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133
[Environmental Health and Preventive Medicine 8, 133–138, September 2003]
Original Article
Validity of Mutagenic Activity as an Indicator of River Water Pollution
H. TSUKATANI1, Y. TANAKA1, N. SERA1, N. SHIMIZU1, S. KITAMORI1 and N. INOUE2
1Fukuoka Institute of Health and Environmental Sciences, Fukuoka, Japan
2Department of Hygiene, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Abstract
Objectives: To investigate if mutagenicity could be expressed by known water pollution indicators,
we determined the mutagenic activity of blue rayon extracts from sampled river water with the Ames
test utilizing new strains of bacteria, and compared the results with those of known indicators of water
pollution.
Methods: Water samples were collected by the blue rayon adsorption method at sixteen sites in six
rivers in the North Kyushu district. The Assay of mutagenicity was carried out using the Ames test.
The test strains were Salmonella typhimurium TA100, YG1024, YG1041 and YG1042. B(a)P, Trp-P-1
and Trp-P-2 were quantified by HPLC. Determinations of SS, BOD, COD, T-N, T-P, DOC, and A260/
DOC were performed.
Results: The extracts from five sampling sites showed higher mutagenicity toward strain YG1024
with or without S9mix, and the extracts from two of these five sites showed higher mutagenicity toward
strain YG1041 with and without S9mix. However, the water pollution indicators did not show specific
trends that were consistent with the mutagenic activity.
Conclusions: Since the mutagenic activity of river water could not be predicted using known
water pollution indicators, we recommend that biological examinations such as mutagenicity tests be
added to the indicators that are currently in use.
Key words: mutagenicity; Ames test; YG strains; river water; sewage
Introduction
Numerous organic pollutants have been found in the water
environment (1–4). Their individual concentrations are very
low, but certain unidentified substances may be mutagenic or
carcinogenic. Detection of the combined effects of different
chemicals of low concentrations is very difficult. Therefore, a
mutagenicity test that is able to evaluate river water syntheti-
cally has been developed (1–5).
New strains of bacteria that show high levels of enzyme
activity and that are sensitive to the mutagenic effects of some
nitro-aromatic compounds have been established (6). Further,
an adsorbent specific for some kinds of mutagens has been
developed (7). Therefore, it is possible to determine mutagenicity
in river water more sensitively and efficiently. To confirm the
importance of the mutagenicity test as a water pollution
indicator, it is necessary to perform the mutagenicity test
repeatedly comparing the results with those of known water
pollution indicators.
Blue rayon is a rayon preparation using copper phthalocy-
anine trisulfonate as a covalently linked ligand (7). It adsorbs
compounds with three or more fused rings. Although this
method does not yield quantitative results, it is a simple method
for extracting chemical compounds and can be applied to a
greater number of samples for river water pollution tests than
other methods (8).
To certify the validity of the mutagenicity test as a water
pollution indicator, we determined the mutagenic activity of
blue rayon extracts from the sampled river water and examined
whether the mutagenicity could be detected from the results
using known water pollution indicators.
Materials and Methods
Sampling
Water samples were collected by the blue rayon adsorption
method as described below, at sixteen sites in six rivers (Tatara
River, Naka River, Mikasa River, Hae River, Doumen River,
and Siragane River) in the North Kyushu district.
Blue rayon in plastic mesh bags (1 g per bag) was hung in
Received Jan. 28 2003/Accepted Jun. 3 2003
Reprint requests to: Hiroko TSUKATANI
Fukuoka Institute of Health and Environmental Sciences, 39 Mukaizano,
Dazaifu, Fukuoka 818-0135, Japan
TEL: +81(92)921-9948, FAX: +81(92)928-1203
Environ. Health Prev. Med. Validity of Mutagenicity as a Water Pollution Indicator
134
the river. Three bags were attached to a wooden float, and the
depth of the hanging bag was about 20 cm. After 24 h, the blue
rayon was taken out, washed with distilled water, dried with
paper towels, and, later on, processed for mutagenic assay and
chemical analysis by HPLC.
While the blue rayon was being hung in the river, we took
water samples from each site and chemically analyzed them to
estimate the condition of river water pollution (water pollution
indicators). The analysis included suspended solid (SS),
biochemical oxygen demand (BOD), chemical oxygen demand
(COD), dissolved organic carbon (DOC), absorbance at
260 nm/DOC (A260/DOC), total nitrogen (T-N), and total phos-
phorus (T-P). The analyses of SS, BOD, COD, T-N and T-P for
waters of rivers or lakes are provided by the water pollution
prevention law in Japan (1970). A260/DOC is useful as a
predictor of trihalomethane (THM) formation potential, which
was defined as the amount of THM formed in the water by
chlorination (9).
The sampling was carried out in March 1997.
Solvent extraction
The dried blue rayon was eluted with methanol-
concentrated ammonia (50:1, 160 ml/g blue rayon) for 30 min
by gentle shaking. The elution was repeated, and eluents were
combined. The solvent was removed by an evaporator. The
samples were then dissolved in dimethylsulfoxide (DMSO) and
sterilized through a 0.45 μm filter.
Mutagenic assay
The Assay of mutagenicity was carried out with the Ames
test (10). The test was performed in duplicate with or without
S9mix. An S9 fraction of phenobarbital/5,6-benzoflavone-
pretreated male Sprague-Dawley (SD) rat liver was purchased
from Oriental Yeast Co. Ltd. (Tokyo, Japan). The S9mix
contained 50 μl of S9 in a total volume of 500 μl. The test
strains were Salmonella typhimurium TA100 and YG1024 for
extracts from all sampled river waters, and YG1041 and
YG1042 for extracts from the Mikasa River. The TA strain was
kindly supplied by Dr. Bruce N. Ames, University of
California, Berkeley, and the YG strains by Dr. T. Nohmi of the
National Institute of Health Sciences. YG1024 is an O-acetyl-
transferase-overproducing derivative of strain TA98, strain
YG1041 is a nitroreductase- and O-acetyltransferase-overpro-
ducing derivative of strain TA98, and strain YG1042 is the
same derivative of TA100 (6). Revertant colonies were counted
using an automatic colony counter (Colony Analyzer CA-9,
Toyo sokki Co. Ltd., Tokyo, Japan). When the dose-response
curve showed a 2-fold or greater increase over the spontaneous
revertants, the mutagenicity of the sample was judged to be
positive. Numbers of revertants per plate of positive controls
were 960 (TA100 -S9mix, AF-2: 2-(2-Furyl)-3-(5-nitro-2-
furyl)acrylamide 0.01 μg), 731 (TA100 +S9mix, B(a)P: benzo(a)
pyrene 1.25 μg), 662 (YG1024 -S9mix, AF-2 0.05 μg), 990
(YG1024 +S9mix, B(a)P 2.5 μg), 37 (YG1041 −S9mix, AF-2
0.025 μg), 360 (YG1041 +S9mix, B(a)P 5 µg), 419 (YG1042
–S9mix, AF-2 0.05 μg), and 523 (YG1042 +S9mix, B(a)P
1.25 μg).
Quantification of B(a)P, Trp-P-1, and Trp-P-2 by HPLC
Chemical substances of extracts from the Mikasa River
were quantified by HPLC with fluorescence detection with a
Waters LC-Module 1, Mightysil ODS column (5 μm particle
size, 250×4.6 mm i.d., Kanto Chemical Co., Inc.), and a Waters
470 scanning fluorescence detector, with mobile phases of 90%
methanol+10% water for benzo(a)pyrene(B(a)P) and 20%
acetonitrile+80% 20 mM phosphoric acid (pH 2) for 3-amino-
1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-
methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The analysis was
performed with a column temperature of 40°C, flow rate of
0.8 ml/min, injection volume of 20 μl, and wavelengths (Ex,
Em) for B(a)P of 370 nm, 420 nm and for Trp-P-1 and Trp-P-2
of 266 nm, 397 nm.
Determination of water pollution indicators
Determinations of SS, BOD, COD, T-N and T-P were
performed according to the testing methods for industrial
wastewater of Japanese Industrial Standard (JIS) K0102 (11).
DOC was determined after passing samples through a 0.45 μm
filter according to the total organic carbon (TOC) method of JIS
K0102. A260/DOC was determined by measuring absorbance at
260 nm through a 50 mm cell, and dividing the value by DOC.
At each sampling site, the determination of each item was
repeated twice, and the results were averaged.
Results
Water pollution indicators
The results of the determination of river water pollution
indicators are shown in Table 1. SS values were higher for the
Hae River than other samples, while BOD and COD values
were highest for the Siragane River. T-N values were higher for
the Mikasa River than the other sites, and T-P values were
highest for the Mikasa River and the Siragane River. There
were no remarkable differences in the DOC and A260/DOC
parameters among the sixteen sampling sites.
Mutagenic activity assessed by the Ames test
The mutagenic activity of the extracts from the sixteen
sites in strains TA100 and YG1024 with and without S9mix is
shown in Table 2. The extracts from sites 3 and 4 of the Tatara
River and site 1 of the Naka River showed mutagenic responses
toward strain YG1024 without S9mix. This result indicates that
these sites were more polluted with frame-shift type direct
mutagens such as nitroarenes. The extracts from sites 2 and 3 of
the Mikasa River showed higher mutagenic responses espe-
cially toward strain YG1024 with S9mix. This result indicates
that these sites were more polluted with frame-shift type
indirect mutagens such as aminoarenes. No sample showed
mutagenic responses toward strain TA100, indicating that the
extracts from blue rayons in the river were weakly polluted by
base-substitution type mutagens.
The mutagenic activity of the extracts from the Mikasa
River in strains TA100, YG1024, YG1041, and YG1042 with
and without S9mix is shown in Table 3. The extracts from all
the sampling sites of the Mikasa River showed higher
mutagenic responses toward strains YG1041 without S9mix
Environ. Health Prev. Med. Validity of Mutagenicity as a Water Pollution Indicator
135
Table 1 River water pollution indices at all sampling sites
Sampling site SS (mg/l) BOD (mg/l) COD (mg/l) DOC (mg/l) A260/DOC (50 mm)-1 T-N (mg/l) T-P (mg/l)
Tatara River 1 10 2.2 2.6 2.02 0.054 2.15 0.03
2 17 1.9 4.0 2.41 0.075 2.35 0.07
3 19 1.7 4.2 3.20 0.058 2.47 0.08
4 46 2.2 2.8 3.70 0.019 1.96 0.06
Naka River 1 17 2.4 3.8 2.52 0.063 3.17 0.08
2 13 1.9 3.4 3.39 0.047 2.00 0.07
Mikasa River 1 3 1.9 9.9 5.89 0.083 19.12 0.73
2 5 2.2 10.2 6.17 0.081 19.98 0.90
3 4 2.5 10.4 5.52 0.090 20.16 0.82
4 8 2.7 9.5 5.40 0.087 17.79 0.81
Hae River 1 88 6.3 11.1 2.93 0.064 4.10 0.28
2 86 4.2 8.6 3.07 0.055 4.30 0.28
Doumen River 1 17 5.6 7.9 4.31 0.060 2.64 0.11
2 13 7.4 11.1 4.16 0.070 2.80 0.14
Siragane River 1 79 35.6 23.7 7.17 0.042 4.81 0.59
2 13 12.8 16.5 6.25 0.077 9.23 0.85
Table 2. Mutagenic activity of extracts from blue rayons in strains TA100 and YG1024
Mutagenic activity (net revertants/g blue rayon)
Sampling TA100 YG1024
site −S9mix +S9mix −S9mix +S9mix
Tatara River 1 —a———
2— — — —
3— — 862 —
4— — 1,068 —
Naka River 1 — — 543 —
2— — — —
Mikasa River 1 — — — —
2 — — — 4,571
3 — — — 2,764
4— — — —
Hae River 1 — — — —
2— — — —
Doumen River 1 — — — —
2— — — —
Siragane River 1 — — — —
2— — — —
a The revertants were less than twice those of the control.
Spontaneous controls: TA100 −S9mix, 157; TA100 +S9mix, 161; YG1024 −S9mix, 214; YG1024 +S9mix, 240 revertants per plate.
Table 3 Mutagenic activity of extracts from the Mikasa River in strains TA100, YG1024, YG1041 and YG1042
Mutagenic activity (net revertants/g blue rayon)
Sampling TA100 YG1024 YG1041 YG1042
site −S9mix +S9mix −S9mix +S9mix −S9mix +S9mix −S9mix +S9mix
1—
a— — — 3,278 — 2,404 —
2 — — — 4,571 2,115 3,185 — —
3 — — — 2,764 1,297 2,693 1,731 —
4 — — — — 2,482 1,661 — —
a The revertants were less than twice those of the control.
Spontaneous controls: TA100 −S9mix, 157; TA100 +S9mix, 161; YG1024 −S9mix, 214; YG1024 +S9mix, 240; YG1041 −S9mix, 12; YG1041 +S9mix,
34; YG1042 −S9mix, 105; YG1042 +S9mix, 92 revertants per plate.
Environ. Health Prev. Med. Validity of Mutagenicity as a Water Pollution Indicator
136
and, at sites 1 and 3, toward YG1042 without S9mix. This
indicates that sites 1 to 4 were polluted with frame-shift and
base-substitution types of direct mutagens such as nitroarenes.
The extracts from sites 2 to 4 showed higher mutagenic
responses toward strain YG1041 with S9mix, indicating that
these sites were more polluted with frame-shift type indirect
mutagens such as aminoarenes.
Quantification of B(a)P, Trp-P-1, and Trp-P-2
Table 4 shows B(a)P, Trp-P-1 and Trp-P-2 concentrations
in the extracts from the Mikasa River measured by fluorescence
HPLC. B(a)P is produced by imperfect combustion and distrib-
uted throughout the environment, therefore B(a)P is regarded as
one of the representative chemicals of environmental pollution
(12, 13). It has been reported that Trp-P-1 and Trp-P-2 are
contained in sewage (4). In the present study, the concentrations
of B(a)P and Trp-P-2 were higher in the extracts from sites 2, 3
and 4 than site 1. This result seems to be caused by the outfall
of a sewage treatment plant located between sites 1 and 2.
However, the chemicals we detected accounted for less than
17% of the total mutagenicity in strains YG1024 and YG1041.
These low rates of contribution would indicate that other
chemicals are contained in the extracts from blue rayons.
Discussion
The mutagenic activity and the seven water pollution
indicators are shown in Fig. 1. The indicators did not show
specific trends that were consistent with the mutagenic activity.
The extracts from sites 2 and 3 of the Mikasa River showed
higher mutagenic responses especially toward strain YG1024
with S9mix, and the extracts of sites 3 and 4 of the Tatara River
and site 1 of the Naka River showed mutagenic responses
toward strain YG1024 without S9mix. This result would indi-
cate that sites 2 and 3 of the Mikasa River were more polluted
Table 4 Quantitative determination of B(a)P, Trp-P-1 and Trp-P-
2 in extracts from the Mikasa River
ng/g blue rayon
Sampling site B(a)P Trp-P-1 Trp-P-2
1n.d.64
2 7 n.d. 13
3 6 n.d. 14
4 7 n.d. 13
n.d.=not detected.
Fig. 1 Chemical characteristics and mutagenic activity of sixteen river water samples.
Environ. Health Prev. Med. Validity of Mutagenicity as a Water Pollution Indicator
137
especially with frame-shift type indirect mutagens such as
aminoarenes. Therefore, to investigate the main cause of the
mutagenicity of the extracts of the Mikasa River, we measured
the mutagenicity toward YG1041 and YG1042 with and
without S9mix, and B(a)P, Trp-P-1 and Trp-P-2 concentrations.
The Location of sampling sites in the Mikasa River is
shown in Fig. 2. This river flows through an area furnished with
sewerage, and a domestic and commercial area is located
downstream. There is outfall from a sewage treatment plant
located between sites 1 and 2 (the capacity of sewage disposal
is 4,170,000 m3/day, the area of sewage disposal is 9,294 ha,
and the population it serves is 687,000). The extracts from sites
2 to 4 showed high mutagenic responses toward strain YG1041
with S9mix. The extracts from all the sampling sites showed
high mutagenic responses toward strains YG1041 without
S9mix and, at sites 1 and 3, toward YG1042 without S9mix.
The concentrations of B(a)P and Trp-P-2 in the extracts from
sites 2, 3 and 4 were higher than those of site 1. The main
source of mutagenic activity in strains YG1024 and YG1041
with S9mix of the extracts from sites 2 to 3 appeared to be the
water discharged into this river from the sewage plant located
between sites 1 and 2 with the possible main causes being the
frame-shift type indirect mutagens such as aminoarenes:
Mutagenic activity in strain YG 1024 with S9mix decreased
from sites 2 to 3, and mutagenic activity in strain YG1041 with
S9mix decreased from sites 2 through 4 in that order. The
extracts from site 1 did not show mutagenic responses toward
strains YG1024 and YG1041 with S9mix. Furthermore, the
concentration of Trp-P-2 was higher in the extracts from sites 2,
3 and 4 than site 1, and Trp-P-2 is mutagenic toward strains
YG1024 and YG1041 with S9mix (4). Mutagenic responses
toward strain YG1041 without S9mix, however, were found in
the extracts from all sites, the possible main cause of mutage-
nicity being nitroarenes. Nitroarenes are ubiquitous environ-
mental pollutants found in diesel emission and airborne particu-
lates (14–16). Manabe et al. (1984) reported the presence of 1-
nitropyrene in wastewater from oil-water separating tanks at
gasoline stations as well as in used crankcase oil, and its
mutagenicity (17). They also reported that the wastewater in the
tank flows out to drains and finally into the wastewater treat-
ment plant or directly into the river. Therefore, the main cause
of the wide-spread mutagenic activity toward strains YG1041
and YG1042 without S9mix in the extracts of the samples
appeared to be direct-acting mutagens, i.e., nitroarenes.
Mutagenic activity and the four water pollution indicators
are correlated in Fig. 3. It is clear that there were no remarkable
differences in the four parameters among sites 1 to 4 of the
Mikasa River. Also, the other three parameters did not show
clear differences (Table 1). In contrast, the extracts from sites 2
and 3 showed higher mutagenic responses especially toward
strains YG1024 and YG1041 with S9mix, and the extracts from
all the sampling sites showed high mutagenic responses toward
strain YG1041 without S9mix and, at sites 1 and 3, toward
YG1042 without S9mix. The mutagenicity determined in this
report would not affect human health. However, water pollution
in small amounts by many kinds of chemicals has been an
important problem. The difference of the results between the
water pollution indicators and the mutagenic activities indicates
that the known water pollution indicators do not reflect the
mutagenic activity of river water. Detection of the combined
effects of different chemicals of low concentration is very
difficult using known water pollution indicators, however, the
Fig. 2 Location of sampling sites in the Mikasa River.
,, and are sampling sites.
Fig. 3 Correlation between mutagenic activity and chemical characteristics of Mikasa River water samples.
Environ. Health Prev. Med. Validity of Mutagenicity as a Water Pollution Indicator
138
mutagenicity test is able to evaluate river water synthetically.
Furthermore, it is possible to predict the main cause of the
mutagenic activities by conducting mutagenicity tests in many
kinds of bacteria. Therefore, the evaluation of river water
mutagenicity seems to be useful and necessary as one of the
indicators of water pollution.
Acknowledgments
This work was supported in part by a Grant-in-Aid for
Cancer Research from the Ministry of Health and Welfare of
Japan and funds under contract with the Environment Agency
of Japan.
References
( 1 ) Bass NG, Albuquerque MB, Ulitzur S, Chartrand A, Acha
CR. Effects of chlorine dioxide on mutagenic activity of Lake
Kinnereth water. Environ. Sci. Technol. 1987; 21: 252–260.
( 2 ) Filipic M. Mutagenicity and toxicity of water extracts from
the Sora River area. Mutat. Res. 1995; 342: 1–8.
( 3 ) Ohe T, Nukaya H. Genotoxic activity of 1-nitropyrene in
water from the Yodo River, Japan. The Science of the Total
Environment 1996; 181: 7–12.
( 4 ) Sasaki Y, Kise H, Kikuchi M. Monitoring and identification
of Ames mutagenic compounds in river water. Environ.
Mutagen Res. (in Japanese) 1996; 18: 21–27.
( 5 ) Vargas VMF, Guidobono RR, Jordao C, Henriques JAP. Use
of two short-term tests to evaluate the genotoxicity of river
water treated with different concentration/extraction proce-
dures. Mutat. Res. 1995; 343: 31–52.
( 6 ) Hagiwara Y, Watanabe M, Oda Y, Sofuni T, Nohmi T. Speci-
ficity and sensitivity of Salmonella typhimurium YG1041 and
YG1042 strains possessing elevated levels of both nitroreduc-
tase and acetyltransferase activity. Mutat. Res. 1993; 291:
171–180.
( 7 ) Sakamoto H, Hayatsu H. A simple method for monitoring
mutagenicity of river water. Mutagens in Yodo River system,
Kyoto-Osaka. Bull. Environ. Contam. Toxicol. 1990; 44:
521–528.
( 8 ) Yamauchi A, Matsumoto N, Nakagawa H, Ohotuka T,
Yamazaki H, Kakiuchi Y. Determination of polycyclic
aromatic hydrocarbons in river waters by blue-cotton
adsorption method. EISEI KAGAKU (in Japanese) 1989;
35(4): 283–290.
( 9 ) Nagafuchi Y, Matsuo H. Studies on a predictor of trihalom-
ethane formation potential in river water. Fukuoka-ken
Hokenkankyoukenkyusyo Nenpou (in Japanese) 1993; 20:
65–69.
(10) Maron DM, Ames BN. Revised methods for the Salmonella
mutagenicity test. Mutat. Res. 1983; 113: 173–215.
(11) Japanese Industrial Standards Committee. The testing meth-
ods for industrial wastewater of Japanese Industrial Standard
(JIS) K0102. 1998. (Published by Japanese Standards Associ-
ation.)
(12) Abe A, Urano K. Characteristics of lethal substances and the
removal effect of molecular sulfur in the Salmonella mutage-
nicity test for river sediments. Mutat. Res. 1996; 351: 61–66.
(13) Ohe T. Monitoring of genotoxicity of river water in high sen-
sitive umu test system using Salmonella typhimurium
NM2009 having high O-acetyltransferase activity. Environ.
Mut. Res. Commun. (in Japanese) 1996; 17: 237–249.
(14) Sera N, Fukuhara K, Miyata N, Tokiwa H. Detection of
nitroazabenzo(a)pyrene derivatives in the semivolatile phase
originating from airborne particulate matter, diesel and
gasoline vehicles. Mutagenesis 1994; 9: 47–52.
(15) Sera N, Kai M, Horikawa K, Fukuhara K, Miyata N, Tokiwa
H. Detection of 3,6-dinitrobenzo(a)pyrene in airborne partic-
ulates. Mutat. Res. 1991; 263: 27–32.
(16) Tokiwa H, Ohnishi Y. Mutagenicity and carcinogenicity of
nitroarenes and their sources in the environment. CRC Crit.
Rev. Toxicol. 1986; 17: 23–60.
(17) Manabe Y, Kinouchi T, Wakisaka K, Tahara I, Ohnishi Y.
Mutagenic 1-nitropyrene in wastewater from oil-separating
tanks of gasoline stations and in used crankcase oil. Environ.
Mutagen 1984; 6: 669–681.
