Emerging Drinking Water Disinfection By-Products Susan D

Emerging Drinking Water Disinfection By-Products Susan D. Richardson

U.S. Environmental Protection Agency, National Exposure Research Laboratory, Athens, GA

U.S. Environmental Protection Agency Office of Research & Development Drinking Water DBPs—What are the Issues?

Concern over possible human health risk:

• Epidemiologic studies: risk of bladder cancer; some cause cancer in laboratory animals

• Recent concerns about possible reproductive & developmental effects (from epi studies)

Goal: Comprehensively identify DBPs formed from different disinfectants, test for toxicity, understand their formation, minimize or eliminate in drinking water Drinking Water DBPs: How are they formed?

DBPs discovered in 1974

Jon Rook Tom Bellar >600 DBPs Identified

Halogenated DBPs Non-halogenated DBPs • Halomethanes • Nitrosamines • Haloacids • Aldehydes • Haloaldehydes • Ketones • Haloketones • Carboxylic acids • Halonitriles • Others • Haloamides • Halonitromethanes • Halofuranones (e.g., MX) • Oxyhalides (e.g., bromate) • Many others >600 DBPs Identified

Halogenated DBPs Non-halogenated DBPs • Halomethanes • Nitrosamines • Haloacids • Aldehydes • Haloaldehydes • Ketones • Haloketones • Carboxylic acids • Halonitriles • Others • Haloamides • Halonitromethanes • Halofuranones (e.g., MX) N-DBPs • Oxyhalides (e.g., bromate) • Many others But, more than 50% still not known….

Unknown 69.9%

HNMs 0.5% HACEs 0.5% HKs 0.9% HALDs 1.8% HANs 0.8% HAAs 11.8%

THMs 13.5%

Halofuranones 0.1% IodoTHMs 0.2%

Nationwide Occurrence Study, Krasner et al., Environ. Sci. Technol. 2006, 40, 7175-7185.

~50% of TOX >1000 Da: Khiari, et al., Proc. 1996 AWWA Water Quality Technology Conference Only 11 DBPs Regulated in U.S.

DBP MCL (µg/L) Total THMs 80 5 Haloacetic acids 60 Bromate 10 Chlorite 1000

Little known about occurrence, toxicity of unregulated DBPs Regulated DBPs do not cause bladder cancer in animals! Only 11 DBPs Regulated in U.S.

DBP MCL (µg/L) Total THMs 80 5 Haloacetic acids 60 Bromate 10 Chlorite 1000

One regulated DBP never tested for cancer -Two unregulated DBPs are carcinogens - Many unregulated DBPs more genotoxic than regulated ones

Richardson, Plewa, Wagner, Schoeny, and DeMarini. Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: A review and roadmap for research. Mutation Research 2007, 636, 178-242. Bladder cancer and drinking water: Pooled analysis

1,5 ) 0-1ug/L 1 >1-5 ug/L >5-25ug/L OR (95% CI (95% OR 0,5 >25-50ug/L >50ug/L 0 Men Women Both OR adjusted by (sex), study, age, smoking status, ever worked in high-risk occupations, heavy coffee consumption and total fluid intake

Villanueva et al., Epidemiology 2004, 15, 357-367. Exposure routes

Inhalation Ingestion Dermal absorption (shower, swimming (water, coffee, tea, (swimming pool, pool, etc.) water-based food and bath, etc.) beverages) Volatile DBP Permeable DBPs e.g. THMs All disinfection by- e.g. THMs, products haloketones, …

TOTAL INTERNAL DOSE

Slide courtesy of Manolis Kogevinas, Centre for Research in Environmental Epidemiology/IMIM, Barcelona Unlike other contaminants that may or may not be present in drinking water…

DBPs are ubiquitous DibromonitromethaneDibromonitromethane——DNADNA AdductsAdducts

600 Control #10 600 Control # 70 Male Rat Liver DNA Female Rat Liver DNA 500 500 Butanol Extracted Butanol Extracted (0.32 *109 ) 9 400 400 (0.24 * 10 )

300 300

1.49 DBNM produces DNA 1.37 1.26 1.16 200 1.26 200 1.49

100 100 adducts in the livers of 0 0 rats after only 30 days 1200 High Dose Male # 58 DNBM 600 High Dose Female # 119 DNBM Liver DNA Butanol Extracted Liver DNA Butanol Extracted 500 of exposure 1000 (7.7 * 109 ) (3.7 * 109 ) 800 400 1.49 600 300 1.37 (in vivo, male and 1.26

200 0.88 400 1.16 1.16 0.88 female rats) 200 100 0 0 High Dose 600 High Dose Male # 59 DNBM 1200 Female # 120 DNBM Liver Tony also now seeing

Liver DNA Butanol Extracted 1.26 500 1000 DNA Butanol (2.3 * 109 ) Extracted (13.6 * 109 ) 400 800 effects in normal 9 P-32 Disintigrations per Minute (DPM) Minute per Disintigrations P-32

300 600 1.4 1.26 0.95 1.12 7 human colon cells 0.88 200 400 1.3 1 1. 2 8 9 0. 5

100 200 0.8 0 0 0 10203040506070 0 10203040506070 Retention Time (min)

Data courtesy of Tony DeAngelo & Leon King, U.S. EPA, NHEERL, RTP, NC Iodo-THMs

THM4 Iodinated THMs

12 CHCl 2I

CHCl 2Br 10

8 g/L)

μ CHClBr 6 CHCl 3 2 THM ( 4 CHClBrI CHClI 2

CHBr 2I 2 CHBrI 2

0 1 Iodinated THMs 3 Cl 2 CHBr 3 2 Cl 3 THM4 4 1 Cl 5 0 Cl 6

Krasner, Weinberg, Richardson, et al., Environ. Sci. Technol. 2006, 40, 7175-7185. Iodo-DBPs Maximized with Chloramines Chlorine: fast fast fast I- + HOCl HOI IO - IO - HOCl 2 HOCl 3 fast iodite iodate NOM NOM Sink for iodide Cl-DBPs iodo-DBPs HOCl also competes for rxn with NOM, so much lower iodo-DBPs with chlorine

Chloramines: HOI also has longer half-life in slow chloraminated waters fast - - - I + NH2Cl HOI X IO2 IO3 iodite iodate fast NOM NOM Cl-DBPs iodo-DBPs Adapted from Bichsel and von Gunten, 1999 and 2000 Genotoxicity of Iodoacetic acid

Plewa et al., Environ. Sci. Technol. 2004 IA also caused developmental effects in mouse embryos (Hunter et al., 1995) Haloamides

H H H O O O Br C Cl C Cl C C NH2 C NH2 C NH2

H H Cl Chloroacetamide Bromoacetamide Dichloroacetamide

H H Cl O O O Br C Cl C Br C C NH2 C NH2 C NH2

Cl Br Cl Bromochloroacetamide Dibromoacetamide Trichloroacetamide

• New class of DBP recently identified

• Nationwide DBP Occurrence Study: up to 14 ug/L; NH2Cl may increase their formation

• Highly genotoxic, cytotoxic Br O

• New iodoamide DBP: Bromoiodoacetamide I CC NH2

- Found in drinking water from 6 states H

Plewa et al., Environ. Sci. Technol. 2008, 42, 955-961. Haloamides--Cytotoxicity

DBP Chemical Class Bromate Tribromopyrrole MX EMS +Control Other DBPs Tribromoacetamide Trichloroacetamide Chloroiodoacetamide Dichloroacetamide Iodoacetamide Bromochloroacetamide Dibromochloroacetamide Diiodoacetamide Bromoiodoacetamide Bromoacetamide Dibromoacetamide Chloroacetamide Haloacetamides Chloroacetonitrile Trichloroacetonitrile Dibromoacetonitrile Bromoacetonitrile Bromochloroacetonitrile Iodoacetonitrile Dichloroacetonitrile Haloacetonitriles Iodoform Chloroform Bromoform Chlorodibromomethane Halomethanes BCNM BDCNM DCNM DBNM BNM TBNM CNM TCNM DBCNM Halonitromethanes 3,3-Dibromo-4-oxopentanoic Acid 3,3-Dibromo-4-oxopentanoic Acid 3-Iodo-3-bromopropenoic Acid 2,3-Dibromopropenoic 2-Iodo-3-bromopropenoic Acid 2-Iodo-3-bromopropenoic Tribromopropenoic AcidTribromopropenoic 3,3-Dibromopropenoic Acid 3,3-Dibromopropenoic Acid 2-Bromo-3-methylbutenedioic 3,3-Bromochloro-4-oxopentanoic Acid 2-Bromobutenedioic Acid Halo Acids BIAA TBAA BDCAA BCAA CAA DCAA IAA BAA TCAA DIAA DBAA DBCAA Haloacetic Acids

10-6 10-5 10-4 10-3 10-2 CHO Cell Cytotoxicity as %C½ Values (~LC50)

Log Molar Concentration (72 h Exposure) December 2006

Data courtesy of Michael Plewa, University of Illinois Haloamides--Genotoxicity

DBP Chemical Class MX EMS +Control Bromate Other DBPs Bromo/iodo Tribromopyrrole amides Tribromoacetamide Chloroiodoacetamide Bromoacetamide Diiodoacetamide Iodoacetamide Trichloroacetamide Chloroacetamide Bromoiodoacetamide Dibromchlorooacetamide Bromochloroacetamide Dibromoacetamide Haloacetamides Chloroacetonitrile Trichloroacetonitrile Dibromoacetonitrile Dichloroacetonitrile Iodoacetonitrile Haloacetonitriles Bromoacetonitrile Bromochloroacetonitrile DBNM TBNM TCNM BCNM BDCNM DCNM CNM Halonitromethanes BNM DBCNM 2,3-Dibromopropenoic Acid 2,3-Dibromopropenoic 3,3-Bromochloro-4-oxopentanoic Acid 3,3-Bromochloro-4-oxopentanoic 3,3-Dibromo-4-oxopentanoic Acid 3,3-Dibromo-4-oxopentanoic 2-Iodo-3-bromopropenoic Acid Halo Acids Acid 2-Bromobutenedioic BCAA DIAA BIAA DBAA IAA TBAA CAA BAA CDBAA Haloacetic Acids

10-6 10-5 10-4 10-3 10-2 Single Cell Gel Electrophoresis Genotoxicity Potency Log Molar Concentration (4 h Exposure)

Not Genotoxic: DCAA, TCAA, BDCAA, Dichloroacetamide, Chloroform Chlorodibromomethane, 3,3-Dibromopropenoic Acid, 3-Iodo-3-bromopropenoic Acid, 2,3,3,Tribromopropenoic Acid December 2006

Data courtesy of Michael Plewa, University of Illinois Genotoxicity of Other DBPs

DBP Chemical Class MX EMS +Control Bromate Other DBPs Bromo/iodo Tribromopyrrole acetonitriles Tribromoacetamide Chloroiodoacetamide Bromoacetamide Diiodoacetamide Iodoacetamide Trichloroacetamide Chloroacetamide Bromoiodoacetamide Dibromchlorooacetamide Bromochloroacetamide Dibromoacetamide Haloacetamides

Bromo- nitromethanes Chloroacetonitrile Trichloroacetonitrile Dibromoacetonitrile Dichloroacetonitrile Iodoacetonitrile Haloacetonitriles Bromoacetonitrile Bromochloroacetonitrile DBNM TBNM TCNM BCNM BDCNM DCNM CNM Halonitromethanes IAA BNM DBCNM 2,3-Dibromopropenoic Acid 2,3-Dibromopropenoic 3,3-Bromochloro-4-oxopentanoic Acid 3,3-Bromochloro-4-oxopentanoic 3,3-Dibromo-4-oxopentanoic Acid 3,3-Dibromo-4-oxopentanoic 2-Iodo-3-bromopropenoic Acid Halo Acids Acid 2-Bromobutenedioic BCAA DIAA BIAA DBAA IAA TBAA CAA BAA CDBAA Haloacetic Acids

10-6 10-5 10-4 10-3 10-2 Single Cell Gel Electrophoresis Genotoxicity Potency Log Molar Concentration (4 h Exposure)

Not Genotoxic: DCAA, TCAA, BDCAA, Dichloroacetamide, Chloroform Chlorodibromomethane, 3,3-Dibromopropenoic Acid, 3-Iodo-3-bromopropenoic Acid, 2,3,3,Tribromopropenoic Acid December 2006

Data courtesy of Michael Plewa, University of Illinois But, all of this toxicity testing is for separate, individual DBPs…

DBPs are really present as MIXTURES

>300 DBPs probably present in glass of water Four Lab Study

Integrated Disinfection By-products Mixtures Research: Toxicological and Chemical Evaluation of Alternative Disinfection Treatment Scenarios A collaborative effort between: NHEERL (National Health and Environmental Effects Research Laboratory), RTP NERL (National Exposure Research Laboratory), Athens NRMRL (National Risk Management Research Laboratory), Cincinnati NCEA (National Center for Environmental Assessment), Cincinnati

Purpose: To address concerns related to potential health effects from exposure to DBPs that cannot be addressed directly from toxicological studies of individual DBPs or simple DBP mixtures In Vitro and In Vivo Toxicological Assays

In vitro: • Reproductive/developmental • Mutagenicity • Carcinogenicity • Neurotoxicity • Metabolism

In vivo: • Reproductive/developmental • Mutagenicity/carcinogenicity • Immunotoxicity • Hepatic/renal toxicity • Neurotoxicity/developmental neurotoxicity • Kinetics/metabolism RO Concentration of DBPs

From Plant (0.5 gpm)

600 gal. 500 gal. 200 CW gal. Ion Exchange

Permeate to Waste

100 gal. 14 gal.

Water is concentrated by RO to maintain the water matrix for rats to be able to drink (can’t give organic solvents to rats!) Full Study Concentration RO Concentration of DBPs

From Plant (0.5 gpm) RAW SURFACE WATER FLOW EQUALIZATION 600 gal. 500 gal. ION EXCHANGE 200 CW gal. COLUMNS Ion BAG Exchange FILTER

Permeate to Waste ULTRAFILTRATION MEMBRANES

100 gal. 14 gal. PERMEATE

CONCENTRATE 1st Phase (1999-2001): ION REVERSE EXCHANGE OSMOSIS COLUMNS MEMBRAMES Cl2 & O3; treated water first Concentrated after

Full Study (2006-2008): Concentrated NOM first

Treated with Cl2 after

1st Phase of study published: Richardson et al., J. Toxicol. Environ. Health 2008, 71, 1165-1186. Other 4-Lab papers also in this special issue General Timeline for Animal Experiments

Exposure Starts GD 2 GD PND PND 0 0 21

Gestation Lactation P0

Born Wean Mate Parturition

Gestation Lact’n F1

Gestation Length Parturition Born Litter Exams Litter Exams Litter Exams Litter Exams Postnatal Day 0 Postnatal Day 6 Day 13 Day 21 F2 Viability Viability Viability Viability Pup weight Pup weight Eye opening Pup weight Anogenital distance Reduce litter size to 8 Nipple retention Wean • 12 litters/group (4 males, 4 females) Assign fate Results

• Good mix of Cl/Br DBPs produced • Most DBPs fairly consistently produced among chlorination events • Most DBPs are stable over time on the rats’ cages Toxicity Results

No effects for: Effects for: • Gestation length • Delayed puberty in F1 females (<1 day) • Prenatal viability • Delayed puberty also seen with regulated DBP mixtures • Postnatal viability • Subtle puberty effect (at 136x) that appears consistent with • Pup Weight dose-response curve of regulated DBPs at 500x, 1000x, 2000x • Eye opening • Increased anogenital distance (AGD) in F1 males • Nipple retention • Males: Sperm counts down 50% • Organ weights • Total litter loss (postnatal) in one F2 litter • Vaginal prolapse in one P0 dam • Dental malocclusion in one F1 litter • Small neurotoxicity effects (motor activity, grip strength) • Inhibited differentiation of human placental trophoblast cells and hCG secretion (both with chlorinated concentrate and regulated DBPs) • Small increase in mutagenicity in chlorinated concentrate • Mammary tumors (regulated DBP mixture) Conclusions

• A thorough examination of reproductive/developmental and other endpoints was predominantly negative

• Some small, subtle effects for chlorinated water concentrate (136x concentration factor may be “on the edge” of ability to see effects)

• Concentration offered by RO a bonus for detecting DBPs present at very low levels (e.g., MX, which is present in drinking water at ng/L levels)

• Combination of comprehensive, qualitative identification work and quantification of 75 DBPs allowed comprehensive assessment of DBPs present in water

• Most DBPs stable on rats’ cages and chlorination events were reproducible

Want to determine the statistical power in this study for detection of the subtle effects noted and do a follow-up study that includes Chlorine vs. Chloramines Formation of iodo-DBPs from X-ray contrast media

HOCl + NOM Iodo-DBPs NH2Cl

Iopamidol

Richardson, Duirk, Lindell, Cornelison, Ternes, presented at Micropol Conference, June 2009 Iodo-DBP Occurrence Study

Iodide (µg/L) Sum iodo-acids Sum iodo-THMs (µg/L) (µg/L) Plant 2 1.0 0.37 4.9

Plant 4 ND 0.10 1.2

Plant 11 1.5 0.21 2.3

Plant 15 ND 0.17 2.4

Detection limit = 0.13 µg/L

Richardson et al., Environ. Sci. Technol. 2008, 42, 8330-8338. Iodo-DBP Occurrence Study

Iodide (µg/L) Sum iodo-acids Sum iodo-THMs (µg/L) (µg/L) Plant 2 1.0 0.37 4.9

Plant 4 ND 0.10 1.2

Plant 11 1.5 0.21 2.3

Plant 15 ND 0.17 2.4

Detection limit = 0.13 µg/L What about other sources of iodine? Iodinated X-ray Contrast Media (ICM)

CH3 OH O NOH

I I O OH

H3CO NH OH NH

I O Iopromide Iopamidol

Iohexol ICM concentrations: rivers, creeks and ground water

100 (> 100 µg/L) Diatrizoate Iopamidol ( 21 µg/L) Iopromide (10 µg/L) Ioxitalamic acid 10 Iothalamic acid ( ) Sum (2.8 µg/L) (1.0 µg/L) µg/L 1 (0.6 µg/L)

0.1

0.01 Rhine Mühlbach Mittelgraben Winkelbach groundwater groundwater

Ternes & Hirsch, Environ. Sci. Technol. (2000) 34, 2741-2748 ICM in U.S. Drinking Water Sources (ng/L)

Iopamidol Iomeprol Iopromide Iohexol Diatrizoate

Plant 1 11 ND ND ND ND Plant 2 510 ND 24 120 93 Plant 4 110 ND 6 49 ND Plant 10 ND ND ND ND ND Plant 11 100 ND ND 85 ND Plant 12 280 ND ND 120 ND Plant 13 ND ND ND ND ND Plant 15 2700 ND 25 ND ND Plant 17 ND ND ND ND ND Plant 19 ND ND ND ND ND Courtesy of Thomas Ternes, Federal Institute of Hydrology, Germany ICM measured using LC/ESI-MS/MS; DLs = 5-20 ng/L ICM in U.S. Drinking Water Sources (ng/L)

Iopamidol Iomeprol Iopromide Iohexol Diatrizoate

HOCl Iodo-DBPs NH2Cl ??

Iopamidol Controlled Laboratory Reactions

Experiments

• React ICM with HOCl, NH2Cl (with and without NOM) • 3 pHs • Follow formation of iodo-DBPs • Identify reaction products and intermediates • Measure genotoxicity

Methods • Iodo-THMs: GC/EI-MS • Iodo-Acids: GC/NCI-MS (with derivatization) • Iopamidol (and other ICM): LC, LC/MS/MS Cristal and Steve • Larger MW products and intermediates: LC/MS/MS • Genotoxicity: Chinese hamster ovary cells, single cell gel electrophoresis Results

HOCl X Iodo-DBPs NH2Cl

Iopamidol

HOCl + NOM Iodo-DBPs 9 NH2Cl

Iodo-THMs & Iopamidol Iodo-Acids Genotoxicity: Comet Assay

The tail moment is the integrated value of DNA density multiplied by the migration distance. The % tail DNA is the amount of DNA that has migrated into the gel from the nucleus.

42 Genotoxicity of Chlorinated Waters Containing Iopamidol

ACC + Iopamidol +HOCl 60 ACC +HOCl

ACC + Iopamidol Average Mean SCGE %Tail DNA Value Average Mean SCGE %Tail DNA

CHO Cells Genomic DNA Damage as the CHO Cells Genomic 0 0 200 400 600 Organic Extracts from Water Samples (Concentration Fold) Roadmap—Where do we go from here?

• Human health effects not solved yet—need more toxicity studies • Studies on route of exposure Have we been looking at the wrong route of exposure? • DBPs are present as complex mixtures—need toxicity studies addressing this • What is in the unidentified fraction—anything of concern? • What about ‘pollutant’ DBPs? • What about DBPs from alternative disinfectants—do we know everything we need to know before plants switch? • Chloramination? UV disinfection? Membrane disinfection? • What about other respiratory/skin effects reported for chloraminated water? Need showering and dermal exposure studies Serious skin rash issues….

“Before” Showering with chloraminated water “After” Showering with chlorinated water at the YMCA in another town Acknowledgments

David DeMarini

Michael Plewa Tony DeAngelo

A few fabulous toxicologists who have helped push this field forward….

Jane Ellen Simmons Also, Mike Narotsky, Sid Hunter, Rex Pegram, …. In closing…

Ever wonder what happens when you have to scale things up for toxicity testing?

(Especially when working with Michael Plewa) The Land of Extraordinarily Large Lab Equipment

Toxicity? 20 L → 1 mL

Chris

Cristal

Steve