Endocrine-disrupting effects of cattle feedlot effluent on an aquatic sentinel species, the fathead minnow - Research

Environmental Health Perspectives, March, 2004 by Edward F. Orlando, Alan S. Kolok, Gerry A. Binzcik, Jennifer L. Gates, Megan k Horton, Christy S. Lambright, L. Earl Gray, Jr., Ana M. Soto, Louis J. Guilette, Jr.

Over the last decade, research has examined the endocrine-disrupting action of various environmental pollutants, including hormones, pharmaceuticals, and surfactants, in sewage treatment plant effluent. Responding to the growth of concentrated animal feeding operations (CAFOs) and the pollutants present in their wastewater (e.g., nutrients, pharmaceuticals, and hormones), the U.S. Environmental Protection Agency developed a new rule that tightens the regulation of CAFOs. In this study, we collected wild fathead minnows (Pimephales promelas) exposed to feedlot effluent (FLE) and observed significant alterations in their reproductive biology. Male fish were demasculinized (having lower testicular testosterone synthesis, altered head morphometrics, and smaller testis size). Defeminization of females, as evidenced by a decreased estrogen:androgen ratio of in vitro steroid hormone synthesis, was also documented. We did not observe characteristics in either male of female fish indicative of exposure to environmental estrogens. Using cells transfected with the human androgen receptor, we detected potent androgenic responses from the FLE. Taken together, our morphologic, endocrinologic, and in vitro gene activation assay data suggest two hypotheses: a) there are potent androgenic substance(s) in the FLE, and/or b) there is a complex mixture of androgenic and estrogenic substances that alter the hypothalamic-pituitary-gonadal axis, inhibiting the release of gonadotropin-releasing hormone or gonadotropins. This is the first study demonstrating that the endocrine and reproductive systems of wild fish can be adversely affected by FLE. Future studies are needed to further investigate the effects of agricultural runoff and to identify the biologically active agents, whether natural or pharmaceutical in origin. Key words: anabelic steroid hormones, aquatic ecosystem health, concentrated animal feeding operation (CAFO), environmental androgens and estrogens, gene expression, HPG axis, hypothalamic-pituitary-gonadal axis, pharmaceuticals and personal care products (PPCPs), Pimephales promelas.

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There has been a great deal of research over the last decade examining the endocrinedisrupting action of various environmental pollutants (Ankley et al. 1997; Guillette and Crain 2000). Much of this research has focused on the ability of chemical pollutants to act as estrogen receptor (ER) or androgen receptor (AR) agonists or antagonists (McLachlan 2001). Most of the compounds studied--pesticides and industrial pollutants--exhibit weak receptor affinities compared with endogenous hormones but can produce endocrine responses both in vitro and in vivo at environmentally relevant doses (Rooney and Guillette 2000; Tyler et al. 1998).

Studies have begun to focus on natural hormones released from animal waste used to fertilize agricultural fields. Significant concentrations of estrogens and androgens have been reported in ponds or streams receiving runoff from fields fertilized with chicken litter (Finlay-Moore et al. 2000; Nichols et al. 1997; Shore et al. 1995). In fact, depending on application rate, concentrations in runoff have been measured as high as 1,280 ng/L (Nichols et al. 1997). Natural hormones, such as estradiol, have also been reported in ponds below cattle holding facilities and have been associated with elevated plasma concentrations of the yolk precursor protein vitellogenin in female turtles (Irwin et al. 2001). Contamination of water systems with endogenous hormones such as 17[beta]-estradiol ([E.sub.2]) and testosterone (T) is not limited to surface waters because [E.sub.2] has been reported in spring water from mantled karst aquifers in agricultural areas (Peterson et al. 2000).

In addition, the presence of endogenous and pharmaceutical estrogens in sewage effluent has been studied as an example of hormonal pollution of the aquatic environment and has been reported as a factor affecting fish development and reproductive activity (Purdom et al. 1994; Tyler et al. 1998). Work performed below sewage treatment plants in Great Britain has documented a significant number of intersex fish compared with rivers with less effluent (Desbrow et al. 1996; Jobling et al. 1998). Furthermore, these studies have reported that many males had elevated levels of estrogen-induced vitellogenin in their blood. This protein does not normally occur in males. Fractionated sewage effluent, derived mostly from domestic sources, exhibited various peaks with estrogenic activity. Those representing ethinyl estradiol and estrone displayed the most potent estrogenic activities (Harries et al. 1996, 1997). In other countries, similar research has supported these observations and extended them by reporting that male fish exposed to sewage effluent not only have detectable plasma vitellogenin concentrations but also display altered plasma concentrations of T and [E.sub.2] (Folmar et al. 1996, 2000; Orlando et al. 1999).

These studies have helped focus attention on the possible detrimental roles of pharmaceutical agents released into the environment. A wide array of pharmaceutical agents, including hormonal mimics, have been reported in sewage and open waters in various countries (Daughton and Ternes 1999; Kolpin et al. 2002; Stumpf et al. 1999; Ternes 1998). These agents include drugs commonly prescribed for the treatment of heart disease, stress, inflammation, bacterial infections (antibiotics), and birth control. Further, veterinary drugs, such as growth promoters and antibiotics, are used extensively in agriculture, but few studies have examined their presence in the environment, although some studies have recently reported the presence of these compounds in groundwater near farms (Peterson et al. 2000). Importantly, no studies have examined the possible effects of these compounds on wildlife exposed to runoff from farms using large concentrations of pharmaceutical agents, such as cattle feedlots.