AssessingAssessing occurrence,occurrence, persistencepersistence andand biologicalbiological effectseffects ofof hormoneshormones releasedreleased fromfrom livestocklivestock wastewaste JocelynJocelyn Hemming,Hemming, MartinMartin Shafer,Shafer, TerenceTerence BarryBarry andand JamesJames SchauerSchauer UniversityUniversity ofof WisconsinWisconsin--MadisonMadison OverallOverall ProjectProject GoalGoal

ToTo determinedetermine thethe presence,presence, persistencepersistence andand biologicalbiological effectseffects ofof naturalnatural andand syntheticsynthetic hormoneshormones thatthat maymay bebe releasedreleased intointo thethe environmentenvironment fromfrom CAFOsCAFOs,, andand evaluateevaluate thethe effectseffects ofof differentdifferent animalanimal wastewaste disposaldisposal practicespractices onon thethe fatefate andand activityactivity ofof thesethese compounds.compounds. SpecificSpecific ObjectivesObjectives

„ CharacterizeCharacterize thethe environmentalenvironmental transporttransport andand fatefate ofof naturalnatural andand syntheticsynthetic steroidsteroid hormoneshormones thatthat accompanyaccompany dischargesdischarges andand thethe disposaldisposal ofof animalanimal wasteswastes fromfrom CAFOsCAFOs inin Wisconsin.Wisconsin. „ EvaluateEvaluate howhow variousvarious animalanimal wastewaste handling/managementhandling/management strategiesstrategies (e.g.,(e.g., lagoonlagoon storagestorage andand sprayingspraying ofof liquidliquid manuremanure vs.vs. deepdeep-- stackingstacking andand fieldfield applicationapplication ofof solidsolid manure)manure) impactimpact thethe transport,transport, fate,fate, potentialpotential exposure,exposure, andand associatedassociated effectseffects ofof dischargeddischarged steroidsteroid hormones.hormones. „ InvestigateInvestigate thethe biologicalbiological effectseffects associatedassociated withwith steroidsteroid hormoneshormones inin animalanimal wastewaste fromfrom CAFOsCAFOs.. ExperimentalExperimental ApproachApproach PartneringPartnering withwith DiscoveryDiscovery FarmsFarms

Established to support and facilitate research on best management practices at operating private farms ExperimentalExperimental ApproachApproach

Study Farm Characteristics and Settings

Location Monitoring Animals Setting (WI) Infrastructure Lafayette 600 steer finishing Un-glaciated. Black 3-surface water flumes and Co. (confined). silt loam over brown auto-samplers in farm 300 acres silt loam. 6% slopes. fields & waterways. Met. station. Buffalo Co. 180 cows, 80-100 Un-glaciated. Silty 2-gauged streams, 2- 1010 acres steers, 32,000 soils. Moderate automated water samplers. pullets slopes. Meteorology station. Dunn Co. 10,000 hog Forested silty soils Extensive soil sampling. 852 acres finishing over sandstone. Lake on-site. Moraine. Iowa Co. 120 cow (organic Un-glaciated. Silty 2-stream + 1-field 480 acres grass-based dairy) soils. Moderate monitoring station. Two slopes. ponds, stream, springs, on- site. Kewaunee 1400 dairy cows, Thin silt loam over 3-surface, 2-tile drain- Co. 4000 permitted CAFO clay loam over flumes and auto-samplers acres fractured dolomite. in farm fields & 4% Slopes. waterways. Met. Sta. Dane Co. 150-200 steer Silty praire soils – Soil Monitoring 2040 acres finishing (semi- ground moraine. Meteorology station. confined). Shallow slopes. ProposedProposed CAFOCAFO--HormoneHormone SamplingSampling ProgramProgram „ EstablishEstablish hormonehormone useuse profileprofile ofof eacheach studystudy farmfarm „ EstablishEstablish wastewaste handlinghandling practicespractices atat eacheach studystudy farmfarm „ ValidateValidate hormonehormone ““fingerprintfingerprint”” inin primaryprimary sourcessources „ DetermineDetermine hormonehormone levelslevels inin receptorsreceptors underunder baselinebaseline hydrologichydrologic conditionsconditions andand variousvarious wastewaste managementmanagement practicespractices andand farmfarm crop/tillagecrop/tillage operationsoperations.. „ DetermineDetermine hormonehormone levelslevels inin receptorsreceptors underunder eventevent hydrologichydrologic conditionsconditions andand variousvarious wastewaste managementmanagement practicespractices andand farmfarm crop/tillagecrop/tillage operations.operations. ExperimentalExperimental ApproachApproach FieldField SamplingSampling StrategyStrategy

„ TileTile Drains/FieldDrains/Field StreamsStreams „ AutomatedAutomated samplerssamplers--eventevent drivendriven „ ShallowShallow SoilSoil-- andand GroundGround WaterWater „ MultiMulti--levellevel piezometerspiezometers toto monitormonitor movementmovement intointo thethe shallowshallow groundground waterwater systemsystem „ SoilsSoils „ WasteWaste StorageStorage FacilitiesFacilities „ FieldField--appliedapplied ManureManure SlurrySlurry ExperimentalExperimental ApproachApproach ReceivingReceiving StreamsStreams --WatershedsWatersheds

„ StreamStream SamplesSamples „ BaselineBaseline „ AutomatedAutomated samplerssamplers--eventevent drivendriven „ StreamStream SedimentsSediments „ <128<128 micronmicron fractionfraction „ AlgaeAlgae „ NativeNative FishFish ExperimentalExperimental ApproachApproach AssessingAssessing WasteWaste Management/UtilizationManagement/Utilization EffectsEffects onon HormonesHormones

„ CAFOCAFO vs.vs. GrazingGrazing

„ TillTill vs.vs. nono--tilltill

„ CropCrop varietyvariety andand typetype

„ SoilSoil typetype andand slopeslope ExperimentalExperimental ApproachApproach TargetTarget HormonesHormones Biological Effect Origin Hormone [CAS #] Estrogen Natural 17β-Estradiol [50-28-2] 17α-Estradiol [57-90-1] Estrone [53-16-7] Estriol [50-27-1] Synthetic Zeranol (α-zearalanol) [26538-44-3] α-Zearalenol [36455-72-8] Zearalanon [5975-78-0] Zearalenon [17924-92-4] Taleranol (β-zearalanol) [42422-68-4]

Androgen Natural Testosterone [58-22-0] 5α-Androstan-17β-ol-3-one (DHT) [521-18-6] Androsterone [53-41-8] 5α-Androstane-3,17-dione [846-46-8] 4-Androstene-3, 17-dione [63-05-8] 11β-Hydroxy-etiocholanolone [739-26-4] 1-Dehydrotestosterone (Boldenone) [846-48-0] 17β-nortestosterone [434-22-0] Synthetic 17β-trenbolone [10161-33-8] 17α-trenbolone [80657-17-6] Progestin Natural Progesterone [57-83-0] Synthetic Melengestrol acetate [2919-66-6]

ExperimentalExperimental ApproachApproach ChemicalChemical AnalysesAnalyses

„ WaterWater SampleSample ExtractionExtraction--EvaluatingEvaluating 22 SPESPE techniquestechniques „ 3M Empore extraction disks (SDB, C18) „ HLB cartridges „ SolidSolid ExtractionExtraction „ Automated Solvent Exchange (ASE) followed by SPE extraction ExperimentalExperimental ApproachApproach ChemicalChemical AnalysesAnalyses

„ HPLC-MS-MS „ Method based on Vanderford et al. 2003

Ion Source Mass Analyzer HPLC ExperimentalExperimental ApproachApproach ChemicalChemical AnalysesAnalyses

„ ExploringExploring preservationpreservation techniquestechniques

„ ExploringExploring recoveriesrecoveries inin variousvarious matricesmatrices Bioassay:Bioassay: EE--ScreenScreen „ MCF-7 breast cancer cell line proliferates in response to estrogenic compounds „ Incubate 5 days with sample

„ Protein dye to indicate cell # 120 „ + control E2 spiked samples 100 80 60 40 20 0 -20 % Maximum Response % Maximum

0.001 0.01 0.1 1 10 100 1000 10000

Estradiol Concentration ng/L Control Estradiol Bioassay:Bioassay: AA--ScreenScreen

„ MCF-7 breast cancer cell line transfected with androgen receptor. Inhibits proliferation in response to androgenic compounds „ Incubate 5 days with sample

„ Protein dye to indicate cell # „ + control: MCF-7 cells

e 120 100 80 60 40 20 0 -20 % Maximum Respons % Maximum 0.01 0.1 1 10 100 1000 10000 Testosterone Concentration ng/L TransgenicTransgenic YeastYeast AssaysAssays • Human ER, AR or PR and corresponding response element linked to ß-galactosidase DNA stably transfected into yeast cell • Incubate overnight with sample (4 hrs PR) • Lyse yeast and perform ß-galactosidase assay

• Normalize activity to yeast density (OD600

e 120 e 120 100 100 80 80 60 60 40 40 20 20 0 0 -20 % Maximum Respons % Maximum

% Maximum Respons % Maximum -20 0.01 0.1 1 10 100 1000 10000 1 10 100 1000 10000 100000 1000000 Estradiol Concentration ng/L Testosterone Concentration ng/L BioassayBioassay comparisoncomparison

Bioassay Reference EC50 Lowest std cmpd ng/L ng/L E-screen Estradiol 2.0 .27 ER-yeast Estradiol 61 14 A-screen DHT 22 14 AR-yeast DHT 1016 290 PR-yeast Progesterone 3145 32

ComparingComparing ChemicalChemical andand BiologicalBiological AnalysesAnalyses

„ Bioassay results reported 17b-estradiol ethinylestradiol estriol as “activities” 120 estrone bisphenol A 100 nonylphenol „ Need to convert chemical 4-octylphenol 4-t-octylphenol analyses to expected 80 testosterone analyses to expected dibutylphthalate activities in order to 60 diethylhexylphthalate compare results 40 20

„ Each chemical has unique Response % Maximum 0 potency potency -20 1E-151E-141E-131E-121E-111E-101E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0.01 „ Assuming additive [Molar] response ComparisonComparison ofof BioassayBioassay andand 140 ChemistryChemistry ResultsResults 120 Total Estrogen Activity BPA OP, t-OP, NP 100 EE2 E1 L 80 E2 E3 60

40 E-screen EEq ng/

20 0 F - E-Screen A - E-Screen E - E-Screen H - E-Screen D - E-Screen C - E-Screen G - E-Screen F - GC-NCI-MS - F A - GC-NCI-MS E - GC-NCI-MS E H - GC-NCI-MS C - GC-NCI-MS C - GC-NCI-MS D G - GC-NCI-MS G HPLCHPLC Fractionation:Fractionation: TIETIE--typetype approachapproach

Samples where activity and chemical analyses are markedly different can be subjected to fractionation followed by bioassay to help identify active components •Interferences WholeWhole fishfish AssaysAssays PartialPartial lifelife--cyclecycle testingtesting ofof specificspecific hormoneshormones

„ Hormones,Hormones, metabolitesmetabolites oror relevantrelevant mixturesmixtures dependingdepending onon datadata gapsgaps „ ExposeExpose breedingbreeding fatheadfathead minnowsminnows „ EndpointsEndpoints includeinclude fecundityfecundity andand secondarysecondary sexsex characteristics,characteristics, plasmaplasma VTG,VTG, hormones.hormones.

Gene Expression

1) Expose adult FHM to 2) Perform sample extracts and full/partial life-cycle showing activity in cell- tests with CAFO based bioassays effluent

24 hrs Remove liver and gonads

Determine Extract RNA cDNA expression of biomarkers Gene Expression Estrogenic Biomarkers: ● Vitellogenin ( males) ● Zona Pellucida (ZP3) ● other? Androgenic Biomarkers : ● Vitellogenin ( females) ● other? Progesteronic ?

ExpectedExpected ResultsResults::

The project will determine the potential for environmental release of hormones: „ under various waste management practices „degradation of hormones and associateassociatedd activities „the persistence of residuals in transport from CAFOs „the relative importance of different natural and synthetic hormones to the biological activity of CAFO wastes and effluents.

The results will assist CAFO operators to optimize management practices that mitigate environmental problems associated with hormones discharged from CAFOs, and help regulators with risk assessment as biologically relevant chemicals will be identified, quantified, and ranked.

Ultimately, the proposed project will help protect sensitive aquatic environments, native species, and humans from wastes associated with CAFOs.