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Mutat Res Gen Tox En 831 (2018) 1–12

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Genotoxicity of disinfection byproducts and disinfected waters: A review of recent literature Constanza Cortésa, Ricard Marcosa,b, a b

T



Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Edifici C, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain CIBER Epidemiología y Salud Pública, ISCIII, Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: Chlorination byproducts Genotoxicity Mutagenicity Comet assay

The presence of water disinfection byproducts (DBPs) in tap water, resulting from disinfection processes involving chlorination or chloramination, increases the mutagenicity of the water and may pose adverse health effects. The topic was reviewed by DeMarini and coworkers in 2007. Here, we review research on the genotoxicity of DBPs published since that time. Studies, primarily using the Salmonella mutagenicity assay, have continued to show that chlorination or chloramination of source waters results in finished, tap, or swimming pool/spa water that is more mutagenic than the original source water. The genotoxic potencies of DBPs in both bacterial and mammalian cells generally rank as iodinated > brominated > chlorinated. Several DBPs are genotoxic in vivo in plants as well as in animals such as the worm Caenorhabditis elegans and the zebrafish Danio rerio. Studies primarily using the comet assay in mammalian cells have identified several non-regulated DBPs as genotoxic. However, the comet assay detects DNA damage that is generally repaired by the cells; thus, genotoxicity data more relevant to persistent mutations, such as chromosomal or gene mutations, are needed for these DBPs. Recent molecular epidemiology has indicated that activation of brominated trihalomethanes by the enzyme GSTT1 and the lack of metabolism of haloacetic acids by a variant of enzyme GSTZ1 are likely causative mechanisms for bladder cancer associated with exposure to chlorinated water. Further studies, especially in vivo, are needed to determine the ability of various DBPs, especially unregulated ones, to induce both gene as well as chromosomal mutations. Such investigations, along with additional molecular epidemiology studies, are required for a comprehensive understanding of the genotoxic and carcinogenic risks associated with DBP exposure.

1. Introduction The implementation of disinfection procedures in public water sources is undoubtedly one of the most important health advances of modern times. In this way, many of the effects associated with waterborne infectious diseases have been significantly reduced, although they persist in many regions worldwide [1]. However, chemical disinfectants react with organic matter and inorganic ions present in source waters, forming new chemical species, water disinfection byproducts (DBPs). In the past 40 years, several studies have examined potential health risks posed by these compounds. Epidemiological evidence links exposure to DBPs to increased risk of bladder cancer and reproductive effects [2,3]. An important milestone in our knowledge on the toxic, genotoxic, and carcinogenic effects of DBPs is the extensive review carried out in 2007 by DeMarini and colleagues [4]. Our present aim is to review subsequently published data. The studies have been classified according

to whether they were obtained using in vitro or in vivo (including some human biomonitoring studies) approaches. In addition, studies have also been divided between those using water samples containing DBPs mixtures, which reflect the actual exposure scenario, and those using individual DBPs, to identify the most hazardous chemicals. 2. DBP classification DBPs were first identified and associated with water disinfection processes in the 1970s [5,6]. By 2000, many of the currently known DBPs had been identified [7,8]. Nevertheless, In the past decade, the analysis of diverse water sources and the implementation of modern analysis techniques such as high-resolution mass spectrometry have led to the identification of several new chemical species [9–11]. To date, the number of reported DBPs is more than 600 [4]. Table 1 summarizes the main classes of known DBPs as well as the range of concentrations found in disinfected waters. As observed, they belong to many chemical

⁎ Corresponding author at: Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. E-mail address: [email protected] (R. Marcos).

https://doi.org/10.1016/j.mrgentox.2018.04.005 Received 27 December 2017; Received in revised form 22 April 2018; Accepted 23 April 2018 Available online 24 April 2018 1383-5718/ © 2018 Elsevier B.V. All rights reserved.

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Table 1 Main groups of DBPs and their levels of occurrence. Occurrence data taken from [4,38,77–81]. Disinfection by-products

Occurrence (μg/L)

HALONITROMETHANES Chloronitromethane, Dichloronitromethane, Trichloronitromethane (Chloropicrin), Bromonitromethane, Dibromonitromethane, Tribromonitromethane (Bromopicrin), Bromochloronitromethane, Bromodichloronitromethane, Dibromochloronitromethane HALOACETIC ACIDS AND OTHER HALOACIDS Chloroacetic acid, Dichloroacetic acid, Trichloroacetic acid. Bromoacetic acid, Dibromoacetic acid, Tribromoacetic acid, Iodoacetic acid, Diiodoacetic acid, Triiodoacetic acid, Bromochloroacetic acid, Bromodichloroacetic acid, Bromoiodoacetic acid, Dibromochloroacetic acid, Chlorodibromoacetic acid TRIHALOMETHANES Chloroform, Bromoform, Dibromochloromethane, Bromodichloromethane, Dichloroiodomethane, Bromochloroiodomethane, Dibromoiodomethane, Chlorodiiodomethane, Bromodiiodomethane, Iodoform, Dichloromethane, Bromochloromethane, Chlorodibromomethane, Dibromomethane OXYHALIDES Bromate (0.2 − 25.1), Chlorate (up to 190), Chlorite (up to 1100) HALOFURANONES MX, Red-MX, Ox-MX, EMX, ZMX, Mucochloric acid, BMX-1, BMX-2, BMX-3, BEMX-1, BEMX-2, BEMX-3 HALOACETONITRILES Chloroacetonitrile, Dichloroacetonitrile, Trichloroacetonitrile, Bromoacetonitrile, Dibromoacetonitrile, Tribromoacetonitrile, Bromochloroacetonitrile, Bromodichloroacetonitrile, Dibromochloroacetonitrile, Iodoacetonitrile HALOKETONES Chloroacetones HALOAMIDES Chloroacetamide, Dichloroacetamide, Trichloroacetamide, Bromoacetamide, Dibromoacetamide, Tribromoacetamide, Bromochloroacetamide, Bromoiodoacetamide, Bromodichloracetamide, Dibromochloroacetamide, Iodoacetamide, Diiodoacetamide, Chloroiodoacetamide HALOAMINES & OTHER AMINES Chloramines, Nitrosamines (NDMA), Heterocyclic amines ALDEHYDES Formaldehyde, Acetaldehyde, Chloroacetaldehyde, Dichloroacetaldehyde, Bromochloroacetaldehyde, Trichloroacetaldehyde (chloral hydrate), Tribromoacetaldehyde Other DBPs Quinones, Cyanogen halides, Chlorophenols, Aldoketoacids, Carboxylic acids, Haloacetates, Halopyrroles, Others

0.1–5

1–2600

0.05–380

0.2–1100 0.08–0.85 MX (0.08–0.85), 0.5–219

10–60 Up to 9.4

1–1180 0.4–497 Formaldehyde (up to 13.7)

The use of other disinfectants has also been associated with formation of different DBP classes. The use of chlorine dioxide (ClO2) is associated with the formation of chlorite, chlorate, and chloride. The use of ozone is related to formation of bromate, formaldehyde, other aldehydes, peroxides, and brominated methane; chloramination procedures may lead to formation of dichloramines, trichloramines, cyanogen chloride, and chloral hydrate [12]. In addition to the disinfectant used, the molecules initially found in the water also influence the DBP species formed. The concentration of organic matter is directly correlated to the concentration of DBPs found in disinfected waters. The presence of aromatic molecules in particular seems to increase the formation of DBPs [13]. Recently, the presence in raw waters of anthropogenic organic compounds such as pharmaceuticals, hormones, pesticides, textile dyes, UV filters, and fuels has led the scientific community to ponder their potential risks. Even though their levels in potable water do not pose a health concern per se, there are concerns regarding the formation of potentially hazardous DBPs during the disinfection process. For instance, analgesics such as

families.

3. Formation of DBPs The presence and abundance of each DBP depends on the disinfectant used, its concentration, and on the spectrum of organic and halogenated molecules present in the source water. The physicochemical characteristics of treated waters also influence the formation of DBPs. Additional variables to be taken into consideration are the contact time and the characteristics of the distribution network. Factors modulating the formation of DBPs are summarized in Table 2. Chlorine is the most commonly used disinfectant, and the relationship between chlorine dose and the amount of organic matter in the treated water is the determining factor behind the by-products that will form. The use of chlorine has been linked to the formation of trihalomethanes (THMs), haloacetic acids (HAAs), halonitromethanes (HNMs), haloacetonitriles, chloramines, chlorophenols, the so-called “mutagen X” (MX), and bromate and chloral hydrate, among others. Table 2 Factors affecting DBP formation. Data taken from [8,82–84]. Factors

Effects

Organic matter in water

DBP formation is proportional to the concentration of NOM Aromatic NOM increases the formation of halogenated DBPs Bromide presence determines the formation of brominated DBPs A basic pH favors the formation of THMs Acidic pH can favor the formation of HAAs Higher temperatures demand the use of higher disinfectant doses Chlorine: THMs, HAAs, HNMs, haloacetonitriles, chloramines, chlorophenols, MX, bromate and chloral hydrate Chlorine dioxide: chlorite, chlorate and chloride Ozonation: bromate, formaldehyde, other aldehydes, hydrogen peroxides and brominated methanes Chloramination: dichloramines, trichloramines, cyanogen chloride and chloral hydrate Residual chlorine in distribution systems favors the formation of HAAs over THMs

Ion presence in water Water pH Water temperature Disinfectant employed

Contact time

2

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acetaminophen (paracetamol) react with chlorine, forming several DBP species [14]. Among the physicochemical characteristics that modulate DBP formation are water pH and temperature. Increased water pH promotes the formation of THMs but leads to a decrease in the levels of HAAs, haloacetonitriles, and haloketones. Increased water temperature requires an increase in disinfectant concentrations to achieve similar disinfection outcomes, resulting in higher rates of DBP formation [8].

heritable damage, and developmental defects [18]. DeMarini and colleagues exhaustively reviewed the genotoxic and carcinogenic effects of DBPs [4]. Here, we review the last ten years of DBP genotoxicity studies, dividing them into in vitro, in vivo, and population studies. Consistent with DeMarini’s review, we have used the term “mutagenic” to refer to changes in DNA sequence, and “genotoxic” to cover mutagenic effects as well as DNA damage. The selected studies have also been classified according to whether they were carried out using water samples (containing a mixture of DBPs) or individual DBPs.

4. Health concerns; genotoxicity evidence

5. In vitro studies

Despite the huge sanitary improvement that water disinfection poses, health concerns exist with regard to DBP formation, especially due to the extensive exposure of the human population to these compounds. The scientific community has assessed their potential adverse effects, in in vivo and in vitro studies [15]. Concomitantly, epidemiological studies have been carried out to assess the effects of disinfected water on the human population. Most of these studies have focused on cancer, especially bladder cancer [16], but other effects may also be observed, such as developmental and reproductive complications [17]. Based on these epidemiological results, several countries and international organizations have issued guidelines and regulations setting the maximum levels of different DBPs, particularly the most abundant ones (Table 3). Despite these efforts, only a few of the currently known DBPs have been analyzed and regulated. Considering the high number of chemical species produced by water disinfection, and the fact that the observed negative health effects could be due to the interaction of two or more DBPs, the evaluation of DBPs hazard using standard animal tests becomes inappropriate, due to time and expense issues. Therefore, most studies assessing DBPs effects have focused on genotoxicity testing. The consequences resulting from DNA damage can occur at low exposure doses and in a single cell and, if the resulting mutation persists, it can be amplified by cell division. Somatic alterations may cause cancer, degenerative conditions, and immune system dysfunction, while in germ cells, DNA damage is linked to reproductive impairment,

Most of the analyses of disinfected water and DBPs genotoxicity studies have been carried out in in vitro models. For mutagenicity analyses, the most widely used systems are auxotrophic (Ames test) strains of Salmonella typhimurium [19]. With mammalian cells, complex genetic abnormalities, such as clastogenic damage, can be measured [20]. Thus, we have divided literature according to the in vitro system used to analyze the potential genotoxicity of DBPs. 5.1. In vitro studies using bacteria Studies with bacterial systems have been extensively used to assess the mutagenic and genotoxic potential of both disinfected water and individual DBPs, as shown in Table 4. 5.1.1. Mutagenicity of disinfected waters Most of the efforts to detect disinfected water mutagenicity in bacteria have focused on comparing the hazards of different water sources and water-disinfection procedures. Daiber and colleagues performed a particularly extensive analysis testing the mutagenicity of 28 samples from raw, finished, swimming pool and spa water from seven different sites in the U.S. [21]. Their results show that, although all disinfected waters present DBPs, brominated waters are more mutagenic than chlorinated waters, likely due to the fact that the Br-DBPs,

Table 3 Summary of legislations and guidelines concerning DBPs. Data taken from [8,82–84]. Country or agency

THMs, ppb

HAAs, ppb

Oxyhalides, ppb

Others, ppb

WHO

Chloroform, 200 Bromoform, 100 BDCM, 60 DBCM, 100

DCAA, 50 TCAA, 20

Bromate, 10 Chlorite, 700

TCA,10

USEPA

TTHM, 80

HAA5, 60

Bromate, 10 Chlorite, 1000

Chlorine, 4000

Europe

TTHM, 100

Bromate, 10

Nitrate, 50000 Nitrite, 500

Canada

TTHM, 100

HAA5, 80

Bromate, 10 Chlorite, 1000

China

TTHM, 100

DCAA, 50 TCAA, 100

Bromate, 10 Chlorite, 70

Japan

TTHM, 100

CAA, 20 DCAA, 40 TCAA, 200

Bromate, 10

Australia

TTHM, 250

CAA, 150 DCAA, 100 TCAA, 100

Bromate, 20 Chlorite, 800

South Africa

Chloroform, 300 Bromoform, 100 BDCM, 100 DBCM, 60

Nitrate, 50000 Nitrite, 3000 Formaldehyde, 500 TCA, 20 Nitrate, 11000 Nitrite, 900 Chlorine, 5000

BDCM (bromodichloromethane), DBCM (dibromochloromethane), TTHM (total trihalomethanes), DCAA (dichloroacetic acid), TCAA (trichloroacetic acid), HAA5 (five HAA: dichloroacetic acid + trichloroacetic acid + chloroacetic acid + bromoacetic acid + dibromoacetic acid), CAA (chloroacetic acid). 3

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Table 4 Bacterial genotoxicity studies. Water source or chemical tested Disinfected waters Pool & spa water samples from different US sites Waters from public pools in Barcelona, Spain Seawater and freshwater pools in France Secondary effluent water from Japan, disinfected with different methods Raw, disinfected and tap water from Wuhan plant, China Chlorine-treated wastewater samples from China Individualized DBPs N-nitrosamines: NDMA, NPIP, NPYR, NMOR, NDPhA N-nitrosamines: NDMA NDEA NMEA, mixture MX MX, IA, DBN, BCN, BA, TCN, DCN, CA, DCA, DBA, CN, TCA and CH CH and BH

S. typhimurium strain

Genetic endpoint

Genotoxicity

Reference

TA100 and RSJ100 TA100 and RSJ100 TA100 TA1535/pSK1002

his reversion his reversion his reversion umuDC-lacZ

Yes, higher in brominated and spa waters Yes Yes, higher in freshwater Yes

[21] [22] [23] [24]

TA1535/pSK1002 TA1535/pSK1002

umuDC-lacZ umuDC-lacZ

Yes Yes, higher in wastewater with high NH3-N concentration

[25] [26]

YG7108 TA97, TA98, TA100 and TA102 TA100 and T98 TA1535/pSK1002

his reversion his reversion

All but NDPhA Only in mixture

[27] [28]

his reversion umuDC-lacZ

MX alone and in combination with MC-LR All but CN and CH

[29] [30]

his reversion

Only BH

[31]

TA97a, TA98, TA100 and TA102

NH3-N (ammonia nitrogen), NDMA(N-nitrosodimethylamine), NDEA (N-nitrosodiethylamine), NMEA (N-nitrosomethylamine), NPIP (N-nitrosopiperidine), NPYR (N-nitrosopyrrolidine), NMOR (N-nitrosomorpholine), NDPhA (N-nitrosodiphenylamine), CH (chloral hydrate), BH (bromal hydrate), MX (mutagen-X), IA (iodoacetic acid), DBN (dibromoacetonitrile), BCN (bromochloroacetonitrile), BA (bromoacetic acid), TCN (trichloroacetonitrile), DCN (dichloroacetonitrile), CA (chloroacetic acid), DCA (dichloroacetic acid), DBA (dibromoacetic acid), CN (chloroacetonitrile), TCA (trichloroacetic acid).

results for MX were more consistent. This halofuranone is highly mutagenic, whether tested alone or in combination with other water contaminants of disinfected water [29]. A second study by Zhang and colleagues also tested the mutagenicity of MX together with other twelve DBPs, showing that MX was the most mutagenic among them [30]. Out of the other twelve DBPs tested, namely six haloacetic acids (chloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoacetic acid, dibromoacetic acid, and iodoacetic acid), five haloacetonitriles (chloroacetonitrile, dichloroacetonitrile, trichloroacetonitrile, dibromoacetonitrile, and bromochloro-acetonitrile), and chloral hydrate, only chloroacetonitrile and chloral hydrate were not genotoxic. Chloral hydrate has proven to be non-mutagenic in a recent study, whereas its brominated congener, bromal hydrate, was positive in the mutagenicity assay [31]. Mutagenicity results obtained for individual DBPs reinforce the data for disinfected-water samples. In general, brominated compounds are more mutagenic than chlorinated ones. Nevertheless, because of the wide variety of mutagenic DBPs, it is difficult to pinpoint the compounds accounting for the biological effects of disinfected-water exposures.

which are more mutagenic, are more abundant in bromine-disinfected water. Another similar study, analyzing the mutagenicity of bromine and chlorine-disinfected pool waters from Barcelona, arrived at a similar conclusion [22]. With regard to recreational waters, spa water was more mutagenic than pool water, independent of the disinfectant used, the only exception being ozonized spa water prepared from well water, which was not mutagenic. Finally, the high use of pools and spas by humans increases the mutagenicity of the water, mostly by increasing the DBP content in water due to human inputs [21,23]. Studies focusing on water treatment have shown interesting results. Reverse osmosis filtration and ozonation remarkably decreased the genotoxic potency of municipal secondary effluents, while chlorination elevated it [24]. A second study, analyzing the raw, finished, and tap water from Wuhan city in China, was consistent with these findings [25], showing that chlorination can increase the mutagenicity of drinking water. Furthermore, the mutagenic effects were higher in January than in July, suggesting a possible climate-related effect. Finally, a study evaluating the effect of dissolved matter in chlorinated wastewater showed that low NH3sNs yielded less genotoxicity after disinfection by chlorination [26]. The same study demonstrated that hydrophilic substances in the organic matter reduced the genotoxicity from chlorination of wastewater with a low NH3sN concentration, while hydrophobic acids increased it. All of these studies found that most of the disinfected water samples were mutagenic. In addition, the comparisons of water samples before and after disinfection showed that the disinfection method used, as well as the physicochemical characteristics of the source water, are factors determining DBPs formation and the mutagenicity of the disinfected water.

5.2. In vitro studies: mammalian cells The recent genotoxicity research on DBPs using mammalian cells in vitro is summarized in Table 5 (studies analyzing disinfected water) and 6 (individual DBPs). These studies have focused mainly on characterizing the genotoxicity of newly discovered species of DBPs and the corresponding mechanisms. 5.2.1. Genotoxicity of disinfected waters Genotoxicity assays in mammalian cell models have been used to test different water sources (Table 5). Most of these studies are based on the analysis of DNA double-strand breaks induced in CHO cells, using the single cell gel electrophoresis assay (comet assay). The results show that UV radiation and chloramination generates lower levels of genotoxic compounds when compared to chlorination and the addition of extra steps of chlorination increase genotoxicity [32–34]. The presence of brominated and iodinated organic molecules in source waters enhances genotoxicity following disinfection [34,35]. The analysis of wastewater effluents demonstrates that genotoxicity dramatically increases after chloramination [36]. When comparing the different

5.1.2. Mutagenicity of individual DBPs In spite of the large number of DBPs formed during the disinfection procedures, studies assessing the mutagenicity of individual DBPs in bacteria have mainly centered on N-nitrosamines and the halofuranone, MX. The two studies focusing on N-nitrosamines gave inconsistent results [27,28]. Although Wagner and colleagues found that, of five Nnitrosamines tested, all but NDPhA are mutagenic, Wang and colleagues found that only the mixture of NDMA, NDEA and NMEA shows mutagenicity. This discrepancy could be due to the different experimental settings, such as the S. typhimurium strains used, the concentrations analyzed, or the genetic endpoint tested. On the other hand, 4

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Table 5 Mammalian cell lines water genotoxicity studies. Water source

Cell line

Genetic endpoint

Genotoxicity

Reference

Swimming pool water concentrates from USA Water from different points of water treatment in China Iopamidol-containing source water after disinfection UV and chlorine disinfected water from Ohio river, USA Chlorine or chloramine disinfected water Raw, disinfected and tap water from Wuhan disinfection plant Wastewater effluent organic matter before and after chlorination

CHO Liver cell L-02

SCGE SCGE

Yes Yes, increased after pre-chlorination and post-chlorination

[85] [32]

CHO CHO

SCGE SCGE

Yes Yes, UV was the less genotoxic disinfection treatment

[35] [33]

CHO HepG2, CHO-K1

SCGE HGPRT mutation, Micronuclei SCGE

Yes, chlorination was the most genotoxic process Yes, chlorination increased genotoxicity

[34] [25]

Chloramination increased genotoxicity, especially in the hydrophobic fraction

[36]

CHO

SCGE (single cell gel electrophoresis, comet assay)

lymphocytes, which observed micronucleus induction [46] and chromosome aberrations [50]. Interestingly, other studies using established cell lines did not show chromosome aberration induction, suggesting that lymphocytes are particularly susceptible to this type of damage [51,52]. Among previously known unregulated classes of DBPs, at least three show the characteristics of human carcinogens: haloacetaldehydes (HALs), hydroxyfuranones (MX compounds), and N-nitrosamines [4]. HALs are highly represented in water, and constitute the second most cytotoxic DBP class, whereas they rank as the second least genotoxic DBP class. However, assessment of HALs in the alkaline comet assay showed that eight of nine tested species are genotoxic to CHO cells [53]. The rank order of genotoxic damage is dibromoacetaldehyde > chloroacetaldehyde ≈ dibromochloroacetaldehyde > tribromoacetaldehyde ≈ bromoacetaldehyde > bromodichloroacetaldehyde > bromochloroacetaldehyde ≈ dichloroacetaldehyde > iodoacetaldehyde, while the only non-genotoxic HAL was trichloroacetaldehyde. This genotoxic damage seems to be associated with oxidative DNA damage, although it is promptly repaired and does not lead to more persistent DNA lesions, such as chromosome aberrations [31,54]. MX compounds received special attention due to the strong mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in bacteria. However, only the mutagenicity of mucochloric acid has been proven so far, possibly due to clastogenic damage [55,56]. Nonetheless, mucobromic acid elicits DNA damage, and MX is genotoxic and clastogenic, suggesting that these two species could present a health hazard [29,55]. Finally, the detection of nitrosodimethylamine (NDMA), a probable human carcinogen, in Canadian water supplies led to increased interest in the genotoxicity of Nnitrosamines [31]. Of seven N-nitrosamines tested in mammalian cell systems, NDMA, N-nitrosopiperidine, N-nitrosomorpholine, and N-nitroso-diphenylamine generated genotoxic lesions, although none of them caused chromosome aberrations [27,28,57]. Other unregulated DBP classes are moderately abundant in disinfected waters, reaching low-μg/L levels [4]. Among these are halonitromethanes (HNMs), acetonitriles (HANs), iodoacids, and haloacetamides (HAMs). HNMs are genotoxic in lymphoid cells, but not mutagenic or clastogenic [56,58]. HANs have proved to cause genetic lesions in CHO and HepG2 cells and, of all the iodoacids tested, only (Z)-3-bromo-3-iodopropenoic acid (Z3B3IPPA) does not give positive results when genotoxicity was assessed in CHO cells with the single-cell gel electrophoresis assay [39,40,59]. Methodical analysis of the genotoxicity of 13 HAMs showed that all except dichloroacetamide cause DNA damage [60]. Activation of antioxidant response element (ARE) signaling, and nuclear accumulation of Rad51 in response to monoHAMs treatment, suggest that DNA double-strand breaks are generated in response to oxidative damage [61]. With improved detection methods, many new species of DBPs have been discovered. Among these are halobenzoquinones (HBQs), which

fractions of organic matter extracted from disinfected water samples, DNA damage was significantly higher in the more hydrophobic fraction, which contains the highest concentration of DBPs, in particular, N-DBPs such as HANs and HAMs. There is a correlation between environmental factors and water genotoxicity; one study [25] demonstrated that the levels of mutagenic and clastogenic effects differ during the calendar year, being higher in January than in July. In summary, the genotoxicity of disinfected water has been demonstrated using mammalian cell culture systems. The generation of genotoxic DBPs depends on the origin of the water sample and the disinfection procedure, with ozonation generating least genotoxic byproducts [37]. Most of the reported studies were based on the comet assay, which detects primary DNA damage. Since much of this damage can be repaired, its significance for human health risk remains to be established (Table 6). 5.2.2. Genotoxicity of individual DBPs The genotoxicity of individual DBPs has been studied using in vitro assays with mammalian cell lines. For trihalomethanes (THMs), the most abundant DBP class, efforts have centered on assessing the genotoxicity of their iodinated forms, identified during the U.S.A. Nationwide Occurrence Study [38]. Most of the iodinated THMs are non-genotoxic in the alkaline comet assay, with the exception of chlorodiiodomethane (CDIM) [39]. Other THMs, such as trichloromethane (TCM), tribromomethane (TBM), bromodichloromethane (BDCM), and dibromochloromethane (DBCM) gave positive results for induction of DNA breaks with the alkaline comet assay [40]. The most extensively studied DBP class has been haloacetic acids (HAAs). Of the chemical species tested, only bromodichloroacetic acid (BDCAA) did not cause genotoxic damage in any of the cell lines tested [40,41]. Plewa and colleagues analyzed the genotoxicity results of HAAs in CHO cells (alkaline comet assay), showing that the rank order of genotoxicity was clearly related to the halogen groups present in the molecule. Iodinated and brominated HAAs were more genotoxic than their chlorinated congeners and monoHAAs produced more DNA lesions than di- or tri-substituted HAAs [41]. GADPH inhibition by HAAs generates mitochondrial damage, which increases intracellular reactive oxygen species (ROS) [42,43]. Thus, oxidative damage could be a mechanism of HAA genotoxicity [44–47]. The DNA repair rate of CHO cells treated with monoHAAs varied, with BAA-treated cells having the slowest DNA repair rate, followed by cells treated with CAA and IAA [48]. These differences suggest that different halogen substitutions induce distinct distributions of DNA lesions. In addition to genotoxic damage detected by the comet assay, monoHAAs (CAA, chloroacetic acid – BAA, bromoacetic acid – IAA, iodoacetic acid), dichloroacetic acid (DCAA) and dibromoacetic acid (DBAA), but not trichloroacetic acid (TCAA), were mutagenic in CHO-K1 cells in the HGPRT gene mutation assay [49]. The mutagenicity of monoHAAs could be due to clastogenic damage, as shown in studies using primary human 5

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Table 6 DBPs genotoxicity studies in mammalian cell lines. Genetic endpoint Chemical family

Species

Cell line

Genotoxic

Trihalomethanes

TCM TBM TIM BDCM DBCM CDIM BCIM BDIM DCIM DBIM

HepG2 HepG2 CHO HepG2 HepG2 CHO CHO CHO CHO CHO

+ + − + + + − − − −

Haloacetic acids

CAA

CHO CHO-K1 HepG2 TK6 Lymphocytes Spermatocytes CHO CHO-K1 HepG2 TK6 Lymphocytes Spermatocytes FH 74 Int CHO CHO-K1 HepG2 TK6 NIH3T3 Spermatocytes Lymphocytes CHO CHO-K1 HepG2 CHO CHO-K1 HepG2 CHO CHO CHO-K1 HepG2 CHO CHO CHO CHO CHO

+

BAA

IAA

DCAA

DBAA

DIAA TCAA

TBAA CDBAA BCAA BIAA BDCAA Haloacetaldehydes

CAL BAL IAL DCAL DBAL TCAL

TBAL

BCAL DBCAL BDCAL Haloacetamides

CAcAm BAcAm IAcAm DCAcAm

Reference Mutagenic

Clastogenic [40] [40] [39] [40] [40] [39] [39] [39] [39] [39]

+ − − +

+ + + + +

− +

+ + + + + +

− −

+ + + −

+ +

+ + + + + − − + + + + + −

CHO CHO CHO CHO CHO CHO CHO-K1 HepG2 TK6 Lymphocytes L5178Y/Tk+/− CHO CHO-K1 TK6 Lymphocytes L5178Y/Tk+/− CHO CHO CHO

+ + + + + − − + + +

+ + +

[53] [53] [53] [53] [53] [53] [31] [40] [51] [54] [46] [53] [31] [51] [54] [56] [53] [53] [53]

+ + + + + + −

[60] [61] [60] [61] [60] [61] [60]

− − − −

+ + + +

− − − −

CHO SW480 CHO SW480 CHO SW480 CHO

[41,43,48] [49] [40] [51] [46,50] [46] [41,43,48] [49] [40] [51] [46,50] [46] [44] [37,41,43,48] [49] [40] [51] [52] [46] [46,50] [41] [49] [40] [41] [49] [40] [39,41] [41] [49] [40] [41] [41] [41] [39] [41]

(continued on next page) 6

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Table 6 (continued) Genetic endpoint Chemical family

Hydroxyfuranones

Species

Cell line

Genotoxic

DBAcAm DIAcAm TCAcAm TBAcAm BCAcAm BDCAcAm BIAcAm DBCAcAm

CHO CHO CHO CHO CHO CHO CHO CHO

+ + + + + + + +

MX

CHO-K1 HepG2 Fibroblasts TK6 Lymphocytes L5178Y/Tk+/− TK6 Lymphocytes L5178Y/Tk+/−

+ + + + +

CHO TK6 Lymphocytes NIH3T3 TK6 Lymphocytes NIH3T3 CHO CHO CHO CHO NIH3T3

+ −

TK6 Lymphocytes L5178Y/Tk+/− TK6 Lymphocytes L5178Y/Tk+/−

+

MBA

MCA

NeNitrosamines

NDMA

NDEA

NPIP NPYR NMOR NDPhA NMEA Halonitromethanes

TCNM

BNM

Reference Mutagenic

Clastogenic [60] [60] [60] [60] [60] [60] [60] [60] +

− − − − −

+ + +

− −

− −

− −

− + − + + − − − − − −

+ −

[29] [40] [86] [55] [55] [56] [55] [55] [56] [27] [56] [57] [28] [56] [57] [28] [27] [27] [27] [27] [28] [58] [58] [56] [58] [58] [56]

NeChloramines

Cl-Glycine Cl-Histamine Cl-Ethanolamine Cl-Lysine Cl-NaAcetyl lysine

WIL2-NS WIL2-NS WIL2-NS WIL2-NS WIL2-NS

+ + + + −

Haloacetonitriles

CAN BAN IAN DCAN

CHO CHO CHO CHO HepG2 CHO HepG2 CHO HepG2

+ + + + + + + + +

[59] [59] [59] [59] [40] [59] [40] [59] [40]

DBAN TCAN

+ + + + −

[87] [87] [87] [87] [87]

Halobenzoquinones

2-CBQ TriCBQ TetraCBQ 2,5-DCBQ 2,6-DCBQ 2,5-DBBQ 2,6-DBBQ 2,3-DIBQ

CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1 CHO-K1

+ + + + + + + +

[62] [62] [62] [62] [62] [62] [62] [62]

Iodoacids

Z3B3IPPA E3B3IPPA E3B2IPPA E2I3MBDA

CHO CHO CHO CHO

− + + +

[39] [39] [39] [39]

Others

Phenazine

HepG2



[64]

(continued on next page)

7

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C. Cortés, R. Marcos

Table 6 (continued) Genetic endpoint Chemical family

Species

Cell line

Genotoxic

T24

+

Reference Mutagenic

Clastogenic [64]

TCM (trichloromethane), TBM (tribromomethane), TIM (triiodemethane), BDCM (bromodichloromethane), DBCM (dibromochloromethane), CDIM (chlorodiiodomethane), BCIM (bromochloroiodomethane), BDIM (bromodiiodomethane), DCIM (dichloroiodomethane), DBIM (dibromoiodomethane), CAA (chloroacetic acid), BAA ([bromoacetic acid), IAA (iodoacetic acid). DCAA (dichloroacetic acid), DBBA (dibromoacetic acid), DIAA (diiodoacetic acid), TCAA (trichloroacetic acid), TBAA (tribromoacetic acid), CDBAA (chlorodibromoacetic acid), BCAA (bromochloroacetic acid), BIAA (bromoiodoacetic acid), BDCAA (bromodichloroacetic acid), CAL (chloroacetaldehyde), BAL (bromoacetaldehyde), IAL (iodoacetaldehyde), DCAL (dichloroacetaldehyde), DBAL (dibromoacetaldehyde), TCAL (trichloroacetaldehyde), TBAL (tribromoacetaldehyde), BCAL (bromochloroacetaldehyde), DBCAL (dibromochloroacetaldehyde), BDCAL (bromodichloroacetaldehyde), CAcAm (chloroacetamide), BAcAm (bromoacetamide), IAcAm (iodoacetamide), DCAcAm (dichloroacetamide), DBAcAm (dibromoacetamide), DIAcAm (diiodoacetamide), TCAcAm (trichloroacetamide), TBAcAm (tribromoacetamide), BCAcAm (bromochloroacetamide), BDCAcAm (bromodichloroacetamide), BIAcAm (bromoiodoacetamide), DBCAcAm (dibromochloroacetamide), MX (mutagen-X), MBA (mucobromic acid), MCA (mucochloric acid), NDMA (N-nitrosodimethylamine), NDEA (N-nitrosodiethylamine), NPIP (N-nitrosopiperidine), NPYR (N-nitrosopyrrolidine), NMOR (N-nitrosomorpholine), NDPhA (N-nitrosodiphenylamine), NMEA (N-nitrosomethylamine), TCNM (trichloronitromethane), BNM (bromonitromethane), CAN (chloroacetonitrile), BAN (bromoacetonitrile), IAN (iodoacetonitrile), DCAN (dichloroacetonitrile), DBAN (dibromoacetonitrile), TCAN (trichloroacetonitrile), 2-CBQ (2-chlorobenzoquinone), triCBQ (trichloro-1-4-benzoquinone), tetraCBQ (tetrachloro-1,4-benzoquinone), 2,5-DCBQ (2,5-dichloro-1,4-benzoquinone), 2,6-DCBQ (2,5-dichloro-1,4-benzoquinone), 2,5-DBBQ (2,5-dibromo-1,4benzoquinone), 2,6-DBBQ (2,6-dibromo-1,4-benzoquinone), 2,3-DIBQ (2,3-di-iodo-1,4-benzoquinone), Z3B3IPPA (Z-3-bromo-3-iodopropenoic acid), E3B3IPPA (E3-bromo-3-iodopropenoic acid), E3B2IPPA (E-3-bromo-2-iodopropenoic acid), E2I3MBDA (E-2-iodo-3-methylbutenedioic acid).

increased micronucleus formation while peracetic acid (PAA) did not. The second study examined whether recycling of combined backwash water could increase genotoxicity, when compared to a conventional chlorine-based method [72]. Although both systems increased the genotoxic and clastogenic damage in zebrafish blood cells, when compared to dechlorinated tap water, the recycling process did not cause more damage than the conventional disinfection process. A population study was performed to assess the possible genotoxicity of swimming pools in humans [73]. Analysis of blood, urine, and exhaled-air samples showed that, after swimming (40 min) in an indoor pool, an increase of micronucleated lymphocytes was observed, which correlated to the exhaled concentration of brominated THMs. However, no detectable DNA damage was observed in blood lymphocytes (comet assay). The increase of exhaled brominated THMs, bromoform in particular, also correlated with increased urine mutagenicity, although there was no increase in urothelial cells micronuclei. The above in vivo studies demonstrate the genotoxicity of specific DBP and disinfected water samples. In vivo studies incorporate metabolism and repair of DNA damage, more closely resembling real-life human exposure scenarios. This in vivo data supports the view that DBPs present a real genotoxic risk to exposed humans.

increase ROS formation and oxidative DNA damage, activating the Nrf/ ARE pathway and the p53 response [47,62]. The N-heterocyclic polycyclic aromatic hydrocarbon phenazine (PZ) was also recently described as a byproduct of water disinfection [63]. Analyses of its genotoxicity have shown that even though PZ elicits DNA damage in bladder cells, such as the T24 cell line, it does not act as a genotoxic agent in hepatic cells such as HepG2, suggesting that DNA damage may depend on cell metabolism [64]. In conclusion, genotoxicity has been detected for some components of all groups of DBPs tested. Data obtained with non-regulated DBPs are particularly relevant, because some of them are potent genotoxicants in cultured mammalian cells. Primary DNA damage is not a robust indicator of risk, and few of the cell systems tested for stable DNA damage showed a genotoxic response. To obtain a more complete picture of DBP genotoxicity in mammalian cells, further studies, examining fixed DNA damage, are needed. 6. In vivo and population studies Most of the studies carried out in animal/plant models have assessed the genotoxicity of single DBPs (Table 7). Among the regulated DBPs, TCM and TBM cause DNA damage in onion tuber roots, leading to chromosome anomalies [65]. A study in zebrafish analyzed the genotoxicity of all four regulated THMs [66]. TCM, CDBM, and BDCM caused DNA damage but TBM did not, even at concentrations higher than in the Allium cepa study. Similar behavior was observed for HAAs, where genotoxicity and clastogenic damage induced by CAA, BAA, DCAA, and DBAA was detected in Vicia faba, but not in Caenorhabditis elegans or Danio rerio [66–68]. Plants may be more susceptible to DBP genotoxicity than are animals. Genotoxic damage induced by N-DBPs such as N-nitrosamines, HNMs, and HANs has also been observed in vivo in species such as C. elegans, D. rerio, Mus musculus, and V. faba [68–70]. Clastogenic damage has only been assessed for three HANs (DCAN, DBAN, and TCAN) and, although all three were DNA damaging in the comet assay, only exposure to TCAN caused chromosome abnormalities [68]. Another two DBPs, DCBQ and sodium bromate (NaBrO3), caused DNA lesions in C. elegans and zebrafish embryos, respectively [66,68]. Only two studies have used in vivo models to determine whether different disinfection methods cause DNA damage. Canistro et al. [71] evaluated whether water from Lake Trasimeno (Italy), treated with three different chemicals at their normal disinfecting concentrations, could affect Cyprinus carpio, the common carp. When clastogenic damage in erythrocytes was analyzed, the chlorine-based disinfectants sodium hypochlorite (NaClO) and chlorine dioxide significantly

7. Summary and conclusions In recent years, numerous advances have been made with regard to the health effects of DBPs. Mutagenicity and DNA damage have been used as surrogate markers of detrimental effects, such as carcinogenesis, in the human population. Special interest has been placed on unregulated DBP classes whose members present characteristics of human carcinogens: aldehydes, hydroxyfuranones, and N-nitrosamines. All of the aldehydes tested showed genotoxicity in at least one of the model systems used, even though they may not generate stable DNA damage, such as chromosome aberrations [31,53,54]. Hydroxyfuranones elicited mutagenic and genotoxic damage in bacteria and cell lines, although only MX, the most potent genotoxicant of the group, caused clastogenic damage [29,48]. On the other hand, even though N-nitrosamines were mutagenic in S. typhimurium, three chemical species were unable to generate DNA damage in mammalian cell systems: NDEA, NPYR, and NMEA [29,57]. NeNitrosamines were the only potentially-carcinogenic DBP class to be tested in vivo and, interestingly, NDMA and NDEA were genotoxic in C. elegans and D. rerio, respectively [67,70]. This data supports previous findings indicating the carcinogenic effect of these classes on animal models, suggesting that more in vivo studies should be carried out in order to establish regulations on exposures to these 8

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Table 7 In vivo DBP genotoxicity studies. Chemical family

Name

Organism

Genetic endpoint

Genotoxicity

Reference

Trihalomethanes

TCM

A. cepa Zebrafish A. cepa

CDBM BDCM

Zebrafish Zebrafish Zebrafish

Yes Yes Yes Yes Yes No Yes Yes

[65]

TBM

SCGE Anaphase-telophase anomalies SCGE SCGE Anaphase-telophase anomalies SCGE SCGE SCGE

CAA

V. faba

BAA

C. elegans V. faba

DCAA

C. elegans V. faba

DBAA

C. elegans V. faba

Yes Yes No Yes Yes No Yes Yes No Yes Yes No No Yes Yes No

Haloacetic acids

[66] [65] [66] [66] [66]

TBAA

Zebrafish

SCGE MN LA-qPCR SCGE MN LA-qPCR SCGE MN LA-qPCR SCGE MN SCGE LA-qPCR SCGE MN SCGE

NeNitrosamines

NDMA NDEA

C. elegans Zebrafish

LA-qPCR SCGE

Yes Yes

[67] [70]

Halonitromethanes

BNM TCNM BCNM

Mouse Mouse Mouse

8-OHdG, ELISA 8-OHdG, ELISA 8-OHdG ELISA

Yes Yes Yes

[69] [69] [69]

Haloacetonitriles

DCAN

V. faba V. faba

TCAN

V. faba

Yes No Yes No Yes Yes

[68]

DBAN

SCGE MN SCGE MN SCGE MN

Halobenzoquinones

DCBQ

C. elegans

LA-qPCR

Yes

[67]

Others

NaBrO3

Zebrafish

SCGE

Yes

[66]

TCAA

Zebrafish C. elegans V. faba

[68] [67] [68] [67] [68] [67] [68] [66] [67] [68] [66]

[68] [68]

TCM (trichloromethane), TBM (tribromomethane), CDBM (chlorodibromomethane), BDCM (bromodichloromethane), CAA (chloroacetic acid), BAA (bromoacetic acid), DCAA (dichloroacetic acid), DBBA (dibromoacetic acid), TCAA (trichloroacetic acid), TBAA (tribromoacetic acid), NDMA (N-nitrosodimethylamine), NDEA (Nnitrosodiethylamine), BNM (bromonitromethane), TCNM (trichloronitromethane), BCNM (bromochloronitromethane), DCAN (dichloroacetonitrile), DBAN (dibromoacetonitrile), TCAN (trichloroacetonitrile), DCBQ (2,6-dichloro-1,4-benzoquinone)., CGE (single cell gel electrophoresis, comet assay), MN (micronucleus), LAqPCR (Long amplicon quantitative PCR assay).

While assessing the genotoxicity of each DBP can give us valuable information on the health risk that they pose individually, these kinds of analyses could underestimate the interactions between different DBPs and between DBPs and the organic matter present in raw water, especially when it is subjected to anthropogenic contaminants [21]. As disinfected water is a complex mixture where many of the DBPs composing the organic halogen fraction remain to be identified, determining the effects of individual DBPs is not sufficient to determine the biological consequences of disinfected water exposure. Therefore, the importance of testing the effects of disinfected waters in situ, in particular, water sources to which a broad population is exposed, such as water from disinfection plants and public swimming pools, becomes clear. Data from epidemiological studies can be relevant to understand the underlying mechanisms leading to genotoxic damage and to propose further approaches in genotoxicity testing. It is interesting to note the data of Cantor et al. [88], who reported that polymorphisms in some enzymes (GSTT1, GSTZ1, and CYP2E19) modified DBP-associated bladder cancer risk. As a general conclusion, the implementation of routine genotoxic analysis could mitigate the risk of disinfected waters by allowing the identification of the best disinfection methods for each type of water source. Nevertheless, possibly some information such as that obtained

potentially carcinogenic compounds [4]. Other N-DBPs have also been extensively studied, mainly those belonging to HAMs, HNMs, and HANs. Of the N-DBPs tested, only DCAcAm did not induce DNA strand breaks; the other HAMs and all the HNMs and HANs tested did [58–60]. HNMs also caused oxidative DNA damage in mice, while HANs were mutagenic in bacteria and caused genotoxicity in V. fava, even though genotoxicity translated into clastogenic damage only in TCAN-exposed plants [30,67,69]. Several authors have performed systematic analyses of members of each DBP class, mainly by assessing genotoxicity in CHO cells by the comet assay [39,53,59,60]. Although this useful screening approach has helped to increase our understanding of genotoxic potencies within and among classes, the assessment of persistent DNA lesions, such as chromosome aberrations or mutations, can mitigate possible overestimation of the risk posed by each DBP. Given the association between mutation and cancer, it is important to introduce the use of carcinogenicity as an end-point when testing the potential hazard of DBPs. Due to the complexity of using mouse models to determine the carcinogenic risk of DBPs, in vitro cell transformation assays have been proposed as suitable alternatives to long-term animal studies [74]. In fact, this approach has been already used to evaluate carcinogenic risk of iodoacetic acid and iodoform [75], as well as halonitromethanes [76]. 9

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using assays which detect primary DNA damage are not relevant enough in terms of risk assessment. This means that the use of assays evaluating chromosome damage should be extended, as well as those using in vivo approaches, although they use model organisms such as C. elegans and zebrafish. Additionally, in vitro approaches assessing carcinogenicity markers of cell transformation are surprisingly lacking. Such studies can provide valuable information linking DBP exposure and cancer induction, as well as detecting DBPs that may be acting as non-genotoxic carcinogens, not detected using the standard genotoxicity assays.

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