Determination of Sex Hormones and Nonylphenol Ethoxylates in the Aqueous Matrixes of Two Pilot-Scale Municipal Wastewater Treatment Plants MAR ESPERANZA, MAKRAM T. SUIDAN,* FUMITAKE NISHIMURA, ZHONG-MIN WANG, AND GEORGE A. SORIAL Department of Civil & Environmental Engineering, University of Cincinnati, Cincinnati, Ohio 45221-0071 ALAN ZAFFIRO Shaw Environmental & Infrastructure, Inc., 11499 Chester Road, Cincinnati, Ohio 45246-4012 PAUL MCCAULEY, RICHARD BRENNER, AND GREGORY SAYLES U.S. Environm ental Protection Agency, NRMRL, Cincinnati, Ohio 45286 Two analytical methods were developed and refined for the detection and quantitation of two groups of endocrine- disrupting chemicals (EDCs) in the liquid matrixes of two pilot- scale municipal wastewater treatment plants. The targeted compounds are seven sex hormones (estradiol, ethinylestradiol, estrone, estriol, testosterone, progesterone, and androstenedione), a group of nonionic surfactants (nonylphenol polyethoxylates), and their biodegradation byproducts nonylphenol and nonylphenol ethoxylates with one, two, and three ethoxylates. Solid phase extraction using C-18 for steroids and graphitized carbon black for the surfactants were used for extraction. HPLC-DAD and GC/ MS were used for quantification. Each of the two 20 L/h pilot-scale plants consists of a primary settling tank followed by a three-stage aeration tank and final clarification. The primary and the waste-activated sludge are digested anaerobically in one plant and aerobically in the other. The pilot plants are fed with a complex synthetic wastewater spiked with the EDCs. Once steady state was reached, liquid samples were collected from four sampling points to obtain the profile for all EDCs along the treatment system. Complete removal from the aqueous phase was obtained for testosterone, androstenedione, and progesterone. Removals for nonylphenol polyethoxylates,estradiol,estrone, and ethinylestradiol from the aqueous phase exceeded 96%, 94%, 52%, and 50%, respectively. Levels of E3 in the liquid phase were low, and no clear conclusions could be drawn concerning its removal. Introduction The term EDCs or endocrine-disrupting chemicals includes diverse groups of heterogeneous contaminants. These com- pounds are reported to interfere with the normal functions of the endocrine system in animals and humans. Observed effects on animals include, among others, changes in sex ratios, decreases in population, behavior modification, and developmental abnormalities. The effects of exposure of humans to EDCs have not been conclusively determined. Studiesreportingdecreasesin sperm counts,reduced fertility, and an increase in occurrence of hormone-dependent cancers have increased both public awareness and scientific and political interest in the subject (1). EDCs are released to the atmosphere as a result of com- bustion and incineration activities (polycyclic aromatic hy- drocarbons (PAHs, dioxins, ...) (2),but the principalsinks for EDCs are rivers, lakes, and other water bodies. Discharges oftreated and untreated wastewater to receivingwater bodies constitute the principalsources for two subgroups ofEDCs s sex hormones and nonylphenol polyethoxylates (NPnEO). More interestingly,municipalwastewater treatment facilities appear to play an important role in the estrogenicity oftheir effluents. Prior to excretion, both natural and synthetic hor- monesare inactivated via sulfation or glucoronidation.Elim- ination occursmainlythrough urine,although smallerquan- tities of sex hormones are also found in feces (1, 3). These inactive hormones are converted into their estrogenic active forms both in sewers and along wastewater treatment trains (4-6). NPnEO are among the third most widely used surfac- tants worldwide.These surfactants do not exhibit estrogenic characteristics. However, partial biodegradation during wastewater treatment generates estrogenic byproducts: non- ylphenol (NP) and nonylphenol ethoxylates with one, two, and three ethoxylate groups (NP1EO, NP2EO, and NP3EO). Extensive research has been conducted on these groups of compounds. Several analytical methods have been pub- lished and validated for the quantitation of sex hormones and NPnEO in environmental samples (see refs 7 and 8 for reviews). Also, an extensive body of data has been compiled on the occurrence and concentrations of these EDCs in municipal wastewater treatment plant effluents, rivers, groundwater, sediments, and sludge (9-12). Data are also available on the biodegradation rates and removalofboth sexhormones and NPnEOfrom wastewater. The data reported were obtained either from lab-scale batch tests in which mixed liquor from a wastewater treatment plant was spiked with the compounds or from influent and effluent samplescollected at full-scale wastewatertreatment facilities (5, 6, 13-16). The limitations of the first studies focus on their applicability to full-scale plants. Problems arisingwhen dealingwith full-scale treatment plants include seasonalchanges(temperature,rainfall),variabilityin influent levels (unexpected discharges),and speciation (conjugated/ unconjugated steroids). Other important issues pertain to analytical difficulties and the level of effort needed for determination of sex hormones in wastewater and sludge matrixes. The goal ofthis investigation is to determine the fate and transport of NPnEO and seven sex hormones in two pilot- scale municipal wastewater treatment plants operated in parallel. The pilot plants provide the opportunity to collect data from 20 L/h continuous-flow systems avoiding the variabilityand analyticaldifficulties associated with full-scale plants. The data reported in this paper pertain to concentra- tions ofthese chemicals in the liquid matrixes and observed removal of these compounds achieved in the pilot plants. *Correspondingauthor phone: (513)556-3695;fax: (513)556-2599; e-mail: makram.suidan@uc.edu. Environ. Sci. Technol. 2004, 38, 3028-3035 3028 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 11, 2004 10.1021/es0350886 CCC: $27.50  2004 American Chemical Society Published on Web 04/27/2004