www.elsevier.com/locate/reviewsmr
Mutation Research 567 (2004) 109–149
Review
Mutagens in surface waters: a review
Takeshi Ohea,*, Tetsushi Watanabeb, Keiji Wakabayashic
aDepartment of Food and Nutrition, Kyoto Women’s University, 35 Kitahiyoshi-cho, Imakumano,
Higashiyama-ku, Kyoto 605-8501, JapanbDepartment of Public Health, Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi,
Yamashina-ku, Kyoto 607-8414, JapancCancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome,
Chuo-ku, Tokyo 104-0045, Japan
Received 29 March 2004; received in revised form 24 August 2004; accepted 25 August 2004
Available online 21 November 2004
Community address: www.elsevier.com/locate/mutres
Abstract
A review of the literature on the mutagenicity/genotoxicity of surface waters is presented in this article. Subheadings of this
article include a description of sample concentration methods, mutagenic/genotoxic bioassay data, and suspected or identified
mutagens in surface waters published in the literature since 1990. Much of the published surface water mutagenicity/
genotoxicity studies employed the Salmonella/mutagenicity test with strains TA98 and/or TA100 with and/or without metabolic
activation. Among all data analyzed, the percentage of positive samples toward TA98 was approximately 15%, both in the
absence and the presence of S9 mix. Those positive toward TA100 were 7%, both with and without S9 mix. The percentage
classified as highly mutagenic (2500–5000 revertants per liter) or extremely mutagenic (more than 5000 revertants per liter) was
approximately 3–5% both towards TA98 and TA100, regardless of the absence or the presence of S9 mix. This analysis
demonstrates that some rivers in the world, especially in Europe, Asia and South America, are contaminated with potent direct-
acting and indirect-acting frameshift-type and base substitution-type mutagens. These rivers are reported to be contaminated by
either partially treated or untreated discharges from chemical industries, petrochemical industries, oil refineries, oil spills, rolling
steel mills, untreated domestic sludges and pesticides runoff. Aquatic organisms such as teleosts and bivalves have also been
used as sentinels to monitor contamination of surface water with genotoxic chemicals. DNA modifications were analyzed for this
purpose. Many studies indicate that the 32P-postlabeling assay, the single cell gel electrophoresis (comet) assay and the
micronucleus test are sensitive enough to monitor genotoxic responses of indigenous aquatic organisms to environmental
pollution. In order to efficiently assess the presence of mutagens in the water, in addition to the chemical analysis, mutagenicity/
genotoxicity assays should be included as additional parameters in water quality monitoring programs. This is because
according to this review they proved to be sensitive and reliable tools in the detection of mutagenic activity in aquatic
environment.
Many attempts to identify the chemicals responsible for the mutagenicity/genotoxicity of surface waters have been reported.
Among these reports, researchers identified heavy metals, PAHs, heterocyclic amines, pesticides and so on. By combining the
blue cotton hanging method as an adsorbent and the O-acetyltransferase-overproducing strain as a sensitive strain for
* Corresponding author. Tel.: +81 75 531 7124; fax: +81 75 531 7170.
E-mail address: [email protected] (T. Ohe).
1383-5742/$ – see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrrev.2004.08.003
T. Ohe et al. / Mutation Research 567 (2004) 109–149110
aminoarenes, Japanese researchers identified two new type of potent frameshift-type mutagens, formed unintentionally, in
several surface waters. One group has a 2-phenylbenzotriazole (PBTA) structure, and seven analogues, PBTA-type mutagens,
were identified in surface waters collected at sites below textile dyeing factories and municipal wastewater treatment plants
treating domestic wastes and effluents. The other one has a polychlorinated biphenyl (PCB) skelton with nitro and amino
substitution group and it was revealed to be 4-amino-3,30-dichloro-5,40-dinitrobiphenyl derived from chemical plants treating
polymers and dye intermediates. However, the identification of major putative mutagenic/genotoxic compounds in most surface
waters with high mutagenic/genotoxic activity in the world have not been performed. Further efforts on chemical isolation and
identification by bioassay-directed chemical analysis should be performed.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Mutagenicity/genotoxicity assays; Mutagens; Surface waters; Polycyclic aromatic hydrocarbons (PAHs); Heterocyclic amines
(HCAs); PBTA-type mutagens; 4-Amino-3,30-dichloro-5,40-dinitrobiphenyl
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
2. Sample concentration of surface waters for mutagenicity/genotoxicity assays . . . . . . . . . . . . . . . . . . . . . . . . 115
3. Review of published mutagenicity/genoxicity assessment data of surface waters . . . . . . . . . . . . . . . . . . . . . . 122
3.1. Salmonella/mutagenicity data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
3.1.1. Mutagenic features of surface waters with Salmonella typhimurium TA98 and TA100 . . . . . . . 122
3.1.2. Mutagenic features of surface waters with nitroreductase- and/or
O-acetyltransferase-overexpressing strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.2. SOS chromotest/umu-test and other bacterial assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
3.3. DNA adduct formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.4. DNA strand breaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
3.5. Micronucleus induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
3.6. Other assessment methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
4. Suspected or identified mutagens in surface waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
5.1. Mutagenic/genotoxic bioassay data on surface waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
5.2. Suspected or identified mutagens/genotoxins in surface waters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
1. Introduction
Surface waters, such as rivers, lakes and seas,
receive large quantities of waste water from industrial,
agricultural, and domestic sources, including muni-
cipal sewage treatment plants. These surface waters,
which contain many unknown compounds, are used as
a source of drinking water, as well as for agricultural,
recreational and religious activities around the world.
Consequently, water pollution can be a serious public
health and aquatic ecosystem problem [1–6]. The US
EPA’s Toxic Release Inventory (TRI) for 2001
T. Ohe et al. / Mutation Research 567 (2004) 109–149 111
Table 1
Toxics release inventory (TRI) total surface water discharges and total air emissions for all chemicals by industry in the United States in the year
of 2001a
Industry type Total water releases (�103 kg) Total air emissions (�103 kg)
Chemical and allied products 26117.1 103348.6
Food and related products 25018.2 25463.3
Primary metal smelting and processing 20262.5 26132.9
Petroleum refining and related industries 7752.9 21849.6
Paper and allied products 7500.9 71283.5
Electric, gas, and sanitary services 1596.5 325492.4
Electronic and other electrical equipment 1332.2 5770.3
Fabricated metal products 790.8 18346.9
Photographic, medical, and optical goods 646.1 3250.9
Coal mining and coal mine services 344.8 348.7
Tobacco products 241.7 1130.3
Metal mining (e.g., Fe, Cu, Pb, Zn, Au, Ag) 193.8 1294.8
Transportation equipment manufacture 89.9 30251.4
Textile mill products 79.6 2603.9
Stone, clay, glass, and concrete products 73.5 14181.8
Leather and leather products 56.6 547.7
Plastic and rubber products 32.2 34973.1
Solvent recovery operations (under RCRAb) 10.7 442.0
Lumber and wood products 9.0 13825.1
Industrial and commercial machinery 8.2 3755.7
Petroleum bulk stations and terminals 5.1 9600.4
Chemical wholesalers 0.8 569.0
Furniture and fixtures 0.3 3548.9
Printing, publishing, and related industries 0.1 8750.2
Apparel <0.1 155.7
No reported SIC code 483.2 1528.3
Miscellaneous manufacturing 16.6 3068.5
Total 100153.0 761763.6
a http://www.epa.gov/triexplorer/industry.htm.b The US Resource Conservation and Recovery Act.
reported that more than 100,000 metric tonnes of
chemicals are released into surface waters and
approximately 762,000 metric tonnes of chemicals
are emitted into the atmosphere annually by industrial
use in the United States as shown in Table 1 [7]. This
data show that large quantities of toxic materials are
routinely released directly or indirectly (via airborne
emission) into aquatic systems after industrial usage.
Table 1 also notes that more than fifty percent of
annual water discharges to aquatic systems come from
the chemical, metal smelting and processing, and
petroleum refining industries. Moreover, 800 metric
tonnes of chemicals released into surface waters and
60,000 metric tonnes of chemicals emitted into the
atmosphere are carcinogens ranked as 1, 2A or 2B
under the IARC classification system, and most of
them are known to have mutagenic and/or clastogenic
activity as shown in Table 2 [8–10]. These carcinogens
are categorized into two types: persistent compounds,
which include metals and polycyclic aromatic
compounds; and volatile compounds. Most chemicals
emitted into the atmosphere eventually reach the
ground or surface waters through deposition, so these
TRI results show that surface waters are readily
contaminated with a variety of known mutagenic or
genotoxic carcinogens.
Mutagenic/genotoxic compounds, including carci-
nogens, whether known or unknown, become the
components of complex environmental mixtures that
can have adverse health effects on humans and
indigenous biota [11]. We know quite a lot about
identified contaminants, and it is relatively easy to
study the sources and fate of those contaminants that
have been identified as priorities for concern and
T. Ohe et al. / Mutation Research 567 (2004) 109–149112
control. Post-emission fate and behavior of polycyclic
aromatic hydrocarbons (PAHs) in complex mixtures
including surface waters have widely investigated
throughout the world, because PAHs are identified
contaminants and are relatively easy to study the
sources and fate [12]. However, few studies have
investigated the identification of novel putative
mutagens and the quantification of their response
concentrations.
On the other hand, the use of short-term bioassays,
which can detect a wide range of chemical substances
that may produce genetic damage, has permitted the
quantification of mutagenic hazard without a priori
information about identity or physical–chemical
property. In studies of the mutagenicity/genotoxicity
Table 2
TRI water releases and air emissions of carcinogens in the United States
Compound Mutagenicity/
clastogenicitya,b
Carcino
Lead compoundsf ## 2B
Formaldehyde +++/## 2A
Nickel compoundsg ++/## 1
Chromium compoundsh +++/## 1
Acetaldehyde +++/## 2B
Arsenic compoundsi +/## 1
1,4-Dioxane +j/# 2B
Cobalt compoundsk ++/## 2B
N,N-Dimethylformamide ++/# 3
Benzene +++/## 1
Chloroform +/## 2B
Catechol ++/# 2B
Polycyclic aromatic compoundsl
Benz(a)anthracene +++/## 2A
Benzo(a)pyrene +++/## 2A
Benzo(b)fluoranthene +/# 2B
Dibenzo(a,h)anthracene ++/## 2A
Indeno(1,2,3-cd)pyrene + 2B
Dibenz(a,h)acridine + 2B
Beryllium compoundsm ++/# 1
Ethylbenzene +n 2B
Epichlorohydrin ++/## 2A
Diaminotoluene (mixed isomers) � 2Bo
Dichloromethane ++/## 2B
Ethylene oxide +++/## 1
Styrene +++/## 2B
Cadmium compoundsp ++/## 1
Creosote ++ 2A
Trichloroetylene + 2A
Vinyl acetate # 2B
Tetrachloroetylene � 2A
1,3-Butadiene ## 2A
of surface water and aquatic biota conducted in the late
1970s, Parry et al. [13] reported on mutagenicity
studies on the tissue of the mussel Mytilus edulis in the
marine environment, and Pelon et al. [14] reported on
the mutagenicity/genotoxicity of Mississippi River
water samples by the Salmonella assay developed by
Ames et al. [15]. Cytogenic damage in fish exposed to
the industrially contaminated Rhine River were also
observed [16,17]. Since 1980, many researchers have
assessed mutagenicity/genotoxicity of surface waters
using a variety of bioassays and analytical methods
from the standpoint of determining the potential
contribution to the mutagenic hazards of treated
drinking water and potential ecological hazard.
Collectively, mutagenicity evaluations of surface
in the year of 2001a
genicityc Total water
releasesd (�103 kg)
Total air
emissionse (�106 kg)
164.3 569.0
152.5 4,800.1
111.1 455.7
80.9 304.4
71.4 5,397.6
64 –
36.9 –
21.8 –
17 242.6
9.6 2,673.8
8.6 647.6
7.8 –
7.4 519.9
4.6 –
4 2,969.9
3.5 –
2.7 –
2.2 9,778.4
2.1 –
1.4 21,077.0
1.1 –
1.1 –
– 3,741.9
– 1,303.9
– 1,213.2
– 973.0
T. Ohe et al. / Mutation Research 567 (2004) 109–149 113
Table 2 (Continued )
Compound Mutagenicity/
clastogenicitya,b
Carcinogenicityc Total water
releasesd (�103 kg)
Total air
emissionse (�106 kg)
Acrylnitrile +++ 2A – 424.5
Chloroprene +++/## 3 – 386.5
Vinyl chloride +++/## 1 – 332.1
Total 776.0 57811.1
a Based on data from references [8–10].b �, compounds for which there is no evidence of mutagenicity or clastogenicity; +, mutagenic in bacterial and/or fungal/yeast cells in vitro;
++, also mutagenic in plants or animal cells in vitro; +++, also mutagenic in the Drosophila melanogaster somatic mutation and recombination
test, and/or sex-linked recessive lethal test, and/or transgenic rodent assays, and/or rodent dominant lethal test. For cytogenetic endpoints, #
refers to substances are clastogenic in in vitro or in vivo assays, ## refers to substances that are clastogenic both in vitro and in vivo. Note: In some
instances conflicting results have been reported in the literature.c IARC classification system: 1—carcinogenic to humans, 2A—probably carcinogenic to humans, 2B—possibly carcinogenic to humans,
3—inadequate or limited evidence of carcinogenicity in experimental animals. IARC monographs on the evaluation of carcinogenic risks to
humans, volumess 11, 15, 16, 23, 32, 47, 49, 52, 54, 58, 60, 62, 63, 71, 73, 77, and supplements 6 and 7. International Agency for Research on
Cancer, Lyon, France.d >1000 kg only.e >3 � 1000 kg only.f Various compounds.g Nickel(II) salts (e.g., NiCl2) and insoluble crystalline nickel (e.g., Ni3S2).h Hexavalent chromium compounds only (e.g., K2Cr2O7, K2CrO4).i Both the +3 and +5 oxidation states are clastogenic in vitro.j Rodent dominant lethal assay only.k Cobalt (II) salts only (e.g., CoCl2).l The TRI lists PACs (polycyclic aromatic compounds) as a category of 19 individual compounds. A list of compounds included is available at
http://www.epa.gov/tri/chemical/chemlist2001.pdf.m Primarily beryllium (II) compounds (e.g., BeSO4).n Animal cells only.o Only 2,4-diaminotoluene evaluated.p Cadmium (II) salts only (e.g., CdCl2).
water provide an indication of potential hazard in the
absence of priority knowledge about the identification
or physical/chemical properties of the putative
toxicants. The Salmonella mutagenicity assay in
particular has been widely used to detect mutagenic
activity in complex environmental mixtures such as
surface waters, especially river waters.
In the early 1990s, Stahl [18], De Flora et al. [19]
and Houk [1] reviewed the genotoxic and/or carcino-
genic hazards of natural waters, the marine environ-
ment, and industrial wastes and effluents. Houk [1]
and Stahl [18] demonstrated that genotoxic organic
compounds can enter surface waters from a wide range
of industrial and municipal sources by summarizing
their genotoxic data performed by short-term genetic
bioassays on literature. They also stressed the
importance of bioassays to detect mutagenicity/
genotoxicity arising from the ubiquity of genotoxic
compounds in the environment and the necessity of the
identification of the sources of contaminants. White
and Rasmussen [4] noted that volumetric emissions
from municipal wastewater treatment plants in large
urban centers often exceed 109 l per day. As a result,
genotoxic loadings from municipal wastewater treat-
ment facilities are often far greater than those of
industrial facilities, and there is a strong relationship
between a measure of human activity (i.e., population)
and surface water genotoxicity. The work of Houk [1]
and White et al. [3–6] implicated a wide range of
industries in the release of complex mutagenic
mixtures for which the identity of the putative
mutagens is not known. On the other hand, some
researchers have reported that conventional waste-
water purification processes do not effectively remove
many chemical contaminants, and treatment may
actually increase the mutagenicity/genotoxicity of
waste waters [2,20–23]. Other studies show a sharp
rise in the mutagenicity/genotoxicity of water samples
collected at sites downstream from wastewater
treatment plants [24,25]. Consequently, the increasing
T. Ohe et al. / Mutation Research 567 (2004) 109–149114
use of contaminated surface waters and an increase in
the magnitude of the contamination pose a serious
problem for the health and welfare of humans and
indigenous aquatic biota. Thus, appropriate bioassay
have been needed for evaluation of surface waters on
potential hazard to human and the water environment.
The purpose of this review is to summarize the state
of the current literature on mutagenicity/genotoxicity
data for surface waters and to lead the most profitable
directions for future research in order to control and
manage effectively our water environment. In this
review, we will focus on a synopsis of the
mutagenicity/genotoxicity assay data in surface
waters in the scientific literature published since
1990. Subheadings include a description of sample
concentration methods, mutagenic/genotoxic bioassay
data, and suspected or identified mutagens in surface
waters. In most cases, surface waters have been
administered in their crude extracts to these biological
test system. Fig. 1 illustrates a breakdown of the
collected surface water mutagenicity/genotoxicity
assays. Results from 178 published mutagenicity/
genotoxicity assays of surface waters were obtained
Fig. 1. Breakdown of mutagenicity/genotoxicity assays for surface water
published since 1990 was summed, and the percentage of each bioassay he
from 128 publications. Published mutagenicity/geno-
toxicity assessments were divided into two major
categories: bacterial assays, including the Salmonella
mutagenicity test, and the SOS Chromotest and
Salmonella umu-test; and aquatic organism and plant
assays, including the micronucleus assay, 32P-post-
labelling, the comet assay and the alkaline unwinding
assay. The 32P-postlabeling assay, DNA strand breaks
and the micronucleus test are unique in that they can
be utilized in the laboratory setting, or they can be
taken to the site for in situ monitoring using fishes or
plants that inhabit regions contaminated by industrial
and municipal wastewater. Genotoxic parameters (e.g.
hepatic DNA adducts) are currently the most valuable
biomarkers for environmental risk assessment and
there are many reports on the studies linking the DNA
damage to subsequent molecular, cellular and tissue-
level alteration of aquatic organisms. In this paper, we
intended to review the studies in which bioassays with
DNA alterations, e.g. mutagenicity, DNA damaging
activity and chromosome aberration, as their end-
points were used to evaluate contamination of surface
water with genotoxic chemicals. The studies on the
s (n = 178). The number of assays used in 128 scientific literatures
ading was calculated. Data sources are provided in Tables 3 and 5–8.
T. Ohe et al. / Mutation Research 567 (2004) 109–149 115
tumor incidence or the incidence of idiopathic lesions,
including oncogene activation, link to mutagens
exposure in aquatic organisms are not cited.
2. Sample concentration of surface waters for
mutagenicity/genotoxicity assays
Mutagenicity/genotoxicity data of surface waters
performed using the bacterial assays are summarized
in Table 3. There are many varieties of monitoring
methods combined with mutagenicity/genotoxicity
tests and selective extraction methodologies for
identifying the possible classes of mutagenic/geno-
toxic organic contaminants in surface waters. A
discussion of different extraction/concentration meth-
ods has been presented in detail by Houk [1] and Stahl
[18]. We describe here briefly the sample concentra-
tion methods used for bacterial mutagencity/geno-
toxicity assays. Although mutagenic potency can be
detected in non-concentrated samples of surface
waters in many cases [30,34,36,44,46–48,83,86,88,
89,92–94], each contaminant is usually present at such
low levels that it is difficult to detect, and therefore
some sort of extraction/concentration method is
required for reliable mutagencity/genotoxicity assess-
ment of surface water samples. Concentration/extrac-
tion methods include liquid–liquid extraction, solid
phase extraction and other types of column chroma-
tography, as well as the blue rayon/cotton hanging
method [102].
Adsorption on Amberlite XAD resins is the most
commonly applied method for concentrating organic
substances from different kinds of surface waters.
XAD resin can generally adsorb a broad class of
mutagenic compounds, including polycyclic aro-
matic hydrocarbons, arylamines, nitro-compounds,
quinolines, anthraquinones, etc. Adsorption, fol-
lowed by elution with organic solvents, is efficient at
extracting all the polar and nonpolar toxic chemicals
and mutagens/genotoxins [103]. Using the XAD
resin column method, many positive results were
observed when those extracts were tested in the
bacterial mutagenicity assays [28,29,31,33–37,40,
42,44,45,50,54,55,57–59,69,72,77,78,87,90,92,93,
95,96].
Liquid–liquid extraction using organic solvents
provides valuable quanititative information and is
widely used. However, the liquid–liquid extracted
water samples showed fewer mutagenic responses
compared with XAD-concentrated ones [54,55,
77,104].
Blue cotton, developed by Hayatsu [102], a solid
matrix bearing covalently linked copper phthalocya-
nine trisulfonate can selectively adsorb polycyclic
planar-type compounds with three or more fused
rings. Sakamoto and Hayatsu [24] collected mutagens
by hanging blue rayon, which contains 2–3 times more
ligands than blue cotton per unit weight, in the Katsura
and Yodo Rivers, Japan. They demonstrated that the
blue rayon hanging method is easy to perform and is
suitable for qualitative screening of the mutagenicity
monitoring of river water. The blue rayon/cotton
hanging method, in which blue rayon or blue cotton as
an adsorbent is hung in the flowing water, has distinct
advantages over the conventional method of transport-
ing large volumes of water to the laboratory for
bioassay. Although this technique is semiquantitative
and cannot provide measures of contamination per
unit volume, it is suitable for collecting large amounts
of target substances and chemicals flowing in the river
for long periods (usually 24 h). It should be noted that
it is a 1-day time-integrated value, as distinguished
from an instant spot value obtainable in the conven-
tional XAD-resin concentration method or liquid–
liquid extraction method [24,52]. In addition, this
method can be easily applied to a wide range of
mutagenicity/genotoxicity monitoring approaches
[65,85] and can collect large quantities of unknown
polycyclic planar chemicals dissolved at ppt-levels
(i.e., ng/l), as can be seen in the case of PBTA-1 and
PBTA-2 [61,64]. For the quantititative determination
of mutagenicity/genotoxicity of surface waters, blue
rayon can be packed in a glass column, and the water
sample is passed through in an identical manner to
that carried out with the XAD resin column
[25,93,105].
The blue-chitin column method, a short column
technique for concentrating mutagens/genotoxins, is
also suitable for qualitative screening of river water
mutagenicity resulting from polycyclics [59,76,106].
Other solid adsorbents including Separon SE [27,38]
and Silica C18 [41,43] have also been utilized to
extract less hydrophilic chemicals in surface waters,
and substances adsorbed on the resins were eluted with
organic solvents. In all cases, organic solvent extracts
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
11
6Table 3
Mutagenicity/genotoxicity summary data of surface waters in bacterial assay
Sample source Preparation method Assay methoda/strain Mutagenic potency
classificationb
Suspected
mutagen/likely
sources
Reference
1. Europe
Llobregat River
(Barcelona, Spain)
Adsorbates on granular activated
carbon/soxhlet extracted with
DCM (non volatile fraction)
Ames assay (plate)/TA98,
TA100
Positive Alklbenzene sulfonate,
polyethoxylated nonyl
phenols and its
brominated derivatives
[26]
Unknown rivers
(Czech Republic)
Separone SE/acetone Ames assay (plate)/TA98 Moderate; TA98 (�S9),
low; TA98 (+S9)
Discharges of chemical
plant with no
wastewater treatment
[27]
River Main, River
Rhine, River
Moselle (Germany)
XAD-7 resin/acetone;
suspended matter
umu assay (microtest/TA1535
pSK1002
Positive – [28]
River Po (Italy) XAD-2/acetone Microsuspension assay/
TA98, TA100
Low; TA98 (�S9, +S9),
TA100 (�S9, +S9)
– [29]
Llobregat River,
Besos River
(Barcelona, Spain)
Rotary evaporation; particulate
matter/DMSO, dissolved
phase/filtration
Ames assay (pre)/TA98, TA100 Extreme; particulate:
TA98 (�S9, +S9), TA100
(�S9, +S9), dissolved:
TA100 (�S9, +S9), high;
dissolved: TA98 (�S9, +S9)
o-Toluidine, nitroquin-
oline, nitroaniline,
dichlorobenzidine,
several aromatic
quinines
[30]
River and sea water
(Venetia, Italy)
XAD-2 resin/DMSO Ames assay (plate)/TA98, TA100 Low, TA98 (�S9, +S9),
TA100 (�S9, +S9)
– [31]
Saale River (Germany) Liquid–liquid extraction/DCM Ames assay (plate)/TA98, TA100 Moderate; TA100 (+S9),
low; TA98 (+S9),
Chemical industry [32]
Rhine River (The
Netherlands)
XAD-4/ethanol, ethanol/CH
(acidic and neutral)
Ames assay (plate)/TA98, TA100 Moderate; TA98 (+S9),
low; TA98 (�S9),
TA100 (�S9, +S9)
– [33]
Sora River (Slovenia) XAD-2/acetone (neutral and
acidic), non-concentrated sample
Ames assay (plate)/TA98, TA100 Low, TA98 (�S9), negative,
TA98 (+S9), TA100
(�S9, +S9)
Municipal waste dump,
untreated effluent,
small local industry,
agricultural area
[34]
Ljubljanica River (Slovenia) XAD-2 resin/acetone (neutral
and acidic)
Ames assay (pre)/TA98, TA100 Moderate; TA100 (�S9,
+S9), low; TA98 (+S9),
negative, TA98 (�S9)
Industry, municipal
waste dump,
agricultural area
[35]
Ljubljaica River, Sora River
(Slovenia)
Non-concentrated sample;
XAD-2/acetone,
DCM (acidic and neutral)
Ames assay (pre)/TA98, TA100 Positive Leaking from the
municipal waste dump
[36]
Schwechat River,
Channel Badner,
Muhlbach
(Austria), Wilga River (Poland)
XAD-2 and XAD-7/acetone Ames assay (plate)/TA98, TA100 Moderate; TA98 (�S9,
+S9), TA100 (�S9, +S9)
Effluents of a
petrohemical plant
[37]
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
11
7
SOS chromotest
Microscreen phage-induction
assay/E. coli WP2sl, E. coli
TH-008
Differential DNA repair test/E.
coli 343/753, 343/765
Labe River (Czech Republic) Separon SE/acetone Ames assay (plate)/TA98 Moderate; TA98,
(�S9, +S9)
– [38]
Elbe River (Germany) Suspended particulate
matter/soxhlet extraction/
toluene, methanol
Ames assay (pre)/TA98 Positive Untreated commercial
sewage, wastewater
from chemical industries
(melting, pesticide,
chlorine industry),
PAH, chlorinated
hydrocarbons
[39]
Ara-test (L-arabinose
resistance test)/S.
typhimurium BA 9
umu-test/S. typhimurium NM2009
Salzach River (Austria) XAD-2, XAD-7 (US EPA
recommended protocol)
Ames assay (plate)/TA98, TA100 Low; TA98 (+S9),
TA100 (+S9)
Waste from community
and industrial sources
[40]
Northern Italian lake (Italy) Sep-Pak Plus C18/methanol,
acetonitrile
Ames assay (plate)/TA98, TA100 Low; TA98 (�S9), TA100
(�S9), negative; TA98
(+S9), TA100 (+S9)
Waste waters from
many small towns
and factories
[41]
Como Lake (Italy) XAD-2/acetone Ames assay (pre)/TA98, TA100 High; TA98 (+S9), low;
TA98 (�S9), TA100 (+S9),
negative; TA100 (�S9)
Increasing chemical
contamination of
the natural aquifers
[42]
Como Lake (Italy) Silica C18/ethyl acetate,
methanol;
lichrolut EN/acetonitrile
Ames assay (plate) Extreme; TA98 (+S9),
moderate; TA98 (�S9),
negative; TA100 (�S9, +S9)
Industrial or agricultural
pollution source
[43]
Rhine River, Elbe River
(Germany)
Non-concentrated sample;
XAD-resin
Ames assay, umu
assay/Salmonella
typhimurium strain
Positive – [44]
Danube River (Austria) Blue rayon hanging method;
XAD-2/hexane, acetone
Ames assay (pre)/TA98, TA100,
YG1024, YG1029
High; YG1024 (+S9) IQ, Trp-P-1, AaC [45]
Ames assay (pre)/YG1024 Positive
Spree, Havel, Stepenitz,
Saale and Rhine
Rivers, Teltow Canal
(Germany)
Direct dilution by medium umu assay/S. typhimurium
TA1535 pSK1002
Positive – [46]
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
11
8
Table 3 (Continued )
Sample source Preparation method Assay methoda/strain Mutagenic potency
classificationb
Suspected
mutagen/likely
sources
Reference
Atlantic Ocean (Lisbon,
Portugal); Aegean
Sea (Paralia, Greece);
Adriatic Sea (Trieste, Italy);
Baltic Sea
(Copenhagen, Denmark);
North Sea (Oslo, Norway)
Filtration Vibrio harvey assay/Vibrio harvey Positive – [47,48]
2. Asia
Yodo River (Japan) Blue rayon hanging
method; blue
rayon batch method
Ames assay (pre)/TA98 High; TA98 (+S9) Unknown 4 potent
mutagen discharged
from sewage plant
[24]
Yodo River (Japan) Blue rayon hanging method Ames assay (pre)/TA98, TA98NR,
TA98/1,8-DNP6
Positive Suspected nitroarene
and aminoarene
[49]
Yodo River (Japan) XAD-2, XAD-4, XAD-8 /
DCM, methanol,
NH4OH (pH 2, 4, 8)
Ames assay (plate)/TA98, TA100 Moderate; TA98 (+S9) Effluents from
sewage plants
[50]
Katsura River (Japan) Blue rayon adsorbate; Sephadex
LH-20/CH, methanol, DCM
Ames assay (pre)/TA98, TA98NR,
TA98/1,8-DNP6, YG1021, YG1024
Positve PAH, effluents
from sewage plants
[51]
Chao Phraya River
and its canal
(Bangkok, Thailand)
Blue rayon hanging method Ames assay (pre)/TA98, TA100,
YG1024, YG1029
High; YG1024 (+S9),
moderate; YG1024 (�S9)
– [52]
Sumida and Ara Rivers
(Tokyo, Japan)
Blue rayon hanging method Ames assay (pre)/TA98, TA100,
YG1024, YG1029
High; YG1024 (+S9),
moderate; YG1024 (�S9)
– [52]
The Seto Inland Sea (Japan) Blue rayon hanging method Ames assay (pre)/TA1024 Moderate; YG2024 (+S9) BaP [53]
Ganga River (India) XAD-4, XAD-8/acetone;
liquid–liquid extraction/hexane
Ames assay (pre)/TA98, TA100,
TA97a, TA102, TA104
Extreme; XAD: TA98 (�S9,
+S9), TA100 (�S9, +S9)
Organochlorinated
and organophosphorus
pesticides
[54]
Moderate; liquid–liquid:
TA98 (�S9), TA100 (+S9),
Low; liquid–liquid: TA98
(+S9), TA100 (�S9, +S9)
Ganga River (India) XAD-4, XAD-8/acetone;
liquid–liquid
extraction/hexane, chloroform
Ames assay (plate)/TA98,
TA100, TA97a, TA102
Moderate; XAD: TA98
(�S9, +S9), TA100
(�S9, +S9)
Pesticides [55]
Low; liquid–liquid: TA98
(�S9, +S9), negative;
TA100 (�S9, +S9)
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
11
9
Yodo River (Japan) Blue rayon column method umu assay/S. typhimurium
NM2009, NM2000
Positive Trp-P-2 [56]
Yodo River (Japan) Blue rayon hanging method;
XAD-2/diethyl ether
umu assay/S. typhimurium
NM2009
Positive – [57]
Yodo River (Japan) XAD-2/diethyl ether umu assay/S. typhimurium
NM2009
Positive 1-NP [58]
Katsura River, Asahi
River (Japan)
XAD-2/diehyl ether;
blue chitin
column/methanol:
conc. ammonia (50:1)
Ames assay (pre)/TA98 moderate (TA98, 9) – [59]
Yodo River (Japan) Blue rayon hanging method umu assay/S. typhimurium
NM2009
Positive Trp-P-1, Trp-P-2,
MeIQx, PhIP
[60]
Nishitakase River (Japan) Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 +S9) PBTA-1 [61]
Taihu Lake (China) Liquid–liquid extraction/acetone Ames test/TA98, TA100 Moderate; TA100 S9,
+S9), low; TA98 S9, +S9)
Domestic sewage,
agricultural and
industrial wastewater
[62]
NakDong River (Korea) Blue rayon hanging method Ames assay (plate)/TA98,
TA97a, YG1041, YG1042
Positive – [63]
Nishitakase River (Japan) Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 +S9) PBTA-2 [64]
Kojima Lake, Asahi River,
Sasagase
River (Okayama, Japan)
blue rayon hanging in
water in a beaker
Ames assay (pre)/YG1024 negative BaP [65]
Lake Baikal (Russia) Blue rayon hanging in
water in a beaker
Ames assay (pre)/YG1024 Positive BaP [65]
Yodo River (Japan) Blue rayon column method Ames assay (pre)/YG1024 Positive PBTA-1, PBTA-2 [25]
Asahi and Sasagase River
(Okayama, Japan)
Blue rayon hanging in
water in a beaker
Ames assay (pre)/YG1024 Negative BaP [65]
Nikko River (Aichi, Japan) Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 +S9) PBTA-3 [66]
Rivers in Japan Blue rayon hanging method Ames assay (pre)/TA100,
YG1024
Extreme; YG1024 +S9),
moderate; YG102 (�S9)
– [67,68]
Yodo River (Japan) Blue rayon hanging method umu assay/S. typhimurium
NM2009, NM2000
Positive Trp-P-2 [69]
Taihu Lake (China) XAD-2 resin/acetone Ames assay (plate)/TA98,
TA100
Moderate; TA98 ( S9, +S9) Discharges from
municipal wastewater
[70]
Nikko River, Uji River (Japan) Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 +S9) PBTA-4 [71]
Tobei River, Asuwa River,
Nishitakase River, Uji
River (Japan)
Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 +S9) PBTA-5, PBTA-6 [72]
Taihu Lake (China) XAD-2 resin/acetone Ames assay (plate)/
TA98, TA100
High; TA98 (�S9 +S9),
negative; TA100 ( S9, +S9)
Illegal deposition
of chemical waste
in the lake
[73]
Ara test/S. typhimurium
BA9
positive
+S
(
(�(�
(
(
(
4
�
(
(
,
�
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
12
0Table 3 (Continued )
Sample source Preparation method Assay methoda/strain Mutagenic potency
classificationb
Suspected
mutagen/likely
sources
Reference
Asuwa River, Katsura
River (Japan)
Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024
(�S9, +S9)
PBTA-7, PBTA-8 [74]
Mawatari River, Asuwa River,
Kitsune River (Fukui, Japan)
Blue rayon hanging method Ames assay (pre)/YG1024,
YG1029
Extreme; YG1024 (+S9) PBTA-3, PBTA-4,
PBTA-6
[75]
Nagara and other rivers
(Japan)
Blue chitin column/
methanol:conc.
ammonia (50:1)
Ames assay (pre)/TA98,
YG1021,
YG1024
Moderate; TA98 (+S9),
negative; TA98 (�S9)
PAHs [76]
Waka River (Wakayama,
Japan)
Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 (�S9) 4-Amino-3,3’-
dichloro-5,4’-
dinitrobiphenyl
[77]
River Yamuna (Mathura,
India)
XAD-4, XAD-8/acetone;
liquid–liquid
extraction/hexane, chloroform
Ames assay (plate)/TA98,
TA100, TA97a, TA102
Extreme; TA98 (�S9, +S9),
high; TA100 (�S9, +S9)
Pesticides,
domestic and
industrial waste
[78]
River Yamuna (downstream
of Agra, India)
XAD-8/DMSO Ames assay (plate)/TA98,
TA100, TA97a, TA102,
TA104
Extreme; TA98 (�S9, +S9),
high; TA100 (�S9, +S9)
Municipal wastes
and the industrial
efluents
[79]
Ames fluctuation test/TA98,
TA100, TA97a, TA102
Positive
Six rivers in North Kyusyu
(Japan)
Blue rayon hanging method Ames assay (plate)/TA100,
YG1024,
YG1041, YG1042
Moderate; YG1024
(�S9, +S9)
BaP, Trp-P-1,
Trp-P-2
[80]
3. North America
Galveston Bay (USA) Blue rayon hanging method Ames assay (pre)/TA98 Positive A collision of
barge tankers
[81]
Yamaska River (Quebec,
Canada)
Flash evaporation (10�) Mutatox test/luminescense
bacterium Photobacterum
Positive – [82]
St. Lawrence River
system (Canada)
Filtered water;
particulates/DCM
SOS Chromotest/E. coli Positive Not correlated with
demonstrated mutagens
such as PAH and
heavy metals
[83]
Aberjona River (MS, USA) Poly(vinylidene difluoride)
filter and
bonded-phase sorbent (CN,
C18)/soxhlet-extracted/DCM,
methanol
The Salmonella typhimurium
forward mutation
assay/Salmonella
typhimurium TM677)
Negative – [84]
St. Lawrence River (Canada);
Providence River,
Charles River
Blue rayon hanging method Ames assay (plate)/TA98, TA100,
YG1024, YG1041,
Moderate; YG1024 (+S9),
low; YG1024 (�S9)
– [85]
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
12
1
Potomac River, Hudson River,
East River (USA)
YG1042
Atlantic Ocean (Annapolis,
MD, USA),
Pacific Ocean (Monerey,
CA, USA)
Filtration Vibrio harvey assay/Vibrio harvey Positive – [48]
4. South America
Cai River, a tributary of the
Guaiba River (Brazil)
Filtration Ames assay (pre)/TA98,
TA100, TA102
Extreme; TA98 (�S +S9),
TA100 (�S9, +S9)
Under the
influence of
petrochemical
industries
[86]
Water courses of Sao Paulo
State (Brasil)
XAD-2 resin/methanol,
DCM
Ames assay (plate)/TA98,
TA100
High; TA98 (+S9), oderate;
TA98 (�S9), negati ;
TA100 (�S9, +S9)
[87]
Cai River, a tributary of the
Guaiba River
(Rio Grande do Sul, Brazil)
Direct assay Ames assay (plate)/TA98 Extreme; TA98 (�S +S9), Affected by the
petrochemical
industrial comples
[88]
Cai River, a tributory of the
Guaiba River (Brazil)
Non-concentrated sample;
liquid–liquid
extraction/DCM (acidic,
basic, neutral);
volatile substances
extraction method
Microsuspension assay/TA98,
TA100, TA102
microscreen phage-
induction assay/E.
coli B/rWP2s(l)
Extreme; TA98 (�S +S9),
TA100 (�S9)
Under the influence
of industrial complex
[89]
Rio Tercero River (Cordoba,
Argentina)
XAD-2/DMSO (pH 2) Ames assay (plate)/TA98, TA100 Low; TA98 (�S9, + 9),
TA100 (�S9, +S9)
– [90]
Canals between Ensenda and
Berisso (Argentina)
Liquid–liquid extraction/DMC
(acidic, neutral)
Ames assay (plate)/TA98, TA100 Extreme; TA98 (+S ,
TA100 (+S9)
Heavily industrialized
area (petrochemical,
oil refinary, steel
rolling mill, petroleum
coke plant, sulfuric
acid plant)
[91]
Matanza-Riachuelo River
(Buenos Aires, (Argentina)
Filtration; XAD-2 Ames assay (plate)/TA98, TA100 Low; TA98 (�S9, + 9),
TA100 (�S9, +S9)
Wastewater from
farming, grazing,
domestic sewage,
industries
[92]
Surface waters in Brazil Liquid–liquid extraction/
DCM; XAD
resin/DCM, methanol;
filtration
Ames assay (plate)/TA98, TA100 Extreme; TA98 (�S
+S9), TA100 (�S9, S9)
– [93]
Sinos River basin (RS, Brasil) Direct concentration
method
Ames assay (pre)/TA1535, TA97,
TA98, TA100, TA102
Low; TA98 (�S9, + 9),
TA100 (�S9, +S9)
Heavy metals and
organic contaminants
[94]
9,
m
ve
9,
9,
S
9)
S
9,
+
S
T. Ohe et al. / Mutation Research 567 (2004) 109–149122
ble
3(C
on
tin
ued
)
mp
leso
urc
eP
rep
arat
ion
met
ho
dA
ssay
met
ho
da/s
trai
nM
uta
gen
icp
ote
ncy
clas
sifi
cati
on
b
Su
spec
ted
muta
gen
/lik
ely
sou
rces
Ref
eren
ce
Mic
rosc
reen
ph
age-
ind
uct
ion
assa
y/E
.
coli
B/r
WP
2s(l
)
Po
siti
ve
Su
rfac
ew
ater
sin
Sao
Pau
lo(B
razi
l)
XA
D4
resi
n/m
eth
ano
l,M
C;
blu
e
rayo
nco
lum
nm
eth
od/m
eth
ano
l:
con
c.am
mo
nia
(50
:1)
Am
esas
say
(pla
te)/
TA
98,
TA
100
Low
;T
A98
(�S
9,
+S
9),
TA
10
0(�
S9
,+
S9
)
–[9
5]
Cri
stai
sR
iver
(Bra
sil)
XA
D-4
/met
han
ol,
DC
M(n
eutr
al)/
met
han
ol,
ethyla
ceta
te(a
cidic
),
Blu
era
yo
nh
ang
ing
met
ho
d
Am
esas
say
(pla
te)/
TA
98,
TA
100,
YG
10
41
,Y
G1
04
2
Mo
der
ate;
TA
98
(�S
9,
+S
9),
low
;T
A1
00
(�S
9,
+S
9)
Ind
ust
rial
effl
uen
t
(tex
tile
dy
ein
g
faci
lity
)
[96
]
M,
dic
hlo
rom
eth
ane
and
PA
H:
po
lycy
clic
arom
atic
hy
dro
carb
on
.T
he
resu
lto
fo
ther
assa
ys
issh
ow
nas
po
siti
ve
or
neg
ativ
e.a
Am
esas
say
iscl
assi
fied
asfo
llow
s:A
mes
assa
y(p
late
),th
est
and
ard
pla
tein
corp
ora
tio
nas
say
[15
,97,9
8];
Am
esas
say
(pre
),th
ep
rein
cub
atio
nm
eth
od
[99
];M
icro
susp
ensi
on
say
[10
0];
Am
esfl
uct
uat
ion
test
[10
1].
bC
lass
ifica
tio
nre
pre
sen
tsth
em
axim
alm
uta
gen
icac
tiv
ity
level
exp
ress
edas
rever
tan
tsp
erli
ter
for
TA
98
and
/or
TA
10
0,a
nd
on
eex
pre
ssed
asre
ver
tan
tsp
erg
blu
era
yo
neq
uiv
alen
t
rY
G1
02
4o
bta
ined
inea
chre
fere
nce
.Cla
ssifi
edas
:lo
w;n
d–
50
0,m
od
erat
e;5
00
–2
50
0,h
igh
;2
50
0–
500
0,e
xtr
eme;
mo
reth
an5
00
0re
ver
tan
tsp
erli
ter
inT
A9
8an
dT
A1
00
[93
],an
d
;n
d–
10
00
,m
od
erat
e;1
00
0–
10,0
00,
hig
h;
10
,00
0–
10
0,0
00
,ex
trem
e;m
ore
than
10
0,0
00
rever
tan
tsp
erg
blu
era
yo
neq
uiv
alen
tin
YG
10
24
[85
].P
aren
thes
issh
ow
sth
ek
ind
of
ain
sw
ith
or
wit
ho
ut
S9
mix
inth
ecl
assi
fica
tio
no
fm
uta
gen
icp
ote
ncy
clas
sifi
cati
on.
Ta
Sa
DC
as fo low
str
were concentrated and exchanged for a solvent that is
compatible with the selected bioassay (e.g., dimethyl
sulfoxide or DMSO).
Grifoll et al. [30] reported that the particulate
matter retained in filter membrane exhibited a stronger
mutagenic activity than the dissolved phase. This
demonstrates that filtration or sterilization via filter
membrane could remove some of the mutagenic
activity. White et al. [5] investigated the sorptive
properties of organic genotoxins in industrial effluents
and revealed that a substantial fraction (up to 99.8%)
of the emitted genotoxicity is associated with
particulate material. Accordingly, the issue of filtra-
tion and sterilization via filtration membrane of
surface waters are important one since a large portion
of the organic pollutants are often adsorbed to the
particulate material.
3. Review of published mutagenicity/genoxicityassessment data of surface waters
3.1. Salmonella/mutagenicity data
3.1.1. Mutagenic features of surface waters with
Salmonella typhimurium TA98 and TA100
There are many assays for detecting the mutagen-
icty/genotoxicity of surface waters, but the utilization
of bioassays with bacteria has proven to be very
effective for monitoring because these assays are
sensitive, inexpensive, reliable, and can be performed
in a short period of time with relatively low cost.
Among the microbial bioassays, the Salmonella
mutagenicity test has been the most widely used for
detecting mutagenicity/genotoxicity in surface waters.
The different responses of the Salmonella strains can
provide information on the classes of mutagens
present in water samples. This test developed by
Ames et al. [15,97,98] is based on the detection of
histidine-independent revertants in selected Salmo-
nella strains after exposure to mutagens with or
without additional activating enzymes. The dose-
response can be quantified by varying sample
concentration and counting revertant colonies per
plate at each concentration. Samples to be tested must
be filter sterilized under normal conditions. It is also
recommended as the standard method in Standard
Methods for the Examination of Water and Waste-
T. Ohe et al. / Mutation Research 567 (2004) 109–149 123
Fig. 2. The percentage of positive and negative results for S. typhimurium TA98 and TA100 for all available observations available from
published data. Data are cited from all observations from the published articles in Table 2. Mutagenicity evaluation employed the ‘‘modified two-
fold rule’’ where positive identification of mutagenicity requires a response at least two-fold greater than the solvent control, plus a clear
concentration–response relationship [109]. In cases where only a single dose was examined, the value was judged to be positive if the mutation
frequency was more than two times the negative control.
water – 20th Edition – by the American Public Health
Association (APHA), American Water Works Asso-
ciation (AWWA) and Water Environment Federation
(WEF) [107]. The test has now been officially
included in the San Paulo State Water Works
Monitoring Program at sites where water is to be
used as a source of drinking water [96] and is the test
method proposed by the U.S. Environmental Protec-
tion Agency for Clean Water Act compliance
monitoring [108]. Much of the published surface
water Salmonella mutagenicity data employed sam-
ples concentrated by direct partitioning into organic
solvents, or adsorption and subsequent solvent elution
to assess the mutagenic potency. Most studies
employed the standard plate-incorporation version
of the assay using strains TA98 and/or TA100 with and
without metabolic activation. Fig. 2 shows the ratio of
positive and negative samples with strains TA98 and
TA100 in the absence and the presence of a metabolic
activation system for all observations cited in Table 3.
Among all data analyzed, the percentage of positive
samples toward TA98 was approximately 15%, both in
the absence and the presence of S9 mix. Positive
TA100 results were 7% both with and without S9 mix.
These observations suggest the predominance of
direct and S9-activated frameshift-type mutagens
rather than direct and S9-activated base-substitu-
tion-type mutagens in surface waters in the world.
T. Ohe et al. / Mutation Research 567 (2004) 109–149124
Table 4
Salmonella typhimurium strains widely used in Ames test for surface waters
Strain Description Source
Frameshift type
TA98 hisD3052, rfa, DuvrB, pKM101 Ames [15]
TA98NR As TA98, but deficient in the classical nitroreductase Rosenkranz [110–111]
TA98/1,8-DNP6 As TA98, but deficient in O-acetyltransferase McCoy [112]
YG1021 TA98 (pYG216): a nitroreductase-overproducing strain Watanabe [113]
YG1024 TA98 (pYG219): an O-acetyltransferase-overproducing strain Watanabe [114]
YG1041 TA98 (pYG233): nitroreductase and O-acetyltransferase-overproducing strain Hagiwara [115]
Base-substituton type
TA100 hisG46, rfa, DuvrB, pKM101 Ames [15]
YG1026 TA100 (pYG216): a nitroreductase-overproducing strain Watanabe [113]
YG1029 TA100 (pYG216): an O-acetyltransferase-overproducing strain Watanabe [114]
YG1042 TA100 (pYG233): nitroreductase and O-acetyltransferase-overproducing strain Hagiwara [115]
Oxidative damage-detecting type
TA102 hisD(G)8476, rfa, pAQ1(hisG428, pKM101) Levin [116]
Table 4 lists the names and genotypes of the
Salmonella typhimurium strains widely used in
Salmonella mutagenicity tests for surface waters.
Based upon possible occurrence analyzed in a 20-
year survey conducted since 1979 by the Environ-
mental Agency of Sao Paulo State in Brasil,
Umbuzeiro et al. [93] proposed boundaries of
mutagenic activity for natural water samples to
compare the distribution of mutagenic potencies,
based on the classification system for industrial wastes
and effluents developed by Houk [1]. The boundaries
are classified as follows: up to 500 revertants per
equivalent liter as ‘‘low’’; from 500 to 2500 revertants
per equivalent liter as ‘‘moderate’’; from 2500 to 5000
revertants per equivalent liter as ‘‘high’’, and more
than 5000 revertants per equivalent liter as ‘‘extreme’’
mutagenic activity. Fig. 3 shows the frequency
distribution of mutagenic potency values for all
available positive data with TA98 and TA100 in the
absence and in the presence of S9 mix according to the
aforementioned mutagenic potency classification.
Among all data analyzed, the percentage ranked as
‘‘high’’ or ‘‘extreme’’ was approximately 3–5% both
for TA98 and TA100, irrespective of the absence or
presence of S9 mix. Some rivers classified as
‘‘extreme’’ showed the maximum mutagenic potency
of more than 10,000 revertants per liter for TA98 and/
or TA100 in the presence or absence of S9 mix. These
results demonstrate that some rivers in Europe, Asia
and South America are contaminated with potent
direct-acting and S9-activated frameshift-type and
base substitution-type mutagens. Those rivers are
reported to be contaminated by either partially treated
or untreated discharges from chemical industries,
petrochemical industries, oil refineries, oil spills,
rolling steel mills, untreated domestic sludges, and
pesticides runoff [30,43,54,78,79,86,88,89,91,93].
Since a detailed discussion of all the Salmonella
mutagenicity test results shown in Table 3 is beyond
the scope of this paper, the description on the
following pages will be restricted to those studies
that recorded mutagenicity levels that would be
classified as extreme.
Grifoll et al. [30] performed a mutagenicity
assessment of the dissolved and particulate phases
of the Besos and Llobregat Rivers, which flow along
populated and industrialized basins near Barcerona,
Spain. Both rivers share domestic, industrial and
agricultural uses and are recipients of a large amount
of untreated effluents. The results indicated that both
rivers are chronically polluted by base substitution and
frameshift mutagens and promutagens. Interestingly,
the particulate (>0.22 mm) phase exhibited a stronger
mutagenic activity than the dissolved phase and the
mutagenic activity of the particulate phase was ranked
as ‘‘extreme’’. They also demonstrated that the base
substituition mutagens remain associated with the
dissolved phase, whereas frameshift mutagens are
T. Ohe et al. / Mutation Research 567 (2004) 109–149 125
Fig
.3.
Fre
quen
cydis
trib
uti
on
of
muta
gen
icpote
nc y
clas
sifi
cati
on
among
posi
tive
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ple
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ith
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um
TA
98
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uta
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nc y
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wer
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pre
ssed
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rms
of
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tants
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.If
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ue
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ng
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yle
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ne a
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ssio
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ial
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ined
inth
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hig
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5000;
extr
eme
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ore
than
5000
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lite
r .
T. Ohe et al. / Mutation Research 567 (2004) 109–149126
more favorably adsorbed to the suspended particulate
phase. This suggests that some frameshift mutagens
might be removed by sterilization via filtration or
filtration of water samples. Guzzella et al. [43]
compared the mutagenicity of water samples from the
Como Lake, Italy, and tried to find the source of
water genotoxins. The result revealed that extreme
mutagenic potency was found with TA98 in the
presence of S9 mix in one water sample collected near
the river mouth in Como Lake more than 5000
revertants per liter and concluded that river influent
was an important source of mutagenic contamination.
However, they reported that lake water samples
did not show any genotoxicity both with Allium
root anaphase aberration assay and Allium root-
micronuclei assay.
Rehana et al. [54] used five different Salmonella
tester strains to compare the mutagenic activity of
water samples from four sites of the Ganga River,
India, using the XAD-resin extraction method and the
liquid–liquid extraction method. Samples always
showed ‘‘extreme’’ mutagenic activity for TA98 and
TA100, both with and without S9 mix. The maximum
activity for each strain was >10,000 revertants per
liter. They also found a similar pattern in the
responsiveness of tester strains for a mixture of
pesticides, suggesting that the mutagenicity of water
extracts may be attributable to the pesticides used in
the upstream region. Aleem and Malik [78] and
Siddiqui and Ahmad [79] reported that XAD-
concentrated water samples from the River Yamuna,
India, was remarkably high for TA98 (classified as
‘‘extreme’’) compared to TA100 (classified as
‘‘high’’), both with and without S9 mix. It was also
reported that XAD-concentrated samples elicited
higher responses than liquid–liquid concentrated
samples, the water samples collected during the
summer exhibited higher mutagenic activity com-
pared with other seasons, and water samples also
contained oxidative (TA102) mutagens. This extreme
mutagenic contamination of the river water is likely to
be derived from a combination of domestic, municipal
and industrial effluents noted at this sampling
site.
Vargas et al. [86,89] reported that extremely potent
activity was observed for TA98 without metabolic
activation among about 100 non-concentrated samples
collected from Caı River, Brasil, an area under the
influence of a petrochemical industrial complex. The
potency ranking was ‘‘extreme’’ for both TA98 and
TA100, with and without metabolic activation. The
highest value was more than 200,000 revertants per
liter for TA98 in the absence of S9 mix compared with
7000 revertants per liter in the presence of S9 mix (the
value was calculated from the data on non-concen-
trated 2-ml sample per plate). The maximum value for
TA100 was more than 50,000 revertants per liter and
more than 10,000 revertants per liter in the absence of
S9 mix and in the presence of S9 mix, respectively. It
was also reported that these non-concentrated samples
lost their activity upon liquid–liquid extraction using
dichloromethane. In turn, they suggested that volatile
compounds were responsible for the mutagenicity that
were lost in the liquid–liquid extraction.
Lemos et al. [88] also reported that water samples
from the Caı River were ranked as ‘‘extreme’’ for
TA98 both with and without S9 mix. The highest
values for TA98 were more than 100,000 and 50,000
revertants per liter in the absence of S9 mix and in the
presence of S9 mix, respectively. Collectively, these
results suggested that volatile substances derived from
petrochemical industries in the area contributed to the
extremely potent mutagenic activity of Caı River
water samples. Investigations carried out by Umbu-
zeiro et al. [83] in a surface water quality monitoring
program analyzed for 20 years in Sao Paulo State in
Brazil demonstrated that 14% of 1007 surface water
samples showed positive mutagenic activity. Among
the positive samples, a total of 81 samples were
analyzed using a dose-response manner. From those
81 samples, 9% were ranked as ‘‘extreme’’ for either
TA98 or TA100 (5400–30,000 revertants per liter). In
addition, the result showed that direct-acting muta-
gens induced frameshift mutations and S9-activated
mutagens induced base substitution mutations. Their
possible pollution sources were petrochemical indus-
trial, oil spill and untreated domestic sludge. In a study
of the Rio Tercero River (Cordova, Argentina) by
Alzuet et al. [91], the presence of S9-activated
mutagens capable of causing base substitution and
frameshift mutations was observed (without S9, data
not available). Maximum activity was observed in
TA98 with S9 mix (8,550,000 revertants per liter),
although moderate activity was found in TA100 with
S9 mix (1000 revertants per liter). The region is a
heavily industrialized and holds the main oil refinery
T. Ohe et al. / Mutation Research 567 (2004) 109–149 127
in the country, several petrochemical industries, a
rolling steel mill and a sulfuric acid plant. Previous
studies have demonstrated the presence of polycyclic
aromatic hydrocarbons in airborne particulate matters,
surface waters and sediments collected in the study
area [117–119].
3.1.2. Mutagenic features of surface waters with
nitroreductase- and/or O-acetyltransferase-
overexpressing strains
S. typhimurium YG1021 and YG1026, strains that
possess high nitroreductase levels, were developed by
introducing plasmids containing the nitroreductase
gene from S. typhimurium TA1535 into TA98 and
TA100, respectively. These strains have been shown to
detect various kinds of mutagenic nitro compounds
much more efficiently than TA98 and TA100 [113].
Watanabe et al. [114] successfully developed addi-
tional new tester strains S. typhimurium YG1024 and
YG1029, strains derived from TA98 and TA100,
respectively, that show remarkably high sensitivity to
both nitroarenes and aromatic amines. S. typhimurium
YG1041 and YG1042 [115], derived from TA98 and
TA100, respectively, have enhanced levels of both
nitroreductase and O-acetyltransferase and are, con-
sequently, highly sensitive to nitroarenes and aromatic
amines. The significantly higher mutagenicity in
the metabolically enhanced diagnostic strains
(e.g.,YG1021, YG1024, YG1026, YG1029, YG1041
and YG1042) in comparison to TA98 or TA100
suggests the presence of aromatic amine-type muta-
gens in the presence of S9 mix and the presence of
aromatic nitro-type mutagens in the absence of S9
mix.
Cerna et al. [38] and Umbuzeiro et al. [98] showed
that YG strains including YG1021, YG1024, YG1041
or YG1042 elicited higher numbers of revertants in
response to effluents and river water samples
(extracted with Separon SE, XAD4, blue rayon or
liquid–liquid method) compared with TA98 or TA100
both with and without metabolic activation. These
indicate the likely presence of aromatic amines and
nitroarenes in the samples tested. Sayato et al. [49,51]
demonstrated that highly sensitive detection of
mutagenicity in surface waters could be effectively
achieved by combining blue cotton/blue rayon as an
effective adsorbent and the new Salmonella tester
strains as a sensitive bioassay. They reported that the
activity of the subfractions, obtained by separating
blue cotton adsorbates collected from the Katsura
River (a tributary of the Yodo River, Japan) via
Sephadex G-25 gel chromatography, was greatly
increased by the addition of metabolic activation,
especially in YG1024, and these fractions showed less
mutagenicty in TA98/1,8-DNP6, suggesting that S9-
activated mutagenic aromatic amines were present in
the Katsura River. Kusamran et al. [52] also reported
that samples obtained by the blue rayon hanging
method from the Chao Phraya river and connected
canals in Bangkok, Thailand, had no significant
mutagenic effect in either TA98 or TA100. However,
samples showed a significantly greater response in
YG1024 than in strain YG1029, especially in the
presence of metabolic activation. These results
indicate that the combination of specific mutagenicity
tests and selective collection methologies can provide
clues to the identity of organic genotoxic pollutants in
surface water samples.
Kataoka et al. [45] reported significantly higher
mutagenicity for YG1024 than for TA98 in the
presence of S9 mix in canal samples collected along
the Danube River, Austria, again suggesting the
presence of aromatic amine mutagens. The occurrence
of base substitution-type mutagenic effects toward
strain TA100 was low or undetectable. In addition,
samples did not elicit a positive response in S.
typhimurium YG1029 with metabolic activation.
Three heterocyclic amines were subsequently identi-
fied in the blue rayon adsorbates. Kira et al. [65]
reported on a simplified handling and transportation
system for monitoring samples from remote sites.
They put blue rayon directly into the sample bottle for
24 h, and blue rayon adsorbed mutagens were
transported to the laboratory. Sampling was performed
in Lake Baikal, Russia and blue rayon adsorbates were
later transported to Okayama, Japan for analysis.
Although the mutagenic potency values in this study
were low (S. typhimurium YG1024 with S9 mix), they
stated that the system might be useful in international
collaborative studies in this area of science.
Ohe et al. [85] employed the blue rayon hanging
method to monitor a wide range of surface water
samples flowing through large metropolitan areas in
North America. Mutagenicity was evaluated using TA
strains and YG strains with and without metabolic
activation. The results demonstrated that YG1024 and
T. Ohe et al. / Mutation Research 567 (2004) 109–149128
YG1041 were much more sensitive than TA98 with S9
mix, and the authors concluded that rivers flowing
through major cities in North America contained
frameshift-type, aromatic amine-like mutagens,
although the levels of mutagenic activity were ranked
as very low compared with data from Thailand and
Japan [52].
Endo et al. [68] collected 541 water samples from
130 rivers in Japan using blue rayon hanging method
between 1996 and 2003 and measured their muta-
genicity by the Ames assay using S. typhimurium
TA100, YG1029 and YG1024 both with and without
S9 mix. The positive ratio was as follows; TA100, �S9
mix: 10%, TA100, +S9 mix: 26%, YG1029, �S9 mix:
29%, YG1029, +S9 mix: 54%, YG1024, �S9 mix:
68% and YG1024, +S9 mix: 87%. Strong mutagenic
activities, i.e. more than 100,000 revertants per gram
blue rayon, were detected for the samples collected
from the Nishitakase, Uji and Katsura Rivers in Kyoto,
the Asuwa, Mawatari and Kitsune Rivers in Fukui and
the Nikko Rivers in Aichi.
Fig. 4 shows frequency distribution results of the
mutagenic potencies on all data available for the
combination of blue rayon hanging method as a
collecting method and YG1024 strain as a bioassay
system. Mutagenic potency was classified as low (up
to 1000 revertants per gram blue rayon equivalent),
moderate (1000–10,000 revertants per gram blue
rayon equivalent), high (10,000–100,000 revertants
Fig. 4. Frequency distribution of mutagenic potency classification in bioass
typhimurium YG1024 strain. Mutagenic potency values are expressed in te
potency is classified as: ND, not detected, low mutagenicity; ND–1000,
100,000, extreme mutagenicity; >100,000 revertants per gram blue rayon
per gram blue rayon equivalent) and extreme (more
than 100,000 revertants per gram blue rayon
equivalent). The percentage of extreme mutagenic
activity was approximately 1% and 19% with S9 mix
and without S9 mix, respectively, as shown in Fig. 4.
Most samples of those were collected from rivers
which received discharges from textile dyeing
factories or sewage plants treating effluents from
textile dyeing factories, and most samples were shown
to contain some PBTA-type mutagens (see Section 4).
Collectively, these data in this section demonstrate
that the blue rayon hanging technique is suitable for
judging the presence of mutagens and identifying
mutagens in surface waters, and that it is suitable in
international collaborative studies of mutagens in
surface waters, since there is no need for transporting
large volumes of water samples to the place where
analysis is performed. Moreover, the combination of
the blue rayon hanging method and the Salmonella test
with metabolically enhanced strains is a simple and
sensitive method to monitor for nitroarene compounds
and aromatic amine mutagens in surface waters.
3.2. SOS chromotest/umu-test and other bacterial
assay
Although the Salmonella/microsome assay has
been widely employed for the detection of mutageni-
city in environmental samples, a variety of other
ay data with a combination of the blue rayon hanging method and S.
rms of revertants per gram blue rayon equivalent (BRE). Mutagenic
moderate mutagenicity; 1000–10,000, high mutagenicity; 10,000–
equivalent (BRE).
T. Ohe et al. / Mutation Research 567 (2004) 109–149 129
assays also exist for investigating complex environ-
mental mixtures. The SOS Chromotest and umu-test
were developed alternatives to the Ames test by
Quillardet et al. [120] and Oda et al. [121],
respectively. An Escherichia coli strain PQ37 or S.
typhimurium strain TA1535/pSK1002, containing a
fusion gene of a b-galactosidase gene (lacZ) and an
SOS response gene, is employed in these assays.
Activation of the SOS repair system by genotoxic
compounds is measured by photometric determination
of the b-galactosidase enzyme activity. The SOS
Chromotest and umu-test are widely used for routine
monitoring of water samples because the results are
available in a single day with minimal advance
preparation. The microplate version of the SOS
Chromotest/umu-test was developed as a rapid and
sensitive screening tool, for the detection of genotox-
ins in surface waters [28,83,122]. A umu-test using an
O-acetyltransferase-overproducing strain has been
applied as a sensitive bioassay to detect the presence
of genotoxicity from nitroarenes and aminoarenes in
surface waters [56–58,60].
The Mutatox test, employing with a dark mutant
strain of luminescent Photobacterium phosphoreum,
the Microscreen phage-induction assay with E. coli
strain [37], the DNA repair assay with E. coli strain
and the Ara-test (L-arabinose resistance mutagenesis
test) with S. typhimurium have been used for screening
surface water samples for genotoxic activity and have
been promoted as candidates for a battery of screening
assays [37,39,82]. Helma et al. [37] evaluated four
bacterial short-term genotoxicity assays, for detecting
the genotoxicity of water samples of different origins.
They concluded based on number of positive response
that the differential DNA repair system was the most
sensitive and the Microscreen assay was the least
sensitive with the SOS Chromotest being equally
sensitive to the Salmonella/microsome assay. Vahl et
al. [39] compared two mutagenicity assays (the Ames
test and the Ara-test) and an SOS induction test for
particulate matter samples of the Elbe River. They
concluded the quantitative response was higher in the
Ara-test. Samples also induced lower genotoxic
potencies in the umu-test than in the mutagenicity
assays.
Vargas et al. [86,89] evaluated the genotoxicity of
river water samples collected from the Sinos River,
Brasil, using the microscreen phage-induction and
Salmonella/microsome assays. They concluded that
the microscreen phage-induction assay was a more
appropriate screening assay for judging the genotoxi-
city of multiple pollutants in water samples in which
both organic compounds and heavy metals were
present.
Since Salmonella survives poorly in unextracted
marine water samples, Czyz et al. [47,48,123] con-
structed genetically modified Vibrio harveyi strains
that produce significantly more neomycin-resistant
mutants, and they found that the Vibrio harveyi test
may be used as an adequate assay for detecting
mutagenic pollution in marine waters due to the greater
sensitivity of the test relative to the Ames assay.
3.3. DNA adduct formation
DNA-adducts in aquatic organisms are effective
molecular dosimeters of genotoxic contaminant
exposure, and the 32P-postlabeling assay has been
used to measure covalent DNA-xenobiotic adducts. In
the 32P-postlabeling assay, DNA is hydrolyzed
enzymatically to 30-monophosphates and DNA
adducts are enriched by the selective removal of
normal nucleotide. The DNA adducts are then labeled
with [32P] phosphate and resulting 32P-labeled DNA
adducts are usually separated by thin-layer chromato-
graphy or high performance liquid chromatography.
Radioactivity of DNA adducts are detected by
autoradiography and liquid scintillation counting,
imaging analysis or a liquid scintillation analyzer.
There is a huge body literature on DNA adducts in
aquatic organisms, including the review by Stei et al.
[124]. The 32P-postlabeling technique is the most
sensitive method for the detection of a wide range of
large hydrophobic compounds bound to DNA, and can
potentially detect one DNA adduct, such as those
derived from polycyclic aromatic compounds (PACs),
in 109–1010 bases. Table 5 summarizes the reports on
DNA adducts in aquatic organisms. In a review of
genotoxic events in some marine fishes, Reichert et al.
[141] documented that DNA adduct levels are a
significant risk factor for certain degenerative and
preneoplastic lesions occurring early in the histogen-
esis of hepatic neoplasms in feral English sole
(Pleuronectes vetulus) from Puget Sound in Washing-
ton, USA, an area which is heavily contaminated with
polycyclic aromatic compounds.
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
13
0
Table 5
Summary of reported studies on DNA adducts in aquatic organisms in vivo analyzed by 32P-postlabeling
Sample source Organism Organ/tissue Suspected mutagens/contaminants Reference
1. Europe
Po River (Italy) Chub (Leuciscus cephalus) Liver AC [125]
Liverpool Bay (UK) Flatfish (Limanda limanda) Liver PAH [126]
Milfold Haven (UK) Teleost, Lipophrys pholis Gill, liver, ovaries, testes oil spill [127]
Tyne Estuary (England) Flounder (Platichthys flesus) Liver PAC [128]
Meuse River (The Netherland) Cryfish (Orconectus limosus) Hepatopancreatic tissue Heavy metal, PCB, pesticides [129]
Angermanalven River (Sweden) Perch (Perca fluviatilis) Hepatic DNA Creosote [130]
Reykjavik Harbor, etc. (Iceland) Shorthorn sculpin (Myoxocephalus scorpius) Liver PAH [131]
Reykjavik Harbor, etc. (Iceland) Mussel (Mytilus edulis) Gill, digestive gland Vessel traffic, PAH, oil spill,
TBT, TPT
[132]
2. Asia
Mediterranean Sea (Turkey) Gray mullet (Oedalechilus labeo, Liza ramada) Livers, blood PAH [133]
Mediterranean Sea (Turkey) Gray mullet (Mugil sp.) Livers, blood PAH [134]
Mediterranean, Black Seas (Turkey) Gray mullet (Mugil sp.) Livers, blood PAH [135]
3. North America
Puget Sound, Washington (USA) English sole (Parophrys vetulus) Liver PAH, PCB [136]
Puget Sound, Washington (USA) Rock sole (Lepidopsetta bilineata) Liver PAH, PCB [136]
Puget Sound, Washington (USA) Starry flounder (Platichthys stellatus) Liver PAH, PCB [136]
Puget Sound, Washington (USA) Chinook salmon (Oncorhynchus tshawytscha) Liver PAH, PCB, DDT [137]
Long Island Sound, Connecticut (USA) Winter flounder (Pseudopleuronectes americanus) Liver AH, PCB [138]
Elizabeth River (USA) Oyster toadfish (Opasanus tau) Liver Creosote [139]
Puget Sound, San Francisco Bay,
San Diego Bay (USA)
English sole (Pleuronectes vetulus) Liver PAH, PCB, DDT, chlordane, dieldrin [140]
Puget Sound, San Francisco Bay,
San Diego Bay (USA)
Starry flounder (Platichthys stellatus) Liver PAH, PCB, DDT, chlordane, dieldrin [140]
Puget Sound, San Francisco Bay,
San Diego Bay (USA)
White croaker (Genyonemus lineatus) Liver PAH, PCB, DDT, chlordane, dieldrin [140]
Charleston Harbor (USA) English sole (Pleuronectes vetulus) Liver PAC [141]
Atlantic Wood site (USA) Mummichog (Fundulus heteroclitus) Liver, anterior kidney,
spleen, blood
Creosote-contaminated site [142]
Fraser River (Canada) Chinook salmon (Oncorhynchus tshawytscha) Liver PCDD, PCDF, PCB [143]
AC: aromatic compound; PAC: polycyclic aromatic compound; PAH: polycyclic aromatic hydrocarbon; PCB: polychlorinated biphenyl; DDT: 4,40-dichlorobiphenyltrichloroethane;
TBT: tributyltin; AH: aromatic hydrocarbon; PCDD: polychlorinated dibenzo-p-dioxin; and PCDF: polychlorinated dibenjofuran.
T. Ohe et al. / Mutation Research 567 (2004) 109–149 131
Wilson et al. [143] measured biological responses
in the liver of juvenile Chinook salmon (Oncor-
hynchus trhawytscha) caught at sites on the upper
Fraser River in British Columbia, Canada to assess the
effects of contaminants on the fish. Juvenile Chinook
salmon on the upper Fraser River had significant
increases in ethoxyresorufin-O-deethylase (EROD)
activity, CYP 1A density and DNA adduct frequency
in comparison to fish from the reference site. There
were strong correlations between EROD activity,
CYP 1A density and DNA adduct concentrations but
no clear correlation between these responses and
polychlorinated dibenzo-p-dioxin, polychlorinated
dibenzofuran or polychlorinated biphenyl (PCB)
concentrations in the fish.
Ericson et al. [132] collected indigenous mussels
(Mytilus edulis) at four sites including Reykjavik
harbor, an area that receives intense traffic from a
variety of small and large vessels, and a reference site
along the south-western coast of Iceland. Additionally,
they transplanted mussels, which were collected at a
reference site, in nylon mesh bags at a depth of 2–6 m
at Reykjavik harbor for 6 weeks. DNA adducts were
subsequently analyzed in the gills and the digestive
gland of the mussels. The highest levels of DNA
adducts were detected in the gills of native mussels
from Reykjavik harbors and several adduct spots were
observed within a diagonal zone on the 32P
postlabelling autoradiograms. In the digestive gland
of the transplanted mussels, a slight but significant
increase in adduct levels up to the same level in the
native mussels from Reykjavik harbor was detected in
winter but not in summer. These results suggest that
the adduct levels found in gills of native mussels
represent adducts that have accumulated during a long
time period.
3.4. DNA strand breaks
DNA strand breaks are potential pre-mutagenic
lesions and are sensitive markers of genotoxic
damage. The most commonly used technique for
DNA strand break detection is the alkaline single cell
gel electrophoresis (comet) assay. This technique
permits the efficient visualization of DNA damage in
individual cells and any cells that have a nucleus can
be used. Nuclear DNA is unwound and electrophor-
esed under alkaline (>pH 13) conditions, and DNA
fragments migrate from the nucleus towards the
anode. The distance and/or amount of DNA migration
from individual nuclei indicate the extent of DNA
damage. Using this high pH level not only helps in the
detection of DNA single-strand breaks, but may also
reveal other classes of DNA damage (e.g. DNA
protein cross-linking, alkali labile sites) and incom-
plete DNA repair. Mitchelmore et al. [144] reviewed
the use of the comet assay for assessing the level of
DNA strand breakage in cells from aquatic species
treated with genotoxic chemicals under laboratory
conditions. A range of genotoxic chemicals yielded
positive effects in various cell types of both vertebrate
and invertebrate aquatic species. Table 6 summarizes
the report on DNA strand breaks in cells from aquatic
organisms treated with surface water samples in vivo
and in vitro. Several of the listed studies examined
responses to pollutants in aquatic species in the field.
Devaux et al. [149] assayed the in vivo response, i.e.
EROD induction and DNA damage, of chub (Leu-
ciscus cephalus) caught in the Rhone and the Ain
Rivers in France. EROD activities and DNA damage
were measured in the livers and the erythrocytes,
respectively. Significantly higher DNA damage,
expressed as tail moments, was found in chub from
two sites of the Rhone River located in an industrial
area. However, no correlation was observed between
EROD activity and DNA damage level.
Rajaguru et al. [133] investigated the genotoxicity
of water samples from the Noyyal River in Tamilnadu,
India, using carp (Cyprinus carpio) by the comet
assay. Immature carp were exposed to water samples
collected from the river at six different locations. DNA
damage was measured as the DNA length:width ratio
of the DNA mass. The ratio in cells from three organs,
i.e., erythrocytes, liver and kidney, of the carp were
measured after 24, 48 and 72 h exposure. Extensive
DNA damage was observed in cells from these organs
exposed to polluted water samples, and the amount of
damage increased with the duration of exposure. The
highest levels of DNA damage were obtained with
samples taken immediately downstream of urban
centers.
Fish cell lines have also been used as in vitro
tools in aquatic toxicology. Schnustein et al. [150]
examined genotoxicity of water samples from the
major German rivers using primary hepatocytes from
zebrafish (Danio rerio). Zebrafish hepatocytes were
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
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–1
49
13
2Table 6
Summary of reported studies on DNA strand breaks and alkaline labile sites in cells from aquatic organisms treated in vivo and in vitro
Sample source Organism/cell Organ/tissue Assay method In vitro/in
vivo
Suspected
mutagens/contaminants
Reference
1. Europe
Istrian coast (Croatia) Mussel (Mytilus galloprovincialis) Haemolymph Alkaline filter elution In vivo – [145]
North Sea (The Netherlands) Seastars (Asterias rubens) Digestive gland Alkaline unwinding assay In vivo PCB, PAH [146]
North Sea (The Netherlands) Dab (Limanda limanda) Liver Alkaline unwinding assay In vivo PCB, PAH [146]
North Sea (The Netherlands) Seastar (Asterias rubens) Pyloric caeca Alkaline unwinding assay In vivo PCB, PAH [147]
North Sea (The Netherlands) Dab (Limanda limanda) Liver Alkaline unwinding assay In vivo PCB, PAH [147]
La Spezia Gulf, Ligurian Sea
(Italy)
Mussel (Mytilus galloprovincialis) Gill Alkaline elution In vivo Sewage, industrial plant [148]
Rhone River (France) Chub (Leuciscus cephalus) Erythrocyte Comet assay In vivo PCB, PAH, heavy metals [149]
Elbe, Rhine Rivers (Germany) Fish cell line RTG-2 and others Comet assay, alkaline
elution, DNA unwinding,
UDS test
In vitro Fluoroquinolonic acid [28]
Rhine, Elbe Rivers (Germany) Zebrafish (Danio rerio) Primary hepatocytes,
gill cells
Comet assay In vitro – [150]
Sava River (Croatia) Mussel (Dreissena polymorpha) Haemocyte Comet assay In vivo Chemical industry, oil
refinery
[151]
Sarno River (Italy) Benthopelagic teleost
(Gambusia holbrooki)
Erythrocyte Comet assay In vivo Industrial, domestic and
agricultural origin
[152]
2. Asia
Yangzi, Hongxing Rivers, etc.
(China)
Human lymphocyte Comet assay In vitro Domestic sewage [153]
Kishon River (Israel) Fish hepatoma cell line RTH-149 Comet assay In vitro Heavy metals, organic
materials
[154]
Noyyal River (India) Carp (Cyprinus carpio) Erythrocyte Comet assay In vivo – [155]
3. North America
New Bedford harbor (USA) Mussel (Mytilus edulis) Gill Alkaline unwinding assay In vivo PCB, PAH, metal, organic
compounds
[156]
Elizabeth River (USA) Oyster (Crassostrea virginica) Gill Alkaline unwinding assay In vivo PCB, PAH, metal, organic
compounds
[156]
East Fork Poplar Creek (USA) Redbreast sunfish (Lepomis auritus) Liver Alkaline unwinding assay In vivo PCB, PAH [157]
Lake Erie, Lake Ontario,
Detroit River (Canada)
Bullhead (Ameiurus nebulosus) Erythrocyte Comet assay In vivo PCB, PAH [158]
Lake Erie (Canada) Carp (Cyprinus carpio) Erythrocyte Comet assay In vivo PCB, PAH [158]
Lake Erie (Canada) Tadpole (Rana clamitans) Erythrocyte Comet assay In vivo Pesticide [159]
Lake Erie (Canada) Tadpole (Bufo americanus) Erythrocyte Comet assay In vivo Pesticide [159]
Small bodies of waters in
Ontario (Canada)
Green frog (Rana clamitans) tadpole Erythrocyte Comet assay In vivo Agricultural activity [160]
Small bodies of waters in
Ontario (Canada)
Frog (Rana pipiens) tadpole Erythrocyte Comet assay In vivo Industrial activity [160]
T. Ohe et al. / Mutation Research 567 (2004) 109–149 133
San
Die
go
Bay
(US
A)
Mu
ssel
(Myt
ilu
sed
uli
s)H
aem
ocy
teC
om
etas
say
Inv
ivo
–[1
61
]
Mar
shC
reek
,E
cart
e
Chan
nel
,et
c.(C
anad
a)
Gre
enfr
og
(Ra
na
cla
mit
an
s)ta
dp
ole
Ery
thro
cyte
Co
met
assa
yIn
viv
o–
[16
2]
Tal
fou
rdC
reek
,T
allg
rass
Pra
irie
dit
ch,
etc.
(Can
ada)
Am
eric
anto
ad(B
ufo
am
eric
an
us)
tad
po
le
Ery
thro
cyte
Co
met
assa
yIn
viv
o–
[16
2]
PC
B:
poly
chlo
rinat
edbip
hen
yl
and
PA
H:
poly
cycl
icar
om
atic
hydro
carb
on.
isolated by a perfusion technique. The water samples
were preincubated with cytochrome P450-competent
S9 preparations from rats for 1 h. Two hundred
milliliters of the S9 mix was used for each well
containing 400 ml of five-times concentrated M199
medium and 1400 ml of the water samples. After
exposure to the water samples for 20 h, cells were
processed in the comet assay. Genotoxicity was
detected for the water samples from the Elbe, Wupper
and Neckar Rivers using the tail moment, relative
DNA contents of head and tail (%DNA) and tail length
as endpoint. However, percentage DNA and tail
moment displayed considerable variability with few
absolute data as compared to tail length. The
parameters tail moment and percentage DNA contents
have been regarded more adequate to precisely
describe a recorded DNA damage [122,127].
3.5. Micronucleus induction
The micronucleus assay is a widely used cytoge-
netic assay for the assessment of in vivo or in vitro
chromosomal damage. In general, the chromosomes
of fish and other aquatic organisms are relatively small
in size and/or high in number. Therefore, the
metaphase analysis of chromosomal aberrations using
these organisms is difficult. However, small size and
large chromosome number does not affect the
performance of the micronucleus assay, so it can be
easily applied to fish or other aquatic organisms. A
recent review by Al Sabti et al. on micronucleus
induction in fish treated with genotoxic chemicals
[163]. Table 7 summarizes the reports on micro-
nucleus induction in aquatic organisms, plants and
cultured cells treated with surface water either in vivo
and in vitro.
Peripheral erythrocytes are most commonly used in
fish micronucleus assays for assessing genotoxic
chemicals [163]. Hayashi et al. [172] examined
micronucleus frequencies in gill cells and RNA-
containing erythrocytes of the funa (Carassius sp.) and
oikawa (Zacco platypus) from the Tomio River in
Nara, Japan. The frequencies were higher and the
variances somewhat smaller in gill cells than in RNA-
containing erythrocytes. Similar results were found in
the hiiragi (Leiognathus nuchalis) and umitanago
(Ditrema temmincki), collected at Mochimune Harbor
in Shizuoka, Japan. Clastogen-treated fish showed
T.
Oh
eet
al./M
uta
tion
Resea
rch5
67
(20
04
)1
09
–1
49
13
4Table 7
Summary of reported studies on micronucleus induction in aquatic organisms, plants and cultured cells treated in vivo and in vitro
Sample source Organism/cell Organ/tissue In vitro/in vivo Suspected mutagens/contaminants Reference
1. Europe
Po River (Italy) Rainbow trout
(Oncorhychus mykiss)
Peripheral blood
erythrocyte
In vivo Urban and industrial sources [164]
Tiber River (Italy) Vicica faba Root tip In vitro – [165]
Salzach River (Austria) Primary rat hepatocyte In vitro Industrial effluent [166]
La Spezia Gulf, Ligurian Sea (Italy) Mussel (Mytilus galloprovincialis) Gill In vivo Sewage, industrial plants [148]
Tiber River (Italy) Barbel (Barbus plebejus) Erythrocyte In vivo – [167]
Astrian rivers (Spain) Brown trout (Salmo trutta) Kidney erythrocyte In vivo Waste waters from towns and villages [168]
Lake water (Italy) Tradescantia clone 4430 Clastogenicity In vitro Waste waters from towns and factories [41]
Como lake (Italy) Onion bulbs Onion root In vitro Industrial or agricultural source [43]
Raices, Ferreria Rivers (Spain) Eel (Anguilla anguilla L.) Reral erythrocyte In vivo Heavy metal [169]
Sava River (Croatia) Mussel (Dreissena polymorpha) Haemocyte In vivo Chemical industry, oil refinery [151]
Sarno River (Italy) Benthopelagic teleost
(Gambusia holbrooki)
Erythrocyte In vivo Industrial, domestic and agricultural origin [152]
2. Asia
Tamagawa River (Japan) Hela/S3 cell In vitro – [170]
Lake Taihu (China) Vicica faba Root tip In vitro Domestic sewage, chemical plants [171]
Lake Taihu (China) Human peripheral lymphocyte In vitro Domestic sewage, chemical plants [171]
Tomio River (Japan) Funa (Carassius sp.) Gill In vivo – [172]
Tomio River (Japan) Oikawa (Zacco platypus) Gill In vivo – [172]
Mochimune Harbor (Japan) Hiiragi (Leiognathus nuchalis) Gill, erythrocyte In vivo – [172]
Mochimune Harbor (Japan) Umitanago (Ditrema temmincki) Gill, erythrocyte In vivo – [172]
Lake Hongzhe (China) Tradescantia clone 03 Plant cutting In vitro – [173]
Lake Dianchi (China) Vicica faba In vitro Municipal sewage, industrial effluent,
farm runoff
[174]
Panlong River (China) Tradescantia clone 4430 Plant cutting In vitro Municipal sewage, industrial effluent [175]
Kui River (China) Vicica faba Root tip In vitro Municipal sewage, industrial effluent [176]
Antai, Baima, Jinan Rivers (China) Tradescantia paludosa clone 03 Plant cutting In vitro – [177]
Lijang River (China) Tradescantia paludosa clone 03 In vitro Industrial effluent, city sewage [178]
Xiaoqing River (China) Vicica faba Root tip In vitro Industrial waste, municipal sewage [179]
Yangzi, Hongxing Rivers, etc. (China) Vicica faba Root tip In vitro Domestic sewage [153]
3. North America
From Virginia to Nova Scotia,
Long Island Sound (USA)
Flounder (Pseudopleuronctes
americanus)
Erythrocyte In vivo Metal, PAH [180]
4. South America
Cai River (Brazil) Cultured human lymphcytes In vitro Petrochemical complex [181]
Los Padres Pond (Argentina) Pisces, Characidae
(Cherodon interuptus)
Erythrocyte In vivo Pesticides use, sewage contamination,
industrial effluents
[182]
PAH: polycyclic aromatic hydrocarbon.
T. Ohe et al. / Mutation Research 567 (2004) 109–149 135
Tab
le8
Su
mm
ary
of
rep
ort
edst
ud
ies
on
gen
oto
xic
ity
of
surf
ace
wat
erex
amin
edin
vit
rob
yst
amen
hai
rm
uta
tio
nas
say,
sist
erch
rom
atid
exch
ange
assa
yan
dch
rom
oso
me
aber
rati
on
test
Sam
ple
sou
rce
Cel
lA
ssay
met
ho
d/e
nd
po
int
Su
spec
ted
mu
tag
ens/
conta
min
ants
Ref
eren
ce
Eu
rope
Sal
zach
Riv
er(A
ust
ria)
Pri
mar
yra
th
epat
ocy
teS
iste
rch
rom
atid
exch
ang
eIn
du
stri
alef
fluen
t[1
66
]
Sal
zach
Riv
er(A
ust
ria)
Pri
mar
yra
th
epat
ocy
teas
say
Ch
rom
oso
mal
aber
rati
on
Ind
ust
rial
effl
uen
t[1
66
]
Com
oL
ake
(Ita
ly)
All
ium
root
Anap
has
eab
erra
tion
assa
yIn
dust
rial
or
agri
cult
ura
lso
urc
e[4
3]
Asi
a Kat
sura
,N
ish
itak
ase,
Kam
oR
iver
s(J
apan
)
Chin
ese
ham
ster
lung
(CH
L)
cell
Sis
ter
chro
mat
idex
chan
ge
Effl
uen
tfr
om
was
tew
ater
trea
tmen
tp
lan
t[1
83
]
Pan
lon
gR
iver
(Ch
ina)
Tra
des
canti
acl
one
44
30
Sta
men
hai
rm
uta
tio
nas
say
Mu
nic
ipal
sew
age,
ind
ust
rial
effl
uen
t[1
75
]
Lij
ang
Riv
er(C
hin
a)Tra
des
canti
acl
one
44
30
Sta
men
hai
rm
uta
tio
nas
say
Ind
ust
rial
effl
uen
t,ci
tyse
wag
e[1
78
]
So
uth
Am
eric
a
Cai
Riv
er(B
razi
l)H
um
anly
mp
ho
cyte
Sis
ter
chro
mat
idex
chan
ge
Pet
roch
emic
alco
mp
lex
[88
]
Afr
ica
Oba
Riv
er(N
iger
ia)
Onio
nbulb
Chro
moso
me
aber
rati
on
inA
lliu
mce
pa
Fae
ces,
leac
hea
tefr
om
refu
sedum
ps,
farm
run
off
[18
4]
higher frequencies of micronucleated cells in gills
than in the erythrocyte population. Sanchez-Galan et
al. [169] examined micronuclei in kidney erythrocytes
in wild brown trout (Salmo trutta) caught in the
Asturias rivers in northern Spain. Brown trout samples
from rivers with high anthropogenic influence
possessed significantly higher mean micronuclei
frequency than ones from more remote rivers.
In order to monitor the genotoxic potential of fresh
water environments, Klobucar et al. [151] transplanted
caged mussels (Dreissena polymorpha) from a
reference site (the Dara River) to four monitoring
sites of different pollution intensity in the Sava River
in northern Croatia. After a month of exposure MN
frequency values increased by more than five-fold
compared to that from the reference site. Results from
the comet assay showed concordance with the
micronucleus assay.
3.6. Other assessment methods
Table 8 summarizes reports on the sister chromatid
exchange (SCE) assay, the chromosomal aberration
test and the Tradescantia stamen hair mutation assay.
SCE induction in cultured Chinese hamster lung
(CHL) cells by blue rayon extracts from the Katsura,
Nishitakase and Kamo Rivers by Ohe et al. [183]
showed that samples collected downstream of waste-
water treatment plants induced higher SCE frequen-
cies than upstream samples, both with and without
metabolic activation. This suggested that the waste-
water effluents from the wastewater treatment plants
were the likely sources of genotoxic chemicals in the
rivers. Eckl [166] reported induction of SCE,
micronuclei and chromosomal aberration in the
primary rat hepatocytes by Salzach River water.
The direct comparison of these three parameters
showed that SCE’s are the most sensitive genotoxic
endpoint induced by water samples from the Salzach
River, followed by micronuclei and chromosomal
aberrations. Since, however, different mechanisms
underlie the formation of these parameters, different
water samples may well induce different cytogenetic
endpoints depending upon the composition of the
samples. Therefore the author concluded that it may
be necessary to determine more than one endpoint in
parallel.
T. Ohe et al. / Mutation Research 567 (2004) 109–149136
In the Tradescantia stamen-hair-mutation (Trad-
SHM) assay, the elevated pink mutation rate in the
inflorescence is an indicator of mutagenicity resulting
from exposure to mutagens in solution. Duan et al.
[153] reported genotoxicity in water from the
Panlonge River examined by two Tradescantia assays,
the Trad-SHM assay and the micronucleus (Trad-
MCN) assay. The plant cuttings bearing young
inflorescences were maintained in water samples for
12 h in the both assays. In both assays, the
genotoxicity of the water samples from the lower
regions of the river were higher than those from the
upper regions. This finding was in accordance with the
accumulation of pollutants in the river as if passes
through an industrial area and receives the discharge
from the municipal sewage of Kunming City, China.
The Trad-MCN assay seemed more sensitive than that
of the Trad-SHM assay in detecting the genotoxicity
of the Panlonge river water. The micronucleus assay
reveals clastogenicity at the chromosomal level, while
the stamen-hair-mutation assay detects gene muta-
tions. Because of the presence of numerous breakage
targets in the chromosomes, the Trad-MCN is more
sensitive than the single locus mutation of the Trad-
SHM assay.
4. Suspected or identified mutagens in surfacewaters
Numerous chemicals are released directly into
surface waters from industrial, domestic and agricul-
tural sources, or following treatment. Surface runoff
and atmospheric deposition also contribute to aquatic
pollution. These xenobiotic contaminants are gener-
ally present in complex mixtures, and many genotoxic
chemicals have been detected. Several heavy metals
including arsenic, cadmium, chromium, nickel and
lead, are known to be genotoxic in vitro [185] and in
vivo [186]. Twenty-five surface water samples
from the St. Lawrence River system in Quebec,
Canada, were analyzed for genotoxicity by the
SOS Chromotest, as well as the concentrations of
several heavy metals [83]. Genotoxic activity was
detected in 14 aqueous fractions of the surface
water without S9 mix, and in 11 with S9 mix.
Genotoxic activity was also detected in seven of the
particulate extracts with S9 mix. Genotoxic heavy
metals, i.e. arsenic, chromium and nickel, were
detected in all the samples at levels of 0.1–3.1 mg/l.
Moreover, genotoxic lead and cadmium were detected
in 18 and 3 samples, respectively. However, none of
the heavy metals was found to be a significant
predictor of surface water.
PAHs are produced by incomplete combustion of
organic matter and are ubiquitous environmental
contaminants. Nagai et al. [76] analyzed 17 PAHs,
including three amino derivatives of PAHs, in seven
river water samples in Gifu, Japan. The highest levels
of PAHs were detected in the water sample from the
Sakai River, Japan. Six PAHs, i.e. phenanthrene,
anthracene, fluoranthene, pyrene, benzo[k]fluor-
anthene and 1-aminopyrene, were detected at con-
centrations rangeing from 2 to 17 ng/l of water, and
the contribution of total PAHs to the observed river
water mutagenicity was estimated to be 0.25%. Kira et
al. [65] found that blue rayon extracts from waters of
Lake Baikal in Russia showed mutagenicity in S.
typhimurium YG1024 with S9 mix and detected
benzo[a]pyrene at a range of 0.13–0.65 ng/l. Nitroar-
enes are also produced by incomplete combustion of
organic substances and are ubiquitous environmental
pollutants found in diesel emission and airborne
particles [187]. Ohe et al. [58] detected 1-nitropyrene,
a direct-acting mutagen in bacterial assays, in water
from the Yodo River, Japan, at a level of 1 ng/l,
accounting for an estimated 1% of the total
genotoxicity activity of XAD-2 river water extracts.
Many mutagenic heterocyclic amines (HCAs)
have been isolated from cooked foods. Ohe [60]
detected 2-amino-3,8-dimethylimidazo[4,5-f]quinox-
aline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-
b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-
b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenyli-
midazo[4,5-b]pyridine (PhIP) in water from the Yodo
River, Japan. Tsukatani et al. [80] reported that the
concentrations of Trp-P-2 were higher in the extracts
at downstream sites from the sewage plant along the
Mikasa River, Kyusyu, Japan than one at its upstream
site. Kataoka et al. [45] also isolated 2-amino-3-
methylimidazo-[4,5-f]quinoline (IQ), 2-amino-9H-
pyrido[2,3-b]indole (AaC) and Trp-P-1 in water from
the Danube River in Vienna, Austria. The concentra-
tion of IQ, Trp-P-1 and AaC was estimated at 1.78,
0.14 and 0.44 ng/g blue rayon equivalent, respectively.
The total amounts of these amines accounted for 26%
T. Ohe et al. / Mutation Research 567 (2004) 109–149 137
of the mutagenicity of blue rayon extracts evaluated by
the Ames test using TA98 with metabolic activation.
Trp-P-1, Trp-P-2, PhIP and MeIQx were found in
human urine [188–190], and Trp-P-1 or Trp-P-2 was
also detected in processed municipal wastewater
[191], river waters [56,59], airborne particles and
rain water [192]. These findings suggest that nightsoil
(i.e., human feces) and sewage treatment plant
effluents are sources of these HCAs. White and
Rasmussen [4] estimated human sanitary waste,
including HCAs, may able to account for substantial
fraction (4–70%) of domestic wastewater genotoxi-
city. In order to elucidate the participation of HCAs in
the surface water genotoxicity, further quantitative
studies should be required.
It was reported that seven PBTA-type compounds,
i.e. 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-
5 - methoxyphenyl] - 5 -amino-7-bromo-4-chloro-2H-
benzotriazole (PBTA-1) [61], 2-[2-(acetylamino)-4-
[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-
amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2)
[64], 2-[2-(acetylamino)-4-[(2-hydroxyethyl)-amino]-
5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-
benzotriazole (PBTA-3) [66], 2-[2-(acetylamino)-4-
amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-
2H-benzotriazole (PBTA-4) [71], 2-[2-(acetylamino)-
4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-
amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6)
[72], 2-[2-(acetylamino)-4-(diethylamino)-5-methox-
yphenyl]-5-amino-7-bromo-4-chloro-2H-benzotria-
zole (PBTA-7) and 2-[2-(acetyl amino)-4-
(diallylamino)-5-methoxyphenyl]-5-amino-7-bromo-
4-chloro-2H-benzotriazole (PBTA-8) [74], were iden-
tified as major mutagens in blue cotton/rayon-
adsorbed substances collected at sites below textile
dyeing factories or municipal water treatment plants
treating domestic waste and effluents from textile
dyeing factories in several rivers in Japan. These
effluents samples showed strong mutagenicity in the
Ames/Salmonella assay. These PBTA-type mutagens
have 2-[2-(acetylamino)-5-methoxyphenyl]-5-amino-
7-bromo-4-chloro-2H-benzotriazole moiety in com-
mon, and show strong mutagenicity in S. typhimurium
YG1024 with S9 mix (Fig. 5). Table 9 summarizes the
amounts of PBTA-type mutagens in river water
samples and their calculated contribution to the total
measured mutagenicity of river water samples. The
highest level of PBTA-type mutagen was detected in
the Asuwa River for PBTA-6 at 468 ng/g of blue
rayon, accounting for 39% of the total mutagenicity of
the river water sample. Based on the synthesis studies,
these PBTA-type mutagens, except for PBTA-6, are
thought to be formed from the corresponding
dinitrophenylazo dyes via reduction with sodium
hydrosulfite and subsequent chlorination with sodium
hydrochlorite (Fig. 6). PBTA-6 is a hydrolyzed
product of 2-[4-[bis(2-acetoxyethyl)amino]-2-(acety-
lamino)-5-methoxyphenyl]-5-amino -7- bromo - 4-
chloro-2H-benzotriazole (PBTA-5) under alkaline
conditions. PBTA-5 was synthesized from the
dinitrophenylazo dye, i.e. 2-[(2-bromo-4,6-dinitro-
phenyl)azo]-5-[bis(2-acetoxyethyl)-amino]-4-meth-
oxyacetanilide (Color Index name Disperse blue
79:1), which is a common dinitrophenylazo dye used
in textile-dyeing factories, by reduction and chlorina-
tion like the other PBTA-type compounds. Indeed,
Morisawa et al. [105] detected four PBTA-type
mutagens in the effluent from a municipal wastewater
treatment plant, where large amounts of wastewater
from textile dyeing factory are treated.
Takamura-Enya et al. [77] identified 4-amino-3,30-dichloro-5,40-dinitrobiphenyl (Fig. 5) as a major
mutagen in the Waka River, Wakayama, Japan. This
chemical accounted for about 50% of the total
mutagenicity of the concentrate from the river water
without S9 mix. Moreover, this polychlorinated
biphenyl derivative activates the human aryl hydro-
carbon receptor-mediated transcription in a lacZ
reporter gene assay with an efficiency almost the
same as that of b-naphthoflavone, a well-known
synthetic aryl hydrocarbon receptor agonist. This
contaminant was assumed to be formed unintention-
ally via postemission modification of drainage water
containing parent chemicals, such as 3,30-dichloro-
benzidine or 3,30-dichloro-4,40-dinitrobiphenyl, which
are known to be raw materials in the manufacture of
polymers and dye intermediates in chemical plants.
There are many reports on the contamination of
river waters with pesticides [54,55,127,193,194], and
some of the pesiticides, such as chlomethoxyfen,
simazine, simetryn and methylparation, are genotoxic
[195–198]. Rehana et al. [54,55] analyzed eight
organochlorine pesticides, i.e. aldrin, a-BHC, DDD,
DDT, dieldrin, endosulfan, endrin and lindane, and
three organophosphorus pesticides, i.e. dimethoate,
2,4-D and methylparathion, in n-hexane extract of
T. Ohe et al. / Mutation Research 567 (2004) 109–149138
Fig. 5. Chemical structures of (A) PBTA-type mutagens and (B) 4-amino-3,30-dichloro-5,40-dinitrobiphenyl, and their mutagenicity in
Salmonella typhimurium YG1024.
water samples collected from the Ganga River, India.
They suggested a significant role of those pesticides in
the mutagenicity of the river water. To better
understand the contamination of the Meuse River in
The Netherlands, Schilderman et al. [129] investigated
water samples and crayfish (Orconectus limosus)
collected at four different locations (Table 5). In the
crayfish, levels of aromatic DNA adducts, heavy metal
residues (Cd, Pd, Cu and Zn), polychlorinated
biphenyl and organochlorine pesticides (hexachlor-
obenzene, dichloro-diphenyl-trichloroethane (DDT),
dichloro-diphenyl-dichloroethylene (DDE)) were
determined in hepatopancreatic tissues. Water
samples were analyzed for polycyclic aromatic
hydrocarbons, heavy metals, and organochlorine
compounds. The concentration of PAHs in water
samples was below the detection limit. The highest
amounts of PCBs, DDT, DDE and Cu were found in
the hepatopancreatic tissues of crayfish captured at the
most downstream site. DNA adduct levels, which may
serve as a dosimeter exposure to DNA damaging
agents such as PAHs and PCBs, were also significantly
higher in hepatopancreatic tissue from the crayfish
from the most downstream site.
As described above, many mutagenic chemicals
have been detected in surface waters. However, the
reports which provide resolute evidences that the
detected chemicals were major mutagens in the
surface waters are quite limited. Bioassay directed
fractionation, in which sensitive biological assays are
combined with various separation methods, is the
promising procedure to elucidate chemicals account
for a substantial portion of the surface water
genotoxicity.
5. Summary
5.1. Mutagenic/genotoxic bioassay data on
surface waters
Thousands of synthetic chemical compounds are
currently registered for use in industry, commerce,
agriculture and the home, and thousands of tonnes of
T. Ohe et al. / Mutation Research 567 (2004) 109–149 139
Table 9
Amounts of PBTA-type mutagens and the ratio of their contribution to the mutagenicity of river water samples
Compound Sampling site Amount (ng/g of blue
rayon or blue cotton)
Contribution ratio (%) Reference
PBTA-1 Nishitakase River 47 21 [61]
Uji River ND NA [105]a
PBTA-2 Nishitakase River 44 17 [64]
Uji River 2–39 0.3–28 [105]a
PBTA-3 Nishitakase River 22 NA [66]
Katsura River 35 NA [66]
Uji River 12–76 6–51 [105]a
Nikko River 140 NA [66]
Mawatari River ND–33 0–17 [75]
Asuwa River ND–59 0–21 [66,75]
Kitsune River ND–27 0–9 [75]
PBTA-4 Nishitakase River 32 NA [71]
Uji River 0.7–63 1–43 [105]a
Uji River 33 NA [71]
Nikko River 21 NA [71]
Mawatari River 0.6–3 0.5–2 [75]
Asuwa River ND–6 0–9 [75]
Kitsune River ND–15 0–7 [75]
PBTA-6 Nishitakase River 21 3 [72]
Katsura River 3 0.6 [72]
Uji River 0.5–45 0.2–14 [105]a
Uji River 0.5–134 13 [74]
Mawatari River 1–122 0.3–17 [75]
Asuwa River ND–468 0–39 [74,75]
Kitsune River ND–32 0–3 [75]
Tobei River 80 2 [72]
PBTA-7 Katsura River 4–51 6–7 [74]
Uji River 8–55 6–16 [74]
Mawatari River 3 0.5 [74]
Asuwa River 4 1 [74]
Kitsune River 55–101 9–16 [74]
PBTA-8 Katsura River 0.2–15 0.6–4 [74]
Uji River 2–31 7 [74]
Asuwa River 1 0.6 [74]
Kitsune River 20–49 7–15 [74]
ND: not detectable, NA: not available.a Data on effluents from a sewage treatment plant.
these are produced annually in the world. Portions of
these chemicals are released either deliberately or
unintentionally into the atmosphere, land, rivers, lakes
and seas, and numerous xenobiotics are ultimately
found in the surface waters and sediments. It has
been estimated that there are approximately 80,000
chemicals in commerce, and the proportion of
mutagens among chemicals in commerce was
approximately 20% [199]. Carcinogens are also
released into the environment [7] and ultimately
migrate into surface waters and accumulate in
sediments. Xenobiotics dissolved or suspended in
water or sediments enter through the gills, the skin, or
the gastrointestinal tract in fish or epidermal cells or
root hairs in plants inhabiting chemically polluted
aquatic environments. Pollack et al. [200] indicated
that environmentally persistent chemicals pose not
only an ecological threat but a health hazard inducing
T. Ohe et al. / Mutation Research 567 (2004) 109–149140
Fig. 6. Chemical synthesis of PBTA-type mutagens from azo dyes.
cancer in humans. Accordingly, the determination of
the potency and quantification of mutagens/carcino-
gens in surface waters is one of the important issues
from the standpoint of genetic hazard to humans and
aquatic ecological significance. However, each che-
mical is usually present at low levels that are very
difficult to determine in surface waters. Accordingly, a
variety of bioassays sensitive to genotoxicants have
been used as an integral tool in the evaluation of the
risk of surface waters as complex mixtures and are
available to aid in the identification of chemicals that
pose a genetic hazard to human health and aquatic
organisms [201–205].
Since the 1980s, more restrict water quality
regulations have been promulgated, and industry,
government and others have spent billions of dollars
to manage the release of toxic substances into the
environment in many countries throughout the
world. Summary data on the Salmonella/mutageni-
city assay obtained in this review showed that
surface waters around the world are heavily
contaminated with mutagenic/genotoxic compounds
originating from either partially treated or untreated
discharges from chemical industries, petrochemical
industries, oil refineries, oil spills, rolling steel mills,
untreated domestic sludges and pesticides runoff.
The predominance of direct and S9-activated frame-
shift-type mutagens rather than direct and S9-
activated base-substitution-type mutagens in surface
waters in the world is also found. These data
demonstrate that mutagenic/genotoxic compounds
are still released into surface waters under current
waste disposal practices through human activities,
including improperly controlled hazardous waste
disposal.
To determine the contamination levels of surface
waters with genotoxic chemicals, numerous methods
have been used, such as chemical analysis, bacterial in
vitro test, and in vivo/in situ genotoxicity tests using
aquatic organisms. Chemical analysis is the most
direct method to prove the existence of specified
substances in surface waters. However, chemical
analysis can evaluate neither the adverse effects of
chemicals nor possible additive, synergistic or
antagonistic events. The use of bioassays, i.e.
biological responses in organisms, has been suggested
as a complement to chemical analysis. Literature
analysis demonstrates that the Salmonella/mutageni-
city assay has been used more often than any other test
system to evaluate the mutagenicity/genotoxicity of
surface waters. Above all, highly sensitive Salmonella
strains having elevated levels of nitroreductase and/or
O-acetyltransferase activity have been applied to
detect the existence of trace levels of aromatic nitro-
type mutagens and/or aromatic amino-type mutagens
in surface waters. Potent mutagenic activity was
observed in many rivers using the combination of O-
acetyltransferase-overexpressing strain as a sensitive
bioassay and blue cotton/rayon as an effective
adsorbent.
Other tests that have been used to assess surface
waters for mutagenic potential include the Micro-
nucleus assay, 32P-postlabelling, the comet assay and
alkaline unwinding assay, using gills or erythrocytes
T. Ohe et al. / Mutation Research 567 (2004) 109–149 141
in aquatic organisms or indigenous plants, and
the SOS Chromotest/umu-test. In ecotoxicological
studies, it is essential to assess the toxic response of
indigenous fauna as indicator species or sentinels of
environmental contamination. The analysis of DNA
modification in aquatic organisms serves as a
promising method to monitor the contamination of
aquatic environments with genotoxic chemicals
because aquatic organisms activate xenobiotics and
respond to genotoxic chemicals at low concentrations.
Many reports documented that the detection of DNA
adducts, DNA strand breaks and micronucleus
induction by 32P-postlabeling assay, the comet assay
and the micronucleus test, respectively, are suitable for
this purpose. These bioassay techniques are highly
sensitive and can measure the cumulative genetic
toxicity caused by all the genotoxic pollutants to
which organisms are exposed in the aquatic environ-
ment. However, field studies examining indigenous
aquatic organisms can be hampered by the mobility of
the sentinel organisms or by the absence of suitable
indigenous animals. Transplantation of aquatic organ-
isms for monitoring exposures to a polluted water
body, e.g. using an in situ cage study, can preclude
these problems and also presents some advantages. By
using transplantation studies, interindividual varia-
bility can be reduced because aquatic organisms with
the same life history and a common genetic back-
ground can be used at similar developmental stages.
Moreover, the data obtained after transplantation
could more specifically reflect the geographical and
temporal conditions of exposure because the site and
length of exposure can be precisely controlled.
However transplantation studies have only recently
been utilized, and additional studies are needed to
establish this methodology.
5.2. Suspected or identified mutagens/genotoxins in
surface waters
Many mutagenic/genotoxic chemicals, such as
heavy metals, PAHs, PCBs, pesticides, were detected
in surface waters. However, most of these chemicals
were not correlated with the observed mutagenicity/
genotoxicity of the surface waters and accounted for
quite limited mutagenic/genotoxic activities.
Depledge also described that only limited evidence
is available to suggest that chemical genotoxins act as
causative agents of the genotoxic disease syndrome
such as neoplasia in marine inverbrates [202]. Several
mutagenic/genotoxic chemicals, such as novel PBTA-
type mutagens and a PCB derivative, were identified
as major mutagens in river waters by the combination
of O-acetyltransferase-overexpressing strain as a
sensitive bioassay and blue cotton/rayon as an
effective adsorbent. Nukaya et al. [61] used a large
volume of blue cotton (27 kg) to collect mutagens
dissolved in trace amounts in river water, leading to
the discovery of two novel potent S9-activated
aromatic amine mutagens at sites below the sewage
plants on the Nishitakase River, one of the tributaries
of the Yodo River system, Japan. Since the structure of
these two mutagens was determined to be a 2-
phenylbenzotriazole (PBTA) compound, they were
named PBTA-1 and PBTA-2. Since one gram blue
rayon hung in the river is capable of collecting about
20-l equivalent of river water [24,105], it was
estimated that the volume of river water equivalent
to 27 kg of blue cotton is equal to 540 cubic meters.
Seven PBTA-type mutagens have by now been
identified from the blue cotton adsorbate samples
collected at sites downstream from sewage plants in
geographically different areas in Japan
[61,64,66,71,74,75]. According to the same method,
a new direct-acting 4-amino-3,30-dichloro-5,40-dini-
trobiphenyl was identified at the site below chemical
plants treating polymers and dye intermediate [77].
These reports demonstrates that the combination of
blue cotton/blue rayon as an effective adsorbent and a
new Salmonella tester strain possessing elevated O-
acetyltransferase levels as a sensitive bioassay led to
the identification of novel mutagens in aqueous
environments.
The efforts on the identification of putative
mutagens in surface waters by bioassay-directed
chemical analysis should be further extended for
better understanding of the risk of adverse effect for
humans and indigenous biota.
6. Conclusion
Mutagenicity/genotoxicity test of complex mix-
tures such as surface waters using variety of bioassays
demonstrates that these environmental mixtures
contain many unidentified and unregulated toxicants
T. Ohe et al. / Mutation Research 567 (2004) 109–149142
which may have carcinogenicity and a risk of
unknown magnitude. It can be concluded that the
analysis of surface waters proved to be an essential
stage of the study to identify areas potentially
contaminated by genotoxic compounds from the
different sources. In literatures analyzed, some rivers
in Europe, Asia and South America show extreme
potency (more than 5000 revertants per liter) towards
Salmonella strains TA98 and TA100 and they are
contaminated with potent direct and S9-activated
frameshift-type and base substitution-type mutagens,
although their major mutagens/genotoxins have not
been clarified. These contamination sources are
supposed to be either partially treated or untreated
discharges from chemical industries, petrochemical
industries, oil refineries, oil spills, rolling steel mills,
untreated domestic sludges and pesticides runoff. In
the future, surface waters in the world will continue to
receive large quantities of discharges including a
variety of undesirable and accidental toxic com-
pounds, owing to further economic development and
technical advancement, and infinite exploitation of
new chemicals.
Thus, appropriate evaluation methods by bioassay
are needed how to effectively assess the relative risks
to humans and the environment from surface waters
and how to effectively manage the risk. Especially, the
response of Salmonella strains that are sensitive to
different chemical classes can help in the identifica-
tion of the classes of genotoxicants present in surface
waters. In order to efficiently assess the presence of
mutagens in the water, in addition to the conventional
chemical analysis, genotoxicity assays should be
included as additional parameters in water quality
monitoring programs. This is because according to
this review they proved to be sensitive and reliable
tools in the detection of mutagenic activity in aquatic
environment.
Although attempts to identify the chemicals
responsible for the mutagenicity/genotoxicity of
surface waters have been reported, newly identified
mutagens are only limited; they are heterocyclic
aromatic amines (HCAs) derived from human feces,
2-phenylbenzotriazole-type (PBTA) compounds
derived from textile dying factories and 4-amino-
3,30-dichloro-5,40-dinitrobiphenyl derived from che-
mical plants treating polymers and dye intermediates.
These reports suggest that any unknown putative
mutagens/genotoxins formed unintentionally through
industrial process will be identified in the future. The
effort on chemical identification of unknown muta-
gens accounting for a substantial portion of the surface
water mutagenicity/genotoxicity using bioassay-
directed chemical analysis should be further expended
for better understanding of the post-emission beha-
vior, the mechanisms of DNA damage and the risk of
adverse effect of the identified toxicants in humans
and indigenous biota. Hopefully, international colla-
borative monitoring studies in this area of science
should be expected for preservation of the aquatic
environment around the world.
Acknowledgements
The authors gratefully acknowledge Paul A. White
of Health Canada, Ottawa for inviting this review and
Virgina Houk of US EPA in Research Triangle Park,
NC for her revision of the English.
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