Assessment of unregulated drinking water risks in U.S. tap water using non-targeted Total Organic Halogen (TOX) analysis

Riley Mulhern, Ph.D. - Department of Environmental Studies, University of Colorado Boulder

1. Overview

This study will use non-targeted analytical methods to assess total organic halogens (TOX) in tap water samples across the U.S. TOX levels in drinking water (including total organic chlorine, bromine, iodine, and fluorine) can provide valuable insight into the overall potential risk of unregulated disinfection byproducts (DBPs) and per- and polyfluoroalkyl substances (PFAS)—two of the most challenging and persistent trace contaminants in drinking water supplies. Treated drinking water samples will be collected from public taps served by water utilities that draw from surface water in each of the 18 major river basins across the continental U.S. This pilot study design will provide new insight into the variability of TOX levels in tap water by watershed and as a function of upstream population. Additionally, TOX levels will be compared to levels of regulated DBPs and PFAS obtained from publicly available data provided to water utility customers in Consumer Confidence Reports. The project will be conducted by students and researchers at the Mulhern laboratory at the University of Colorado Boulder. The initial sampling effort is expected to be completed within 12 months, with opportunities for follow-on studies to increase scope and impact. All data collected through this study will be transparently shared with the public through an open-access data repository.

2. Introduction

The provision of public drinking water is essential to the protection of public health. Advancements in municipal water treatment in the first half of the 20^th century contributed to the largest decline in urban mortality in U.S. history.¹ The passage of the Safe Drinking Water Act (SDWA) in 1974 also created a uniform regulatory framework for protecting drinking water quality across the nation, significantly improving water safety.²

Even so, as scientific understanding of drinking water quality has advanced, regulations have not kept pace. Every five years, the U.S. Environmental Protection Agency (EPA) conducts a review of unregulated chemical and microbial contaminants in drinking water across the nation, known as the Contaminant Candidate List (CCL). These are contaminants that are “known or anticipated to occur in public water systems” but do not have enforcement limits. Nearly 200 chemical contaminants and 25 microbial contaminants have appeared on the CCL since 1998 but only two of these have ever been regulated. As a result, many potential, known drinking water contaminants are not required to be monitored or treated by water utilities, leaving many consumers in the dark around emerging or unknown water quality risks and contributing to declining trust in public water.^3,4 Public distrust is exacerbated by poor quality Consumer Confidence Reports (CCRs) issued by some utilities to their customers, lacking clear communication or complete information about drinking water contaminants.⁵

Two of the most challenging contaminants in drinking water include disinfection byproducts (DBPs) and per- and polyfluoroalkyl substances (PFAS). Rather than distinct chemicals, these are large contaminant classes, or groupings of many different potentially toxic chemicals in drinking water. DBPs, which are formed by the reaction of disinfectants with organic matter in water, are a class of over 700 compounds with more still being discovered.^6–8 They are ubiquitous in drinking water and are associated with bladder cancer, birth defects, and miscarriage.⁹ PFAS or “forever chemicals,” which are used as industrial surfactants and in many consumer applications to make products water- and grease-proof, may include thousands or even millions of different chemical structures, depending on how the class is defined.¹⁰ Certain PFAS are linked to cancer, suppressed immune response, and increased cholesterol.¹¹ Under the SDWA, water utilities are only required to monitor, treat for, and report on nine DBPs (four trihalomethanes and five haloacetic acids) and six PFAS, even though evidence exists that unregulated compounds and mixtures of compounds may pose even greater risk.^9,12

Between DBPs and PFAS alone, it would take decades or centuries to adequately assess, quantify, regulate, and treat the thousands of unknowns in drinking water by addressing one chemical at a time. An alternative approach, however, is to use bulk organic parameters in drinking water to indicate the overall DBP or PFAS content. This can be accomplished through non-targeted analysis of total organic halogens (a quantity often referred to as TOX). Halogens are a group of nonmetallic elements in the periodic table including chlorine, bromine, iodine, and fluorine, that, when bonded to carbon, create the chemical structures of DBPs and PFAS. DBPs are typically made up of organic chlorine, bromine, and iodine while PFAS contain organically bonded fluorine. Thus, measurement of the total organic chlorine, total organic bromine, total organic iodine, and total organic fluorine (together, TOX) can be a powerful tool for assessing potential drinking water risks beyond the small subset of currently regulated contaminants.

In this study, we propose to analyze TOX in treated drinking water samples from 18 cities representing the 18 major river basins across the continental U.S. and compare the results to publicly available water quality data and CCRs to assess unregulated drinking water risks. The primary research questions we aim to address is: how do TOX levels in treated tap water compare to regulated contaminant levels and how do they vary across the U.S.?

We hypothesize that:

H1: TOX levels in tap water across the U.S. will exceed the sum of individually regulated DBPs and PFAS reported in utility CCRs.

H2: After controlling for differences in water treatment, tap water TOX levels will be higher in cities relying on surface water sources with larger populations in the upstream catchment area (indicating greater upstream wastewater influence) compared to cities relying on surface water sources with smaller upstream populations.

Impact Statement. These results will provide novel insights into the magnitude and spatial variability of unregulated DBPs and PFAS in drinking water across the U.S., uncovering potential gaps in current reporting and identifying watersheds where greater health risks could exist. By providing a transparent side-by-side comparison of bulk DBP and PFAS risk in different cities and watersheds across the U.S., we aim to equip local public health agencies, water professionals, community groups, and water consumers with the data needed to guide future study and inform health-protective decision-making. Long term, this research could serve as the basis for larger epidemiological studies related to bulk TOX parameters to advance our understanding of the human health impacts of complex mixtures of trace level contaminants in drinking water.

3. Methods

Sample Site Selection. Samples will be collected from drinking water outlets in public buildings (e.g., libraries or university campuses) in cities relying on surface water in each of the 18 major river basins across the continental U.S. as defined by two-digit hydrologic unit codes (HUC). Figure 1 shows the 18 basins and an approximate, proposed sample location near a major discharge point of the basin to capture the surface water impact of the largest possible upstream area. The exact sample locations/cities will be finalized at the start of the project based on in-depth analysis of nearby cities relying on surface water for public drinking water supply.

Figure 1. Map of the 18 major river basins based on HUC 2 codes in the continental U.S. and locations of potential sampling sites (and the adjacent river) at the outlet of each basin. Sampling sites will be finalized at the project start based on a detailed analysis of eligible cities relying on surface water for public drinking water supply.

Sampling Recruitment. Specific sampling sites within each city selected above will be identified through an internet search of public buildings in the area. Suitable candidates will be contacted through phone calls and email to invite them to help collect tap water samples at their location through mail-out sample kits. Kits will be shipped to the sample site with a pre-paid overnight return shipping. Participants at each site who support the sampling will receive a $50 gift card paid for with project funds as an incentive and will receive a letter with the water quality results from their location.

Sampling Protocol. Sample kits mailed to each site will include high density polyethylene (HDPE) sample bottles packed in insulated shipping containers. Mail-out sampling kits using a participatory science approach have been shown to yield high quality results without significant sample contamination concerns.^13–15 For quality assurance, reagent grade water will also be shipped to each field site with instructions for collecting a field blank to ensure that no contamination results from the sampling process.

Analytical Methods. TOX will be analyzed by Combustion Ion Chromatography (CIC) after adsorption onto a clean carbon media following the German Institute for Standardization (DIN) standard 38409-59.^16,17 An overview of the process can be seen in Figure 2. Briefly, aqueous samples are first passed through activated carbon to separate and concentrate organically bound halogens from inorganic halogens in the sample. The solid carbon media is then combusted at a temperature of over 1000 degrees Celsius to break down the organic bonds and each of the inorganic halogens are analyzed by ion chromatography to quantify the adsorbable organic fluorine (AOF), chlorine (AOCl), bromine (AOBr), and iodine (AOI). These adsorbable quantities of halogenated organics are the best representation of overall TOX levels using currently available methods. Additionally, water samples will also be analyzed for inorganic fluoride, which can interfere with the measurement of total organic fluorine if levels are too high, by ion chromatography following EPA Method 300.1.¹⁸ Sample processing to extract halogenated organic compounds onto the carbon media will be conducted at Dr. Mulhern’s laboratory at the University of Colorado Boulder using a Phromochrom SPE-03 automated solid phase extraction set-up. The carbon media will then be analyzed by CIC using state-of-the-science instrumentation at the analytical chemistry laboratories of RTI International, a non-profit research institute.

Figure 2. Overview of the approximate analytical method to detect total organic halogens (TOX) in drinking water samples. Figure produced by Metrohm.¹⁶

Publicly Available Water Quality Data. Publicly available water quality data will be obtained for each sampling site from the U.S. Environmental Protection Agency’s Safe Drinking Water Information System (SDWIS) and from local CCRs. These data will be used to compare the reported levels of DBPs and PFAS against the measured levels of TOX.

Statistical Approach and Power Analysis. TOX results will be compared to publicly available DBP and PFAS data for each site from required water utility monitoring. Since health-based thresholds for TOX in drinking water do not yet exist, TOX data will be normalized by the maximum detected values to assign a relative health risk score between 0 and 1 for each site. The sum of regulated trihalomethanes and haloacetic acids will be compared against the AOCl and AOBr results. Meanwhile, AOI represents the unregulated fraction of iodinated DBPs, but this will still serve as an important comparison against the regulated fraction, especially since iodinated DBPs may contribute more to the overall toxicity.^9,19 PFAS data will be compared to the AOF results, which represent both PFAS and other fluorinated organic compounds originating from pesticides and/or pharmaceuticals in drinking water.

Additionally, the results will be analyzed using multiple regression analysis to assess the effect of upstream population on overall TOX levels while controlling for differences in water treatment. While this study will provide only a small sample of preliminary data, 18 tap water samples from across the U.S. will yield an adequate sample size to detect a large effect (Cohen’s f² = 0.35; power = 80%; alpha = 0.05) of upstream population on downstream TOX levels, if one exists. Even this small sample size, however, will serve as valuable pilot to guide future sampling efforts and power more robust statistical explorations.

Ethics and Data Management. All data from this study will be shared in an open, transparent repository in accordance with ResearchHub’s Open Science Commitment. This work is exempt from the University of Colorado Boulder’s Institutional Review Board approval as it does not include human subjects.

4. Budget

Anticipated costs to complete this pilot study include laboratory and sampling supplies, shipping costs for mail-out sample kits, analytical expenses, participant incentives (i.e., gift cards), and personnel costs for logistics, sample processing, data analysis, and administrative tasks. These expenses are summarized in Table 1 below. Any additional funding raised through the ResearchHub platform above the budgeted $50,000 would be used to expand the study to additional sampling sites within each watershed, increasing the spatial representation and statistical power of the analysis.

Table 1. Summary of estimated costs to conduct pilot TOX sampling in 18 sites across the U.S.

Item Description	Quantity	Unit cost	Total
Sample bottles (Per site)	18	$50.00	$900.00
Sample shipping outbound (Per site, Ground)	18	$50.00	$900.00
Sample shipping inbound (Per Site, Priority Overnight)	18	$300.00	$5,400.00
Laboratory supplies and reagents (Per site)	18	$80.00	$1,440.00
Total organic halogens analysis by CIC	18	$200.00	$3,600.00
Inorganic fluoride analysis	18	$11.75	$211.50
Participant sampling incentives (Gift cards)	18	$50.00	$900.00
Professional research assistant (12 mos, 0.4 FTE)	1	$24,000.00	$24,000.00
Professional research assistant fringe (12 mos, 14.2%)	1	$8,520.00	$8,520.00
Administration and accounting	1	$4,128.50	$4,128.50
		Total	$50,000.00

5. Timeline

The estimated project timeline and specific project tasks are shown in Table 2. The primary sample collection and analysis tasks are expected to be completed within 12 months of the project start date, with potential for expansion of the sampling effort with future funding. Dissemination of the pilot results will occur at the project close.

Table 2. Estimated project timeline in months.

Project Task

Sep 2025

Oct 2025

Nov 2025

Dec 2025

Jan 2026

Feb 2026

Mar 2026

Apr 2026

May 2026

Jun 2026

Jul 2026

Aug 2026

1. TOX analytical method development and testing

X

X

X

X

2. Sampling site identification and recruitment

X

X

X

X

X

X

3. Sample collection

X

X

X

X

X

X

4. Sample analysis

X

X

X

X

X

5. Data analysis

X

X

X

6. Research Team and Qualifications

This project will be led by Dr. Riley Mulhern, Assistant Professor of Environmental Studies and Public Health at the University of Colorado Boulder (www.equalwaterlab.com). Dr. Mulhern has extensive experience with field-based water quality studies, community engagement, and participatory science projects. He has authored or co-authored over 20 peer-reviewed studies focused on water quality, water treatment, and health risk assessment and teaches classes about water quality and environmental health. Examples of relevant work include:

MacDonald Gibson, J. et al. Effect of Community Water Service on Lead in Drinking Water in an Environmental Justice Community. Environmental Science & Technology (2023) doi:10.1021/acs.est.3c01341.
Mattos, K. J. et al. Reaching those left behind: knowledge gaps, challenges, and approaches to achieving SDG 6 in high-income countries. Journal of Water, Sanitation and Hygiene for Development (2021) doi:10.2166/washdev.2021.057.
Jones, C. H. et al. Quantitative Relative Chemical Assessment to Support Risk Frameworks for Water Recycling. ACS EST Water (2025) doi:10.1021/acsestwater.4c01140.
Wood, E. et al. PFAS in Rural U.S. Well Water: Using Participatory Science to Identify and Communicate Results to Address Risks. Environmental Science & Technology. (2025) doi:10.1021/acs.est.5c02521.
Mulhern, R. E. et al. Improved decision making for water lead testing in U.S. child care facilities using machine-learned Bayesian networks. Environmental Science & Technology (2023) doi:10.1021/acs.est.2c07477.

Dr. Mulhern will be supported by a part-time professional research assistant to manage sample site selection, recruitment, sampling logistics, sample processing, and coordination with the external analytical chemistry team at RTI International.

7. References

1. Cutler, D. & Miller, G. The Role of Public Health Improvements in Health Advances: The Twentieth-Century United States. Demography 42, 1–22 (2005).

2. Weinmeyer, R., Norling, A., Kawarski, M. & Higgins, E. The Safe Drinking Water Act of 1974 and Its Role in Providing Access to Safe Drinking Water in the United States. AMA J. Ethics 19, 1018–1026 (2017).

3. Jaffee, D. Unequal trust: Bottled water consumption, distrust in tap water, and economic and racial inequality in the United States. WIREs Water 11, e1700 (2024).

4. Yang, E., Butcher, D. A., Edwards, M. A. & Faust, K. M. Exploring the Relationship between Public Trust toward the Water Sector and the Use of Bottled Water within US Shrinking Cities. J. Water Resour. Plan. Manag. 149, 04022071 (2023).

5. Iuliano, K. et al. “Is my tap water safe to drink?”: Examining the quality of a national sample of United States drinking water reports. Int. J. Hyg. Environ. Health 265, 114536 (2025).

6. Kristiana, I., McDonald, S. & Joll, C. A. The forest or the trees: a critical review on the analysis of total organic halogen (TOX) in drinking waters and its utility as a water quality parameter. Environ. Sci. Water Res. Technol. 6, 2313–2330 (2020).

7. Fairey, J. L. et al. Chloronitramide anion is a decomposition product of inorganic chloramines. Science 386, 882–887 (2024).

8. McCurry, D. L. The chloramine dilemma. Science 386, 851–852 (2024).

9. Allen, J. M. et al. Drivers of Disinfection Byproduct Cytotoxicity in U.S. Drinking Water: Should Other DBPs Be Considered for Regulation? Environ. Sci. Technol. 56, 392–402 (2022).

10. Schymanski, E. L. et al. Per- and Polyfluoroalkyl Substances (PFAS) in PubChem: 7 Million and Growing. Environ. Sci. Technol. 57, 16918–16928 (2023).

11. Sunderland, E. M. et al. A review of the pathways of human exposure to poly- and perfluoroalkyl substances (PFASs) and present understanding of health effects. J. Expo. Sci. Environ. Epidemiol. 29, 131–147 (2019).

12. Ríos-Bonilla, K. M. et al. Neurotoxic Effects of Mixtures of Perfluoroalkyl Substances (PFAS) at Environmental and Human Blood Concentrations. Environ. Sci. Technol. 58, 16774–16784 (2024).

13. Wood, E. et al. PFAS in Rural U.S. Well Water: Using Participatory Science to Identify and Communicate Results to Address Risks. Environ. Sci. Technol. (2025) doi:10.1021/acs.est.5c02521.

14. Hoponick Redmon, J. et al. A Participatory Science Approach to Evaluating Factors Associated with the Occurrence of Metals and PFAS in Guatemala City Tap Water. Int. J. Environ. Res. Public. Health 19, 6004 (2022).

15. Sinkway, T. D. et al. Crowdsourcing citizens for statewide mapping of per- and polyfluoroalkyl substances (PFAS) in Florida drinking water. Sci. Total Environ. 926, 171932 (2024).

16. Metrohm. Analyzing halogenated organic compounds with CIC according to DIN 38409-59. https://www.metrohm.com/en/discover/blog/20-21/analyzing-halogenated-organic-compounds-with-cic-according-to-di.html (2022).

17. DIN. DIN 38409-59 German standard methods for the examination of water, waste water and sludge - Parameters characterizing effects and substances (group H) - Part 59: Determination of adsorbable organically bound fluorine, chlorine, bromine and iodine (AOF, AOCl, AOBr, AOI) using combustion and subsequent ion chromatographic measurement (H 59). https://dx.doi.org/10.31030/3373494 (2022).

18. Pfaff, J. D., Hautman, D. P. & Munch, D. METHOD 300.1 DETERMINATION OF INORGANIC ANIONS IN DRINKING WATER BY ION CHROMATOGRAPHY. (1999).

19. 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. 636, 178–242 (2007).

Assessment of unregulated drinking water risks in U.S. tap water using non-targeted Total Organic Halogen (TOX) analysis

Assessment of unregulated drinking water risks in U.S. tap water using non-targeted Total Organic Halogen (TOX) analysis

Funding Progress

Funders

Peer Reviews

Related Funding

Request for Proposals - Comprehensive Analysis of Water Quality in Urban U.S.

Nonprofit Recipient

Topics

DOI