Chemical and biological characterization of newly discovered iodoacid drinking water disinfection byproducts

Article abstract of DOI:10.1021/es049971v

Iodoacid drinking water disinfection byproducts (DBPs) were recently uncovered in drinking water samples from source water with a high bromide/iodide concentration that was disinfected with chloramines. The purpose of this paper is to report the analytical chemical identification of iodoacetic acid (IA) and other iodoacids in drinking water samples, to address the cytotoxicity and genotoxicity of IA in Salmonella typhimurium and mammalian cells, and to report a structure-function analysis of IA with its chlorinated and brominated monohalogenated analogues. The iodoacid DBPs were identified as iodoacetic acid, bromoiodoacetic acid, (Z)- and (E)-3-bromo-3-iodopropenoic acid, and (E)-2-iodo-3-methylbutenedioic acid. IA represents a new class (iodoacid DBPs) of highly toxic drinking water contaminants. The cytotoxicity of IA in S. typhimurium was 2.9× and 53.5× higher than bromoacetic acid (BA) and chloroacetic acid (CA), respectively. A similar trend was found with cytotoxicity in Chinese hamster ovary (CHO) cells; IA was 3.2× and 287.5× more potent than BA and CA, respectively. This rank order was also expressed in its genotoxicity with IA being 2.6× and 523.3× more mutagenic in S. typhimurium strain TA100 than BA and CA, respectively. IA was 2.0× more genotoxic than BA and 47.2× more genotoxicthan CA in CHO cells. The rank order of the toxicity of these monohalogenated acetic acids is correlated with the electrophilic reactivity of the DBPs. IA is the most toxic and genotoxic DBP in mammalian cells reported in the literature. These data suggest that chloraminated drinking waters that have high bromide and iodide source waters may contain these iodoacids and most likely other iodo-DBPs. Ultimately, it will be important to know the levels at which these iodoacids occur in drinking water in order to assess the potential for adverse environmental and human health risks.

Full text of DOI:10.1021/es049971v

Research
waters that have high bromide and iodide source waters
may contain these iodoacids and most likely other iodo-DBPs.
Ultimately, it will be important to know the levels at
which these iodoacids occur in drinking water in order to
assess the potential for adverse environmental and
human health risks.
Chemical and Biological
Characterization of Newly
Discovered Iodoacid Drinking Water
Disinfection Byproducts
M I C H A E L J . P L E W A * A N D
E L I Z A B E T H D . W A G N E R
Introduction
Each day approximately 250 000 public water purification
facilities in the United States provide over 1.3 × 1010 L of
high-quality, safe drinking water to 90% of the population
(1). The production and distribution of disinfected water was
a profound public health triumph of the twentieth century
that significantly reduced infections by waterborne microbial
pathogens. The disinfection of drinking water in public
facilities primarily uses chemical disinfectants such as
chlorine, chloramines, ozone, and chlorine dioxide (2). These
disinfectants are strong oxidants that convert naturally
occurring and synthetic organic material along with bromide
and iodide in the raw water into chemical disinfection
byproducts (DBPs). Over 500 DBPs have been identified;
however, each disinfection method generates a different suite
and distribution of DBPs (3).While reducing the public health
risk of acute infection by waterborne pathogens, the un-
intended generation of DBPs poses a chronic health risk.
DBPs represent an important class of environmentally
hazardous chemicals that carry long-term human health
implications (3-5). Epidemiological studies demonstrate that
individuals who consume chlorinated drinking water have
an elevated risk of cancer of the bladder, stomach, pancreas,
kidney, and rectum as well as Hodgkin’s and non-Hodgkin’s
lymphoma (6-8). DBPs also have been linked to reproductive
and developmental effects, including the induction of
spontaneous abortions (9, 10).
College of Agricultural, Consumer and Environmental
Sciences, Department of Crop Sciences, University of Illinois
at Urbana-Champaign, Urbana, Illinois 61801
S U S A N D . R I C H A R D S O N A N D
A L F R E D D . T H R U S T O N , J R .
National Exposure Research Laboratory, U.S. Environmental
Protection Agency, Athens, Georgia 30605
YI N - T A K W O O
Risk Assessment Division, Office of Pollution Prevention and
Toxics, U.S. Environmental Protection Agency,
Washington, D.C. 20460
A . B R U C E M C K A G U E
CanSyn Chemical Corporation, 200 College Street,
Toronto, Ontario M5S 3E5, Canada
Iodoacid drinking water disinfection byproducts (DBPs)
were recently uncovered in drinking water samples from
source water with a high bromide/iodide concentration that
was disinfected with chloramines. The purpose of this
paper is to report the analytical chemical identification of
iodoacetic acid (IA) and other iodoacids in drinking
water samples, to address the cytotoxicity and genotoxicity
of IA in Salmonella typhimurium and mammalian cells,
and to report a structure-function analysis of IA with its
chlorinated and brominated monohalogenated analogues.
The iodoacid DBPs were identified as iodoacetic acid,
bromoiodoacetic acid, (Z)- and (E)-3-bromo-3-iodopropenoic
acid, and (E)-2-iodo-3-methylbutenedioic acid. IA represents
a new class (iodoacid DBPs) of highly toxic drinking
water contaminants. The cytotoxicity of IA in S. typhimurium
was 2.9× and 53.5× higher than bromoacetic acid (BA)
and chloroacetic acid (CA), respectively. A similar trend was
found with cytotoxicity in Chinese hamster ovary (CHO)
cells; IA was 3.2× and 287.5× more potent than BA and
CA, respectively. This rank order was also expressed in its
genotoxicity with IA being 2.6× and 523.3× more mutagenic
in S. typhimurium strain TA100 than BA and CA, respectively.
IA was 2.0× more genotoxic than BA and 47.2× more
genotoxic than CA in CHO cells. The rank order of the toxicity
of these monohalogenated acetic acids is correlated
with the electrophilic reactivity of the DBPs. IA is the most
toxic and genotoxic DBP in mammalian cells reported in
the literature. These data suggest that chloraminated drinking
Although chlorine has been used for over 100 years as a
water disinfectant, the majority of DBPs present in drinking
water have yet to be chemically defined (5, 11). Identified
DBPs account for less than 50% of the total organic halide
(TOX) produced in chlorinated drinking water (12-14).
Percentages of known DBPs are even smaller for drinking
water treated with alternative disinfectants, with only 17.1,
8.3, and 28.4% of the TOX accounted for in waters treated
with chloramines, ozone, or chlorine dioxide, respectively
(14). Disinfectants used alone or in combination generate
novel DBPs (such as 2,3,5-tribromopyrrole) that express toxic
and genotoxic activity (15).
The haloacetic acids represent the second largest group
of DBPs generated by water disinfection. An estimated 55 000
t of trichloroacetic acid alone are generated annually (16).
Bromide is naturally present in many source waters that
results in an increase in bromine-containing DBPs after water
disinfection with chlorine, chloramine, or ozone (15, 17, 18).
The chlorinated and brominated haloacetic acids have been
evaluated for their mutagenicity and cytotoxicity in Salmo-
nella typhimurium (19-21). We published an analysis of the
induction of chronic and acute cytotoxicity as well as the
induction of genomic DNA damage in mammalian cells by
specific DBPs (22). In general, the brominated haloacetic
acids were significantly more cytotoxic and genotoxic than
their chlorinated analogues. In a recent U.S. nationwide
occurrence study, iodoacid DBPs were uncovered in drinking
water samples from source water with a high bromide
* Corresponding author phone: (217)333-3614; fax: (217)333-8046;
e-mail: mplewa@uiuc.edu.
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Published on Web 08/17/2004
2004 Am erican Chem ical Society
VOL. 38, NO. 18, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4 7 1 3

concentration that was disinfected with chloramines (23).
Gas chromatography/ mass spectrometry (GC/ MS) was used
to tentatively identify those DBPs as iodoacetic acid, bromo-
iodoacetic acid, bromoiodopropenoic acid (two isomers),
and 2-iodo-3-methylbutenedioic acid. Identifications have
since been confirmed for iodoacetic acid and bromoiodo-
acetic acid. The specific isomers of (Z)- and (E)-3-bromo-
3-iodopropenoic acid and (E)-2-iodo-3-methylbutenedioic
acid were confirmed (results reported here). The identifica-
tion of these iodoacids is important toxicologically, as
iodoacetic acid (IA) has been shown to induce neural tube
closure defects and other developmental abnormalities in
mouse embryos (24, 25).
Bacterial and Mam m alian Cells. The S. typhimurium
tester strain, TA100 (hisG46, rfa; ∆uvrB-bio; pKM101, ampr)
was stored as frozen cultures in Luria-Bonner broth (LB)
plus 10% DMSO at -80 °C (30). Clone 11-4-8 of the transgenic
Chinese hamster ovary (CHO) cell line AS52 was the
mammalian cell system used in this research (31). The CHO
cells were maintained in Ham’s F12 medium containing 5%
fetal bovine serum (FBS) and grown in 100 mm glass culture
plates at 37 °C in an atmosphere of 5% CO2 in air. For long-
term storage, the cells were frozen in FBS:DMSO (9:1, v/ v)
and kept at -80 °C.
Sa lmonella Microplate Cytotoxicity Assay. The detailed
procedures for the Salmonella microplate cytotoxicity assay
were published (21), and an expanded description of the
assay can be found in the Supporting Information. This assay
measured the cytotoxicity of the DBPs during a growth period
of ∼3 cell divisions. A concurrent negative control of bacteria
without DBP exposure was included with each microplate,
and the blank-corrected data for the negative control was set
at 100% growth. The blank-corrected data for each DBP
concentration was converted into a percentage of the negative
control. In general, each DBP concentration was replicated
4-8 times per microplate, and each experiment was repeated
a minimum of 2 times.
IA is the focus of this paper because it may represent a
new class (iodoacid DBPs) of potent drinking water con-
taminants. The purpose of this paper is (i) to report the
analytical chemical identification of IA and other iodoacids
in drinking water samples, (ii) to address the cytotoxicity
and genotoxicity of IA in S. typhimurium and mammalian
cells, and (iii) to report a structure-function analysis of IA
with its chlorinated and brominated monohalogenated
analogues. The health risks of DBPs are poorly understood
and are compounded by the lack of a comparative database
on the quantitative toxicity of DBPs.
Sa lmonella Preincubation Mutagenicity Assay. The
detailed procedures for the Salmonella mutagenicity assay
were published (21), and an expanded description of the
assay can be found in the Supporting Information. The
induction of histidine revertants per 5 × 108 cells treated for
each DBP concentration was used to generate the concen-
tration-response data and to calculate the mutagenic
potency. The mutagenicity experiments were repeated 3
times.
Mam m alian Cell Microplate Cytotoxicity Assay. The
detailed procedures for the CHO cell microplate cytotoxicity
assay were published (32) and can be found in the Supporting
Information. This assay measures the reduction in cell density
as a function of DBP concentration over a period of ∼3 cell
divisions (72 h). The blank-corrected absorbency value of
the negative control (cells with medium only) was set at 100%.
The absorbency for each treatment group well was converted
into a percentage of the negative control. For each DBP
concentration 8 replicate wells were analyzed per experiment,
and the experiments were repeated twice.
Single Cell Gel Electrophoresis (SCGE) Assay. The
procedures for the CHO cell microplate SCGE assay were
published (22, 32), and they can be found in the Supporting
Information. SCGE is a sensitive assay that quantitatively
determines genomic DNA damage and predictive carcino-
genic potency. After the CHO cells were treated with the
DBP for 4 h, acute cytotoxicity with a vital dye was measured
(33). A computerized image analysis system (Komet 3.1,
Kinetic Imaging Ltd., Liverpool, UK) was used to measure
various SCGE parameters (i.e., % tail DNA and tail moment)
of 25 randomly chosen nuclei per slide. The tail moment
(integrated value of migrated DNA density multiplied by the
migration distance) was used as the primary measure of DNA
damage. Each experiment was repeated 3 times.
Safety and Data Analysis. Manipulations of toxic and
mutagenic chemicals were conducted using disposable
papers and gloves in a certified biological/ chemical safety
hood. Data from the experiments were transferred to Excel
spreadsheets (Microsoft Corp., Redmond, WA) and analyzed
using the statistical and graphical functions of SigmaPlot 8,
SigmaStat 3, and Table Curve 4.03 (SPSS Inc., Chicago, IL).
The tail moment values in the SCGE assay were not normally
distributed and violated the requirements for analysis by
parametric statistics. The median tail moment value for each
slide was determined, and the data were averaged. Averaged
median values express a normal distribution according to
Experimental Section
Drinking Water Analysis. Drinking water samples discussed
in this paper were collected from a full-scale drinking water
treatment plant in the United States that treated a source
water with moderate levels of bromide (0.15 mg/ L) and total
organic carbon (7.01 mg/ L) with chloramines only (chlorine
and ammonia were added simultaneously). Samples (39 L
each) were collected in November 2001 and December 2003
at the treatment plant in 2-L Teflon bottles, shipped overnight
to the U.S. EPA laboratory the following day, and extracted
and concentrated using XAD resins. Details regarding the
drinking water treatment and concentration can be found
in Supporting Information. Methylation derivatizations with
BF3-methanol were used to enable the detection of carboxylic
acid DBPs with GC/ MS (15). GC/ MS analyses with electron
ionization (EI) were performed on a Micromass Autospec II
high-resolution mass spectrometer equipped with an Agilent
6890 gas chromatograph using conditions that can be found
in Supporting Information.
Chem icals and Reagents. Bromoacetic acid and chlo-
roacetic acid were purchased from Fluka Chemical Co.
(Buchs, Switzerland). IA was purchased from Aldrich Chemi-
cal Co. (Milwaukee, WI). Bromoiodoacetic acid was prepared
by the reaction of diiodoacetic acid (26) with bromine in
water. Bromine (2.0 g, 12.5 mmol) was added to a stirred
suspension of finely ground diiodoacetic acid (3.1 g, 10 mmol)
in water (10 mL) over 10 min. The resultant solution was
stirred at room temperature for 2.5 h and then evaporated
to give a red oil. Extraction with chloroform, concentration,
and cooling of the extract gave bromoiodoacetic acid (1.0 g),
mp 82-84 °C. The purity of the product by GC using flame
ionization detection was 75% and contained 10% dibro-
moacetic acid and 15% diiodoacetic acid. A mixture of 85%
(Z)- and 15% (E)-3-bromo-3-iodopropenoic acid was pre-
pared from 3-bromopropiolic acid (27) by addition of
hydrogen iodide (28). The dimethyl ester of (E)-2-iodo-3-
methylbutenedioic acid was prepared by the sequential
addition of methyl copper-magnesium bromide and iodine
to dimethylacetylenedicarboxylate (29). Hydrolysis of the
dimethyl ester by refluxing with 10% hydrochloric acid gave
(E)-2-iodo-3-methylbutenedioic acid, mp 80-81 °C, after
crystallization from hexane. The DBPs were dissolved in
dimethyl sulfoxide (DMSO) and stored at -22 °C in sealed,
sterile glass vials.
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FIGURE 1. Mass spectra for iodoacids (identified in their methyl ester forms).
the central limit theorem (34). The averaged median tail
moment values obtained from repeated experiments were
used with a one-way analysis of variance test (35). If a
significant F value of P e 0.05 was obtained, a Holm-Sidak
multiple comparison versus the control group analysis was
conducted. The power of the test statistic (â) was g0.8 at R
) 0.05.
to evaluate the toxicity of important new classes of DBPs,
such as these iodoacids, which have just been identified for
the first time in drinking water.
Analytical Chem istry. Five iodoacidssIA, bromoiodo-
acetic acid, (Z)- and (E)-3-bromo-3-iodopropenoic acid, and
(E)-2-iodo-3-methylbutenedioic acidswere identified in their
methyl ester forms using GC/ MS. Figure 1 shows their EI
mass spectra; empirical formulas shown above the molecular
ions and fragments were obtained using high-resolution MS
(10000 resolution). These iodoacids were found in the finished
drinking waters treated by chloramination (Figure 2) and
Results and Discussion
The chemistry and toxicity of DBPs have been investigated
for more than a quarter of a century. However, it is important
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FIGURE 2. GC/MS chromatogram of chloraminated drinking water sample.
were not in the corresponding raw water blanks. As a result,
they were determined to be DBPs of the treatment process.
These iodinated DBPs were likely formed by the reaction of
monochloramine with naturally occurring iodide to form
hypoiodous acid (HOI), which in turn reacts with natural
organic matter (36, 37) to form the iodoacids. Because none
of the iodoacid methyl esters were present in the NIST or
Wiley Library Databases, extensive interpretation of their
mass spectra was performed to generate possible structures
for the unknown compounds. Unlike other halogenated
compounds, such as brominated and chlorinated compounds
that are easily recognizable from their distinctive isotopic
patterns, the presence of iodine is not easy to detect in mass
spectra since iodine has only one naturally occurring
isotope: 127 Da. A compound with one bromine in its
structure will give a doublet isotopic pattern, which is due
to the overlap of the two naturally occurring bromine isotopes
(79 and 81 Da). For example, the presence of one bromine
atom is indicated from the mass spectrum of bromoiodo-
acetic acid methyl ester (Figure 1). The molecular ion doublet
at m/ z 278/ 280 is due to the overlap of the two bromine
isotopes. On the other hand, the presence of iodine is not
immediately obvious in this spectrum nor in the spectra of
the other iodoacids (Figure 1). However, it is fortunate that
the molecular ions of these methyl esters were present. It
was evident that the highest m/ z ions in these mass spectra
were the molecular ions (and not fragment ions), due to the
loss of 31 Da (for the monoacid esters), and in some cases
59 Da, which is common for carboxylic methyl esters. The
presence of the m/ z 59 ion in each of these mass spectra also
supports the assignment of these as carboxylic acid methyl
esters. The diacid ester ((E)-2-iodo-3-methylbutenedioic acid
dimethyl ester) showed the characteristic loss of 59 Da and
the presence of m/ z 59 but underwent a rearrangement to
lose 32 Da (CH3OH) instead of the more common 31 Da
(OCH3). Once the molecular ions were known, and it was
likely that these were carboxylic acid methyl esters, then the
presence of iodine could be uncovered from careful inspec-
tion of the mass spectra. For example, in most cases, there
was a small ion at m/ z 127, representing the I+ ion, and often
the loss of iodine (M - I)+ was often present at substantial
relative abundance.
From the low resolution EI-MS data, tentative structures
were proposed. For the first two iodoacid methyl esters
(iodoacetic acid methyl ester and bromoiodoacetic acid
methyl ester), only a single isomer was possible. However,
several structural isomers were possible for the remaining
iodoacids (six different isomers were possible for the two
bromoiodopropenoic acid methyl ester isomers observed,
and two isomers were possible for the single 2-iodo-3-
FIGURE 3. Possible structures for bromoiodopropenoic acid methyl
ester and iodomethylbutenedioic acid dimethyl ester isomers.
methylbutenedioic acid dimethyl ester isomer observed).
These structures are illustrated in Figure 3. High-resolution
MS data supported the empirical formulas for these tentative
structural assignments and the presence of iodine in their
structures, with exact mass data of 199.939 (observed) and
199.933 (theoretical) for iodoacetic acid methyl ester, 277.849
(observed) and 277.844 (theoretical) for bromoiodoacetic acid
methyl ester, 289.850 (observed) and 289.844 (theoretical)
for (Z)-3-bromo-3-iodopropenoic acid methyl ester, and
283.957 (observed) and 283.955 (theoretical) for (E)-2-iodo-
3-methylbutenedioic acid dimethyl ester. The mass spectral
signal of (E)-3-bromo-3-iodopropenoic acid methyl ester was
too weak to obtain high resolution data. After we tentatively
identified these iodoacids, we purchased the IA standard
and synthesized the remaining standards and methylated
these standards to check their mass spectra and GC retention
times against those in our drinking water samples. Because
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4 7 1 6 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 18, 2004

methyl ester also matched those in our drinking water
samples, confirming their identities. After their initial dis-
covery in drinking water samples collected for the nationwide
occurrence study, we have since resampled this location (in
December 2003) and continue to observe these five iodoacids
in the chloraminated drinking water.
Analytical Biology. We present new results of the cyto-
toxicity of IA and bromoacetic acid (BA) in S. typhimurium.
Previously published cytotoxicity data for chloroacetic acid
(CA) are used for comparison (21). The mutagenicity of IA
in S. typhimurium was determined in this current study;
comparative data for BA and CA were previously published
(21). The CHO cell cytotoxicity of IA, BA, and CA were
determined in this study. The SCGE analysis of IA in CHO
cells was determined in the current study; the SCGE data for
BA and CA were published (22).
Cytotoxicity and Mutagenicity in S. typhimurium. The
Salmonella microplate cytotoxicity assay is a quantitative
measurement of the repression of bacterial growth induced
by a test agent. In Figure 4, the ordinate is expressed in two
units of measurement. The concentration (expressed as µM)
is the concentration of the haloacetic acid present in the
reaction tube. The concentration (expressed as µM-h)
included the 1-h exposure to the haloacetic acid concentra-
tion in the reaction tube plus the 210 min in the microplate
well at half of its original concentration. This is due to the
addition of 2× concentrated LB to each microplate well. The
use of these two measurements allows for the comparison
of the relative viability of the S. typhimurium cells between
the cytotoxicity assay and the preincubation mutagenicity
assay (21).
The chronic cytotoxicity of IA was evaluated within a
concentration range from 10 µM to 1 mM. Each of the 20
concentrations was replicated from 4 to 12 times within two
independent experiments (Figure 4). At concentrations above
100 µM, IA induced significant cytotoxicity as compared to
the negative control; the %C1/ 2 value was 303 µM (Table 1).
The %C1/ 2 value is the concentration of the test agent,
determined from a regression analysis of the data, that
induced a cell density of 50% as compared to the concurrent
negative control. For BA the experiments were conducted in
triplicate with 24 repeats at each concentration. The %C1/ 2
value for BA was 881 µM with concentrations above 216 µM
significantly different from the negative control (Table 1).
The %C1/ 2 value for CA was 16.2 mM (21).
FIGURE 4. Induction of cytotoxicity in S. typhimurium by iodoacetic,
bromoacetic, and chloroacetic acids. The ordinate is expressed (i)
as the µM concentration of the haloacetic acid present in the
reaction tube and (ii) as the µM-h, which included the 1-h exposure
to the haloacetic acid concentration in the reaction tube plus the
210 min in the microplate well at half of its original concentration.
The abscissa expresses the percent of bacterial growth as a
percentage of the concurrent negative control for each haloacetic
acid.
the mass spectra for the bromoiodopropenoic acid methyl
ester isomers and the iodomethylbutenedioic acid dimethyl
ester isomers were expected to be nearly identical and the
GC retention times were expected to be very similar, all of
the isomers except those of the 2-bromo-3-iodopropenoic
acid and (Z)-2-iodo-3-methylbutenedioic acid were synthe-
sized to ensure correct identifications. Results did reveal
nearly identical mass spectra and similar retention times for
the bromoiodopropenoic acid methyl ester isomers and the
2-iodo-3-methylbutenedioic acid dimethyl ester isomers.
However, unequivocal matches were obtained for (Z)- and
(E)-3-bromo-3-iodopropenoic acid methyl ester and (E)-2-
iodo-3-methylbutenedioic acid dimethyl ester, confirming
their identities. GC/ MS retention times and mass spectra of
iodoacetic acid methyl ester and bromoiodoacetic acid
IA was analyzed for its direct-acting mutagenicity with S.
typhimurium strain TA100 for reversion at the hisG46 allele.
The mutagenicity of IA was assayed using a preincubation
TABLE 1. Chronic Cytotoxicity Analysis of Iodoacetic, Bromoacetic, and Chloroacetic Acids in Salmonella typhimurium TA100
or Chinese Hamster Ovary AS52 Cells
1
range of significant
concentration-response (µM)
r 2 of
%C /2
ANOVA
test statistic
haloacetic acid
regression analysisa
(µM)b
Salmonella typhimurium Cells
iodoacetic acid
100-1000
0.99
0.98
0.97
303
881
F20,163 ) 303.47
P < 0.001
brom oacetic acid
chloroacetic acidc
507-2260
F14,372 ) 256.98
P < 0.001
800-100 000
16 200
F14,73 ) 891.32
P < 0.001
CHO Cells
iodoacetic acid
brom oacetic acid
chloroacetic acid
0.500-12.0
2.00-25.0
300-2000
0.97
0.99
0.99
2.95
9.56
848
F13,122 ) 54.71
P < 0.001
F10,165 ) 94.45
P < 0.001
F8,127 ) 37.62
P < 0.001
a
b
Regression analysis was used to determ ine the %C1/2 value from the cytotoxicity concentration-response curve. The %C1/2 value is the
c
concentration of the test agent that reduces the cell density by 50% as com pared to the concurrent negative control. Sum m arized from ref 21.
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TABLE 2. Genotoxicity of Iodoacetic, Bromoacetic, and Chloroacetic Acids in Salmonella typhimurium TA100 or Chinese
Hamster Ovary AS52 Cells
r 2 of
genotoxic
potencyb
ANOVA
test statistic
haloacetic acid
regression analysisa
Salmonella typhimurium TA100 Mutagenicity
iodoacetic acid
0.82
0.98
0.92
14 129 revertants/µm ol
5 465 revertants/µm ol
27 revertants/µm ol
F13, 79 ) 59.02
P < 0.001
brom oacetic acidc
chloroacetic acidc
F8, 69 ) 72.12
P < 0.001
F12, 104 ) 53.49
P < 0.001
CHO Cell Single Cell Gel Electrophoresis
iodoacetic acid
0.99
0.99
0.99
8.70 µM
17.0 µM
411 µM
F9, 49 ) 33.90
P < 0.001
brom oacetic acidd
chloroacetic acidd
F8, 50 ) 77.47
P < 0.001
F8, 23 ) 20.13
P < 0.001
a
The regression analysis represents the linear section of the concentration-response curve for S. typhim urium . For the CHO cell SCGE assay,
the r represents the fit of the regression analysis for the concentration-response curve used to calculate the genotoxic potency value. b For S.
2
typhim urium , the m utagenic potency is expressed as the induced revertants per µm ole of the test agent. For the CHO cell SCGE assay, the genotoxic
c
potency is the concentration of the test agent at the m idpoint of the tail m om ent concentration-response curve. Sum m arized from ref 21.
d
Sum m arized from ref 22.
FIGURE 6. Concentration-response curves illustrating the chronic
cytotoxicity of iodoacetic, bromoacetic, and chloroacetic acids in
CHO cells after 72-h exposure.
FIGURE 5. Concentration-response curves illustrating the relative
mutagenicity of iodoacetic, bromoacetic, and chloroacetic acids
in S. typhimurium strain TA100.
procedure with a treatment time of 1 h in a concentration
range of 25-300 µM with three independent experiments.
As illustrated by the µM-h scale in Figure 4, this concentration
range was not cytotoxic. A significant increase in the induced
revertants per 5 × 108 cells plated was observed at IA
concentrations of 70 µM and above (Table 2). The average
spontaneous reversion frequency was 130 revertants per 5
× 108 cells plated; 300 µM IA induced 919 revertants per 5
× 108 cells plated (Figure 5).
calculated for each concentration in the linear region of the
DBP concentration-response curve and an average mu-
tagenic potency value was determined (Table 2). With a
mutagenic potency of 14 129 revertants/ µmol, IA is the most
potent haloacetic acid analyzed in S. typhimurium.
Cytotoxicity and Genotoxicity in CHO Cells. Although
BA and CA were previously evaluated for chronic cytotoxicity
in CHO cells (22), we made a modification to the microplate
assay and analyzed the monohalogenated acetic acids. IA
was analyzed in a concentration range from 100 nM to 12
µM and induced a significant amount of cytotoxicity at
concentrations of 500 nM and above. BA was cytotoxic in the
concentration range from 2 to 25 µM, while CA was cytotoxic
in a range from 300 µM to 2 mM. The %C1/ 2 values for IA,
BA, and CA were 2.95, 9.56, and 848 µM, respectively (Table
1, Figure 6).
To directly compare the mutagenicity of IA with BA and
CA in S. typhimurium, we calculated the induced mutagenic
potency of each agent. The mutagenic potency was calculated
for the haloacetic acid concentrations that expressed a
significant increase over their corresponding negative control.
The mutagenicity concentration-response curves were
analyzed by running a linear regression for specific regions
2
of the curve and the coefficient of linearity (r ) for each agent
IA was a potent inducer of genomic DNA damage in CHO
cells. We measured two indices of DNA damage, the % tail
DNA (the amount of DNA migrating from the nucleus into
the gel), and the SCGE tail moment (the product of the
amount of migrated DNA and the distance). IA induced a
significant concentration response in the mean % tail DNA
is presented in Table 2. The induced revertants per plate
were calculated by subtracting the average negative control
revertant frequency from the treatment revertant frequency.
Using these data the induced revertants per reaction tube
were determined. The induced revertants per micromole were
9
4 7 1 8 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 18, 2004

TABLE 3. Summary of Physicochemical Properties of
Monohaloacetic Acids
bond length, bond dissociation energy,
physicochemical properties
of monohaloacetic acidsa
and relative SN2 reactivity of C-X
bond based on alkyl halidesb
length dis. energy relative
compd log P pK ELUMO C-X
(Å)
(kcal/mol)
SN2
a
CA
BA
IA
0.38 2.82 0.126 C-Cl 1.77
0.52 2.90 0.111 C-Br 1.93
0.91 3.12 0.091 C-I 2.14
78.5
65.9
57.4
1
50
150-250
a
Calculated log P and ELUMO and m easured pKa sum m arized from
ref 42. The m easured log P of CA and BA are 0.22 and 0.41, respectively
b
(54). Sum m arized from ref 41.
TABLE 4. Pearson Correlation Analysis of Physicochemical and
Toxicological Measurements of Monohaloacetic Acids
cytotoxicity (r)
genotoxicity (r)
CHO
physicochemical
parameters
FIGURE 7. Comparison of the genomic DNA damage induced by
iodoacetic acid using two SCGE measurement parameters: median
tail moment and the percentage of fragmented DNA migrated into
the gel (% tail DNA).
Salmonella CHO Salmonella
log P
pKa
ELUMO
-0.73
-0.73
0.84
-0.84
0.07
-0.71
-0.71
0.83
-0.83
0.09
0.99
0.99
-0.99
-0.99
0.53
-0.72
-0.72
0.83
-0.84
0.08
bond length (Å)
C-X dissociation energy
relative SN2
-0.71
-0.69
0.99
-0.70
S. typhimurium and CHO cells, the log P of the un-ionized
monohaloacetic acids follows the order of IA > BA > CA. The
correlation coefficients (r) for cytotoxicity (%C1/ 2) and the
log P values were similar for S. typhimurium and CHO cells
(Table 4). A direct correlation also results between geno-
toxicity and log P. The higher mutagenic potency values for
S. typhimurium express increased mutagenic activity. Lower
SCGE genotoxic potency values for CHO cells indicate
stronger DNA-damaging capacity and thus an inverse cor-
relation with log P.
The lipophilicity of and cell permeability to monohalo-
acetic acids can be substantially decreased by ionization,
which is determined by their pKa and the pH of the medium.
The fraction ( f ) of un-ionized monohaloacetic acids can be
calculated by the formula (38):
f ) 1/ (1 + 10(pH-pK )
)
a
FIGURE 8. Concentration-response curves illustrating the relative
levels of genomic DNA damage induced by iodoacetic, bromoacetic,
and chloroacetic acids.
Chemicals with higher pKa values are less likely to be ionized.
For the monohaloacetic acids the ranking of pKa follows the
order of IA > BA > CA. A correlation among the pKa and the
cytotoxicity and genotoxicity of the monohalogenated acetic
acids was observed (Table 4). However, the relatively minor
difference in pKa (Table 3) does not appear to be a major
contributing factor to the substantial difference in the relative
genotoxic/ cytotoxic potency. Furthermore, since the experi-
mental conditions were maintained around neutral pH, all
three monohaloacetic acids should be mostly in the ionized
form. It is possible that facilitated or active membrane
transport of anionic monohaloacetates may be an additional
mechanism for cellular uptake. The experimental conditions
were maintained around neutral pH, thus the monohaloacetic
acids should be in an ionized form, and facilitated or active
membrane transport of anionic monohaloacetates may be
a mechanism for cellular uptake. There is some evidence of
active transport of CA and BA across synthetic membranes
(39), but the relevance to biological membranes remains to
be studied. Tissue distribution studies in rats (40) showed
significant accumulation of CA and IA in the kidney and
liver. However, a percutaneous absorption study using
human skin sections showed very poor permeability to CA
and BA around neutral pH (38).
in the range from 5 to 20 µM (Figure 7). Likewise a significant
increase in the average median tail moment values was
observed from 5 to 20 µM (Figure 7, Table 2). The SCGE
genotoxic potency is the concentration of the test agent that
is at the midpoint of the rising slope of the tail moment
concentration-response curve. This value was determined
after regressing the data within the concentration range that
was not acutely cytotoxic. The genotoxic potency of IA was
8.70 µM (Table 2), and it expressed the highest genotoxicity
of the three monohaloacetic acids (Figure 8).
Com parative Responses and Possible Mechanism s of
Action. The cytotoxicity and genotoxicity of the mono-
haloacetic acids is expected to be related to the cellular uptake
and transport of the chemicals and their subsequent chemical
interaction with cellular macromolecules. Table 3 sum-
marizes several relevant physicochemical properties and
parameters that may be useful in understanding the mecha-
nistic basis of the observed data.
The ability of the monohaloacetic acids to cross cell
membranes is dependent on their lipophilicity, the degree
of ionization, and possible transport mechanisms. Consistent
with the rank order of their cytotoxicity and genotoxicity in
9
VOL. 38, NO. 18, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4 7 1 9

The chemical reactivity of monohaloacetic acids is
expected to be similar to that of methyl halides. With the
exception of methyl fluoride, methyl halides are potent
alkylating agents that react by an SN2 type of mechanism.
The reactivity of methyl halides is primarily dependent on
the carbon-halogen bond dissociation energy, which in turn
is related to the bond length. Since the atomic size of the
halogen follows the order I > Br > Cl, the length of carbon-
halogen bond increases and the bond dissociation energy
decreases (Table 3) accordingly. Polarizability and delocal-
ization of the electron cloud also contribute to making iodine
a better leaving group than bromine and a much better
leaving group than chlorine. Typically, the SN2 reactivity of
an alkyl iodide is 3-5× greater than an alkyl bromide, which
in turn is 50×greater than an alkyl chloride (41). Interestingly,
this relative SN2 reactivity is moderately correlated with
cytotoxicity in S. typhimurium and CHO cells, as well as
genotoxicity in CHO cells and mutagenic potency in S.
typhimurium (Table 4). This correlation is remarkably similar
to that observed in developmental toxicity using mammalian
whole embryo cultures (24, 42). A comparison of the potency
of developmental toxicity with the calculated lowest un-
occupied molecular orbital (ELUMO) of monohaloacetic acids
showed an inverse relationship, supporting the view that
electrophilic reactivity played an important role (42). This
relationship was also observed with the cytotoxicity and
genotoxicity expressed in both cell types (increased revertants/
µmole for S. typhimurium and decreased µM concentration
for the genotoxic potency measurement with CHO cells)
(Table 4).
of protective nucleophiles are rendered much more sus-
ceptible to other electrophilic carcinogens. In this respect,
it would be important to closely monitor the occurrence of
IA in drinking water and study the combination effects of IA
with other DBP genotoxins.
Im plications and Future Directions. Given the high
cytotoxic and genotoxic potency of IA in S. typhimurium
and CHO cells, further work is planned for the other four
iodoacids that have been identified as DBPs. Efforts are
currently underway to purify the synthetic standards of
bromoiodoacetic acid, (Z)-3-bromo-3-iodopropenoic acid,
and (E)-3-bromo-3-iodopropenoic acid, so that their purities
will be sufficiently high to conduct definitive cytotoxicity
and genotoxicity studies. We are in the process of synthesizing
(E)-2-iodo-3-methylbutenedioic acid in a pure, diacid form
for biological analysis (initially, the dimethyl ester was
obtained due to difficulty in synthesizing the diacid). Research
is planned to obtain quantitative concentration data for these
iodoacids in drinking water. In the nationwide occurrence
study, these iodoacids were found only in drinking water
from one location that was treated with chloramines (and
whose source waters were high in bromide/ iodide) (23).
Interestingly, the highest levels of iodinated THMs (tri-
halomethanes) (dichloroiodomethane, bromochloroiodo-
methane, dibromoiodomethane, chlorodiiodomethane, and
bromodiiodomethane) were also observed in this same
drinking water at a total of 18.7 µg/ L (which was 81% of the
concentration of the four regulated THMsschloroform,
brom oform , brom odichlorom ethane, and chlorodibro-
momethane) (23). These findings are consistent with earlier
work by Bichsel and von Gunten that predicted an increased
formation of iodinated DBPs in chloraminated drinking water,
based on the very slow rate of iodide oxidation to iodate by
chloramines, which allows hypoiodous acid (HOI) to ac-
cumulate and react with natural organic matter to form
iodinated DBPs (36, 37). Thus, it is likely that the highest
levels of iodoacids (and other iodo-DBPs) will not be in
chlorinated drinking waters but in chloraminated drinking
waters. This is in stark contrast to the effect chloramine
disinfection has on the formation of the regulated chloro/
bromo-THMs and haloacetic acids, where chloramination
significantly reduces their levels compared to chlorination.
As a result, chloraminated drinking waters that have high
bromide and iodide source waters will be targeted for this
future quantitative occurrence work to determine whether
the iodoacids commonly occur in high-iodide, chloraminated
drinking waters and at what levels they are present. Ulti-
mately, it will be important to know the levels at which these
iodoacids occur in order to assess the potential for adverse
environmental and human health risks. It should be noted
that the iodoacids identified in this study were from a plant
using chloramines only. For plants that have a significant
free chlorine contact time before the addition of ammonia
(to form chloramines), iodo-DBP formation may not be a
problem.
The toxic consequence of chemical interaction between
monohaloacetic acids and cellular constituents is dependent
on the sensitivity of the target tissue and the overall effect
on the whole organism. Like methyl halides, monohaloacetic
acids are expected to be relatively soft electrophiles, which
preferentially react with soft nucleophiles, such as thiol
groups of cysteinyl residues in proteins and glutathione (43).
IA is well-known as a metabolic inhibitor of glycolysis because
of inhibition of sulfhydryl rich glyceraldehyde-3-phosphate
dehydrogenase and can cause acute toxicity (40) via poisoning
of the heart and nerve cells (44, 45) as well as chromosome
aberrations due to the depletion of adenosine 5′-triphosphate
(ATP) (46, 47). Both IA and methyl iodide are potent depletors
of cellular glutathione (40, 48, 49), which is the key protective
nucleophile against cytotoxicity, oxidative stress, and elec-
trophilic attacks on the hard nucleophilic sites on purines
and pyrimidines. The level of intracellular glutathione is also
a key regulator for the induction of stress-activated signal
transduction pathways (50).
The finding that IA is the most potent genotoxic DBP in
mammalian cells may raise substantial concern as a potential
health hazard. IA has also been shown to be the most potent
haloacid in vitro toxicant in mammalian embryo cultures
(42). The carcinogenic potential of IA remains to be studied.
A limited study in 1953 showed that IA was a tumorigenesis
promoter or cocarcinogen on mouse skin (51). No other long-
term carcinogenesis studies have been conducted. The closely
related CA has been tested by the National Toxicology
Program (NTP) (52) (1992) by oral exposure in rats and mice
and shown to have no carcinogenic activity. The ability to
detect any potential carcinogenic activity of CA may have
been complicated by its acute myocardial toxicity. Although
IA is 1.8× more acutely toxic than CA in rats (40), the
substantially higher genotoxicity of IA (around 2 orders of
magnitude) may shift the delicate balance toward higher
potential for carcinogenic activity. Another closely related
compound, methyl iodide, is carcinogenic (53). Beyond
concern for IA as an individual chemical, the marked
depletion of cellular glutathione by IA is also a great concern
in the presence of other carcinogenic DBPs. Cells stripped
Acknowledgments
The authors thank Kearney T. W. Gunsalus and Danny
Ramirez for their assistance with the S. typhimurium
cytotoxicity and mutagenicity analysis of IA. We would also
thank Gene Crumley and Terrance Floyd at the U.S. EPA
National Exposure Research Laboratory for their assistance
with extractions, derivatizations, and analyses. Finally, we
wish to gratefully acknowledge the assistance of Dr. M. P.
Sudhakaran and the City of Corpus Christi Water Department
for providing drinking water samples and operational and
water quality data. Without their generous cooperation, this
study would not have been possible. This research was funded
in part by U.S. EPA Grant CR 83069501 (to M.J.P.). This paper
has been reviewed in accordance with the U.S. Environmental
9
4 7 2 0 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 18, 2004

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