Analysis of Estrogens in Wastewater Effluent with the Addition of Humic Substances

1. Analysis of Estrogens in Wastewater Effluent with the Addition of Humic Substances Kelly Giffear Dr. Victoria Del Gaizo Moore Department of Chemistry Elon University

2. Overview • Background • Wastewater treatment plants • Endocrine-disruptor compounds • EPA’s response • Past Research • Current Research • Conclusions • Future Experiments

3. Background http://madisonplus.com/mp-daily/breast-cancer-awareness/ http://www.topnews.in/health/diseases/prostate-cancer http://www.choosehope.com/product/ribbon-awareness-car-magnets

4. Image from biosolids.com.au

5. Endocrine Disruptors (EDC) • Any compound that interferes with normal hormone function • Class of compounds defined by biological function rather than chemical nature • Mechanisms of action • Mimic • Block • Stimulate • Destroy

6. EDCs • Hormones • Estrogens • Polychlorinated Biphenyls • Pesticides • Heavy Metals • Surfactants • AlkylphenolEthoxylates • Nitrate – potential EDC

7. Effects of EDCs • Endocrine disruptors (including estrogens) have been found to: • Affect growth, development, reproduction in plants and animals • Cause intersexuality in fish • Affect sex determination in turtles • Cause egg shell thinning in birds

8. Survey of US waters Location of 139 stream sampling sites. Koplin, D.W.; Furlong, E.T.; Meyer, M.T.; Thurman, E.M.; Zaugg, S.D.; Barber, L.B.; Buxton, H.T. Environ. Sci. Technol. 2002, 36, 1202-1211.

9. Survey of US Waters Frequency of detection of organic wastewater contaminants by general use category. Number of compounds in each category shown above bar. Koplin, D.W.; Furlong, E.T.; Meyer, M.T.; Thurman, E.M.; Zaugg, S.D.; Barber, L.B.; Buxton, H.T. Environ. Sci. Technol. 2002, 36, 1202-1211.

10. Estrogen Structures O H O H C H C H 3 3 17 O H H H H H H H H O H O 17b-Estradiol (E2) Estriol (E1) O O H C H C H 3 3 C H 17 H H H H H H H O H O Estrone (E4) 17a-Ethynyl Estradiol (E3)

11. EPA Response • The EPA does not regulate estrogen levels in water • Due to lack of regulation, wastewater treatment may or may not be removing estrogens • In 1996, the EPA identified endocrine disruption as one of its top six research priorities LTG 1: Provide a better understanding LTG 2: Determine the extent of impact LTG 3: Support testing program. www.epa.gov/endocrine/

12. Past Research • Solid-phase extraction (SPE)/high performance liquid chromatography (HPLC) analysis • Speciation and concentrations of each estrogen • Extracted estrogens from clean water and wastewater effluent • On site sample extraction • Elution in lab • Collected on Alltech high-flow C18 cartridges

13. 17b-estradiol 17a-ethynyl estradiol estrone estriol 5 mAu 0 1 2 3 4 5 6 7 8 Time (min) Spiked Milli-Q Sample Concentrations in mg/L Dr. Ponton, unpublished data

14. Peak Area vs. Concentration E1 E2 E4 E3 6.0 mg/L 20.0 mg/L 50 AU 80.0 mg/L 0 2 4 6 8 10 Time (min) Dr. Ponton, unpublished data

15. estriol b-estradiol 17a-ethynyl estradiol estrone Calibration Curve 20 18 16 14 12 Peak Area 10 R2 0.9996 n = 3 to 5 8 6 4 2 0 0 20 40 60 80 100 Concentration (mg/L) Dr. Ponton, unpublished data

16. Wastewater Effluent Sampling Site • Graham wastewater treatment facility • Sample Storage • Acidified to pH ~3 • Stored at 4° C

17. 50 mAu 0 2 4 6 8 10 12 14 16 18 20 Time (min) Wastewater Effluent Chromatogram • No estrogens extracted • Peak observed has unknown identity Dr. Ponton, unpublished data

18. 5 mAu 0 2 4 6 8 10 12 14 16 18 20 Time (min) Spiked WW Effluent Chromatogram • 10 mg/L water sample concentration • No estrogens detected Dr. Ponton, unpublished data

19. 3 hypotheses • Irreversible binding to something • Reversible binding/equilibrium • Not bound, but blocking activity and masking concentration

20. Tannic Acid2 Alginic Acid2 Humic Substances to Blame? Humic Acid1 1 Stevenson F.J. (1982): Humus chemistry. Genesis, composition, reactions. John Wiley and Sons. 2 http://www.genome.jp/ Yamamoto, H. Environ. Sci. Technol. 2003, 37, 2646-2657

21. Project Goals • Are humic substances interfering with estrogenic activity in wastewater? • Clean water and wastewater samples • If so, how are humic substances interfering with estrogenic activity? • Methology

22. Yeast β-Galactosidase Assay • Needed an assay effectively assess estrogens in wastewater using a colormetric scale • Kit from ThermoScientific • Baker’s Yeast • From Dr. Balsiger (University of Texas) • Deleted PDR5 gene • Insert pG/ER and pUCΔSS-ERE plasmids

23. Assay Protocol • Grow yeast cultures in SC-UW media overnight at 37°C • Dilute cultures to OD650=0.08-0.1 • Add treatments (estrogen and/or humic substances) • Incubate at 37°C for 2 hours • Record OD650 and A450 for baseline data • Add assay buffer • Record OD650and A450 at 15 and 30 mins.

24. Are recombinant yeast estrogen sensitive? 1 = Media and Yeast; 2= Media, Yeast, Estrogen; n=6

25. Do Humic Substances have estrogenic activity? Relative concentrations of HS 1=0.1;2=0.01;3=0.001;4=0.0001; n=1

26. Preliminary results with Humic Substances n=1

27. Conclusions • Yeast incubated with estrogen are 3-5 times as sensitive than yeast without estrogen • Humic substances do not inherently have estrogenic activity • Humic substance added to estrogen decreases the estrogenic activity ten-fold

28. Future Experiments • Repeating experiments in wastewater effluent • Does protocol translate in true environment setting • Spiked samples vs. natural samples • ELISA assay with pure water spiked with estrogen and humic substances • How do estrogen and humic substances bind • ELISA assay with wastewater effluent • Spiked samples vs. natural samples

29. Acknowledgements Funding Elon University Chemistry Department Elon University Lumen Scholars Program ElonCollege Fellows Program Mentors Dr. Victoria Del Gaizo Moore Dr. Lisa M. Ponton Yeast Dr. H.A. Balsiger Samples Graham Wastewater Treatment Facility