Impacts of hormone loads associated with concentrated animal feeding operations on fish communities and fish sex differentiation
The occurrence of endocrine disrupting chemicals (EDCs) in concentrated animal feed operation (CAFO) waste, and the potential effects of these chemicals on aquatic ecosystems have been of recent concern, especially in large agricultural regions. The waste from CAFOs is often applied as efficient fertilizer to nearby agricultural fields, resulting in the runoff of hormones, pesticides, and nutrients into adjacent aquatic systems. There is a lack of research on the potential impacts of this runoff on fish in these systems, as well as a lack of tools available to use in investigating these impacts on early life stage fish. We utilized field and laboratory studies to investigate CAFO associated mixtures of chemicals on fish. We also characterized baseline expression of genes involved in sex differentiation in fathead minnows (Pimephales promales) to be used in assessing endocrine disruption during development. A field study was conducted in which fish community structure, reproductive condition, and growth were assessed in aquatic ditches adjacent to agricultural fields land-applied with CAFO waste. Fish species richness and index of biodiversity were significantly lower in these ditches compared to those in a reference creek with no CAFO impacts. We also found fish in the CAFO ditches had faster somatic growth than those in the reference creek. An in situ exposure of CAFO-impacted ditchwater was conducted on fathead minnows from embryos (< 24 h post fertilization, hpf) to 45 d post hatch (dph). These fish show significantly skew sex ratio toward males (60%) compared to those in a control group (49%). These fish also were significantly larger. Using a new technique to identify sex in early life stages, a study was conducted to evaluate sex-specific gene expression changes throughout development in fathead minnows for the first time. Six genes involved in sex differentiation and gonadal development were evaluated including: dmrt1, cyp19a, cyp17, star, esr1, ar. These data were used as baseline information to assess sex-specific changes in gene expression after exposure (5 ng/L) to a synthetic estrogen (17α-ethinylestradiol) and a synthetic androgen (17β-trenbolone) during a sensitive time in development (10–20 dph). Mostly, those genes upstream in the steroidogenesis process were effected (cyp17 and star), and esr1 was an indicator estrogen exposure. Similar patterns of expression changes were seen in an exposure of fathead minnows from embryo (< 24 hpf) to 20 dph to a mixture of chemicals in a laboratory setting similar to those found at CAFO impacted sites. Juveniles from this laboratory study of CAFO chemical mixture sampled at 45 dph did not have an altered sex ratio, but the majority of males in the exposure group did have the presence of ovarian cavities (not present in the control males) indicating a feminizing effect from this exposure. In conclusion, our results indicate that the fish community at a reference stream was more diverse compared to CAFO impacted sites. There was also evidence of faster somatic growth in fish at the CAFO sites, and there were indications they may be investing less in reproductive growth. We observed signs of endocrine disruption in both laboratory and in situ exposures to CAFO associated mixtures of chemicals (i.e. decreased resproductive conditions, increased growth, and gene expression changes). However, the results of these studies show contrasting effects, demonstrating the sensitivity of endocrine effects to differences in overall composition of estrogen and androgen concentrations in these mixtures. We were able to use a new molecular sex marker to establish the first sex-specific baseline expression of a small suit of genes involved in sex differentiation in fathead minnow. This tool was further utilized to assess sex-specific gene expression changes in response to hormone exposure in early life stage fathead minnows. These baseline data allowed us to further confirm the estrogenic effect of the CAFO chemical mixture used in our laboratory exposure study. The studies presented here can serve as a starting point to gain a better understanding of how CAFO associated hormones and chemical mixtures impact endocrine function in fish during development.
Sepulveda, Purdue University.
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