Grantee Research Project Results
Final 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 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 was to determine the presence, persistence and biological effects of natural and synthetic hormones that may be released into the environment from concentrated animal feeding 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 were 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.
Summary/Accomplishments (Outputs/Outcomes):
Year 1 efforts focused on the validation of the sample collection, extraction and analytical techniques. A critical component of the overall study (Goal 1) was to determine levels of the target suite of hormones in runoff and subsurface tile drainage from selected sites on each of the cooperating Discovery Farms. In addition, measurements of hormone pools in on-farm sources and receptors were required. Therefore, our studies focused on the analytical challenges of measuring hormones at nanogram per liter (ng/L) concentrations in these complex environmental matrices. Our original plan for collecting the runoff/tile drainage samples was not viable from the perspective of hormone stability, as nearly all of the hormones that we had targeted for analysis were rapidly degraded from field-collected runoff samples. Therefore, we investigated the use of sodium azide, hydrochloric acid (HCl) and sulfuric acid (H2SO4) to inhibit the degradation of hormones and estrogenic and androgenic activities in samples of surface water runoff from cattle manure-amended fields during storage at 4°C. Hormones and hormone metabolites were extracted using solid phase extraction and analyzed using high performance liquid chromatography with tandem mass spectrometry. Estrogenic and androgenic activities were assessed by E-screen and A-screen, respectively. Results indicated significant degradation of estrogenic, androgenic and progestogenic hormones and activities, which was likely due to microbial activity, within hours of sample collection. The inclusion of internal standards provides a means to account for hormone losses due to extraction inefficiency and to some extent degradation. However, internal standards are unable to adequately account for significant losses and are not available for E-screen and A-screen. Sodium azide did not adequately inhibit androgen degradation at the concentration used (1 g·L-1). Acid preservation (HCl or H2SO4, pH 2) stabilized the estrogenic and androgenic activities and coupling acid preservation with the use of internal standards resulted in reliable and accurate recovery of a suite of androgens, estrogens and progestogens for up to 14 d of storage at 4°C (Havens, et al., 2010).
Efforts also were focused on optimizing extraction procedures for hormones associated with solids, including manure and soils. We evaluated the efficacy of three techniques (accelerated solvent extraction [ASE], soxhlet and sonication) for the extraction of a large suite of estrogens, androgens and progestogens, as well as their metabolites, from soils of varying characteristics (e.g., particle size, organic content, cation exchange capacity). The stability of hormones spiked into these soils and stored for 30, 90 and 180 days at -20°C also was investigated. Four experimental matrices (reagent sand, Antigo silt loam, soil with high clay content and soil with high organic content) were spiked with 50 μL of 10 μg·mL-1 (in methanol; final concentration 100 μg·kg-1) of a mix of hormones, hormone metabolites and isotopically-labeled standards (ISTDs). After equilibration, the spiked hormones were extracted, in triplicate, by ASE, soxhlet and sonication and analyzed using liquid chromatography with tandem mass spectrometry. The average recoveries of the 19 analytes extracted from all four matrices were 71± 18%, 69 ± 24% and 56 ± 34% for ASE, sonication and soxhlet, respectively. The recoveries of estriol-d3, 17β-estradiol-d5, α-zearalenol-d4 and testosterone-d5 spiked into antigo silt loam significantly decreased within 30 days of storage (Havens, 2011).
Year 2 and 3 efforts focused on collecting and analyzing samples from the Discovery Farms sites. An additional farm was added to the project: UW-Platteville’s Pioneer Farm (Objectives 2 and 3).
Surface water runoff and soil from three cattle and dairy farms that utilize best management practices were evaluated for hormone concentrations of a large suite of hormones (23 natural and synthetic compounds including estrogens, androgens and progestogens), estrogenic activity and nutrient concentrations. Surface water runoff was collected from weirs collocated with United States Geological Survey monitoring stations at the edge of each of six study fields from March 2008 to March 2010 and was analyzed for hormone concentrations using liquid chromatography with tandem mass spectrometry and for estrogenic activity using the E-screen bioassay. The majority of the runoff events occurred in February and March when the soil was frozen. Progesterone and 4-androstenedione were the most frequently detected hormones (42% and 36%, respectively) in runoff and occurred at concentrations greater than 100 ng·L-1. Most of the high progesterone and 4-androstenedione concentrations were found at the field that sustained cattle grazing during the winter and were likely due to frozen soil and cattle congregation near the monitoring station. Estrogens were detected in 19% of the runoff samples collected. Zearalenone, an estrogenic compound of fungal origin, also was found in some samples. Estrogenic activity was detected in runoff samples at estradiol equivalent concentrations ranging from 0.09 to 133 ng·L-1. The progesterone metabolite, 17,20-dihydroxyrogesterone, was the most frequently detected (14%) hormone in the collected soil samples. The hormones detected at the highest concentrations in soil samples were 5α-androstan-3,17-dione and 17β-trenbolone. The hormone concentrations in soils samples were generally less than 10 μg·kg-1. All soil samples had estradiol equivalent concentrations less than 1.0 μg·kg-1 (Havens, 2011).
Our efforts in exploring the ecological effects of steroid hormones (Objective 4) began with an experiment testing the hypothesis that effluent from a cattle-finishing operation in which the animals are administered Synovex implants containing trenbolone acetate (TNB), would have a negative impact on egg production in fathead minnows. The treatments were continuous 3-week exposure to the following doses of CAFO effluent: 0%, 0.1%, 0.3%, 1%, and 3%. The 100% CAFO effluent was collected from a holding lagoon at the CAFO study site. There was no significant effect of CAFO effluent on the number of eggs produced by fathead minnows. 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 due to rapid degradation of hormones associated with the liquid manure.
We then studied hormones that we found in the highest concentrations in our field sampling that did not have published information on their effects in fathead minnows: progesterone and 4-androstenedione.
In an effort to better understand the potential endocrine disrupting effects of progesterone in male fish, computer assisted sperm analysis (CASA) was used to evaluate the effects of this steroid on sperm motility in the fathead minnow (Pimephales promelas). The rationale for focusing on sperm motility is that certain progestins have been shown to bind to surface membrane receptors on fish spermatozoa and increase sperm swimming velocity. It was hypothesized, therefore, that sperm swimming velocity might be a useful indicator of progestin exposure in fish. Adult male fathead minnows (ages 6–12 months) were exposed to environmentally relevant doses of progesterone, both longer-term (1 week, in vivo exposure) and short-term (minutes, in vitro exposure). Sperm then were video recorded and analyzed by CASA. When fathead minnows were continuously exposed for 1 week to low levels of progesterone in vivo there was a significant dose-dependent reduction in sperm motility. There was no effect of short-term progesterone exposure on fathead minnow sperm swimming characteristics. Additional research is required to elucidate the mechanism by which progesterone alters sperm swimming in the fathead minnow (Murack, et al., 2011).
Experiments then were conducted to evaluate the effects of short-term exposure to environmentally relevant concentrations of progesterone on reproduction and embryonic development in the fathead minnow (Pimephales promelas). For the reproduction assay, groups of reproductively mature fish were exposed for 21 d to nominal concentrations of 0, 10, 100, and 1000 ng/L progesterone in a flow through system, and various key reproductive endpoints (e.g., egg number, fertilization success) were quantified throughout the exposure period. The embryonic development assay consisted of incubating fathead minnow eggs in static culture to quantify the effects of progesterone on early development and hatching success. Progesterone caused dose-dependent decreases in fecundity and fertility, and also significantly reduced GSI (gonadosomatic index) and vitellogenin gene induction in females. There were no effects of progesterone on early embryonic development or hatching success. Progesterone may be a significant endocrine disrupting chemical in fish (DeQuattro, et al., submitted).
4-Androstenedione (A4) is an active androgen in fishes and is a substrate in the biosynthetic pathways of other key fish steroid hormones (e.g., testosterone and estradiol-17β). Because this hormone was found in runoff samples from manure-amended fields, we explored whether exposure to exogenous A4, at ecologically relevant concentrations, adversely affects fish reproduction and early development. Reproductively mature fathead minnows were exposed for 26 days to nominal concentrations of 0, 10, 100, and 1000 ng/L A4 in a flow through system and various key reproductive endpoints (e.g., fecundity, fertilization success) were measured. Fertilized fathead minnow eggs were exposed to nominal concentrations of A4, in static cultures, to assess early development and hatching success. At termination of the fish short term reproduction assay, adults were euthanized, animals were weighed for whole body mass, and secondary sexual characteristics were assessed for abnormalities. Livers were collected to measure Vtg mRNA expression. Gonads were collected and weighed for GSI determination. A4 exposure significantly decreased male GSI but had no significant effect on fecundity and female GSI. There were no noticeable effects on fertilization, secondary sexual characteristics, and development hatchability.
Conclusions:
This research was undertaken to explore whether hormones from livestock production contribute to endocrine disruption in the environment. Our early finding that the hormones are rapidly degraded in runoff suggests minimal impact on aquatic organisms. Efforts that prevent manure from entering waterways (e.g., avoiding spreading manure on frozen soil, buffer strips) also will likely prevent hormones from entering waterways. Although the majority of our runoff and soil samples did not exhibit high levels of hormones, the two hormones that were most frequently found were progesterone and 4-androstenedione. These two hormones have not been studied to the extent that many other hormones have, especially in terms of occurrence and reproductive endpoints in fish. The finding that progesterone decreases egg production in fathead minnows is an important discovery as this has not been documented in the literature. Zearalenone also was found in some runoff samples at concentrations high enough to contribute to the estrogenic activity in the E-screen bioassay. Future work should explore the effects of zearalenone on fish reproductive endpoints.
Journal Articles on this Report : 6 Displayed | Download in RIS Format
Other project views: | All 27 publications | 6 publications in selected types | All 6 journal articles |
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DeQuattro ZA, Peissig EJ, Antkiewicz DS, Lundgren EJ, Hedman CJ, Hemming JDC, Barry TP. Effects of progesterone on reproduction and embryonic development in the fathead minnow (Pimephales promelas). Environmental Toxicology and Chemistry 2012;31(4):851-856. |
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DeQuattro ZA, Hemming JDC, Barry TP. Effects of androstenedione exposure on fathead minnow (Pimephales promelas) reproduction and embryonic development. Environmental Toxicology and Chemistry 2015;34(11):2549-2554. |
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Havens SM, Hedman CJ, Hemming JDC, Mieritz MG, Shafer MM, Schauer JJ. Stability, preservation, and quantification of hormones and estrogenic and androgenic activities in surface water runoff. Environmental Toxicology and Chemistry 2010;29(11):2481-2490. |
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Havens SM, Hedman CJ, Hemming JDC, Mieritz MG, Shafer MM, Schauer JJ. Comparison of accelerated solvent extraction, soxhlet and sonication techniques for the extraction of estrogens, androgens and progestogens from soils. Journal of Agricultural Chemistry and Environment 2014;3(3):103-120. |
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Havens SM, Hedman CJ, Hemming JDC, Mieritz MG, Shafer MM, Schauer JJ. Influence of acidification on the partitioning of steroid hormones among filtrate, filter media, and retained particulate matter. Journal of Environmental Quality 2016;45(5):1776-1781. |
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Murack PJ, Parrish J, Barry TP. Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas). Aquatic Toxicology 2011;104(1-2):121-125. |
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Relevant Websites:
http://uwdiscoveryfarms.org/ Exit
http://www.uwplatt.edu/pioneerfarm/ Exit
http://www.slh.wisc.edu/ Exit
Progress 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.