Grantee Research Project Results
Final Report: An Integrated Approach to Developing a Total Facility Estrogen Budget at a Swine Farrowing CAFO
EPA Grant Number: R833420Title: An Integrated Approach to Developing a Total Facility Estrogen Budget at a Swine Farrowing CAFO
Investigators: Kullman, Seth W. , Meyer, M. T. , Reckhow, Kenneth H.
Institution: North Carolina State University , Nicholas School of the Environment and Earth Sciences , Organic Geochemistry Research Laboratory
EPA Project Officer: Aja, Hayley
Project Period: February 1, 2008 through January 31, 2013 (Extended to January 31, 2014)
Project Amount: $663,532
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:
Little information is available regarding the concentration, release, fate and transport of estrogenic compounds in animal waste treatment and storage facilities. Naturally occurring estrogens in animal wastes present an emerging risk to terrestrial and aquatic environments through their potential release and action as endocrine disruptors. Given the trend in agriculture toward concentrated animal feeding operations and the extensive volume of waste generated, potential for environmental impact is plausible. Yet large data gaps exist in the overall understanding of generation of natural hormones, their fate throughout these facilities and factors governing transport to environmental matrices. In this project, we address these data gaps and have developed a comprehensive assessment of hormonal loads from a prototypic swine CAFO including quantitative information regarding reproductive status and hormone excretion by individual animals, the stability of natural hormones in open pit holding lagoons, and fate and attenuation of natural hormones spray field application of swine waste as fertilizer. These studies are organized through evaluating hormone fate and transport within defined facility compartment and operational units. By creating a hierarchical structure, assessments of natural hormones fate was “parameterized” and used for input into a Bayesian network model. Three specific aims were addressed: (1) establish “total facility estrogen budget” based upon composite measurements of natural hormones throughout a swine farrowing (CAFO); (2) develop a Bayesian network model that characterized causal relationships for a total facility estrogen budget in a probabilistic manner; and (3) assess the estrogenic activity of CAFO extracts in an in vivo medaka fish model. Our experimental approach was designed as “proof of principle” to generate a comprehensive understanding of the generation, fate and transport and putative biological effects of natural hormones derived from animal feeding operations.
Figure 1. Project field site, showing barns, lagoons, and surrounding sprayfields of the two separate farms. Lagoon and barns that were sampled for this project are indicated in Figure 2.3, below. Source: Google Earth Version 5.1.3533.1731.
Summary/Accomplishments (Outputs/Outcomes):
Given the prevalence of animal feeding operations (AFOs) in the modern agricultural landscape, the need to understand the behavior of emerging contaminants on these operations is unprecedented. The first major objective of this dissertation was to assess the occurrence and mass flux of two major classes of natural manure-borne endocrine disrupting compounds, steroid hormones and phytoestrogens, in the waste of a prototypical North Carolina swine sow operation. In addition to the assessment of analyte concentrations using LC/MS-MS, which was performed at the U.S. Geological Survey Organic Geochemistry Research Laboratory, the estrogenic activity of these samples was tracked using two in vitro estrogen screening assays: the yeast estrogen screen (YES) and the T47D-KBluc.
Comprehensive Assessment of Hormones, Phytoestrogens, and Estrogenic Activity in an Anaerobic Swine Waste Lagoon:
In aim 1 of this study, the distribution of steroid hormones, phytoestrogens, and estrogenic activity was thoroughly characterized within the anaerobic waste lagoon of a typical commercial swine sow operation. Three independent rounds of sampling were conducted in June 2009, April 2010, and February 2011. Thirty-seven analytes in lagoon slurry and sludge were assessed using LC/MS-MS, and yeast estrogen screen was used to determine estrogenic activity. Of the hormone analytes, steroidal estrogens were more abundant than androgens or progesterone, with estrone being the predominant estrogen species. Conjugated hormones were detected only at low levels. The isoflavone metabolite equol was by far the predominant phytoestrogen species, with daidzein, genistein, formononetin, and coumestrol present at lower levels. Phytoestrogens were often more abundant than steroidal estrogens, but contributed minimally towards total estrogenic activity. Analytes were significantly elevated in the solid phases of the lagoon; although low observed log KOC values suggest enhanced solubility in the aqueous phase, perhaps due to dissolved or colloidal organic carbon. The association with the solid phase, as well as recalcitrance of analytes to anaerobic degradation, results in a markedly elevated load of analytes and estrogenic activity within lagoon sludge. Overall, findings emphasize the importance of adsorption and transformation processes in governing the fate of these compounds in lagoon waste, which is ultimately used for broadcast application as a fertilizer.
Figure 2. Analyte concentrations (LC/MS-MS results) and EEQs (YES results) in lagoon slurry and sludge (liquid, grey bars; solids black bars).
Mass Flux of Hormones, Phytoestrogens, and Estrogenic Activity on a Swine Sow Operation:
As a second component of aim 1, the influx, transformation, and attenuation of steroid hormones, phytoestrogens, and estrogenic activity was assessed across all units of a prototypical swine sow operation. In urine and feces of sows across four defined reproductive stages, a variety of free and conjugated estrogen and androgen species were detected, and progesterone was generally the most abundant steroid hormone overall. Excreta also contained phytoestrogens indicative of a soy-based diet—particularly the isoflavones daidzein, genistein, and equol. During storage in barn pits and in the anaerobic lagoon, conjugated hormones dissipated, and concentrations of androgens and progesterone were sharply attenuated. Waste storage also appeared to favor the formation of estrone and equol as transformation products of steroidal estrogens and isoflavones, respectively. Following the land application of lagoon slurry to agricultural soils, all analytes and estrogenic activity exhibited initial attenuation within 2 days. However, compounds including estrone, androstenedione, progesterone, and equol were still detected in soil at 61-days post-application. Estrogenic activity in the yeast estrogen screen and T47D-KBluc in vitro assays tracked well with reported analyte concentrations, although concordance between these assays and LC/MS-MS results varied depending upon sample type. Across samples, estrone was determined to be the most estrogenically important analyte in the sow waste. Results provide comprehensive insight into the fate of these analytes in this widely used waste management system.
Figure 3. Flow of waste through the barns, lagoon, and sprayfield on this prototypical swine flow operation.
A Bayesian Network Model for Assessing Natural Estrogen Fate and Transport in a Swine Waste Lagoon:
Commercial swine waste lagoons are regarded as a major reservoir of natural estrogens, which have the potential to produce adverse physiological effects on exposed aquatic organisms and wildlife. However, there remains limited understanding of the complex mechanisms of physical, chemical, and biological processes that govern the fate and transport of natural estrogens within an anaerobic swine lagoon. To improve lagoon management and ultimately help control the offsite transport of these compounds from swine operations, in aim 2 we developed a probabilistic Bayesian network model to assess natural estrogen fate and budget and then compared against data collected from a commercial swine field site. In general, the model was able to describe the estrogen fate and budget in both the slurry and sludge stores within the swine lagoon. Sensitivity analysis within the model, demonstrated that the estrogen input loading from the associated barn facility was the most important factor in controlling estrogen concentrations within the lagoon slurry storage, while the settling rate was the most significant factor in the lagoon sludge storage. The degradation reactions were shown to be minor in both stores based on prediction of average total estrogen concentrations. Management scenario evaluations demonstrated that the best possible management options to reduce estrogen levels in the lagoon are either to adjust the estrogen input loading from swine barn facilities or to effectively enhancing estrogen bonding with suspended solids through the use of organic polymers or inorganic coagulants.
Figure 4. Mass balance equations for total estrogens with major mechanisms in the lagoon slurry and sludge layers.
Bridging the Gap from Screening Assays to Estrogenic Effects in Fish: Potential Roles of Multiple Estrogen Receptor Subtypes:
The third aim of the study was designed to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants, and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens, or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hERα. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hERα and hERβ, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ERβ subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ER subtypes are critically involved in vitellogenesis and choriogenesis, with the ERα:ERβ ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures.
Figure 5. Competitive ligand binding of steroidal estrogens and swine lagoon extract to medaka estrogen receptors.
Bio-analytical screening assays:
Throughout this study, estrogen screening assays (YES and T47D-KBluc) were used as tools to track estrogenic activity on the swine operation, and to determine the relative contribution of individual analytes to total estrogenic activity within each unit of the farm. Although matrix interference was found to be a confounding issue for certain subsets of samples (in particular, feces and barn flush solids), a strong correlation was observed overall between assay-derived 17β-estradiol equivalents (EEQs) and LC/MS-MS results, affirming the utility of these assays for assessing the presence of estrogenic compounds in a range of environmental samples.
Figure 6. Concentration-response curves for representative steroidal estrogens, phytoestrogens, and androgens in the YES assay, demonstrating the relative estrogenic potency of these compounds.
Limitations: One notable limitation of our AFO study was the inability to assess surface runoff from the sprayfield soil, which would have provided considerable information regarding the mobility and ultimate environmental risk posed by these compounds. In our laboratory’s original proposal for this project, we had planned to conduct field sampling at a different commercial swine sow AFO, which had groundwater wells and adjacent surface waters that we intended to monitor for analytes and estrogenic activity. However, our permission to use that field site was withdrawn by the landowner prior to the onset of our study in 2008, and we were forced to modify our approach. We were very fortunate to gain access to our current field site, from which all data in this study was collected; however, this site does not have drains, wells, or any surface water from which to monitor analyte mobility. Based on our experience as well as that of others, we feel that much could be gained if incentives and/or protection were provided to landowners to promote the use of commercial AFOs for scientific research. Nevertheless, the results from our study do provide pertinent information on the persistence of manure-borne EDCs in agricultural soils.
Conclusions:
Results from this study provide useful information on the mass flux of hormones, phytoestrogens, and estrogenic activity through the waste management system of a swine sow AFO. As the design of our field site is prototypical for swine AFOs in the southeastern and midwestern United States, these findings may be highly pertinent to other operations of this type, and allow insight into potential effects of operational practices (e.g., holding of waste in barn pits, sludge accumulation, slurry land application) on the mass loading of these compounds. In regards to assessing potential effects on aquatic populations, our results additionally provide insight into the translational capacities of in vitro screening assays for ecological risk assessment. Given the global push towards the development of high-throughput assays for the screening of toxicants, perhaps more studies of this nature are necessitated in order to anchor these assays to actual biological effects in exposed organisms.
Journal Articles on this Report : 6 Displayed | Download in RIS Format
Other project views: | All 28 publications | 8 publications in selected types | All 8 journal articles |
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Knuth M, Mahapatra D, Jima D, Wan D, Hammock B, Law M, Kullman S. Vitamin D deficiency serves as a precursor to stunted growth and central adiposity in zebrafish. NATURE 2020;10(1):16032 |
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Knuth M, Stutts W, Ritter M, Garrard K, Kullman S. Vitamin D deficiency promotes accumulation of bioactive lipids and increased endocannabinoid tone in zebrafish. JOURNAL OF LIPID RESEARCH 2021;62:100142 |
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Lee B, Kullman SW, Yost EE, Meyer MT, Worley-Davis L, Williams CM, Reckhow KH. Predicting characteristics of rainfall driven estrogen runoff and transport from swine AFO spray fields. Science of The Total Environment 2015;532:571-580. |
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Lee Pow CSD, Yost EE, Aday DD, Kullman SW. Sharing the roles: an assessment of Japanese medaka estrogen receptors in vitellogenin induction. Environmental Science & Technology 2016;50(16):8886-8895. |
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Yost EE, Meyer MT, Dietze JE, Meissner BM, Worley-Davis L, Williams CM, Lee B, Kullman SW. Comprehensive assessment of hormones, phytoestrogens, and estrogenic activity in an anaerobic swine waste lagoon. Environmental Science & Technology 2013;47(23):13781-13790. |
R833420 (2011) R833420 (Final) |
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Yost EE, Lee Pow C, Hawkins MB, Kullman SW. Bridging the gap from screening assays to estrogenic effects in fish: potential roles of multiple estrogen receptor subtypes. Environmental Science & Technology 2014;48(9):5211-5219. |
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Supplemental Keywords:
CAFO, AFO, natural hormones, estrogenProgress 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.
Project Research Results
- 2012 Progress Report
- 2011 Progress Report
- 2010 Progress Report
- 2009 Progress Report
- 2008 Progress Report
- Original Abstract
8 journal articles for this project