The mechanism(s) of estrogenic compound removal during physical and biological wastewater treatment processes is not fully understood. Here, we compare overall input and output fluxes of 17b-estradiol (E2), and total estrogenic activity at six Arizona municipal wastewater treatment facilities utilizing different treatment processes.  The objectives of this work were to determine 1) the overall fate of E2 relative to total estrogenic activity during wastewater treatment and 2) distinguish the importance of aqueous-phase and adsorbed contaminants as determinants of total estrogenic activity in wastewater.  A total mass balance approach was used, in which liquid and solid samples were obtained as appropriate from plant influent, plant effluent, and digested sludge. 

Liquid samples were extracted on C18 resin; sludge and biosolid samples were processed using microwave-assisted extraction.   E2 was measured using an immuno-based (ELISA) assay, estrogenic activity was quantified by the yeast estrogen screen (YES bioassay). 
Overall reduction of total estrogenic activity through the six plants ranged from 26 to 99%, whereas E2 removal ranged from 23 to 91%.  Total estrogenic activity increased during anaerobic sludge digestion despite low levels of E2 in anaerobically digested sludge.  It is possible that anaerobic treatment converted ethoxylated alkylphenols to more potent estrogens (octylphenol and nonylphenol).  Decreased removal efficiencies of E2 were observed in those facilities without anaerobic digestion, whereas overall total estrogenic activity removals were equal or greater to other plants with anaerobic digestion.