Environ. Sci. Technol.

2007, 41, 160-165

non- or slowly biodegradable influent substrates, degradation

Effect of Ammonia Nitrogen and intermediates, end products, and soluble microbial products
Dissolved Organic Matter Fractions (4). On one hand, some of the DOM are toxic themselves (4,
5), on the other hand, some forms of DOM react with chlorine
on the Genotoxicity of Wastewater to form toxic DBPs (2, 4). Therefore, only measuring the
formation of typical DBPs is not sufficient to investigate the
Effluent during Chlorine Disinfection effect of chlorination on the ecological safety of wastewater.
The application of short-term genotoxicity tests for
LI-SHA WANG, HONG-YING HU,* AND wastewater is helpful in monitoring genotoxic DBPs and in
CHAO WANG checking for the presence of unknown substances with
Environmental Simulation and Pollution Control State Key genotoxic properties (6-7). Although some researches have
Joint Laboratory, Department of Environmental Science and studied the effect of chlorination on wastewater with the
Engineering, Tsinghua University, Beijing 100084, PR China Ames test, there are no consistent conclusions. Studies by
Meier (8) and Crebelli (9) demonstrated that chlorination of
secondary effluents did not play a substantial part in
mutagenicity. However, Meier’s (10) later study and Fukui’s
Chlorine is a widely used disinfectant which prevents the (11) study showed that chlorination resulted in substantial
spread of harmful pathogens when reusing wastewater, increases in mutagenicity in some wastewater. Research
but harmful byproducts might be formed and cause adverse conducted by Nakamuro (12) on the Yodo river in Japan
ecological and health effects. In this study, the potential which had received the effluent of a wastewater treatment
plant indicated that chlorination could reduce mutagenicity.
effects of chlorination on the genotoxicity of different
The inconsistencies among these results suggest that chlo-
biologically treated wastewater samples were investigated rination can influence wastewater genotoxicity in a way which
using the umu test. For the first time, ammonia nitrogen (NH3s is specific to the wastewater samples used. The aim of this
N) was found to significantly influence genotoxicity study was, therefore, to investigate the effect of chlorine
during wastewater chlorination. After chlorination, the disinfection on genotoxicity for different kinds of biologically
genotoxicity decreased in wastewater with a low NH3sN treated wastewater and also to clarify the frequency of
concentration (<10∼20 mg/L), but it increased notably genotoxic effects, by using the in vitro umu test with the
in wastewater with a high NH3sN concentration (>10∼20 genetically modified strain TA1535/pSK1002 of S. typhimu-
mg/L). By fractionating the DOM (dissolved organic rium which allows to measure the induction of SOS repair
matter) in wastewater into different fractions, it was response after contact with genotoxic compounds.
found that the hydrophilic substances (HIS) fraction of
DOM was the key fraction involved in decreasing genotoxicity Materials and Methods
during the chlorination of wastewater with a low NH3s Wastewater Samples. Wastewater samples used in this study
N concentration, while the hydrophobic acids (HOA) fraction were collected from the effluents of different domestic
of DOM was the key fraction involved in increasing wastewater treatment and reclamation plants before disin-
genotoxicity during chlorination of wastewater with a fection, in which activated sludge process (AS), anaerobic-
anoxic-oxic process (AAO) and membrane bioreactor (MB)
high NH3sN concentration. Furthermore, fluorescence
were used as the treatment methods. The samples were
spectroscopy analysis on different fractions indicated that immediately delivered to the laboratory after sampling and
some free or combined aromatic amino acids might filtered to eliminate any suspended solid before use.
produce highly genotoxic byproducts during the chlorination Chlorination Treatment. Chlorine disinfection was con-
of wastewater with a high NH3sN content, and this was ducted within 24 h of sampling. A series of 600 mL glass
then demonstrated through experiments on the chlorination bottles with Teflon inner plugs were prepared, and each bottle
of free aromatic amino acids. was filled with about 580 mL of the sample wastewater. The
pH of the samples was adjusted to 7.0 ( 0.2 with 1 M H2SO4
or 2 M NaOH solution, then 12.5 mL of buffer solution (34.0
Introduction g/L KH2PO4 and 35.5 g/L Na2HPO4) was added to keep the
pH stable during disinfection. Different concentrations of
Wastewater reclamation and reuse is a viable and attractive
available chlorine were added into the corresponding reaction
approach to solving water shortages in many countries. In
bottles by using a NaClO solution with a concentration of 5
order to prevent the spread of harmful pathogens in reclaimed
g available chlorine per liter. The bottles were sealed and
wastewater, chlorine disinfection has been widely used, but
kept in a dark isothermal chamber (20 °C) for 30 min. All of
many researchers have reported that chlorine could react
the chemical reagents used were of an analytical purity.
with dissolved organic matter (DOM) to produce numerous
disinfection byproducts (DBPs) with genotoxic, mutagenic Water Quality Measurements. The pH value of the
and/or carcinogenic activity (1-3). wastewater samples was measured in the field during the
For chlorination of drinking water, most researchers sampling process, and other parameters were measured after
focused on the reaction of chlorine with natural organic sampling and filtration. Concentration of chemical oxygen
matter (NOM) in the formation of typical DBPs, such as demand (COD) was analyzed by a closed reflux digestion
trihalomethanes (THMs) and haloacetic acids (HAAs). How- and a titrimetric method. Concentration of ammonia nitrogen
ever, compared to drinking water, there are many more types (NH3sN) was determined by colorimetry using the nessler-
of DOM in biologically treated wastewater, including residual ization method. Concentration of dissolved organic carbon
(DOC) was detected using a TOC analyzer (model TOC-5000A,
* Corresponding author phone: (+86-10)6279-4005; fax: (+86- Shimadzu, Japan). UV absorbance at 254 nm (UV254) was
10)6277-1472; e-mail: [email protected]. measured with a photospectrometer (model UV-2401, Shi-
160 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 1, 2007 10.1021/es0616635 CCC: $37.00  2007 American Chemical Society
Published on Web 11/18/2006
madzu, Japan). Residual chlorine was measured by DPD
colorimetric method (13). TABLE 1. Characteristics of Wastewater Samples Used in This
Concentration of Wastewater Samples. Wastewater Study
samples before and after disinfection treatments were COD NH3sN DOC UV254 genotoxicitya
acidified to pH 2 with 2 M H2SO4, and passed through resin samples pH (mg/L) (mg/L) (mg/L) (m-1) (µg 4-NQO/L-sample)
cartridges which were filled with 1 g of CHP20P resin
A(AAO) 8.3 10.2 1.1 5.6 16.4 13.8 ( 0.7
(Mitsubishi Chemical Corporation, Japan). The cartridges
B(AS) 8.1 12.0 23.3 10.6 15.9 4.5 ( 0.6
had previously been washed with 10 mL of methanol, 10 mL C(AS) 7.8 11.5 14.6 5.7 14.2 10.1 ( 0.4
of acetone and 20 mL of distilled water. Five hundred D(AS) 7.9 6.1 4.5 7.4 13.3 20.9 ( 2.9
milliliters of each sample was loaded on a cartridge, then E(MB) 8.3 6.7 2.7 5.7 12.9 12.5 ( 1.4
dried under a flow of nitrogen and eluted with 4 mL of F(AS) 8.2 24.4 3.8 16.8 15.1 56.5 ( 3.7
acetone. The eluate was reduced to a small volume by vacuum G(MB) 7.8 10.4 0.9 7.0 13.8 5.9 ( 0.5
evaporation and then dried under a nitrogen flow. The dry a Mean ( Standard deviation (based on triplicate analyses). AS,
residue was dissolved in 2 mL of dimethylsulfoxide (DMSO) activated sludge process; AAO, anaerobic-anoxic-oxic process; MB,
and stored in the dark at -20 °C before used in the umu test. membrane bioreactor.
Umu Test. The umu test for genotoxicity was performed
with Salmonella typhimurium TA1535/pSK1002 without S9
activation according to ISO 13829 (14). Concentrated samples
were submitted to the test and carried out in triplicates.
During each test, a series of 4-NQO (4-nitroquinoline-N-
oxide) reference samples with different concentrations were
run concurrently to obtain the dose-effect curve of 4-NQO.
The genotoxicity of the concentrated samples was standard-
ized to an equivalent 4-NQO concentration and it was then
divided by concentration ratio to get the genotoxicity value
of the samples before concentration.
Fractionation. Methods for isolating hydrophilic sub-
stances (HIS), hydrophobic acids (HOA), hydrophobic neu-
trals (HON), and hydrophobic bases (HOB) fractions of DOM
are specified by Huang (15) and briefly described as follows:
Five liters of wastewater sample passed through XAD 8
column and the column was back-flushed with 0.1 N HCl to FIGURE 1. Change in genotoxicity of wastewater samples after
elute HOB fraction. Then the previous effluent from the chlorination with differing chlorine dosages. Error bars represent
column was acidified to pH 2 and passed through the column the standard deviation based on triplicate analyses. Characteristics
again. The effluent was HIS fraction and the column was of samples Α∼E are shown in Table 1.
back-flushed with 0.1 N NaOH to elute HOA fraction. Finally,
HON fraction adsorbed in the XAD 8 resin was Soxhlet-
extracted with anhydrous methanol. After fractionation, the TABLE 2. DOC and UV254 of the Fractions of Samples F and Ga
pH value of each fraction was adjusted to 7.0 ( 0.2 and then sample F sample G
MilliQ water was added to each fraction to increase the
DOC (mg/L) UV254 (m-1) DOC (mg/L) UV254 (m-1)
volume to 5 liters.
Fluorescence Spectroscopy. Fluorescence spectra were HIS 5.5 7.9 4.1 8.8
recorded on a fluorescence spectrophotometer (model HOA 5.1 5.3 1.6 4.6
F-2500, Hitachi, Japan). Three-dimensional spectra were HON 2.7 1.8 0.7 0.5
obtained by measuring the emission spectra in the range HOB 3.5 1.5 0.6 0.1
from 240 to 600 nm repeatedly, and at the excitation a HIS, hydrophilic substances; HOA, hydrophobic acids; HON,

wavelengths from 220 to 420 nm, at 5 nm intervals in the hydrophobic neutrals; HOB, hydrophobic bases.
excitation domain. Spectra were then concatenated into an
excitation emission matrix (EEM). The three-dimensional
plots and contour maps were produced using the OriginPro increased, and finally, the genotoxicity of sample C did not
7.5 program. All contour maps were plotted using the same change that much. After analyzing their water quality data
scale range of fluorescence intensities and number of (Table 1), it is found that the NH3sN concentration of samples
contours. A, D, and E was much lower than that of sample B, and that
the NH3sN concentration of sample C was midway between
Results and Discussion them. The NH3sN concentration was, therefore, hypoth-
esized to be the key parameter resulting in the differing
Characteristics of the Wastewater Samples Used. Table 1
patterns of genotoxic change during chlorination.
shows the characteristics and genotoxicity of wastewater
samples Α∼G. Their genotoxicity ranged within equivalent To investigate the effects of ammonia nitrogen, NH4Cl
of 4.5-56.5 µg of 4-NQO per liter, and there were no obvious was added to samples D and E which had low NH3sN
relationships between the genotoxicity and the value of COD, concentrations originally. After samples D and E with altered
NH3-N, DOC, and UV254. Since the effluents from biological NH3sN concentrations were disinfected with 10 and 30 mg/L
wastewater treatment systems contain a variety of types of of chlorine for 30 min, both the genotoxicity and the total
DOM (4), the genotoxicity was considered to derive from residual chlorine were measured (see Figure 2). From Figure
complex mixtures and, therefore, could not easily be 2(a) and (b), it can be seen that the genotoxicity of chlorinated
estimated from the conventional water quality indices. wastewater D and E did increase obviously with an increasing
Effect of Chlorination on Wastewater Genotoxicity. The NH3sN concentration. When NH3sN was low (<10∼20 mg/
genotoxicity of samples Α∼E after disinfection with differing L), the genotoxicity after disinfection was lower than that
chlorine dosages is shown in Figure 1. It can be seen that before disinfection, and when NH3-N was high (>10∼20
with chlorine dosage increasing, the genotoxicity of samples mg/L), the genotoxicity after disinfection was higher than
A, D, and E decreased, while the genotoxicity of sample B that before disinfection.

VOL. 41, NO. 1, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 161

FIGURE 2. Changes in the genotoxicity and total residual chlorine of samples D and E with altered ammonia nitrogen concentrations after
chlorination. (a) Genotoxicity of sample D, dashed line represents the genotoxicity of sample D before chlorination. (b) Genotoxicity of
sample E, dashed line represents the genotoxicity of sample E before chlorination. Different scales are used on the vertical axes of (a)
and (b) panels. (c) Total residual chlorine of sample D. (d) Total residual chlorine of sample E. Error bars represent the standard deviation
based on triplicate analyses.

FIGURE 3. Changes in genotoxicity after chlorination of sample F, sample G and their fractions following the addition of differing amounts
of ammonia nitrogen. (a) Genotoxicity of sample F and its fractions. (b) Genotoxicity of sample G and its fractions. SF, sample F without
fractionation; SG, sample G without fractionation; HIS, hydrophilic substances; HOA, hydrophobic acids; HON, hydrophobic neutrals; HOB,
hydrophobic bases. Error bars represent the standard deviation based on triplicate analyses.

During chlorination, ammonia nitrogen quickly reacted under a low NH3sN concentration could reduce the geno-
with free chlorine and changed it to combined chlorines toxicity of wastewater, while disinfection of the wastewater
(chloramines). Because the reactivity of combined chlorines with free chlorine under a high NH3sN concentration might
is much weaker than that of free chlorine, combined chlorines increase the genotoxicity.
were more slowly consumed during the reaction with DOM It should be noted that a recently discovered DBP,
in wastewater. This is the reason why the total residual iodoacetic acid, found in U.S. drinking water treated with
chlorine after 30 min chlorination increased with an increas- chloramines turned out to be the most toxic DBP ever found
ing NH3sN concentration as shown in Figure 2(c) and (d). (18). Choi (19) pointed out that chloramines could produce
Because of the weak reactivity of combined chlorines, N-nitrosodimethylamine as a DBP. Zheng (20) reported that
ammonia nitrogen was reported to decrease the formation chlorination of publicly owned treatment works secondary
of typical DBPs, such as THMs and HAAs during chlorination effluent containing residual ammonia could lead to chlo-
of drinking water and wastewater (16, 17). However, the ramination of organic compounds and the resulting produc-
results in this study suggest that disinfection with free chlorine tion of cyanogen chloride and free cyanide. All of these

162 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 1, 2007

FIGURE 4. Fluorescence spectroscopy of fractions of sample F before chlorination. HIS, hydrophilic substances; HOA, hydrophobic acids;
HON, hydrophobic neutrals; HOB, hydrophobic bases.

FIGURE 5. Fluorescence spectroscopy of fractions of sample G before chlorination. HIS, hydrophilic substances; HOA, hydrophobic acids;
HON, hydrophobic neutrals; HOB, hydrophobic bases.

findings warned us that chlorination with a high NH3sN Effect of Chlorination on the Change in Genotoxicity of
concentration or chloramination of wastewater might be Different DOM Fractions. In order to discover the main
potentially harmful to the ecological system. precursors in causing the changes in wastewater genotoxicity,

VOL. 41, NO. 1, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 163

TABLE 3. EEM Peaks for the Fractions of Samples F and G
sample F sample G
Exmax/Emmax Intensity at Exmax/Emmax Exmax/Emmax Intensity at Exmax/Emmax
peak (nm/nm) (AU) (nm/nm) (AU) substance
HIS1 280/314 708.5 275/308 969.2 soluble microbial byproduct-like
HIS2 285/412 1173.0 humic/fulvic acid-like
HIS3 320/405 922.4 325/408 1400.0 humic/fulvic acid-like
HOA1 225/295 1312.8 225/301 690.8 tyrosine-like, aromatic protein
HOA2 230/347 1870.1 230/349 1101.0 tryptophan-like, aromatic protein
HOA3 280/315 1028.8 275/307 859.3 soluble microbial byproduct-like
HOA4 275/355 1225.3 280/364 694.5 soluble microbial byproduct-like
HON1 225/306 186.8 tyrosine-like, aromatic protein
HON2 225/341 1635.0 230/348 837.7 tryptophan-like, aromatic protein
HON3 280/314 893.4 275/308 671.1 soluble microbial byproduct-like
HON4 275/338 962.5 280/348 337.7 soluble microbial byproduct-like
HON5 265/446 376.6 humic/fulvic acid-like
HOB1 225/294 389.0 tyrosine-like, aromatic protein
HOB2 225/342 1058.0 225/343 162.1 tryptophan-like, aromatic protein
HOB3 275/308 528.5 275/308 609.0 soluble microbial byproduct-like

samples F and G were fractionated into four fractions. For

both sample F and sample G, HIS and HOA were dominant
in the DOC and UV254 value (Table 2). DOC is typically used
as an aggregate measure for the content of organic matters
and UV254 mainly reflects the content of unsaturated aromatic
organics which are able to react with chlorine and produce
DBPs (21). Therefore, HIS and HOA were considered to be
the two main parts of DOM which might result in the change
in genotoxicity.
With the aim of studying the effect of ammonia nitrogen
on changes in genotoxicity during chlorination, NH4Cl was
added to sample F and each of its fractions to get an NH3sN
concentration of 3.8 mg/L (the same as that in sample F) and
10 mg/L, and NH4Cl was added to sample G and each of its
fractions to get an NH3sN concentration of 0.9 mg/L (the FIGURE 6. Effect of chlorination on the genotoxicity of three aromatic
same as that in sample G) and 30 mg/L. After the samples amino acids in the presence of different amounts of NH3sN. Phe,
were chlorinated with 10 mg/L of chlorine for 30 min, the Phenylalanine; Tyr, Tyrosine; Try, Tryptophan. Error bars represent
genotoxicity was measured as shown in Figure 3. the standard deviation based on triplicate analyses.
From Figure 3(a) and (b), it can be clearly seen that the
change in genotoxicity for samples F and G was consistent more aromatic protein than other fractions. Since some
with the previous result in this paper. For the four fractions amino acids were reported to produce much more cyanogen
from sample F and sample G, the genotoxicity of HIS and halides when NH3sN concentrations are higher (23), it is
HOA was higher than that of HON and HOB before possible for some of the free or combined amino acids to
chlorination, and during chlorination, the change in geno- produce more genotoxic byproducts when NH3sN concen-
toxicity of HIS and HOA was greater than that of HON and trations are higher and thus result in an increase in
HOB, which proves the conclusion above that HIS and HOA genotoxicity.
are the two main fractions causing the change in genotoxicity. Genotoxicity of Twenty Amino Acids after Chlorination
After chlorination with a low NH3sN concentration, the With and Without NH3sN. Twenty amino acids: glycine,
genotoxicity of both HIS and HOA decreased noticeably, while alanine, valine, leucine, isoleucine, methionine, phenylala-
when NH3sN increased to a relatively high concentration, nine, tyrosine, tryptophan, serine, proline, threonine, cys-
the genotoxicity of HIS still decreased, but the genotoxicity teine, asparagine, glutamine, lysine, histidine, arginine,
of HOA increased noticeably. This indicated that HIS played aspartate, and glutamate were dissolved in MilliQ water to
a key role in the decrease of genotoxicity during chlorination a concentration of 0.1 mmol/L and NH4Cl was added to get
with a low NH3sN concentration and HOA played a key role NH3sN concentrations of 0, 50, and 150 mmol/L. Then all
in the increase of genotoxicity during chlorination with a the solutions were chlorinated with 10 mmol/L chlorine for
high NH3sN concentration. 30 min, and the genotoxicity of the chlorinated amino acids
Fluorescence Spectroscopy of the Different DOM Frac- was measured. Only three aromatic amino acids, phenyla-
tions. In order to find out why the genotoxicity of different lanine (Phe), tyrosine (Tyr), and tryptophan (Try) displayed
fractions changed so nonuniformly, EEM was used to an obvious genotoxicity after chlorination (see Figure 6). It
characterize the fractions of samples F and G. The contour could be seen that when NH3-N concentration was higher,
maps of the results are shown in Figure 4 and Figure 5. It can the genotoxicity of Phe did not change much, while the
be seen that the there were different peaks for different genotoxicity of Tyr and Try increased noticeably. These results
fractions. The position of Exmax/Emmax and the intensity at validated the supposition that after chlorination, more
Exmax/Emmax for these peaks is shown in Table 3. The genotoxic chlorinated amino acids could be formed when
substances which the peaks represent are also listed in Table NH3sN concentration was higher. Furthermore, because
3 according to the literature (22). some PBTA (2-phenylbenzotriazole) compounds, another
From Table 3, it was found that HIS contained more kind of chlorinated amines, were also found to be highly
humic/fulvic acid than other fractions, and HOA contained genotoxic (24), further efforts on genotoxicity evaluation and

164 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 1, 2007

chemical identification of chlorinated amines should be (12) Nakamuro, K.; Ueno, H.; Sayato, Y. Mutagenic activity of organic
performed. concentrates from municipal River water and sewage effluent
after chlorination or ozonation. Water Sci. Technol. 1989, 21,
1895-1898.
Acknowledgments (13) APHA, AWWA, WEF. Standard methods for the examination of
This study was supported by National Natural Science Fund water and wastewater, 19th ed., 4500-Cl G. DPD Colorimetric
of China (NSFC) and NSFC-JST joint-project (nos. 20510076, Method; Eaton, A. D., Clesceri, L. S., Greenberg, A. E., Eds.;
20477021, and 20277025). We are grateful to Dongbin Wei APHA: Washington DC, 1995; pp 45-47.
(14) International Standard Organisation. Water QualitysDeter-
and Jie Tian for their helpful comments on this manuscript. mination of the Genotoxicity of Water and Waste Water Using
the umu-Test, 1st ed., ISO 13829; Geneva: Switzerland, 2000;
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