2008 Progress Report: Transport/Fate/Ecological Effects of Steroids from Poultry Litter & Evaluations of Existing/Novel Management Strategies

EPA Grant Number: R833418
Title: Transport/Fate/Ecological Effects of Steroids from Poultry Litter & Evaluations of Existing/Novel Management Strategies
Investigators: Fisher, Daniel J. , Kane, Andrew S. , Klauda, Ronald J. , Staver, Kenneth , VanVeld, Peter , Yonkos, Lance T.
Institution: Wye Research and Education Center , Maryland Department of Natural Resources , School of Medicine at the University of Maryland , Virginia Institute of Marine Science
EPA Project Officer: McOliver, Cynthia
Project Period: August 1, 2007 through June 30, 2010 (Extended to July 31, 2012)
Project Period Covered by this Report: July 1, 2007 through November 30,2008
Project Amount: $694,736
RFA: Fate and Effects of Hormones in Waste from Concentrated Animal Feeding Operations (CAFOS) (2006) RFA Text |  Recipients Lists
Research Category: Endocrine Disruptors , Health , Safer Chemicals

Objective:

(1) Multiple litter sources will be screened for fecal steroids;

(2) Steroids in aqueous litter mixtures (lab generated and field collected) will be monitored over time to determine degradation rates and pathways;

(3) Fish will be exposed to aqueous litter mixtures in laboratory assays to determine the affects of steroid degradation on bioactivity;

(4) Influences of agricultural management practices on steroid transport to surface waters will be investigated;

(5) Maryland Biological Stream Survey protocols will be applied to agriculturally impaired watersheds to assess possible community and population level disturbances resulting from fecal steroid exposure.

(6) Largemouth bass will be collected from lakes for determination of endocrine disruptive effects, primarily blood plasma vitellogenin and gonadal abnormalities (intersex);

(7) Male fathead minnows will be caged in lakes that receive runoff from poultry litter amended fields to look for induction of VTG.

Progress Summary:

The award was converted to a cooperative agreement to allow participation of U.S. EPA scientists who currently are working on endocrine disruption issues. Dr. Steve Hutchins and his group at the EPA's National Risk Management Research Laboratory in Ada, Oklahoma, analyzed aqueous samples for steroids using his GC-MS method. Dr. Jim Lazorchak and his group at the EPA's Molecular Indicator Research Branch in Cincinnati, Ohio, analyzed fish livers for vitellogenin (VTG) using a new VTG mRNA gene expression assay. Dr. Vickie Wilson of the EPA's National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division in Research Triangle Park, North Carolina, analyzed aqueous samples for estrogenicity using a novel in-vitro estrogen-inducible reporter-gene.
 
(1) Multiple litter sources will be screened for fecal steroids. Selected samples of broiler litter from the Delmarva Peninsula have been analyzed for steroids. Nine samples were collected and analyzed using an RIA method. The average 17β-estradiol concentration in these samples was 93.1 ng/g dry litter (S.D> = 32.77). The broiler litter used for the 2008 watershed application discussed below had an estradiol concentration of 101 ng/g dry litter.
 
(2) Steroids in aqueous litter mixtures (lab generated and field collected) will be monitored over time to determine degradation rates and pathways. Poultry litter was applied to a 33 acre no-till agricultural watershed in the summer of 2008. Runoff from this watershed was collected and analyzed for 17β-estradiol, estrone, 17α-estradiol, and estriol using GC-MS. Rain events produced runoff from this watershed 2 days post application and then 8 days post application. The majority of the estrogen runoff from these watersheds was estrone. Estriol was never detected in any runoff sample. After the first event the runoff contained 32 ng/L estrone, 9.2 ng/L 17β-estradiol, and 1.7 ng/L 17α-estradiol. Runoff from the second event contained only estrone at a concentration of 9.2 ng/L. When runoff from the first event was held and allowed to age, there was a rapid loss of 17β-estradiol with concentrations becoming nondetectable in 2 days. Estrone degraded more slowly with detectable levels (8.5 ng/L) remaining after 16 days. Results from this first year of the study indicate that the major estrogen in runoff from poultry litter amended watersheds is estrone and that estrone remains in aqueous solution for up to 16 days.
 
Estrogenicity of aqueous samples from the runoff and aging experiments were analyzed using a novel in-vitro estrogen-inducible reporter-gene assay. This is a very sensitive measure of an aqueous sample's estrogenicity. Results indicate that aqueous samples from the receiving pond mentioned above were still estrogenic after 16 days. In addition, samples from the no-till aging experiment were still estrogenic after 32 days. This indicates that, although these samples showed low levels of measurable estrogens, they were still estrogenic.
 
(3) Fish will be exposed to aqueous litter mixtures in laboratory assays to determine the affects of steroid degradation on bioactivity. Adult male fathead minnows were exposed in the laboratory to aged flume runoff from the no-till watershed and runoff that had been frozen to preserve any estrogenic compounds at levels found in the original runoff. Because of high, possibly toxic ammonia concentrations in the direct runoff from the litter (25 mg/L), the flume runoff was diluted 5-fold prior to the fish exposures. In addition, fish were exposed to water from the pond receiving the runoff from this no-till watershed. Analysis of fish for VTG was conducted using a blood plasma ELISA and a new liver VTG mRNA gene expression assay. VTG mRNA gene expression should provide a more rapid response to the presence of estrogens because the gene must first be turned on before VTG is produced in the liver and is present in circulating blood plasma. Results from the gene assay showed that VTG was detected in fish exposed for 9 days and in fish exposed from 4 to 8 days, indicating that the estrogenic effect from poultry litter is still present even after aging. In addition, VTG was measured in the fathead minnow blood plasma and mRNA after 9 days in both preserved and aged samples.
 
(4) Influences of agricultural management practices on steroid transport to surface waters will be investigated. Poultry litter also was applied to a 33 acre watershed adjacent to that used in (2) above. This watershed was tilled using a turbo-till apparatus that roughens the surface and buries some of the litter an inch or two under the surface. Two rain events occurred after litter application (2 above). During the first event, there were measurable concentrations of estrogens from the no-till watershed (2 above) but no runoff from the turbo-till watershed indicating the increased water holding capacity of the soil after turbo-tillage. In runoff from the second event, only estrone was detected in runoff from both watersheds. A composite sample of runoff from the no-till watershed had a concentration of 9.2 ng/L estrone, while a similar sample from the turbo-till watershed had an estrone concentration of only 3.9 ng/L. The turbo-till method reduced steroid concentrations by about 50%. Nutrient runoff also was reduced by using turbo-till. For example, both dissolved and total phosphorous runoff loads were reduced by 80% in May and 60% in June by using turbo-till. This shows that simple changes in tillage strategies can have dramatic effects in runoff losses of water soluble compounds.
 
(5) Maryland Biological Stream Survey (MBSS) protocols will be applied to agriculturally impaired watersheds to assess possible community and population level disturbances resulting from fecal steroid exposure. In the spring of 2007 and 2008, WREC and Maryland DNR scientists sampled five streams on the Delmarva Peninsula using the MBSS protocol. These streams were in areas where past WREC studies had found measurable levels of aqueous steroids following poultry litter application to adjacent fields. The fish and benthic community structure of these streams currently is being analyzed by Maryland DNR scientists. In addition, land use patterns, stream bank stability and water quality also were determined. Results of these analyses will be compared to sentinel MBSS sites routinely sampled by Maryland DNR.
 
(6) Largemouth bass will be collected from lakes for determination of endocrine disruptive effects, primarily blood plasma vitellogenin and gonadal abnormalities (intersex). Smallmouth and largemouth bass are species sensitive to the development of intersex in male fish. Because small streams on the Delmarva Peninsula have no smallmouth bass, our sampling effort for intersex has been concentrated on largemouth bass. In addition, the small streams on Delmarva that Maryland DNR samples for its MBSS program have very few largemouth bass. Because of this, in the spring of 2008, lakes in MD and DE on the Delmarva Peninsula with large populations of largemouth bass were sampled. These lakes receive runoff from watersheds that have a history of poultry litter application. Results indicate that no bass from these lakes had measurable levels of VTG. All of the lakes sampled did have intersex, as measured by the presence of oocytes (eggs) in male gonads. The frequency of occurrence of bass with intersex ranged between 33% and 100%, but the number of oocytes in each intersex bass was quite low, ranging from 2.4 to 11 depending on the lake sampled. This indicates a lower level of intersex than that found by the U.S. Geological Survey in its Potomac and Shenandoah River studies.
 
(7) Male fathead minnows will be caged in lakes that receive runoff from poultry litter amended fields to look for induction of VTG. Fish were caged in the MD lakes sampled in (6) above for 4 and 9 days. No VTG was detected in any caged fish.

Future Activities:

A laboratory experiment will be conducted with fathead minnows using pure estradiol and a mix of pure estradiol and pure testosterone to determine whether the presence of testosterone affects the induction of VTG in male and female fish. A second application of poultry litter will be conducted on the experimental watersheds at WREC. As in Year 1, no-till and turbo-till tillage practices will be compared for differences in loss of water soluble compounds including steroids and nutrients. This will give us 2 years of comparison of these different tillage practices. Fish will be exposed to runoff from the no-till watersheds at various times as it is allowed to age. Steroid concentrations (estradiol, estrone and testosterone) will be tracked, including some conjugates, as the litter ages. VTG will be measured in male fish to determine whether the litter loses its estrogenicity over time. In addition, estrogenicity changes over time will be monitored using the in-vitro estrogen-inducible reporter-gene. Largemouth bass will be sampled in a river system on the Delmarva Peninsula for intersex.

Journal Articles:

No journal articles submitted with this report: View all 9 publications for this project

Supplemental Keywords:

Steroids, nutrients, endocrine disruption, largemouth bass (Micropterus salmoides), fathead minnow (Pimephales promelas), intersex, plasma VTG, VTG mRNA, in-vitro estrogen-inducible reporter-gene, tillage management practices, EDC, endocrine disruptor

Progress and Final Reports:

Original Abstract
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011 Progress Report
  • Final Report