Toxicology Centre

Date of presentation Title or place of presentation

Unregulated halogenated disinfection by-products: Temporal trends in occurrence, NOM precursors,

and in vitro toxicity

Tena Watts Hui Peng, John Giesy, & Paul Jones

November 14, 2017 Society of Environmental Toxicology and Chemistry

Minneapolis, MN

Toxicology Centre

Date of presentation Title or place of presentation

Introduction • Water disinfection: the removal of pathogens from drinking water by use of physical or chemical

technologies • In the early 1970s, trihalomethanes were the first DBPs identified, with maximum allowable

concentrations regulated in 1979

Cl2

NH2Cl ClO2 O3 UV

Humic acid example Hydrophobic or hydrophilic

Acidic, basic, neutral

Br - I -

NO2 -

NH3 Disinfection By-Products (DBPs)

Natural Organic Matter Disinfectant Inorganic

Precursors

Toxicology Centre

Date of presentation Title or place of presentation

Current Understanding of DBPs and Challenges

• THM exposure correlated to increased rates of bladder cancer and adverse pregnancy outcomes1-3 • ~700 halogenated DBPs identified, with hundreds more that have been detected4,5 • Many unregulated DBPs have enhanced toxicities6-10

• Iodinated > brominated > chlorinated • Low abundances, standards unavailable, complex mixtures subject to matrix effect

Trihalomethanes (THMs) US EPA maximum annual average: 0.08 mg/L

Haloacetic acids (HAAs) US EPA maximum annual average: 0.06 mg/L

Toxicology Centre

Date of presentation Title or place of presentation Tena Watts November 14, 2017

Research Questions

• What unregulated brominated (Br-DBPs) and iodinated (I-DBPs) disinfection by-products exist in Saskatchewan drinking water?

• What relationships can be described between the temporal trends of NOM precursors and

unregulated DBPs?

• What endpoint is suitable for assessing the toxicity of real DBP mixtures?

Toxicology Centre

Date of presentation Title or place of presentation

Method Development for the Detection of Unknown Br- and I-DBPs

LC-HRMS • HPLC column (Amide and C18) • Q Exactive™ Quadrupole-Orbitrap™

Mass Spectrometer • Negative mode electrospray

(ESI) and atmospheric pressure chemical ionization (APCI)

2L of chlorinated water 2000x concentrated Solid phase extraction: • HLB, C18, and WAX • pH 7 and pH 2

Non-redundant library DIPIC-Frag method; Accurate mass, isotopic profiles,

MS2 spectra, chemometrics

• Buffalo Pound Water Treatment Plant (Moose Jaw, Saskatchewan, Canada) • Source water is a small eutrophic lake with elevated concentrations of bromide

Sample Preparation Instrumentation Data Analysis Library of Results

Toxicology Centre

Date of presentation Title or place of presentation

Frequency distributions of method precision for (A) Br-DBPs and (B) I-DBPs under different sample pretreatment method. Precision was calculated as relative standard deviation of each DBP to the mean value of abundances.

Heat map and hierarchical clustering of peak abundances of (A) Br-DBPs and (B) I-DBPs identified by use of various sample pretreatment conditions. Color indicates log-transformed abundances of DBPs.

• Library of 553 Br-DBPs and 112 I-DBPs • Best coverage of compounds and precision of

method (~10% variation) with HLB_pH2, Amide column, ESI (-) mode

Method Development for the Detection of Unknown Br- and I-DBPs

Toxicology Centre

Date of presentation Title or place of presentation

Results • Predicted structures for 41/50 and 9/10 most abundant Br- and I-DBPs, respectively • Several high-abundance Br- and I-DBPs containing nitrogen or sulfur • Trihalo-hydroxy-cyclopentene-diones (trihalo-HCDs)11 and sulfonic acids12

Toxicology Centre

Date of presentation Title or place of presentation Tena Watts November 14, 2017

Prince Albert Water Treatment Plant

• Three stages: Raw, Clearwell, Treated • April 2016 – March 2017 monthly sampling

• No data for August due to oil spill

Raw Water

Physical screen

Coagulation with poly-aluminum chloride

Powdered activated carbon

Flocculation

Sedimentation

Clarification Pretreatment

KMNO4 oxidation of

organics

Pre-chlorine dose

Silica sand/ anthracite

Filtration Clearwell

UV

Post-chlorine dose

Disinfection

Reservoir

Fully Treated

North Saskatchewan

River

Distribution System

Toxicology Centre

Date of presentation Title or place of presentation

Results: Disinfection By-Products

Raw Raw Raw Clearwell Clearwell Clearwell Finished Finished Finished

• Three trends exist for the formation of DBPs during the conventional water treatment process • These trends are consistent among monthly samples

Finished Clearwell Raw Finished Clearwell Raw

SO

OHO

Cl

Br

OO

Br

Cl2

OH

Toxicology Centre

Date of presentation Title or place of presentation

Results: Disinfection By-Products

0 2.0×106 4.0×106 6.0×106 8.0×106 1.0×1070

5.0×106

1.0×107

1.5×107

2.0×107

R2=0.94p<0.001

C5O3ClBr2

C5O

3Cl 2

Br

0 1.0×106 2.0×106 3.0×1060

5.0×106

1.0×107

1.5×107

R2=0.71p<0.001

C2NO3SClBr

CH

O3S

ClB

r

Correlation matrix of the abundances of DBPs shows that there are many different precursors and reaction pathways.

(A) Correlation between analogs with differing halogen substitutions

(B) Correlation between compounds containing sulfur

Toxicology Centre

Date of presentation Title or place of presentation

Results: Disinfection By-Products

SO

OHO

Cl

Br

OO

Br

Cl2

OH

• Seasonal trends exist for unregulated DBPs

A M J J S O N D J F M A M J J S O N D J F M A M J J S O N D J F M A M J J S O N D J F M

Lowest abundance in fall Greatest abundance in fall Greatest abundance in spring Greatest abundance in early winter

Toxicology Centre

Date of presentation Title or place of presentation

Results: Natural Organic Matter

Finished Clearwell Raw

C18H21O10

Finished Clearwell Raw

C17H27O3S

Finished Clearwell Raw

• General trend of decreasing NOM through the treatment process, consistent across months • Decrease can occur primarily before or after clearwell stage (UV and major chlorine dose)

Toxicology Centre

Date of presentation Title or place of presentation

A M J J S O N D J F M A M J J S O N D J F M A M J J S O N D J F M A M J J S O N D J F M

A M J J S O N D J F M A M J J S O N D J F M A M J J S O N D J F M A M J J S O N D J F M

Results: Temporal Trends Natural Organic Matter

DBPs

Toxicology Centre

Date of presentation Title or place of presentation

Results: Precursors of Sulfonic DBPs • Only 2/61 sulfonic DBPs closely followed trends of sulfur-containing NOM • Sulfonic DBPs were strongly correlated to concentration of bromide

SO

OHO

Cl

Br

0 4 8 12 16 20 240

1000000

2000000

3000000

0.0

200000.0

400000.0

600000.0

800000.0CHSO3ClBrC7H4SO6

Sample ID

CH

O3S

ClBr C

7H4SO

6

0 1.0×106 2.0×106 3.0×1060

50

100

150

200

R2=0.77p<0.001

CHSO3ClBr

Bro

mid

e(µ

g/L)

Toxicology Centre

Date of presentation Title or place of presentation

Methods: Toxicity Assessment

CHO-K1 Crystal violet dye (595 nm)

MCF-7 transfected with luciferase tagged Nrf-2

MTT cytotoxicity assay

Steadylite plus reporter gene assay system • Fold-induction compared to negative

control

72 h CHO-K1 Cytotoxicity Assay

16 h Nrf-2 Oxidative Stress Assay

Concentration range: 0.46875x – 60x

Concentrations: 7.5x, 15x, 30x

• April, June, September, November, January, March • Raw, clearwell, treated

Toxicology Centre

Date of presentation Title or place of presentation

Raw Clearwell Treated

April 56.989 ± 1.919 60.884 ± 3.601 54.511 ± 1.830

June 28.493 ± 2.365 57.809 ± 8.073 28.587 ± 1.833

September 50.219 ± 1.956 42.822 ± 1.688 30.125 ± 1.965

November 40.529 ± 1.111 37.051 ± 1.209 12.434 ± 1.216

January 77.009 ± 13.242 70.583 ± 5.329 24.350 ± 1.388

March 84.602 ± 15.703 40.018 ± 2.119 15.429 ± 1.281

72 h CHO-K1 Cytotoxicity of Water Samples Collected at the PAWTP: LC50 reported as mean ± standard deviation, with a colour gradient to show the most cytotoxic fractions with a darker shade of blue.

Dependent 2-group Wilcoxon Signed Rank Test (Mann-Whitney):

Raw vs. Clearwell p-value = 0.5625

Clearwell vs. Treated p-value = 0.03125*

Raw vs. Treated p-value = 0.0625

Results: 72 h Cytotoxicity Assay

Toxicology Centre

Date of presentation Title or place of presentation

Results: 16 h Nrf-2 Oxidative Stress Assay

* ** *

* ** *

* * *

*

* **

*

**

**

* *

* *

** **

**

**

**

**

**

**

**

* Significant at α=0.05 ** Significant at α=0.01

Dependent 2-group Wilcoxon Signed Rank Test (Mann-Whitney):

Raw vs. Clearwell p-value = 0.2188

Clearwell vs. Treated p-value = 0.03125*

Raw vs. Treated

p-value = 0.03125*

Toxicology Centre

Date of presentation Title or place of presentation

Pearson Correlation Analysis • April, June, September, November, January, March

• LC50 cytotoxicity values and induction of oxidative stress at 15 x • Bromide, temperature, turbidity, % UV transmittance, chlorine doses • DOM: total ion abundance, abundances of CHO, CHOS, and CHON compounds • DBPs: Br-DBPs, I-DBPs, sulfonic acids

• Oxidative stress and cytotoxicity were only significantly correlated for raw water

• Toxicities of raw water and clearwell water could not predict the toxicities of finished water

R2 = 0.6891 p-value = 0.04084*

Toxicology Centre

Date of presentation Title or place of presentation

• High concentrations of unregulated DBPs, with three different trends in formation/reduction

• Some seasonal trends exist for DBPs, but there are

many possible precursors and reaction pathways

• Most sulfonic DBPs were more strongly correlated to concentrations of Br than to S-NOM

• Toxicity of raw water could not predict the toxicity of finished water; disinfection alters the toxic pathways

• Oxidative stress is the better endpoint to compare the toxicities of DBP mixtures

Big Picture

Raw Raw Raw Clearwell Clearwell Clearwell Finished Finished Finished

A M J J S O N D J F M A M J J S O N D J F M

Toxicology Centre

Date of presentation Title or place of presentation

Thank you • Dr. Paul D. Jones • Dr. Hui Peng • Dr. John P. Giesy • Les Dickson • Dr. Lynn Weber • Dan Conrad • Andy Busse • Jim Hevdebo

Toxicology Centre

Date of presentation Title or place of presentation

(1) Costet, N.; Garlantézec, R.; Monfort, C.; Rouget, F.; Gagnière, B.; Chevrier, C.; Cordier, S. Environmental and urinary markers of prenatal exposure to drinking water disinfection by-products, fetal growth, and duration of gestation in the PELAGIE birth cohort (Brittany, France, 2002-2006). Am. J. Epidemiol. 2012, 175 (4), 263–275. (2) Cantor, K. P.; Lynch, C. F.; Hildesheim, M. E.; Dosemeci, M.; Lubin, J.; Alavanja, M.; Craun, G. Drinking water source and chlorination byproducts. I. Risk of bladder cancer. Epidemiology 1998, 9 (1), 21–28. (3) Villanueva, C. M.; Fernández, F.; Malats, N.; Grimalt, J. O.; Kogevinas, M. Meta-analysis of studies on individual consumption of chlorinated drinking water and bladder cancer. J. Epidemiol. Community Heal. 2003, 57, 166--173. (4) Gonsior, M.; Schmitt-Kopplin, P.; Stavklint, H.; Richardson, S. D.; Hertkorn, N.; Bastviken, D. Changes in dissolved organic matter during the treatment processes of a drinking water plant in Sweden and formation of previously unknown disinfection byproducts. Environ. Sci. Technol. 2014, 48 (21), 12714–12722. (5) Yang, M.; Zhang, X. Current trends in the analysis and identification of emerging disinfection byproducts. Trends in Environmental Analytical Chemistry. 2016, 10, 24–34. (6) Richardson, S. D.; Plewa, M. J.; Wagner, E. D.; Schoeny, R.; DeMarini, D. M. Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: A review and roadmap for research. Mutat. Res. - Rev. Mutat. Res. 2007, 636 (1–3), 178–242. (7) Plewa, M. J.; Muellner, M. G.; Richardson, S. D.; Fasano, F.; Buettner, K. M.; Woo, Y. T.; Mckague, a. B.; Wagner, E. D. Occurrence, synthesis, and mammalian cell cytotoxicity and genotoxicity of haloacetamides: An emerging class of nitrogenous drinking water disinfection byproducts. Environ. Sci. Technol. 2008, 42 (3), 955–961. (8) Richardson, S. D.; Fasano, F.; Ellington, J. J.; Crumley, F. G.; Buettner, K. M.; Evans, J. J.; Blount, B. C.; Silva, L. K.; Waite, T. J.; Luther, G. W.; Mckague, a. B.; Miltner, R. J.; Wagner, E. D.; Plewa, M. J. Occurrence and mammalian cell toxicity of iodinated disinfection byproducts in drinking water. Environ. Sci. Technol. 2008, 42 (22), 8330–8338. (9) Li, J.; Moe, B.; Vemula, S.; Wang, W.; Li, X. F. Emerging disinfection byproducts, halobenzoquinones: Effects of isomeric structure and halogen substitution on cytotoxicity, formation of reactive oxygen species, and genotoxicity. Environ. Sci. Technol. 2016, 50 (13), 6744–6752. (10) Zhang, X.; Echigo, S.; Minear, R. a; Plewa, M. J. Characterization and comparison of disinfection by-products of four major disinfectants. Am. Chem. Soc. 2000, 299–314. (11) Pan, Y., Li, W., Li, A., Zhou, Q., Shi, P., & Wang, Y. (2016a). A New Group of Disinfection Byproducts in Drinking Water: Trihalo-hydroxy-cyclopentene-diones. Environmental Science & Technology, 50(14), 7344–7352. (12) Zahn, D.; Frömel, T.; Knepper, T. P. Halogenated methanesulfonic acids: A new class of organic micropollutants in the water cycle. Water Res. 2016, 101, 292–299.

References

Toxicology Centre

Date of presentation Title or place of presentation

Toxicology Centre

Date of presentation Title or place of presentation

0 5.0×106 1.0×107 1.5×107 2.0×1070

50

100

150

R2=0.02p=0.41

C5O3Cl2Br

Bro

mid

e

OO

Br

Cl2

OH

Toxicology Centre

Date of presentation Title or place of presentation

0 4 8 12 16 20 240

1000000

2000000

3000000

0.0

200000.0

400000.0

600000.0

800000.0CHSO3ClBrC7H4SO6

Sample ID

CH

O3S

ClBr C

7H4SO

6

50 100 150 200 0

50

100

Rel

ativ

e A

bund

ance

82.9949

126.9850

170.9753

S

S

COOH

S

COOHHOOC

214.9651

S

COOHHOOCCOOH

Toxicology Centre

Date of presentation Title or place of presentation

Results: Dissolved Organic Matter • Direct injection on Orbitrap HF instrument

Total

Total ion abundance of dissolved organic matter compounds detected in water collected at the raw, clearwell, and treated stages of treatment.

Ion abundance of dissolved organic matter compounds detected in raw water, stacked based on the presence of S or N in the predicted formula.