forensic ecotoxicology: in search of endocrine disrupting … · 2016-01-22 ·...

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


pH 9.0filter, sparge, C18


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


NP1EC

NP1EC

alkylate

CH2N2


C5H11 C

C2H5

CH3

OH2C C

O

O CH3


C6H13 C

CH3

CH3

OH2C C

O

O CH3


alkylate

CH2N2

C5H11 C

C2H5

CH3

OH2C C

O

O CH3


C6H13 C

CH3

CH3

OH2C C

O

O CH3


C

C2H5

CH3

OH2C C

O

O CH3

Chemical Formula: C13H17O3+

Molecular Weight: 221

NP1EC

C

CH3

CH3

OH2C C

O

O CH3



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


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



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



C

CH3

CH3

H2C

H2C

H2CC

O

O OH2C C O

O

CH3H3C


C

CH3

CH3

OH2C C O

O

CH3

CH2H2C

H2C

H2C

H2CC

O

OH3C



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


C

CH3

CH3

OH2C C O

O

CH3

CH2H2C

H2C

H2C

H2CC

O

OH3C



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



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


C

C2H5

CH3

OH2C C O

O

CH3

CH2H2C

H2C

H2C

H2CC

O

OH3C



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




C6H13

C

C2H5

CH3

C5H11 OH2C

H2C O

H2C C

O

O CH3


C

C2H5

CH3

OH2C

H2C O

H2C C

O

O CH3



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


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


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)

forensic ecotoxicology: in search of endocrine disrupting … · 2016-01-22 ·...

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