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Estrogenic Activity in the Environment : Municipal Wastewater Effluent , River , Ponds , and Wetlands

Estrogenic activity of regional water samples was evaluated. Samples obtained fromwetlands and ponds involved in various agricultural land uses, from three river sites over four seasons, and from municipal wastewater effluent held in storage lagoons were evaluated. The estrogen-responsive cell line MCF-7 BOS was used in the E-screen assay to determine 17b-estradiol equivalents (E2 Eq) of water samples extracted by solid-phase extraction. Estrogenic activity in surrounding wetlands and ponds from different land uses was not different, with 10 M E2 Eq (0.3 ppt). Estrogenic activity of Red River samples was within the same range as wetland–pond samples. The highest activity was found downstream from municipal wastewater treatment effluent discharge sites, in winter when river flow was lowest (approximately 63 10 ME2 Eq). Results showed that 7 of 20 wetland–pond samples and 5 of 12 river samples were below the limits of quantitation (approximately 3 3 10 M E2 Eq). Toxicity was found in fall and summer river samples upstream from municipal wastewater release sites. The timing of toxicity did not coincide to the presence of elevated fecal coliforms. Estrogenic activity in wastewater effluent from lagoons decreased over time (approximately 25 to 5 3 10 M E2 Eq) with an apparent half-life of 8 d for one lagoon. The median concentration of detectable estrogenic activity in regional water samples was approximately 50-fold less than the median 17b-estradiol concentration of estradiol detected in some U.S. streams in previous studies. WITH ADVANCES in technology and instrumentation, scientists are now able to detect chemical residues in parts per trillion (ppt). While Kolpin et al. (2002) reported that 82 organic wastewater contaminants were present in streams across the United States, the biological consequences of many of these chemicals at the concentrations detected are unknown. Also unknown are the biological effects of mixtures of these chemicals on organisms. Endocrine disruption as a result of anthropogenic activity has become a relatively recent concern, initiated by the work of Guillette, Jr. et al. (1994) reporting that alligators in contaminated Florida lakes had abnormal gonad development and sex hormone concentrations. Other studies (Harries et al., 1996; Allen et al., 1999) have found contaminated waters to be associated with vitellogenin production (a protein normally only produced in the liver of female fish in response to estradiol) and intersex gonads (oocytes present in testes) in male fish. Organismal research conducted at environmental sites of concern requires extended study periods and is expensive, labor intensive, and subject to vandalism. Several in vitro assay systems have been developed to assess estrogenic activity. One type of assay is receptor based. These assays indicate receptor binding or estradiol displacement (which may or may not result in downstream activation or inhibition). Another type of assay uses cells (either mammalian or yeast) transfected with DNA for estradiol receptors. The feedback mechanisms that would normally be present in cells responsive to estradiol would not be found in yeast. Transfected mammalian cells would not have receptor numbers equivalent to unaltered cells. Therefore these assay types do not reflect true cellular potential. Some of the limitations associated with risk assessment of estrogenic compounds using these types of assays were addressed by Safe et al. (2002). The in vitro E-screen assay developed by Soto et al. (1995) allows for determination of estrogenic activity of samples using a human mammary carcinoma cell line that is dependent on estrogen receptor activity for proliferation. These cells have not been genetically manipulated by scientists, and therefore reflect their natural physiological state (first conceding that they are derived from a carcinoma and not ‘‘normal’’ tissue). The assumption is that unaltered cells in whole organisms, with estrogen receptors, have the cellular potential to react in a similar manner. The E-screen assesses a biological endpoint (cell proliferation), so not only does a substance need to bind to the receptor, it must be capable of eliciting a coordinated sequence of events stimulated by estradiol binding. Estrogen-independent proliferation can be established by co-incubation of sample with an estrogen receptor antagonist. Unlike enzyme-linked immunosorbent assays (ELISAs), which are designed for their chemical specificity and lack of cross-reactivity, the E-screen can detect activity in environmental samples without any information as to the chemical structure of compounds present. Currently environmental samples are being screened using an ELISA for estradiol, which would not detect many compounds in environmental samples that have been shown to have estrogenic activity (e.g., Peterson et al., 2000). Before agricultural and industrial practices can be evaluated for their effects on estrogenicity of environmental waters, an idea of ‘‘indigenous’’ levels of activity (from natural as well as anthropogenic activity) in our water supplies must be established. Therefore this study evaluated the estrogenic activity of water samples from wetlands and ponds from USDA-ARS, Animal Metabolism Unit, Biosciences Research Laboratory, 1605 Albrecht Boulevard, Fargo, ND 58105. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. Received 6 Dec. 2004. *Corresponding author (shappeln@fargo.ars.usda.gov). Published in J. Environ. Qual. 35:122–132 (2006). Technical Reports: Surface Water Quality doi:10.2134/jeq2004.0464 a ASA, CSSA, SSSA 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: CRP, Conservation Reserve Program; ELISA, enzyme-linked immunosorbent assay; E2, 17b-estradiol; E2 Eq, 17b-estradiol equivalents; LC–MS/MS, liquid chromatography–tandem mass spectrometry; MWWTP, municipal wastewater treatment plant; RR, Red River; TSS, total suspended solids. R e p ro d u c e d fr o m J o u rn a l o f E n v ir o n m e n ta l Q u a lit y . P u b lis h e d b y A S A , C S S A , a n d S S S A . A ll c o p y ri g h ts re s e rv e d . 122 Published online January 3, 2006