Travis Hanselman Ph.D. exit seminar

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

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

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

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

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

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

9. FDMW UF Dairy Research Unit

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 methods • However, 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 results

11. FDMW (20 mL),n=4 extract w/ ether (20 mL) O H Sub sample ether (4 mL) evaporate to dryness and reconstitute with buffer H O ß-Estradiol EIA1 EIA2 EIA3 Immunoassay Experiment • Sample preparation according to a published method for dairy wastes (Raman et al., 2001) • Quality control experiment • Comparison of three commercially-available ß-estradiol EIAs

12. Immunoassay Description Description EIA1 EIA2 EIA3 Assay principle Competitive Competitive Competitive Table 2. Description and cross-reactivity of three immunoassays. Estradiol Antibody Rabbit poly Rabbit poly Rabbit poly Matrix TBS Serum Serum Conjugate/Enzyme E2-ALP E2-Biotin/SHRP E2-HRP Substrate p-NPP TMB TMB Range (pg mL-1) 0-30,000 0-6,000 0-2,000 MDL (pg mL-1) 29 7 10 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 Cross-reactivity (%) ß-Estradiol 100 100 100 α-Estradiol 0.1 0.3 0.3 Estrone 4.6 1.4 2.1 Estriol 0.5 1.1 1.5

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

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

15. Why different and which is right? • Quality control data (accuracy, precision, linearity) • Calibration accuracy “pure” ß-estradiol solutions (all assays reported the same concentrations) • Duplicate precision was quite good (CV<13%) • Dilution and spiked recovery (linear deviation) • The 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……………

16. Method Development for GC-MS • A 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-1 • Immunoassay of FDMW showed ng L-1 • Needed 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. 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 Method Development

19. FDMW estrone α-estradiol ß-estradiol estriol --------------------recovery, % (RSD)----------------- 1 92 (5) 96 (6) 116 (5) 90 (9) 2 104 (5) 105 (5) 134 (8) 99 (9 ) 3 105 (2) 93 (5) 121 (2) 109 (5) Table 3. Five day recovery of 40 ng from FDMW. 4 107 (7) 94 (10) 139 (8) 107 (10) 5 Avg. 98 (7) 94 (9) 114 (8) 90 (12) 101 (5) 96 (7) 125 (6) 99 (9) Method Performance • GC-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 spike

20. Estrogen recovered (ng) α-estradiol 100% estrone 99% ß-estradiol 122% estriol 92% Estrogen added (ng) Method Performance

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

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

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

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

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

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

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

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

29. Survey of Estrogens in Water • No 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-1

30. Summary & Conclusions • The accurate measurement of estrogens in environmental samples is a challenging task • Data 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-MS • The 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 resources

31. Acknowledgments Committee • Don Graetz, Chair • Ann Wilkie, CoChair • Tom Obreza • Nancy Denslow Also… • Nancy Szabo and the ATCL staff • Dawn Lucas • Funding provided by the School of Natural Resources and Environment Mini-grants Program

32. Questions?