Environ. Sci. Technol. 2008, 42, 955–961
Occurrence, Synthesis, and Mammalian Cell Cytotoxicity and Genotoxicity of Haloacetamides: An Emerging Class of Nitrogenous Drinking Water Disinfection Byproducts M I C H A E L J . P L E W A , * ,† MARK G. MUELLNER,† SUSAN D. RICHARDSON,‡ F R A N C E S C A F A S A N O , ‡,⊥ KATHERINE M. BUETTNER,‡ YIN-TAK WOO,§ A. BRUCE MCKAGUE,| AND ELIZABETH D. WAGNER† College of Agricultural, Consumer and Environmental Sciences, Department of Crop Sciences, University of Illinois at Urbana–Champaign, 1101 West Peabody Drive, Urbana, Illinois 61801, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30605, Risk Assessment Division, Office of Pollution Prevention and Toxics, U.S. Environmental Protection Agency, Washington, DC 20460, and CanSyn Chemical Corp., 200 College Street, Toronto, Canada M5S 3E5
Received July 16, 2007. Revised manuscript received October 24, 2007. Accepted October 29, 2007.
The haloacetamides, a class of emerging nitrogenous drinking water disinfection byproduct (DBPs), were analyzed for their chronic cytotoxicity and for the induction of genomic DNA damage in Chinese hamster ovary cells. The rank order for cytotoxicity of 13 haloacetamides was DIAcAm > IAcAm > BAcAm > TBAcAm > BIAcAm > DBCAcAm > CIAcAm > BDCAcAm > DBAcAm > BCAcAm > CAcAm > DCAcAm > TCAcAm. The rank order of their genotoxicity was TBAcAm > DIAcAm ≈ IAcAm > BAcAm > DBCAcAm > BIAcAm > BDCAcAm > CIAcAm > BCAcAm > DBAcAm > CAcAm > TCAcAm. DCAcAm was not genotoxic. Cytotoxicity and genotoxicity were primarily determined by the leaving tendency of the halogens and followed the order I > Br >> Cl. With the exception of brominated trihaloacetamides, most of the toxicity rank order was consistent with structure–activity relationship expectations. For di- and trihaloacetamides, the presence of at least one good leaving halogen group (I or Br but not Cl) appears to be critical for significant toxic activity. Log P was not a factor for monohaloacetamides but may play a role in the genotoxicity of trihaloacetamides and possible activation of dihaloacetamides by intracellular GSH and -SH compounds. * Corresponding author phone: (217) 333-3614; e-mail mplewa@ uiuc.edu. † University of Illinois at Urbana–Champaign. ‡ National Exposure Research Laboratory, U.S. Environmental Protection Agency. § Office of Pollution Prevention and Toxics, U.S. Environmental Protection Agency. | CanSyn Chemical Corp. ⊥ Currently at the University of Torino, Torino, Italy 10125. 10.1021/es071754h CCC: $40.75
Published on Web 12/12/2007
2008 American Chemical Society
With the advent of the U.S. EPA Stage 2 DBP regulations, water utilities are considering the use of disinfectants that are alternatives to chlorine. The use of these alternative disinfectants will shift the distribution of DBP chemical classes. The emergence of new, highly toxic iodinated, nitrogenous DBPs, as illustrated by the discovery of bromoiodoacetamide as a new DBP, underscores the importance of comparative toxicity studies to assist in the overall goal of safer drinking water practice.
Introduction When a disinfectant reacts with natural organic matter and/ or bromide/iodide in raw water, drinking water disinfection byproducts (DBPs) are unintentionally formed (1). Although the acute benefits of drinking water disinfection are universally acknowledged, there is concern about adverse health risks due to long-term DBP exposure (2, 3). Epidemiology studies provide moderate evidence for DBP associations with adverse pregnancy outcomes (4–8); specific DBPs are mutagens, carcinogens, teratogens, or developmental toxicants (9–18). In 2006, the U.S. Environmental Protection Agency (EPA) promulgated the Stage 2 Disinfectants/Disinfection Byproducts Rule (19). In order to comply with this Rule, a number of utilities are switching from chlorine to chloramine disinfection; this may increase nitrogenous disinfection byproducts (N-DBPs), such as acetamides. N-DBPs were cited as research priorities by the U.S. EPA (20, 21) and in a recent review on the occurrence, toxicity, and research priorities on emerging DBPs (22). Acetamides have been identified as DBPs from drinking water treatment plants and from laboratory studies (23–26). Haloamides were recently quantified in a Nationwide Occurrence Study of priority, unregulated DBPs (21). Chloro-, bromo-, dichloro-, dibromo-, and trichloroacetamide were found in finished drinking water from several locations (maximum of 14 µg/L) from water treated with chlorine dioxide-chlorine-chloramines. As with the formation of haloacetonitriles (27), there is preliminary evidence that chloramination may increase their formation. Because nitriles can hydrolyze to form haloamides (28, 29), it is possible that the haloamides are hydrolysis products of the corresponding haloacetonitriles, which are commonly found as DBPs. The analyzed haloacetamides and their abbreviations are listed in Table 1. There is little information on the genotoxicity and carcinogenicity of haloamides. These agents react with cellular protein thiols and are prototypical alkylating agents inducing a multitude of biological responses, including apoptosis and necrosis (30). IAcAm was a cocarcinogen in a mouse skin assay (31) and enhanced nitrosamide-induced tumors in rats (32, 33). The objective of our research was to quantitatively analyze and compare the chronic cytotoxicity and the acute genomic DNA damaging capacity of 13 haloacetamides using in vitro mammalian cell assays extensively used in DBP research (27, 34–38). In addition, we identified the occurrence of a new iodinated DBP, bromoiodoacetamide, in drinking water.
Materials And Methods Reagents. General reagents were purchased from Fisher Scientific Co. (Itasca, IL) and Sigma Chemical Co. (St. Louis, MO). Media and fetal bovine serum (FBS) were purchased from Hyclone Laboratories (Logan, UT). The haloacetamides were dissolved in dimethyl sulfoxide (DMSO) and stored at -22 °C in sterile glass vials. Sources and purities of the haloacetamides are presented in Table 1. VOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Characteristics of the Haloacetamides Analyzed in This Study haloacetamide
abbreviation
CAS no.
chemical formula
MWa
source and purity
iodoacetamide diiodoacetamide bromoiodoacetamide chloroiodoacetamide bromoacetamide dibromoacetamide tribromoacetamide bromochloroacetamide dibromochloroacetamide bromodichloroacetamide chloroacetamide dichloroacetamide trichloroacetamide
IAcAm DIAcAm BIAcAm CIAcAm BAcAm DBAcAm TBAcAm BCAcAm DBCAcAm BDCAcAm CAcAm DCAcAm TCAcAm
144–48–9 5875–23–0 62872–36–0 62872–35–9 683–57–8 598–70–9 594–47–8 62872–34–8 855878–13–6 98137–00–9 79–07–2 683–72–7 594–65–0
C2H4INO C2H3I2NO C2H3BrINO C2H3ClINO C2H4BrNO C2H3Br2NO C2H2Br3NO C2H3BrClNO C2H2Br2ClNO C2H2BrCl2NO C2H4ClNO C2H3Cl2NO C2H2Cl3NO
184.96 310.85 263.856 219.405 137.96 216.86 295.75 172.41 251.305 206.85 93.51 127.96 162.40
Sigma-Aldrich, >97% CanSyn Chem Corp., 99% CanSyn Chem Corp., 85%b CanSyn Chem Corp., >95% Sigma-Aldrich, 98% CanSyn Chem Corp., >95% CanSyn Chem Corp., >95% CanSyn Chem Corp., 95% CanSyn Chem Corp., >95% CanSyn Chem Corp., >95% Sigma-Aldrich, >95% Sigma-Aldrich, 98% Sigma-Aldrich, 99%
a
MW ) molecular weight.
b
7.5% each of dibromoacetamide and diiodoacetamide as impurities.
Preparation of Haloacetamides. Eight haloacetamides were synthesized. DBAcAm was prepared from ethyl dibromoacetate by reaction with ammonium hydroxide (39). BCAcAm was prepared from bromochloroacetic acid by conversion to the ethyl ester followed by reaction with ammonium hydroxide (39). BDCAcAm and DBCAcAm were prepared from the corresponding acids (40, 41) by conversion to the methyl esters with BF3/MeOH followed by reaction with ammonium hydroxide (40). TBAcAm was prepared from the acid in a similar manner. BIAcAm was similarly prepared from bromoiodoacetic acid to give colorless material, mp 181–183 °C. The purity of the product by gas chromatography (GC) using flame ionization detection was 85% and contained 7.5% each of DBAcAm and DIAcAm as impurities. CIAcAm was prepared from methyl chloroiodoacetate (42) and DIAcAm was prepared from diiodoacetic acid (43) via the methyl ester, by similar reaction with ammonium hydroxide. Chinese Hamster Ovary Cells. Chinese hamster ovary (CHO) cells, line AS52, clone 11-4-8 were used (44). We employed this cell line in previous DBP toxicity studies (27, 34–38, 45, 46). The CHO cells were maintained in Ham’s F12 medium containing 5% FBS at 37 °C in a humidified atmosphere of 5% CO2. CHO Cell Chronic Cytotoxicity Assay. This calibrated assay measures the reduction in cell density as a function of DBP concentration over a period of approximately 3 cell divisions (72 h) (27, 34–38, 45, 46). Detailed procedures with statistical analysis are presented in the Supporting Information. Within each experiment, 10 haloacetamide concentrations were analyzed with 8 replicate microplate wells for each concentration. Each experiment was repeated 2–3×. Thus, the cytotoxicity of each concentration was evaluated with 16-24 individual cell cultures. Single Cell Gel Electrophoresis Assay. Single cell gel electrophoresis (SCGE) quantitatively measures genomic DNA damage induced in individual nuclei of treated cells (47). We employed this calibrated assay to determine the genotoxicity of DBPs (27, 34–38, 45, 46). Detailed procedures and statistical analysis are presented in the Supporting Information. CHO cells were exposed to a haloacetamide for 4 h at 37 °C, 5% CO2. Each experiment consisted of a negative control, a positive control (3.8 mM ethylmethanesulfonate), and 9 haloacetamide concentrations. The concentration range was determined by the acute cytotoxicity of the haloacetamide. In general, each concentration was evaluated with 2 microgels with 25 randomly chosen nuclei per microgel. The experiments were repeated a minimum of 3× with 6 microgels per concentration. Drinking Water Analysis. Drinking water samples were collected from full-scale drinking water treatment plants in the United States that use chloramines for disinfection. One plant used chlorine for disinfection. Details regarding the 956
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drinking water treatment, concentration, and analysis can be found in the Supporting Information. GC/mass spectrometry (MS) analyses with electron ionization (EI) were carried out on an Agilent 6890 GC interfaced to a WatersMicromass Autospec II high resolution, double focusing mass spectrometer at 1000 resolution.
Results and Discussion Identification and Occurrence of a New Iodinated Acetamide. We identified BIAcAm as a DBP for the first time in drinking water from 12 treatment plants (of a total of 23 analyzed) and located in 6 U.S. states. One plant used chlorine for disinfection; 22 plants used chloramination. These plants had source waters with relatively high natural bromide and iodide levels. Three of the plants had very small amounts of BIAcAm in their raw waters, at levels 500 × lower than the finished waters. The other 9 plants only had BIAcAm in their finished waters, and not in their corresponding raw waters. Using GC with selected ion monitoring-MS, we did not detect IAcAm, CIAcAm, or DIAcAm. The EI mass spectrum of BIAcAm is shown in Figure 1. Selected ion monitoring of 5 key ions (m/z 127, 136, 138, 220, and 263) was used to identify this compound in the drinking water extracts. A match of these ions with a match of the GC retention time was used to confirm its presence. All haloamides measured expressed distinctive GC/MS chromatographic peak shapes (Figure 1, Supporting Information). This distinctive “tailing” peak shape appears to be due to surface reactions of the haloamides in the EI ion source and provided further confirmation of BIAcAm. All of the haloamides show a prominent peak at m/z 44, which represents the amide group (Figure 1). The presence of bromine and iodine is evident in the mass spectrum of BIAcAm, with 1-bromine doublets present at m/z 263/265, 220/222, and 136/138, and the presence of iodine at m/z 127 and loss of iodine at m/z 136/138. Chlorinated and brominated forms were measured in drinking water previously (21), but this is the first report of an iodinated amide. Naturally occurring bromide and iodide contribute to the formation of brominated and iodinated DBPs (25, 37, 38, 48–51). There is evidence that chloramination increases the formation of iodinated DBPs (37, 52, 53). Therefore, while this is the first report of an iodinated amide DBP, it is not surprising that iodo-amides would form in source waters with high bromide/iodide and chloramine disinfection. As mentioned earlier, it is possible that BIAcAm and other haloamides are hydrolysis products of the corresponding haloacetonitriles, which are commonly found as DBPs. The amides can undergo further hydrolysis to form carboxylic acids, but this reaction requires longer reaction times and higher temperatures than the initial conversion to the amide (29).
FIGURE 1. EI mass spectrum of bromoiodoacetamide.
FIGURE 2. Comparison of the CHO cell chronic cytotoxicity concentration–response curves of 13 haloacetamides. CHO Cell Cytotoxicity. Data from individual experiments were normalized to the averaged percent of the concurrent negative control; these data were plotted as a concentration–response curve (Figure 2). An ANOVA test was conducted with normalized data representing each microplate well. If a significant F value of P e 0.05 was obtained, a Holm-Sidak multiple comparison analysis was conducted. The power of the test statistic (1-β) was maintained at g0.8 at R ) 0.05. The lowest concentration that induced a significant cytotoxic response ranged from 25 nM (DIAcAm) to 800 µM (DCAcAm) (Table 2). Regression analysis was conducted on each concentration–response curve; the coefficient of determination (R2) ranged from 0.95 to 0.99. From these concentration–response curves, the %C½ value was calculated (Table 2). The %C½ value is the concentration that induced a cell density of 50% as compared to the concurrent negative control. The %C½ values ranged from 678 nM (DIAcAm) to 2.05 mM (TCAcAm). The rank order for cytotoxicity of the 13 haloacetamides based on their %C½ values was DIAcAm
> IAcAm > BAcAm > TBAcAm > BIAcAm > DBCAcAm > CIAcAm > BDCAcAm > DBAcAm > BCAcAm > CAcAm > DCAcAm > TCAcAm. CHO Cell Genotoxicity. Acute genomic DNA damage was measured as SCGE tail moment values. Acute cytotoxicity was determined with trypan blue vital dye; data were used within the range that contained g70% viable cells (Table 2, Figure 3). The data were plotted, and regression analysis was used to fit the curve; the coefficient of determination (R2) ranged from 0.97 to 0.99. SCGE tail moment values are not normally distributed, thus the median tail moment value for each microgel was determined and averaged. Averaged median values express normal distributions according to the Central Limit theorem (54) and were used with an ANOVA test. If a significant F value of P e 0.05 was obtained, a Holm-Sidak multiple comparison analysis was conducted (1-β g 0.8 at R ) 0.05, Table 2). The lowest concentration that induced a significant SCGE genotoxic response ranged from 25 µM for DIAcAm, BIAcAm, BAcAm, and DBCAcAm VOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Summary Comparison of the CHO Cell Chronic Cytotoxicity and Acute Genotoxicity of the Haloacetamides chemical
lowest toxic conc. (M)a
%C½ Value (M)b
R 2c
lowest genotox. conc. (M)d
iodoacetamide diiodoacetamide bromoiodoacetamide chloroiodoacetamide bromoacetamide dibromoacetamide tribromoacetamide bromochloroacetamide dibromochloroacetamide bromodichloroacetamide chloroacetamide dichloroacetamide trichloroacetamide
5.00 × 10-7 2.50 × 10-8 2.00 × 10-6 2.00 × 10-6 0.50 × 10-6 2.50 × 10-6 2.00 × 10-6 1.00 × 10-6 1.00 × 10-6 2.00 × 10-6 7.50 × 10-5 8.00 × 10-4 5.00 × 10-4
1.42 × 10-6 6.78 × 10-7 3.81 × 10-6g 5.97 × 10-6 1.89 × 10-6 1.22 × 10-5 3.14 × 10-6 1.71 × 10-5 4.75 × 10-6 8.68 × 10-6 1.48 × 10-4 1.92 × 10-3 2.05 × 10-3
0.98 0.98 0.98 0.96 0.99 0.99 0.97 0.98 0.96 0.98 0.98 0.95 0.96
3.00 × 10-5 2.50 × 10-5 2.50 × 10-5 2.00 × 10-4 2.50 × 10-5 5.00 × 10-4 3.00 × 10-5 4.00 × 10-4 2.50 × 10-5 7.50 × 10-5 7.50 × 10-4 NA 5.00 × 10-3
genotox. potency (M)e
R 2f
3.41 × 10-5 3.39 × 10-5 7.21 × 10-5h 3.02 × 10-4 3.68 × 10-5 7.44 × 10-4 3.25 × 10-5 5.83 × 10-4 6.94 × 10-5 1.46 × 10-4 1.38 × 10-3 NS >1 × 10-2 6.54 × 10-3
0.99 0.98 0.99 0.99 0.99 0.99 0.97 0.99 0.98 0.99 0.99 NA 0.98
a Lowest toxic concentration was the lowest concentration of the haloacetamide in the concentration–response curve that induced a significant reduction in cell density as compared to the negative control. b The %C1/2 value is the concentration of the haloacetamide, determined from a regression analysis of the data, that induced a cell density of 50% as compared to the concurrent negative control. c R2 is the coefficient of determination for the regression analysis upon which the %C1/2 value was calculated. d The lowest genotoxic concentration was the lowest concentration of the haloacetamide in the concentration–response curve that induced a significant amount of genomic DNA damage as compared to the negative control. e The SCGE genotoxic potency is the haloacetamide concentration that was calculated, using regression analysis, at the midpoint of the curve within the concentration range that expressed above 70% cell viability of the treated cells. f R2 is the coefficient of determination for the regression analysis upon which the genotoxic potency value was calculated. g The calculated %C1/2 value for BIAcAm alone assuming an additive model for the DIAcAm and DBAcAm contaminants was 3.35 × 10-6 M. h The calculated SCGE genotoxic potency value for BIAcAm alone assuming an additive model for the DIAcAm and DBAcAm contaminants was 1.62 × 10-5 M. NA ) not applicable; NS ) not statistically significant from the negative control.
FIGURE 3. Comparison of the CHO cell acute genotoxicity concentration–response curves of 13 haloacetamides. to 5 mM for TCAcAm. The SCGE genotoxic potency was calculated at the midpoint of the concentration–response curve, and ranged from 32.5 µM for TBAcAm to 6.5 mM for TCAcAm (Table 2). The rank order of genotoxic potency from most potent to least was TBAcAm > DIAcAm ≈ IAcAm > BAcAm > DBCAcAm > BIAcAm > BDCAcAm > CIAcAm > BCAcAm > DBAcAm > CAcAm > TCAcAm. DCAcAm was not genotoxic (Table 2). Structure–Activity Relationships (SARs) and Factors Affecting the Toxicity of Haloacetamides. The haloacetamides have or may generate a number of electrophilic reactivities: (i) for monohaloacetamides, alkylation by the SN2 reaction, inducing the displacement of a halogen atom 958
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at the R carbon, (ii) for dihaloacetamides, the potential generation of highly reactive R-halothioether electrophilic intermediates by cellular glutathione GSH or -SH compounds, (iii) for trihaloacetamides, nucleophilic attack at the electrophilic carbonyl carbon to yield trihalomethyl carbanions, which in turn, may lead to trihalomethanes as well as electrophilic dihalocarbene intermediates. In addition to chemical reactivity, the capacity to cross cell membranes is an important factor for toxicity. The logarithm of the octanol–water partition coefficient (log P) is a measure of lipophilicity which correlates with cell permeability. Log P increased with the degree of halogenation and with the size
TABLE 3. Estimated and Measured log P and Combined Toxicity Values of the Haloacetamides Studied compound iodoacetamide bromoacetamide chloroacetamide diiodoacetamide dibromoacetamide dichloroacetamide bromoiodoacetamide chloroiodoacetamide bromochloroacetamide tribromoacetamide dibromochloroacetamide bromodichloroacetamide trichloroacetamide
estimated log Pa
measured log P
monohaloacetamides -0.08 -0.19c; -0.15d -0.49 -0.52c,d -0.58 -0.53c; -0.59d dihaloacetamides 0.92 0.09 0.18d -0.09 0.19c; -0.03d 0.50 0.41 0.00 trihaloacetamides 1.10 1.01 0.92 0.83 1.04c; 0.79d
combined toxicity valueb 5.63 × 104 5.17 × 104 1.31 × 103 5.78 × 104 2.64 × 103 1.68 × 102 1.02 × 105e 6.49 × 103 3.33 × 103 5.61 × 104 2.70 × 104 1.29 × 104 2.33 × 102
a Calculated using the KOWWIN program (version 1.67) developed by U.S. EPA using the atom/fragment approach (program available at http://www.epa.gov/oppt/newchems/pubs/sustainablefutures.htm). b The Combined Toxicity Index is the reciprocal of the averaged %C1/2 and the SCGE genotoxic potency values. A larger Toxicity Index value indicates greater overall toxicity. c Based on data compiled by (62). d Based on data compiled by (63). e Calculation based on the estimated BIAcAm values alone.
of the halogen. Only the estimated log P values were used in the present study (Table 3). For the 13 haloacetamides, CHO cell chronic cytotoxicity and acute genotoxicity were highly and significantly correlated (r ) 0.99; P < 0.001). The rank order and relative activities follow: monohaloacetamides (cytotoxicity and genotoxicity), I > Br >> Cl; dihaloacetamides (cytotoxicity), I2 > IBr > ICl > Br2 > BrCl >> Cl2, and (genotoxicity), I2 > IBr > ICl > BrCl > Br2; Cl2 was inactive; trihaloacetamides (cytotoxicity and genotoxicity) Br3 > Br2Cl > BrCl2 >> Cl3. The rank order and relative activity of the monohaloacetamides are related to their SN2 reactivity. Owing to increasing bond length and decreasing dissociation energy, the leaving tendency of the halogen in alkyl halides followed the order I > Br >> Cl. The SN2 reactivity of an alkyl iodide was 3–5× greater than alkyl bromide, which was 50× greater than alkyl chloride (37, 55). The cytotoxicity of IAcAm was 1.3× greater than BAcAm, which was 78× greater than CAcAm. IAcAm was more genotoxic than BAcAm, which was 38× more potent than CAcAm. Log P does not play a major role; the small difference in the log P of BAcAm vs CAcAm cannot account for the large difference in relative activity. Consistent with the relative leaving tendencies of the halogen, dihaloacetamides containing the most iodo group(s) expressed the greatest combined cyto- and genotoxicity indices, followed by bromo group(s) and chloro group(s) (Table 3). DCAcAm was weakly cytotoxic and was not genotoxic. These results are difficult to explain by SN2 reactivity alone but may involve the activation of dihaloacetamides by intracellular GSH or -SH compounds, which displace one halogen and form highly reactive R-halothioether electrophilic intermediates. The key element of this reaction is the presence of at least one halogen with good leaving tendency. With GSH-mediated activation, the weak activity of DCAcAm and similar combined toxicity indices between DBAcAm vs BCAcAm may be expected (Table 3). The estimated log P values followed the order I2 > IBr > ICl > Br2 > BrCl > Cl2. This relative order is identical to their cytotoxicity and genotoxicity. Log P may play a more important role in the activity of dihaloacetamides by affecting cellular uptake. The cytotoxicity and genotoxicity of trihaloacetamides decreased with a decrease in the number of bromo groups. The cytotoxicity of TCAcAm was lower than TBAcAm by almost 3 orders of magnitude; this confirmed results in
human leukemia P388 cells (56). Only one bromo group was required for potent cytotoxicity; the %C1/2 values of TBAcAm, DBCAcAm, and BDCAcAm were within the same order of magnitude. In contrast, the decrease in genotoxic potency with a decrease in the number of bromo groups was more gradual (Table 2). The cytotoxicity and genotoxicity of trihaloacetamides could be partially explained by electrophilic reactivity at the carbonyl carbon as well as the possible release of electrophilic dihalocarbene intermediates (see discussion above). Alternatively, it is possible that reductive dehalogenation may yield cytotoxic free radicals; this pathway and the metabolic competency of the CHO cells have only been partially defined (57). Glutathione S-transferase theta 1-1 (GST T1-1) catalyzes preferential activation of brominated trihalomethanes to genotoxic intermediates (58, 59); the possible role of GST T1-1 in the activation of trihaloacetamides in CHO cells remains to be explored. Comparison of the Toxicity of Haloacetamides to Other DBP Classes. We compared the mammalian cell cytotoxic potencies, and genotoxic potencies between the haloacetamides and other DBP chemical classes, by calculating the CHO cell chronic cytotoxicity and acute genotoxicity indices (Figure 4). The cytotoxicity index was determined by calculating the median %C 1/2 value of all of the individual members of a single class of DBPs. The reciprocal was taken of this number so that a larger value was equated with that of higher cytotoxic potency. The genotoxicity index was determined by calculating the median SCGE genotoxicity potency value from the individual members within a single class of DBPs. The reciprocal was taken of this number so that a larger value was equated with that of higher genotoxicity. As a class, the haloacetamides were 99× more cytotoxic than 13 haloacetic acids (60), 142× more cytotoxic than the 5 regulated haloacetic acids (35), 2× more cytotoxic than the haloacetonitriles (27), and 1.4× more cytotoxic than the halonitromethanes (36). The haloacetamides were 19× more genotoxic than 13 haloacetic acids (60), 12× more genotoxic than the 5 regulated haloacetic acids (35), and 2.2× more genotoxic than the halonitromethanes (36). The haloacetamides were slightly less genotoxic (0.9×) than the haloacetonitriles (27). With the enforcement of the U.S. EPA Stage 2 DBP regulations, water utilities are considering the use of disinfectants that are alternatives to chlorine. The use of these alternative disinfectants will shift the distribution of DBP VOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Comparison of the CHO cell chronic cytotoxicity index values and the CHO genotoxicity index values for a series of DBP chemical classes. chemical classes (21, 37, 61). The emergence of new, highly toxic iodinated, nitrogenous DBPs, such as BIAcAm, underscores the importance of comparative toxicity studies to assist in the overall goal of safer drinking water practice.
Acknowledgments This paper is dedicated to Professor Saburo Matsui in celebration of his service to and retirement from Kyoto University and to recognize his outstanding contributions in environmental chemistry and engineering. We thank Gene Crumley for assistance with drinking water extractions and MS analyses, Ed Sverko of Environment Canada for providing an analysis using an inert source, and Fred Menger of Emory University for helpful discussions. We are especially grateful to Prof. Marco Vincenti of the University of Torino (Italy) for his support of F.F. and promoting her collaboration with EPA-Athens on this study. This research was funded in part by AwwaRF Grant 3089, U.S. EPA Cooperative Agreement CR83069501, and Illinois-Indiana Sea Grant R/WF-09-06 (M.J.P. and E.D.W.). M.M. was supported by T32 ES07326 (NIEHS). We appreciate the support by the Center of Advanced Materials for the Purification of Water with Systems, a National Science Foundation Science and Technology Center, under Award CTS-0120978. This paper has been reviewed in accordance with the U.S. EPA’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.
Note Added after ASAP Publication This paper was published ASAP December 12, 2007 with an error in the first paragraph of the Results and Discussion section; the corrected version published ASAP December 27, 2007.
Supporting Information Available Detailed information on the sampling, extraction and concentration of drinking water; the subsequent GC/MS analysis; and the biological assays. This information is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Richardson, S. D. Drinking water disinfection by-products. In The Encyclopedia of Environmental Analysis and Remediation; Wiley: New York, 1998; Vol. 3, pp 1398-1421. (2) Richardson, S. D.; Simmons, J. E.; Rice, G. Disinfection byproducts; the next generation. Environ. Sci. Technol. 2002, 36, 198A– 205A. 960
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