Halonitromethane Drinking Water Disinfection Byproducts: Chemical Characterization and Mammalian Cell Cytotoxicity and Genotoxicity

Article abstract of DOI:10.1021/es030477l

Halonitromethanes are drinking water disinfection byproducts that have recently received a high priority for health effects research from the U.S. Environmental Protection Agency (EPA). Our purpose was to identify and synthesize where necessary the mixed halonitromethanes and to determine the chronic cytotoxicity and the acute genotoxicity of these agents in mammalian cells. The halonitromethanes included bromonitromethane (BNM), dibromonitromethane (DBNM), tribromonitromethane (TBNM), bromochloronitromethane (BCNM), dibromochloronitromethane (DBCNM), bromodichloronitromethane (BDCNM), chloronitromethane (CNM), dichloronitromethane (DCNM), and trichloronitromethane (TCNM). Low- and high-resolution gas chromatography/mass spectrometry (GC/MS) was used to identify the mixed chloro-bromonitromethanes in finished drinking waters, and analytical standards that were not commercially available were synthesized (BDCNM, DBCNM, TBNM, CNM, DCNM, BCNM). The rank order of their chronic cytotoxicity (72 h exposure) to Chinese hamster ovary (CHO) cells was DBNM > DBCNM > BNM > TBNM > BDCNM > BCNM > DCNM > CNM > TCNM. The rank order to induce genomic DNA damage in CHO cells was DBNM > BDCNM > TBNM > TCNM > BNM > DBCNM > BCNM > DCNM > CNM. The brominated nitromethanes were more cytotoxic and genotoxic than their chlorinated analogues. This research demonstrated the integration of the procedures for the analytical chemistry and analytical biology when working with limited amounts of sample. The halonitromethanes are potent mammalian cell cytotoxins and genotoxins and may pose a hazard to the public health and the environment.

Full text of DOI:10.1021/es030477l

Environ. Sci. Technol. 2004, 38, 62-68
Introduction
Halonitromethane Drinking Water
Disinfection Byproducts: Chemical
Characterization and Mammalian
Cell Cytotoxicity and Genotoxicity
The disinfection of drinking water was a major public health
improvement during the 20th century that reduced the
transmission of deadly waterborne diseases (1). Drinking
water disinfection byproducts (DBPs) are an unintended
consequence of chemical disinfection and are formed during
water treatment when a disinfectant reacts with organic
matter and/ or bromide that is naturally present in the water.
In 1974 the first DBPs were identified in chlorinated drinking
water (2). Two years later, a U.S. EPA survey showed that
chloroform and other trihalomethanes (THMs) were ubiq-
uitous in chlorinated drinking water (3); the same year, the
National Cancer Institute published results linking chloro-
form to cancer in laboratory animals (4). The U.S. EPA
regulated chloroform and other THMs under the Safe
Drinking Water Act (5). In almost 30 years following the
identification of chloroform, hundreds of DBPs have been
identified (6). Chloropicrin (trichloronitromethane) was the
first of the halonitromethanes to be identified as a DBP and
has been measured in several previous studies (7-10),
including EPA’s Information Collection Rule monitoring effort
(11). The first brominated halonitromethane, bromopicrin
(tribromonitromethane), was identified approximately 10
years ago and found at e2 µg/ L levels in a high-bromide
water that was ozonated (12). Two other brominated
analogues, dibromochloronitromethane and dichlorobro-
monitromethane, had been found previously in reactions of
chlorine with model compounds containing nitro groups
(13) but had not yet been identified as DBPs in drinking
water. In 1999, additional brominated halonitromethanes
(bromonitromethane and dibromonitromethane) were iden-
tified in waters treated with ozone-chlorine and chlorine
alone, along with bromopicrin and chloropicrin (14, 15). Most
of these were also found in drinking water treated with
chlorine or chloramines only (without ozone) but at much
lower levels, indicating that ozone may play an important
role in their formation. Earlier Hoigne´ and Bader also
observed this effect of ozone on the increased formation of
chloropicrin (7). In addition, when water with elevated
bromide levels (1 ppm) was treated with ozone-chlorine or
ozone-chloramines, a shift to more brominated species was
observed (15). With low bromide levels, chlorinated species
dominated (chloro-, dichloro-, and trichloronitromethane);
with elevated bromide levels, tremendous increases were
observed for brominated species (bromo-, dibromo-, bro-
mochloro-, bromodichloro-, dibromochloro-, and tribro-
monitromethane) (15). Although there is some limited
knowledge now regarding the formation of these halo-
nitromethanes in drinking water, little is known about their
occurrence and toxicity.
M I C H A E L J . P L E W A , *
E L I Z A B E T H D . W A G N E R , A N D
P A U L I N A J A Z W I E R S K A
Department of Crop Sciences, College of Agricultural,
Consumer and Environmental Sciences, University of Illinois
at Urbana-Champaign, 1101 West Peabody Drive,
Urbana, Illinois 61801
S U S A N D . R I C H A R D S O N A N D
P A U L H . C H E N
National Exposure Research Laboratory, U.S. Environmental
Protection Agency, Athens, Georgia 30605
A . B R U C E M C K A G U E
CanSyn Chem. Corp., 200 College Street,
Toronto, Ontario M5S 3E5, Canada
Halonitromethanes are drinking water disinfection
byproducts that have recently received a high priority for
health effects research from the U.S. Environmental
Protection Agency (EPA). Our purpose was to identify and
synthesize where necessary the mixed halonitromethanes
and to determine the chronic cytotoxicity and the acute
genotoxicity of these agents in mammalian cells. The
halonitromethanes included bromonitromethane (BNM),
dibromonitromethane (DBNM), tribromonitromethane (TBNM),
bromochloronitromethane (BCNM), dibromochloroni-
tromethane (DBCNM), bromodichloronitromethane (BDCNM),
chloronitromethane (CNM), dichloronitromethane (DCNM),
and trichloronitromethane (TCNM). Low- and high-
resolution gas chromatography/mass spectrometry
(GC/MS) was used to identify the mixed chloro-bromo-
nitromethanes in finished drinking waters, and analytical
standards that were not commercially available were
synthesized (BDCNM, DBCNM, TBNM, CNM, DCNM, BCNM).
The rank order of their chronic cytotoxicity (72 h exposure)
to Chinese hamster ovary (CHO) cells was DBNM >
DBCNM > BNM > TBNM > BDCNM > BCNM > DCNM
> CNM > TCNM. The rank order to induce genomic
DNA damage in CHO cells was DBNM > BDCNM > TBNM
> TCNM > BNM > DBCNM > BCNM > DCNM >
CNM. The brominated nitromethanes were more cytotoxic
and genotoxic than their chlorinated analogues. This
research demonstrated the integration of the procedures
for the analytical chemistry and analytical biology when
working with limited amounts of sample. The haloni-
tromethanes are potent mammalian cell cytotoxins and
genotoxins and may pose a hazard to the public health and
the environment.
To target DBPs for future health effects studies, the U.S.
EPA prioritized a list of ∼500 DBPs according to predicted
adverse health effects (16). The three bromonitromethanes
that had been identified at the time (bromo-, dibromo-, and
tribromonitromethane) received the highest ranking and
were subsequently included in a nationwide occurrence
study. Shortly following this ranking, the remaining five
halonitromethanes (chloro-, dichloro-, bromochloro-, bromo-
dichloro-, dibromochloronitromethane) were identified in
ozone-chlorine-treated waters (results reported here) and
were added as target analytes to the nationwide occurrence
study. Quantitative methods developed for the measurement
of halonitromethanes in the nationwide occurrence study
involved the use of liquid-liquid extraction-GC-electron
capture detection (ECD) as the primary method, with solid-
phase extraction-GC/ MS and purge-and-trap-GC/ MS used
* Corresponding author phone: (217)333-3614; fax: (217)333-
8046; e-mail: mplewa@uiuc.edu.
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6 2 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 1, 2004
10.1021/es030477l CCC: $27.50
2004 Am erican Chem ical Society
Published on Web 11/27/2003

for confirmation of selected species (17). Results from the
nationwide study supported the finding that ozonation
plays an important role in trihalonitromethane formation.
Preozonation, in many cases, increased the formation of
trihalonitromethanes (following treatment with secondary
chloramines) (18). Halonitromethanes ranged from 0.1 to 3
µg/ L, with tribromo-, bromodichloro-, dibromochloro-,
dibromo-, bromochloro-, and bromonitromethane observed
(waters high in bromide were targeted in this study) (17, 18).
The genotoxicity of three halonitromethanesschloro-
nitromethane, dichloronitromethane, and trichloronitro-
methaneshave been previously reported (19-22). These
halonitromethanes were more mutagenic in Salmonella
typhimurium than the corresponding halomethanes, and
the bromo-substituted nitromethanes were generally more
mutagenic than their chlorinated analogues (23, 24). We
reported the first preliminary data on the chronic cytotoxicity
of bromo-, dibromo-, and tribromonitromethane and the
acute genotoxicity of bromo- and dibromonitromethane in
mammalian cells (25).
nitromethane was prepared from the ammonium salt of
methyl nitroacetate, as reported for the ethyl ester (30), as
follows: sulfuryl chloride (7.4 g, 0.55 mol) in ether (10 mL)
was added to a stirred suspension of the powdered salt (6.8
g, 0.05 mol) in ether (50 mL) at room temperature. After 15
min, the ammonium chloride was filtered and the filtrate
washed with water, dried, and concentrated to give crude
methyl chloronitroacetate (7 g). The crude product was
refluxed with 10% hydrochloric acid (HCL) (14 mL) with
stirring for 1.5 h. The product was cooled, extracted with
ether, washed with water, and dried. The ether was removed
by distillation through a 10 cm Vigreux column, the column
rem oved, and the product distilled to give chloroni-
tromethane (1.6 g), bp 122-123 °C (lit. bp 122-123 °C) (31).
Dichloronitromethane was prepared from the ammonium
salt of methyl nitroacetate as follows: chlorine (3.6 g, 0.05
mol) was bubbled into a stirred solution of the salt (6.8 g,
0.05 mol) in water (60 mL) over 10 min at room temperature.
After another 10 min, the insoluble product oil was extracted
with ether, washed with water, and dried. Anhydrous
ammonia was bubbled into the ether solution of the product,
and the ammonium salts of unreacted methyl nitroacetate
and methyl chloronitroacetate were filtered. The salts were
dissolved in water and chlorinated again as described above.
The ether extract of the second chlorination was treated again
with anhydrous ammonia to remove unreacted nitroacetates.
This filtrate was combined with the filtrate from the first
chlorination, and the combined filtrates were washed with
water, dried, and concentrated to give crude methyl dichlo-
ronitroacetate (6.3 g). The crude product was refluxed with
20% HCL (13 mL) with stirring for 3.5 h. The product was
processed as described above for chloronitromethane to give
dichloronitromethane (2.2 g), bp 108-110 °C (lit. bp 110-
112 °C, 108-110 °C) (32). Bromochloronitromethane was
prepared as follows: anhydrous ammonia was bubbled into
an ether solution of crude methyl chloronitroacetate (6.1 g,
0.04 mol) prepared as described above. The resultant
ammonium salt (6.5 g) was dissolved in water (65 mL) and
treated with bromine (6.4 g, 0.04 mol) with stirring at room
temperature. After 10 min, the insoluble product oil was
extracted with ether, dried, and concentrated to give crude
methyl bromochloronitroacetate (7 g). The product was
refluxed with 20% sulfuric acid (15 mL) with stirring for 3 h.
After workup as described above for chloronitromethane and
removal of most of the ether by distillation through a 10 cm
Vigreux column, the residue (5 g) was fractionated on silica
gel (150 g) and the product eluted with 9:1 pentane:ether.
The solvent was distilled through the column, the column
removed, and the product distilled to give bromochloroni-
tromethane (0.7 g), bp 94-97 °C at 160 mm. Purities of these
synthesized halonitromethanes by GC using flame ionization
detection were as follows: chloronitromethane (97%), dichlo-
ronitromethane (99%), bromodichloronitromethane (92%),
dibromochloronitromethane (97%), tribromonitromethane
(96%), and bromochloronitromethane (90%).
The objectives of this research were to (i) identify the
mixed chloro-bromo-nitromethanes in drinking water, (ii)
synthesize halonitromethane analytical standards that were
not commercially available, (iii) report the analytical chem-
istry for the analysis and identification of this class of DBPs,
(iv) analyze and rank order the cytotoxic potency of these
agents in mammalian cells, and, (v) determine the genotoxic
potency of the halonitromethanes in mammalian cells.
Experimental Section
Drinking Water Sam ples. Drinking water samples treated
with ozone-chlorine, ozone-chloramines, chloramine, and
chlorine were collected from a pilot plant in Jefferson Parish,
LA, which used Mississippi River water as the raw water source
(2.6-3.0 mg/ L total organic carbon; 0.054 mg/ L bromide).
Samples (75 L for analytical identification work) were acidified
to pH 2 and concentrated immediately after collection using
XAD-8 over XAD-2 resins, eluted with ethyl acetate and
concentrated to 1 mL using rotary evaporation followed by
evaporation under nitrogen (14).
GC/MS Analysis. GC/ MS analyses with electron ionization
(EI) were performed on a VG 70-SEQ high-resolution mass
spectrometer equipped with a HP 5890A gas chromatograph.
The high-resolution mass spectrometer was operated at an
accelerating voltage of 8 kV and at resolutions of 1 000 and
10 000 for low- and high-resolution experiments, respectively.
Perfluorokerosene (pfk) was used as the mass calibrant.
Injections of 1-2 µL of the extracts (or standard solutions in
ethyl acetate) were introduced via a split/ splitless injector
onto a J&W Scientific DB-5 column (30 m, 0.25 mm i.d., 0.25
µm film thickness). The GC temperature program consisted
of an initial temperature of 35 °C for 4 min, followed by a rate
increase of 9 °C/ min to 285 °C, which was held for 30 min.
An injection port temperature of 170 °C and a GC/ MS transfer
line temperature of 200 °C were used to analyze trihaloni-
tromethanes (to prevent thermal decomposition) (26).
Chem ical Standards. Bromonitromethane (90% pure) and
trichloronitromethane (98% pure) were purchased from
Aldrich Chemical Co. (St. Louis, MO). Dibromonitromethane
(98% pure) was provided by Majestic Research (Athens, GA)
(15). Bromodichloronitromethane and dibromochloroni-
tromethane were prepared by the addition of nitromethane
to commercial bleach, to which a solution of bromine in 6
M NaOH had been previously added (27). The products were
separated by fractional distillation under reduced pressure.
Tribromonitromethane was prepared by bromination of
nitromethane in 1 M NaOH (28). The reaction product was
isolated by steam distillation to give colorless material, bp
85-86 °C at 20 mm (lit. 85 °C at 17 mm) (29). Chloro-
Cell Culture Medium and Biologicals. General laboratory
reagents were purchased from Fisher Scientific Co. (Itasca,
IL) and Sigma Chemical Co. (St Louis, MO). Media supplies
and fetal bovine serum (FBS) were purchased from Hyclone
Laboratories (Logan, UT). Clone 11-4-8 of Chinese hamster
ovary (CHO) cell line AS52 were maintained in Ham’s F12
medium containing 5% FBS, 1% antibiotics (100 U/ mL
sodium penicillin G, 100 µg/ mL streptomycin sulfate, 0.25
µg mL amphotericin B, 0.85% saline), and 1% glutamine at
37 °C in a humidified atmosphere of 5% CO2 (33). For long-
term storage, the cells were frozen in FBS:dimethyl sulfoxide
(9:1, v/ v) and kept at -80 °C.
Microplate Cytotoxicity Assay. Chronic cytotoxicity to
mammalian cells was measured using a modification of an
assay we developed for the analysis of DBPs (25, 34). Flat-
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FIGURE 1. Electron ionization mass spectra for bromochloronitromethane (a), dichloronitromethane (b), and chloronitromethane (c).
bottom, tissue culture 96-well microplates were employed;
eight replicate wells were prepared for each concentration
of a specific DBP. Eight wells were reserved for the blank
control consisting of 200 µL of F12 medium + 5% FBS. The
negative control consisted of eight wells containing 100 µL
of a titered CHO cell suspension (3 × 104 cells/ mL) plus 100
µL F12 + FBS. The wells for the remaining columns contained
3000 CHO cells, F12 + FBS, and a known concentration of
a DBP for a total of 200 µL. To prevent crossover contamina-
tion between wells due to volatilization of the test agent, a
sheet of sterile AlumnaSeal (RPI Corporation, Mt. Prospect,
IL) was pressed over the wells before the microplate was
covered. The plate was placed on a rocking platform for 10
min to uniformly distribute the cells, and the microplate was
placed in a tissue culture incubator for 72 h. Each well was
gently aspirated, fixed in 100% methanol for 10 min, and
stained for 30 min with a 1% crystal violet solution in 50%
methanol. The plate was gently washed, and 50 µL of DMSO
was added to each well for 30 min. The plate was analyzed
in a BioRad microplate reader at 595 nm. The data were
automatically recorded and transferred to an Excel spread-
sheet in a microcomputer connected to the microplate reader.
Single-Cell Gel Electrophoresis (SCGE) Assay. The day
before treatment, 2 × 104 CHO cells were added to each
microplate well in 200 µL of F12 + 5% FBS and incubated.
The next day the cells were washed with Hank’s balanced
salt solution (HBSS) and treated with the halonitromethane
in F12 medium without FBS in a total volume of 25 µL for
4 h at 37 °C, 5% CO2. The wells were covered with sterile
AlumnaSeal. After incubation, the cells were washed 2× with
HBSS and harvested with 50 µL of 0.005% trypsin + 53 µM
EDTA. The trypsin was inactivated with 70 µL of F12 + FBS.
A 10 µL aliquot was removed to measure acute cytotoxicity
using 10 µL of 0.05% trypan blue vital dye in phosphate-
buffered saline (PBS). SCGE data were not used if the acute
cytotoxicity exceeded 30%. The remaining cell suspension
from each well was embedded in a layer of low-melting point
agarose prepared with PBS upon clear microscope slides that
were previously coated with a layer of 1% normal melting
point agarose prepared with deionized water and dried
overnight. The detailed methods for preparing and electro-
phoresing the SCGE slides were published previously (34).
The cellular membranes were removed by an overnight
immersion in lysing solution at 4 °C. The slides were placed
in an alkaline buffer (pH 13.5) in an electrophoresis tank,
and the DNA was denatured for 20 min. The slides were
electrophoresed at 25 V, 300 mA (0.72 V/ cm) for 40 min at
4 °C. The slides were removed, neutralized with Tris buffer,
pH 7.5, rinsed in cold water, dehydrated in cold methanol,
dried at 50 °C, and stored at room temperature in a covered
slide box. For analysis, the slides were hydrated in cold water
for 20 min and stained with 65 µL of 2 µg/ mL ethidium
bromide for 3 min. The slides were rinsed in cold water and
analyzed with a Zeiss fluorescence microscope with an
excitation filter of BP 546/ 10 nm and a barrier filter of 590
nm. For each experiment two slides were prepared per
treatment group. The slides were coded, and 25 randomly
chosen nuclei were analyzed in each slide using a charge-
coupled device camera. A computerized image analysis
system (Komet version 3.1, Kinetic Imaging Ltd., Liverpool,
U.K.) was employed to determine the tail moment (integrated
value of migrated DNA density multiplied by the migration
distance) of the nuclei as an index of DNA damage. The
digitalized data were automatically transferred to a computer-
based spreadsheet for subsequent statistical analysis.
Safety and Data Handling. Manipulations of toxic and
mutagenic chemicals were conducted in certified biological/
chemical safety hoods. In general, for the chronic cytotoxicity
assays, the experiments were repeated at least twice with a
minimum of eight independent replicates for each chemical
concentration per experiment, and for the SCGE assay three
experiments were conducted with six slides analyzed per
treatment group. The median tail moment value for each
slide was determined, and the data from all of the slides
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TABLE 1. Important Mass Spectral Ions for Halonitromethanes
compd
monoisotopic mol wt
important MS ions (m/z, relative abundance)
brom onitrom ethane
chloronitrom ethane
139
95
30(41), 43(4), 44(13), 46(10), 79(10), 81(10),93(100), 95(95)
43(9), 44(10), 46(12), 49(100), 51(42)
brom ochloronitrom ethane
173
43(23), 46(9), 47(18), 48(38), 49(11), 50(15), 79(13), 81(13), 92(12),
94(12), 127(87), 129(100), 131(37)
dibrom onitrom ethane
217
43(18), 46(10), 79(30), 81(30), 92(45), 94(45), 122(4), 124(4), 158(2),
160(4), 162(2), 171(67), 173(100), 175(63)
dichloronitrom ethane
brom odichloronitrom ethane
129
207
43(22), 46(8), 47(15), 48(33), 49(7), 50(12), 83(100), 85(71), 87(13)
36(12), 46(20), 47(41), 49(19), 61(20), 63(12), 77(8), 79(17), 81(15),
82(36), 84(25), 91(8), 93(8), 126(17), 128(23), 130(9), 161(70),
163(100), 165(55), 167(13)
dibrom ochloronitrom ethane
251
295
163
36(7), 46(11), 47(27), 49(12), 61(6), 63(4), 79(20), 81(20), 91(18),
93(18), 105(4), 107(4), 126(30), 128(34), 130(11), 170(6), 172(10),
174(7), 205(55), 207(100), 209(77), 211(24)
36(13), 38(5), 46(6), 79(22), 81(22), 91(35), 93(35), 105(6), 107(7),
121(10), 123(10), 170(24), 172(42), 174(23), 249(44), 251(100),
253(98), 255(41)
tribrom onitrom ethane (brom opicrin)
trichloronitrom ethane (chloropicrin)
46(1), 47(28), 49(9), 61(10), 63(8), 65(2), 82(33), 84(21), 86(4),
117(100), 119(96), 121(30)
TABLE 2. CHO Cell Chronic Cytotoxicity Induced by the Halonitromethanes
1
compd
ANOVA test statistic
%C /2 value (M)a
r2 b
rank order
brom onitrom ethane
F11,68 ) 23.19
P e 0.001
F8,78 ) 56.60
P e 0.001
F9,17 ) 19.72
P e 0.001
F16,95 ) 41.53
P e 0.001
F16,47 ) 35.24
P e 0.001
F19,91 ) 16.54
P e 0.001
F18,47 ) 15.24
P e 0.001
F31,96 ) 10.25
P e 0.001
7.06 × 10-6
6.09 × 10-6
8.57 × 10-6
5.29 × 10-4
3.73 × 10-4
5.36 × 10-4
4.05 × 10-5
1.32 × 10-5
6.88 × 10-6
0.85
3
dibrom onitrom ethane
tribrom onitrom ethane
chloronitrom ethane
0.99
0.99
0.87
0.91
0.94
0.82
0.81
0.90
1
4
8
7
9
6
5
2
dichloronitrom ethane
trichloronitrom ethane
brom ochloronitrom ethane
brom odichloronitrom ethane
dibrom ochloronitrom ethane
F28,78 ) 29.21
P e 0.001
a
b
The %C1/2 value is the chem ical concentration that induced a 50% reduction of the cell density as com pared to the negative control. r2 refers
to the fit of the regression analysis upon which the %C1/2 value was calculated.
representing each halonitromethane concentration were
averaged. Averaged median values express a normal distri-
bution according to the central limit theorem and were used
with a one-way analysis of variance test (35). If a significant
F value of P e 0.05 was obtained, a Dunnett’s multiple
comparison versus the control group analysis was conducted.
In most cases, the power of the test statistic (â) was g0.8 at
R ) 0.05. To analyze the strength of association between
pairs of variables, we used the Pearson Product Moment
Correlation test.
not trivial. Only bromonitromethane and trichloro-
nitromethane were found in the NIST or Wiley spectral library
database. The spectra generated here were recently submitted
to NIST for inclusion in an upcoming database release.
Another complicating factor was that none of the haloni-
tromethanes show molecular ions in their mass spectra; the
molecular weight information and the overall composition
of the structures are missing. Each of these compounds loses
•
a NO2 , leaving the remaining fragment as the highest mass
+
ion (e.g., tribromonitromethane loses NO2• to form a CBr3
as the highest mass ion). There is only the slightest hint of
the presence of the NO2 in their structures, with small (usually
e10% relative abundance) m/ z 46 ions in their mass spectra.
The presence of another ion at m/ z 43 also helped to identify
these compounds. This ion was present in the mono- and
dihalonitromethanes and is typically a C3H7 group for most
compounds. However, the m/ z 43 ion in this case was due
to the uncommon CHNO+ ion, as determined by high-
resolution EI-MS (observed exact mass ) 43.006 Da, theo-
retical mass for CHNO ) 43.006 Da; theoretical mass for
C3H7 ) 43.055 Da). These data suggested the presence of an
NO2 group in the structure of these compounds. Mass spectra
of three halonitromethanes (bromochloro-, dichloro-, and
chloronitromethane) are shown in Figure 1; mass spectra of
the other halonitromethanes can be found in a recent
Results and Discussion
The chemistry and toxicity of DBPs have been investigated
for over a quarter century. However, many important classes
of DBPs, such as the halonitromethanes, have not been
adequately studied for their cytotoxicity and genotoxicity.
We integrated the analytical chemistry with the analytical
biology in the first comprehensive evaluation of the toxicity
and genotoxicity of halonitromethane DBPs using an in vitro
mammalian cell system.
Analytical Chem istry. The first identification of chloro-,
dichloro-, bromochloro-, bromodichloro-, and dibromo-
chloronitromethane in drinking water is reported here. They
were primarily found in waters treated with ozone-chlorine.
In general, the identification of the halonitromethanes was
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VOL. 38, NO. 1, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 6 5

FIGURE 2. Log-linear plot of the chronic CHO cell cytotoxicity induced by the halonitromethanes and the positive controls EMS and
potassium bromate. The statistical analysis of these data are presented in Table 2.
FIGURE 3. Log-linear plot of the SCGE tail moment data showing genomic DNA damage induced by the halonitromethanes and the positive
controls EMS and potassium bromate. The statistical analysis of these data are presented in Table 3.
publication that details their thermal decomposition behavior
when analyzed by GC or GC/ MS (26). Table 1 lists important
mass spectral ions for the halonitromethanes. After we
tentatively identified nine halonitromethanes, we purchased
standards for the commercially available bromonitromethane
and trichloronitromethane and synthesized the others. GC/
MS retention times and mass spectra of the pure standards
matched those in our drinking water samples, confirming
their identities.
The trihalonitromethanes required special conditions for
detection. They are thermally unstable and decompose under
commonly used GC injection port temperatures (200-250
°C) (12), forming radical ions and radical reaction products
(26). The trihalonitromethanes also thermally decompose in
the GC/ MS transfer line at commonly used transfer line
temperatures (g250 °C), producing mass spectra that are
mixtures of the undecomposed parent compound and
decomposition products. For example, at a GC/ MS transfer
line temperature of 290 °C, tribromonitromethane exhibited
a mixed mass spectrum composed of tribromonitromethane
and bromoform. The trihalonitromethanes also show unusual
[fragment + 1]+ ions in their mass spectra, which can
complicate their identification. We recommend that low GC
injection port temperatures (e170 °C) and low transfer line
temperatures (e225 °C) be used for their analysis (26). This
behavior was observed only for the trihalonitromethanes and
not for the mono- or dihalonitromethanes.
Brominated nitromethanes were also found in the recent
U.S. Nationwide DBP Occurrence Study. Individual haloni-
tromethanes ranged from 0.1 to 3 µg/ L, with tribromo-,
9
6 6 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 1, 2004

TABLE 3. CHO Cell Genotoxicity Induced by the Halonitromethanes
compd
ANOVA test statistic
SCGE genotoxic potency (M)a
r2 b
rank order
brom onitrom ethane
F14,42 ) 85.96
P e 0.001
1.36 × 10-4
0.88
5
dibrom onitrom ethane
tribrom onitrom ethane
chloronitrom ethane
F11,54 ) 39.43
2.62 × 10-5
6.99 × 10-5
2.15 × 10-3
4.21 × 10-4
9.34 × 10-5
1.65 × 10-4
6.32 × 10-5
1.43 × 10-4
0.94
0.95
0.99
0.97
0.86
0.97
0.98
0.95
1
3
9
8
4
7
2
6
P e 0.001
F14,43 ) 9.02
P e 0.001
F9,27 ) 60.58
P e 0.001
F8,36 ) 61.21
P e 0.001
F10,20 ) 25.76
P e 0.001
F7,22 ) 30.42
P e 0.001
F6,14 ) 13.3
P e 0.001
F8,29 ) 9.02
P e 0.001
dichloronitrom ethane
trichloronitrom ethane
brom ochloronitrom ethane
brom odichloronitrom ethane
dibrom ochloronitrom ethane
a
b
The genotoxic potency is the chem ical concentration at the m idpoint of the SCGE concentration-response curve. r2 refers to the fit of the
regression analysis upon which the SCGE genotoxic potency value was calculated.
bromodichloro-, dibromochloro-, dibromo-, bromochloro-,
and bromonitromethane observed (waters high in bromide
were targeted in this study) (17, 18). It is likely that bromo-
and mixed chloro-bromo-nitromethanes are present in
many drinking waters treated with ozone-chlorine, ozone-
chloramines, chlorine, or chloramines and that they have
been largely undetected due to the difficulty in identifying
them, the lack of analytical standards, and the special
instrumental conditions required for their identification.
Analytical Biology. For the first time, the chronic cell
cytotoxicity and acute genotoxicity in a mammalian system
of a complete set of bromo- and chloro-substituted halo-
nitromethanes were determined. The microplate cytotoxicity
assay measured the reduction in cell density as a function
of the halonitromethane concentration over a 72 h period.
A reduction in cell density could be due to a disruption in
the cell cycle, growth retardation, or cell toxicity. We refer
to the halonitromethane concentration that causes a 50%
reduction of the cell density as compared to the negative
control as the %C1/ 2 value rather than the LC50.
We compared the chronic cytotoxicity of the halonitro-
methanes in CHO cells with qualitative cytotoxicity reported
for S. typhimurium (23, 24) and found no significant
correlation (r ) 0.60; P ) 0.09). This is similar to data
published with the bromo- and chloro-substituted haloacetic
acids in that one cannot use Salmonella cytotoxicity data to
predict mammalian cell cytotoxicity (34, 36).
There are very few studies on the genotoxicity of haloni-
tromethanes in mammals or mammalian cells. SCGE is a
very sensitive assay that can quantitatively determine ge-
nomic DNA damage (37). The halonitromethanes were potent
mammalian cell genotoxins (Figure 3). The SCGE genotoxic
potency was calculated for each halonitromethane as the
midpoint in the concentration-response curves (Figure 3,
Table 3). All nine of the halonitromethanes exceeded the
genotoxicity of the positive control mutagen EMS in a range
from 260× for dibromonitromethane to 3× for chloro-
nitromethane. The halonitromethanes were more potent
DNA-damaging agents than potassium bromate. Potassium
bromate was reported as an oxidative stress-inducing geno-
toxin (38) and animal carcinogen (39). The genotoxic potency
of potassium brom ate was 7.2 m M (34); the haloni-
tromethanes were between 275× and 3× more genotoxic
(dibromonitromethane and chloronitromethane, respec-
tively). From a structure-function perspective, the bromi-
nated nitrom ethanes and the m ixed brom o-chloro-
nitromethanes were more genotoxic than the chlorinated
nitromethanes. The mono-, di-, and tribrominated ni-
tromethanes were 16×, 16×, and 1.3× more genotoxic than
their chlorinated analogues. A comparison of the mutage-
nicity of these halonitromethanes in S. typhimurium (23, 24)
and genomic DNA damage in CHO cells exhibited no
significant correlation (r ) -0.003; P ) 0.99). These data
indicate that mutagenicity in S. typhimurium cannot predict
genotoxicity in mammalian cells, and this conclusion agrees
with that reported for the haloacetic acids (34). The haloni-
tromethane DBPs, especially the bromo-substituted forms,
are potent mammalian cell genotoxins and may pose a hazard
to the public health and the environment.
The data from repeated cytotoxicity experiments were
averaged and plotted (Figure 2), and regression analysis was
used to calculate the %C1/ 2 value for each halonitromethane
(Table 2). There was an approximately 100-fold range in the
%C1/ 2 values from
a value of 6.09 µM for dibromo-
nitromethane to 536 µM for trichloronitromethane (Table
2). All of the halonitromethanes were more cytotoxic than
the positive controls ethylmethanesulfonate (EMS) (%C1/ 2
)4.19 mM) and potassium bromate (%C1/ 2 )964 µM) (Figure
2). The rank order in declining toxicity demonstrated that
the brominated nitromethanes were more cytotoxic than
their chlorinated analogues. It appeared that the multi-
brominated halonitromethanes were the most toxic. For both
the bromo- and chloronitromethanes, the rank order in
declining cytotoxicity was the di-, mono-, and trihalogenated
forms (Table 2). In previous work we found that the
brominated haloacetic acids were more cytotoxic than their
chlorinated analogues (34). In all cases, the halonitromethanes
were more cytotoxic than the corresponding haloacetic acids.
Bromonitromethane and chloronitromethane were 1.25×
and 1.8× more cytotoxic than bromoacetic acid and chloro-
acetic acid, respectively. Dichloronitromethane and trichloro-
nitromethane were more cytotoxic than dichloroacetic acid
and trichloroacetic acid (29.5× and 32.6×, respectively). The
results were the most striking for dibromonitromethane and
tribromonitromethane, 83× and 116× more cytotoxic than
dibromoacetic acid and tribromoacetic acid.
Concentrations observed in the nationwide occurrence
study (17, 18) can help to put the relative toxicities of the
halonitromethanes in perspective. For example, the maxi-
mum occurrence of the monobrominated halonitromethane
species (bromonitromethane) was 0.3 µg/ L, whereas the
maximum occurrence of the corresponding haloacetic acid
(bromoacetic acid) was 1.6 µg/ L. For this maximum occur-
rence, bromoacetic acid was 5.3× higher in concentration,
9
VOL. 38, NO. 1, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 6 7

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but bromonitromethane was 1.26× more cytotoxic and 8×
more genotoxic. Similarly, the maximum occurrence of the
dichloronitromethane was approximately 1 µg/ L, as com-
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Acknowledgments
This paper has been reviewed in accordance with the U.S.
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Received for review May 22, 2003. Revised manuscript re-
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ES030477L
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