ORIGINAL ARTICLE Removal of Aqueous 17a-Ethinylestradiol by Rhodococcus Species Devin O’Grady, Sarah Evangelista, and Viviane Yargeau* Department of Chemical Engineering, McGill University, Montreal, Quebec, Canada. Received: August 21, 2008 Accepted in revised form: June 18, 2009 Abstract Pharmaceutical substances such as 17a-ethinylestradiol (EE2), the active component of the contraceptive pill, are often not eliminated during wastewater treatment and are persistent in the environment. Using solid-phase extraction and chromatography methods for analyzing low concentrations of EE2 and metabolites, biodegrad- ability of EE2 was studied using four bacteria grown under aerobic conditions in the absence or presence of a readily available carbon source (2.5 g=L of adipic acid or glucose). Preliminary identification of produced metabolites was also completed. In presence of a cosubstrate, Rhodococcus erythropolis removed EE2 up to 47% of its initial concentration (1.4 and 0.5 mg=L) in 13 h, while it removed only 10% after 75 h without a cosubstrate. In the presence of a cosubstrate, Rhodococcus equi removed EE2 up to 39% of its initial concentration (1.4 mg=L) after 65 h, whereas no significant EE2 removal was observed using Rhodococcus rhodochrous and Rhodococcus zopfii. Preliminary analysis of EE2 transformation products confirmed the presence of phenol and a compound of high molecular weight (331 amu). These results improve our understanding of the effect of common biological wastewater treatment variables such as bacteria strains, acclimation, and presence of cosubstrate, on the removal of EE2. Key words: 17a-ethinylestradiol; Rhodococcus erythropolis; Rhodococcus equi; Rhodococcus zopfii; Rhodococcus rhodo- chrous Introduction S everal studies have shown that natural and synthetic estrogens are often not eliminated during wastewater treatment, and additionally are not biodegraded in the envi- ronment (Ternes et al., 1999b; Heberer, 2002; Jurgens et al., 2002; Yoshimoto et al., 2004;). 17a-Ethinylestradiol (EE2), a synthetic estrogen used in the oral contraceptive pill, has been detected in ecologically relevant amounts in sewage effluents, surface water, river water, bed sediments, and in activated and digested sludge (Weber et al., 2005). In 10 Canadian municipal wastewater effluents (MWWEs), EE2 was dis- charged into the environment at concentrations of up to 42 ng=L (Ternes et al., 1999b), with a median concentration of 9 ng=L. The maximum concentration of EE2 from four MWWEs in Michigan was measured to be 0.8 ng=L (Parrott and Blunt, 2005). Baronti et al. (2000) measured EE2 concen- trations up to 13 ng=L in untreated sewages in the Tiber River in Rome, Italy. Ternes et al. (1999b) also measured estrogen levels in 16 German municipal sewage treatment effluents and found concentrations of EE2 up to 15 ng=L. Last, is that EE2 was detected in groundwater and in raw and purified drinking water in Germany up to concentrations of 2.4 ng=L (Heberer, 2002). In fish, as in all other vertebrates, estrogens play an important role in many reproductive and develop- mental processes. For example, less than 1 ng=L EE2 and even as little as 0.1 ng=L can stimulate male rainbow trout to pro- duce an egg yolk protein called vitellogenin normally only associated with sexually mature female ( Jurgens et al., 2002); and 4 ng=L caused male fathead minnows failure to develop normal secondary sexual characteristics such as gonad de- velopment, sex determination, and reproductive maturity ( Jurgens et al., 2002). In vivo tests have demonstrated that EE2 is approximately 11–27 times more potent than the natural female sex hormone 17b-estradiol (E 2 ) (Huber et al., 2004), suggesting that EE2 is one of the most important endocrine disruptors found in the environment. The presence of estrogens, detected wherever sought, and the identification of their negative impact on aquatic systems stimulated research on the fate of EE2 during wastewater treatment. The removal of estrogens in activated sludge treatment has been investigated in previous studies. Urase and Kikuta (2005) examined the removal of E 2 , estrone (E 1 ), and EE2, among other pharmaceuticals, by activated sludge with batch experiments using a lab-scale reactor. The EE2 concentration remained relatively stable and exhibited the highest adsorption rate (as high as 60% of the initial concentration after 20 min), whereas the majority of the *Corresponding author: Department of Chemical Engineering, McGill University, 3610 University Street, Montreal, Quebec, Canada H3A 2B2. Phone: 514-398-2273; Fax: 514-398-6678; E-mail: viviane .yargeau@mcgill.ca ENVIRONMENTAL ENGINEERING SCIENCE Volume 26, Number 9, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ees.2008.0272 1393