1. Determination of Natural Steroidal Estrogens in Flushed Dairy Manure Wastewater and Surface and Ground Water Travis Hanselman Ph.D. exit seminar

2. What are estrogens? Where do they come from? Why are they a concern?

3. What are estrogens? A group of natural steroid hormones with an aromatic A-ring as a distinctive part of their tetracyclic molecular frameworkA group of natural steroid hormones with an aromatic A-ring as a distinctive part of their tetracyclic molecular framework Estradiol Estrone HO O H HO O O HO O H H Estriol

4. Physicochemical Properties Estrogens are nonvolatile, moderately hydrophobic, nonionic compounds with low water solubilityEstrogens are nonvolatile, moderately hydrophobic, nonionic compounds with low water solubility Table 1. Estrogen characteristics MW=Molecular weight; K ow =Octanol-water partition coefficient; VP=Vapor pressure; †, Solubility in water

5. Where do estrogens come from? Estrogens are excreted naturally in the urine and feces of humans, wildlife and livestock; especially from femalesEstrogens are excreted naturally in the urine and feces of humans, wildlife and livestock; especially from females

6. Why are estrogens a concern? Endocrine disruptionEndocrine disruption –Feminization of male aquatic vertebrate species –  -Estradiol is biologically active at 10-100 ng L -1

7. 1.Development of methods for measuring estrogens in livestock wastes and surface and ground water resources 2.Surveys of estrogens in waterways 3.Characterization of estrogens in livestock wastes 4.Estrogen fate in manure, soil, and water 5.Wildlife and/or test organisms should be studied for evidence of reproductive abnormalities 6.Other biologically-active agents in livestock wastes such as androgens, gestagens, growth promoters, and antibiotics need to be characterized Critical Research Needs

8. Measure estrogens in flushed dairy manure wastewater (FDMW) and surface and ground waterMeasure estrogens in flushed dairy manure wastewater (FDMW) and surface and ground water Research Objective FDMW

9. UF Dairy Research Unit FDMW

10. Few “Ideal” Analytical Options Previous researchers have relied heavily on enzyme immunoassay (EIA) methods due to their commercial availability, ease of use, pg mL -1 detection limits, and a lack of alternative quantitation methodsPrevious researchers have relied heavily on enzyme immunoassay (EIA) methods due to their commercial availability, ease of use, pg mL -1 detection limits, and a lack of alternative quantitation methods However, EIAs can suffer from false-positive and false-negative interferences due to cross-reactivity and matrix effects associated with humic substancesHowever, EIAs can suffer from false-positive and false-negative interferences due to cross-reactivity and matrix effects associated with humic substances Thus, depending on sample complexity and EIA reagents, antibodies, and protocol, a potential exists for different EIAs to yield different or inaccurate resultsThus, depending on sample complexity and EIA reagents, antibodies, and protocol, a potential exists for different EIAs to yield different or inaccurate results

11. Comparison of three commercially-available ß-estradiol EIAsComparison of three commercially-available ß-estradiol EIAs Immunoassay Experiment FDMW (20 mL),n=4 extract w/ ether (20 mL) Sub sample ether (4 mL) evaporate to dryness and reconstitute with buffer EIA1EIA2EIA3 ß-Estradiol HO O H Sample preparation according to a published method for dairy wastes (Raman et al., 2001)Sample preparation according to a published method for dairy wastes (Raman et al., 2001) Quality control experimentQuality control experiment

12. Immunoassay Description DescriptionEIA1EIA2EIA3 Assay principleCompetitive Estradiol AntibodyRabbit poly MatrixTBSSerum Conjugate/EnzymeE2-ALPE2-Biotin/SHRPE2-HRP Substratep-NPPTMB Range (pg mL -1 )0-30,0000-6,0000-2,000 MDL (pg mL -1 ) 29 7 10 Cross-reactivity (%) ß-Estradiol100 α-Estradiol 0.1 0.3 Estrone 4.6 1.4 2.1 Estriol 0.5 1.1 1.5 E2, estradiol; ALP, alkaline phosphatase; SHRP, streptavidin horseradish peroxidase; HRP, horseradish peroxidase; p-NPP, p-nitrophenol phosphate; TMB, tetramethylbenzidine; MDL, minimum detection limit. TBS, Tris-buffered saline containing proteins, detergents, and azide Table 2. Description and cross-reactivity of three immunoassays.

13. FDMW 1 17 ß-Estradiol (ng L -1 ) a b ab Immunoassay Comparison

14. FDMW 2 17 ß-Estradiol (ng L -1 ) a b b Immunoassay Comparison

15. Why different and which is right? Quality control data (accuracy, precision, linearity)Quality control data (accuracy, precision, linearity) Calibration accuracy “pure” ß-estradiol solutions (all assays reported the same concentrations)Calibration accuracy “pure” ß-estradiol solutions (all assays reported the same concentrations) Duplicate precision was quite good (CV<13%)Duplicate precision was quite good (CV<13%) Dilution and spiked recovery (linear deviation)Dilution and spiked recovery (linear deviation) The QC results suggested that coextracted humic substances affected one or more EIAsThe QC results suggested that coextracted humic substances affected one or more EIAs Ideally, EIA disagreement can be reconciled with a more conclusive technique like gas chromatography-mass spectrometry (GC-MS), but……………Ideally, EIA disagreement can be reconciled with a more conclusive technique like gas chromatography-mass spectrometry (GC-MS), but……………

16. Method Development for GC-MS A sensitive method for GC-MS analysis of estrogens in dairy wastes not availableA sensitive method for GC-MS analysis of estrogens in dairy wastes not available Raman et al., 2001 reported GC-MS method detection limits of ~10 μg L -1Raman et al., 2001 reported GC-MS method detection limits of ~10 μg L -1 Immunoassay of FDMW showed ng L -1Immunoassay of FDMW showed ng L -1 Needed to develop a method for measuring endogenous estrogen concentrations by GC-MSNeeded to develop a method for measuring endogenous estrogen concentrations by GC-MS

17. Method Development for GC-MS A difficult, expensive, frustrating, trial and error process……

18. Method Development FDMW (40 mL) centrifuged 15,000 g Ultrasonic MOH extraction of solids, 2x10 mL; centrifuge 4,000 g combine supernatants Filtration 20 μm Carbograph SPE extraction MOH:MC elution and evaporation C18 SPE purification Acetone elution and filtration 0.2 μm GC-MS EIA 1 EIA 2

19. Method Performance GC-MS recovery and reproducibility experimentGC-MS recovery and reproducibility experiment Recovery of estrogens spiked into FDMW over 5- days of sampling; 40 ng fixed level and 20, 40, 60, and 80 ng variable spikeRecovery of estrogens spiked into FDMW over 5- days of sampling; 40 ng fixed level and 20, 40, 60, and 80 ng variable spike --------------------recovery, % (RSD)----------------- 1 92 (5) 92 (5) 96 (6) 96 (6) 116 (5) 116 (5) 90 (9) 90 (9) 2 104 (5) 104 (5) 105 (5) 105 (5) 134 (8) 134 (8) 99 (9 ) 99 (9 ) 3 105 (2) 105 (2) 93 (5) 93 (5) 121 (2) 121 (2) 109 (5) 109 (5) 4 107 (7) 107 (7) 94 (10) 94 (10) 139 (8) 139 (8) 107 (10) 107 (10) 5Avg. 98 (7) 98 (7) 94 (9) 94 (9) 114 (8) 114 (8) 90 (12) 90 (12) FDMWestroneα-estradiolß-estradiolestriol 101 (5) 96 (7) 125 (6) 99 (9) Table 3. Five day recovery of 40 ng from FDMW.

20. Method Performance Estrogen recovered (ng ) Estrogen added (ng) α-estradiol100%estrone99% ß-estradiol122%estriol92%

21. GC-MS FDMW Concentrations Estrogen (ng L -1 ) FDMW estrone α-estradiol ß-estradiol estriol-ND

22. GC-MS vs. EIA 1 17ß-estradiol ( ng L -1 ) FDMW * *

23. GC-MS vs. EIA 2 17ß-estradiol ( ng L -1 ) FDMW * * * * *

24. Are dairy manure estrogens finding their way into surface and ground water?

25. Surface Water Preliminary GC-MS method development and survey experimentPreliminary GC-MS method development and survey experiment 4 sampling locations, 1 impacted4 sampling locations, 1 impacted

26. Ground Water 3 FDEP monitoring wells, 1 “other” well3 FDEP monitoring wells, 1 “other” well

27. Method Development Method adaptationMethod adaptation Spiked recovery of 40 ng of estrogens from surface and ground waterSpiked recovery of 40 ng of estrogens from surface and ground water Carbograph SPE extraction MOH:DCM elution and evaporation 200 mL water samples filtration 20 μm C18 SPE purification Acetone elution and filtration 0.2 μm GC-MS

28. Method Performance Positive interference of the GC-MS signals was observed for water samples; particularly for estronePositive interference of the GC-MS signals was observed for water samples; particularly for estrone Table 4. Estimated recovery of estrogens from water.

29. Survey of Estrogens in Water No measurable concentrations of α-estradiol, ß- estradiol, or estriol in any of the surface or ground water samplesNo measurable concentrations of α-estradiol, ß- estradiol, or estriol in any of the surface or ground water samples Estrone was detected in one surface water sample and one ground water sample, <60 ng L -1Estrone was detected in one surface water sample and one ground water sample, <60 ng L -1

30. Summary & Conclusions The accurate measurement of estrogens in environmental samples is a challenging taskThe accurate measurement of estrogens in environmental samples is a challenging task Data acquired from EIAs should be interpreted cautiously and with strict quality control protocolData acquired from EIAs should be interpreted cautiously and with strict quality control protocol A method was developed which enabled ng L -1 measurements of estrogens in FDMW and surface and ground water by GC-MSA method was developed which enabled ng L -1 measurements of estrogens in FDMW and surface and ground water by GC-MS The survey of surface and ground water at the dairy did not reveal any gross contamination by estrogens at the time of samplingThe survey of surface and ground water at the dairy did not reveal any gross contamination by estrogens at the time of sampling The method contributes a foundation for future research involving estrogen analysis of livestock wastes and water resourcesThe method contributes a foundation for future research involving estrogen analysis of livestock wastes and water resources

31. AcknowledgmentsCommittee Don Graetz, ChairDon Graetz, Chair Ann Wilkie, CoChairAnn Wilkie, CoChair Tom ObrezaTom Obreza Nancy DenslowNancy DenslowAlso… Nancy Szabo and the ATCL staffNancy Szabo and the ATCL staff Dawn LucasDawn Lucas Funding provided by the School of Natural Resources and Environment Mini-grants ProgramFunding provided by the School of Natural Resources and Environment Mini-grants Program

32. Questions?