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Occurrence and in vitro toxicity of disinfection by-products in Saskatchewan drinking water treatment plants

Tena Watts Hui Peng, John P. Giesy, & Paul D. Jones

University of Saskatchewan Society of Environmental Toxicology and Chemistry

Prairie Northern Chapter June 16, 2017

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Outline

• Introduction

• Buffalo Pound Water Treatment Plant • Method development for the detection of

Br- and I-DBPs • Toxicity throughout the treatment

process

• Prince Albert Water Treatment Plant • Comparison to Buffalo Pound • Correlation analysis

• Conclusion

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Introduction

Natural Organic Matter Disinfectant

Cl2

NH2Cl ClO2 O3 UV

Humic acid example

Inorganic Precursors

Br - I -

NO2 -

NH3 Disinfection By-Products (DBPs)

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

• Early 1970s, trihalomethanes were the first identified DBPs with maximum allowable concentrations regulated in 1979

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Current Understanding of DBPs

• >700 identified DBPs and more that remain unknown5 • Many unregulated DBPs have enhanced toxicities

• iodinated > brominated > chlorinated6-8 • nitrogenous > carbonaceous9,10

• Health Canada regulates 4 trihalomethanes (THMs; 0.1 mg/L) and 5 haloacetic acids (HAAs; 0.08 mg/L)1 • THMs correlated with increased rates of bladder cancer and adverse

pregnancy outcomes2-4

1 Health Canada. http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/sum_guide-res_recom/index-eng.php (2014). 2 Costet, N., et al. Am. J. Epidemiol. (2012). 3 Cantor, K.P., et al. Epidemiology. (1998). 4

Villanueva, C.M., et al. J. Epidemiol. Community Health. (2003). 5 Yang, M., et al. Environ. Sci. Technol. (2016). 6 Yang, Y., et al. Environ. Sci. Technol. (2014). 7 Richardson, S.D., et al. Mutat. Res.-Rev. Mutat. Res. (2007). 8 Richardson, S.D., et al. Environ. Sci. Technol. (2008). 9 Plewa, M.J., et al. Environ. Sci. Technol. (2008). 10 Muellner, M.G., et al. Environ. Sci. Technol. (2007).

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Research Questions

1. What brominated and iodinated compounds are yet to be identified in drinking water and how can we screen for them? • Q Exactive UHRMS - DIPIC-Frag method

2. Are current treatment technologies at BPWTP removing DBPs and their associated toxicities?

3. Can we predict the toxicity of chlorinated water that contains complex DBP mixtures?

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• Source water is quite eutrophic and contains high concentration of bromide (Br-)

• October 8, 2015: raw water intake, after initial chlorination, post-flocculation, pre-GAC filtration (post-sand filtration), and finished water (post-GAC, second chlorine dosage)

• April 15, 2016: no initial chlorination, GAC filter offline; raw water intake, clearwell, and finished (chlorinated) stages

Buffalo Pound Water Treatment Plant (BPWTP)

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8 • Source water is the North Saskatchewan River • Less biological material

• Coagulation, flocculation, sand filtration, UV filtration, and chlorination

• Monthly samples collected over one year at raw water intake, clearwell (after UV filtration), and finished water stages (April 2016 to March 2017) • No data for August due to Husky oil spill

Prince Albert Water Treatment Plant (PAWTP)

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

Mass Spectrometer • Electrospray (ESI) and

Atmospheric pressure chemical ionization (APCI)

Methods: Detection of unknown Br- and I-DBPs (BPWTP) Mass spectrometry library

Solid phase extraction: • HLB, C18, and WAX • pH 7 and pH 2

Non-redundant library

DIPIC-Frag method; Accurate mass, isotopic profiles,

chemometrics, MS2 spectra

2 L samples of chlorinated water

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• Library of 553 Br- and 112 I-DBPs • Predicted structures for 41/50 and 9/10 most abundant Br- and I-DBPs • Several high-abundance Br- and I-DBPs containing nitrogen or sulfur

• Methanesulfonic acids11

Results: Detection of Br- and I-DBPs in Chlorinated Water (BPWTP)

(B)

SO

OHO

Cl

BrSO

OHO

Cl

I

11 Zahn, D., et al. Water Res. (2016).

500

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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

• Greatest coverage of Br- and I-DBPs – adjustment to pH 2; HLB cartridge

72 h CHO-K1 Cytotoxicity Assay

16 h Nrf-2 Oxidative Stress Assay

Concentration range: 0.46875x – 60x

Concentrations: 7.5x, 15x, 30x

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Raw Chlorinated Post-Flocculation Pre-GAC (Filtered) Finished

LC50(mean ± se) 17.298 ± 0.836 11.611 ± 0.576 43.797 ± 1.588 33.874 ± 1.006 66.703 ± 1.746 Residual standard error: 5.811 (118 df)

Cytotoxicity at Each Stage of Treatment (BPWTP; Oct 2015)

• Raw water induced cytotoxicity independent of DBPs • Chlorination slightly increased cytotoxicity, but as water moves

through the treatment process, cytotoxicity is reduced

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• Cytotoxicity was observed in chlorinated samples • Significant differences (α=0.05) in oxidative stress were found

between chlorinated and finished water (3.75x, 7.5x, and 15x), pre-GAC and finished water (7.5x and 15x), and chlorinated and post-floc water (3.75x)

Oxidative Stress Response at Each Stage of Treatment (BPWTP; Oct 2015)

(A) Induction of the Nrf-2 oxidative stress pathway in MCF-7 cells (16 h) by water collected at BPWTP at each stage of treatment (* significant at α=0.05 in one sample t-test against µ=1). (B) 16 h MTT cytotoxicity assay of the same samples.

(A) (B)

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Does the treatment process increase oxidative stress? BP-Raw vs. BP-Finished p-value: 0.001525** PA-Raw vs. PA-Finished p-value: 0.09082 Does chlorination increase oxidative stress? BP-Clearwell vs. BP-Finished p-value: 0.00422** PA-Clearwell vs. PA-Finished p-value: 0.3648 Do stages prior to chlorination (coagulation & filtration) alter the oxidative stress of raw water? BP-Raw vs. BP-Clearwell p-value: 0.1459 PA-Raw vs. PA-Clearwell p-value: 0.7367

Induction of the Nrf-2 oxidative stress pathway in MCF-7 cells (16 h) by raw, clearwell, and finished water collected from BPWTP and PAWTP in April 2016 (* significant at α=0.05 and ** significant at α=0.01 in one-sample t-test against µ=1).

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Comparing Oxidative Stress at BPWTP and PAWTP (Apr 2016)

• The treatment process, and specifically chlorination, increased the oxidative stress of water at BPWTP but not PAWTP

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Raw Clearwell Finished

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 LC50 values (mean ± se) of raw, clearwell, and finished water samples from PAWTP. Colour gradient represents most cytotoxic extracts with darker shades of blue.

Dependent 2-group Wilcoxon Signed Rank Test (Mann-Whitney): Raw vs. Clearwell: p-value = 0.5625 Clearwell vs. Finished: p-value = 0.03125* Raw vs. Finished: p-value = 0.0625

Cytotoxicity of Water Collected from PAWTP

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Raw Clearwell Finished

April 1.536 ± 0.0487** 1.873 ± 0.1930* 2.190 ± 0.0816**

June 1.541 ± 0.1139* 1.380 ± 0.0723* 3.589 ± 0.1177**

September 1.353 ± 0.0389** 1.454 ± 0.0567** 2.960 ± 0.1821**

November 1.530 ± 0.0944* 1.967 ± 0.0200** 3.303 ± 0.2894**

January 1.338 ± 0.1116* 1.281 ± 0.0634* 2.190 ± 0.1827**

March 1.193 ± 0.0683* 1.372 ± 0.1070* 2.683 ± 0.0833** Fold induction of the Nrf-2 oxidative stress pathway (mean ± sd; n=3) by raw, clearwell, and finished water from PAWTP. Colour gradient represents samples with the greatest induction with darker blues. Significance was evaluated in a one-sample t-test against µ=1 (*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. Finished: p-value = 0.03125* Raw vs. Finished: p-value = 0.03125*

Oxidative Stress of Water Collected from PAWTP

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(A) Correlation analysis of water parameters and toxicities for water collected in April, June, September, November, January, and March from the PAWTP. (B) Significant correlations at α=0.05.

Correlation Analysis Can we predict the toxicity of real DBP mixtures?

• Oxidative stress & cytotoxicity of raw water • UV transmittance & oxidative stress of raw water • Turbidity of raw water & cytotoxicity of finished water • Bromide in clearwell water & oxidative stress of finished water

• 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

(B) (A)

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• DIPIC-Frag method was robust for detecting Br- and I-DBPs • Established a library with 553 Br- and 112 I-DBPs

• The current treatment process at BPWTP, specifically the GAC

filter, is removing cytotoxic and oxidative stress inducing compounds

• Finished water at PAWTP was not significantly more cytotoxic than raw water, however, it did induce greater oxidative stress • Oxidative stress may be a more selective endpoint than

cytotoxicity for DBP mixtures

• In univariate analyses, raw water parameters were not strong predictors for the toxicity of finished water (DBP mixtures)

• Raw water toxicity could not predict finished water toxicity

Conclusions

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• Dan Conrad at the Buffalo Pound Water Treatment Plant, and Andy Busse and Jim Hevdebo at the Prince Albert Water Treatment Plant for their collaboration

• Hui Peng • Paul Jones, John Giesy, Les Dickson, & Lynn Weber • Funding from NSERC CREATE for Water Security & The Global

Institute for Water Security

Thank you!