Factors on the formation of strong mutagen [3-chloro-4-(dichloromethyl)- 5-hydroxy-2(5H)-furanone] MX by chlorination of syringaldehyde

Article abstract of DOI:10.1016/S0043-1354(00)00175-5

Syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde) was chlorinated in the laboratory at different reaction time, temperature, pH and chlorine dose, and MX was determined and quantified by GC/MS. The finding suggests that the optimal chlorination conditions are as follows: temperature = 298 K, pH = 5, Cl:syrin. (mol:mol) = 9:1. We also found that the formation of MX increased with the syringaldehyde concentration in a linear relationship. In addition, when syringaldehyde was chlorinated with a different chlorine dose in an alkaline condition, no MX was detected. This study will conduce to elucidating the kinetic process for the formation of MX from syringaldehyde and will be of great help for better control of MX in drinking water. Syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde) was chlorinated in the laboratory at different reaction time, temperature, pH and chlorine dose, and MX was determined and quantified by GC/MS. The finding suggests that the optimal chlorination conditions are as follows: temperature = 298 K, pH = 5, Cl:syrin. (mol:mol) = 9:1. We also found that the formation of MX increased with the syringaldehyde concentration in a linear relationship. In addition, when syringaldehyde was chlorinated with a different chlorine dose in an alkaline condition, no MX was detected. This study will conduce to elucidating the kinetic process for the formation of MX from syringaldehyde and will be of great help for better control of MX in drinking water. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0043-1354(00)00175-5

Wat. Res. Vol. 34, No. 17, pp. 4313±4317, 2000
7 2000 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(00)00175-5
0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
RESEARCH NOTE
FACTORS ON THE FORMATION OF STRONG MUTAGEN
[3-CHLORO-4-(DICHLOROMETHYL)-5-HYDROXY-2(5H)-
FURANONE] MX BY CHLORINATION OF
SYRINGALDEHYDE
YANG CHENGYONG1, CHEN ZHUO1, ZOU HUIXIAN1*, LU JUNHE1 and
ZHANG JINQI2
1Department of Environmental Science and Engineering, State Key Laboratory of Pollution Control
and Resource Reuse, Nanjing University, Nanjing, 210093, People's Republic of China and
2Department of Chemistry, Nanjing University, Nanjing, 210093, People's Republic of China
(First received 1 September 1999; accepted in revised form 4 February 2000)
AbstractÐSyringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde) was chlorinated in the laboratory at
di€erent reaction time, temperature, pH and chlorine dose, and MX was determined and quanti®ed by
GC/MS. The ®nding suggests that the optimal chlorination conditions are as follows:
temperature=298 K, pH=5, Cl:syrin. (mol:mol)=9:1. We also found that the formation of MX
increased with the syringaldehyde concentration in
a linear relationship. In addition, when
syringaldehyde was chlorinated with a di€erent chlorine dose in an alkaline condition, no MX was
detected. This study will conduce to elucidating the kinetic process for the formation of MX from
syringaldehyde and will be of great help for better control of MX in drinking water. 7 2000 Elsevier
Science Ltd. All rights reserved
Key wordsÐmutagen, MX, drinking water, precursor, syringaldehyde, chlorination
INTRODUCTION
tionated organic matter from Taihu Lake by sorp-
tion on a series of resin absorbents according to the
MX, a by-product of the chemical reactions that
method of Leenheer (1981) with some modi®-
occur in chlorinated drinking water, was found to
be one of the most potent direct-acting mutagens
by GC/MS, and characterized humic substances by
ever tested in Ames tests strain TA 100 (Hemming
et al., 1986), and accounted for up to 67% of the
overall mutagenity in chlorine disinfected drinking
cations, compared the yield of MX of each fraction
analyzing their oxidation products. From that study
(Huixian et al., 1999), we presumed and selected
probable precursors, and found the yield of MX
produced by syringaldehyde per mole was highest.
MX undergoes several reactions and transform-
waters (Smeds et al., 1997). MX was also found to
be a mutagen in bacteria (Holmbom et al., 1984)
and mammalian cells (Jansson and Hyttinen, 1994),
ations in water. The aqueous stability of the com-
to be a potent carcinogen and cause tumors at
pound has been studied by Backlund et al. (1989).
The reactions/transitions, which are highly pH
doses in both male and female rats (Komulainen et
al., 1997), and to induce DNA damage in HL-60
dependent, include tautomeric transformations from
ring to dissociated open chain form, isomerization
cells (Marsteinstredet et al., 1997). It is generally
assumed that MX is a product of the reaction of
of MX to its E-form and degradation of MX. In
this work, we have further studied the in¯uence of
chlorine with humic substances (Backlund et al.,
1988, 1989; Kronberg et al., 1988; Charles et al.,
chlorination (reaction time, temperature, pH and
chlorine dose) on the formation of MX by GC/MS
1992; Langvik and Hormi, 1994), phenolic com-
pounds (Langvik et al., 1991a), and some amino
acids (Horth et al., 1990). Xu et al. (1997a,b) frac-
in a solution containing 15 mg syringaldehyde and
the relationship between the yield of MX and syrin-
galdehyde concentration. It is conducive to screen-
ing the precursor of MX to a larger extent and
elucidating the kinetic process of the formation of
*Author to whom all correspondence should be addressed.
Fax: +86-25-3707304; e-mail: zhangjq@nju.edu.cn
4313

4314
Yang Chengyong et al.
umn was 30 m  0.25 mm  0.25 mm HP-5 fused silica
capillary column. Identi®cation of methylated MX was
based on position matching of retention time and some
characteristic ions, m/z 147, 107, 199, 201 and 203 ions
(Huixian et al., 1995).
MX. And the results may be also helpful for the
better control of MX formation in drinking water.
MATERIALS AND METHODS
Chlorination of syringaldehyde
RESULTS AND DISCUSSION
An 0.2 l bu€er solution containing 15 mg of syringalde-
hyde was chlorinated by the addition of freshly prepared
sodium hypochlorite/hypochlorous acid solution in glass
bottle. Chlorination was carried out in the dark. The fol-
lowing ranges of conditions were studied: pH, 2±
9.5(pH=2±4: CH3COONa and HCl; pH=5: CH3COONa
and CH3COOH; pH=6±9.5: H3PO4, NaH2PO4 and
Na2HPO4); temperature, 278±303 K; reaction time, 2±24 h;
syringaldehyde, 3±15 mg; Cl:syrin. (mol:mol) ratio, 3.0±
19±18.0.
Disappearance of syringaldehyde after chlorination
At the following ranges of conditions: pH, 2±9.5;
temperature, 278±303 K; reaction time, 2 h; syrin-
galdehyde, 15 mg; Cl:syrin. (mol:mol) ratio, 3.0±
18.0, more than 99% syringaldehyde disappeared.
At the chlorination condition (Cl:syrin.) (mol:mol)
ratio, 6.0; time, 5 min; pH, 5; temperature, 298 K),
syringaldehyde disappeared nearly to 99%. It
means that syringaldehyde reacts with chlorine very
easily.
Analysis of syringaldehyde
After chlorination, the aqueous solution was quenched
with a certain quantity of NaAsO2 and was extracted
three times with 20, 10, 10 ml of dichloromethane. The or-
ganic phase was concentrated to a small volume by rotary
vacuum evaporator. Quantitative analysis was performed
with gas chromatography-mass spectrometry by the in-
ternal standard method in selective ion monitoring. Identi-
®cation of syringaldehyde was based on position matching
of retention time and some characteristic ions, m/z 96,
111, 139, 167, 182 ions.
In¯uence of reaction time
Changes of yields of MX with time are shown in
Figs 1 and 2. Figure 1 shows that MX increases
with the reaction time at Cl:syrin. (mol:mol) ratio
of 6.0 in six di€erent temperatures (278±303). As
syringaldehyde reacted with chlorine in a short
time, there is no direct relationship between the dis-
appearance of syringaldehyde and the formation of
MX. In Fig. 2, MX decreases with the reaction time
at Cl:syrin. (mol:mol) ratio of 9.0±18.0, which
Analysis of MX
After chlorination, the aqueous solution was quenched
with a certain quantity of NaAsO2 and was extracted
three times with 20, 10, 10 ml of newly distilled diethyl
ether at pH=2. The organic phase was concentrated to a suggested that MX is formatted rapidly and
small volume by rotary vacuum evaporator and evapor-
degraded with excess chlorine at higher Cl:syrin.
ated to dryness by a gentle stream of nitrogen gas. The
ratio (Schenck et al., 1990).
extracts were methylated by 0.6 ml saturated BF3 in
methanol in a 958C water bath for 1 h. The reaction mix-
ture was neutralized with 1 ml 2% (w/v) NaHCO3, and
In¯uence of chlorine dose
then extracted three times with 0.5, 0.4, 0.3 ml n-hexane
sequentially. The n-hexane extract was concentrated to
0.1 ml under a gentle nitrogen gas ¯ow for later analysis.
Quantitative analysis was performed with gas chromatog-
raphy-mass spectrometry by the internal standard method
in selective ion monitoring. The gas chromatography col-
The e€ect of the applied chlorine dose on the for-
mation of MX was investigated using a wide range
of chlorine to syringaldehyde mole ratio from 3:1 to
18:1 (Fig. 3). The formation of MX is found to
Fig. 1. Formation of MX as a function of reaction time.
(pH=5, Cl:syrin.=6:1).
Fig. 2. Formation of MX as a function of reaction time.
(pH=5, T = 298 K).

MX formation by chlorinating syringaldehyde
4315
chlorine and produces additional compounds. The
formation of MX is usually favored by chlorination
at acidic conditions, but as to easy degrading
organics such as syringaldehyde, the MX yield
decreased at a lower pH condition. And at alkaline
and neutral conditions, we cannot detect the MX.
In this case, the syringaldehyde reacts with chlorine
and produces other compounds than MX. And at
pH=7.2, the formed MX reacts quickly with chlor-
ine (Langvik et al., 1991b). Further study in this
area is needed.
In¯uence of temperature
The range of drinking water temperature is usual
according to the real chlorination temperature of
our drinking water. In this study, we used six di€er-
ent temperatures (278, 283, 288, 293, 298 and
303 K) to investigate the formation of MX. The
yield of MX against T was plotted in Fig. 5. At
lower temperatures (278 or 283 K), the formation
of MX is small. As the reaction temperature
increases, the formation of MX becomes more
Fig. 3. Formation of MX as a function of chlorine dose
(pH=5, T = 298 K).
depend strongly on the applied chlorine concen- rapid and reaches the largest quantity at 298 K, but
tration: it increased as the mole ratio was increased when the temperature increases continually, the for-
to 9:1, and decreased as the weight ratio was mation of MX becomes less. It may be because that
increased to 18:1. This may indicate that excess at high temperatures the procedure of the formation
chlorine reacts further with formed MX and pro- of MX by the chlorination of syringaldehyde
duces other compounds (Schenck et al., 1990).
becomes more complicated and comprises more
unknown reactions.
In¯uence of pH
In¯uence of syringaldehyde concentration
The e€ect of pH on MX formation is very com-
plex due to the complicated reaction mechanism
Five 0.2 l bu€er solution (pH=5.0) containing
(Fig. 4). At acidic conditions, we can detect the di€erent amount of syringaldehyde (3, 6, 7.5, 9 and
compound of MX. And the optimal pH value for 12 mg) was chlorinated at a chlorine:syringaldehyde
the formation of MX is 5.0, which is contradictory ratio of 9:1 and 298 K. Two contact times (2 and
to the results reported by Langvik et al. (1991a). It 4 h) are investigated. Figure 6 shows the linear re-
may be because that when the pH value decreases, lationship between MX and the concentration of
MX produced from syringaldehyde reacts with
Fig. 4. Formation of MX as a function of pH (T = 298 K, Fig. 5. Formation of MX as a function of temperature.
Cl:syrin.=9:1).
(pH=5, Cl:syrin.=6:1).

4316
Yang Chengyong et al.
mation of Ames mutagenicity and of the strong bac-
terial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-
2(5H)-furanon and other halogenated compounds
during disinfection of humic water. Chemosphere 17,
1329±1336.
Backlund P., Wondergem E., Voogd K. and de Jong Ad
(1989) In¯uence of chlorination pH and chlorine dose
on the formation of mutagenic activity and the strong
bacterial
mutagen
3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanon (MX) in water. Chemosphere 18,
1903±1911.
Charles M. J., Gong C., Kannlgantl R. and Marbury G.
D. (1992) High-resolution mass spectrometry method
for the analysis of 3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanon in water. Environ. Sci. Technol.
26, 1030±1035.
Hemming J., Holmbom B., Reunanen M. and Kronberg
L. (1986) Determination of the strong mutagen 3-
chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in
chlorinated drinking and humic waters. Chemosphere
15(5), 549±556.
Holmbom B. R., Voss R. H., Mortimer R. D. and Wong
A. (1984) Fractionation, isolation, and characterization
of ames-mutagenic compound present in kraft chlori-
nation e‚uents. Environ. Sci. Technol. 18, 333.
Horth H., Fielding M., Jams H. A., Thomas M. J., Gib-
son T. and Wilcox P. (1990) Production of organic
chemical and mutagens during chlorination of amino
acids in water. In Water Chlorination: Chemistry En-
vironmental Impacts and Health E€ects, vol. 6, eds R. L.
Joley, L. W. Condie, J. D. Johnson, S. Katz, R. A.
Minnear, J. S. Mattice and V. A. Jacobs, pp. 107±125.
Lewis Publishers, Chelsea, MI, USA.
Fig. 6. Formation of MX as a function of syrin. concen-
tration (pH=5, T = 298 K, Cl:syrin.=9:1).
syringaldehyde. It indicates that the formation of
MX per weight of syringaldehyde is constant and
does not change with the concentration of syringal-
dehyde in the other conditions.
Huixian Z., Xu X., Jinqi Z. and Zhen Z. (1995) The deter-
mination of strong mutagen MX[3-chloro-4-(dichloro-
methyl)-5-hydroxy-2(5H)-furanone] in drinking water in
China. Chemosphere 30, 2219±2225.
Huixian Z., Juhne L., Zhuo C., Chengyong Y. and Jinqi
Z. (2000) Screening the precursor of strong mutagen [3-
chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone]
MX From Chlorinated Water. Wat. Res. 34, 225±229.
Jansson K. and Hyttinen J. M. (1994) Induction of gene
mutation in mammalian cells by 3-chloro-4-(dichloro-
methyl)-5-hydroxy-2(5H)-furanone in (MX) a chlorine
disinfection by-products in drinking water. Mutat. Res.
322, 129±132.
Komulainen H., Kosma V. M., Vaittinen S. L., Vartiainen
T., Korhonen E. K., Lotjonen S., Tuominen R. K. and
Tuomisto (1997) Carcinogenicity of the drinking water
mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-
furanone in the rat. Journal of the National Cancer Insti-
tute 89(12), 848±856.
Kronberg L., Holmbom B., Reunanen M. and Tikkanen
L. (1988) Identi®cation and quanti®cation of the Ames
mutagenic compound 3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanone and of its geometric isomer E-
2-chloro-3(dichloromethyl)-4-oxobutenoic acid in chlor-
ine-treated humic water and drinking water extracts.
Environ. Sci. Technol. 22, 1097±1103.
CONCLUSION
According to our previous research work (Xu et
al., 1997a,b; Huixian et al., 1999), syringaldehyde is
the major producer of oxidized humic materials and
on the basis of our result of screening the precur-
sors of MX, syringaldehyde is the compound which
is chlorinated to yield the maximum MX at the
same chlorination conditions. From our study, the
relationship between MX and the concentration of
syringaldehyde is linear and the optimal conditions
for the formation of MX by chlorination of syrin-
galdehyde are as follows: pH, 5.0; Cl:syrin. (mol/
mol), 9.1: T, 298 K and t, 6 h. Syringaldehyde
reacts with chlorine very quickly and there is no
direct relationship between the disappearance of
syringaldehyde and the formation of MX. The most
ecient way to decrease the MX of drinking water
is to make our endeavor to wipe o€ the humic ma-
terials before chlorination. By altering the chlori-
nation conditions, we can also reduce the formation
of MX. The results of this study will also be helpful
for further studying the kinetic process of the for-
mation of MX and be conducive to screening the
precursors of MX to a larger extent.
Langvik V. A., Hormi O., Tikkanen L. and Holmbom B.
(1991a) Formation of the mutagen 3-chloro-4-(dichloro-
methyl)-5-hydroxy-2(5H)-furanone and related com-
pounds by chlorination of phenolic compounds.
Chemosphere 22, 547±555.
Langvik V. A., Holmbom B. and Tikkanen L. (1991b)
Reactivity of the mutagen 3-chloro-4-(dichloromethyl)-
5-hydroxy-2(5H)-furanone (MX). Chemosphere 23, 873±
880.
AcknowledgementsÐThis study is funded by the National
Natural Sciences Foundation of the P.R. of China.
Langvik V. A. and Hormi O. (1994) Possible reaction
pathway for the formation of 3-chloro-4-(dichloro-
methyl)-5-hydroxy-2(5H)-furanone (MX). Chemosphere
28, 1111±1117.
REFERENCES
Backlund P., Kronberg L. and Tikkanen L. (1988) For-
Leenheer J. A. (1981) Comprehensive approach to pre-

MX formation by chlorinating syringaldehyde
4317
parative isolation and fractionation of dissolved organic Smeds A., Vartiainen T., Makipaakkanen J. and Kronberg
carbon from natural waters and waste waters. Environ.
Sci. Technol. 15, 578±587.
L. (1997) Concentration of Ames mutagenic chlorohy-
droxyfuranones and related compounds in drinking
waters. Environ. Sci. Technol. 31, 1033±1039.
Xu X., Lin L., Huixian Z., Yongbin L., Liansheng W. and
Jinqi Z. (1997a) Study on the precursors of strong
mutagen [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-
furanone] by chlorination of fractions from di€erent
waters. Chemosphere 35, 1709±1716.
Xu X., Huixian Z. and Jinqi Z. (1997b) Formation of
strong mutagen [3-chloro-4-(dichloromethyl)-5-hydroxy-
2(5H)-furanone] MX by chlorination of fractions of
lake water. Wat. Res. 31, 1021±1026.
Marsteinstredet U., Brunborg G., Bjoeras M., Soederlund
E., Seeberg E., Kronberg L. and Holme J. A. (1997)
DNA damage induced by 3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanone (MX) in HL-60 cells and puri-
®ed DNA in vitro. Mutation Research 390(12), 171±178.
Schenck K. M., Meier J. R., Ringhand H. P. and Kop¯er
F. C. (1990) Recovery of 3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanone from water samples on XAD
resins and the e€ect of chlorine on its mutagenicity.
Environ. Sci. Technol. 24, 863±867.