Grantee Research Project Results
2008 Progress Report: Assessing Occurrence, Persistence and Biological Effects of Hormones Released from Livestock Waste
EPA Grant Number: R833421Title: 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, 2007 through June 30,2008
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:
Year 1 efforts necessarily focused on the validation of the sample collection, extraction and analytical techniques. A critical component of the overall study is to determine levels of the target suite of hormones in runoff and subsurface tile drainage from selected sites on each of the cooperating farms. In addition, measurements of hormone pools in on-farm sources and receptors are required. Our studies have therefore focused on the analytical challenges of measuring hormones at nanogram per liter (ng/L) concentrations in these complex environmental matrices. Our studies over the past 9 months have demonstrated that nearly all of the hormones that we are targeting for analysis were rapidly degraded from field-collected runoff samples. We subsequently determined through an extensive series of experiments that this loss was primarily a microbial degradation phenomenon, and was relevant on time-scales of hours to days. Even grab samples that we collected and immediately filtered and stored frozen showed substantial loss of hormones. Therefore, we devoted much of the last quarter of calendar year 2007 and athe first quarter of calendar year 2008 to developing appropriate methods to collect and stabilize the samples designated for hormone characterization.
From this work we determined that immediate acidification (using hydrochloric acid or sulfuric acid) or sodium azide works to prevent hormone loss. Additionally, the use of dueterated internal standards (dISTD) has proven essential for accurate HPLC-MS/MS quantification. We have secured 11 dISTDs out of 23 target compounds and will be obtaining 3 more.
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. An additional focus this year has been building and deploying the appropriate sampling infrastructure (especially event sensitive) at the participating Discovery Farms sites and progress has been steady.
Spring (2008) 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.
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. 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.
Preliminary results indicated that juvenile fish exposed for 3 days to 3% CAFO effluent had significantly higher baseline and stress-induced blood concentrations of cortisol. An advantage of using the stress response as an indicator of contaminant exposure in fish is that it eliminates the inherent variability associated with using reproductive endpoints, and the reliance on reproductively mature individuals.
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).
Future Activities:
Future activities will focus on (a) continuing our implementation of the sampling strategy at the Discovery Farms; (b) finalizing extraction and clean-up steps for solid samples; 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). 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. Selected compounds also may 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 projectSupplemental Keywords:
bioassay, LC-MS-MS, fathead minnow, vitellogenin, endocrine disruption, reproduction, manure CAFO, CAFO effluentRelevant Websites:
http://www.uwdiscoveryfarms.org/ ExitProgress and Final Reports:
Original AbstractThe 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.