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Ana M. Soto,¹ Janine M. Calabro,¹ Nancy V. Prechtl,¹ Alice Y. Yau,² Edward F. Orlando,³ Andreas Daxenberger,⁴ Alan S. Kolok,⁵ Louis J. Guillette, Jr.,⁶ Bruno le Bizec,⁷ Iris G. Lange,⁴ and Carlos Sonnenschein¹

¹Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts, USA; ²Southwest Research Institute, San Antonio, Texas, USA; ³Biology Department, St. Mary's College of Maryland, St. Mary's City, Maryland, USA; ⁴Institut für Physiologie, Forschungszentum für Milch und Lebensmittel, Technischen Universität München-Weihenstephan, Freising-Weihenstephan, Germany; ⁵Department of Biology, University of Nebraska at Omaha, Omaha, Nebraska, USA; ⁶Department of Zoology, University of Florida, Gainesville, Florida, USA; ⁷LABERCA-Laboratoire National de Référence, Ecole Nationale Vétérinaire de Nantes, Nantes, France

Abstract
Studies reveal that surface waters worldwide are contaminated with hormonally active agents, many released from sewage treatment plants. Another potential source of aquatic hormonal contamination is livestock feedlot effluent. In this study, we assessed whether feedlot effluent contaminates watercourses by measuring a) total androgenic [methyltrienolone (R1881) equivalents] and estrogenic (17ß-estradiol equivalents) activity using the A-SCREEN and E-SCREEN bioassays and b) concentrations of anabolic agents via gas chromatography-mass spectroscopy and enzyme-based immunoassays. Water samples were collected over 3 years from up to six sites [all confluent with the Elkhorn River, Nebraska, USA: a feedlot retention pond (site 1) , a site downstream from site 1 (site 2) , a stream with intermediate livestock impact (site 3) , and three sites with no observable livestock impact (sites 4-6) ] and two sources of tap water. In 1999, samples from site 1 contained 9.6 pM R1881 equivalents and 1.7 pM 17ß-estradiol equivalents. Site 2 samples had estrogen levels similar to those in site 1 samples but lower androgen levels (3.8 pM R1881 equivalents) . Androgen levels in site 3 samples were similar to those in site 2 samples, whereas estrogen levels decreased to 0.7 pM 17ß-estradiol equivalents. At site 6, androgen levels were approximately half those found at site 3, and estrogen levels were comparable with those at site 3. Sampling in later years was limited to fewer sites because of drought and lack of permission to access one site. Instrumental analysis revealed estrone but no significant levels of resorcylic acid lactones or trenbolone metabolites. Tap water was devoid of hormonal activity. We conclude that feedlot effluents contain sufficient levels of hormonally active agents to warrant further investigation of possible effects on aquatic ecosystem health. Key words: agricultural runoff, anabolic steroid hormones, aquatic ecosystem health, A-SCREEN, concentrated animal feeding operations (CAFOs) , environmental androgens, environmental estrogens, E-SCREEN, personal care products, pharmaceuticals. Environ Health Perspect 112:346-352 (2004) . doi:10.1289/ehp.6590 available via http://dx.doi.org/ [Online 1 December 2003]

Address correspondence to A.M. Soto, Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111 USA. Telephone: (617) 636-6954. Fax: (617) 636-3971. E-mail: ana.soto@tufts.edu

We thank D. Maume for help with high resolution GS-MS estrogen measurement methodology and C. Michaelson and C. Wieloch for skilled technical assistance.

This work was supported by the European Commission (contracts BIO4-CT98-4757 and FAIR-CT98-4752) .

The authors declare they have no competing financial interests.

Received 14 July 2003 ; accepted 1 December 2003.