Disinfection Byproducts Confirmed over 50 Years: Systematic Curation, Modes of Toxic Action, and Toxicogenomics

Abstract

Research on water disinfection byproducts (DBPs) over the past 50 years has unveiled more than 700 DBPs, many of which pose unrecognized risks to human health. Despite significant progress, systematic integration of the chemical curation of the confirmed DBPs with emerging toxicological paradigms is missing. This review systematically curates 716 confirmed DBPs reported from 1974 to 2024, offering a comprehensive resource to support risk identification and prioritization. We introduce a multiple classification framework based on molecular scaffolds, functional groups, and halogenation patterns, enabling researchers to map structural diversity to toxicological profiles. The modes of action applied in DBP research outlined here include nonspecific baseline toxicity, receptor- or enzyme-specific interactions, and reactive mechanisms such as oxidative stress and genotoxicity. Furthermore, we provide a system-level toxicogenomic view of DBP-induced toxicity, which has progressed from static single- and multiomics approaches to emerging time-resolved omics. These approaches reveal DBP-induced perturbations: the genome defines susceptibility, the transcriptome reflects expression levels, the proteome indicates functional execution, and the metabolome captures phenotypic outcomes. This review underscores critical knowledge gaps while charting future directions for DBP risk assessment and regulation and supports global efforts toward clean and safe drinking water.

1. Introduction

Water disinfection is an effective tool for the protection of public health, significantly curbing waterborne diseases, such as cholera and typhoid. Since the first implementation of continuous chlorine disinfection in 1908, substantial progress has been made in ensuring safe drinking water. This success is rightly celebrated as one of the greatest public health triumphs of the 20th century. In 1974, Bellar et al. discovered that trihalomethanes (THMs), including chloroform, bromodichloromethane, dibromochloromethane, and bromoform, were formed as disinfection byproducts (DBPs) through the reactions of chlorine with natural organic matter (NOM). This finding marked the beginning of the identification of DBPs and investigations into their associated health risks. Regulatory agencies worldwide, including the United States Environmental Protection Agency (US EPA), the European Union, and the World Health Organization, have since established guidelines to mitigate the risks associated with DBPs. Despite great efforts, the emergence of new DBPs driven by diminishing pristine source water continues to challenge drinking water safety. ,

Over the past half-century, the number of identified DBPs has grown significantly, from the initial few THMs to over 600 documented compounds. , Identification of new DBPs has been enabled by advancements in high-resolution mass spectrometry (HRMS) analytical techniques, expanding from GC–MS to LC–MS , and supercritical fluid chromatography–mass spectrometry (SFC–MS), , as well as cutting-edge data analysis strategies (e.g., IodoFinder machine learning). However, despite extensive efforts in DBP identification and profiling, critical knowledge gaps persist. To date, a comprehensive, nonredundant DBP inventory that includes uniquely identified compounds, computational identifiers, and associated physicochemical properties is still lacking. This limitation hinders research progress in toxicological, analytical, and computational studies.

The formation of diverse DBPs raises concerns about potential health risks. DBP toxicity studies initially focused on regulated compounds such as THMs and haloacetic acids (HAAs), , with an emphasis on cytotoxicity and genotoxicity. − However, as understanding deepened, research expanded to include diverse toxicity trials and multiple endpoints across various DBP species. For instance, recent studies have revealed that trihalophenols (THPs) exhibit potent immunotoxicity, iodoacetic acid (IAA) acts as an endocrine disruptor, , and halobenzoquinones (HBQs) induce developmental malformation. Nitrosamines (NAs), a group of nitrogenous DBPs (N-DBPs), have been demonstrated to cause metabolic activation and promote tumor formation, indicating their carcinogenic potential. These findings reflect an evolving toxicological landscape in which emerging DBPs have previously unrecognized health effects. Understanding DBP-mediated toxicities through mechanistic insights is essential for achieving early molecular-level warning signals.

Modern toxicological research has shifted from static toxicity endpoints to high throughput omics approaches, providing critical multilayer molecular insights of DBP toxicities. Transcriptomics and metabolomics techniques capture gene expression changes and metabolic disruptions following DBP exposure. − However, these characterizations capture only a fraction of the vast diversity of toxicity. DBPs possess diverse chemical features, ranging from varied chemical structures and functional groups to different reactivity profiles. These complexities hinder data interpretation and obscure insights into toxicity mechanisms, leaving substantial gaps in our understanding of their adverse health effects.

To address these challenges, this review aims to compile a comprehensive and updated list of DBPs confirmed by authentic chemical standards as reported from 1974 to 2024. We will systematically evaluate studies of DBP modes of toxic action and present a system-level toxicogenomic view of DBP toxicity. By integrating data from diverse studies, this review seeks to generate new insights and guide future research toward safer water treatment practices and enhanced public health protection.

2. Curation of Confirmed DBP Dataset: 1974–2024

Climate change is diminishing pristine source water for drinking water treatment, which has driven the emergence of diverse DBPs and challenges their identification and risk assessment. Compiling the diverse chemical properties of emerging DBPs enables systematic classification and computational analysis. For example, the dataset of over 200 DBPs enabled the determination of carcinogenic potential using mechanism-based structure–activity relationships (SAR) analysis. Similarly, a comprehensive dataset comprising over 1000 DBPs facilitated the assessment of endocrine-disrupting potential via high-throughput SAR analysis across 18 toxicity endpoints.

A comprehensive DBP inventory is essential for future toxicological studies. Successive efforts have been made to establish a DBP library in the past 50 years. − Notably, Richardson compiled a comprehensive list of 622 DBPs in 2011. The list provides the names of DBP generated under different disinfection processes and their classifications based on functional groups and halogenation patterns. However, due to the lack of unique identification information, inconsistent naming conventions lead to redundant entries. A predictive toxicology study extracted 5638 chemicals for modes of action (MOA) classification, but about 40% were removed due to duplicates and mismatches of chemical names or simplified molecular input line entry system (SMILES), underscoring the need for data accuracy and uniqueness. Recently, Yang et al. updated the DBP dataset of 6310 (until 2022) available on the “DBPs Information” website (https://dbps.com.cn/main). The dataset includes 651 confirmed DBPs, 1478 tentatively identified DBPs, and 4142 proposed entries. This dataset provides detailed chemical information on DBPs, including molecular and structural formulas, molecular weights, CAS numbers, and carbon frameworks, in addition to data on water sources, disinfection processes, functional groups, and halogenation patterns. Nonetheless, the recent dataset lacks a batch-download function, restricting its application in big data analysis.

To facilitate efficient tracking of DBP characteristics, sources, and multiple classifications, we compiled an open-access database of 716 confirmed DBPs from the literature spanning 1974–2024 (Figure and Table S1). This new inventory includes standardized International Union of Pure and Applied Chemistry (IUPAC) names, updated CAS numbers from authoritative sources, and supplemental computational identifying information (InChIKey and SMILES) to ensure unique identification of each DBP and eliminate redundant entries. Detailed chemical information and literature were obtained from a range of available databases, including the “DBPs Information” website, recent DBP studies (i.e., 2022–2024), PubChem (https://pubchem.ncbi.nlm.nih.gov/), CompTox Dashboard (https://comptox.epa.gov/dashboard/), Reaxys (https://www.reaxys.com/#/search/quick/query), and CAS SciFinder (https://scifinder-n.cas.org/). Emerging DBPs that may be confirmed in future studies can be easily incorporated into our dataset. All related information is accessible through cited publications in this review (Table S1initially sorted by CAS numbers for ease of searching). Moreover, because each disinfection method generates distinct DBP profiles, a systematic documentation of information on water sources and disinfection processes is summarized in the Supporting Information (Note S1).

1.

Sankey diagram depicting the 716 confirmed DBPs identified from 1974 to 2024. This diagram integrates multiple classifications of DBPs. Sample sources: real drinking water, simulated water, swimming pool water, spa water, and wastewater. Disinfection processes: categorized by chlorination, chloramination, bromination, chlorine dioxide disinfection, ozonation- and UV photolysis-mediated processes, combinations of chlorination/chloramination/chlorine dioxide disinfection, and other methods. Composition: differentiating carbon- and nitrogen-based organic DBPs as well as inorganic DBPs. Structural groups: aromatic, aliphatic, and alicyclic compounds. Functional groups: carboxylic acids, phenols, aldehydes, ketones, amides, nitriles, alkanes, quinones, amines, nitrosamines, nitromethanes, esters, alkenes, aromatic compounds, ethers, alcohols, and others. Halogenation: comparing nonhalogenated DBPs with halogenated ones, specifically chlorinated (Cl-DBPs), brominated (Br-DBPs), and iodinated (I-DBPs). Detailed data are listed in Table S1.

DBPs are classified into multiple groups for their structure-dependent toxicity studies. Classically, Plewa et al. demonstrated that N-DBPs exhibited significantly greater toxicity than carbonaceous DBPs (C-DBPs). − Across various halogenated DBP classes, the order iodo- > bromo- > chloro-DBPs (I-DBPs > Br-DBPs > Cl-DBPs) was followed in most cases, as determined using Chinese hamster ovary (CHO) cell assays. Similarly, Yang et al. reported that aromatic and alicyclic DBPs exhibited stronger endocrine-disrupting effects than their aliphatic and heterocyclic counterparts. Inorganic and organic DBPs display synergistic cytotoxicity in binary mixtures.

DBP toxicity is structure-dependent in terms of specific toxicity endpoints. For example, haloacetonitriles (HANs) showed various toxicity potencies in different toxicity assays. In acute DNA damage detected by the Comet assay, the potential order was IAN > BAN > DBAN; in chronic cytotoxicity detected by assays using CHO cells, the order was DBAN > IAN ≈ BAN. In this case, DBAN exhibited greater chronic cytotoxic effects than IAN, suggesting that generalizing I-DBPs as consistently more toxic than Br-DBPs and Cl-DBPs across various halogenation patterns is overly simplistic and potentially misleading. Similarly, although N-DBPs are often reported to exhibit greater toxicity than their carbonaceous counterparts, this conclusion is drawn from assays on a relatively small number of DBPs out of >700 confirmed and thus risks oversimplifying the structural and mechanistic diversity within the DBP universe. Equally limiting is the field’s historical dependence on CHO cells: while convenient and effective for assessment of disinfection methods, this single in vitro system lacks key metabolic capabilities and tissue-specific context, offering only a narrow window into toxicity pathways. Moreover, Pan et al. considered both structural and functional aspects when comparing 11 cyclic and 9 aliphatic DBP groups. They found that aliphatic and cyclic DBPs presented variable potency at different toxicity endpoints. Aliphatic DBPs exhibited a higher cytotoxicity in HepG2 cells, whereas cyclic DBPs showed significantly greater developmental and acute toxicity in zebrafish embryos. These results suggest that neither aliphatic nor cyclic DBPs can be universally classified as more toxic; instead, their toxicity depends on the specific biological context being evaluated. Overreliance on individual classification schemes, such as elemental composition, halogenation, or structural groupings, risks overlooking critical determinants of DBP toxicity.

2.1. C-DBPs and N-DBPs

C-DBPs form during disinfection processes via reactions between disinfectants and carbon-rich precursors. The major classes of C-DBPs include THMs, HAAs, haloketones (HKs), HBQs, and halophenols (HPs). N-DBPs are formed most commonly during chlorination and chloramination. The toxicity of N-DBPs varies widely because of their structural diversity and varying reactivity in cells. Thus, N-DBPs are classified into high-, moderate-, and low-toxicity groups. High-toxicity N-DBPs typically possess reactive nitrogen groups or aromatic conjugated systems that promote metabolic activation and covalent binding to DNA, leading to strong genotoxic effects. For example, NAs can form DNA-alkylating agents after metabolic activation. , Halonitromethanes (HNMs) and halonitrophenols (HNPs) generate nitro radicals that induce oxidative DNA damage. − HANs with cyano groups (–CN) exhibit high genotoxicity linked to alkylation potential, modulated by glutathione. , Haloamides (HAs) produce reactive amines and carbamates via hydrolysis, while halo-tyrosyl dipeptides disrupt DNA replication through covalent modification. Additionally, halo-tyrosyl dipeptides can form covalent modifications of nucleic acids and proteins, interfering with DNA replication and posing mutagenic risks. Cyanides inhibit cytochrome C oxidase, resulting in acute respiratory and cardiovascular toxicity. , Moderate-toxicity N-DBPs contain nitrogen heterocycles or nitrogen–carbon bonds that can become reactive, leading to oxidative stress and cellular damage. Examples include haloimidazoles, which form electrophilic intermediates, monoHAAs and monohaloacetamides (monoHAcAms) which create protein adducts, and indoles or imides which generate reactive oxygen species (ROS). Low-toxicity N-DBPs, such as thiazoles and halocarbazoles, possess stable aromatic or conjugated structures, making them less prone to metabolic activation and generally less toxic.

2.2. Differences among Aromatic, Aliphatic, and Alicyclic DBPs

The differences among aromatic, aliphatic, and alicyclic DBPs primarily pertain to their organic carbon skeletons. Such a classification overview has been specifically discussed in previous reviews. In short, aromatic DBPs feature a planar cyclic structure following Hückel’s rule, which confers unique electronic properties and enhances stability. In contrast, aliphatic DBPs possess open-chain or branched saturated structures, while alicyclic DBPs exhibit cyclic, nonaromatic frameworks.

Alicyclic DBPs represent a minor subset (57/716; 8%) within the overall DBP family (Table S1). In our investigated list, the major alicyclic DBPs include HBQs, halofuranones (also known as mutagen DBPs and analogues; MX-DBPs), certain alicyclic ketones, and alicyclic NAs. Additionally, several emerging alicyclic DBPs have been identified, such as halocyclopentadienes (HCPDs), lactones, lactams, imides, and haloiminoquinones. Among these, HBQs have received more attention than other alicyclic DBP groups. Li and colleagues have studied HBQs extensively , examining their cytotoxicity, genotoxicity, developmental toxicity, neurotoxicity, and other toxic effects. ,,− The best known of MX-DBPs is 3-chloro-4-(dichloromethyl)-5-hydroxy-2­(5H) furanone, which has been predicted by quantitative structure–activity relationship (QSAR) to be a potential carcinogen. HCPDs are an important emerging class of alicyclic DBPs. Their predicted bioconcentration factors range from 384 to 3980, which is 2–3 orders of magnitude higher than those of regulated DBPs (such as THMs and HAAs) and high-risk unregulated DBPs (HBQs, HANs, HAA, HNMs, haloacetaldehydes, I-THMs, and I-HAAs). Notably, 1,2,3,4,5,5-hexachloro-1,3-cyclopentadiene is about 100,000 × more toxic than THMs and HAAs. Additionally, previous studies have shown that alicyclic DBPs exhibit notably high acute toxicity in fish, Daphnia magna, and algae, comparaed to their aromatic counterparts. An in-depth investigation of alicyclic DBPs is warranted to fully understand their strong toxicological effects.

As many as 354 confirmed aromatic DBPs are compiled in this dataset. The main classes are phenol-DBPs (e.g., phenols, HNPs, and HPs) and various benzene-containing DBPs. This aromatic group also includes aromatic amides (e.g., halobenzamides and halophenylacetamides), halobenzaldehydes (HBzALs), (halo)­styrenes, (halo)­anilines, phenylalanines, anisoles, halophenylacetonitriles (HPANs), and halobenzothiazoles (HBTs). Polycyclic compounds such as halocarbazoles (HCBzs), haloquinone imides (HQIs), and halonaphthoquinones (HNQs) also belong here. Some, such as halohydroxybenzonitriles (HHBNs), feature both benzene and hydroxyl groups, combining characteristics of benzene derivatives. In contrast, heterocyclic aromatic DBPs do not contain a classical benzene ring. This group includes halopyrroles, haloimidazoles, halopyridinols, and halotriazines.

Aliphatic DBPs are documented in a total of 297 entries. The typical aliphatic DBPs include alkanes, HAcAms, HNMs, and HANs, as well as haloacetaldehydes (HAcALs) and ketoaldehydes. Other compounds, such as organotins, are also included. The aliphatic DBPs generally result in lower molecular complexity, distinct volatility, and solubility characteristics compared to aromatic DBPs. Some DBPs cannot be neatly classified into either group. Examples include ketones, alcohols, carboxylic acids, and NAs.

2.3. Roles of Various Chemical Functional Groups of DBPs

Functional groups dictate the chemical behavior, reactivity, and toxicological profiles of DBPs. In the dataset, DBPs are categorized by major functional groups and listed in descending order of abundance: carboxylic acids, phenols, aldehydes, ketones, amides, nitriles, alkanes, quinones, amines, nitrosamines, nitromethanes, esters, alkenes, aromatics, ethers, alcohols, and other groups (Figure and Table S1). This is a particularly important classification to emphasize, as functional groups can serve as key determinants of chemical toxicity. They also offer an alternative to traditional chemical class-based approaches for predictive toxicity modeling, especially in explaining MOAs. For example, ecological SAR analysis often relies on chemical functional group classifications to infer toxicological behaviors.

Carboxylic acids (e.g., HAAs) and their ionized forms are water-soluble but poorly membrane-permeable, limiting bioaccumulation yet still contributing to systemic exposure. Aldehydes and ketones, such as HKs and HAcALs, are electrophilic and form protein or DNA adducts, underpinning their cytotoxic and genotoxic potential. , Haloalkanes, including THMs, are among the most studied DBPs. Their lipophilicity favors partitioning into fatty tissues, while their low reactivity masks the need for metabolic activation pathways such as GSTT1-mediated genotoxicity. , Esters, ethers, and alcohols, though less reactive, can undergo hydrolysis or oxidation, acting as precursors to more toxic DBP species under environmental or physiological conditions.

In summary, each chemical functional group defines the intrinsic chemical reactivity of DBPs and determines their key physical and biochemical properties, such as solubility, partitioning, and the ability to undergo metabolic transformation. These factors collectively shape the toxicological profiles of DBPs and are critical for understanding how DBPs interact with environmental and biological systems. Despite well-established links between certain functional groups and toxicity endpoints, few studies systematically quantify the relative toxic contributions of multiple co-occurring groups, which limits the predictability of current SAR models.

2.4. Halogenation versus Non-Halogenation

Halogenation is crucial to the biological reactivity of DBPs. Halogens strongly withdraw electrons, increasing the electrophilicity of molecules. As a result, compared to their nonhalogenated analogues, halogenated DBPs become more reactive with biological nucleophiles such as protein thiols and DNA nucleobases, potentially leading to genotoxic effects. Incorporating halogens redistributes electron density within the molecule, creating reactive centers prone to nucleophilic attack via mechanisms such as Michael addition. Certain nonhalogenated oxygenated DBPs, most notably α,β-unsaturated carbonyls (e.g., acrolein), also exhibit high electrophilicity and readily form adducts with nucleophiles such as thiol groups. Thus, although halogenation often enhances DBP reactivity, other structurally diverse electrophilic DBPs can likewise induce nucleophile-targeting activity and genotoxicity. Moreover, the type, number, and position of the halogen atoms significantly shape the toxicological profile of DBPs. Comparative studies have demonstrated a general trend in the toxic potency: I-DBPs > Br-DBPs > Cl-DBPs. This trend is attributed, in part, to the differential generation of ROS. Iodine and bromine atoms, due to their greater polarizability and lower bond dissociation energies, facilitate the formation of reactive intermediates and free radicals under physiological conditions. These intermediates can disrupt redox homeostasis, leading to DNA damage, lipid peroxidation, and mitochondrial dysfunction.

3. Modes of Toxic Action of DBPs

This comprehensive DBP inventory highlights remarkable progress in identification over the past few decades. However, such inventories alone cannot resolve the critical question of which DBPs are the primary toxicity drivers and why. Addressing this gap requires a mechanistic understanding of how structurally diverse DBPs interact with biological systems. Specifically, DBP exposure elicits a wide spectrum of toxic responses, from baseline membrane perturbations to receptor-specific disruptions and reactive oxidative damagekey elements of their toxicological MOAs. To organize these responses, we adopt a MOA framework, adapted from Escher et al., − which categorizes toxic effects into nonspecific, specific, and reactive pathways (Figure ). This section critically evaluates these MOAs in the context of DBPs, identifies current knowledge gaps, and proposes priorities for future research.

2.

Representative modes of action (MOAs) commonly observed in studies of DBPs. This figure summarizes the major MOAs of DBP-induced toxicity: (I) nonspecific toxicity (baseline toxicity) via mitochondrial dysfunction leading to apoptosis and necrosis; (II) specific toxicity through xenobiotic-sensing and hormone-disrupting nuclear receptors, neurotransmitter receptors interference, as well as metabolic enzyme inhibition; and (III) reactive toxicity, including genotoxicity (direct DNA binding and repair inhibition) and oxidative stress (DNA, lipid, and protein oxidation). Pathways such as Nrf2, p53, and NF-κB mediate adaptive or adverse outcomes. The timeline reflects evolving mechanistic insights from 1974 to 2024. Created in BioRender. Xiang, T. (2025) https://BioRender.com/7qfiur8.

3.1. Nonspecific Toxicity: Baseline Toxicity

Nonspecific toxicity, also termed baseline toxicity, is a fundamental toxicological mechanism particularly relevant to DBPs with high lipophilicity and membrane affinity, such as THMs, HAAs, and HBQs. These DBPs can integrate into the lipid bilayers of cell membranes, compromising membrane integrity and cellular homeostasis. Reported effects include narcosis, mitochondrial inhibition, and loss of organelle compartmentalization, which disrupt the cellular energy balance and ultimately trigger cytotoxicity such as apoptosis or necrosis.

Several bioassays have been applied to evaluate the baseline toxicity of DBPs. The Microtox assay, which uses bioluminescence inhibition in Vibrio fischerias a proxy for membrane disruption and impaired metabolic activity, , has been employed to assess chloroform and other THMs, revealing dose-dependent cytotoxicity. , Similar bacterial-based assays, such as ToxScreen and BLT-Screen, which use Photobacterium leiognathi, have demonstrated comparable results for chlorinated and brominated DBPs. However, the limited metabolic capacity of prokaryotic models compared to that of eukaryotic cells reduces their suitability for assessing DBPs that undergo metabolic activation. To improve biological relevance, various mammalian-cell-based assays have been introduced to investigate baseline toxicity. For example, endpoints such as lactate dehydrogenase leakage, MTT reduction, crystal violet uptake, neutral red uptake, and other related endpoints have been applied to DBPs. Human-derived cell lines (e.g., human embryonic kidney cells; HEK293) provide enhanced physiological relevance, though variability in metabolic competence and cell-specific sensitivity to DBP subclasses complicates interstudy comparability.

Importantly, the term “baseline toxicity” is often misunderstood with general “cytotoxicity” in DBP literature, leading to misinterpretation. Baseline toxicity involves nonspecific chemical accumulation in membranes, disrupting their integrity and impairing functions such as mitochondrial respiration, whereas cytotoxicity encompasses both specific (e.g., receptor-mediated) and nonspecific pathways of adverse cellular effects. This distinction is essential for interpreting assay results, particularly when structurally diverse DBPs may engage in multiple toxicity pathways. Furthermore, baseline toxicity is often measured alongside specific assays to validate assay integrity and rule out cytotoxic interference, yet such data are rarely reported explicitly. , Despite its foundational role in toxicology, baseline toxicity remains under-reported in DBP studies and is often dismissed as background noise rather than recognized as a mechanistic signal. This oversight limits assay selection, risk prioritization, and mechanistic understanding. Future research should integrate baseline toxicity endpoints with mechanistic assays, particularly under environmentally relevant low-dose, chronic exposure conditions, to distinguish nonspecific membrane interference from specific pathways and improve DBP risk assessments.

3.2. Specific Toxicity: Receptor and Enzyme-Mediated Toxicity

Specific toxicity arises from targeted interactions between DBPs and biological macromolecules, particularly receptors and enzymes. Receptor-mediated interactions are especially critical due to their potential to trigger long-lasting systemic effects even at low concentrations, similar to the environmental concentrations of DBPs. Based on their toxicological roles, DBP-affected receptors can be broadly grouped into three main categories: xenobiotic-sensing receptors, endocrine-related nuclear receptors, and neurotransmitter receptors.

The first group comprises xenobiotic-sensing receptors, including the aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptor (PPAR), pregnane X receptor (PXR), and retinoid X receptor (RXR). These receptors regulate detoxification, lipid metabolism, and cytochrome P450 expression. Planar aromatic compounds, such as halophenolic DBPs, including chlorinated benzenes and iodinated phenols, may function as AhR agonists. Notably, chlorination of bisphenol analogues (e.g., BPS and BPF) significantly enhances their disruptive effects on PPARγ. , Similarly, BPA chlorination byproducts have been shown to increase the antagonistic activity toward RXRβ. Molecular docking studies suggest these compounds may act through similar binding mechanisms as endogenous RXR ligands. However, structure–activity relationships for PXR and PPAR remain largely undefined across different DBP subclasses, limiting predictive modeling.

The second category comprises endocrine-related nuclear receptors such as androgen receptors, estrogen receptors, and thyroid receptors. The androgen system, along with estrogen and thyroid systems, was prioritized in the 1996 amendment to the US Safe Drinking Water Act, yet it remains largely understudied in environmental endocrine disruption. Halogenated DBPs, such as halophenols, iodoacetic acids, and halogenated resorcinol, have been shown to have affinity for these receptors, contributing to endocrine disruption and reproductive abnormalities. ,, Molecular docking revealed that DBP binding to estro-androgenic receptors is driven by hydrophobicity, hydrogen bonding, halogenation, and van der Waals forces, with interaction strength and molecular volume correlating to endocrine-disrupting potency. Iodinated and polyhalogenated DBPs exhibited stronger binding forces and, consequently, more pronounced endocrine activity, following the trend: I-DBPs > Br-DBPs > Cl-DBPs. However, it is acknowledged that these findings are based on a limited set of compounds, and potential outliers may influence the observed trend.

The third category involves neurotransmitter receptors such as nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid (GABA) receptors, which regulate neural signaling. DBP-induced interference with these targets has been linked to developmental neurotoxicity and synaptic dysfunction. Neonicotinoids achieve insect selectivity by exploiting structural differences (e.g., nitroimines, cyanoimines, or nitromethylenes) in nAChRs, with their negatively charged groups favoring insect over mammalian receptors, though the long-term effects on humans remain unclear. Diazepam and 2-methylamino-5-chlorobenzophenone (MACB), a known chlorinated DBP, disrupted GABA pathway-related amino acid homeostasis and stimulated neurogenesis, potentially leading to excitotoxicity. While evidence in this domain remains limited, such receptor interactions warrant further investigation, especially in vulnerable developmental stages.

In addition to receptor-specific targets, enzyme interference represents another important toxicological mechanism. For instance, certain HAAs and their metabolites (e.g., fluoroacetate) can inhibit aconitase activity in the tricarboxylic acid (TCA) cycle by forming fluorocitrate, leading to ATP depletion, oxidative stress, and hepatotoxic or neurotoxic effects. These mitochondrial disruptions underscore the multisystemic consequences of specific DBPs beyond canonical receptor signaling. Ultimately, this potentially leads to hepatotoxicity, neurobehavioral abnormalities, developmental toxicity, and even death.

Taken together, receptor- and enzyme-mediated specific-target toxicities represent critical MOAs for DBPs. Cross-pathway interactions among these mechanisms further complicate the DBP toxicology. For example, RXR forms heterodimers with PPARs, linking endocrine disruption to metabolic effects, while oxidative stress induced by mitochondrial dysfunction may secondarily alter nuclear receptor signaling. Despite these connections, most studies examine DBP actions in isolation, leaving the integrative toxicological landscape largely unexplored. However, the current understanding is limited by narrow chemical coverage, a lack of cross-pathway integration, and underdeveloped SAR frameworks. Advancing this field requires systematic evaluations across DBP subclasses, incorporation of multitarget screening strategies, and validation under environmentally relevant exposure conditions to reveal key toxicity drivers and inform mechanism-based risk assessment.

3.3. Reactive Toxicity: Genotoxicity and Oxidative Stress

Reactive toxicity represents a critical MOA for DBPs, arising from their inherent electrophilic reactivity or their ability to generate ROS. These pathways lead to genotoxicity, either directly via covalent DNA binding or indirectly through oxidative stress, and can also trigger broader cytotoxic consequences. Regulatory toxicology traditionally distinguishes between direct-acting genotoxicants, assumed to have no safe exposure threshold, and indirect-acting agents, which may exhibit dose-dependent effects. However, most DBP studies have not clearly delineated these mechanistic distinctions, limiting both toxicological interpretation and risk relevance. , This distinction is crucial for defining exposure thresholds and prioritizing emerging DBPs for risk assessment. Clarifying the nature of genotoxic mechanisms is essential for establishing exposure thresholds and prioritizing emerging DBPs for hazard control.

Direct genotoxicity involves the covalent binding of electrophilic DBPs or their metabolites to DNA, resulting in adduct formation, cross-linking, or strand breaks. For instance, IAAs and iodoform have been shown to induce DNA damage in mouse embryo fibroblast cells (NIH3T3 cell line) through assays such as the cytokinesis-block micronucleus (CBMN) assay and the Comet assay. Herein, mutagenicity is one of the most used endpoints for assessing direct genotoxicity. For instance, N-DBPs such as HANs and HNMs alkylate DNA bases and trigger mutagenic events detectable by assays such as the Ames test. These findings suggest that direct genotoxicity is plausible for specific DBP subclasses, especially those with strong electrophilic centers and a high nucleophilic affinity.

Indirect genotoxicity, by contrast, arises through secondary mechanisms such as ROS overproduction, impaired DNA repair, and checkpoint disruption. These processes, despite lacking direct DNA binding, can lead to mutations, chromosomal instability, and genome-wide perturbations. A representative case is dibromoacetonitrile, a well-characterized indirect genotoxicant, which has been confirmed to have two distinct mechanisms of indirect genotoxicity: (i) impairing nucleotide excision repair and suppressing histone γ-H2AX phosphorylation, and (ii) perturbing replication checkpoint signaling through Chk1 inhibition. Notably, the soft electrophilic features of many DBPs suggest that indirect genotoxicity may be a more plausible route under environmentally relevant conditions. In support of this, a recent study reported that 98% of 50 tested DBPs activated the Nrf2-ARE oxidative stress response, highlighting a widespread propensity for redox-mediated DNA damage, particularly under low-dose chronic exposures.

Some DBPs, particularly HBQs, have dual genotoxic mechanisms, simultaneously forming DNA/protein adducts and inducing ROS-mediated damage. For example, tetrachlorobenzoquinone and 1,4-benzoquinone have demonstrated both covalent reactivity and oxidative DNA damage. These dual modes exemplify the complexity of DBP-induced genotoxicity and highlight the need for an integrated mechanistic evaluation framework.

Oxidative stress represents another critical facet of reactive toxicity. ROS can induce DNA damage, protein oxidation, and lipid peroxidation, playing a central role in mediating both genotoxic and cytotoxic effects. At the genotoxic level, ROS oxidizes DNA bases, causes DNA strand breaks, and impairs DNA repair mechanisms, ultimately leading to mutations, chromosomal aberrations, and genomic instability. HBQs have been shown to induce oxidative stress. Li et al. studied their genotoxicity via 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in 24 h, identifying 2,6-dichlorobenzoquinone and trichlorobenzoquinone as the most potent. While informative, such short-term markers may not reveal the delayed or adaptive outcomes that could alter risk profiles under real-world exposure durations. While oxidative stress has been widely implicated in DBP-induced toxicity, the cellular responses it elicits are highly dynamic, context-specific, and cell-type dependent, making them difficult to capture accurately using standard in vitro assays. In physiological systems, oxidative stress is not merely a static insult; it triggers complex signaling cascades involving feedback regulation, compensatory repair pathways, and adaptive remodeling that unfold over time. However, most toxicity assessments rely on simplified in vitro models, typically acute exposures (e.g., ≤24 h) in immortalized cell lines, which fail to replicate these biological complexities.

At the cytotoxic level, excessive ROS disrupts membranes, inactivates enzymes, impairs mitochondrial function, and induces apoptosis or necrosis. DBPs deplete antioxidant defenses, such as glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). GSH acts as a key intracellular ROS scavenger, and its depletion compromises the cell’s ability to neutralize oxidative insults. For example, halohydroxybenzonitriles (HHBNs) lower GSH levels and inhibit antioxidant enzyme activity, leading to cumulative oxidative stress and downstream apoptosis. Malondialdehyde (MDA), a terminal product of lipid peroxidation, is commonly used as a biomarker of oxidative stress severity. For instance, the chlorinated MX-DBP has been shown to elevate MDA levels and reduce GSH levels, suggesting a link between redox imbalance and DBP cytotoxicity. Yet, whether these effects persist under long-term exposure or are mitigated by adaptive responses remains poorly understood.

Importantly, oxidative insults can activate transcriptional factors such as the Nrf2-ARE, p53, and NF-kB pathways, initiating antioxidant and detoxification gene expression. These adaptive processes require intact metabolic networks and longer observation windows, factors that are often absent in standard acute assays. Consequently, failure to consider exposure duration, redox kinetics, and cellular adaptation risks overestimating or mischaracterizing the toxic potential of DBPs.

Collectively, reactive toxicity, including both genotoxic and oxidative pathways, represents a pivotal MOA for many DBPs. However, current assessments lack sufficient mechanistic resolution, particularly in distinguishing between direct and indirect effects. The prevalence of oxidative stress as a central mediator also calls for redox-sensitive assays that capture time-dependent and cell-specific responses. Moving forward, integrating multimechanistic frameworks and environmentally relevant exposure models is essential to accurately characterize DBP toxicity and support mechanism-based risk assessment.

4. Advancing Toxicogenomic Strategies on DBP Research

Conventional in vitro and in vivo assays have revealed that many DBPs elicit toxicological endpoints such as cytotoxicity and genotoxicity, providing a foundation for current risk assessments. However, these assays are often performed under high-dose, single-DBP exposures and tend to capture only late-stage, irreversible outcomes. Such an approach limits the sensitivity to detect early molecular perturbations that precede overt toxicity. Under environmentally relevant low-dose and chronic DBP exposures, traditional endpoints may therefore be inadequate to answer the central question: through what molecular events do DBPs ultimately endanger human health?

Toxicogenomic strategies possess high sensitivity in detecting early biological responses. They allow for the comprehensive profiling of genome, transcriptome, proteome, and metabolome changes across multiple biological levels and species, from microbes to mammals. Despite their promise, existing toxicogenomic studies of DBP research often remain fragmented and lack mechanistic integration. In this section, we discuss approaches from static single- and multiomics to dynamic time-resolved toxicogenomics, tailored for DBP research (Figure ). Representative studies illustrate how each strategy unravels the mechanistic details.

3.

Schematic diagram of representative DBP-induced toxicogenomics approaches. This figure illustrates the classical static omics and dynamic time-resolved toxicogenomics tailored for DBP research. Classical toxicogenomics encompass genome, epigenome, transcriptome, epitranscriptome, proteome, and metabolome analysis. Time-resolved omics track toxicological responses over time, capturing key phases such as early stress, cumulative damage, and potential recovery, thereby offering mechanistic insights across exposure scenarios, from acute to chronic, and short- to long-term. Created in BioRender. Xiang, T. (2025) https://BioRender.com/1x8amtv.

4.1. Classical Single-Omics Approaches

The genome defines an individual’s genetic susceptibility to DBP exposure, providing the foundation for identifying risk-associated variants and interindividual variability. Genomic approaches, such as single nucleotide polymorphism (SNP)-based genome-wide association studies, have identified potential gene–environment interactions, for example, between THM exposure and the rs907611 SNP in relation to bladder cancer susceptibility. Genetic polymorphisms in critical metabolizing enzymes influence the DBP-related bladder cancer risk. However, most DBP-related genomic studies remain focused on associations rather than causality and often fail to elucidate how genetic variants influence biological pathways, such as xenobiotic metabolism, DNA repair pathways, or oxidative stress responses, in response to DBP exposure.

The epigenome mediates long-term or heritable effects of DBPs through mechanisms such as DNA methylation and histone modifications, bridging environmental exposure with chronic health risks. Chronic exposure to THMs, for instance, has been associated with epigenetic perturbations. Individuals with lifetime exposure above 85 μg/L exhibited differential DNA methylation at 29 CpG sites (cytosine–phosphate–guanine dinucleotides), implicating transcriptional repression of tumor suppressor genes (e.g., RB1) and activation of oncogenes (e.g., SOX2), suggesting potential links to colorectal and bladder cancers. Global DNA hypomethylation further contributes to genomic instability and apoptosis, reinforcing the plausibility of epigenetic mechanisms in DBP-mediated carcinogenesis. However, most studies are based on cross-sectional designs, limiting causal inference and temporal resolution. Experimental evidence supports these associations; for example, bromate (BrO₃ ^–) exposure induced dose- and time-dependent histone acetylation at the p21 promoter in normal rat kidney cells detected by chromatin immunoprecipitation (ChIP) assay, correlating with increased p21 expression. This finding provides a clear example of how DBP exposure can be quantitatively linked to epigenetic regulation, enabling dose–response evaluation within the framework of epigenomics.

The transcriptome captures dynamic gene expression changes in response to DBPs, offering early insights into molecular disturbances and activated toxicity pathways. For example, RNA-sequencing (RNA-seq) of human T24 bladder cancer cells exposed to HBQs revealed a marked upregulation of genes associated with oxidative stress, particularly those regulated by the Nrf2 antioxidant pathway, along with increased expression of pro-apoptotic genes, indicating concurrent activation of defense and cell death programs. Similarly, exposure to BAAs in zebrafish resulted in the downregulation of genes related to visual transduction, alongside the activation of ferroptosis and apoptosis pathways, suggesting a mechanistic link between transcriptomic changes and neurodevelopmental toxicity. These examples demonstrate how alterations in RNA abundance can be systematically mapped to specific toxicological pathways. However, current studies largely rely on bulk RNA sequencing, which averages gene expression across a mixed cell population. This obscures cell-type-specific responses and may miss critical subpopulation vulnerabilities. Emerging techniques such as single-cell RNA-seq and spatial transcriptomics offer the potential to resolve cellular heterogeneity and spatial gradients of toxic effects. However, their application in environmental health research, especially in the context of DBP studies, remains in the early exploratory stage and is still limited. Moreover, transcriptomic shifts do not always translate into functional outcomes; without parallel validation at the proteomic or phenotypic level, such data may lead to incomplete or misleading interpretations.

The epitranscriptome regulates post-transcriptional RNA modifications, enabling rapid cellular responses to acute DBP exposure and short-term toxic effects, which is different from the relatively stable and long-term regulatory role of the epigenome. For example, RNA N ₆-methyladenosine (m⁶A) modification serves as a key regulatory mechanism for rapid cellular responses to environmental stimuli. Exposure to 2,4,6-THPs elevated m⁶A methylation levels and methyltransferase (especially Mettl3) abundance in mouse macrophages by m⁶A-seq, suggesting that immune suppression may be linked to significant changes in RNA m⁶A modifications. Apart from m⁶A, other classic reversible or irreversible epitranscriptomic modifications, such as 5-methylcytosine (m⁵C), N1-methyladenosine (m¹A), and pseudouridine (Ψ), have not yet been reported in DBP studies, highlighting the need for broader and more comprehensive toxicogenomic investigations in this field.

The proteome, as the direct executor of protein-level changes that mediate cellular function in response to DBP exposure, provides a functional readout complementary to transcript-based analysis. For example, DBP-induced immune modulation has been associated with the activation of C-type lectin receptor signaling, while exposure to HBQs and IAAs has been linked to disrupted glutathione metabolism and oxidative stress sensitization. In addition, chlorine-based disinfection conditions induce the upregulation of stress-response proteins, particularly under nutrient-limited environments. These findings highlight how DBP exposure can reprogram proteomic networks related to antioxidant defense, immune regulation, and metabolic adaptation. However, current proteomic studies largely emphasize changes in protein abundance, with limited attention to deeper regulatory levels such as post-translational modifications, subcellular localization, and protein–protein interactions. These dimensions are critical for understanding the mechanistic consequences of DBP exposure on protein functions. To advance this field, more targeted and integrative proteomic strategies are needed, ones that not only quantify proteins but also capture their functional states, interaction networks, and pathway-level dynamics in response to DBPs.

The metabolome reflects the most immediate phenotypic consequences of DBP exposure, capturing oxidative stress, energy disruption, and pathway-level alterations as the final layer of the toxic response. In mammalian models, monoHAcAms have been shown to induce hepatic oxidative stress and perturb amino acid and lipid metabolism in mice, while bromoacetonitriles disrupted bile acid pathways and mitochondrial energy production. In human populations, metabolomics integrated with urinary THM biomarkers revealed novel metabolites such as cinnamoylglycine and 1-methylurate, which were inversely associated with type II diabetes, suggesting possible protective metabolic adaptations. At the microbial level, nontargeted metabolomics of Microcystis aeruginosa exposed to 2,6-DCBQ identified 208 differential metabolites, with major disruptions in ABC transporters, two-component systems, and folate biosynthesis pathways, highlighting the broad metabolic reprogramming triggered by DBPs. Despite these advances, many metabolomic findings remain correlative, and current pathway enrichment analyses often rely on static reference databases that fail to reflect exposure-specific or cell-type-specific metabolic adaptations.

4.2. Integrative Multi-Omics Approaches

While individual omics provide snapshots of DBP-induced changes, they often yield fragmented datasets lacking mechanistic cohesion. Multiomics approaches provide a systems-level view of DBP-induced toxicity: the genome defines susceptibility, the transcriptome reveals regulatory responses, the proteome reflects functional activity, and the metabolome captures phenotypic consequences. For instance, in a study integrating metabolomics and transcriptomics, a 12 week exposure to THPs was shown to markedly disrupt the glutathione metabolism pathway, suggesting impaired hepatic antioxidant and detoxification capacities. Nevertheless, challenges remain due to the lack of integrative analytical frameworks that are capable of linking multiomics data across spatial and temporal scales. Current efforts must move beyond “omics layering” toward systems-level analysis that maps DBP-induced perturbations within dynamic biological networks.

4.3. Time-Resolved Toxicogenomics

DBPs typically have been detected at low concentrations over long periods. Relying only on static toxicity end points , may miss subtle but biologically meaningful effects. However, current omics applications are still predominantly limited to static analysis at a single time point, also called snapshot omics. DBP-induced toxicity is typically gradual, involving delayed effects, temporary compensation, or irreversible damage that emerges from prolonged exposure. Static omics cannot capture time-dependent transitions, thus limiting mechanistic insight into how early molecular perturbations evolve into adverse phenotypes. In future studies to address this gap, time-resolved omics strategies are critically needed. Temporal transcriptomic or metabolomic profiling can track the activation, regulation, and resolution of cellular pathways over time. For instance, early stress responses, such as inflammation or oxidative stress, may be transient and reversible, whereas prolonged activation may result in chronic dysfunction or disease development. These approaches offer valuable insights into stage-specific transitions and the temporal evolution of toxicity; however, they are still underutilized in DBP studies.

Despite its promise, toxicogenomics currently faces several bottlenecks. First, overlapping transcriptomic and morphometric signatures obscure genuine molecular events; coupling high-throughput omics with targeted molecular assays, such as receptor-reporter systems or minimal-pathway perturbations, would provide specific pathway anchors. Second, the vast dimensionality of multiomics datasets predisposes models to overfitting; consensus-network pruning combined with stringent cross-validation is essential to retain only reproducible functional modules. Third, adverse effect repositories remain sparse in DBP-specific molecular events, necessitating the continuous collection of validated events to enrich these mechanistic taxonomies.

5. Concluding Remarks and Perspectives

Drinking water disinfection is the most effective means to minimize the microbial risk and prevent waterborne disease. However, the disinfection processes used to kill microbial pathogens produce a variety of DBPs through reactions between the disinfectants and NOM in water. ,− Furthermore, climate change increases unpredictable extreme events such as flooding and wildfires, which affect the quality of source water. Minimizing both microbial and chemical risks is critical to achieving “Clean Water”, one of the top Sustainable Development Goals (SDG) identified by the United Nations (UN) (https://sdgs.un.org/goals).

Why does DBP research remain relevant and important to protecting public health after 50 years of research? What DBPs are responsible for the observed human health effects? How do we critically analyze the wealth of available information, build on advances made in the past 50 years, and address the remaining challenges in drinking water disinfection and unintended DBP formation?

Human epidemiological studies conducted in several countries have consistently shown an increased risk of bladder cancer associated with the consumption of drinking water disinfected by chlorine. − However, these epidemiological studies did not measure or identify the actual DBPs in drinking water. Thus, the specific DBPs in disinfected water that contribute to increased bladder cancer risk are not known.

The total amount of organic halogens, a surrogate of halogenated DBPs, was repeatedly measured in chlorinated water. However, only about 30% of the total organic halogens detected in treated drinking water have been accounted for in the identified DBPs, and the majority (70%) are unknown. ,,,,,, Faced with this knowledge gap, regulatory agencies, such as the US EPA and Health Canada, regulate only a few DBPs that are easily quantifiable. ,,− , The US EPA has established regulatory limits for 11 DBPs: bromate, chlorite, five HAAs, and four (total) THMs. Regulation of this small number of DBPs is not because they are known to cause adverse health effects at concentrations typically found in drinking water, but because they serve as surrogates to the complex mixture of DBPs in chlorinated drinking water. None of these regulated DBPs has sufficient potency to account for the cancer risks observed in epidemiological or animal studies. , Despite good intentions for public health protection, the current regulatory guidelines for DBPs do not address the issue of the actual DBPs with the potential to cause adverse health effects, including bladder cancer, colorectal cancer, and birth defects. ,− ,,,

5.1. Confirmed DBPs and Beyond

What drives the correlation between consumption of chlorinated drinking water and bladder cancer, i.e., what are the ″toxicity drivers” in disinfected drinking water? Identification of new DBPs and determination of primary toxicity drivers remain active and important areas of research. − The 716 DBPs summarized in Table S1 have each been identified and confirmed using authentic chemical compounds (standards). They represent a fraction of all possible DBPs that could be formed, depending on the disinfection processes and source water characteristics. Beyond the 716 confirmed DBPs and thousands with tentative identifications, many yet-unknown byproducts remain to be discovered. Advances in reaction-prediction modeling, nontarget HRMS analysis, and artificial intelligence (AI)-driven big-data analytics are steadily uncovering these unknown byproducts, yet turning those discoveries into actionable risk management requires a well-defined triage framework. We advocate a concise, three-tier prioritization framework, learning complex structure-toxicity associations from massive chemical–biological activity data: (1) flag DBPs with the predicted high occurrence frequency and concentrations in drinking water through monitoring or exposure modeling; (2) rank DBPs in silico using QSAR, ToxCast, or MIE-affinity models, reserving top scorers for targeted bioassays; and (3) cluster DBPs by structural similarity, advancing the most hazardous clusters to mixture-interaction and ADME studies (i.e., absorption, distribution, metabolism, and excretion).

Additionally, a lack of quantitative concentrations of many DBPs in drinking water has limited the progress in conducting epidemiological studies, particularly on new DBPs, and determining the potential of these DBPs as toxicity drivers. To establish dose–response relations in toxicological studies, researchers often use concentrations (doses) of DBPs much higher than their concentrations (exposure levels) measured in disinfected drinking water. This difference could be on the order of magnitude. Although extrapolation from effects observed at higher doses to potential health risks estimated for lower exposure levels is a common practice, it involves large uncertainties. A mechanistic understanding of the various toxicities of DBPs will provide a scientific rationale for extrapolation approaches (linear vs nonlinear, threshold or no threshold).

5.2. Toxicity of Individual DBPs and Their Mixtures

Generally, current research on DBP toxicity faces four major bottlenecks. First, most bioassays focus on a narrow spectrum of toxicity end points, such as cytotoxicity and genotoxicity, leaving key mechanisms unexplored. Expanding end points to include endocrine, immune, neurodevelopmental, and metabolic effects, coupled with multiomics profiling, will reveal hidden hazards and pinpoint true toxicity drivers. Second, the absence of an overarching mechanistic framework hampers extrapolation and regulation; adopting pathway-based toxicology anchored in adverse effects will evaluate the mechanism-driven risk. There is also uncertainty in extrapolating from the results of in vitro studies to the potential in vivo outcomes. Cellular responses, while mechanistically informative, may not predict organism-level health consequences. Innovative models, including organoid systems, 3D co-cultures, and organ-on-a-chip devices, offer physiologically relevant, organ-level responses that could ultimately replace traditional animal experiments. Integrating advanced physiologically based pharmacokinetic (PBPK) modeling with in vitro to in vivo extrapolation sharpens predictions by accounting for absorption, first-pass metabolism, and saturation, yielding more reliable administered-equivalent doses and free in vivo concentrations.

A toxicological understanding of DBP mixtures is limited. Most toxicological studies test each individual DBP compound under controlled conditions and provide useful dose–response information. In real-world scenarios, DBPs exist as complex mixtures (Table S1) whose composition and reactivity fluctuate with water chemistry. ,, Conventional one-compound studies underestimate the health risks of these dynamic “cocktails”. Mixture toxicity addresses this gap by profiling unknown environmental mixtures (e.g., drinking water, wastewater, and swimming pools) and defined synthetic mixtures (known spiked DBP mixtures). Tracking the main toxicity drivers contributing to mixtures enables mechanistic dissection of pathway convergence and synergy between DBPs, revealing how low-potency compounds amplify the effects of potent congeners. However, mixture studies are still rare and focus on a small subset of DBPs. Mixture-toxicity research should address three key facets: (1) elucidating the underlying interaction mechanisms, (2) mapping how those mechanisms shift across dose levels, and (3) identifying the DBPs that act as primary toxicity drivers. The recent reported mixture effects generally fall into additive, synergistic, or antagonistic domains. Depending on the toxicity end point, interaction patterns migrate along a continuum, from concentration-addition (CA) to synergy, or antagonism, or hybrids thereof. For example, in CHO cells, DBP mixtures show additive chronic cytotoxicity but nonadditive acute genotoxicity. , Binary mixtures of aromatic halogenated DBPs span the full spectrum from CA to strong synergy and antagonism in bacterial growth-inhibition assays. However, dose-dependent transitions are evident: when concentrations drop into the low nanomolar range and cellular defenses become limiting, mixtures switch from CA to pronounced nonadditive cytotoxicity. Typically, only a few toxicity drivers that dominate the bioassay-equivalent quantity, account for most of the hazard, leaving the bulk of co-occurring DBPs toxicologically silent. Effect-directed analysis, which integrates tiered fractionation, HRMS and targeted bioassays, now enables rapid interrogation of thousands of halogen-containing features to pinpoint these key toxicity drivers. ,

Overall, an integrated four-pillar strategy for future research may enhance knowledge translation from the accelerating discovery of DBPs to concrete public-health protection. First, nontarget HRMS, reaction-prediction modeling, and AI-enabled analytics should feed a concentration-driven, structure-alert, pathway-quantified triage framework that pinpoints the most probable toxicity drivers. Second, environmentally relevant, low-dose mixture assays guided by pathway-based toxicology and toxicogenomics that track specific adverse effects should be emphasized over high-dose single-DBP tests. Third, next-generation human-relevant platforms are needed to generate mechanism-anchored, animal-free data acceptable to regulators. Fourth, mixture-toxicity patterns should be rigorously mapped to isolate the DBPs that dominate the overall risk. By closing the loop among discovery science, risk assessment, and disinfection engineering, this integrated approach offers the best path toward “microbe-safe and DBP-smart” drinking water and advances the UN SDG of Clean Water for all.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.4c13900.

• Compilation of confirmed 716 DBPs (1974–2024) (XLSX)

• Disinfection strategies application on different water sources (PDF)

T.X. led the original draft writing, manuscript review and editing, data curation, visualization, and formal analysis. J.L. and X.Y. contributed to data curation, visualization, and formal analysis. D.Z. and Q.S. assisted with manuscript review and editing. X.-F.L. and X.C.L. conceptualized the study, supervised the project, acquired funding, and contributed to manuscript review and editing, formal analysis, and data curation.

The authors declare no competing financial interest.

Published as part of Environmental Science & Technology special issue “Celebrating the 50th Anniversary of the Discovery of Drinking Water Disinfection Byproducts”.