Environ. Sci. Technol. 2003, 37, 3782-3793
Tribromopyrrole, Brominated Acids, and Other Disinfection Byproducts Produced by Disinfection of Drinking Water Rich in Bromide SUSAN D. RICHARDSON* AND ALFRED D. THRUSTON, JR. National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30605 CHAIM RAV-ACHA, LUDMILA GROISMAN, INNA POPILEVSKY, OLGA JURAEV, AND VICTOR GLEZER The Research Laboratory of Water Quality, Israel Ministry of Health, P.O.B. 8255, Tel-Aviv 61082, Israel A. BRUCE MCKAGUE CanSyn Chem. Corporation, Toronto, Ontario M5S 3E5, Canada MICHAEL J. PLEWA AND ELIZABETH D. WAGNER College of Agricultural, Consumer, and Environmental Sciences, Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Using gas chromatography/mass spectrometry (GC/MS), we investigated the formation of disinfection byproducts (DBPs) from high bromide waters (2 mg/L) treated with chlorine or chlorine dioxide used in combination with chlorine and chloramines. This study represents the first comprehensive investigation of DBPs formed by chlorine dioxide under high bromide conditions. Drinking water from full-scale treatment plants in Israel was studied, along with source water (Sea of Galilee) treated under carefully controlled laboratory conditions. Select DBPs (trihalomethanes, haloacetic acids, aldehydes, chlorite, chlorate, and bromate) were quantified. Many of the DBPs identified have not been previously reported, and several of the identifications were confirmed through the analysis of authentic standards. Elevated bromide levels in the source water caused a significant shift in speciation to bromine-containing DBPs; bromoform and dibromoacetic acid were the dominant DBPs observed, with very few chlorine-containing compounds found. Iodo-trihalomethanes were also identified, as well as a number of new brominated carboxylic acids and 2,3,5-tribromopyrrole, which represents the first time a halogenated pyrrole has been reported as a DBP. Most of the bromine-containing DBPs were formed during pre-chlorination at the initial reservoir, and were not formed by chlorine dioxide itself. An exception was the iodoTHMs, which appeared to be formed by a combination of chlorine dioxide with chloramines or chlorine (either added deliberately or as an impurity in the chlorine dioxide). * Corresponding author phone: (706)355-8304; fax: (706)355-8302; e-mail:
[email protected]. 3782
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 17, 2003
A separate laboratory study was also conducted to quantitatively determine the contribution of fulvic acids and humic acids (from isolated natural organic matter in the Sea of Galilee) as precursor material to several of the DBPs identified. Results showed that fulvic acid plays a greater role in the formation of THMs, haloacetic acids, and aldehydes, but 2,3,5-tribromopyrrole was produced primarily from humic acid. Because this was the first time a halopyrrole has been identified as a DBP, 2,3,5tribromopyrrole was tested for mammalian cell cytotoxicity and genotoxicity. In comparison to other DBPs, 2,3,5tribromopyrrole was 8×, 4.5×, and 16× more cytotoxic than dibromoacetic acid, 3-chloro-4-(dichloromethyl)-5hydroxy-2-[5H]-furanone [MX], and potassium bromate, respectively. 2,3,5-Tribromopyrrole also induced acute genomic damage, with a genotoxic potency (299 µM) similar to that of MX.
Introduction Because of tightened regulations and health concerns over chlorinated byproducts in drinking water, many drinking water utilities are switching from chlorine to alternative disinfectants, such as ozone, chlorine dioxide, and chloramines. However, much less is known about the DBPs from these alternative disinfectants than those from chlorine. Chlorine dioxide is a popular alternative because it does not produce appreciable levels of trihalomethanes (THMs), and is often used in situations where the THM regulations would be exceeded if chlorine were used. It is estimated that more than 500 drinking water treatment plants in the U.S. use chlorine dioxide in drinking water treatment. A comprehensive review on the aqueous reactions of chlorine dioxide has been published recently by Rav-Acha (1). Although it is well-known that chlorine dioxide does not produce substantial levels of THMs, very little is known about other DBPs produced by chlorine dioxide in actual drinking water plants. And, even less is known about the formation of chlorine dioxide DBPs in the presence of high bromide levels. Bromide is naturally present in many source waters across the U. S. and other parts of the world, and when it is present it has caused an increase in bromine-containing DBPs (upon chlorine, chloramine, and ozone disinfection) (2, 3-8). Because bromine-containing compounds are generally more carcinogenic than their chlorinated analogues (9), the U.S. EPA is interested in discovering what DBPs are formed by chlorine dioxide, as well as by combination of chlorine dioxide with chlorine or chloramines, under these high bromide conditions. Drinking water in Israel was initially studied because its source water (the Sea of Galilee) has natural levels of bromide (∼2 mg/L) among the highest in the world for surface water, and chlorine dioxide is used for disinfection at full-scale treatment plants. Source water is pumped from the Sea of Galilee (a freshwater lake, also called Lake Kinereth), treated with pre-chlorination (for initial algae control) in the first reservoir, transported to another reservoir for treatment with chlorine dioxide and chloramines, and distributed through a 300-mile-long distribution system which reaches as far as the Negev Desert (Figure 1). When the National Water Carrier (NWC) was first established in the mid-1960s, chlorine was used as the sole disinfectant; however, when high bromoform levels were observed in the mid-1970s (sometimes exceeding 100 µg/L), it was decided to switch from chlorine to chlorine 10.1021/es030339w CCC: $25.00
2003 American Chemical Society Published on Web 08/05/2003
FIGURE 1. Map showing the Israel National Water Carrier (NWC) system. dioxide as the primary disinfectant to lower these levels. When chlorine dioxide was used at this point as the sole disinfectant (at 1 mg/L), there were problems maintaining an adequate disinfectant residual throughout the long distribution system (which allowed bacterial regrowth). Because there was a limit to the amount of chlorine dioxide that could be applied (due to the continuous formation of chlorite, which would exceed Israel’s maximum contaminant level of 500 µg/L), it was decided to use chloramines as a secondary disinfectant (following primary disinfection with chlorine dioxide) to maintain disinfection in the distribution system. Most DBP studies published in the literature involve the measurement of pre-selected, targeted chemicals; in contrast, we carried out more comprehensive studies, attempting to identify all organic DBPs formed in the water through the use of a combination of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) techniques. In addition, several DBPs (chlorite, chlorate, bromate, THMs, haloacetic acids (HAAs), and some aldehydes) were quantified in drinking water samples, and one of the new DBPs identified (2,3,5tribromopyrrole) was tested for cytotoxicity and genotoxicity.
Materials and Methods Drinking Water Treatments. Chlorine dioxide treated drinking water was collected from two full-scale plants in Israel, both of which use Sea of Galilee (Lake Kinereth) raw source waters. The larger of these plants also applies chloramines along with chlorine dioxide to maintain disinfection in the distribution system of the NWC and uses a pretreatment with chlorine for algae control at its initial reservoir (Figure 1). Water was collected from three sampling points as follows (Figure 1): (1) the Sea of Galilee at the entrance to the NWC (after screen filtration); (2) entrance to Eshkol Reservoir, after
pre-chlorination for algae control (applied at the Tsalmon Reservoir); and (3) exit of Eshkol Reservoir, after the main treatment plant (which applied either chlorine dioxidechloramine or chlorine dioxide-chlorine). The smaller plant used only chlorine dioxide disinfection, which enabled the study of DBPs formed by chlorine dioxide disinfection alone; however, small amounts of free chlorine impurities can also be present. Ambient bromide levels were approximately 2 mg/L (ppm) throughout the study. Chlorine dioxide was generated at the two plants through the oxidation of sodium chlorite by chlorine gas. At the larger plant, prechlorine doses ranged from 0.8 to 1.6 ppm; chlorine dioxide doses ranged from 0.6 to 0.85 ppm; chloramine doses were approximately 0.8 ppm (through the in situ addition of ammonia followed by chlorine); and chlorine doses (when used as a secondary treatment with chlorine dioxide) were approximately 1.3 ppm. Details of specific doses for each sampling, residual disinfectant levels at the time of sampling, exact bromide levels, and source water quality characteristics can be found in the Supporting Information. In addition to the samples collected at these two drinking water treatment plants, a separate, controlled laboratory study was performed, where pure chlorine dioxide s devoid of any traces of chlorine s was generated in the laboratory by the method of Masschelein (10), by the reaction of sodium chlorite and acetic anhydride, and used to treat the same source water under controlled conditions. This enabled the distinction of those DBPs that were formed by chlorine dioxide itself from those that were formed by the combination of chlorine dioxide and chlorine/chloramines or from chlorine impurities present in chlorine dioxide generated at the full-scale plants. Doses were approximately 1.0 ppm chlorine dioxide. In another laboratory experiment, pure chlorine dioxide was used with chlorine to treat the same source water to mimic a corresponding treatment at the larger full-scale plant. In this experiment, doses of 0.6 ppm chlorine dioxide and 1.4 ppm chlorine were applied to the source water. Five samplings were conducted over the course of two years: December 1998, May 1999, September 1999, November 1999, and July 2000. During two of the five samplings (Sept. 1999 and July 2000), the larger treatment plant switched from chlorine dioxide-chloramine to chlorine dioxidechlorine, so this combination was also studied. Another controlled laboratory study was conducted to quantitatively determine the contribution of fulvic acids and humic acids (from isolated natural organic matter in the Sea of Galilee) as precursor material to several of the DBPs identified. For this work, fulvic acid and humic acid were isolated from the Sea of Galilee (from water and sediment, respectively) and reacted with chlorine and chlorine dioxide in the laboratory. Fulvic acid was isolated from the Sea of Galilee according to a method by Thurman and Malcolm (11). Lake water (150 L) was acidified to pH 2 with HCl and passed through a 60 × 5 cm column filled with 1200 mL of Superlite DAX-8 resin (Supelco). The resins were cleaned by washing them with 0.1 N NaOH (5×), followed by sequential Soxhlet extractions with methanol, diethyl ether, acetonitrile, and methanol again. The column was then washed with doubly distilled water until the TOC was < 0.1 ppm, and was then washed with 0.1 N HCl. Finally, the column was eluted in reverse with 0.1 N NaOH, and acidified back to pH 2. The same procedure was repeated on a smaller column, and the final eluent was acidified to precipitate humic material, which was removed by centrifugation. The supernatant was made basic with 0.1 N NaOH and passed through a column containing cationic exchange resins (AG-MP-50), which was then eluted with distilled water. The eluent was freeze-dried, and the resulting fulvic acid (150 mg) gave the following elementary analysis: C, 52.5%; H, 6.20%; N, 1.26%. VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3783
TABLE 1. DBPs (Organics) Identifieda sampling 2 (May 99)
sampling 3 (Sept 99)
Cl2
chloroform bromodichloromethane dibromochloromethane bromoform dichloroiodomethane chlorobromoiodomethaned dibromoiodomethaned chlorodiiodomethane bromodiiodomethane iodoform
x x x x x x -
x x x x x -
Halomethanes x x x x x x x x -
x x x x x -
x x x x x x x
x -
x x x x -
x x x x x -
x x -
x x x -
bromoacetic acid bromochloroacetic acid dibromoacetic acid tribromoacetic acid 2-bromobutanoic acidd trans-4-bromo-2-butenoic acid cis-4-bromo-2-butenoic acid 2,2-dibromopropanoic acid 3,3-dibromopropenoic acidd trans-2,3-dibromopropenoic acid cis-2,3-dibromopropenoic acid 2,2-dibromobutanoic acid trans-2,3-dibromo-2-butenoic acidd 3,3-dibromo-4-oxopentanoic acidd tribromopropenoic acidd
x x x x x x x x x x x x x x
x x x x x x x x x x x x x
Haloacidsb x x x x x x x x x x x x x x x x x x x x x
x x x x x x x x x x x
x x x x x x x x x
x -
x x x -
x x -
x -
x x -
cis-2-bromobutenedioic acid trans-2-bromobutenedioic acid cis-2-bromo-3-methylbutenedioic acid trans-2,3-dibromobutenedioic acidd
x x x x
x x x x
x x x x
x x -
x x x x
x x -
x x -
-
-
-
-
dibromobenzoic acide dibromo-2-aminobenzoic acide dibromotriaminobenzoic acide
x x x
x x x
x x x
x x x
x x x
x x x
-
-
-
-
-
bromoacetonitriled 3-bromopropanenitrilec bromochloroacetonitrile dibromoacetonitriled
x
x x x
Halonitriles x x
x x
x
x
x x
x x x x
x -
x x x
2-bromo-2-methylpropanald bromobutenal dibromoacetaldehyde bromopentadienald,f tribromoacetaldehyde
-
x x -
Haloaldehydes x x x x
x x x x
-
x -
x -
x x -
-
-
bromopropanoned 1,1-dibromopropanoned 1,3-dibromopropanoned 1,1,1-tribromopropanoned 1,1,3-tribromopropanone 1,1,3,3-tetrabromopropanone 3-bromo-4-methyl-3-penten-2-onec
x x -
x x x x x -
Haloketones x x x x x x -
x x
x
x
x x
x x
-
x -
dibromonitromethaned
-
x
Halonitromethanes x -
-
-
-
-
-
-
DBP identified
1,4-dibromo-2,3-butanediolc
-
12 h later
Cl2
lab ClO2
lab ClO2 + Cl2
-
-
-
-
-
-
-
-
-
-
x
-
-
x
-
-
-
x
-
x
Haloaromatics x -
x -
-
x
x
-
x
x
2,3,5-tribromopyrrolec,d
-
-
Halopyrroles -
9
Cl2
ClO2 + Cl2
-
x
3784
lab ClO2
x
2-bromoethanol acetate 2,3-dibromopropyl acetatec
-
ClO2 small plant
Haloalcohols x Haloacetates x x -
dichlorophenol 4-bromo-2-methoxy-5-methylphenolc,e
ClO2 + Cl2
sampling 5 (July 00)
ClO2 + NH2Cl
-
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 17, 2003
TABLE 1. (Continued) sampling 2 (May 99) DBP identified
Cl2
ClO2 + NH2Cl
tribromothiophene formaldehydef acetaldehydef acetonef 2-hexanonef glyoxalf methyl glyoxalf
-
x -
12 h later x -
sampling 3 (Sept 99) Cl2
ClO2 + Cl2
ClO2 small plant
Non-Halogenated DBPs x -
sampling 5 (July 00) lab ClO2 -
Cl2
ClO2 + Cl2
lab ClO2
lab ClO2 + Cl2
x x x x
x x x -
x x x x
x x x x
a Compounds in italics were confirmed through the analysis of purchased or synthesized standards; an ‘x’ denotes that the compound was identified in the particular sample; an ‘-’ denotes that it was not found. b Identified as methyl esters. c Present in library database (NIST or Wiley). d High-resolution data available; supports identification. e Exact isomer uncertain. f Identified as PFBHA derivatives.
Because of the small concentration of humic acid in the Sea of Galilee (about 85% is fulvic), the humics were isolated from the lake’s sediment rather than from the water according to a procedure published by De Serra and Schnitzer (12). Crude lake sediment (160 g, dry weight) was decalcified with sulfuric acid and dissolved in 1.6 L of 0.5 N NaOH. After filtration through glass wool, the supernatant was acidified to pH 2, and the humic matter was precipitated. The precipitant was treated with HCl/HF to remove minerals, and the resulting humic acid (500 mg) gave the following analysis: C, 47.52%; H, 6.05%; N, 7.52%. “Synthetic water” was created by dissolving the isolated fulvic acid or the humic acid material in distilled water having the same alkalinity (125 mg/L), hardness (226 mg/L), ionic strength (400 mg/L), and pH (7.8) as the Sea of Galilee water. Ca(HCO3)2 was used for alkalinity, CaCl2 and MgCl2 were used for hardness (taking into account the calcium used already for alkalinity), NaBr was used for bromide, and the ionic strength was accomplished by adding NaCl. All chemicals were of analytical grade, >99% purity. Fulvic acid (17.5 mg/L as C), humic acid (17.5 mg/L as C), and bromide (10 mg/L) concentrations, as well as oxidants concentrations, were increased by a factor of 5 over the natural conditions to provide increased levels of DBPs to aid in their detection and quantitation. Synthetic waters were treated with 5 mg/L of chlorine or 5 mg/L of chlorine dioxide and allowed to react for 24 h. At the end of the reaction period, the oxidants were quenched with sodium sulfite. THMs, HAAs, and aldehydes were quantified by methods given below. Blanks, which did not contain the fulvic or humic acids, were also analyzed. Sample Concentration. Treated samples were quenched with sodium sulfite prior to concentration. A 40-L portion of each water was acidified to pH 2 and concentrated using XAD resins (XAD-8 over XAD-2). The resins were eluted with ethyl acetate, and the ethyl acetate eluents were dried with sodium sulfate and rotoevaporated to 1 mL. XAD resins were cleaned by Soxhlet extraction prior to use, according to a previously published procedure (13). Quantitative Analyses. Bromide, chlorite, and chlorate were quantified for two samplings (1 and 3) using EPA Method 300.1 (14). Bromate was quantified by a selective anion concentration (SAC) ion chromatography method that permits lower limits of quantitation for bromate (0.20 µg/L) than EPA Method 300.1 (15). Trihalomethanes (THMs) were analyzed according to EPA Method 524.2 (16), using a Finnigan Magnum GC-mass spectrometer connected to a Tekmar Purge & Trap 3000 concentrator and a Tekmar Precept II autosampler. Haloacetic acids were analyzed according to EPA Method 552 (16) on a ThermoQuest/ Finnigan Polaris GC-mass spectrometer. Carbonyl compounds were quantified using a modification of EPA Method
556 (in which GC with electron capture detection was replaced by GC/MS) (14). 2,3,5-Tribromopyrrole was quantified (using a ThermoQuest/Finnigan Magnum GC-mass spectrometer) through the comparison of sample peak areas with a calibration curve generated using a synthetic sample of 2,3,5-tribromopyrrole, which was prepared as described by Gilow and Burton (17) (synthesis described in Supporting Information). The yield of 2,3,5-tribromopyrrole was about 98%, with 2% of 2,3,4-tribromopyrrole. The two isomers, which have similar mass spectra, can be easily separated because of their different GC retention times (8.35 and 10.42 min, respectively). Qualitative GC/MS Analyses. For the qualitative, unknown DBP identifications, low- and high-resolution GC/ electron ionization (EI)-MS and GC/chemical ionization (CI)-MS analyses were performed on a hybrid high-resolution mass spectrometer (VG 70-SEQ, Micromass, Inc.) equipped with a GC (model 5890A, Hewlett-Packard/Agilent). The high-resolution mass spectrometer was operated at an accelerating voltage of 8 kV. Low-resolution analyses were carried out at 1000 resolution and high-resolution analyses were carried out at 10 000 resolution. Positive CI experiments were accomplished by using methane gas. Injections of 1-2 µL of the extract were introduced via a split/splitless injector onto a GC column (DB-5, 30 m × 0.25 mm id, 0.25 µm film thickness, J&W Scientific/Agilent). The GC temperature program consisted of an initial temperature of 35 °C, which was held for 4 min, followed by an increase at a rate of 9 °C/min to 285 °C, which was held for 30 min. Transfer lines were held at 280 °C, and the injection port was controlled at 250 °C. Derivatizations. Pentafluorobenzylhydroxylamine (PFBHA) derivatizations were used to extract and aid in the identification of relatively polar carbonyl-containing compounds. For this work, 750 mL of treated water (and raw water as a control) was derivatized with PFBHA according to a procedure published by Sclimenti et al. (18). Methylation derivatizations with BF3/methanol were used to aid in identifying carboxylic acids (19). Chemical Standards and Cell Culture Medium. The following chemicals were prepared synthetically: 2,3,5tribromopyrrole, 2,2-dibromopropanoic acid, 3,3-dibromopropenoic acid, cis- and trans-2,3-dibromopropenoic acid, 2,2-dibromobutanoic acid, trans-2,3-dibromo-2-butenoic acid, tribromopropenoic acid, trans-2,3-dibromobutenedioic acid, 3,3-dibromo-4-oxopentanoic acid, and cis- and trans2-bromo-3-methyl-2-butenedioic acid dimethyl ester. Details of these synthesis procedures can be obtained in the Supporting Information. All other chemicals and reagents were either purchased at the highest level of purity from Aldrich, Sigma, Supelco, or Fisher Scientific, or were previously synthesized and reported elsewhere (20). Media VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3785
TABLE 2. DBP Concentrations, µg/La sampling 2 (May 99)
sampling 3 (Sept 99) lab ClO2
Cl2
ClO2 + Cl2
lab ClO2
lab ClO2 + Cl2
raw ND ND ND ND
ND 0.3 3.7 55
ND 0.20 2.2 57
ND ND ND ND
ND 0.3 3.5 67
2.5 ND -
ND ND ND ND ND ND ND ND ND
ND ND ND ND 12 ND 1 ND ND
ND ND ND ND 14.1 ND 1.6 ND ND
ND ND ND ND ND ND ND ND ND
ND ND ND ND 8.2 ND ND ND ND
-
2.9 2.6 ND ND
5.2 3.3 4.9 2.0
6.8 4.5 3.7 ND
16 3.5 4.2 4.2
31 3.5 3.5 4.0
Cl2
ClO2 + NH2Cl
12 h later
chloroform bromodichloromethane dibromochloromethane bromoform
0.35 0.64 9.39 57
ND 0.21 2.4 19.5
ND 0.24 2.95 21
Halomethanes ND ND ND ND 0.30 0.40 ND 1.9 2.1 ND 59 61
ND ND ND 2.1
ND ND ND ND
chloroacetic acid dichloroacetic acid trichloroacetic acid bromoacetic acid dibromoacetic acid tribromoacetic acid bromochloroacetic acid bromodichloroacetic acid chlorodibromoacetic acid
38.7 3.9 -
12.5 1.4 -
12.5 1.8 -
Haloacetic Acids ND 22.9 23.3 ND ND ND -
ND -
-
-
-
DBP identified
formaldehyde acetaldehyde glyoxal methyl glyoxal a
raw
-
ClO2 + Cl2
sampling 5 (July 00)
ClO2 small plant
Cl2
Aldehydes -
-
-
ND: Not detected; an “-” denotes that the compound was not measured.
supplies and fetal bovine serum (FBS) were purchased from Hyclone Laboratories (Logan, UT). The transgenic Chinese hamster ovary (CHO) cell line AS52 clone 11-4-8 was used in this research (21). The CHO cells were maintained in Ham’s F12 medium containing 5% FBS and grown in 100-mm glass culture plates at 37 °C in an atmosphere of 5% CO2 in air. Microplate Chronic Mammalian Cell Cytotoxicity Assay and Single Cell Gel Electrophoresis Assay. The mammalian cell cytotoxicity assay was conducted as presented by Plewa et al. (22) with a modification. After staining the cells with crystal violet the microplates were gently washed with tap water and tapped dry. Into each well 50 µL of dimethyl sulfoxide was added, and the stain was extracted from the cell membranes for 30 min. The microplates were then analyzed with a microplate reader. The single-cell gel electrophoresis assay was conducted as previously described (22). Further details can be found in the Supporting Information.
Results and Discussion Table 1 lists the DBPs that were observed in Samplings 2, 3, and 5. A complete list of DBPs identified in all of the samplings can be found in the Supporting Information. DBPs shown in italics were confirmed through the analysis of authentic chemical standards (purchased or synthesized); all other identifications should be considered tentative. The criteria we used for listing an identified compound as a DBP was its presence in the treated samples in quantities at least 2 to 3 times greater than that in the untreated, raw water (as judged by comparing chromatographic peak areas) (20). However, most of the halogenated DBPs observed in these samples were observed only in the treated samples, and were not detected in the raw, untreated water. Library database searching (of the NIST and Wiley database) was used initially to aid in the identification of several compounds; however, many DBPs were not present in either of the databases. In those cases, and in cases where a library match was insufficient to offer a tentative identification, high resolution EIMS was used to provide key empirical formula information on the molecular ion and fragments, CIMS was used to provide molecular weight information when molecular ions were missing in the EI mass spectra, and mass spectra were extensively interpreted to provide tentative chemical iden3786
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 17, 2003
tifications. Methylation derivatizations were important for revealing the presence of several new bromo-acid DBPs, and PFBHA derivatizations revealed the presence of a few small aldehydes and ketones. In addition to the DBPs that were qualitatively identified, several DBPs and other chemical species were also quantified. Representative results are shown in Table 2. Complete results of all samplings can be found in the Supporting Information. Halogenated Organic DBPs. Most of the halogenated compounds judged to be true DBPs were generally observed in more than one of the five samplings. Examples of these include THMs (with bromoform as the dominant DBP), dibromoacetic acid (another major DBP), tribromoacetic acid, several other brominated acids, dibromoacetonitrile, 2-bromo2-methylpropanal, 2,3,5-tribromopyrrole, and other DBPs listed in Table 1. Besides the traditional THMs (chloroform, bromoform, bromodichloromethane, and chlorodibromomethane), several iodo-THMs were also identified (Table 1). The iodide level is approximately 17-18 ppb in the Sea of Galilee (23), so the formation of these iodo-THMs was not surprising. Although iodo-THMs have been identified previously (24-33), they are not commonly reported. This is partly due to the fact that they are not regulated and are somewhat difficult to identify s some of them (dibromoiodomethane, chlorodiiodomethane, and bromodiiodomethane) are not present in any of the spectral library databases (NIST or Wiley), and standards for most are not commercially available. Chlorine-containing DBPs that are usually dominant under low-bromide conditions (for chlorine and chloramine disinfection) s chloroform and dichloroacetic acid s were found at very low concentrations or not at all in these samples, with a shift to bromoform and dibromoacetic acid occurring under these high-bromide conditions. Thus, the high bromide content in the source water was making a major impact on the speciation of the DBPs. There was some tribromoacetic acid identified and quantified in these samples, but it is likely the concentrations of tribromoacetic acid actually produced were higher than those quantified because some of the tribromoacetic acid originally produced was converted to bromoform due to the relatively high pH (>8.0) of the water (the conversion of tribromoacetic acid to bromoform is basecatalyzed (34)). Because iodo-THMs were found in these
FIGURE 2. GC/MS chromatogram of drinking water treated with pre-chlorine (second Sampling) methylated extract showing brominated acids identified. DBPs denoted with an asterisk were confirmed through the analysis of authentic chemical standards. samples, a thorough search was made for any iodo-HAAs that might have been formed. To do this, it was necessary to analyze the methylated samples for the corresponding methyl esters, and as none of these iodo-methyl esters are in the library databases, their mass spectra were predicted on the basis of spectra of the corresponding bromo- and bromochloro-methyl esters. For example, all of these bromoand bromochloro-acetic acid methyl esters (mono-, di-, and tri-halogenated) show mass spectral losses of m/z 31 (loss of OCH3 group) and m/z 59 (COOCH3 group), and monoand di-halogenated esters also show a loss of one halogen from the molecular ion. Thus, the corresponding masses of these fragments for the iodo-compounds were calculated, and these ions were used along with the molecular ions to create reconstructed ion chromatograms, which would show the appearance of these ions if they were present in the samples. However, none of the 10 possible iodinated methyl esters was found in any of the samples studied. Therefore, it appears that either they were not formed, or some may have initially formed but were not stable in the high pH water of the Sea of Galilee. Five iodo-acids have been recently identified in drinking water from a U. S. Nationwide DBP Occurrence Study (iodoacetic acid, iodobromoacetic acid, 2 isomers of iodobromopropenoic acid, and 2-iodo-3-methylbutenedioic acid) (35). Most of the THMs, bromo-acids, and other halogencontaining DBPs observed were produced during the prechlorination at the initial Tsalmon Reservoir (for algae control) and subsequent chlorine dioxide treatment did not significantly increase their concentrations. The exceptions were the iodo-THMs that were formed predominantly by chlorine dioxide and chlorine dioxide-chloramine treatment. Only traces of two iodo-THMs (dibromoiodomethane and dichloroiodomethane) were found in the pre-chlorinated reservoir water, and dichloroiodomethane was seen in the pre-chlorinated water in only one of the samplings. None of the THMs (except a trace amount of dibromoiodomethane) was found in the pure, laboratory chlorine dioxide-treated water. Therefore, the combination of chlorine dioxide with chloramines or chlorine (deliberately added or chlorine impurities in the chlorine dioxide) may be important for their formation. In a recently conducted U. S. Nationwide DBP Occurrence Study, a drinking water plant that used chloramines (and no pre-chlorination) was found to produce the highest levels of iodo-THMs (35). It is likely that significant reductions in THMs, bromo-acids, and other halogencontaining DBPs could be achieved if chlorine dioxide was
applied before chlorine or used in the place of chlorine in the anti-algae pretreatment process. Although dibromoacetic acid appears to be formed mostly by pre-chlorination, laboratory chlorine dioxide treatments also show that chlorine dioxide may contribute (to a lesser extent) to its formation. For example, source waters treated with pure laboratory-generated chlorine dioxide yielded 2.5 and 1.2 µg/L of dibromoacetic acid, respectively, in September and November 1999 (Table 2 and Supporting Information). The corresponding chlorinated analogue, dichloroacetic acid, has been reported as a chlorine dioxide DBP in a previous study by Colclough et al. (36), in which pure chlorine dioxide was reacted in a carefully controlled laboratory study with natural aquatic fulvic acids. Seasonal variations in DBPs were also observed. Higher levels of THMs and HAAs were observed in the warmer months (May, September, and July) as compared to the colder months (December and November) (Table 2 and Supporting Information). For example, bromoform levels formed by prechlorination were 57, 59, and 55 µg/L, respectively, for Samplings 2, 3, and 5 (May, September, and July). By comparison, bromoform levels were 23.3 and 44 µg/L for Sampling 1 and 4 (December and November). Qualitatively, a larger number of brominated DBPs was also observed in drinking water treated in those warmer months (Table 1 and Supporting Information). This can be attributed to both the higher temperature (increased reaction rate of disinfectant species with natural organic matter) and to the presence of Peridinium algae blooms that occur in the spring, summer, and fall months (which contribute to increased levels of natural organic matter). In this study, several new brominated acids were identified that have not been previously reported in drinking water (Table 1). Figure 2 shows a representative GC/MS chromatogram containing new brominated acids (a methylated drinking water extract). The new bromo-acids identified include acids such as 2-bromobutanoic acid, 3,3-dibromopropenoic acid, tribromopropenoic acid, and trans-2,3dibromobutenedioic acid, representing mono-, di-, and tribrominated species and mono- and di-acid species. Figure 3 shows example spectra of cis-2,3-dibromopropenoic acid methyl ester and tribromopropenoic acid methyl ester. Table 3 lists key mass spectral ions for the bromo-acids that are not present in the library databases. One of the more unusual bromo-acids is 3,3-dibromo-4-oxopentanoic acid. A related compound, 5,5,5-trichloro-4-oxopentanoic acid has been reported previously in drinking water treated with chlorine VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3787
FIGURE 3. Mass spectra of DBPs identified as cis-2,3-dibromopropenoic acid methyl ester and tribromopropenoic acid methyl ester.
TABLE 3. Important Mass Spectral Ions for New Bromo-Acids (in Methyl Ester Form) MW
important MS ions (m/z, relative abundance)
2-bromobutanoic acid methyl ester trans-4-bromo-2-butenoic acid methyl ester cis-4-bromo-2-butenoic acid methyl ester 2,2-dibromopropanoic acid methyl ester
180 178 178 244
3,3-dibromopropenoic acid methyl ester
242
trans-2,3-dibromopropenoic acid methyl ester cis-2,3-dibromopropenoic acid methyl ester 2,2-dibromobutanoic acid methyl ester trans-2,3-dibromo-2-butenoic acid methyl ester 3,3-dibromo-4-oxopentanoic acid methyl ester
242 242 258 256 286
tribromopropenoic acid methyl ester
320
59(100), 101(50), 121(20), 123(20), 152(50), 154(50), 180(0.1), 182(0.1) 85(80), 99(50), 119(40), 121(40), 147(60), 149(65), 178(30), 180(32) 99(40), 119(25), 121(25), 147(60), 149(55), 178(65), 180(60) 43(50), 165(100), 167(98), 185(45), 187(80), 189(42), 244(0.2), 246(0.4), 248(0.2) 183(0.8), 185(1.5), 187(0.8), 211(50), 213(100), 215(50), 242(1.2), 244(3), 246(1.2) 163(100), 165(95), 211(45), 213(90), 215(44), 242(25), 244(50), 246(24) 163(100), 165(95), 211(40), 213(80), 215(38), 242(20), 244(40), 246(19) 179(73), 181(72), 199(12), 201(24), 203(12), 258(0.5), 260(1), 262(0.5) 177(98), 179(100), 225(26), 227(50), 229(25), 256(15), 258(10), 260(14) 43(100), 244(11), 246(22), 248(11), 255(4), 257(10), 259(4), 286(3), 288(6), 290(3) 241(50), 243(100), 245(49), 289(25), 291(70), 293(68), 295(24), 320(10), 322(28), 324(30), 326(10) 59(80), 191(96), 193(100), 222(15), 224(15) 59(70), 176(16), 177(15), 178(17), 179(14), 204(98), 205(40), 206(100), 207(38), 236(10), 238(10) 59(55), 221(35), 223(34), 269(46), 271(100), 273(45), 300(20), 302(38), 304(18)
compound
trans-2-bromobutenedioic acid dimethyl ester 222 cis-2-bromo-3-methylbutenedioic acid dimethyl ester 236 trans-2,3-dibromobutenedioic acid dimethyl ester
300
(37). Correspondingly, chloro-propenoic, -butenoic, -butanoic, and -butenedioic acids have also been reported in chlorinated drinking water samples (37). Therefore, most of the new bromo-acids reported here are related to chloroacids previously found to be DBPs. Elevated levels of bromide in the source water are responsible for the dominance of these brominated species in these drinking waters. In addition, we have also recently observed most of these bromoacids in waters in the United States treated with ozone-postchlorine, as part of a recently completed U. S. Nationwide DBP Occurrence Study (35, 38), and we have also seen a number of these bromo-acids in a study involving the pilotplant treatment of bromine enriched waters (from Ohio) with either chlorine or ozone-chlorine (39). Thus, the formation of these bromo-acids is not unique to the high bromide source waters of Israel; they are likely to be formed in many other chlorinated (or ozone-chlorinated) waters that contain relatively high ambient bromide levels. In the pilot plant samples treated with pre-ozone followed by chlorine, the use of pre-ozonation appeared to actually increase the formation of the bromo-acids (39). 3788
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 17, 2003
A particularly interesting brominated DBP identified was 2,3,5-tribromopyrrole. To our knowledge, this is the first time that a halogenated pyrrole has been observed as a drinking water DBP (for any disinfectant). It was found in samples treated with chlorine dioxide-chlorine and chlorine dioxidechloramine. A trace was also found in the laboratory chlorine dioxide treatment. However, it appears that the combination of chlorine dioxide and chloramines or chlorine may play a more important role in its formation. To quantify tribromopyrrole in the water samples it was synthesized, as described in Supporting Information. According to the chemical preference (40), 2,3,5-tribromopyrrole was obtained in 98% while the 2,3,4 isomer was obtained in 2% only. The synthetic product was used to prepare a calibration curve and to quantify it in a special sampling conducted in January 2002 (in waters treated with chlorine dioxide-chloramine) at 0.05 µg/L. Comparisons to previously analyzed internal standards allowed an estimation for the concentration of 2,3,5-tribromopyrrole in the July 2001 sampling, revealing it to be approximately the same order of magnitude in the finished drinking water.
FIGURE 4. Mass spectrum of DBP identified as 2,3,5-tribromopyrrole. The mass spectrum of 2,3,5-tribromopyrrole is shown in Figure 4. Its identification will serve to illustrate the procedure used to identify other DBPs in this study. In the case of 2,3,5tribromopyrrole, a library match of its spectrum was obtained with the Wiley database. It was not present in the NIST library database. In contrast, most of the DBPs identified in this study were not present in any of the spectral libraries, which necessitated additional manual interpretation of other spectra. If this assignment is correct, then the ion clusters at m/z 301/303/305/307, 222/224/226, 195/197/199, 116/ 118, and 79/81 would represent (1) the molecular ion, (2) loss of Br from the molecular ion, (3) loss of the NHCBr group, (4) loss of Br from the m/z 195/197/199 ion, and (5) the Br+ ion, respectively. High-resolution EIMS confirmed the empirical formula of C4H2NBr3 for the molecular ion with an exact mass of 300.7703 (within 0.003 Da of the theoretical value). Its structure was further confirmed through a match of the mass spectrum and GC retention time with an authentic standard. Another brominated DBP of interest was 1,1,3,3-tetrabromopropanone, which was observed in three of the drinking water samplings. Its chlorinated analogues1,1,3,3tetrachloropropanoneshad been found as one of only two chlorine-containing DBPs identified in a previous chlorine dioxide DBP study in the U. S. where there was low-bromide source water (13). When source waters (Sea of Galilee) were treated with pure, laboratory-generated chlorine dioxide (Sampling 4), 1,1,3,3-tetrabromopropanone was observed; therefore, results indicate that chlorine dioxide plays a role in its formation. 1,1,3,3-Tetrabromopropanone has also been found as a DBP from ozone-chloramine treatment (4, 20). Nonhalogenated DBPs. A few nonhalogenated aldehydes and ketones were also formed, including formaldehyde, acetaldehyde, glyoxal, methylglyoxal, and acetone. These
carbonyl compounds are typically observed as DBPs of ozone, however, it is clear that chlorine dioxide also produces them. Previous studies have also indicated that chlorine dioxide can produce aldehydes as DBPs (41, 42). They were observed not only in samples from the larger full-scale treatment plant that uses chlorine dioxide-chloramine, but were also found at the smaller treatment plant that uses chlorine dioxide only and in the water treated in the laboratory with pure chlorine dioxide (Tables 1 and 2 and Supporting Information). For example, formaldehyde, acetaldehyde, glyoxal, and methyl glyoxal were found at levels of 16, 3.5, 4.2, and 4.2 µg/L, respectively, in source waters treated with pure chlorine dioxide in the laboratory (Table 2). PFBHA derivatization with GC/MS was used to qualitatively identify the aldehydes and ketones. PFBHA derivatization was also used to quantify four of these carbonyl species (Table 2 and Supporting Information). 2,4-Dinitrophenylhydrazine derivatization with liquid chromatography (LC)/electrospray ionization (ESI)MS detection was also used to try to uncover additional highly polar DBPs (43); however, for these samples, LC/MS did not reveal any new polar DBPs that were not also found with the PFBHA-GC/MS procedure. Surprisingly, no nonhalogenated carboxylic acids were formed as DBPs in any of the samples from Israel. There were several carboxylic acids present in the water samples, but they were also found at the same levels as in the raw, untreated water. Thus, they were not judged to be DBPs. In our previous study of chlorine dioxide treated drinking water in Evansville, IN, the majority of the DBPs formed were nonhalogenated carboxylic acids (13). Other studies of chlorine dioxide treated drinking water and studies involving the reaction of chlorine dioxide with humic material have also reported the formation of carboxylic acids (37), so this result was surprising for these samples. This may be due to VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3789
TABLE 4. Inorganic DBPs Quantified, µg/L Sampling 1 (Dec 98) DBP
raw
Cl2
ClO2 + NH2Cl
chlorite chlorate bromate
e5.0 e5.0 e0.20
e5.0 e5.0 e0.20
340 36.1 0.41
Sampling 3 (Sept 99)
reservoir 42 h later
ClO2 small plant
raw
Cl2
ClO2 + Cl2
reservoir 11 h later
ClO2 small plant
376 49.6 0.52
577 52.1 e0.20
e5.0 e5.0 e0.2
e5.0 e5.0 3.15
110 58 0.42
66 64 0.42
190 85 e0.2
differences in the natural organic matter present in the Sea of Galilee or due to the high bromide content. Finally, in the first sampling taken in the winter of 1998, a host of bromine-containing aromatic compounds was observed in water treated with chlorine dioxide and also water treated with chlorine dioxide-chloramine (Supporting Information) (44). These brominated compounds were not seen in the raw water blanks. However, because of their unusual nature and because they were not present in any of the later samplings, we investigated the possibility that they could be reaction products with the resin material, and not true DBPs. We tested this by passing a mixture of the active oxidants and other chemicals in purified water over new XAD-8/2 resins (from a new jar that had not been subsequently cleaned by Soxhlet extraction), and eluted with ethyl acetate, as was done in the winter 1998 sampling. The chemicals included ammonium chloride and chlorine bleach (to make chloramines), sodium chlorite, sodium bromide, potassium bromate, and sodium chlorate (active chlorine dioxide was not included because it was measured at 0.0 ppm at the time of the sampling). The resulting GC/MS analysis of this sample did show the presence of most of these brominated aromatic species observed in the drinking water sampling of winter 1998. Therefore, we believe these brominated aromatics are artifacts, and not true DBPs. The brominated aromatics are likely formed by the reaction of the active oxidant species with styrene and other resin impurities that are present in new resin samples. No other brominated species, besides these brominated aromatics, were found to be resin artifacts. It is not apparent why the extensive Soxhlet extraction cleaning did not sufficiently remove these impurities from the resins in the first sampling; we have subsequently observed resin impurities in at least one other study where the resins had also been pre-cleaned. Inorganic DBPs. Inorganic DBPs identified included chlorite, chlorate, and bromate (Table 4). Chlorite and chlorate are well documented inorganic DBPs from chlorine dioxide treatment, and chlorite is currently regulated at 1.0 mg/L in the United States under the Stage 1 Disinfectants (D)/DBP Rule (45). Bromate is primarily viewed as a DBP from the ozonation of waters containing natural bromide, but it has been shown to form under unusual circumstances when chlorine dioxide disinfection is carried out in the presence of sunlight (46). Bromate has been shown to cause cancer in laboratory animals (47) and is currently regulated at 10 µg/L in the United States (45). In our samplings, the initial anti-algae pre-chlorination did not produce any chlorite or chlorate; these were clearly the result of treatment with chlorine dioxide (Table 4). In at least one case (Sampling 1), levels of chlorite exceeded the Israel maximum contaminant level (MCL) of 500 µg/L. On the other hand, bromate was found in waters treated with chlorine, and not in those treated by chlorine dioxide. The highest level observed was 3.15 µg/L, which was found in Sampling 3 (Table 4). Minor concentrations of bromate (0.4-0.5 µg/L) were observed when chlorine dioxide was used together with chloramines, but the presence of bromate is likely due to use of chlorine to generate chloramines. DBPs Formed by Natural Fulvic/Humic Acids. A separate, controlled laboratory study was conducted to quantitatively 3790
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 17, 2003
FIGURE 5. Formation of THMs from natural isolated fulvic and humic acids. determine the contribution of fulvic acids and humic acids (from isolated natural organic matter in the Sea of Galilee) as precursor material to several of the DBPs identified. For this work, fulvic acid and humic acid were isolated from the Sea of Galilee (from water and sediment, respectively) and reacted with chlorine or chlorine dioxide in the laboratory using “synthetic water” that had the same alkalinity, hardness, ionic strength, and pH as the Sea of Galilee, but 5 times the fulvic/humic content and bromide level to allow enhanced DBP formation (see Materials and Methods Section for details). THMs, HAAs, aldehydes, and 2,3,5-tribromopyrrole were quantified in this study. Results show that fulvic acid is the primary precursor for the formation of bromoform, chloroform, and dibromoacetic acid (levels of 300, 47, and 28 µg/L, respectively, when reacted with chlorine), whereas formaldehyde shows a somewhat greater contribution from humic acid (Figures 5, 6, and 7). Results for other DBPs indicate that both fulvic and humic acid precursors give rise to their formation. However, when the smaller percentage contribution of humic acid is considered for the natural source water (humic acid content is only 15% of the total humic content of the Sea of Galilee; fulvic content is 85%), humic acid probably plays a very small role in the formation of many of the THMs, HAAs, and aldehydes. On the other hand, 2,3,5-tribromopyrrole appears to be produced primarily by humic acid, and particularly when treated with a mixture of chlorine dioxide and chlorine (measured at a concentration of 0.04 µg/L for humic acid treatment, and 0.007 µg/L for fulvic acid treatment). It is interesting to note that a soil humic model proposed by Schulten and Schnitzer (48) that was based on 13C NMR, pyrolysis, and oxidative degradation data, includes a pyrrole group in its structure. In addition, the elementary analysis (C, H, N, X) for these natural humic and fulvic acids shows a greater contribution from N in the humic acid as compared to that in the fulvic acid. The finding of 2,3,5-tribromopyrrole at significant levels only when humic acid was reacted with a mixture of both chlorine dioxide and chlorine supports the observation of 2,3,5-tribromopyrrole in full-scale plant treatments involving combinations of chlorine dioxide and
FIGURE 6. Formation of HAAs from natural isolated fulvic and humic acids (DBAA ) dibromoacetic acid, BAA ) bromoacetic acid, BCAA ) bromochloroacetic acid, TCAA ) trichloroacetic acid, and DCAA ) dichloroacetic acid).
FIGURE 7. Formation of aldehydes from natural isolated fulvic and humic acids. chlorine (Samplings 3 and 5) or chlorine dioxide and chloramines (Sampling 4), as well as findings from a controlled laboratory reaction of chlorine dioxide and chlorine (Sampling 5) with Sea of Galilee source water (Table 1 and Supporting Information). In none of the samplings was 2,3,5-tribromopyrrole observed in pre-chlorinated waters (with chlorine treatment only). Thus, it appears that the combination of chlorine dioxide and chlorine is necessary for its formation. Cytotoxicity and Genotoxicity of 2,3,5-Tribromopyrrole. Because this represented the first time a halogenated pyrrole has been identified as a DBP, we were interested in evaluating its cytotoxicity and genotoxicity to determine whether it may have potential health risks. For this work, a mammalian cell cytotoxicity assay and a single-cell gel electrophoresis (SCGE) assay were used. The mammalian cell cytotoxicity assay provides a measure of the toxic impact of a chemical in which cells are continuously exposed throughout several cell divisions. Because standard plating methods are laborious and time-consuming, and require large amounts of sample, we developed a rapid, semiautomatic microplate-based cytotoxicity assay (22, 49). This assay assesses the ability of Chinese hamster ovary (CHO) cells (or other mammalian
FIGURE 8. Chronic cytotoxic and acute genotoxic responses of CHO cells to 2,3,5-tribromopyrrole (TBP). (A) Cytotoxicity concentration-response curve of CHO cells after exposure to TBP for 72 h. (B) Concentration-response curves illustrating acute genomic DNA damage induced by TBP. The gray boxes represent the induction of the SCGE % tail DNA as a function of TBP concentration, and the open circles represent the SCGE tail moment values. The pictorial inserts in panel B illustrate fluorescent images of (top) a nucleus from a cell exposed to 350 µM TBP and (bottom) nuclei from the negative control. cells) to survive and replicate during chronic exposure to a wide concentration range of the chemical agent. On the other hand, the SCGE assay quantitatively measures genomic DNA damage, detecting single-strand and double-strand breaks, DNA cross-links, alkali-labile sites, and incomplete excision repair sites in individual nuclei. This assay is very sensitive and has a high correlation in identifying mutagens and carcinogens (21, 50, 51). In the SCGE assay, cells are exposed to the chemical agent, the nuclei are subjected to electrophoresis and stained; nuclei that contain DNA damage exhibit a characteristic “comet tail” appearance when viewed by fluorescence microscopy. The amount of DNA that migrates from the nucleus is a direct measure of DNA damage. With a computer-aided digital camera, these data can be quantified and statistically analyzed. The chronic cytotoxicity assay revealed 2,3,5-tribromopyrrole (TBP) to be a strong cytotoxin in CHO cells. Over the concentration range of 1-100 µM (with 8 to 24 replicate tests per concentration), TBP induced a significant chronic toxic effect to CHO cells at concentrations above 10 µM (F18, 221 ) 110.1; P e 0.001). The CHO cell cytotoxicity curve is shown in Figure 8A. The %C1/2 value (concentration of TBP that induced a 50% reduction in cell density as compared to the concurrent negative control) was 60.6 µM. Compared to VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3791
TABLE 5. Comparison of CHO Cell Chronic Cytotoxicity and Acute Genotoxicity of 2,3,5-Tribromopyrrole and Other DBPs/Toxic Agentsa
chemical bromoacetic acid dibromoacetic acid tribromoacetic acid chloroacetic acid 2,3,5-tribromopyrrole 3-chloro-4-(dichloro-methyl)-5hydroxy-2[5H]-furanone (MX) potassium bromate ethylmethanesulfonate
chronic cytotoxicity %C1/2 (mM)
SCGE genotoxic potency (mM)
0.009 0.500 1.000 0.944 0.061 0.275
0.017 1.756 2.456 0.411 0.299 0.244
0.963 4.250
7.195 6.057
a Data are from Plewa et al. (22) except for that of 2,3,5-tribromopyrrole; %C1/2 is the concentration of the chemical that induces 50% of the cell density as compared to the negative control; SCGE genotoxic potency is the concentration of the chemical at the midpoint of the SCGE tail moment concentration-response curve.
other DBPs, TBP was approximately 8×, 4.5×, and 16× more cytotoxic than dibromoacetic acid, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), or potassium bromate, respectively (Table 5). Ethylmethanesulfonate was used as the positive control in these studies and TBP was nearly 70× more cytotoxic than this positive control to CHO cells. Correspondingly, TBP induced acute genomic DNA damage to CHO cells in a concentration-dependent manner in the SCGE genotoxicity assay (Figure 8B). The number of replicate SCGE slides ranged from 2 to 8 for each of the 11 TBP concentrations analyzed. The acute cytotoxicity of the exposed CHO cells ranged from 100 to 93% viable cells. After a 4-h exposure to a concentration range of 10-350 µM, a significant increase in the amount of genomic DNA migration was observed (F11,45 ) 89.2; P e 0.001). At concentrations above 150 µM, the SCGE % tail DNA values increased significantly. The SCGE tail moment measurement also showed a significant concentration-dependent increase in genomic DNA damage (F11,45 ) 83.4; P e 0.001) with TBP concentrations above 250 µM. The midpoint of the concentration-response curve is used to represent the SCGE genotoxic potency; regression analysis of the tail moment data indicated that the genotoxic potency was 299 µM (r2 ) 0.96) for TBP. This genotoxicity is similar to that of MX and more than 20× more genotoxic than the positive control, EMS. In conclusion, the elevated bromide levels in the water from Israel contributed to the formation of many brominated DBPs, including a new halogenated pyrrole DBP, 2,3,5tribromopyrrole, which was shown to be strongly cytotoxic and genotoxic to mammalian cells. Very few chlorinecontaining DBPs were observed. Bromoform and dibromoacetic acid were the dominant DBPs observed, and a number of other interesting brominated compounds were identified. Iodo-THMs were also found, but no iodo-HAAs were observed. Most of the THMs, HAAs, and other brominecontaining DBPs observed were produced during prechlorination at the initial reservoir. The exceptions were the iodo-THMs and 2,3,5-tribromopyrrole that were formed predominantly by chlorine dioxide-chloramine or chlorine dioxide-chlorine treatment. It is likely that significant reductions in THMs, HAAs, and other bromine-containing DBPs could be achieved if chlorine dioxide was applied before chlorine or used in the place of chlorine in the anti-algae pretreatment. A few nonhalogenated DBPs were formed, including formaldehyde, acetaldehyde, glyoxal, methyl glyoxal, acetone, and 2-hexanone, which are traditionally found as ozonation 3792
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 17, 2003
byproducts. These were formed not only at the larger plant that uses chlorine dioxide-chloramine treatment, but were also formed in water from the smaller plant that uses chlorine dioxide only and in the laboratory chlorine dioxide treated samples. No nonhalogenated carboxylic acids were found, which was surprising, given that they were the predominant DBPs produced in an earlier study of chlorine dioxide DBPs in the U. S. (13). Results of laboratory reactions with isolated fulvic and humic acid from the Sea of Galilee showed that fulvic acid plays a greater role in the formation of THMs, haloacetic acids, and aldehydes, but 2,3,5-tribromopyrrole is produced primarily from humic acid.
Acknowledgments We thank Gene Crumley and Terrance Floyd at the U.S. EPA National Exposure Research Laboratory for their assistance with the extractions, derivatizations, and analyses. We gratefully acknowledge Dan Hautman and Herb Brass of the U.S. EPA’s Office of Groundwater and Drinking Water in Cincinnati, OH, for providing quantitative inorganic analyses. We would like to thank Francesc Ventura of AGBAR (Barcelona, Spain) for sharing initial standards of iodo-THMs and synthesis methods with us. We would also like to thank Dr. Hilla Ben-David and Dr. Orna Dreazen, the present and former Directors of the Public Health Laboratories in TelAviv, for their encouragement and support, as well as Dr. Danielle Perl-Treves for her assistance with aldehyde analyses. And, last but not least, we acknowledge Mekorot Ltd.s the Water Company of Israelsfor supporting the samplings at various stations along the National Water Carrier, and especially Engineer Alon Tarkenitz of this company, for his important advice and assistance. This paper has been reviewed in accordance with the U.S. Environmental Protection Agency’s peer and administrative review policies and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the U.S. EPA.
Supporting Information Available Synthesis of chemical standards, structures of halogenated aromatic compounds observed, table of disinfectant doses and residual disinfectant levels for drinking water treatment, table of source water quality characteristics, comprehensive list of DBPs identified, and DBP concentrations for samplings 1 and 4. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Rav-Acha, Ch. Transformation of Pollutants by Chlorine Dioxide in Water. In The Handbook of Environmental Chemistry; Hutzinger, O., Ed.; Springer-Verlag: New York, 1998; Vol. V, p 62. (2) Rook, J. J. Water Treat. Exam. 1974, 23 (2), 234. (3) Rav-Acha, Ch.; Choshen, E.; Serri, A.; Limoni, B. Environ. Pollut. B 1985, 10, 47. (4) Richardson, S. D.; Thruston, A. D., Jr.; Caughran, T. V.; Chen, P. H.; Collette, T. W.; Floyd, T. L.; Schenck, K. M.; Lykins, B. W., Jr. Environ. Sci. Technol. 1999, 33, 3378. (5) Shukairy, H. M.; Miltner, R. J.; Summers, R. S. J. Am. Water Works Assoc. 1994, 86 (6), 72. (6) Rook, J. J.; Gras, A. A.; van der Heijden, B. G.; de Wee, J. J. Environ. Sci. Health 1978, A13, 91. (7) Cooper, W. J.; Zika, R. G.; Steinhauer, M. S. J. Am. Water Works Assoc. 1985, 77 (4), 116. (8) Krasner, S. W.; McGuire, M. J.; Jacangelo, J. G.; Patania, N. J.; Reagan, K. M.; Aieta, E. M. J. Am. Water Works Assoc. 1989, 81, 41. (9) Disinfectants and Disinfectant Byproducts, Environmental Health Criteria 216. International Programme on Chemical Safety, World Health Organization: Geneva, 2000. (10) Masschelein, W. J. In Chlorine Dioxide: Chemistry and Environmental Impact of Oxychlorine Compounds; Rice, R. G., Ed.; Ann Arbor Science: Ann Arbor, MI, 1979.
(11) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Technol. 1981, 15, 463. (12) De Serra, M. O.; Schnitzer, M. Can. J. Soil Sci. 1972, 52, 365. (13) Richardson, S. D.; Thruston, A. D., Jr.; Collette, T. W.; Patterson, K. S.; Lykins, B. W., Jr.; Majetich, G.; Zhang, Y. Environ. Sci. Technol. 1994, 28, 592. (14) Methods for the Determination of Organic and Inorganic Compounds in Drinking Water, Vol. 1; Technical Support Center, Office of Groundwater and Drinking Water and the National Exposure Research Laboratory, Office of Research and Development, U. S. EPA: Cincinnati, OH, August 2000; EPA/815-R00-014. (15) Hautman, D. P. Proceedings of the American Water Works Association Water Quality Technology Conference; American Water Works Association: Denver, CO, 1992. (16) Methods for the Determination of Organic Compounds in Drinking Water, Supplement 1; Environmental Monitoring Systems Laboratory, Office of Research and Development, U. S. EPA: Cincinnati, OH, July 1990; EPA/600/4-90020. (17) Gilow, H. M.; Burton, D. E. J. Org. Chem. 1981, 46, 2221. (18) Sclimenti, M. J., Krasner, S. W., Glaze, W. H., and Weinberg, H. S. Proceedings of the American Water Works Association Water Quality Technology Conference; American Water Works Association: Denver, CO, 1990. (19) Kanniganti, R.; Johnson, J. D.; Ball, L. M.; Charles, M. J. Environ. Sci. Technol. 1992, 26, 1998. (20) Richardson, S. D.; Thruston, A. D., Jr.; Caughran, T. V.; Chen, P. H.; Collette, T. W.; Floyd, T. L.; Schenck, K. M.; Lykins, B. W., Jr. Environ. Sci. Technol. 1999, 33, 3368. (21) Wagner, E. D.; Rayburn, A. L.; Anderson, D.; Plewa, M. J. Environ. Mol. Mutagen. 1998, 32, 360. (22) Plewa, M. J.; Kargalioglu, Y.; Vankerk, D.; Minear, R. A.; Wagner, E. D. Environ. Mol. Mutagen. 2002, 40, 134. (23) Israel Geological Institute, personal communication. (24) Brass, H. J.; Feige, M. A.; Halloran, T.; Mello, J. W.; Munch, D.; Thomas, R. F. The National Organic Monitoring Survey: Samplings and Analyses for Purgeable Organic Compounds. In Drinking Water Quality Enhancement Through Source Protection; Pojasek, R. B., Ed.; Ann Arbor Science: Ann Arbor, MI, 1977; p 393. (25) Thomas, R. F.; Weisner, M. J.; Brass, H. J. The Fifth Trihalomethane: Dichloroiodomethane, Its Stability and Occurrences in Chlorinated Drinking Water. In Water Chlorination Environmental Impact and Health Effects; Jolley, R. J., Brungs, W. A., Cumming, R. B., Eds.; Ann Arbor Science: Ann Arbor, MI, 1980; Vol. 3, p 161. (26) Brass, H. J.; Kingsley, B. A. Quality Assurance Programs in the Analysis of Trace Organic Contaminants. In Organic Carcinogens in Drinking Water; Ram, N. M., Calabrese, E., Christman, R. F., Eds.; John Wiley & Sons: New York, 1986; p. 173. (27) Bichsel, Y.; von Gunten, U. Environ. Sci. Technol. 2000, 34 (13), 2784. (28) Cancho, B.; Ventura, F.; Galceran, M.; Diaz, A.; Ricart, S. Water Res. 2000, 34 (13), 3380. (29) Coleman, W. H.; Lingg, R. D.; Melton, R. G.; Klopfler, F. C. The Occurrence of Volatile Organics in Five Drinking Water Supplies Using Gas Chromatography-Mass Spectrometry. In Identification and Analysis of Organic Pollutants in Water; Keith, L. H., Ed.; Ann Arbor Science: Ann Arbor, MI, 1976; p. 305. (30) Glaze, W. H.; Henderson, J. E.; Smith, G. Analysis of New Chlorinated Organic Compounds Formed by Chlorination of Municipal Wastewater. In Water Chlorination Environmental Impact and Health Effects; Jolley, R. J., Ed.; Ann Arbor Science: Ann Arbor, MI. 1975; Vol. 1, p 139. (31) Hanai, Y., Saitou, A., Mio, N. Bull. Inst. Environ. Sci. Technol. Yokohama Natl. Univ. 1996, 22, 11-16.
(32) Hansson, R. C.; Henderson, M. J.; Jack, P.; Taylor, R. D. Water Res. 1987, 21, 1265. (33) Kleopfer, R. D. Analysis of drinking water for organic compounds. In Identification and Analysis of Organic Pollutants in Water; Keith, L. H., Ed.; Ann Arbor Science: Ann Arbor, MI, 1976; p. 305. (34) Heller-Grossman, L.; Manka, J.; Limoni-Releis, B.; Rebhun, M. Water Res. 1993, 27 (8), 1323. (35) Weinberg, H. S.; Krasner, S. W.; Richardson, S. D.; Thruston, A. D., Jr. The Occurrence of Disinfection By-Products (DBPs) of Health Concern in Drinking Water: Results of a Nationwide DBP Occurrence Study; U. S. Environmental Protection Agency, National Exposure Research Laboratory, Athens, GA, 2002; EPA/ 600/R-02/068. (36) Colclough, C. A.; Johnson, J. D.; Christman, R. F.; Millington, D. S. In Water Chlorination: Environmental Impact and Health Effects; Jolley, R. L., Brungs, W. A., Cotruvo, J. A., Cumming, R. B., Mattice, J. S., Jacobs, V. A., Eds.; Ann Arbor Science: Ann Arbor, MI, 1983; Vol. 4, Chapter 15. (37) Richardson, S. D. Drinking Water Disinfection Byproducts. In The Encyclopedia of Environmental Analysis and Remediation; Meyers, R. A., Ed.; John Wiley & Sons: New York, 1998; Vol. 3, p 1398. (38) Krasner, S. E.; Pastor, S.; Chinn, R.; Sclimenti, M. J.; Weinberg, H. S.; Richardson, S. D.; Thruston, A. D., Jr. Proceedings of the American Water Works Association Water Quality Technology Conference; American Water Works Association: Denver, CO, 2001. (39) Simmons, J. E.; Richardson, S. D.; Schenck, K. M.; Speth, T. F.; Miltner, R. J. Environ. Mol. Mutagen. 2002, 39 (Suppl. 33), 59. (40) Jones, R. A.; Bean, G. P. The Chemistry of Pyrroles. Academic Press: London, 1977. (41) Rav-Acha, Ch.; Choshen, E.; Sarel, S. Helv. Chim. Acta 1986, 69, 1728. (42) Rav-Acha, Ch.; Choshen, E. Environ. Sci. Technol. 1987, 21, 1069. (43) Richardson, S. D.; Caughran, T. V.; Poiger, T.; Guo, Y.; Crumley, F. G. Ozone Sci. Eng. 2000, 22(6), 653. (44) Richardson, S. D.; Thruston, A. D., Jr.; Caughran, T. V.; Chen, P. H.; Collette, T. W.; Schenck, K. M.; Lykins, B. W., Jr.; RavAcha, Ch.; Glezer, V. Water Air Soil Pollut. 2000, 123 (1), 95. (45) National Primary Drinking Water Regulations: Disinfectants and Disinfection Byproducts; Final Rule. Fed. Regist. 16 Dec 1998, 63 (241), 69389. (46) Gordon, G.; Emmert, G. L.; Bubnis, B. Proceedings of the American Water Works Association Water Quality Technology Conference; American Water Works Association: Denver, CO, 1995; Part 2, p 1985. (47) Kurokawa, Y.; Hayashi, Y.; Maekawa, A.; Takahashi, M.; Kokubo, T.; Odashima, S. J. Natl. Cancer Inst. 1983, 71, 965. (48) Schulten, H.-R.; Schnitzer, M. Naturwissenschaften 1993, 80, 29. (49) Plewa, M. J.; Kargalioglu, Y.; Vankerk, D.; Minear, R. A.; Wagner, E. D. Water Sci. Technol. 2000, 42, 109. (50) Anderson, D.; Yu, T.-W.; McGregor, D. B. Mutagenesis 1998, 13, 539. (51) Tice, R. R.; Agurell, E.; Anderson, D.; Burlinson, B.; Hartmann, A.; Kobayashi, H.; Miyamae, Y.; Rojas, E.; Ryu, J.-C.; Sasaki, Y. F. Environ. Mol. Mutagen. 2000, 35, 206.
Received for review January 28, 2003. Revised manuscript received April 23, 2003. Accepted June 9, 2003. ES030339W
VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3793
