The United States (US) is the largest producer of livestock products, and over the past few decades the farming in the US has shifted heavily towards concentrated animal feeding operations (CAFOs), in order to meet global demand (FAO, 2016; Long et al., 2018). The US Department of Agriculture (USDA) estimated that livestock and poultry produced over 1.1 billion tons of wet weight manure in 2007 (US EPA, 2013). Manure produced on CAFOs is typically stored in wastewater lagoons which function as holding reservoirs or anabolic reactors, and the resultant wastewater generally does not receive any treatment prior to its land application (Bradford et al., 2008). Manure applied as fertilizer has been shown to improve soil condition for plant growth (Kapkiyai et al., 1999) and to increase the organic content of soil (Sommerfeldt & Chang, 1987). Although manure generated by CAFOs can be used as fertilizer, its runoff from agricultural fields presents environmental risks with regard to contaminants in these wastes, including excessive nutrients (Jongbloed & Lenis, 1998), salts (Hao & Chang, 2003), heavy metals (Barker & Zublena, 1995), microbial pathogens (Schets et al., 2005), antibiotics (Gilchrist et al., 2007), and natural and synthetic hormones (Liu et al., 2012).
The use of natural and synthetic hormones in CAFO agricultural practices is common, with over 90% of cattle on feedlots in the US treated with one or more growth-promoting steroids or steroid-like compounds (Gadd et al., 2010; USDA, 2000). For example, implants containing the anabolic steroid trenbolone acetate are subcutaneously inserted into cows, and two active metabolites, 17α -trenbolone (17α –TB) and 17β- trenbolone (TB), are excreted from the cow (Schiffer et al., 2001). The synthetic estrogen, 17α -ethynylestradiol (EE2), is used to increase livestock productivity (i.e. feed use efficiency), to treat diseases, and to regulate breeding in cattle (Gadd et al., 2010). While EE2 is released from CAFOs, the major source of EE2 in the environment is from human urine, as EE2 is the active ingredient in most birth control pills (Pauwels et al., 2008). Animal waste containing TB and EE2 can enter waterways through runoff from land application of manure or through leakage or spillage from manure lagoons (Burkholder et al., 2007). Analyses of water samples from a beef CAFO reported concentrations of 125 ng/L 17α –TB and 20 ng/L TB in a discharge drain that collects runoff, and 50 ng/L 17α –TB and 7 ng/L TB in a receiving stream located 381 meters downstream (Durhan et al., 2006). In waste storage lagoons, EE2 was detected at 195 ± 59.8 ng/L but was not detected in receiving streams (Liu et al., 2012). Although the present study focuses on the synthetic hormones EE2 and TB, farmers often use several other pharmaceuticals (i.e. methyl testosterone, dexamethasone, medroxyprogesterone, and norgestrel), based on their needs and desired production outcomes (Liu et al., 2012).
Another chemical of concern involving CAFO operations is atrazine (Leet et al., 2012). Atrazine is one of the most commonly used herbicides in agricultural practices and is frequently detected in streams in the US (Gilliom et al., 2006). Since CAFOs are often located close to agricultural fields, those fields are likely to have received both atrazine and CAFO manure, and runoff is likely to discharge both veterinary pharmaceuticals and herbicides into surface waters - especially following rain events (Stoeckel et al., 2012). Thus, CAFO runoff varies in chemical nature, yet the introduction of compounds into the environment occurs routinely (Bradford et al., 2008).
Exposure to compounds present in CAFO runoff can interfere with the hypothalamus-pituitary-gonadal axis leading to dysregulation of spermatogenesis and oogenesis (Bringolf et al., 2004; Tillitt et al., 2010). For example, fathead minnows (Pimephales promelas) exposed to 20 ng/L EE2 had fewer mature spermatozoa, with mainly spermatogonia and spermatocytes in the testes (Salierno & Kane, 2009). Additionally, ovotestis formation was reported in female western mosquitofish (Gambusia affinis) exposed to 1 or 10 µg/L TB for 28 days when exposure started at the fry stage, but not when exposure started at the adult stage, suggesting a crucial window of sensitivity to androgenic compounds (Sone et al., 2005). Lastly, fathead minnows exposed to 5 and 50 µg/L atrazine during active reproduction exhibited decreased egg production and induced gonadal abnormalities such as genetic males with testicular oocytes and ovaries with undifferentiated germ cells (Tillitt et al., 2010).
Traditionally, studies on aquatic organisms have analyzed the effects of individual compounds of CAFO runoff, however only a few investigations have studied the results for multiple constituents of CAFO runoff (Huang et al., 2012; Leet et al., 2015). Chemicals occur in the environment as mixtures, thus highlighting the necessity to conduct laboratory studies with mixtures in order to simulate real world environmental conditions. The present study used environmentally relevant concentrations of EE2, TB, and atrazine and their mixture to simulate CAFO runoff to assess whether exposure during the period of sexual differentiation in western mosquitofish (starting at 2–7 days post hatch [DPH]) would affect gonadal development. To evaluate gonadal development, the fish were dissected as adults and the ovaries and testes were histologically analyzed and compared to those of unexposed fish.