Potential of Concentrated Animal Feed Operations (CAFOs)To Contribute Estrogens to the Environment Principal Investigator: Stephen R. Hutchins US EPA NRMRL, Subsurface Protection and Remediation Division Robert S. Kerr Environmental Research Center, P.O. Box 1198, Ada, OK 74820 Phone: (580) 436-8563 Fax: (580) 436-8703 email: hutchins.steve@epa.gov Cooperative Agreement Support: James N. Dumont and David M. Janz Department of Zoology, Oklahoma State University, 430 Life Sciences West, Oklahoma State University, Stillwater, OK 74078 Phone: (405) 744-9683 Fax: (405) 744-7824 email: dumontj@okstate.edu Phone: (405) 744-7593 Fax: (405) 744-7824 email: djanz@okstate.edu Contract Support: G. Peter Breidenbach and Dennis D. Fine ManTech Environmental Services, Inc, Robert S. Kerr Environmental Research Center, P.O. Box 1198, Ada, OK 74820Phone: (580) 436-8668 Fax: (580) 436-8501 email: breidenbach.peter@epa.gov Phone: (580) 436-8669 Fax: (580) 436-8501 email: fine.dennis@epa.gov Concentrated Animal Feed Operations (CAFOs) • In the United States, an estimated 376,000 animal feed operations confine animals, generating approximately 128 billion pounds of manure each year • A facility is an animal feed operation (AFO) if animals are stabled/confined, or fed/maintained, for 45 days or more within any 12-month period, and the facility does not produce any crops, vegetation, or forage growth • Concentrated animal feed operations (CAFOs) are the largest of these and are regulated under the Clean Water Act. CAFOs are generally considered to be operations with more than 1000 animal units (AU) Manure Available for Land Application, 1997* (chart) Percentage Shareby > 1000 AU Operations: Row 1 Sector: Cattle, Total Manure (billion pounds): 32.9 Percentage Shareby > 1000 AU Operations: 83% Row 2 Sector: Dairy, Total Manure (billion pounds): 45.5 Percentage Shareby > 1000 AU Operations: 23% Row 3 Sector: Swine, Total Manure (billion pounds): 16.3 Percentage Shareby > 1000 AU Operations: 55% Row 4 Sector: Poultry, Total Manure (billion pounds): 33.5 Percentage Shareby > 1000 AU Operations: 49% *Office of Wastewater Management (Office of Water/USEPA) Website: (http://cfpub1.epa.gov/npdes/home/cfm) Environmental Estrogens • Refer to a wide range of anthropogenic or naturally occurring compounds that elicit estrogenic responses by mimicking endogenous estradiol • Natural Estrogens: -17ß-Estradiol (“Estradiol”), Estriol, Estrone, Equilin, Equilenin, Genistein • Synthetic Estrogens: -Ethinyl estradiol, Mestranol, Diethylstilbestrol •Other Compounds -o, p’–DDT, Nonylphenol, Bisphenol A Graphic images of the following chemicals: Estradiol, Estriol, Ethinyl Estradiol, Estrogen “Core”, Estrone Comparison of Estrogenic Activity in Terms of EC50 Measured by Yeast Estrogen Screen* Natural Estrogens Substance: Estradiol, Relative Ratio of Estrogenic Activity: 1.0 Substance: Estrone, Relative Ratio of Estrogenic Activity: 0.21 Substance: Estriol, Relative Ratio of Estrogenic Activity: 0.0013 Substance: 17ß-Estradiol-3-Sulfate, Relative Ratio of Estrogenic Activity: 0.000053 Phytoestrogens Substance: Genistein, Relative Ratio of Estrogenic Activity: Other Compounds Substance: Nonylphenol, Relative Ratio of Estrogenic Activity: Substance: Bisphenol A, Relative Ratio of Estrogenic Activity: *Matsui et al, 2000 Comparison of Estrogenic Activity in Terms of Plasma VitellogeninInduction* Substance: Minimum Aqueous Concentration Required for Vitellogenin Induction: Natural Estrogens Substance: Estrone, Minimum Aqueous Concentration Required for Vitellogenin Induction: 100 ng/L Substance: Estradiol, Minimum Aqueous Concentration Required for Vitellogenin Induction: 10 ng/L Synthetic Estrogens Substance: Ethinyl Estradiol, Minimum Aqueous Concentration Required for Vitellogenin Induction: 2 ng/L *Arcand-Hoy et al, 1998 Use and Expected Environmental Impact of Estrogenic Pharmaceuticals Prescribed and Sold in the U.S.* Human Use Pharmaceutical Estrogen Product: Oral Contraceptives (ethinyl estradiol, mestranol), Calculated Estrogen Use: 88 kg/yr, Expected Introduction Concentrations to the Aquatic Environment: 2.2 ng/L Pharmaceutical Estrogen Product: Hormone Replacement Therapy (conjugated estrogens), Calculated Estrogen Use: 1700 kg/yr, Expected Introduction Concentrations to the Aquatic Environment: 42 ng/L Animal Use (Cattle Only) Pharmaceutical Estrogen Product: Growth-Enhancement (estradiol), Calculated Estrogen Use: 580 kg/yr, Expected Introduction Concentrations to the Aquatic Environment: 14 ng/L *Arcand-Hoy et al, 1998 CAFO Contributions of Estrogens - Cattle • Growth Hormones-Estrogens (estradiol, estradiol benzoate) -Androgens (trenbolone acetate, testosterone propionate) -Progestins (progesterone) • For cattle, the estradiol concentration in the urine averages 13 ng/L • Estimated that at least 90% of feedlot cattle slaughtered in 1995 were administered growth-enhancing hormones • Cattle subjected to growth hormones generate urine with estradiol concentrations five-to sixfold greater CAFO Contributions of Estrogens - Poultry • Average estimated hormone concentrations per kg dry weight litter: -14 µg estrogens (estradiol, estrone) in male broilers-65 µg estrogens (estradiol, estrone) in female broilers-133 µg testosterone in male and female broilers • Field study shows sizeable edge-of-field losses of estradiol (20-2530 ng/L) and testosterone (10-1830 ng/L) in runoff from litter-amended grasslands (Finlay-Moore et al, 2000) • No growth hormones added: natural production of estrogens and testosterone • In 1998, the U.S. poultry industry produced almost eight billionbroilers with a total production of almost 12 billion kg litter•Estimated estrogen production: 160,000 –760,000 kg/year CAFO Contributions of Estrogens - Swine • No growth hormones added: natural production of estrogens and testosterone • Estrogen production in swine: ????????????????? Evaluation of CAFO Lagoon Effluents for EDC Activity using Bioassays Principal Investigator: James N. Dumont Department of Zoology 430 Life Sciences West Oklahoma State University Stillwater, OK 74078 Phone: (405) 744-9683 Fax: (405) 744-7824 email: dumontj@okstate.edu Co-Principal Investigator: David M. Janz Department of Zoology 430 Life Sciences West Oklahoma State University Stillwater, OK 74078 Phone: (405) 744-7593 Fax: (405) 744-7824 email: djanz@okstate.edu OSU CAFO EDC Study - Objective The objective of this study is to evaluate lagoon samples from swine, beef, and dairy CAFOs for possible EDC activity, using a variety of tests based upon the African Clawed Frog (Xenopus laevis) image of OSU Swine Lagoon image of OSU Beef Lagoon image of OSU Dairy Lagoon OSU CAFO EDC Study –Test Description FETAX (Frog Embryo Teratogenesis Assay – Xenopus) XTRA (XenopusTail Resorption Assay) Adult Male Frog Exposure - Vitellogen in Expression and Estradiol/Testosterone Changes OSU CAFO EDC Study –Test DescriptionFETAX -Frog Embryo Teratogenesis Assay - Xenopus • 96-hr assay that uses early stage Xenopusembryos (Stages 8-11, approximately 8-12 hours old) •Measures growth, mortality, and malformation for detection of potential developmentally toxic compounds and mixtures • Standardized Protocol (ASTM Standard Guide E 1439-91) • Not used for detection of EDC activity per se; needed for screening samples for potential toxicity to determine allowable concentrations OSU CAFO EDC Study –Test DescriptionXTRA –XenopusTail Resorption Assay • Tail resorption controlled by thyroxin –measures disruption of thyroid gland • Uses Stage 56/57 (approximately 38/40-day old) Xenopuslarvae • Four replicate tanks, 10 larvae per tank, water changed twice per week • Larvae photographed every other day; tail length measured by SigmaScan software • Test duration 15-20+ days (until metamorphosis is complete) OSU CAFO EDC Study –Test Description Adult Male Frog Exposure – Vitellogenin Expression and Estradiol/Testosterone Changes •Vitellogenin Analysis – Exposure of male oviparous vertebrates to natural and synthetic estrogens can induce synthesis of the phospholipoglycoproteinyolk precursor vitellogenin •Estradiol/Testosterone Analyses – Indicator of alterations in reproductive endocrine homeostasis Exposure: Four groups of 5 adult male Xenopusexposed for 21 days • Untreated Controls–reared in charcoal-filtered water • Positive Plasma Controls–reared in charcoal-filtered water; intraperitoneal injection of 1 mg/kg ethinylestradiol on Days 1, 3, and 6 • Positive Aqueous Controls–reared in charcoal-filtered water with 1 mg/L ethinylestradiol • Test Group–reared in CAFO lagoon effluentAnalyses • After exposure, plasma prepared and assayed for vitellogenin using Western immunoblotting and enzyme-linked immunoassay (ELISA), and assayed for estradiol and testosterone using enzyme-linked immunoassay (ELISA) OSU CAFO EDC Study –Conclusions Conclusions are Preliminary–Analyses Pending • Although the swine effluent lagoon is quite toxic, none of the lagoons have exhibited significant EDC activity, at least based on these bioassays • EDC activity may betruly insignificant in these CAFO lagoon effluents • However: - These lagoons may not be truly representative of large-scale commercial operations - EDC effects on steroid hormone homeostaesis may be more pronounced under long-term exposure OSU CAFO EDC Study – Poster Presentation 22ndAnnual SETAC Meeting, November 11-15, Baltimore, MD • Lagoon Water from Confined Animal Feed Operations and Amphibian Development Dumont, J.N.*, Oklahoma State University, Stillwater, OK Hutchins, S.R., U.S. EPA (NRMRL/SPRD), Ada, OK • Endocrine Modulating Effects of Lagoon Water from Confined Animal Feed Operations on Amphibians Weber, L.P.*, Dumont, J.N., and Janz, D.M., OSU, Stillwater, OK Selcer, K.W., Duquesne University, Pittsburgh, PA Hutchins, S.R., U.S. EPA (NRMRL/SPRD), Ada, OK Analysis of Environmental Estrogens in Swine Wastewater, using ELISA, LC/MS/MS, and GC/MS G. Peter Breidenbach Research Microbiologist ManTechEnvironmental Services, Inc. Robert S. Kerr Environmental Research Center P.O. Box 1198 Ada, OK 74820 Phone: (580) 436-8668 Fax: (580) 436-8501 email: breidenbach.peter@epa.gov Dennis D. Fine Analytical Chemist ManTech Environmental Services, Inc. Robert S. Kerr Environmental Research Center P.O. Box 1198 Ada, OK 74820 Phone: (580) 436-8669 Fax: (580) 436-8501 email: fine.dennis@epa.gov Environmental Estrogen Analysis – Objective • The objective of this study is to develop a protocol for screening and analyzing swine lagoon effluent and ground water for estrogens at environmental levels (ng/L) • Screening will be done using enzyme-linked immunoassay (ELISA) specific for estradiol. Positive samples will then be analyzed for individual estrogens • Individual estrogens will be analyzed by LC/MS/MS • LC/MS/MS Interferences - switch to GC/MS Environmental Estrogen Analysis – ELISA Screen Initial Procedure • Solid phase extraction of 250-mL sample using 6-mL ENVI-CARB SPE cartridge • Sequential washing with water, methanol/acetic acid, and methanol • Elution with methylene chloride/methanol • Evaporation to dryness; resuspension with water/methanol to 500 µL • Direct analysis of 20-µL aliquots by ELISA for estradiol•Estimate of estradiol concentration using external calibration curve Environmental Estrogen Analysis – ELISA Screen Preliminary Findings • Cross-reactivity: higher concentrations of other estrogens will produce a similar positive response in the estradiol ELISA screen - Estriol –Response is 1% that of estradiol - Estrone–Response is 1% that of estradiol - Ethinyl estradiol –Response is 0.2% that of estradiol • Swine lagoon effluent appears to exert a positive interference, in that estimated estradiol concentrations can be much higher than those confirmed by direct LC/MS/MS analysis Environmental Estrogen Analysis – LC/MS/MS Analysis Initial Procedure (Finnegan TSQ 7000 Mass spectrometer) • Analyze same SPE extract prepared for ELISA screening • Micro-liquid chromatography electrospray MS/MS method • 2-µL sample loop injection onto 5-µL Zorbax C18SB packed capillary column • Gradient elution with acetonitrile/water • Addition of ammonium hydroxide to column eluent prior to electrospray source to abstract phenoxy proton from the estrogen • Quantitate estrogen concentrations using calibration curves with estrone-d4as the internal standard Preliminary Results of Swine Lagoon Estrogen Analysis (all concentrations in ng/L original water sample) Chart contact Mr. Hutchins for description Environmental Estrogen Analysis –LC/MS/MS Analysis Potential Problems • LC/MS/MS analysis shows numerous organic compounds that elute inearly part of chromatograph that may cause positive response with ELISA screen • LC clean-up of complex samples (e.g., swine lagoon effluents) prior to ELISA screen, using gel permeation chromatography and silica gelprep to remove early-eluting interferences • Estrogenic response in LC/MS/MS (electrospray) system is suppressed when high organic interferences coelutewith compounds of interest (insufficient clean-up) Possible Solutions • Evaluate other SPE cartridges and/or alternate analytical techniques Pig graphic saying "So...What did you finally end up with??" Environmental Estrogen Analysis – SPE Method Final Procedure • Solid phase extraction of 500-mLwater sample or 25-mLswine lagoon effluent sample using OASIS HLB SPE cartridge • Sequential washing with water/methanol, water, and methanol/aqueous ammonium hydroxide • Elution with MTBE/methanol • Evaporation to dryness; - for ELISA screen, resuspendwith methanol to 250 µL and dilute 1:2 with water-for GC/MS analysis, resuspendwith acetone to 1000 µL Environmental Estrogen Analysis – ELISA Screen Final Procedure - used with water samples only • Direct analysis of 20-µL aliquots by ELISA for estradiol • Estimate of estradiolconcentration using external calibration curve • Concentration factor = 1000; detection limit ~ 0.05 ng/L estradiolin original water sample Environmental Estrogen Analysis – GC/MS Analysis Final Procedure (Finnigan 4600 Mass Spectrometer) • Prepare pentafluorobenzylderivatives of phenolicgroups and trimethylsilylderivatives of hydroxygroups • Analyze the derivatizedestrogens by GC/MS using a J&W DB5-MS capillary column and negative ion chemical ionization mass spectrometry • Quantitateestrogen concentrations using internal calibration curves and estrone-d4, estradiol-d3and ethinyl estradiol-d4as internal standards • Concentration factor = 25 (lagoon effluent) and 1000 (ground water); detection limit ~20 ng/L estrogen in lagoon effluent and ~0.5 ng/L in ground water Recovery of Estrogens Spiked in Distilled Water and in Swine Waste Lagoon Samples Distilled Water Spiked at 1 µg/L: Lagoon Effluent Spiked at 1 µg/L (duplicates): Row 1 % Recovery: 7-a-Methylestrone (surrogate), Distilled Water Spiked at 2ng/L: 110, Distilled Water Spiked at 1 µg/L: 85.2, Lagoon Effluent Spiked at 1 µg/L (duplicates): 86.2 87.4 Row 2 % Recovery: Estrone, Distilled Water Spiked at 2ng/L: 110, Distilled Water Spiked at 1 µg/L: 83.2, Lagoon Effluent Spiked at 1 µg/L (duplicates): 68.8 58.8 Row 3 % Recovery: Estradiol, Distilled Water Spiked at 2ng/L: 160, Distilled Water Spiked at 1 µg/L: 84.0, Lagoon Effluent Spiked at 1 µg/L (duplicates): 73.6 83.2 Row 4 % Recovery: Ethinyl estradiol, Distilled Water Spiked at 2ng/L: 110, Distilled Water Spiked at 1 µg/L: 90.8, Lagoon Effluent Spiked at 1 µg/L (duplicates): 86.0 88.0 Row 5 % Recovery: Estriol, Distilled Water Spiked at 2ng/L: 210, Distilled Water Spiked at 1 µg/L: 115, Lagoon Effluent Spiked at 1 µg/L (duplicates): 75.4 109 Environmental Estrogen Analysis – GC/MS Analysis Continuing Work •Investigate increasing concentrations of derivatizingreagents to improve quantitation of estriol •Improve recoveries of estrogens by using deactivated glassware and increasing concentrations of derivatizingagents End of text