A Study of Endocrine A Study of Endocrine Disrupting Compounds in Disrupting Compounds in

WastewaterWastewater

Arthur HoArthur HoSpacegrant Mentor: Dr. Bob ArnoldSpacegrant Mentor: Dr. Bob Arnold

Chemical and Environmental Chemical and Environmental EngineeringEngineering

University of ArizonaUniversity of Arizona

Effects in AnimalsEffects in Animals

Kidd et al., 2007

USGS Surface Water ReconnaissanceUSGS Surface Water Reconnaissance(Kolpin (Kolpin et alet al., 2002)., 2002)

• 139 Sites

• 95 Compounds

Comparison of 4 Compounds:

Compound (Use and Effect) CMAX (µg/L) Days to Med Dose

• Caffeine (stimulant) 5.7 5.7 x 103 (Excedrin Migraine)

• Ibuprofen (anti-inflammatory) 1.0 105 (Motrin)

• 17α-ethinyl estradiol (oral contraceptive) 0.27 37 (Alesse-21)

• 17β-estradiol (vertebrate hormone) 0.093 (Phoenix) 5.3 x 103 (Activella)

• 4-nonylphenol (surfactant metabolite) 40 (Tucson) 4.6 x 10-4 as estrogenic E2, EE2

Examples of Estrogenic Examples of Estrogenic CompoundsCompounds

Chemical StructureMolecular

WeightLog Kow

Relative Estrogenic Activity

(YES bioassay)

17-estradiol (E2)

 272.39

 3.94

 1.0

17-ethinyl estradiol (EE2)

296.40 4.15 1.32

Estrone (E1)270.37 3.43 0.20

Estriol (E3)288.39 2.81 0.01

Nonylphenol (NP)

220.354.48

2×10-4

Octylphenol (OP)

206.33 4.12 3×10-4

Bisphenol A (BPA)

228.29 3.40 8×10-5

Estrogenic Activity at JWPCPEstrogenic Activity at JWPCP

Effluent: 0.12 mols EE2 equivalents/day

Dewatered Cake: 0.26 mols EE2

equivalents /day

Influent:1.66 mols EE2

equivalents/day

Removal of estrogenic activity through the whole treatment plant = [1.656 – (0.1178 + 0.26)] / 1.656 = 77%

Cytoplasm

E

R

R E

E

Cytoplasm

= Estrogen

= Receptor

Yeast Estrogen/Androgen Screen

R

hER/hAR

-gal

CPRGyellow

CPRred

RE

lac-z

Yeast Estrogen Screen (YES) AssayYeast Estrogen Screen (YES) Assay

17- Ethinylestradiol

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

1.E-13 1.E-12 1.E-11 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06

Molar concentration in well

A570 n

m - A

630 n

m

EC50=1.1 x 10-10 M

Cytoplasm

E

R

R E

E

Cytoplasm

= Estrogen

= Receptor

Yeast Estrogen/Androgen Screen

R

hER/hAR

-gal

CPRGyellow

CPRred

RE

lac-z

AgonismAgonism

Anti-estrogens: Mechanism of Anti-estrogens: Mechanism of ActionAction

AntagonismAntagonismR E R A

RE

RA

-gal

No protein production

Tamoxifen is an Anti-Tamoxifen is an Anti-estrogenestrogen

•Known anti-estrogens: (hydroxy) tamoxifen, chlorinated hormones,

•Known anti-androgens: 4-,4’-DDE, flutamide

01020304050

60708090

100

1.00E-07 1.00E-06 1.00E-05

Tamoxifen concentration (M)

Per

cent

Rel

ativ

e E

stro

geni

c A

ctiv

ity

1 nM EE2

TXFN + 1 nM EE2

MethodsMethods Obtaining Wastewater samplesObtaining Wastewater samples Extraction of OrganicsExtraction of Organics Preparation of organicsPreparation of organics Yeast Estrogen AssayYeast Estrogen Assay

RESULTSRESULTS Anti-Estrogen Test: EXTRACT from RAW SLUDGE SOLIDS

0

10

20

30

40

50

60

70

80

90

1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00

Fraction of extract in well

% r

el B

-gala

cto

sid

ase

20% MeOH eluent

50% MeOH eluent

80% MeOH eluent

NP w ater w / 1nM EE2

Anti-estrogen test: EXTRACT from RAW SLUDGE LIQUID

0

10

20

30

40

50

60

70

80

90

100

1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00

Fraction of extract in well

% r

el. B

-gal

acto

sida

se

20% MeOH eluent

50% MeOH eluent

80% MeOH eluent

NP water w/ 1nM EE2

RAW SLUDGE SOLID EXTRACTS ARE HIGHLY ESTROGENIC

RAW SLUDGE LIQUID EXTRACTS ARE ANTI-ESTROGENIC

RESULTSRESULTSAnti-estrogen test: EXTRACT from DIGESTED SLUDGE SOLID

0

10

20

30

40

50

60

70

80

90

100

1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00

Fraction of extract in well

% r

el. B

-gal

acto

sida

se

20% MeOH eluent

50% MeOH eluent

80% MeOH eluent

NP water w/ 1nM EE2

Anti-estrogen test: EXTRACT from DIGESTER SLUDGE LIQUID

0

10

20

30

40

50

60

70

80

1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00

Fraction of extract in well

% B

-ga

lac

tos

ida

se

20% MeOH eluent

50% MeOH eluent

80% MeOH eluent

NP water w/ 1nM EE2

DIGESTED SLUDGE SOLIDS LOST ESTROGENIC ACTIVITY

DIGESTED SLUDGE LIQUIDS HIGHLY ANTI-ESTROGENIC

AcknowledgementsAcknowledgements

Dave Newman & Sondra TeskeDave Newman & Sondra Teske

Susan BrewSusan Brew

Matt Giardiana and AzSGC StaffMatt Giardiana and AzSGC Staff

Bingfeng DongBingfeng Dong