Toxicological Relevance of EDCs and
Pharmaceuticals in Drinking Water
AwwaRF Project 3085 – WRF 04-003
Shane Snyder, Ph.D.
Applied R&D Center - SNWA
©2008 AwwaRF. ALL RIGHTS RESERVED
Director
Ron Zegers
Manager
Dave Rexing
Administrative Support
Linda Parker
Jereena Bosket
Research Chemists
Doug Mawhinney, Ph.D.
Oscar Quinones
Rebecca Trenholm
Brett Vanderford
Janie Zeigler
©2008 AwwaRF. ALL RIGHTS RESERVED
Post-Doctoral Researchers
Fernando Rosario, Ph.D.
Mark Benotti, Ph.D.
Ben Stanford, Ph.D.
Water Quality Analysts
Spencer Porter
Shannon Ferguson
Process Enhancement
Eric Wert, P.E.
Julia Lew
Research Interns
Tony Baik (U. of Buffalo)
Elaine Go (UNLV)
Christy Meza (UNLV)
Sarper Sarp (GIST)
Mei Xin, Ph.D. (UNR)
©2008 AwwaRF. ALL RIGHTS RESERVED
History
©2008 AwwaRF. ALL RIGHTS RESERVED
ENDOCRINOLOGY
September 1951, Volume 49, Number 3
Estrogenic, Androgenic and Gonadotrophic Activity in
Wheat Germ Oil
Ezra Levin, John F. Burns, and V.K. Collins
VioBin Laboratories, Monticello, Illinois
Proc. Soc. Exptl. Biol. Med. – 1952
Estrogenic Action of Some DDT Analogues. (19903)
A.L. Fisher, H.H. Keasling, and F.W. Schueler
Department of Pharmacology, College of Medicine, State University of
Iowa, Iowa City
©2008 AwwaRF. ALL RIGHTS RESERVED
Developments in Industrial Microbiology – 1970
Steroid Hormones as Water Pollutants
I.
Metabolism of Natural and Synthetic Ovulation-Inhibiting Hormones by
Microorganisms of Activated Sludge and Primary Settled Sewage
Henry H. Tabak and Robert L. Bunch
U.S. Department of the Interior, Federal Water Pollution Control Administration,
Advanced Waster Treatment Research Laboratory, Cincinnati, Ohio
Our Knowledge of the fate of steroids normally present in domestic wastewater is
rather meager. A better understanding of steroid biodegradation was
sought. The data obtained showed that the susceptibility of the natural
and synthetic ovulation-inhibiting steroids varied as to the rate of
oxidation by the microorganisms of activated sludge. The synthetic
estrogen and progestin components of oral contraceptives exhibited
greater overall resistance to microbial degradation than the natural
hormones.
©2008 AwwaRF. ALL RIGHTS RESERVED
©2008 AwwaRF. ALL RIGHTS RESERVED
©2008 AwwaRF. ALL RIGHTS RESERVED
Drugged Waters
Does it matter that pharmaceuticals are turning up in
water supplies? By Janet Raloff
Treated municipal wastewater entering a Swiss stream. Treatment
plants have not been designed to remove excreted drugs before
releasing their effluent into public waterways.
March 21, 1998, Science News, Vol. 153
©2008 AwwaRF. ALL RIGHTS RESERVED
Objective
To inform water utilities, regulators, the scientific
community, and the public with information
regarding the occurrence and health relevance
of EDCs and pharmaceuticals in drinking water.
These data provide guidance for allocating
resources responsibly. Meaningful treatment
goals and analytical reporting limits must be
based on human health protection.
©2008 AwwaRF. ALL RIGHTS RESERVED
Core Team
Principal Investigators:
Shane Snyder
Richard Pleus
Gretchen Bruce
Erin Snyder
Jocelyn Hemming
Public Information:
JC Davis
Roger Buehrer
Administration:
David Rexing
AwwaRF Project Manager: Djanette Khiari
PAC:
Pankaj Parekh
Charles Staples
Steve Macaulay
Mike Wehner
©2008 AwwaRF. ALL RIGHTS RESERVED
Project Scope
•
Identify tracers for pharmaceuticals and suspected EDCs
relevant to drinking water
– Based on occurrence, toxicity, and analytical capabilities
•
•
•
•
•
Screen variety of source and drinking waters
Conduct comparative assessment of estrogenicity
Assess toxicology of pharmaceuticals
Assess toxicology of suspected EDCs
Communication guidance for utilities
©2008 AwwaRF. ALL RIGHTS RESERVED
Site Selection
©2008 AwwaRF. ALL RIGHTS RESERVED
Samples collected per time zone
195
43
88
17 Participating Utilities
©2008 AwwaRF. ALL RIGHTS RESERVED
Compound Selection
©2008 AwwaRF. ALL RIGHTS RESERVED
Pharmaceuticals
Pharmaceuticals
Synonym(s)
Use
MRL (ng/L)
Atenolol
Tenormin
Beta-blocker
0.25
Atorvastatin
Lipitor
Antilipidemic
0.25
o-Hydroxy atorvastatin
Atorvastatin metabolite
0.50
p-Hydroxy atorvastatin
Atorvastatin metabolite
0.50
Carbamazepine
Tegretol
Anticonvulsant
0.50
Diazepam
Valium
Tranquilizer
0.25
Diclofenac
Voltaren
NSAID
0.25
Enalapril
Renitec, Vasotec
ACE Inhibitor
0.25
Fluoxetine
Prozac
Antidepressant
0.50
Fluoxetine metabolite
0.50
Norfluoxetine
Gemfibrozil
Lopid
Antilipidemic
0.25
Meprobamate
Miltown
Anti-anxiety
0.25
Naproxen
Aleve
NSAID
0.50
Phenytoin
Dilantin
Antiepileptic
1.0
Risperidone
Risperidal
Antipsychotic
1.0
Simvastatin
Zocor
Antilipidemic
0.25
Simvastatin metabolite
0.25
Antibiotic
0.25
Triclosan
Antimicrobial
1.0
Trimethoprim
Antibiotic
0.25
Simvastatin hydroxy acid
Sulfamethoxazole
Bactrim
©2008 AwwaRF. ALL RIGHTS RESERVED
Potential EDCs
Potential EDCs
Atrazine
MRL (ng/L)
0.25
Potential EDCs
Galaxolide
MRL (ng/L)
25
Benzophenone
25
Linuron
BHA
25
Methoxychlor
10
BHT
25
Metolachlor
10
α-BHC
10
Musk Ketone
25
β-BHC
10
Nonylphenol
100
γ-BHC (Lindane)
10
Octachlorstyrene
10
δ-BHC
10
Octylphenol
25
Bisphenol A
5.0
TCEP
50
Butylbenzylphthalate
50
TCPP
50
DEET
25
Tonalide
25
Diazinon
10
Traseolide
25
Bis(2-ethylhexyl)phthalate
100
Vinclozolin
10
Atrazine
Diazinon
Musk Ketone
©2008 AwwaRF. ALL RIGHTS RESERVED
0.50
Vinclozolin
Steroids and Phytoestrogens
Steroids
Synonym
E2
Human estrogen
0.50
Estrone
E1
Human estrogen
0.20
Synthetic birth control
1.0
Human estrogen
0.50
Human androgen
0.50
EE2
Progesterone
Biochanin A
Chrysin
MRL (ng/L)
Estradiol
Ethynylestradiol
Estradiol
Source
Testosterone
T
Phytoestrogens
Source(s)
MRL (ng/L)
Apigenin
Leafy plants
1.0
Biochanin A
Legumes and red clover
1.0
Chrysin
Passiflora caerula (passion flower)
1.0
Coumestrol
Alfalfa
1.0
Daidzein
Legumes and red clover
1.0
Equol
Daidzein metabolite
10
Formononetin
Clover
1.0
Genistein
Legumes and red clover
1.0
Glycitein
Legumes
1.0
Matairesinol
Oilseeds (such as sesame)
5.0
Naringenin
Citrus fruits and tomatoes
1.0
©2008 AwwaRF. ALL RIGHTS RESERVED
Testosterone
Formononetin
Genistein
Analytical Methods
©2008 AwwaRF. ALL RIGHTS RESERVED
Analytical Methods
– Sample Collection/Preservation
•
•
•
•
•
1 Liter, pre-cleaned, silanized amber glass bottles
Preserved with 0.1% sodium azide
Quenched with 50 mg/L ascorbic acid
Custom shipping containers
Samples stored at 4 °C until extraction
– Sample Preparation and Extractions
• Samples filtered when necessary
– 0.45 µm GF/F filter
• Automated solid phase extraction
– Utility water samples
– Most food/beverages
milk
– Instrument Analysis
• GC-MS/MS and LC-MS/MS
• Quantification: External, internal and
isotope dilution calibration
coffee
©2008 AwwaRF. ALL RIGHTS RESERVED
beer
Isotope Dilution
•
D
D
MW 314
F
D
D
D
D
D
D
D
D
Atoms are replaced with stable, heavier isotopes
12C
F
Æ 13C,
14N
Æ 15N,
1H
Æ 2H (deuterium)
F
MW 309
F
•
Little interference, not abundant in nature
•
Isotopes virtually identical in all chemical characteristics
to native compound Æ act as a “perfect” internal
standard
•
Change in MW Æ Can be differentiated in MS detectors
H
O
H
H
H
H
N
H
F
F
O
N
H
Fluoxetine (-d5)
•
Samples spiked with isotopes prior to extraction
•
Correct for loss of native compound due to:
–
–
–
–
Matrix adsorption
Preparation/Extraction loss
Matrix suppression (LC-MS)
Change in sensitivity due to
active sites (GC-MS)
©2008 AwwaRF. ALL RIGHTS RESERVED
Calibration
Standard
Extracted Wastewater
Effluent Sample
©2008 AwwaRF. ALL RIGHTS RESERVED
Trimethoprim
Triclosan
Sulfamethoxazole
Naproxen
Meprobamate
Gemfibrozil
Fluoxetine
Dilantin
Diclofenac
Diazepam
Carbamazepine
Atrazine
Surface Water Spiked @ 25 ng/L
Isotope Dilution
External Calibration
120%
100%
80%
60%
40%
20%
0%
Wastewater Influent
Recoveries in Various Waters
Using Isotope Dilution
Wastewater Effluent
Raw Drinking Water
Finished Drinking Water
120%
100%
80%
60%
40%
20%
Trimethoprim
Triclosan
Naproxen
Sulfamethoxazole
©2008 AwwaRF. ALL RIGHTS RESERVED
Meprobamate
Gemfibrozil
Fluoxetine
Dilantin
Diclofenac
Diazepam
Carbamazepine
Atrazine
0%
Analysis of Pharmaceuticals in Water by Isotope
Dilution Liquid Chromatography/Tandem Mass
Spectrometry
Brett J. Vanderford and Shane A. Snyder
Southern Nevada Water Authority, 1350 Richard Bunker Avenue, Henderson,
Nevada 89015
Published in Environ. Sci. Technol. 2006, 40, 7312-7320
Broad range analysis of endocrine disruptors and
pharmaceuticals using gas chromatography and
liquid chromatography tandem mass spectrometry
Rebecca A. Trenholm, Brett J. Vanderford, Janie C. Holady, David J. Rexing,
Shane A. Snyder
Published by Elsevier, Chemosphere, 65 (2006) 1990-1998
©2008 AwwaRF. ALL RIGHTS RESERVED
E-screen Assay
(Dr. Jocelyn Hemming – WSLH)
• Human breast cancer cells proliferate in response to
estrogenic compounds
• Calibrated using estrogen (E2) and results reported as
estradiol equivalents (EEq)
Control
Exposed
©2008 AwwaRF. ALL RIGHTS RESERVED
QA / QC
– Travel/Field Blanks
• At least 1-2 per sampling event (1 per 10 samples collected)
– Laboratory Extractions
• 1 unspiked and 1 spiked DI water extracted with each batch of samples
• At least 1 sample duplicate and matrix spike extracted with each sampling event
– Instrument Blanks/CCVs
• Blank solvent injections performed at least every 6 samples
• Instrument continuing calibration verifications (CCVs) performed every 6 samples
– E-screen Analysis
• DI blank, spike (17β-estradiol) and duplicate sample extracted with each batch of samples
• Additional matrix spike (17β-estradiol ) performed with food extractions
©2008 AwwaRF. ALL RIGHTS RESERVED
Results
©2008 AwwaRF. ALL RIGHTS RESERVED
Travel and Laboratory Blanks
Field Blanks (n=28)
Laboratory Blanks (n=40)
Max
Median
(ng/L)
(ng/L)
#
(%)
Atenolol
1.1
0.9
2
5.0
7.1
Triclosan
3.2
2.1
2
5.0
1
3.6
Estrone
0.4
0.4
1
2.5
0.68
1
3.6
Genistein
1.4
1.4
1
2.5
0.63
0.63
1
3.6
Daidzein
1.1
1.1
1
2.5
Atrazine
0.29
0.29
1
3.6
Naringenin
2.5
2.5
1
2.5
Bisphenol A
5.5
5.5
1
3.6
Dioctyl phthalate
130
130
1
2.5
Triclosan
1.0
1.0
1
3.6
Progesterone
0.68
0.68
1
3.6
Daidzein
1.4
1.4
1
3.6
Naringenin
1.6
1.6
1
3.6
BHT
44
44
1
3.6
Compound
Max
Median
Frequency
(ng/L)
(ng/L)
#
(%)
Risperidone
2.3
2.0
2
7.1
Butylbenzyl phthalate
59
55
2
Sulfamethoxazole
0.41
0.41
Fluoxetine
0.68
Norfluoxetine
Compound
©2008 AwwaRF. ALL RIGHTS RESERVED
Frequency
Target Compounds
Pharmaceuticals (20)
Atenolol
Atorvastatin
o-Hydroxy atorvastatin
p-Hydroxy atorvastatin
Carbamazepine
Diazepam
Diclofenac
Dilantin
Enalapril
Fluoxetine
Norfluoxetine
Gemfibrozil
Meprobamate
Naproxen
Risperidone
Simvastatin
Simvastatin hydroxy acid
Sulfamethoxazole
Triclosan
Trimethoprim
Potential EDCs (26)
Atrazine
Benzophenone
BHA
BHT
α-BHC
β-BHC
γ-BHC
δ-BHC
Bisphenol A
Butylbenzyl phthalate
DEET
Diazinon
Dioctyl phthalate
Galaxolide
Linuron
Methoxychlor
Metolachlor
Musk ketone
Nonylphenol
Octachlorostyrene
Octylphenol
TCEP
TCPP
Tonalide
Traseolide
Vinclozolin
Steroid Hormones (5) Phytoestrogens (11)
Estradiol
Estrone
Ethinylestradiol
Progesterone
Testosterone
©2008 AwwaRF. ALL RIGHTS RESERVED
Apigenin
Biochanin A
Chrysin
Coumestrol
Daidzein
Equol
Formononetin
Genistein
Glycitein
Matairesinol
Naringenin
Detected in Raw Water*
Pharmaceuticals
Atenolol
Atorvastatin
o-Hydroxy atorvastatin
p-Hydroxy atorvastatin
Carbamazepine
Diazepam
Diclofenac
Dilantin
Enalapril
Fluoxetine
Norfluoxetine
Gemfibrozil
Meprobamate
Naproxen
Risperidone
Simvastatin
Simvastatin hydroxy acid
Sulfamethoxazole
Triclosan
Trimethoprim
Potential EDCs
Atrazine
Benzophenone
BHA
BHT
α-BHC
β-BHC
γ-BHC
δ-BHC
Bisphenol A
Butylbenzyl phthalate
DEET
Diazinon
Dioctyl phthalate
Galaxolide
Linuron
Methoxychlor
Metolachlor
Musk ketone
Nonylphenol
Octachlorostyrene
Octylphenol
TCEP
TCPP
Tonalide
Traseolide
Vinclozolin
Steroid Hormones
Estradiol
Estrone
Ethinylestradiol
Progesterone
Testosterone
©2008 AwwaRF. ALL RIGHTS RESERVED
Phytoestrogens
Apigenin
Biochanin A
Chrysin
Coumestrol
Daidzein
Equol
Formononetin
Genistein
Glycitein
Matairesinol
Naringenin
* In at least 20% of samples
Raw Intake for Drinking Water Facilities (n=19, *n=17)
Compound
Max (ng/L)
Median (ng/L)
Frequency (%)
Sulfamethoxazole
110
12
89
Meprobamate
73
8.2
84
Atrazine
870
32
79
Carbamazepine
51
4.1
79
Estrone
0.94
0.33
79
Dilantin
29
5.0
74
Atenolol
36
2.2
63
Gemfibrozil
24
2.2
58
Naproxen
32
0.93
58
Trimethoprim
11
0.75
58
TCEP
530
120
53
TCPP (Fyrol PCF)
720
180
42
Metolachlor
81
17
37
Apigenin *
3.0
1.8
35
DEET
110
85
32
Triclosan
6.4
3.0
32
Dioctyl phthalate
170
110
26
Linuron
9.3
4.1
26
Formononetin *
6.1
3.3
24
Genistein *
4.3
2.2
24
Diclofenac
1.2
1.1
21
Galaxolide
48
36
21
Nonylphenol
130
110
21
©2008 AwwaRF.3.1
ALL RIGHTS RESERVED 2.1
21
Progesterone
Target Compounds
Pharmaceuticals (20)
Atenolol
Atorvastatin
o-Hydroxy atorvastatin
p-Hydroxy atorvastatin
Carbamazepine
Diazepam
Diclofenac
Dilantin
Enalapril
Fluoxetine
Norfluoxetine
Gemfibrozil
Meprobamate
Naproxen
Risperidone
Simvastatin
Simvastatin hydroxy acid
Sulfamethoxazole
Triclosan
Trimethoprim
Potential EDCs (26)
Atrazine
Benzophenone
BHA
BHT
α-BHC
β-BHC
γ-BHC
δ-BHC
Bisphenol A
Butylbenzyl phthalate
DEET
Diazinon
Dioctyl phthalate
Galaxolide
Linuron
Methoxychlor
Metolachlor
Musk ketone
Nonylphenol
Octachlorostyrene
Octylphenol
TCEP
TCPP
Tonalide
Traseolide
Vinclozolin
Steroid Hormones (5) Phytoestrogens (11)
Estradiol
Estrone
Ethinylestradiol
Progesterone
Testosterone
©2008 AwwaRF. ALL RIGHTS RESERVED
Apigenin
Biochanin A
Chrysin
Coumestrol
Daidzein
Equol
Formononetin
Genistein
Glycitein
Matairesinol
Naringenin
Detected in Drinking Water*
Pharmaceuticals
Atenolol
Atorvastatin
o-Hydroxy atorvastatin
p-Hydroxy atorvastatin
Carbamazepine
Diazepam
Diclofenac
Dilantin
Enalapril
Fluoxetine
Norfluoxetine
Gemfibrozil
Meprobamate
Naproxen
Risperidone
Simvastatin
Simvastatin hydroxy acid
Sulfamethoxazole
Triclosan
Trimethoprim
Potential EDCs
Atrazine
Benzophenone
BHA
BHT
α-BHC
β-BHC
γ-BHC
δ-BHC
Bisphenol A
Butylbenzyl phthalate
DEET
Diazinon
Dioctyl phthalate
Galaxolide
Linuron
Methoxychlor
Metolachlor
Musk ketone
Nonylphenol
Octachlorostyrene
Octylphenol
TCEP
TCPP
Tonalide
Traseolide
Vinclozolin
Steroid Hormones
Estradiol
Estrone
Ethinylestradiol
Progesterone
Testosterone
©2008 AwwaRF. ALL RIGHTS RESERVED
Phytoestrogens
Apigenin
Biochanin A
Chrysin
Coumestrol
Daidzein
Equol
Formononetin
Genistein
Glycitein
Matairesinol
Naringenin
* In at least 20% of samples
Finished Water for Drinking Water Treatment Facility (n=18)
Compound
Max (ng/L)
Median (ng/L)
Frequency (%)
Atrazine*
870
49
83
Meprobamate
42
5.7
78
Dilantin
19
6.2
56
Atenolol
18
1.2
44
Carbamazepine
18
6.0
44
Gemfibrozil
2.1
0.48
39
TCEP
470
120
39
DEET
93
63
33
Metolachlor
27
16
33
TCPP (Fyrol PCF)
510
210
28
Sulfamethoxazole
3.0
0.39
22
*Atrazine is regulated under the Safe Drinking Water Act with an MCL of 3000 ng/L
©2008 AwwaRF. ALL RIGHTS RESERVED
Food / Beverages
©2008 AwwaRF. ALL RIGHTS RESERVED
Sample Matrices
• Food/beverage items
– Bottled water
– Soy sauce
– Beer
– Tea
– Coffee
– Apple/vegetable juice
– Milk (cow/soy based)
– Baby formula (cow/soy based)
©2008 AwwaRF. ALL RIGHTS RESERVED
E-screen Results (EEq ng/L) 1700
350
4000
300
300
250
200
150
100
50
©2008 AwwaRF. ALL RIGHTS RESERVED
Co
ffe
e
Be
er
13
So
yM
il k
*
6.0
So
yS
au
So
ce
yB
as
ed
Fo
rm
ula
*
4.6
2.9
WW
2.5
2n
d
0.73 0.75 0.77
Ap
ple
Ju
Mi
ice
lk
Ba
se
dF
or
mu
la
Fin
ish
ed
Wa
ter
Gr
ee
nT
ea
Ve
ge
tab
le
Ju
ice
Mi
lk
Wa
ter
Bo
ttle
d
Co
w
nd
0
0.57
EEq Comparison
(Max Finished Water)
≈
1 cup coffee
(17 ng/L, 240 mL)
5.3 Liters Finished
Drinking Water
(0.77 ng/L)
©2008 AwwaRF. ALL RIGHTS RESERVED
EEq Comparison
≈
Soy Baby Formula
(1700 ng/L, 4 oz Bottle)
265 Liters of Finished Drinking Water
(0.77 ng/L)
©2008 AwwaRF. ALL RIGHTS RESERVED
Daily Intake of EEq (ng) Comparison
• “Typical” day for average adult, per serving
Breakfast
Lunch
2 Cups coffee = 8.2 ng
Glass of milk = 0.1 ng
Glass of Juice = 0.2 ng
Dinner
Sushi w/ soy
sauce = 4.5 ng
Glass of beer = 2.2 ng
Cup of tea = 0.7 ng
• Est. Total Daily intake of EEq = 15.9 ng
©2008 AwwaRF. ALL RIGHTS RESERVED
Conclusions
©2008 AwwaRF. ALL RIGHTS RESERVED
What will we find at pg/L, fg/L, åg/L?
Why do we view
EDC/PPCPs differently?
Consider perchlorate…
We must determine
toxicological relevance!
©2008 AwwaRF. ALL RIGHTS RESERVED
Conclusions
•
•
•
•
•
Trace amounts of steroids and pharmaceuticals have been reported in
water for more than 30 years
Robust analytical methods are capable of accurately detecting and
quantifying chemicals in water at levels < 0.000000001 g/L
Only 11 of 62 target compounds were detected in finished drinking
water (>20% frequency)
– Atrazine had highest frequency at 83%, but at less than 1/3rd the MCL
– If MRLs were 10 ng/L, then 9 of 62 would have been detected
– If MRLs were 100 ng/L, then 3 of 62 would have been detected
– If MRLs were 1000 ng/L, then no compounds would have been detected
Exposure to estrogenic chemicals in diet are far greater than in
drinking water
Toxicological relevance is critical in order to establish meaningful
treatment and analytical goals
©2008 AwwaRF. ALL RIGHTS RESERVED