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Analysis of Estrogens in Wastewater Effluent with the Addition of Humic Substances. Kelly Giffear Dr. Victoria Del Gaizo Moore Department of Chemistry Elon University. Overview. Background Wastewater treatment plants Endocrine-disruptor compounds EPA’s response Past Research

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analysis of estrogens in wastewater effluent with the addition of humic substances

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

Kelly Giffear

Dr. Victoria Del Gaizo Moore

Department of Chemistry

Elon University

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

endocrine disruptors edc
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
slide6
EDCs
  • Hormones
    • Estrogens
  • Polychlorinated Biphenyls
  • Pesticides
  • Heavy Metals
  • Surfactants
    • AlkylphenolEthoxylates
  • Nitrate – potential EDC
effects of edcs
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
survey of us waters
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.

survey of us waters1
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.

estrogen structures
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)

epa response
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/

past research
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
spiked milli q sample

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

slide14

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

calibration curve

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

wastewater effluent sampling site
Wastewater Effluent Sampling Site
  • Graham wastewater treatment facility
  • Sample Storage
    • Acidified to pH ~3
    • Stored at 4° C
wastewater effluent chromatogram

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

spiked ww effluent c hromatogram

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

3 hypotheses
3 hypotheses
  • Irreversible binding to something
  • Reversible binding/equilibrium
  • Not bound, but blocking activity and masking concentration
humic substances to blame

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

project goals
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
yeast galactosidase assay
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
assay protocol
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.
are recombinant yeast estrogen sensitive
Are recombinant yeast estrogen sensitive?

1 = Media and Yeast; 2= Media, Yeast, Estrogen; n=6

do humic substances have estrogenic activity
Do Humic Substances have estrogenic activity?

Relative concentrations of HS 1=0.1;2=0.01;3=0.001;4=0.0001; n=1

conclusions
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
future experiments
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
acknowledgements
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