2009 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: August 1, 2008 through July 31,2010
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


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

(2) Steroids in aqueous litter mixtures (laboratory-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:

(1) Tillage practices can have a dramatic effect on runoff of water-soluble contaminants from poultry litter amended watersheds. The Turbo-till method, which buries some of the litter after application and roughens the soil surface, reduced steroid concentrations (Estrone - E1 and 17-β estradiol – E2) and estrogenicity (EEQ) in runoff compared to runoff from a No-till field. This Turbo-till practice also reduced the runoff of nitrogen and phosphorous compared to No-till application of litter (Figure 1);


Figure 1. Monthly nutrient runoff loads from NT and TT watersheds (2008, 2009, 2010).

(2) Results from laboratory microcosms containing runoff from poultry litter amended fields suggest that there are a number of dynamic processes occurring that effect E1, E2 and Vtg in fathead minnows. At a minimum, conjugated E1 becomes deconjugated and free E1 and E2 degrades. These processes are both the result of microbial action. Changes in the relative concentrations of E1 and E2 over time due to this microbial activity might explain differences in measured estrogens and, therefore, estrogenic activity since E2 is much more bioactive and causes greater induction of Vtg in fish than E1.


Figure 2. 2008 NT flume mesocosm changes in estrogens, estrogenicity, and fathead minnow Vtg over time. Red borders indicate fathead minnow exposure periods.



Figure 3. 2009 NT flume mesocosm changes in estrogens, estrogenicity, and fathead minnow Vtg over time. Red borders indicate fathead minnow exposure periods.

(3) In 2008, occurrence of testicular oocytes in male largemouth bass from six lakes and ponds on the Delmarva Peninsula ranged from 33% to 100%, with a mean across all sites of 63% (Table 1). Severity, estimated by number of oocytes/histological section, was generally very low. This is the first finding of intersex on the peninsula. Resampling of bass in 2009 from Tuckahoe Lake showed the occurrence of intersex varied from 0% (May) to 33% (August) (Table 2). Sampling from a large river on the peninsula (Pocomoke River) showed a constant occurrence of intersex (20%) in bass collected on both sample occasions. Severity was also low as in 2008, with no male fish having more than 4 oocytes detected;

Table 1:Delmarva Peninsula in 2008

 Table 2: Delmarva Peninsula in 2009

(4) In 2009, 20 male largemouth bass from fishing tournaments were examined and no testicular oocytes were found. These fish were caught in the Potomac River and in Mattawoman Creek, a tributary of the Potomac. In 2010, 24 tournament mortality fish caught in the Potomac River during spring tournaments were examined. Testicular oocytes were found in 4%.

(5) In 2010, laboratory experiments using different poultry litters and fathead minnows produced similar responses to those seen in runoff mesocosm studies from 2008 and 2009. Initial E1 levels were low but increased substantially before decreasing over the remaining study interval. Concentrations of E2 followed the same pattern but to a lesser degree reflecting lower initial concentrations. Based on the Perdue litter treatment, which was subsampled at frequent intervals, the time to peak E1 was Day 6, somewhat later than in mesocosm studies. This may reflect differences in microbial communities between field runoff and laboratory generated litter treatments. All three litters examined in 2010 induced Vtg gene expression and blood plasma protein production in male fathead minnows. Tysons, with the highest measured estrogens of the three litters, induced the greatest vitellogenic response. Perdue and Allens had similar responses despite apparently higher estrogen levels in the Allens treatment.

Figure 4. 2010 changes in estrogens (GC/MS) and fathead minnow Vtg (blood plasma and Vtg mRNA) over time. Red border indicates fathead minnow exposure period.


Future Activities:

All activities proposed in the original cooperative agreement have been completed. In addition, additional CAFO steroid work proposed in conjunction with the new Harry R. Hughes Center for Agro-Ecology, Inc. (USDA) antibiotics project funded in 2010 has been mostly completed. Estrogen samples from these 2010 experiments are currently being analyzed by LC/MS/MS and RIA for comparison with the GC/MS analyses already completed. Estrogenicity (EEQ) samples are currently being analyzed using an in-vitro estrogen-inducible reporter-gene assay. These EEQ samples will be compared to the estrogen analyses to determine relationships between the change in estrone and 17-β estradiol concentrations over time and estrogenicity. Estrogenicity in fish as measured by Vtg induction will be compared to the EEQ results to determine the relationship between an aquatic endpoint and a human cell line endpoint.


Blazer VS, Iwanowicz LR, Iwanowicz DD, Smith DR, Young JA, Hedrick JD, Foster SW, Reeser SJ. Intersex (testicular oocytes) in smallmouth bass Micropterus dolomieu from the Potomac River and selected nearby drainages. Journal of Aquatic Animal Health 2007;19:242-253.

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

Relevant Websites:

Progress and Final Reports:

Original Abstract
  • 2008 Progress Report
  • 2010 Progress Report
  • 2011 Progress Report
  • Final Report