Forensic Ecotoxicology: In Search of Endocrine Disrupting Compounds in
Municipal Wastewater
D.A. Birkholz, PhD, P.Chem
Right solutions….
….Right partner
Acknowlegements
Preston Kylmatycki, ALS Laboratory Group, Edmonton
Jarrod Roberts, ALS Laboratory Group, Edmonton
Lydia Chiasson, HydroQual Laboratories, Calgary
Stephen Goudey, HydroQual Laboratories, Calgary
Grace Novak, City of Edmonton, Goldbar Wastewater Treatment Plant,
Endocrine Responses in Treated Municipal Effluent
• Have been reported• Particularly, vitellogenin
induction in male fish• Feminization of fish “roach”
has been reported• Potential candidates include:
nonylphenol, bisphenol A, nonylphenol ethoxylates, nonylphenol carboxylates, pharmaceuticals, etc.
Potential Endocrine Disrupting Compounds• Pharmaceuticals • Steroid hormones • Alkyphenol, ethoxylates and carboxylates and nonylphenol• Perfluorosulfonates - butane, hexane, octane• Perfluorcarboxylates - hexane, heptane, octane, nonane, decane, undecane, dodecane• Perfluorooctanesulfonamides (methyl, ethyl) • Fluorotelomer acids (hexyl, octyl, decyl) • Phthalate esters• Brominated flame retardents• Chlorinated dioxins/furans• Brominated dioxins/furans • Congener specific PCBs• Organochlorine pesticides • Triclosan• Personal care products (e.g. musks MX, MK, HHCB, AHTN, ATII)• Bisphenol A• Oxybenzone• Metals: lead, mercury, cadmium, etc• TOTAL ANALYTICAL COST $$$$ MUCHO DOLLARS!
Endocrine Disrupting Compounds
• Chemical analyses for this list is prohibitively expensive• Even if we obtained the data, how would we interpret the
results: not enough relevant toxicological data.• What about cumulative effects including synergism?• How realistic is the list of EDCs?• Are there other EDCs present which may be of greater
concern?• Chemistry is limiting because we have to focus on
specific analytes and use selected ion monitoring procedures. As a result may be blind to other EDCswhich may be present. Responses have been seen at 1 ng/L for 17β-estradiol so analyses have to be very sensitive.
Forensic Approach
separate
identify
manage
Forensic Method
pH 3filter, sparge, C18
25%, 50%, 75%, 100%MeOH fractions off column
Ambient filter, sparge, C18
25%, 50%, 75%, 100%MeOH fractions off column
pH 9.0filter, sparge, C18
25%, 50%, 75%, 100%MeOH fractions off column
detailed chemistry rationalize confirmatory testing
Retest
3 aliquots
fractionate chemical characterization
select biotests no effect - end
confirm effects
Approach
Look forpatterns in effects
pH 3, ambient & 9
Endocrine Tests for Estrogens
• A number of cell based in vitro assays have been applied which uses cells transfected with an estrogen receptor controlled reporter gene.
• Among these is the YES assay using yeast cells transfected with the human estrogen receptor (ER) and the reporter gene b -galactosidase . Estrogenic substances, which bind to the ER, bind to the estrogen responding elements causing the prouction of the ß-galactosidase enzyme which is measured colorimetrically.
• Exposure of male Fathead minnow and measurement of the yolk sac protein vitellogenin.
• Tested final effluent from Edmonton Goldbar wastewater treatment facility
Results and Discussion
25573.8-fold
Effluent Females
10016.7-fold
Effluent Males
668Control Females
6Control Males
FatheadMinnowAssays
Plasma Vitellogenin (µg/mL)
Results and Discussionun
treat
ed
aera
ted
filter
ed
25%
MeO
H
50%
MeO
H
75%
MeO
H
100%
MeO
H
100%
MeO
H
C18
effl
uent
pH 3pH 7
pH 11
0
0.5
1
1.5
2
2.5
3
3.5YE
S R
espo
nse
in E
2 Eq
uiva
lent
s (n
g/L)
70 % Recovery !
10.00 15.00 20.00 25.00 30.00 35.00 40.000
1000000
2000000
3000000
4000000
5000000
6000000Abundance
Time-->
TIC: L261742-1 50% MeOH pH 3.0
10.00 15.00 20.00 25.00 30.00 35.00 40.000
1000000
2000000
3000000
4000000
5000000
6000000
AbundanceTIC: L261742-1 pH amb 50% MeOH
Time--> 10.00 15.00 20.00 25.00 30.00 35.00 40.000
1000000
2000000
3000000
4000000
5000000
6000000
Abundance
TIC: L261742-1 pH 9 50% MeOH
diisooctylphthalate
diisooctylphthalateISTD
diisooctylphthalateISTD
ISTD
(replib) 1,2-Benzenedicarboxylic acid, d iisooctyl ester20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
0
50
100
29
4157
70
8393
104121 132
149
167
279
O
O
O
O
20 40 60 80 100 120 140 160 180 200 220 240 260 280 3000
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
m/z-->
Abundance
Scan 3166 (26.572 min): 149
167
57
43 71
11383 2792920713297 191179 267227241253 293
19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance TIC: L261742-1 pH amb 50% MeOH
TIC: L261742-MB pH amb 50% MeOH
Diisooctyl phthalate
Blank contribution
22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.500
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
Time-->
Abundance Ion 207.00 L261742 pH 3 Diazo
Ion 207.00 L261742 pH 3 Extract
Ion 207.00 L261742 pH 3 Acetylated
23.0023.5024.0024.5025.0025.5026.0026.5027.0027.5028.0028.5029.000
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
Time-->
Abundance Ion 251.00 L261742 pH 3.0 Diazo
Ion 251.00 L261742 pH 3.0 extract
Ion 251.00 L261742 pH 3.0 acetylated
50% MeOH Fraction After Alkylation
• The appearance of responses at m/z 207 and 251 is consistent with the presence of nonylphenoxyacetic acid (NP1EC) and nonylphenoxyethoxyaceticacid (NP2EC)
• These should have molecular ions of m/z 292 and 336 respectively
• The largest peaks did not have these molecular ions which raised the question as to what the compounds were?
12.5013.0013.5014.0014.5015.0015.5016.0016.5017.0017.5018.0018.5019.000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
400000
420000
Time-->
Abundance
Ion 207.00 (206.70 to 207.70): 0101003.D\data.ms
17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.000
50000
100000
150000
200000
250000
300000
350000
400000
450000
Time-->
Abundance
Ion 251.00 (250.70 to 251.70): 0101003.D\data.ms
20 40 60 80 100 120 140 160 180 200 220 240 260 280 3000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
m/z-->
Abundance
Scan 2710 (23.620 min): 6101034.D (-2705) (-)207
91 1175941 193147 179 22177 134105 159 27729 308235 261249 293
If NP1EC expect m/z 292 not 308
23.0023.5024.0024.5025.0025.5026.0026.5027.0027.5028.0028.500
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
Time-->
Abundance
Ion 251.00 L261742 pH 3.0 diazo Ion 251.00 L261742 pH ambient diazo
Ion 251.00 L261742 pH 9 diazo
50% MeOH Alkylated
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
m/z-->
Abundance
Scan 3011 (25.568 min): 6101034.D117
251
59
135
9145
73 35216129321177 207191 223 337237 273 305289
If NP2EC expect m/z 336 not 352
Phase II Study
• 100 L of wastewater extracted at pH 3.0
• Concentration factor 2000-fold
• Previous concentration factor 500-fold
• Yes response for 100L 14 vs 3.4 ~ 4-fold
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.000
130000
Time-->
Abundance
Ion 149.00 100L pH 3.0 No derivatization
10.00 15.00 20.00 25.00 30.00 35.00 40.000
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000170000
Time-->
Ion 149.00 2L pH 3.0 No derivatization
Abundance
2000-fold
500-fold
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.000
200000
400000
600000
800000
1000000
Time-->
Abundance
Ion 207.00 2 L 500-fold concentration
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.000
200000
400000
600000
800000
1000000
Time-->
AbundanceIon 207.00 100 L 2000-fold concentration
24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.500
200000
400000
600000
800000
1000000
Time-->
Abundance
Ion 251.00 2L 500-fold concentration
24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.500
200000
400000
600000
800000
1000000
Time-->
Ion 251.00 100 L 2000-fold concentration
Abundance
Increasing toxicity, persistence and bioconcentration
C9H19 OCH2CH2
C9H19 O CH2CH2O CH2COOH8
C9H19 OCH2CH2
NP9EO
NP9EC NP8EO
C9H19 OCH2CH2OCH2COOH
C9H19 OCH2COOH
C9H19 OCH2CH2OCH2CH2OH
C9H19 OCH2CH2OH
NP2EC
NP1EC
OH9
OH8
NP2EO
NP1EOC9H19 OH
NP
C9H19
OCH2
H2C
OHn
Nonylphenol ethoxylates
C9H19
OCH2
H2C
OCH2COOH
Nonylphenol carboxylates
n
WILL ALKYLATE with CH2N2
WILL NOT ALKYLATE with CH2N2
Because responses were only observed in the 50% MeOH fraction after alkylation we must be dealing with nonylphenol carboxylates
C5H11 C
C2H5
CH3
OH2C C
O
O H
Chemical Formula: C17H26O3Molecular Weight: 278
C6H13 C
CH3
CH3
OH2C C
O
O H
Chemical Formula: C17H26O3Molecular Weight: 278
NP1EC
NP1EC
alkylate
CH2N2
Chemical Formula: C17H26O3Molecular Weight: 278
C5H11 C
C2H5
CH3
OH2C C
O
O CH3
Chemical Formula: C18H28O3Molecular Weight: 292
C6H13 C
CH3
CH3
OH2C C
O
O CH3
Chemical Formula: C18H28O3Molecular Weight: 292
alkylate
CH2N2
C5H11 C
C2H5
CH3
OH2C C
O
O CH3
Chemical Formula: C18H28O3Molecular Weight: 292
C6H13 C
CH3
CH3
OH2C C
O
O CH3
Chemical Formula: C18H28O3Molecular Weight: 292
C
C2H5
CH3
OH2C C
O
O CH3
Chemical Formula: C13H17O3+
Molecular Weight: 221
NP1EC
C
CH3
CH3
OH2C C
O
O CH3
Chemical Formula: C12H15O3+
Molecular Weight: 207
NP1EC
15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.000
50000
100000
150000
200000
250000
300000
350000
400000
Time-->
AbundanceIon 207.00
15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.000
50000
100000
150000
200000
250000
300000
350000
400000
Time-->
AbundanceIon 221.00
C6H13 C
CH3
CH3
OH2C C
O
O CH3
C5H11 C
C2H5
CH3
OH2C C
O
O CH3
NP1EC
50% MeOH
pH 3.0
18.80 19.00 19.20 19.40 19.60 19.80 20.00 20.20 20.40 20.60 20.800
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
38000
40000
42000
44000
46000
48000
50000
52000
Time-->
Abundance
Nonylphenol monoethoxylate
C9H19
OCH2
H2C
OH
Chemical Formula: C17H28O2Molecular Weight: 264
PRECURSOR
14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
400000
420000
Time-->
Abundance Ion 207.00 50% MeOH fraction after alkylation
C6H13 C
CH3
CH3
OH2C C
O
O CH3
C
CH3
CH3
OH2C C
O
O CH3
Chemical Formula: C12H15O3+
Molecular Weight: 207
16.40 16.60 16.80 17.00 17.20 17.40 17.60 17.80 18.00 18.20 18.40 18.600
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000
9500
Time-->
Abundance
Ion 207.00
Ion 308.00
C
CH3
CH3
OH2C C O
O
CH3
Chemical Formula: C12H15O3+
Molecular Weight: 207
C
CH3
CH3
H2C
H2C
H2CC
O
O OH2C C O
O
CH3H3C
Chemical Formula: C17H24O5Molecular Weight: 308
C
CH3
CH3
OH2C C O
O
CH3
CH2H2C
H2C
H2C
H2CC
O
OH3C
Chemical Formula: C12H15O3+
Molecular Weight: 207
dm-CA6P1EC
Carboxyalkylphenol monoethoxycarboxylates (CAP1ECs)
17.80 17.90 18.00 18.10 18.20 18.30 18.40 18.50 18.60 18.70 18.800
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000
9500
Time-->
Abundance
Ion 207.00Ion 336.00
C
CH3
CH3
H2C
H2C
H2C
H2C
H2CC
O
O OH2C C O
O
CH3H3C
Chemical Formula: C19H28O5Molecular Weight: 336
C
CH3
CH3
OH2C C O
O
CH3
CH2H2C
H2C
H2C
H2CC
O
OH3C
Chemical Formula: C12H15O3+
Molecular Weight: 207
dm-CA8P1EC
Dicarboxylicacidmetabolites
15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
400000
420000
Time-->
Abundance
Ion 207.00 (206.70 to 207.70): 0101003.D\data.ms
OP1
EC
NP1
ECO
P1E C
NP1
ECN
P1EC C
A8P1
ECO
P1EC
OP1
ECC
A6P1
EC
DP 1
EC
CA6
P1EC
CA6
P 1E C
CA8
P1EC
CA 8
P1EC
CA
8P1E
CC
A8P 1
E CC
A8P
1EC
CA
8P1E
C CA 8
P1EC
DP1
EC
CA
7P1E
C
15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Time-->
Abundance Ion 221.00 (220.70 to 221.70): 0101003.D\data.ms
C5H11 C
C2H5
CH3
OH2C C
O
O CH3
NP1EC
C
C2H5
CH3
OH2C C
O
O CH3
Chemical Formula: C13H17O3+
Molecular Weight: 221
17.85 17.90 17.95 18.00 18.05 18.10 18.15 18.200
1000
2000
3000
4000
5000
6000
Time-->
Ion 221.00 (220.70 to 221.70): 0101003.D\data.ms (*)
Abundance
Ion 308.00 (307.70 to 308.70): 0101003.D\data.ms (*)
C
C2H5
CH3
H2C
H2CC
O
O OH2C C O
O
CH3H3C
Chemical Formula: C17H24O5Molecular Weight: 308
C
C2H5
CH3
OH2C C O
O
CH3
CH2H2C
H2C
H2C
H2CC
O
OH3C
Chemical Formula: C13H17O3+
Molecular Weight: 221
dm-CA6P1EC
Dicarboxylic acid metabolites
15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.500
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Time-->
Abundance
Ion 221.00 (220.70 to 221.70): 0101003.D\data.ms
NP 1E
C
CA8P1EC
NP1
E CN
P 1E
C
CA
8P1E
CD
P1EC
DP 1
EC
CA
6P1E
CC
A6P
1EC
CA
7P1E
C
CA
9P1E
C
CA
8P1E
C
C
CH3
CH3
C6H13 OH2C
H2C O
H2C C
O
O CH3
C
CH3
CH3
OH2C
H2C O
H2C C
O
O CH3
Chemical Formula: C20H32O4Molecular Weight: 336
Chemical Formula: C14H19O4+
Molecular Weight: 251
C6H13
C
C2H5
CH3
C5H11 OH2C
H2C O
H2C C
O
O CH3
Chemical Formula: C20H32O4Molecular Weight: 336
C
C2H5
CH3
OH2C
H2C O
H2C C
O
O CH3
Chemical Formula: C15H21O4+
Molecular Weight: 265
C5H11
NP2EC
NP2EC
17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.000
50000100000150000200000250000300000350000400000450000
Time-->
Abundance
Ion 251.00
17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.000
50000100000150000200000250000300000350000400000450000
Time-->
Ion 265.00
Abundance
C
CH3
CH3
OH2C
H2C O
H2C C
O
O CH3
C
C2H5
CH3
OH2C
H2C O
H2C C
O
O CH3
17.6017.7017.8017.9018.0018.1018.2018.3018.4018.5018.6018.700
500
10001500
2000
2500
3000
3500
4000
4500
5000
55006000
6500
Time-->
Abundance
Ion 251.00 (250.70 to 251.70): 0101003.D\data.ms (*)Ion 322.00 (321.70 to 322.70): 0101003.D\data.ms (*)
C
CH3
CH3
C5H11 OH2C
H2C O
H2C C
O
O CH3
OP2EC
17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.000
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
Time-->
Ion 251.00
Ion 352.00
OCH2CH2OCH2COOCH3C
CH3
CH3
H2C
H2C
H2CC
O
H3CO
Chemical Formula: C19H28O6Molecular Weight: 352
CA6P2EC
19.90 20.00 20.10 20.20 20.30 20.40 20.50 20.60 20.70 20.80 20.90 21.000
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000
9500
Time-->
Abundance
Ion 251.00
Ion 380.00
OCH2CH2OCH2COOCH3C
CH3
CH3
H3COOCC5H10
Chemical Formula: C21H32O6Molecular Weight: 380
CA8P2EC
17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.000
50000
100000
150000
200000
250000
300000
350000
400000
450000
Time-->
Abundance
Ion 251.00 (250.70 to 251.70): 0101003.D\data.ms
OP
2 EC
NP
2 EC
CA
6 P2E
CC
A5P
2EC
CA6
P2E
C
CA8P2EC
OP
2EC
CA
8P2E
CC
A8P
2EC
CA
7 P2E
C
18.00 18.50 19.00 19.50 20.00 20.50 21.000
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Time-->
Abundance
Ion 265.00 (264.70 to 265.70): 0101003.D\ data.msN
P2E
C CA8
P2E
C
NP2EC
CA5
P2E
C
CA 8
P2E
C
DP
2EC
CA6
P2E
CC
A7P
2EC
CA8
P2E
C
CA8P2EC
CA6
P2E
C
OP
2EC
CA
9P2E
C
Conclusions• YES response for 50% MeOH fraction at pH ambient and pH 9.0 best
explained by diisooctyl phthalate. Diisooctyl phthalate cannot explain the entire response.
• YES responses for 50% MeOH fraction at pH 3.0 cannot be explained by diisooctyl phthalate.
• YES responses for 50% MeOH fraction at pH 3.0 likely due to nonylphenol carboxylates and nonylphenol dicarboxylates
• Unknowns extracted at pH 3.0 require alkylation (diazomethane) to form the corresponding methyl esters – supportive of carboxylates
• Homologous series of responses observed at m/z 207 and 221. Major metabolites found include: CA8P1EC, CA6P1EC , NP1EC. Dominated by CA81PEC
•Homologous series of responses observed at m/z 251 and 265. Major metabolites found include: NP2EC, CA6P2EC and CA8P2EC. Dominatedby CA6P2EC and CA8P2EC
•The major metabolites found in the 50% MeOH extract were CA8P1EC, CA6P2EC and CA8P2EC
•These are dicarboxylates formed by carboxylation of the alkyl side chain and the terminal ethoxy group in the nonylphenol ethoxylate (NP1EO and NP2EO)
•The dicarboxylates have been reported in treatment plant effluents and rivers. But their endocrine disrupting properties have not been assessed.
•Our finding suggests that these compounds may be responsible forobserved endocrine responses in treated effluent
Conclusions
C9H19 OH2C
H2C O CH2CH2OH
n = 20-30
NPnEO
C9H19 O CH2CH2OCH2COOH
NP2EC
O CH2CH2OCH2COOHC8H16C
O
HO
CA8P2EC
C9H19 O CH2COOH
NP1EC
HOOCC8H16 O CH2COOH
CA8P1EC
O CH2CH2OCH2COOHC6H12C
O
HO
CA6P2EC
HOOCC6H12 O CH2COOH
CA6P1EC
45 - 64%
37 - 73%
17 - 43%
17 - 53%
41 - 46% 35 - 41%
Reference: Di Corcia et al (2000). Environ. Sci. Technol. 34: 3914-3919
CAPECs(Carboxyl Alkyl Phenol Ethoxy
Carboxylates)
• Laboratory biodegradation tests show that CAPECs with 3-8 carbons are recalcitrant. (Di Corcia et al (2000)
• Persisted in test liquor 5-months after their generation (Di Corcia et al (2000)
• Analysis of sewage treatment plant effluent showed CAPECs as a total accounted for 63% of the total A9PE breakdown products leaving the plant. (Di Corcia et al (2000)
•Of the CAPEC class, the carboxyalkylphenol monoethoxycarboxylates(CAP1ECs) and carboxyalkylphenol diethoxycarboxylates (CAP2ECs) are the most common (Hoai et al, 2004).
•Recent publications suggest that NPnECs and NPnEOs are endocrine disrupting (Sumpter and Johnson (2005); Fenet et al (2003); Sheahan et al (2002); Sapozhnikova et al (2005) and Isobe and Takada (2004).
•Our forensic approach suggests that CAPECs not NPnECs and NPnEOsare responsible for the EDC responses based upon a bioassay directed forensic approach
•This approach demonstrates that a bioassay directed approach can provide more meaningful information than chemical analyses alone.
CAPECs(Carboxyl Alkyl Phenol Ethoxy Carboxylates)