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: ooe@kyoto-wu.ac.jp (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

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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

09

–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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