Occurrence, synthesis, and mammalian cell cytotoxicity and genotoxicity of haloacetamides: An emerging class of nitrogenous drinking water disinfection byproducts

Article abstract of DOI:10.1021/es071754h

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. 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.

Full text of DOI:10.1021/es071754h

Environ. Sci. Technol. 2008, 42, 955–961
With the advent of the U.S. EPA Stage 2 DBP regulations,
Occurrence, Synthesis, and
Mammalian Cell Cytotoxicity and
Genotoxicity of Haloacetamides: An
Emerging Class of Nitrogenous
Drinking Water Disinfection
Byproducts
water utilities are considering the use of disinfectants that are
alternativestochlorine. Theuseofthesealternativedisinfectants
willshiftthedistributionofDBPchemicalclasses. Theemergence
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 uni-
versally 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 mu-
tagens, 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 Oc-
currence 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.
M I C H A E L J . P L E W A , * , †
M A R K G . M U E L L N E R , †
S U S A N D . R I C H A R D S O N , ‡
F R A N C E S C A F A S A N O , ‡ ,
K A T H E R I N E M . B U E T T N E R , ‡
Y I N - T A K W O O , § A . B R U C E M C K A G U E , | A N D
E L I Z A B E T H D . W A G N E R †
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
theexceptionofbrominatedtrihaloacetamides, mostofthetoxicity
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.
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
* Corresponding author phone: (217) 333-3614; e-mail mplewa@
uiuc.edu.
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.
† 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.
9
10.1021/es071754h CCC: $40.75
2008 American Chemical Society
VOL. 42, NO. 3, 2008
/
ENVIRONMENTAL SCIENCE & TECHNOLOGY 955
Published on Web 12/12/2007

TABLE 1. Characteristics of the Haloacetamides Analyzed in This Study
haloacetamide
iodoacetamide
abbreviation
CAS no.
chemical formula
MWa
source and purity
IAcAm
144–48–9
C2H4INO
C2H3I2NO
184.96
310.85
263.856
219.405
137.96
216.86
295.75
172.41
251.305
206.85
93.51
Sigma-Aldrich, >97%
diiodoacetamide
DIAcAm
BIAcAm
CIAcAm
BAcAm
DBAcAm
TBAcAm
BCAcAm
DBCAcAm
BDCAcAm
CAcAm
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
CanSyn Chem Corp., 99%
CanSyn Chem Corp., 85%b
CanSyn Chem Corp., >95%
Sigma-Aldrich, 98%
bromoiodoacetamide
chloroiodoacetamide
bromoacetamide
dibromoacetamide
tribromoacetamide
bromochloroacetamide
dibromochloroacetamide
bromodichloroacetamide
chloroacetamide
C2H3BrINO
C2H3ClINO
C2H4BrNO
C2H3Br2NO
C2H2Br3NO
C2H3BrClNO
C2H2Br2ClNO
C2H2BrCl2NO
C2H4ClNO
C2H3Cl2NO
C2H2Cl3NO
CanSyn Chem Corp., >95%
CanSyn Chem Corp., >95%
CanSyn Chem Corp., 95%
CanSyn Chem Corp., >95%
CanSyn Chem Corp., >95%
Sigma-Aldrich, >95%
dichloroacetamide
trichloroacetamide
DCAcAm
TCAcAm
127.96
162.40
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 dibro-
moacetate 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 Informa-
tion. Within each experiment, 10 haloacetamide concentra-
tions 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 treatment, concentration, and analysis can
be found in the Supporting Information. GC/mass spec-
trometry (MS) analyses with electron ionization (EI) were
carried out on an Agilent 6890 GC interfaced to a Waters-
Micromass Autospec II high resolution, double focusing mass
spectrometer at 1000 resolution.
Results and Discussion
Identification and Occurrence of a New Iodinated Aceta-
mide. 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 haloa-
mides measured expressed distinctive GC/MS chromato-
graphic 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 chlorami-
nation 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 cor-
responding 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).
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
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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 concentra-
tion–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-ꢀ) 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 determi-
nation (R2) ranged from 0.95 to 0.99. From these concen-
tration–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-ꢀ 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
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TABLE 2. Summary Comparison of the CHO Cell Chronic Cytotoxicity and Acute Genotoxicity of the Haloacetamides
lowest toxic
conc. (M)a
lowest genotox.
conc. (M)d
c
f
chemical
%C½ Value (M)b
R2
genotox. potency (M)e
R2
iodoacetamide
diiodoacetamide
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
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
3.41 × 10-5
0.99
0.98
0.99
0.99
0.99
0.99
0.97
0.99
0.98
0.99
0.99
NA
6.78 × 10-7
3.39 × 10-5
g
h
bromoiodoacetamide
chloroiodoacetamide
bromoacetamide
dibromoacetamide
tribromoacetamide
bromochloroacetamide
dibromochloroacetamide
bromodichloroacetamide
chloroacetamide
3.81 × 10-6
7.21 × 10-5
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
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
dichloroacetamide
trichloroacetamide
5.00 × 10-3
0.98
a Lowest toxic concentration was the lowest concentration of the haloacetamide in the concentration–response curve that
b
induced a significant reduction in cell density as compared to the negative control. 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
c
the concurrent negative control. 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,
f
at the midpoint of the curve within the concentration range that expressed above 70% cell viability of the treated cells. 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 haloaceta-
mides have or may generate a number of electrophilic
reactivities: (i) for monohaloacetamides, alkylation by the
SN2 reaction, inducing the displacement of a halogen atom
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 elec-
trophilic 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
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TABLE 3. Estimated and Measured log P and Combined Toxicity Values of the Haloacetamides Studied
compound
estimated log Pa
measured log P
combined toxicity valueb
monohaloacetamides
iodoacetamide
bromoacetamide
chloroacetamide
-0.08
-0.49
-0.58
-0.19c; -0.15d
-0.52c,d
5.63 × 104
5.17 × 104
1.31 × 103
-0.53c; -0.59d
dihaloacetamides
diiodoacetamide
0.92
0.09
-0.09
0.50
5.78 × 104
2.64 × 103
1.68 × 102
dibromoacetamide
dichloroacetamide
bromoiodoacetamide
chloroiodoacetamide
bromochloroacetamide
0.18d
0.19c; -0.03d
e
1.02 × 105
0.41
6.49 × 103
0.00
3.33 × 103
trihaloacetamides
tribromoacetamide
1.10
1.01
0.92
0.83
5.61 × 104
2.70 × 104
1.29 × 104
2.33 × 102
dibromochloroacetamide
bromodichloroacetamide
trichloroacetamide
1.04c; 0.79d
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).
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 electro-
philic 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 trihaloaceta-
mides 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 haloaceta-
mides 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 %C1/2 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 geno-
toxicity. 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).
For the 13 haloacetamides, CHO cell chronic cytotoxicity
and acute genotoxicity were highly and significantly cor-
related (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 monohaloac-
etamides 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 dihaloac-
etamides by intracellular GSH or -SH compounds, which
displace one halogen and form highly reactive R-halothio-
ether 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
With the enforcement of the U.S. EPA Stage 2 DBP
regulations, water utilities are considering the use of dis-
infectants that are alternatives to chlorine. The use of these
alternative disinfectants will shift the distribution of DBP
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Acknowledgments
This paper is dedicated to Professor Saburo Matsui in celebra-
tion 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
aninertsource, andFredMengerofEmoryUniversityforhelpful
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
SeaGrantR/WF-09-06(M.J.P. andE.D.W.). M.M. wassupported
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 Technol-
ogy Center, under Award CTS-0120978. This paper has been
reviewed in accordance with the U.S. EPA’s peer and admin-
istrative 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
errorinthefirstparagraphoftheResultsandDiscussionsection;
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.
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