Biosolids are the nutrient-rich organic residual materials resulting from the treatment of domestic sewage at a wastewater treatment facility, and are increasingly land-applied for agricultural and land-reclamation purposes as part of the wastewater management process. While the presence and fate of estrogenic endocrine-disruptors (eEDCs) in wastewater has been extensively studied, much less focus has been given to examining the presence and fate of eEDCs during biosolids treatment. In particular, little work has been done to measure the net estrogenic potency of biosolids using in vitro bioassays, such as the Yeast Estrogen Screen (YES) assay. This is despite the fact that widespread land-application of biosolids provides for the direct introduction of eEDCs into terrestrial and aquatic environments. The relative scarcity of bioassay-based net estrogenicity data for sludges and biosolids is in large part due to the analytical challenges involved in working with such a complex sample matrix. Comprehensive sampling at wastewater treatment plants in Guelph and London, ON, demonstrated that the estrogenicity of anaerobically-treated biosolids is considerably lower (12.0-19.7 ng/g estradiol-equivalents) than that reported in earlier published studies. The results of the present study were made possible due to the development of a sample preparation methodology that overcame the toxic effects that sludge and biosolid samples typically exert on yeast cells in the YES assay. An anti-estrogenicity assay was also applied for the first time to sludges/biosolids to measure the extent to which antagonistic compounds ‘block’ the response of the YES assay. The results of these tests suggest that although the net estrogenicity of anaerobically treated solids is indeed low, up to twice the amount of estrogenicity measured by the YES assay may be masked in biosolids by the presence of antagonistic compounds. While aerobic treatment conditions reduced net estrogenicity to at-or-below detectable levels, net estrogenicity remained relatively constant throughout the unit processes of the anaerobic treatment train. Biosolid ageing during storage led to an overall decrease in net estrogenicity of both conventionally-treated “restricted use” and advanced-treated “unrestricted use” anaerobic biosolids. However, levels of net estrogenicity were observed to spike during the early stages of storage, particularly under freeze/thaw conditions. / Natural Science and Engineering Research Council of Canada (NSERC) PGS-D3 scholarship, Water Environment Association of Ontario, Canadian Water Network

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