogenic activities were observed in two fractions in which E1 and E2 elution were individually confirmed. Also, the contribution by unknown substances to the activities for the sewage samples was estimated to be as small as between 6.7 and 9.4%.