Grantee Research Project Results

2009 Progress Report: Assessing Occurrence, Persistence and Biological Effects of Hormones Released from Livestock Waste

EPA Grant Number: R833421
Title: Assessing Occurrence, Persistence and Biological Effects of Hormones Released from Livestock Waste
Investigators: Hemming, Jocelyn , Schauer, James J. , Shafer, Martin M. , Barry, Terence
Institution: University of Wisconsin - Madison
EPA Project Officer: Aja, Hayley
Project Period: July 1, 2007 through June 30, 2010 (Extended to June 30, 2011)
Project Period Covered by this Report: July 1, 2008 through June 30,2009
Project Amount: $699,543
RFA: Fate and Effects of Hormones in Waste from Concentrated Animal Feeding Operations (CAFOS) (2006) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Human Health , Safer Chemicals

Objective:

The overall goal is to determine the presence, persistence and biological effects of natural and synthetic hormones that may be released into the environment from concentrated animal feed operations (CAFOs), and evaluate the effects of different animal waste disposal practices on the fate and activity of these compounds. This research will help to evaluate whether CAFO waste is an important source of endocrine disrupting chemicals in the environment. The specific objectives are to:

Identify and quantify the suite of estrogenic, androgenic and progesteronic compounds associated with various types of intensive animal farming.
Characterize the environmental transport and fate of natural and synthetic steroid hormones that accompany discharges and the disposal of animal wastes from CAFOs in Wisconsin.
Evaluate how various animal waste handling/management strategies (e.g., lagoon storage and spraying of liquid manure vs. deep-stacking and field application of solid manure) impact the transport, fate, potential exposure, and associated effects of steroid hormones discharged from CAFOs.
Investigate the ecological effects associated with steroid hormones in animal waste from CAFOs using reproductive, developmental and gene expression endpoints in fathead minnows.

Progress Summary:

Initial efforts necessarily focused on the validation of the sample collection, extraction and analytical techniques. Our early studies demonstrated that nearly all of the hormones that we are targeting for analysis were rapidly degraded from field-collected runoff samples. From this work we determined that immediate acidification works to prevent hormone loss. Additionally, the use of deuterated internal standards (dISTD) has proved essential for accurate HPLC-MS/MS quantification. We have secured 14 dISTDs out of 23 target compounds.

CAFO samples are being collected from participants in the Wisconsin “Discovery Farms” program, which is a consortium of privately owned farms working with university and government researchers to study the viability, effectiveness, and economics of environmental regulations on large-scale farming enterprises. In addition, samples are being collected from University of Wisconsin-Platteville’s Pioneer Farms. Including all sites, nearly 50 events (e.g. rain or snow melt) have been sampled. Additionally soil samples have been collected from the monitored fields.

Spring (08) Snowmelt Runoff Samples

In the spring of 2008, grab samples were collected during snow melt event flows from three Discovery Farm sites: a steer facility (surface water), a dairy (subsurface tiles), and a dairy and steer facility (3 surface water sites, 1 subsurface tile site).

Seven hormones were identified and quantified using HPLC-MS/MS in the snow-melt grab samples. These include: estradiol, a-zearalanol, zearalenone, 4-androstene-3,17-dione, 17b-nortestosterone, progesterone, and 17,20-dihydroxyprogesterone. Levels of most hormones were <100 ng/L, though concentrations of the most prevalent hormones (progesterone, 17,20-dihydroxy-progesteone, and zearalenone) ranged up to 350 ng/L. Estrogenic activity from the snow melt samples were also determined using the E-screen bioassay. In this assay, the only target compounds that should elicit a response would be estriol, estradiol, estrone and the zearanols. In general, trends in the E-screen bioassay were consistent with the chemical profiles of the samples.

Preliminary results indicate similar patterns of hormones found in runoff samples collected with composite samplers during the 2009 snowmelt.

Fathead Minnow Exposures

Three percent CAFO effluent (from a steer facility) had no effect on the number of eggs produced by fathead minnows during a three week exposure. In addition, the levels of vitellogenin expression in the exposed males were similar to their respective controls. The levels of hormones may have been too low to cause an effect due to the dilution of effluent (necessary to prevent ammonia toxicity to the fish) or rapid degradation of hormones associated with the liquid manure.

In vitro studies were conducted with mature fathead minnow oocytes to evaluate the impact of pure trenbolone acetate (TNB) on final oocyte maturation. TNB inhibited final oocyte maturation in a dose-dependent manner suggesting that hormones associated with CAFO effluent may have an impact on oocyte maturation without necessarily altering the number of eggs laid per female. Therefore, given these findings, we will carefully quantify fertilization percentage in future exposure studies. In developing fathead minnow embryos, trenbolone acetate caused dose-dependant mortalities and developmental abnormalities (e.g., pericardial edema).

We completed a study using Computer Assisted Sperm Analysis (CASA) to investigate the acute effects of various progestins on sperm motility in fathead minnows. Semen samples were exposed short-term in vitro to various doses of progesterone, 17a,20b-P, or melengestrol acetate (MA). The sperm were activated, video recorded, and analyzed for the following parameters using CASA: percent motility, curvilinear velocity, velocity average path, straight line velocity, and percent linearity. Preliminarily, short-term progesterone exposure had a slight, but significant effect on fathead minnow sperm motility.

Future Activities:

Future activities will focus on: (a) preparing manuscripts for publication on the preservation of runoff samples and extraction optimization of soil samples; (b) completing sample collection from Discovery and Pioneer Farm sites; and (c) characterizing the target hormone suite with HPLC-MS/MS and hormonal activity with in vitro bioassays (A-screen, E-screen and transfected yeast). Additional work will be conducted on fractionating extracts for a TIE-type approach for identifying compounds responsible for hormone activity in the sample extracts. Future work with fish will investigate the effects of other steroids associated with CAFO effluent, alone and in combination, especially on fathead oocyte maturation and embryonic development. Three week adult exposures with reproductive and gene expression endpoints are also planned. Selected compounds may also be tested to evaluate their effects on developing zebrafish embryos to take advantage of this model species.

Journal Articles:

No journal articles submitted with this report: View all 27 publications for this project

Supplemental Keywords:

bioassay, LC-MS-MS, fathead minnow, vitellogenin, endocrine disruption, reproduction, manure.

Relevant Websites:

http://www.uwdiscoveryfarms.org/

http://www.uwplatt.edu/pioneerfarm/

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.