Mutagens in Surface Waters: a Review

Mutation Research 567 (2004) 109–149 www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres 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, Japan bDepartment of Public Health, Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan cCancer 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

Abstract

A review of the literature on the mutagenicity/genotoxicity of surface waters is presented in this article. Subheadings of this article include a description of sample concentration methods, mutagenic/genotoxic bioassay data, and suspected or identified mutagens in surface waters published in the literature since 1990. Much of the published surface water mutagenicity/ genotoxicity studies employed the Salmonella/mutagenicity test with strains TA98 and/or TA100 with and/or without metabolic activation. Among all data analyzed, the percentage of positive samples toward TA98 was approximately 15%, both in the absence and the presence of S9 mix. Those positive toward TA100 were 7%, both with and without S9 mix. The percentage classified as highly mutagenic (2500–5000 revertants per liter) or extremely mutagenic (more than 5000 revertants per liter) was approximately 3–5% both towards TA98 and TA100, regardless of the absence or the presence of S9 mix. This analysis demonstrates that some rivers in the world, especially in Europe, Asia and South America, are contaminated with potent direct- acting and indirect-acting frameshift-type and base substitution-type mutagens. These rivers are reported to be contaminated by either partially treated or untreated discharges from chemical industries, petrochemical industries, oil refineries, oil spills, rolling steel mills, untreated domestic sludges and pesticides runoff. Aquatic organisms such as teleosts and bivalves have also been used as sentinels to monitor contamination of surface water with genotoxic chemicals. DNA modifications were analyzed for this purpose. Many studies indicate that the 32P-postlabeling assay, the single cell gel electrophoresis (comet) assay and the micronucleus test are sensitive enough to monitor genotoxic responses of indigenous aquatic organisms to environmental pollution. In order to efficiently assess the presence of mutagens in the water, in addition to the chemical analysis, mutagenicity/ genotoxicity assays should be included as additional parameters in water quality monitoring programs. This is because according to this review they proved to be sensitive and reliable tools in the detection of mutagenic activity in aquatic environment. Many attempts to identify the chemicals responsible for the mutagenicity/genotoxicity of surface waters have been reported. Among these reports, researchers identified heavy metals, PAHs, heterocyclic amines, pesticides and so on. By combining the blue cotton hanging method as an adsorbent and the O-acetyltransferase-overproducing strain as a sensitive strain for

* Corresponding author. Tel.: +81 75 531 7124; fax: +81 75 531 7170. E-mail address: [email protected] (T. Ohe).

1383-5742/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2004.08.003 110 T. Ohe et al. / Mutation Research 567 (2004) 109–149 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 which contain many unknown compounds, are used as a source of drinking water, as well as for agricultural, Surface waters, such as rivers, lakes and seas, recreational and religious activities around the world. receive large quantities of waste water from industrial, Consequently, water pollution can be a serious public agricultural, and domestic sources, including muni- health and aquatic ecosystem problem [1–6]. The US cipal sewage treatment plants. These surface waters, 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 activity as shown in Table 2 [8–10]. These carcinogens chemicals are released into surface waters and are categorized into two types: persistent compounds, approximately 762,000 metric tonnes of chemicals which include metals and polycyclic aromatic are emitted into the atmosphere annually by industrial compounds; and volatile compounds. Most chemicals use in the United States as shown in Table 1 [7]. This emitted into the atmosphere eventually reach the data show that large quantities of toxic materials are ground or surface waters through deposition, so these routinely released directly or indirectly (via airborne TRI results show that surface waters are readily emission) into aquatic systems after industrial usage. contaminated with a variety of known mutagenic or Table 1 also notes that more than fifty percent of genotoxic carcinogens. annual water discharges to aquatic systems come from Mutagenic/genotoxic compounds, including carci- the chemical, metal smelting and processing, and nogens, whether known or unknown, become the petroleum refining industries. Moreover, 800 metric components of complex environmental mixtures that tonnes of chemicals released into surface waters and can have adverse health effects on humans and 60,000 metric tonnes of chemicals emitted into the indigenous biota [11]. We know quite a lot about atmosphere are carcinogens ranked as 1, 2A or 2B identified contaminants, and it is relatively easy to under the IARC classification system, and most of study the sources and fate of those contaminants that them are known to have mutagenic and/or clastogenic have been identified as priorities for concern and 112 T. Ohe et al. / Mutation Research 567 (2004) 109–149 control. Post-emission fate and behavior of polycyclic of surface water and aquatic biota conducted in the late aromatic hydrocarbons (PAHs) in complex mixtures 1970s, Parry et al. [13] reported on mutagenicity including surface waters have widely investigated studies on the tissue of the mussel Mytilus edulis in the throughout the world, because PAHs are identified marine environment, and Pelon et al. [14] reported on contaminants and are relatively easy to study the the mutagenicity/genotoxicity of Mississippi River sources and fate [12]. However, few studies have water samples by the Salmonella assay developed by investigated the identification of novel putative Ames et al. [15]. Cytogenic damage in fish exposed to mutagens and the quantification of their response the industrially contaminated Rhine River were also concentrations. observed [16,17]. Since 1980, many researchers have On the other hand, the use of short-term bioassays, assessed mutagenicity/genotoxicity of surface waters which can detect a wide range of chemical substances using a variety of bioassays and analytical methods that may produce genetic damage, has permitted the from the standpoint of determining the potential quantification of mutagenic hazard without a priori contribution to the mutagenic hazards of treated information about identity or physical–chemical drinking water and potential ecological hazard. property. In studies of the mutagenicity/genotoxicity Collectively, mutagenicity evaluations of surface

Table 2 TRI water releases and air emissions of carcinogens in the United States in the year of 2001a Compound Mutagenicity/ Carcinogenicityc Total water Total air clastogenicitya,b releasesd (103 kg) emissionse (106 kg) Lead compoundsf ## 2B 164.3 569.0 Formaldehyde +++/## 2A 152.5 4,800.1 Nickel compoundsg ++/## 1 111.1 455.7 Chromium compoundsh +++/## 1 80.9 304.4 Acetaldehyde +++/## 2B 71.4 5,397.6 Arsenic compoundsi +/## 1 64 – 1,4-Dioxane +j/# 2B 36.9 – Cobalt compoundsk ++/## 2B 21.8 – N,N-Dimethylformamide ++/# 3 17 242.6 Benzene +++/## 1 9.6 2,673.8 Chloroform +/## 2B 8.6 647.6 Catechol ++/# 2B 7.8 – Polycyclic aromatic compoundsl 7.4 519.9 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 4.6 – Ethylbenzene +n 2B 4 2,969.9 Epichlorohydrin ++/## 2A 3.5 – Diaminotoluene (mixed isomers) 2Bo 2.7 – Dichloromethane ++/## 2B 2.2 9,778.4 Ethylene oxide +++/## 1 2.1 – Styrene +++/## 2B 1.4 21,077.0 Cadmium compoundsp ++/## 1 1.1 – Creosote ++ 2A 1.1 – Trichloroetylene + 2A – 3,741.9 Vinyl acetate # 2B – 1,303.9 Tetrachloroetylene 2A – 1,213.2 1,3-Butadiene ## 2A – 973.0 T. Ohe et al. / Mutation Research 567 (2004) 109–149 113

Table 2 (Continued ) Compound Mutagenicity/ Carcinogenicityc Total water Total air clastogenicitya,b releasesd (103 kg) 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 and Rasmussen [4] noted that volumetric emissions absence of priority knowledge about the identification from municipal wastewater treatment plants in large or physical/chemical properties of the putative urban centers often exceed 109 l per day. As a result, toxicants. The Salmonella mutagenicity assay in genotoxic loadings from municipal wastewater treat- particular has been widely used to detect mutagenic ment facilities are often far greater than those of activity in complex environmental mixtures such as industrial facilities, and there is a strong relationship surface waters, especially river waters. between a measure of human activity (i.e., population) In the early 1990s, Stahl [18], De Flora et al. [19] and surface water genotoxicity. The work of Houk [1] and Houk [1] reviewed the genotoxic and/or carcino- and White et al. [3–6] implicated a wide range of genic hazards of natural waters, the marine environ- industries in the release of complex mutagenic ment, and industrial wastes and effluents. Houk [1] mixtures for which the identity of the putative and Stahl [18] demonstrated that genotoxic organic mutagens is not known. On the other hand, some compounds can enter surface waters from a wide range researchers have reported that conventional waste- of industrial and municipal sources by summarizing water purification processes do not effectively remove their genotoxic data performed by short-term genetic many chemical contaminants, and treatment may bioassays on literature. They also stressed the actually increase the mutagenicity/genotoxicity of importance of bioassays to detect mutagenicity/ waste waters [2,20–23]. Other studies show a sharp genotoxicity arising from the ubiquity of genotoxic rise in the mutagenicity/genotoxicity of water samples compounds in the environment and the necessity of the collected at sites downstream from wastewater identification of the sources of contaminants. White treatment plants [24,25]. Consequently, the increasing 114 T. Ohe et al. / Mutation Research 567 (2004) 109–149 use of contaminated surface waters and an increase in from 128 publications. Published mutagenicity/geno- the magnitude of the contamination pose a serious toxicity assessments were divided into two major problem for the health and welfare of humans and categories: bacterial assays, including the Salmonella indigenous aquatic biota. Thus, appropriate bioassay mutagenicity test, and the SOS Chromotest and have been needed for evaluation of surface waters on Salmonella umu-test; and aquatic organism and plant potential hazard to human and the water environment. assays, including the micronucleus assay, 32P-post- The purpose of this review is to summarize the state labelling, the comet assay and the alkaline unwinding of the current literature on mutagenicity/genotoxicity assay. The 32P-postlabeling assay, DNA strand breaks data for surface waters and to lead the most profitable and the micronucleus test are unique in that they can directions for future research in order to control and be utilized in the laboratory setting, or they can be manage effectively our water environment. In this taken to the site for in situ monitoring using fishes or review, we will focus on a synopsis of the plants that inhabit regions contaminated by industrial mutagenicity/genotoxicity assay data in surface and municipal wastewater. Genotoxic parameters (e.g. waters in the scientific literature published since hepatic DNA adducts) are currently the most valuable 1990. Subheadings include a description of sample biomarkers for environmental risk assessment and concentration methods, mutagenic/genotoxic bioassay there are many reports on the studies linking the DNA data, and suspected or identified mutagens in surface damage to subsequent molecular, cellular and tissue- waters. In most cases, surface waters have been level alteration of aquatic organisms. In this paper, we administered in their crude extracts to these biological intended to review the studies in which bioassays with test system. Fig. 1 illustrates a breakdown of the DNA alterations, e.g. mutagenicity, DNA damaging collected surface water mutagenicity/genotoxicity activity and chromosome aberration, as their end- assays. Results from 178 published mutagenicity/ points were used to evaluate contamination of surface genotoxicity assays of surface waters were obtained water with genotoxic chemicals. The studies on the

Fig. 1. Breakdown of mutagenicity/genotoxicity assays for surface waters (n = 178). The number of assays used in 128 scientific literatures published since 1990 was summed, and the percentage of each bioassay heading 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, widely used. However, the liquid–liquid extracted including oncogene activation, link to mutagens water samples showed fewer mutagenic responses exposure in aquatic organisms are not cited. compared with XAD-concentrated ones [54,55, 77,104]. Blue cotton, developed by Hayatsu [102], a solid 2. Sample concentration of surface waters for matrix bearing covalently linked copper phthalocya- mutagenicity/genotoxicity assays nine trisulfonate can selectively adsorb polycyclic planar-type compounds with three or more fused Mutagenicity/genotoxicity data of surface waters rings. Sakamoto and Hayatsu [24] collected mutagens performed using the bacterial assays are summarized by hanging blue rayon, which contains 2–3 times more in Table 3. There are many varieties of monitoring ligands than blue cotton per unit weight, in the Katsura methods combined with mutagenicity/genotoxicity and Yodo Rivers, Japan. They demonstrated that the tests and selective extraction methodologies for blue rayon hanging method is easy to perform and is identifying the possible classes of mutagenic/geno- suitable for qualitative screening of the mutagenicity toxic organic contaminants in surface waters. A monitoring of river water. The blue rayon/cotton discussion of different extraction/concentration meth- hanging method, in which blue rayon or blue cotton as ods has been presented in detail by Houk [1] and Stahl an adsorbent is hung in the flowing water, has distinct [18]. We describe here briefly the sample concentra- advantages over the conventional method of transport- tion methods used for bacterial mutagencity/geno- ing large volumes of water to the laboratory for toxicity assays. Although mutagenic potency can be bioassay. Although this technique is semiquantitative detected in non-concentrated samples of surface and cannot provide measures of contamination per waters in many cases [30,34,36,44,46–48,83,86,88, unit volume, it is suitable for collecting large amounts 89,92–94], each contaminant is usually present at such of target substances and chemicals flowing in the river low levels that it is difficult to detect, and therefore for long periods (usually 24 h). It should be noted that some sort of extraction/concentration method is it is a 1-day time-integrated value, as distinguished required for reliable mutagencity/genotoxicity assess- from an instant spot value obtainable in the conven- ment of surface water samples. Concentration/extrac- tional XAD-resin concentration method or liquid– tion methods include liquid–liquid extraction, solid liquid extraction method [24,52]. In addition, this phase extraction and other types of column chroma- method can be easily applied to a wide range of tography, as well as the blue rayon/cotton hanging mutagenicity/genotoxicity monitoring approaches method [102]. [65,85] and can collect large quantities of unknown Adsorption on Amberlite XAD resins is the most polycyclic planar chemicals dissolved at ppt-levels commonly applied method for concentrating organic (i.e., ng/l), as can be seen in the case of PBTA-1 and substances from different kinds of surface waters. PBTA-2 [61,64]. For the quantititative determination XAD resin can generally adsorb a broad class of of mutagenicity/genotoxicity of surface waters, blue mutagenic compounds, including polycyclic aro- rayon can be packed in a glass column, and the water matic hydrocarbons, arylamines, nitro-compounds, sample is passed through in an identical manner to quinolines, anthraquinones, etc. Adsorption, fol- that carried out with the XAD resin column lowed by elution with organic solvents, is efficient at [25,93,105]. extracting all the polar and nonpolar toxic chemicals The blue-chitin column method, a short column and mutagens/genotoxins [103]. Using the XAD technique for concentrating mutagens/genotoxins, is resin column method, many positive results were also suitable for qualitative screening of river water observed when those extracts were tested in the mutagenicity resulting from polycyclics [59,76,106]. bacterial mutagenicity assays [28,29,31,33–37,40, Other solid adsorbents including Separon SE [27,38] 42,44,45,50,54,55,57–59,69,72,77,78,87,90,92,93, and Silica C18 [41,43] have also been utilized to 95,96]. extract less hydrophilic chemicals in surface waters, Liquid–liquid extraction using organic solvents and substances adsorbed on the resins were eluted with provides valuable quanititative information and is organic solvents. In all cases, organic solvent extracts 116 T. Ohe et al. / Mutation Research 567 (2004) 109–149 [26] [29] Reference [27] [28] [30] [31] [32] [35] [36] [33] [34] [37] uent, fl uents of a fl -Toluidine, nitroquin- polyethoxylated nonyl phenols and its brominated derivatives – Suspected mutagen/likely sources Discharges of chemical plant with no wastewater treatment – o oline, nitroaniline, dichlorobenzidine, several aromatic quinines – Chemical industry Municipal waste dump, untreated ef Industry, municipal waste dump, agricultural area municipal waste dump – small local industry, agricultural area Ef petrohemical plant S9, S9), S9, S9, +S9) S9) S9, +S9) S9, +S9), S9, +S9), S9), negative, S9), b S9, +S9) S9, +S9), high; S9, +S9) S9, +S9) S9, +S9), TA100 cation fi S9, +S9), dissolved: S9, +S9) Positive Alklbenzene sulfonate, Low; TA98 ( TA100 ( classi low; TA98 (+S9) Positive TA98 ( ( TA100 ( dissolved: TA98 ( TA100 ( low; TA98 (+S9), +S9), low; TA98 (+S9), negative, TA98 ( low; TA98 ( TA98 (+S9), TA100 ( TA100 ( +S9), TA100 ( /strain Mutagenic potency a assay (microtest/TA1535 Ames assay (plate)/TA98, TA100 TA98, TA100 umu pSK1002 Ames assay (pre)/TA98, TA100 Extreme; particulate: Ames assay (plate)/TA98, TA100 Moderate; TA98 (+S9), Ames assay (plate)/TA98, TA100 Low, TA98 ( Ames assay (pre)/TA98, TA100 Moderate; TA100 ( Ames assay (pre)/TA98, TA100 Positive Leaking from the liquid extraction/DCM Ames assay (plate)/TA98, TA100 Moderate; TA100 (+S9), ltration – fi Adsorbates on granular activated carbon/soxhlet extracted with DCM (non volatile fraction) Separone SE/acetone Ames assay (plate)/TA98 Moderate; TA98 ( Rotary evaporation; particulate matter/DMSO, dissolved phase/ XAD-7 resin/acetone; suspended matter XAD-2 resin/DMSO Ames assay (plate)/TA98, TA100 Low, TA98 ( XAD-4/ethanol, ethanol/CH (acidic and neutral) acidic), non-concentrated sample and acidic) Non-concentrated sample; XAD-2/acetone, DCM (acidic and neutral) XAD-2 and XAD-7/acetone Ames assay (plate)/TA98, TA100 Moderate; TA98 ( (Barcelona, Spain) (Czech Republic) Besos River (Barcelona, Spain) Rhine, River Moselle (Germany) (Venetia, Italy) Netherlands) (Slovenia) Channel Badner, Muhlbach Llobregat River Unknown rivers Llobregat River, River Main, River River Po (Italy) XAD-2/acetoneRiver and sea water Microsuspension assay/ Saale River (Germany)Rhine River (The Liquid Ljubljaica River, Sora River Schwechat River, Sora River (Slovenia) XAD-2/acetoneLjubljanica (neutral River and (Slovenia) XAD-2 resin/acetone (neutral Table 3 Mutagenicity/genotoxicity summary data of surface watersSample in source bacterial assay Preparation method Assay method 1. Europe (Austria), Wilga River (Poland) T. Ohe et al. / Mutation Research 567 (2004) 109–149 117 [40] [38] [39] [41] [45] [42] [43] [44] [46] C a Waste from community and industrial sources sewage, wastewater from chemical industries (melting, pesticide, chlorine industry), PAH, chlorinated hydrocarbons – Waste waters from many small towns and factories Industrial or agricultural pollution source Increasing chemical contamination of the natural aquifers – – S9, +S9) S9) S9), S9), TA100 S9), TA100 (+S9), S9, +S9) S9), negative; TA98 TA100 (+S9) ( ( (+S9), TA100 (+S9) moderate; TA98 ( negative; TA100 ( TA98 ( negative; TA100 ( High; YG1024 (+S9)Positive IQ, Trp-P-1, A Positive E. NM2009 E. coli , l S. WP2s BA 9 strain umu S. typhimurium -arabinose L S. typhimurium E. coli Salmonella assay/ 343/753, 343/765 -test/ SOS chromotest Microscreen phage-induction assay/ TH-008 Differential DNA repair test/ coli Ames assay (plate)/TA98, TA100 Low; TA98 ( Ames assay (plate)/TA98, TA100 Low; TA98 (+S9), resistance test)/ typhimurium umu Ames assay (pre)/TA98Ara-test ( Positive Untreated commercial Ames assay (pre)/YG1024 Positive Ames assay (plate) Extreme; TA98 (+S9), Ames assay (pre)/TA98, TA100, YG1024, YG1029 umu TA1535 pSK1002 Ames assay, assay/ typhimurium acetonitrile recommended protocol) matter/soxhlet extraction/ toluene, methanol methanol; lichrolut EN/acetonitrile XAD-2/hexane, acetone Direct dilution by medium Non-concentrated sample; XAD-resin Saale and Rhine Rivers, Teltow Canal (Germany) (Germany) Labe River (Czech Republic) Separon SE/acetone Ames assay (plate)/TA98 Moderate; TA98, Northern Italian lake (Italy) Sep-Pak Plus C18/methanol, Salzach River (Austria) XAD-2, XAD-7 (US EPA Elbe River (Germany) Suspended particulate Como Lake (Italy) XAD-2/acetone Ames assay (pre)/TA98, TA100 High; TA98 (+S9), low; Spree, Havel, Stepenitz, Como Lake (Italy)Rhine River, Elbe River Silica C18/ethyl acetate, Danube River (Austria) Blue rayon hanging method; 118 T. Ohe et al. / Mutation Research 567 (2004) 109–149 [24] [50] [51] [52] [52] [53] [54] [55] Reference [47,48] [49] uents fl uents from fl mutagen discharged from sewage plant sewage plants from sewage plants – – Organochlorinated and organophosphorus pesticides Pesticides Suspected mutagen/likely sources – and aminoarene S9, S9) S9) liquid: S9, +S9) – S9, +S9) liquid: TA98 liquid: TA98 b – – S9, +S9) S9), TA100 (+S9), cation fi S9, +S9), TA100 S9, +S9) S9, +S9), negative; PositveHigh; YG1024 (+S9), moderate; YG1024 ( PAH, ef High; YG1024 (+S9), moderate; YG1024 ( Moderate; liquid TA98 ( Low; liquid (+S9), TA100 ( Moderate; XAD: TA98 ( ( Low; liquid ( TA100 ( Positive Positive Suspected nitroarene classi Extreme; XAD: TA98 ( +S9), TA100 ( Vibrio harvey /strain Mutagenic potency assay/ a Ames assay (plate)/TA98, TA100 Moderate; TA98 (+S9)Ames assay (pre)/TA98, TA98NR, TA98/1,8-DNP6, YG1021, YG1024 Ef YG1024, YG1029 YG1024, YG1029 Ames assay (plate)/TA98, TA100, TA97a, TA102 Vibrio harvey Ames assay (pre)/TA98TA98/1,8-DNP6 High; TA98 (+S9) Unknown 4 potent Ames assay (pre)/TA98, TA100, TA97a, TA102, TA104 liquid extraction/hexane liquid – – OH (pH 2, 4, 8) 4 LH-20/CH, methanol, DCM Blue rayon hanging method Ames assay (pre)/TA98, TA100, Blue rayon hanging method Ames assay (pre)/TA98, TA100, liquid liquid extraction/hexane, chloroform Filtration method; blue rayon batch method DCM, methanol, NH ) Continued and its canal (Bangkok, Thailand) (Tokyo, Japan) Portugal); Aegean Sea (Paralia, Greece); Baltic Sea (Copenhagen, Denmark); North Sea (Oslo, Norway) Yodo River (Japan) Blue rayon hanging method Ames assay (pre)/TA98, TA98NR, Katsura River (Japan)Chao Phraya River Blue rayon adsorbate; Sephadex Sumida and Ara Rivers The Seto Inland Sea (Japan)Ganga River (India) Blue rayon hanging method Ames assay (pre)/TA1024 XAD-4, XAD-8/acetone; Ganga River (India) Moderate; YG2024 (+S9) BaP XAD-4, XAD-8/acetone; Adriatic Sea (Trieste, Italy); Yodo River (Japan)Yodo Blue River rayon (Japan) hanging XAD-2, XAD-4, XAD-8 / Sample source Preparation method Assay method Table 3 ( Atlantic Ocean (Lisbon, 2. Asia T. Ohe et al. / Mutation Research 567 (2004) 109–149 119 [65] [25] [65] [66] [67,68] [69] [70] [71] [72] [73] [65] [60] [61] [63] [64] [59] [62] [57] [58] [56] BaP – municipal wastewater Illegal deposition of chemical waste in the lake BaP MeIQx, PhIP – – Domestic sewage, agricultural and industrial wastewater – S9) S9, S9, +S9) S9, +S9) S9, +S9) Discharges from S9, +S9), PositiveModerate; TA98 ( Trp-P-2 High; TA98 ( negative; TA100 ( positive Extreme; YG1024 (+S9), moderate; YG1024 ( Positive Trp-P-1, Trp-P-2, +S9), low; TA98 ( Positive Positive 1-NP PositivePositive Trp-P-2 S. typhimurium S. typhimurium S. typhimurium S. typhimurium S. typhimurium S. typhimurium assay/ assay/ assay/ Ames assay (pre)/YG1024 Negative BaP umu NM2009, NM2000 TA100 TA98, TA100 Ara test/ BA9 Ames assay (pre)/YG1024 Positive YG1024 Ames assay (pre)/YG1024 negative umu NM2009 TA97a, YG1041, YG1042 umu NM2009 Ames assay (pre)/TA98 moderate (TA98, +S9) NM2009, NM2000 umu assay/ NM2009 liquid extraction/acetone Ames test/TA98, TA100 Moderate; TA100 ( – Blue rayon hanging in water in a beaker Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 (+S9) PBTA-5, PBTA-6 water in a beaker blue rayon hanging in water in a beaker XAD-2/diehyl ether; blue chitin column/methanol: conc. ammonia (50:1) XAD-2/diethyl ether Rivers in Japan Blue rayon hanging method Ames assay (pre)/TA100, (Okayama, Japan) Nishitakase River, Uji River (Japan) Sasagase River (Okayama, Japan) River (Japan) Yodo River (Japan)Asahi and Sasagase River Nikko River (Aichi, Japan) Blue rayon column method Blue rayon hanging method Ames assay (pre)/YG1024 Ames assay (pre)/YG1024 Positive Extreme; YG1024 (+S9) PBTA-3 PBTA-1, PBTA-2 Yodo River (Japan)Taihu Lake (China)Nikko River, Uji River (Japan)Tobei Blue River, rayon Asuwa hanging River, Blue method rayon hanging method XAD-2 resin/acetoneTaihu Lake Ames (China) assay (pre)/YG1024 Ames assay (plate)/TA98, Extreme; XAD-2 YG1024 resin/acetone (+S9) PBTA-4 Ames assay (plate)/ Lake Baikal (Russia) Blue rayon hanging in Nishitakase River (Japan)Taihu Lake (China) Blue rayon hanging method Liquid Ames assay (pre)/YG1024 Extreme; YG1024 (+S9) PBTA-1 Nishitakase River (Japan)Kojima Lake, Asahi River, Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 (+S9) PBTA-2 Yodo River (Japan) Blue rayon hanging method NakDong River (Korea) Blue rayon hanging method Ames assay (plate)/TA98, Yodo River (Japan)Katsura River, Asahi XAD-2/diethyl ether Yodo River (Japan) Blue rayon column method umu assay/ Yodo River (Japan) Blue rayon hanging method; 120 T. Ohe et al. / Mutation Research 567 (2004) 109–149 Reference [74] [75] [76] [77] [78] [79] [81] [80] [82] [83] [84] [85] - - ’ ’ uents fl Suspected mutagen/likely sources PBTA-7, PBTA-8 PBTA-6 PAHs dinitrobiphenyl dichloro-5,4 Pesticides, domestic and industrial waste Municipal wastes and the industrial e barge tankers BaP, Trp-P-1, Trp-P-2 – demonstrated mutagens such as PAH and heavy metals – – S9) 4-Amino-3,3 S9, +S9), S9, +S9), S9) S9) S9, +S9) S9, +S9) b cation fi S9, +S9) S9, +S9) classi ( Extreme; YG1024 (+S9) PBTA-3, PBTA-4, Moderate; TA98 (+S9), negative; TA98 ( Extreme; TA98 ( high; TA100 ( Extreme; TA98 ( high; TA100 ( Positive Moderate; YG1024 ( Positive Positive Not correlated with Negative Moderate; YG1024 (+S9), low; YG1024 ( E. coli /strain Mutagenic potency a TM677) luminescense uctuation test/TA98, fl Salmonella / Salmonella typhimurium YG1029 Ames assay (pre)/TA98, YG1021, YG1024 Ames assay (plate)/TA98, TA100, TA97a, TA102 TA100, TA97a, TA102, TA104 Ames TA100, TA97a, TA102 YG1024, YG1041, YG1042 bacterium Photobacterum SOS Chromotest/ The forward mutation assay typhimurium YG1024, YG1041, ) Mutatox test/ uoride) fl liquid – lter and Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 Blue rayon hanging method Ames assay (pre)/YG1024, Blue chitin column/ methanol:conc. ammonia (50:1) Blue rayon hanging method Ames assay (pre)/YG1024 Extreme; YG1024 ( XAD-4, XAD-8/acetone; liquid extraction/hexane, chloroform XAD-8/DMSO Ames assay (plate)/TA98, Blue rayon hanging method Ames assay (plate)/TA100, Flash evaporation (10 Filtered water; particulates/DCM fi bonded-phase sorbent (CN, C18)/soxhlet-extracted/DCM, methanol Blue rayon hanging method Ames assay (plate)/TA98, TA100, ) Continued River (Japan) Kitsune River (Fukui, Japan) (Japan) Japan) India) of Agra, India) (Japan) Canada) system (Canada) Providence River, Charles River Asuwa River, Katsura Mawatari River, Asuwa River, Nagara and other rivers Waka River (Wakayama, River Yamuna (Mathura, River Yamuna (downstream Six rivers in North Kyusyu Galveston Bay (USA)Yamaska River (Quebec, Blue rayon hanging method Ames assay (pre)/TA98 Positive A collision of St. Lawrence River Aberjona River (MS, USA) Poly(vinylidene di St. Lawrence River (Canada); Table 3 ( Sample source Preparation method Assay method 3. North America T. Ohe et al. / Mutation Research 567 (2004) 109–149 121 [48] [86] [87] [88] [89] [90] [91] [92] [93] [94] uence fl nary, steel fi uence of fl – Under the in petrochemical industries petrochemical industrial comples Under the in of industrial complex – Heavily industrialized area (petrochemical, oil re rolling mill, petroleum coke plant, sulfuric acid plant) Wastewater from farming, grazing, domestic sewage, industries – Heavy metals and organic contaminants S9, +S9), S9, +S9), Affected by the S9, +S9), S9, S9, +S9) S9, +S9), S9, +S9), S9, +S9), S9, +S9) S9, +S9) S9) S9, +S9) S9, +S9) S9, +S9) S9), negative; Positive Extreme; TA98 ( TA100 ( High; TA98 (+S9), moderate; TA98 ( TA100 ( Extreme; TA98 ( TA100 ( TA100 ( TA100 (+S9) TA100 ( +S9), TA100 ( Low; TA98 ( TA100 ( Vibrio harvey E. ) assay/ l B/rWP2s( YG1042 Vibrio harvey TA100, TA102 Ames assay (plate)/TA98, TA100 Microsuspension assay/TA98, TA100, TA102 microscreen phage- induction assay/ coli Ames assay (plate)/TA98, TA100 Extreme; TA98 (+S9), Ames assay (plate)/TA98, TA100 Extreme; TA98 ( Ames assay (pre)/TA1535, TA97, TA98, TA100, TA102 liquid extraction/DMC liquid extraction/ liquid – – – ltration Filtration Ames assay (pre)/TA98, Filtration XAD-2 resin/methanol, DCM Direct assay Ames assay (plate)/TA98 Extreme; TA98 ( Non-concentrated sample; liquid extraction/DCM (acidic, basic, neutral); volatile substances extraction method XAD-2/DMSO (pH 2) Ames assay (plate)/TA98, TA100 Low; TA98 ( Liquid (acidic, neutral) Filtration; XAD-2 Ames assay (plate)/TA98, TA100 Low; TA98 ( DCM; XAD resin/DCM, methanol; fi method c Ocean (Monerey, fi East River (USA) Guaiba River (Brazil) MD, USA), Paci CA, USA) State (Brasil) Guaiba River (Rio Grande do Sul, Brazil) Guaiba River (Brazil) Argentina) Berisso (Argentina) (Buenos Aires, (Argentina) Potomac River, Hudson River, Cai River, a tributary of the Atlantic Ocean (Annapolis, Water courses of Sao Paulo Cai River, a tributary of the Cai River, a tributory of the Rio Tercero River (Cordoba, Canals between Ensenda and Matanza-Riachuelo River Surface waters in Brazil Liquid Sinos River basin (RS, Brasil) Direct concentration 4. South America 122 T. Ohe et al. / Mutation Research 567 (2004) 109–149

were concentrated and exchanged for a solvent that is , and compatible with the selected bioassay (e.g., dimethyl [93] [96] Reference [95] sulfoxide or DMSO). Grifoll et al. [30] reported that the particulate ; Microsuspension

uent matter retained in ﬁlter membrane exhibited a stronger ﬂ blue rayon equivalent

[99] mutagenic activity than the dissolved phase. This demonstrates that filtration or sterilization via filter membrane could remove some of the mutagenic . Parenthesis shows the kind of Industrial ef (textile dyeing facility) Suspected mutagen/likely sources – activity. White et al. [5] investigated the sorptive [85] properties of organic genotoxins in industrial effluents and revealed that a substantial fraction (up to 99.8%) of the emitted genotoxicity is associated with S9, +S9) S9, particulate material. Accordingly, the issue of filtra-

S9, +S9), tion and sterilization via ﬁltration membrane of b surface waters are important one since a large portion S9, +S9)

of the organic pollutants are often adsorbed to the cation ﬁ particulate material. Positive Moderate; TA98 ( +S9), low; TA100 ( classi TA100 ( ; Ames assay (pre), the preincubation method

E. 3. Review of published mutagenicity/genoxicity

5000, extreme; more than 5000 revertants per liter in TA98 and TA100 assessment data of surface waters [15,97,98] –

3.1. Salmonella/mutagenicity data

3.1.1. Mutagenic features of surface waters with ) /strain Mutagenic potency l a 2500, high; 2500 cation. Salmonella typhimurium TA98 and TA100 – ﬁ There are many assays for detecting the mutagenicty/genotoxicity of surface waters, but the utilization B/rWP2s( of bioassays with bacteria has proven to be very Microscreen phage-induction assay/ coli Ames assay (plate)/TA98, TA100 Low; TA98 ( Ames assay (plate)/TA98, TA100, YG1041, YG1042 effective for monitoring because these assays are sensitive, inexpensive, reliable, and can be performed 500, moderate; 500 – in a short period of time with relatively low cost. Among the microbial bioassays, the Salmonella 100,000, extreme; more than 100,000 revertants per g blue rayon equivalent in YG1024 – mutagenicity test has been the most widely used for detecting mutagenicity/genotoxicity in surface waters. ed as: low; nd ﬁ cation of mutagenic potency classi

ﬁ The different responses of the Salmonella strains can

. provide information on the classes of mutagens present in water samples. This test developed by [101] Ames et al. [15,97,98] is based on the detection of 10,000, high; 10,000 – XAD4 resin/methanol, MC; blue rayon column method/methanol: conc. ammonia (50:1) methanol, ethylacetate (acidic), Blue rayon hanging method histidine-independent revertants in selected Salmo-

ed as follows: Ames assay (plate), the standard plate incorporation assay nella strains after exposure to mutagens with or fi without additional activating enzymes. The dose- uctuation test ) fl response can be quantified by varying sample concentration and counting revertant colonies per cation represents the maximal mutagenic activity level expressed as revertants per liter for TA98 and/or TA100, and one expressed as revertants per g ; Ames

ﬁ plate at each concentration. Samples to be tested must Continued 1000, moderate; 1000

– be ﬁlter sterilized under normal conditions. It is also [100] Paulo (Brazil) Classi Ames assay is classi recommended as the standard method in Standard a b Surface waters in Sao Cristais River (Brasil) XAD-4/methanol, DCM (neutral)/ Table 3 ( Sample source Preparation method Assay method assay for YG1024 obtained in each reference. Classi low; nd DCM, dichloromethane and PAH: polycyclic aromatic hydrocarbon. The result of other assays is shown as positive or negative. strains with or without S9 mix in the classi 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 employed the standard plate-incorporation version Association (APHA), American Water Works Asso- of the assay using strains TA98 and/or TA100 with and ciation (AWWA) and Water Environment Federation without metabolic activation. Fig. 2 shows the ratio of (WEF) [107]. The test has now been ofﬁcially positive and negative samples with strains TA98 and included in the Sa˜n Paulo State Water Works TA100 in the absence and the presence of a metabolic Monitoring Program at sites where water is to be activation system for all observations cited in Table 3. used as a source of drinking water [96] and is the test Among all data analyzed, the percentage of positive method proposed by the U.S. Environmental Protec- samples toward TA98 was approximately 15%, both in tion Agency for Clean Water Act compliance the absence and the presence of S9 mix. Positive monitoring [108]. Much of the published surface TA100 results were 7% both with and without S9 mix. water Salmonella mutagenicity data employed sam- These observations suggest the predominance of ples concentrated by direct partitioning into organic direct and S9-activated frameshift-type mutagens solvents, or adsorption and subsequent solvent elution rather than direct and S9-activated base-substitu- to assess the mutagenic potency. Most studies tion-type mutagens in surface waters in the world. 124 T. Ohe et al. / Mutation Research 567 (2004) 109–149

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 and South America are contaminated with potent Salmonella typhimurium strains widely used in direct-acting and S9-activated frameshift-type and Salmonella mutagenicity tests for surface waters. base substitution-type mutagens. Those rivers are Based upon possible occurrence analyzed in a 20- reported to be contaminated by either partially treated year survey conducted since 1979 by the Environ- or untreated discharges from chemical industries, mental Agency of Saõ Paulo State in Brasil, petrochemical industries, oil refineries, oil spills, Umbuzeiro et al. [93] proposed boundaries of rolling steel mills, untreated domestic sludges, and mutagenic activity for natural water samples to pesticides runoff [30,43,54,78,79,86,88,89,91,93]. compare the distribution of mutagenic potencies, Since a detailed discussion of all the Salmonella based on the classification system for industrial wastes mutagenicity test results shown in Table 3 is beyond and effluents developed by Houk [1]. The boundaries the scope of this paper, the description on the are classified as follows: up to 500 revertants per following pages will be restricted to those studies equivalent liter as ‘‘low’’; from 500 to 2500 revertants that recorded mutagenicity levels that would be per equivalent liter as ‘‘moderate’’; from 2500 to 5000 classified as extreme. revertants per equivalent liter as ‘‘high’’, and more Grifoll et al. [30] performed a mutagenicity than 5000 revertants per equivalent liter as ‘‘extreme’’ assessment of the dissolved and particulate phases mutagenic activity. Fig. 3 shows the frequency of the Besos and Llobregat Rivers, which flow along distribution of mutagenic potency values for all populated and industrialized basins near Barcerona, available positive data with TA98 and TA100 in the Spain. Both rivers share domestic, industrial and absence and in the presence of S9 mix according to the agricultural uses and are recipients of a large amount aforementioned mutagenic potency classification. of untreated effluents. The results indicated that both Among all data analyzed, the percentage ranked as rivers are chronically polluted by base substitution and ‘‘high’’ or ‘‘extreme’’ was approximately 3–5% both frameshift mutagens and promutagens. Interestingly, for TA98 and TA100, irrespective of the absence or the particulate (>0.22 mm) phase exhibited a stronger presence of S9 mix. Some rivers classified as mutagenic activity than the dissolved phase and the ‘‘extreme’’ showed the maximum mutagenic potency mutagenic activity of the particulate phase was ranked of more than 10,000 revertants per liter for TA98 and/ as ‘‘extreme’’. They also demonstrated that the base or TA100 in the presence or absence of S9 mix. These substituition mutagens remain associated with the results demonstrate that some rivers in Europe, Asia dissolved phase, whereas frameshift mutagens are

T. Ohe et al. / Mutation Research 567 (2004) 109–149 125

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respons of the Fig. 126 T. Ohe et al. / Mutation Research 567 (2004) 109–149 more favorably adsorbed to the suspended particulate influence of a petrochemical industrial complex. The phase. This suggests that some frameshift mutagens potency ranking was ‘‘extreme’’ for both TA98 and might be removed by sterilization via filtration or TA100, with and without metabolic activation. The filtration of water samples. Guzzella et al. [43] highest value was more than 200,000 revertants per compared the mutagenicity of water samples from the liter for TA98 in the absence of S9 mix compared with Como Lake, Italy, and tried to find the source of 7000 revertants per liter in the presence of S9 mix (the water genotoxins. The result revealed that extreme value was calculated from the data on non-concen- mutagenic potency was found with TA98 in the trated 2-ml sample per plate). The maximum value for presence of S9 mix in one water sample collected near TA100 was more than 50,000 revertants per liter and the river mouth in Como Lake more than 5000 more than 10,000 revertants per liter in the absence of revertants per liter and concluded that river influent S9 mix and in the presence of S9 mix, respectively. It was an important source of mutagenic contamination. was also reported that these non-concentrated samples However, they reported that lake water samples lost their activity upon liquid–liquid extraction using did not show any genotoxicity both with Allium dichloromethane. In turn, they suggested that volatile root anaphase aberration assay and Allium root- compounds were responsible for the mutagenicity that micronuclei assay. were lost in the liquid–liquid extraction. Rehana et al. [54] used five different Salmonella Lemos et al. [88] also reported that water samples tester strains to compare the mutagenic activity of from the Ca´ı River were ranked as ‘‘extreme’’ for water samples from four sites of the Ganga River, TA98 both with and without S9 mix. The highest India, using the XAD-resin extraction method and the values for TA98 were more than 100,000 and 50,000 liquid–liquid extraction method. Samples always revertants per liter in the absence of S9 mix and in the showed ‘‘extreme’’ mutagenic activity for TA98 and presence of S9 mix, respectively. Collectively, these TA100, both with and without S9 mix. The maximum results suggested that volatile substances derived from activity for each strain was >10,000 revertants per petrochemical industries in the area contributed to the liter. They also found a similar pattern in the extremely potent mutagenic activity of Ca´ı River responsiveness of tester strains for a mixture of water samples. Investigations carried out by Umbu- pesticides, suggesting that the mutagenicity of water zeiro et al. [83] in a surface water quality monitoring extracts may be attributable to the pesticides used in program analyzed for 20 years in Saõ Paulo State in the upstream region. Aleem and Malik [78] and Brazil demonstrated that 14% of 1007 surface water Siddiqui and Ahmad [79] reported that XAD- samples showed positive mutagenic activity. Among concentrated water samples from the River Yamuna, the positive samples, a total of 81 samples were India, was remarkably high for TA98 (classified as analyzed using a dose-response manner. From those ‘‘extreme’’) compared to TA100 (classified as 81 samples, 9% were ranked as ‘‘extreme’’ for either ‘‘high’’), both with and without S9 mix. It was also TA98 or TA100 (5400–30,000 revertants per liter). In reported that XAD-concentrated samples elicited addition, the result showed that direct-acting muta- higher responses than liquid–liquid concentrated gens induced frameshift mutations and S9-activated samples, the water samples collected during the mutagens induced base substitution mutations. Their summer exhibited higher mutagenic activity com- possible pollution sources were petrochemical indus- pared with other seasons, and water samples also trial, oil spill and untreated domestic sludge. In a study contained oxidative (TA102) mutagens. This extreme of the Rio Tercero River (Cordova, Argentina) by mutagenic contamination of the river water is likely to Alzuet et al. [91], the presence of S9-activated be derived from a combination of domestic, municipal mutagens capable of causing base substitution and and industrial effluents noted at this sampling frameshift mutations was observed (without S9, data site. not available). Maximum activity was observed in Vargas et al. [86,89] reported that extremely potent TA98 with S9 mix (8,550,000 revertants per liter), activity was observed for TA98 without metabolic although moderate activity was found in TA100 with activation among about 100 non-concentrated samples S9 mix (1000 revertants per liter). The region is a collected from Ca´ı River, Brasil, an area under the 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 activity of the subfractions, obtained by separating rolling steel mill and a sulfuric acid plant. Previous blue cotton adsorbates collected from the Katsura studies have demonstrated the presence of polycyclic River (a tributary of the Yodo River, Japan) via aromatic hydrocarbons in airborne particulate matters, Sephadex G-25 gel chromatography, was greatly surface waters and sediments collected in the study increased by the addition of metabolic activation, area [117–119]. especially in YG1024, and these fractions showed less mutagenicty in TA98/1,8-DNP6, suggesting that S9- 3.1.2. Mutagenic features of surface waters with activated mutagenic aromatic amines were present in nitroreductase- and/or O-acetyltransferase- the Katsura River. Kusamran et al. [52] also reported overexpressing strains that samples obtained by the blue rayon hanging S. typhimurium YG1021 and YG1026, strains that method from the Chao Phraya river and connected possess high nitroreductase levels, were developed by canals in Bangkok, Thailand, had no significant introducing plasmids containing the nitroreductase mutagenic effect in either TA98 or TA100. However, gene from S. typhimurium TA1535 into TA98 and samples showed a significantly greater response in TA100, respectively. These strains have been shown to YG1024 than in strain YG1029, especially in the detect various kinds of mutagenic nitro compounds presence of metabolic activation. These results much more efficiently than TA98 and TA100 [113]. indicate that the combination of specific mutagenicity Watanabe et al. [114] successfully developed addi- tests and selective collection methologies can provide tional new tester strains S. typhimurium YG1024 and clues to the identity of organic genotoxic pollutants in YG1029, strains derived from TA98 and TA100, surface water samples. respectively, that show remarkably high sensitivity to Kataoka et al. [45] reported significantly higher both nitroarenes and aromatic amines. S. typhimurium mutagenicity for YG1024 than for TA98 in the YG1041 and YG1042 [115], derived from TA98 and presence of S9 mix in canal samples collected along TA100, respectively, have enhanced levels of both the Danube River, Austria, again suggesting the nitroreductase and O-acetyltransferase and are, con- presence of aromatic amine mutagens. The occurrence sequently, highly sensitive to nitroarenes and aromatic of base substitution-type mutagenic effects toward amines. The significantly higher mutagenicity in strain TA100 was low or undetectable. In addition, the metabolically enhanced diagnostic strains samples did not elicit a positive response in S. (e.g.,YG1021, YG1024, YG1026, YG1029, YG1041 typhimurium YG1029 with metabolic activation. and YG1042) in comparison to TA98 or TA100 Three heterocyclic amines were subsequently identi- suggests the presence of aromatic amine-type muta- fied in the blue rayon adsorbates. Kira et al. [65] gens in the presence of S9 mix and the presence of reported on a simplified handling and transportation aromatic nitro-type mutagens in the absence of S9 system for monitoring samples from remote sites. mix. They put blue rayon directly into the sample bottle for Cˇ erna´ et al. [38] and Umbuzeiro et al. [98] showed 24 h, and blue rayon adsorbed mutagens were that YG strains including YG1021, YG1024, YG1041 transported to the laboratory. Sampling was performed or YG1042 elicited higher numbers of revertants in in Lake Baikal, Russia and blue rayon adsorbates were response to effluents and river water samples later transported to Okayama, Japan for analysis. (extracted with Separon SE, XAD4, blue rayon or Although the mutagenic potency values in this study liquid–liquid method) compared with TA98 or TA100 were low (S. typhimurium YG1024 with S9 mix), they both with and without metabolic activation. These stated that the system might be useful in international indicate the likely presence of aromatic amines and collaborative studies in this area of science. nitroarenes in the samples tested. Sayato et al. [49,51] Ohe et al. [85] employed the blue rayon hanging demonstrated that highly sensitive detection of method to monitor a wide range of surface water mutagenicity in surface waters could be effectively samples flowing through large metropolitan areas in achieved by combining blue cotton/blue rayon as an North America. Mutagenicity was evaluated using TA effective adsorbent and the new Salmonella tester strains and YG strains with and without metabolic strains as a sensitive bioassay. They reported that the activation. The results demonstrated that YG1024 and 128 T. Ohe et al. / Mutation Research 567 (2004) 109–149

YG1041 were much more sensitive than TA98 with S9 per gram blue rayon equivalent) and extreme (more mix, and the authors concluded that rivers flowing than 100,000 revertants per gram blue rayon through major cities in North America contained equivalent). The percentage of extreme mutagenic frameshift-type, aromatic amine-like mutagens, activity was approximately 1% and 19% with S9 mix although the levels of mutagenic activity were ranked and without S9 mix, respectively, as shown in Fig. 4. as very low compared with data from Thailand and Most samples of those were collected from rivers Japan [52]. which received discharges from textile dyeing Endo et al. [68] collected 541 water samples from factories or sewage plants treating effluents from 130 rivers in Japan using blue rayon hanging method textile dyeing factories, and most samples were shown between 1996 and 2003 and measured their muta- to contain some PBTA-type mutagens (see Section 4). genicity by the Ames assay using S. typhimurium Collectively, these data in this section demonstrate TA100, YG1029 and YG1024 both with and without that the blue rayon hanging technique is suitable for S9 mix. The positive ratio was as follows; TA100, S9 judging the presence of mutagens and identifying mix: 10%, TA100, +S9 mix: 26%, YG1029, S9 mix: mutagens in surface waters, and that it is suitable in 29%, YG1029, +S9 mix: 54%, YG1024, S9 mix: international collaborative studies of mutagens in 68% and YG1024, +S9 mix: 87%. Strong mutagenic surface waters, since there is no need for transporting activities, i.e. more than 100,000 revertants per gram large volumes of water samples to the place where blue rayon, were detected for the samples collected analysis is performed. Moreover, the combination of from the Nishitakase, Uji and Katsura Rivers in Kyoto, the blue rayon hanging method and the Salmonella test the Asuwa, Mawatari and Kitsune Rivers in Fukui and with metabolically enhanced strains is a simple and the Nikko Rivers in Aichi. sensitive method to monitor for nitroarene compounds Fig. 4 shows frequency distribution results of the and aromatic amine mutagens in surface waters. mutagenic potencies on all data available for the combination of blue rayon hanging method as a 3.2. SOS chromotest/umu-test and other bacterial collecting method and YG1024 strain as a bioassay assay system. Mutagenic potency was classified as low (up to 1000 revertants per gram blue rayon equivalent), Although the Salmonella/microsome assay has moderate (1000–10,000 revertants per gram blue been widely employed for the detection of mutageni- rayon equivalent), high (10,000–100,000 revertants city in environmental samples, a variety of other

Fig. 4. Frequency distribution of mutagenic potency classification in bioassay data with a combination of the blue rayon hanging method and S. typhimurium YG1024 strain. Mutagenic potency values are expressed in terms of revertants per gram blue rayon equivalent (BRE). Mutagenic potency is classified as: ND, not detected, low mutagenicity; ND–1000, moderate mutagenicity; 1000–10,000, high mutagenicity; 10,000– 100,000, extreme mutagenicity; >100,000 revertants per gram blue rayon equivalent (BRE). T. Ohe et al. / Mutation Research 567 (2004) 109–149 129 assays also exist for investigating complex environ- Salmonella/microsome assays. They concluded that mental mixtures. The SOS Chromotest and umu-test the microscreen phage-induction assay was a more were developed alternatives to the Ames test by appropriate screening assay for judging the genotoxi- Quillardet et al. [120] and Oda et al. [121], city of multiple pollutants in water samples in which respectively. An Escherichia coli strain PQ37 or S. both organic compounds and heavy metals were typhimurium strain TA1535/pSK1002, containing a present. fusion gene of a b-galactosidase gene (lacZ) and an Since Salmonella survives poorly in unextracted SOS response gene, is employed in these assays. marine water samples, Czyz˙ et al. [47,48,123] con- Activation of the SOS repair system by genotoxic structed genetically modified Vibrio harveyi strains compounds is measured by photometric determination that produce significantly more neomycin-resistant of the b-galactosidase enzyme activity. The SOS mutants, and they found that the Vibrio harveyi test Chromotest and umu-test are widely used for routine may be used as an adequate assay for detecting monitoring of water samples because the results are mutagenic pollution in marine waters due to the greater available in a single day with minimal advance sensitivity of the test relative to the Ames assay. preparation. The microplate version of the SOS Chromotest/umu-test was developed as a rapid and 3.3. DNA adduct formation sensitive screening tool, for the detection of genotoxins in surface waters [28,83,122].Aumu-test using an DNA-adducts in aquatic organisms are effective O-acetyltransferase-overproducing strain has been molecular dosimeters of genotoxic contaminant applied as a sensitive bioassay to detect the presence exposure, and the 32P-postlabeling assay has been of genotoxicity from nitroarenes and aminoarenes in used to measure covalent DNA-xenobiotic adducts. In surface waters [56–58,60]. the 32P-postlabeling assay, DNA is hydrolyzed The Mutatox test, employing with a dark mutant enzymatically to 30-monophosphates and DNA strain of luminescent Photobacterium phosphoreum, adducts are enriched by the selective removal of the Microscreen phage-induction assay with E. coli normal nucleotide. The DNA adducts are then labeled strain [37], the DNA repair assay with E. coli strain with [32P] phosphate and resulting 32P-labeled DNA and the Ara-test (L-arabinose resistance mutagenesis adducts are usually separated by thin-layer chromato- test) with S. typhimurium have been used for screening graphy or high performance liquid chromatography. surface water samples for genotoxic activity and have Radioactivity of DNA adducts are detected by been promoted as candidates for a battery of screening autoradiography and liquid scintillation counting, assays [37,39,82]. Helma et al. [37] evaluated four imaging analysis or a liquid scintillation analyzer. bacterial short-term genotoxicity assays, for detecting There is a huge body literature on DNA adducts in the genotoxicity of water samples of different origins. aquatic organisms, including the review by Stei et al. They concluded based on number of positive response [124]. The 32P-postlabeling technique is the most that the differential DNA repair system was the most sensitive method for the detection of a wide range of sensitive and the Microscreen assay was the least large hydrophobic compounds bound to DNA, and can sensitive with the SOS Chromotest being equally potentially detect one DNA adduct, such as those sensitive to the Salmonella/microsome assay. Vahl et derived from polycyclic aromatic compounds (PACs), al. [39] compared two mutagenicity assays (the Ames in 109–1010 bases. Table 5 summarizes the reports on test and the Ara-test) and an SOS induction test for DNA adducts in aquatic organisms. In a review of particulate matter samples of the Elbe River. They genotoxic events in some marine fishes, Reichert et al. concluded the quantitative response was higher in the [141] documented that DNA adduct levels are a Ara-test. Samples also induced lower genotoxic significant risk factor for certain degenerative and potencies in the umu-test than in the mutagenicity preneoplastic lesions occurring early in the histogen- assays. esis of hepatic neoplasms in feral English sole Vargas et al. [86,89] evaluated the genotoxicity of (Pleuronectes vetulus) from Puget Sound in Washing- river water samples collected from the Sinos River, ton, USA, an area which is heavily contaminated with Brasil, using the microscreen phage-induction and polycyclic aromatic compounds. 130 T. Ohe et al. / Mutation Research 567 (2004) 109–149 [141] [142] [143] [140] [140] [140] [136] [137] [138] [139] [136] [136] [135] [134] [133] [128] [129] [130] [131] [132] [125] [126] [127] -dichlorobiphenyltrichloroethane; 0 c, PAH, oil spill, fi Creosote-contaminated site TBT, TPT spleen, blood Gill, liver, ovaries, testes oil spill ) Liver AH, PCB ) Livers, blood PAH P-postlabeling ) Liver PCDD, PCDF, PCB ) Liver PAH, PCB, DDT 32 ) Liver PAH ) Liver PAH, PCB, DDT, chlordane, dieldrin ) Liver PAH, PCB, DDT, chlordane, dieldrin ) Liver PAH, PCB ) Liver, anterior kidney, -dioxin; and PCDF: polychlorinated dibenjofuran. ) Liver PAC ) Liver PAH, PCB, DDT, chlordane, dieldrin Liza ramada ) Liver PAH, PCB p , ) Liver PAH, PCB ) Liver Creosote ) Hepatopancreatic tissue Heavy metal, PCB, pesticides ) Liver PAC ) Liver PAH )LiverAC ) Hepatic DNA Creosote ) Gill, digestive gland Vessel traf sp.) Livers, blood PAH sp.) Livers, blood PAH Myoxocephalus scorpius Oncorhynchus tshawytscha Oncorhynchus tshawytscha Pseudopleuronectes americanus Opasanus tau Platichthys stellatus Platichthys stellatus Genyonemus lineatus Fundulus heteroclitus Pleuronectes vetulus Pleuronectes vetulus Parophrys vetulus Mugil Mugil Oedalechilus labeo sh ( Lepidopsetta bilineata fi Platichthys flesus Limanda limanda ounder ( Orconectus limosus Mytilus edulis Lipophrys pholis ounder ( ounder ( fl Perca fluviatilis Leuciscus cephalus fl fl sh ( sh ( fi fi Starry White croaker ( English sole ( Atlantic Wood site (USA)Fraser River (Canada) Mummichog ( Chinook salmon ( Charleston Harbor (USA) English sole ( Puget Sound, San FranciscoSan Bay, Diego Bay (USA) Puget Sound, San FranciscoSan Bay, Diego Bay (USA) Puget Sound, Washington (USA)Long Island Sound, ConnecticutElizabeth (USA) River (USA)Puget Winter Sound, San Francisco ChinookSan Bay, salmon Diego ( Bay (USA) Oyster toad Puget Sound, Washington (USA)Puget Sound, Washington (USA) Rock sole ( Starry Puget Sound, Washington (USA) English sole ( Mediterranean, Black Seas (Turkey) Gray mullet ( Mediterranean Sea (Turkey) Gray mullet ( Meuse River (The Netherland)Angermanalven River (Sweden)Reykjavik Harbor, etc. (Iceland)Reykjavik Harbor, etc. (Iceland) Cry Perch ( Shorthorn sculpinMediterranean ( Sea (Turkey) Mussel ( Gray mullet ( Po River (Italy)Liverpool Bay (UK)Milfold Haven (UK)Tyne Estuary (England) Flat Chub ( Flounder Teleost, ( AC: aromatic compound; PAC: polycyclic aromatic compound; PAH: polycyclic aromatic hydrocarbon; PCB: polychlorinated biphenyl; DDT: 4,4 3. North America 2. Asia Table 5 Summary of reported studies on DNA adducts in aquatic organisms in vivo analyzed by Sample source1. Europe Organism Organ/tissue Suspected mutagens/contaminants Reference TBT: tributyltin; AH: aromatic hydrocarbon; PCDD: polychlorinated dibenzo- T. Ohe et al. / Mutation Research 567 (2004) 109–149 131

Wilson et al. [143] measured biological responses fragments migrate from the nucleus towards the in the liver of juvenile Chinook salmon (Oncor- anode. The distance and/or amount of DNA migration hynchus trhawytscha) caught at sites on the upper from individual nuclei indicate the extent of DNA Fraser River in British Columbia, Canada to assess the damage. Using this high pH level not only helps in the effects of contaminants on the fish. Juvenile Chinook detection of DNA single-strand breaks, but may also salmon on the upper Fraser River had significant reveal other classes of DNA damage (e.g. DNA increases in ethoxyresorufin-O-deethylase (EROD) protein cross-linking, alkali labile sites) and incom- activity, CYP 1A density and DNA adduct frequency plete DNA repair. Mitchelmore et al. [144] reviewed in comparison to fish from the reference site. There the use of the comet assay for assessing the level of were strong correlations between EROD activity, DNA strand breakage in cells from aquatic species CYP 1A density and DNA adduct concentrations but treated with genotoxic chemicals under laboratory no clear correlation between these responses and conditions. A range of genotoxic chemicals yielded polychlorinated dibenzo-p-dioxin, polychlorinated positive effects in various cell types of both vertebrate dibenzofuran or polychlorinated biphenyl (PCB) and invertebrate aquatic species. Table 6 summarizes concentrations in the fish. the report on DNA strand breaks in cells from aquatic Ericson et al. [132] collected indigenous mussels organisms treated with surface water samples in vivo (Mytilus edulis) at four sites including Reykjavik and in vitro. Several of the listed studies examined harbor, an area that receives intense traffic from a responses to pollutants in aquatic species in the field. variety of small and large vessels, and a reference site Devaux et al. [149] assayed the in vivo response, i.e. along the south-western coast of Iceland. Additionally, EROD induction and DNA damage, of chub (Leu- they transplanted mussels, which were collected at a ciscus cephalus) caught in the Rhone and the Ain reference site, in nylon mesh bags at a depth of 2–6m Rivers in France. EROD activities and DNA damage at Reykjavik harbor for 6 weeks. DNA adducts were were measured in the livers and the erythrocytes, subsequently analyzed in the gills and the digestive respectively. Significantly higher DNA damage, gland of the mussels. The highest levels of DNA expressed as tail moments, was found in chub from adducts were detected in the gills of native mussels two sites of the Rhone River located in an industrial from Reykjavik harbors and several adduct spots were area. However, no correlation was observed between observed within a diagonal zone on the 32P EROD activity and DNA damage level. postlabelling autoradiograms. In the digestive gland Rajaguru et al. [133] investigated the genotoxicity of the transplanted mussels, a slight but significant of water samples from the Noyyal River in Tamilnadu, increase in adduct levels up to the same level in the India, using carp (Cyprinus carpio) by the comet native mussels from Reykjavik harbor was detected in assay. Immature carp were exposed to water samples winter but not in summer. These results suggest that collected from the river at six different locations. DNA the adduct levels found in gills of native mussels damage was measured as the DNA length:width ratio represent adducts that have accumulated during a long of the DNA mass. The ratio in cells from three organs, time period. i.e., erythrocytes, liver and kidney, of the carp were measured after 24, 48 and 72 h exposure. Extensive 3.4. DNA strand breaks DNA damage was observed in cells from these organs exposed to polluted water samples, and the amount of DNA strand breaks are potential pre-mutagenic damage increased with the duration of exposure. The lesions and are sensitive markers of genotoxic highest levels of DNA damage were obtained with damage. The most commonly used technique for samples taken immediately downstream of urban DNA strand break detection is the alkaline single cell centers. gel electrophoresis (comet) assay. This technique Fish cell lines have also been used as in vitro permits the efficient visualization of DNA damage in tools in aquatic toxicology. Schnustein et al. [150] individual cells and any cells that have a nucleus can examined genotoxicity of water samples from the be used. Nuclear DNA is unwound and electrophor- major German rivers using primary hepatocytes from esed under alkaline (>pH 13) conditions, and DNA zebrafish (Danio rerio). Zebrafish hepatocytes were 132 T. Ohe et al. / Mutation Research 567 (2004) 109–149 Reference [147] [147] [152] [146] [146] [148] [149] [28] [150] [151] [145] [153] [154] [155] [156] [159] [156] [157] [159] [160] [158] [158] [160] nery fi Suspected mutagens/contaminants agricultural origin – re – materials – compounds compounds vivo In vitro Fluoroquinolonic acid lter elution In vivo fi elution, DNA unwinding, UDS test Comet assay In vitro Erythrocyte Comet assay In vivo Industrial, domestic and gill cells ) Liver Alkaline unwinding assay In vivo PCB, PAH ) Haemolymph Alkaline ) Gill Alkaline elution In vivo Sewage, industrial plant ) Haemocyte Comet assay In vivo Chemical industry, oil ) Erythrocyte Comet assay In vivo PCB, PAH ) Gill Alkaline unwinding assay In vivo PCB, PAH, metal, organic ) tadpole Erythrocyte Comet assay In vivo Agricultural activity ) Erythrocyte Comet assay In vivo Pesticide ) Erythrocyte Comet assay In vivo PCB, PAH, heavy metals ) Digestive gland Alkaline unwinding assay In vivo PCB, PAH ) Erythrocyte Comet assay In vivo Pesticide ) Pyloric caeca Alkaline unwinding assay In vivo PCB, PAH ) Liver Alkaline unwinding assay In vivo PCB, PAH ) Liver Alkaline unwinding assay In vivo PCB, PAH ) Gill Alkaline unwinding assay In vivo PCB, PAH, metal, organic ) Primary hepatocytes, ) Erythrocyte Comet assay In vivo ) Erythrocyte Comet assay In vivo PCB, PAH ) tadpole Erythrocyte Comet assay In vivo Industrial activity Lepomis auritus sh ( fi Rana clamitans Danio rerio Ameiurus nebulosus Asterias rubens Bufo americanus Rana clamitans Asterias rubens Mytilus galloprovincialis Mytilus galloprovincialis Dreissena polymorpha Mytilus edulis Crassostrea virginica sh ( Leuciscus cephalus Cyprinus carpio Cyprinus carpio Rana pipiens fi Limanda limanda Limanda limanda Mussel ( (Gambusia holbrooki) Human lymphocyte Comet assay In vitro Domestic sewage Green frog ( Bullhead ( Frog ( (Italy) (China) Ontario (Canada) Detroit River (Canada) Ontario (Canada) North Sea (The Netherlands)La Spezia Gulf, Ligurian Sea Dab ( North Sea (The Netherlands)North Sea (The Netherlands) Dab ( Seastar ( Istrian coast (Croatia) Mussel ( Rhone River (France)Elbe, Rhine Rivers (Germany) Fish cell line RTG-2 and othersRhine, Chub Elbe ( Rivers (Germany) Zebra Sava River (Croatia) Mussel ( Comet assay, alkaline Yangzi, Hongxing Rivers, etc. Sarno River (Italy) Benthopelagic teleost North Sea (The Netherlands) Seastars ( Kishon River (Israel)Noyyal River (India) Fish hepatoma cell line RTH-149 Carp ( Comet assay In vitro Heavy metals, organic New Bedford harbor (USA) Mussel ( Elizabeth River (USA) Oyster ( Small bodies of waters in East Fork Poplar CreekLake (USA) Erie, Lake Redbreast Ontario, sun Lake Erie (Canada) Tadpole ( Small bodies of waters in Lake Erie (Canada)Lake Erie (Canada) Carp ( Tadpole ( Table 6 Summary of reported studies on DNASample strand source breaks and alkaline labile sites in cells from aquatic organisms treated in vivo and in vitro Organism/cell Organ/tissue Assay method In vitro/in 1. Europe 2. Asia 3. North America T. Ohe et al. / Mutation Research 567 (2004) 109–149 133

isolated by a perfusion technique. The water samples were preincubated with cytochrome P450-competent [162] [162] [161] 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 cytogenetic 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 Erythrocyte Comet assay In vivo Erythrocyte Comet assay In vivo 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

) Haemocyte Comet assay In vivo induction in ﬁsh treated with genotoxic chemicals [163]. Table 7 summarizes the reports on micro-

Bufo nucleus induction in aquatic organisms, plants and

Rana cultured cells treated with surface water either in vivo ) ) tadpole

Mytilus edulis and in vitro. Peripheral erythrocytes are most commonly used in ﬁsh micronucleus assays for assessing genotoxic American toad ( americanus tadpole Green frog ( clamitans 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 Prairie ditch, etc. (Canada) Channel, etc. (Canada) (Ditrema temmincki), collected at Mochimune Harbor Talfourd Creek, Tallgrass Marsh Creek, Ecarte San Diego Bay (USA) Mussel (

PCB: polychlorinated biphenyl and PAH: polycyclic aromatic hydrocarbon. in Shizuoka, Japan. Clastogen-treated fish showed 134 T. Ohe et al. / Mutation Research 567 (2004) 109–149 [167] [168] [41] [43] [169] [164] [165] [166] [148] [151] [152] [170] [171] [171] [172] [172] [173] [174] [175] [176] [177] [178] [179] [153] [172] [172] [180] [181] [182] uent, uent uent fl fl fl nery fi uent uent, city sewage uents fl fl fl – – – – – – – farm runoff – – industrial ef In vivo Urban and industrial sources In vitro Municipal sewage, industrial ef Peripheral blood erythrocyte Root tip In vitro Erythrocyte In vivo Industrial, domestic and agricultural origin Root tip In vitro Domestic sewage, chemical plants Root tipRoot tipRoot tip In vitro In vitro Municipal sewage, In industrial vitro ef Industrial waste, municipal sewage Domestic sewage Erythrocyte In vivo Metal, PAH Erythrocyte In vivo Pesticides use, sewage contamination, ) Gill In vivo Sewage, industrial plants ) Gill, erythrocyte In vivo ) Haemocyte In vivo Chemical industry, oil re ) Gill, erythrocyte In vivo ) Kidney erythrocyte In vivo Waste waters from towns and villages .) Reral erythrocyte In vivo Heavy metal ) Gill In vivo ) Erythrocyte In vivo clone 03clone 03 Plant cutting In vitro In vitro Industrial ef ) ) sp.) Gill In vivo Salmo trutta clone 4430 Clastogenicity In vitro Waste waters from towns and factories clone 03clone 4430 Plant cutting Plant cutting In vitro In vitro Municipal sewage, industrial ef Ditrema temmincki ) Pseudopleuronctes Zacco platypus Dreissena polymorpha Mytilus galloprovincialis Leiognathus nuchalis Barbus plebejus Carassius Anguilla anguilla L Oncorhychus mykiss Cherodon interuptus Tradescantia ( Vicica faba (Gambusia holbrooki) Vicica faba Tradescantia Vicica faba Tradescantia Vicica faba Tradescantia paludosa Tradescantia paludosa Vicica faba Vicica faba americanus Flounder ( ( Long Island Sound (USA) Astrian rivers (Spain)Lake water (Italy) Como lake (Italy)Raices, Ferreria Rivers (Spain)Sava River (Croatia) Brown Eel trout ( ( Onion bulbs Mussel ( Onion root In vitro Industrial or agricultural source Po River (Italy)Tiber River (Italy) Salzach River (Austria) Rainbow trout Primary rat hepatocyte In vitro Industrial ef La Spezia Gulf, LigurianTiber Sea River (Italy) (Italy) Mussel ( Barbel ( Sarno River (Italy) Benthopelagic teleost Tamagawa River (Japan) Hela/S3 cell In vitro Lake Taihu (China) Lake Taihu (China)Tomio River (Japan)Mochimune Harbor (Japan)Lake Hongzhe (China) Lake Dianchi (China) Human peripheralPanlong lymphocyte Funa River ( (China) UmitanagoKui ( River (China) Antai, Baima, Jinan RiversLijang (China) River (China) Xiaoqing River (China) Yangzi, Hongxing Rivers, etc. (China) In vitro Domestic sewage, chemical plants Tomio River (Japan)Mochimune Harbor (Japan) Hiiragi ( Oikawa ( From Virginia to Nova Scotia, Los Padres Pond (Argentina) Pisces, Characidae Cai River (Brazil) Cultured human lymphcytes In vitro Petrochemical complex Table 7 Summary of reported studies on micronucleusSample induction source in aquatic organisms, plants1. and Europe cultured cells treated in vivo and in vitro Organism/cell Organ/tissue In vitro/in vivo Suspected mutagens/contaminants Reference 2. Asia 3. North America 4. South America PAH: polycyclic aromatic hydrocarbon. T. Ohe et al. / Mutation Research 567 (2004) 109–149 135 higher frequencies of micronucleated cells in gills than in the erythrocyte population. Sanchez-Galan et [184] [88] [178] [166] [43] [183] [175] al. [169] examined micronuclei in kidney erythrocytes [166] in wild brown trout (Salmo trutta) caught in the Asturias rivers in northern Spain. Brown trout samples from rivers with high anthropogenic influence omosome aberration test uent

possessed significantly higher mean micronuclei fl 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 uent, city sewage uent reference site (the Dara River) to four monitoring uent fl fl sites of different pollution intensity in the Sava River fl in northern Croatia. After a month of exposure MN uent from wastewater treatment plant

frequency values increased by more than ﬁve-fold ﬂ compared to that from the reference site. Results from Faeces, leacheate from refusefarm dumps, runoff the comet assay showed concordance with the micronucleus assay.

3.6. Other assessment methods Allium cepa

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 wastewater treatment plants induced higher SCE frequencies than upstream samples, both with and without metabolic activation. This suggested that the wastewater 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 clone 4430clone 4430 Stamen hair mutation assay Stamen hair mutation assay Municipal sewage, industrial ef Industrial ef primary rat hepatocytes by Salzach River water. The direct comparison of these three parameters Tradescantia Tradescantia showed that SCE’s are the most sensitive genotoxic Chinese hamster lung (CHL) cell Sister chromatid exchange Ef 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 Kamo Rivers (Japan) Oba River (Nigeria) Onion bulb Chromosome aberration in Cai River (Brazil) Human lymphocyte Sister chromatid exchange Petrochemical complex Katsura, Nishitakase, Como Lake (Italy) AlliumPanlong root River (China) Lijang River (China) Anaphase aberration assay Industrial or agricultural source Salzach River (Austria) Primary rat hepatocyte assay Chromosomal aberration Industrial ef Salzach River (Austria) Primary rat hepatocyte Sister chromatid exchange Industrial ef Africa South America Asia Table 8 Summary of reported studies on genotoxicity of surface water examined in vitro by stamen hair mutation assay, sister chromatid exchange assay and chr parallel. Sample sourceEurope Cell Assay method/endpoint Suspected mutagens/contaminants Reference 136 T. Ohe et al. / Mutation Research 567 (2004) 109–149

In the Tradescantia stamen-hair-mutation (Trad- metals, i.e. arsenic, chromium and nickel, were SHM) assay, the elevated pink mutation rate in the detected in all the samples at levels of 0.1–3.1 mg/l. inflorescence is an indicator of mutagenicity resulting Moreover, genotoxic lead and cadmium were detected from exposure to mutagens in solution. Duan et al. in 18 and 3 samples, respectively. However, none of [153] reported genotoxicity in water from the the heavy metals was found to be a significant Panlonge River examined by two Tradescantia assays, predictor of surface water. the Trad-SHM assay and the micronucleus (Trad- PAHs are produced by incomplete combustion of MCN) assay. The plant cuttings bearing young organic matter and are ubiquitous environmental inflorescences were maintained in water samples for contaminants. Nagai et al. [76] analyzed 17 PAHs, 12 h in the both assays. In both assays, the including three amino derivatives of PAHs, in seven genotoxicity of the water samples from the lower river water samples in Gifu, Japan. The highest levels regions of the river were higher than those from the of PAHs were detected in the water sample from the upper regions. This finding was in accordance with the Sakai River, Japan. Six PAHs, i.e. phenanthrene, accumulation of pollutants in the river as if passes anthracene, fluoranthene, pyrene, benzo[k]fluor- through an industrial area and receives the discharge anthene and 1-aminopyrene, were detected at con- from the municipal sewage of Kunming City, China. centrations rangeing from 2 to 17 ng/l of water, and The Trad-MCN assay seemed more sensitive than that the contribution of total PAHs to the observed river of the Trad-SHM assay in detecting the genotoxicity water mutagenicity was estimated to be 0.25%. Kira et of the Panlonge river water. The micronucleus assay al. [65] found that blue rayon extracts from waters of reveals clastogenicity at the chromosomal level, while Lake Baikal in Russia showed mutagenicity in S. the stamen-hair-mutation assay detects gene muta- typhimurium YG1024 with S9 mix and detected tions. Because of the presence of numerous breakage benzo[a]pyrene at a range of 0.13–0.65 ng/l. Nitroar- targets in the chromosomes, the Trad-MCN is more enes are also produced by incomplete combustion of sensitive than the single locus mutation of the Trad- organic substances and are ubiquitous environmental SHM assay. 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 4. Suspected or identified mutagens in surface from the Yodo River, Japan, at a level of 1 ng/l, waters accounting for an estimated 1% of the total genotoxicity activity of XAD-2 river water extracts. Numerous chemicals are released directly into Many mutagenic heterocyclic amines (HCAs) surface waters from industrial, domestic and agricul- have been isolated from cooked foods. Ohe [60] tural sources, or following treatment. Surface runoff detected 2-amino-3,8-dimethylimidazo[4,5-f]quinox- and atmospheric deposition also contribute to aquatic aline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3- pollution. These xenobiotic contaminants are gener- b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3- ally present in complex mixtures, and many genotoxic b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenyli- chemicals have been detected. Several heavy metals midazo[4,5-b]pyridine (PhIP) in water from the Yodo including arsenic, cadmium, chromium, nickel and River, Japan. Tsukatani et al. [80] reported that the lead, are known to be genotoxic in vitro [185] and in concentrations of Trp-P-2 were higher in the extracts vivo [186]. Twenty-five surface water samples at downstream sites from the sewage plant along the from the St. Lawrence River system in Quebec, Mikasa River, Kyusyu, Japan than one at its upstream Canada, were analyzed for genotoxicity by the site. Kataoka et al. [45] also isolated 2-amino-3- SOS Chromotest, as well as the concentrations of methylimidazo-[4,5-f]quinoline (IQ), 2-amino-9H- several heavy metals [83]. Genotoxic activity was pyrido[2,3-b]indole (AaC) and Trp-P-1 in water from detected in 14 aqueous fractions of the surface the Danube River in Vienna, Austria. The concentra- water without S9 mix, and in 11 with S9 mix. tion of IQ, Trp-P-1 and AaC was estimated at 1.78, Genotoxic activity was also detected in seven of the 0.14 and 0.44 ng/g blue rayon equivalent, respectively. particulate extracts with S9 mix. Genotoxic heavy 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 Asuwa River for PBTA-6 at 468 ng/g of blue the Ames test using TA98 with metabolic activation. rayon, accounting for 39% of the total mutagenicity of Trp-P-1, Trp-P-2, PhIP and MeIQx were found in the river water sample. Based on the synthesis studies, human urine [188–190], and Trp-P-1 or Trp-P-2 was these PBTA-type mutagens, except for PBTA-6, are also detected in processed municipal wastewater thought to be formed from the corresponding [191], river waters [56,59], airborne particles and dinitrophenylazo dyes via reduction with sodium rain water [192]. These findings suggest that nightsoil hydrosulfite and subsequent chlorination with sodium (i.e., human feces) and sewage treatment plant hydrochlorite (Fig. 6). PBTA-6 is a hydrolyzed effluents are sources of these HCAs. White and product of 2-[4-[bis(2-acetoxyethyl)amino]-2-(acety- Rasmussen [4] estimated human sanitary waste, lamino)-5-methoxyphenyl]-5-amino-7-bromo-4- including HCAs, may able to account for substantial chloro-2H-benzotriazole (PBTA-5) under alkaline fraction (4–70%) of domestic wastewater genotoxi- conditions. PBTA-5 was synthesized from the city. In order to elucidate the participation of HCAs in dinitrophenylazo dye, i.e. 2-[(2-bromo-4,6-dinitro- the surface water genotoxicity, further quantitative phenyl)azo]-5-[bis(2-acetoxyethyl)-amino]-4-meth- studies should be required. oxyacetanilide (Color Index name Disperse blue It was reported that seven PBTA-type compounds, 79:1), which is a common dinitrophenylazo dye used i.e. 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]- in textile-dyeing factories, by reduction and chlorina- 5 - methoxyphenyl] - 5 -amino-7-bromo-4-chloro-2H- tion like the other PBTA-type compounds. Indeed, benzotriazole (PBTA-1) [61], 2-[2-(acetylamino)-4- Morisawa et al. [105] detected four PBTA-type [N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- mutagens in the effluent from a municipal wastewater amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2) treatment plant, where large amounts of wastewater [64], 2-[2-(acetylamino)-4-[(2-hydroxyethyl)-amino]- from textile dyeing factory are treated. 5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H- Takamura-Enya et al. [77] identified 4-amino-3,30- benzotriazole (PBTA-3) [66], 2-[2-(acetylamino)-4- dichloro-5,40-dinitrobiphenyl (Fig. 5) as a major amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro- mutagen in the Waka River, Wakayama, Japan. This 2H-benzotriazole (PBTA-4) [71], 2-[2-(acetylamino)- chemical accounted for about 50% of the total 4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5- mutagenicity of the concentrate from the river water amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) without S9 mix. Moreover, this polychlorinated [72], 2-[2-(acetylamino)-4-(diethylamino)-5-methox- biphenyl derivative activates the human aryl hydro- yphenyl]-5-amino-7-bromo-4-chloro-2H-benzotria- carbon receptor-mediated transcription in a lacZ zole (PBTA-7) and 2-[2-(acetyl amino)-4- reporter gene assay with an efficiency almost the (diallylamino)-5-methoxyphenyl]-5-amino-7-bromo- same as that of b-naphthoflavone, a well-known 4-chloro-2H-benzotriazole (PBTA-8) [74], were iden- synthetic aryl hydrocarbon receptor agonist. This tified as major mutagens in blue cotton/rayon- contaminant was assumed to be formed unintention- adsorbed substances collected at sites below textile ally via postemission modification of drainage water dyeing factories or municipal water treatment plants containing parent chemicals, such as 3,30-dichloro- treating domestic waste and effluents from textile benzidine or 3,30-dichloro-4,40-dinitrobiphenyl, which dyeing factories in several rivers in Japan. These are known to be raw materials in the manufacture of effluents samples showed strong mutagenicity in the polymers and dye intermediates in chemical plants. Ames/Salmonella assay. These PBTA-type mutagens There are many reports on the contamination of have 2-[2-(acetylamino)-5-methoxyphenyl]-5-amino- river waters with pesticides [54,55,127,193,194], and 7-bromo-4-chloro-2H-benzotriazole moiety in com- some of the pesiticides, such as chlomethoxyfen, mon, and show strong mutagenicity in S. typhimurium simazine, simetryn and methylparation, are genotoxic YG1024 with S9 mix (Fig. 5). Table 9 summarizes the [195–198]. Rehana et al. [54,55] analyzed eight amounts of PBTA-type mutagens in river water organochlorine pesticides, i.e. aldrin, a-BHC, DDD, samples and their calculated contribution to the total DDT, dieldrin, endosulfan, endrin and lindane, and measured mutagenicity of river water samples. The three organophosphorus pesticides, i.e. dimethoate, highest level of PBTA-type mutagen was detected in 2,4-D and methylparathion, in n-hexane extract of 138 T. Ohe et al. / Mutation Research 567 (2004) 109–149

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. higher in hepatopancreatic tissue from the crayfish They suggested a significant role of those pesticides in from the most downstream site. the mutagenicity of the river water. To better As described above, many mutagenic chemicals understand the contamination of the Meuse River in have been detected in surface waters. However, the The Netherlands, Schilderman et al. [129] investigated reports which provide resolute evidences that the water samples and crayfish (Orconectus limosus) detected chemicals were major mutagens in the collected at four different locations (Table 5). In the surface waters are quite limited. Bioassay directed crayfish, levels of aromatic DNA adducts, heavy metal fractionation, in which sensitive biological assays are residues (Cd, Pd, Cu and Zn), polychlorinated combined with various separation methods, is the biphenyl and organochlorine pesticides (hexachlor- promising procedure to elucidate chemicals account obenzene, dichloro-diphenyl-trichloroethane (DDT), for a substantial portion of the surface water dichloro-diphenyl-dichloroethylene (DDE)) were genotoxicity. determined in hepatopancreatic tissues. Water samples were analyzed for polycyclic aromatic hydrocarbons, heavy metals, and organochlorine 5. Summary compounds. The concentration of PAHs in water samples was below the detection limit. The highest 5.1. Mutagenic/genotoxic bioassay data on amounts of PCBs, DDT, DDE and Cu were found in surface waters the hepatopancreatic tissues of crayfish captured at the most downstream site. DNA adduct levels, which may Thousands of synthetic chemical compounds are serve as a dosimeter exposure to DNA damaging currently registered for use in industry, commerce, agents such as PAHs and PCBs, were also significantly 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 Contribution ratio (%) Reference rayon or blue cotton) 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–60–9 [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 released into the environment [7] and ultimately these chemicals are released either deliberately or migrate into surface waters and accumulate in unintentionally into the atmosphere, land, rivers, lakes sediments. Xenobiotics dissolved or suspended in and seas, and numerous xenobiotics are ultimately water or sediments enter through the gills, the skin, or found in the surface waters and sediments. It has the gastrointestinal tract in ﬁsh or epidermal cells or been estimated that there are approximately 80,000 root hairs in plants inhabiting chemically polluted chemicals in commerce, and the proportion of aquatic environments. Pollack et al. [200] indicated mutagens among chemicals in commerce was that environmentally persistent chemicals pose not approximately 20% [199]. Carcinogens are also only an ecological threat but a health hazard inducing 140 T. Ohe et al. / Mutation Research 567 (2004) 109–149

Fig. 6. Chemical synthesis of PBTA-type mutagens from azo dyes. cancer in humans. Accordingly, the determination of waste disposal practices through human activities, the potency and quantification of mutagens/carcino- including improperly controlled hazardous waste gens in surface waters is one of the important issues disposal. from the standpoint of genetic hazard to humans and To determine the contamination levels of surface aquatic ecological significance. However, each che- waters with genotoxic chemicals, numerous methods mical is usually present at low levels that are very have been used, such as chemical analysis, bacterial in difficult to determine in surface waters. Accordingly, a vitro test, and in vivo/in situ genotoxicity tests using variety of bioassays sensitive to genotoxicants have aquatic organisms. Chemical analysis is the most been used as an integral tool in the evaluation of the direct method to prove the existence of specified risk of surface waters as complex mixtures and are substances in surface waters. However, chemical available to aid in the identification of chemicals that analysis can evaluate neither the adverse effects of pose a genetic hazard to human health and aquatic chemicals nor possible additive, synergistic or organisms [201–205]. antagonistic events. The use of bioassays, i.e. Since the 1980s, more restrict water quality biological responses in organisms, has been suggested regulations have been promulgated, and industry, as a complement to chemical analysis. Literature government and others have spent billions of dollars analysis demonstrates that the Salmonella/mutageni- to manage the release of toxic substances into the city assay has been used more often than any other test environment in many countries throughout the system to evaluate the mutagenicity/genotoxicity of world. Summary data on the Salmonella/mutageni- surface waters. Above all, highly sensitive Salmonella city assay obtained in this review showed that strains having elevated levels of nitroreductase and/or surface waters around the world are heavily O-acetyltransferase activity have been applied to contaminated with mutagenic/genotoxic compounds detect the existence of trace levels of aromatic nitro- originating from either partially treated or untreated type mutagens and/or aromatic amino-type mutagens discharges from chemical industries, petrochemical in surface waters. Potent mutagenic activity was industries, oil refineries, oil spills, rolling steel mills, observed in many rivers using the combination of O- untreated domestic sludges and pesticides runoff. acetyltransferase-overexpressing strain as a sensitive The predominance of direct and S9-activated frame- bioassay and blue cotton/rayon as an effective shift-type mutagens rather than direct and S9- adsorbent. activated base-substitution-type mutagens in surface Other tests that have been used to assess surface waters in the world is also found. These data waters for mutagenic potential include the Micro- demonstrate that mutagenic/genotoxic compounds nucleus assay, 32P-postlabelling, the comet assay and are still released into surface waters under current 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 causative agents of the genotoxic disease syndrome the SOS Chromotest/umu-test. In ecotoxicological such as neoplasia in marine inverbrates [202]. Several studies, it is essential to assess the toxic response of mutagenic/genotoxic chemicals, such as novel PBTA- indigenous fauna as indicator species or sentinels of type mutagens and a PCB derivative, were identified environmental contamination. The analysis of DNA as major mutagens in river waters by the combination modification in aquatic organisms serves as a of O-acetyltransferase-overexpressing strain as a promising method to monitor the contamination of sensitive bioassay and blue cotton/rayon as an aquatic environments with genotoxic chemicals effective adsorbent. Nukaya et al. [61] used a large because aquatic organisms activate xenobiotics and volume of blue cotton (27 kg) to collect mutagens respond to genotoxic chemicals at low concentrations. dissolved in trace amounts in river water, leading to Many reports documented that the detection of DNA the discovery of two novel potent S9-activated adducts, DNA strand breaks and micronucleus aromatic amine mutagens at sites below the sewage induction by 32P-postlabeling assay, the comet assay plants on the Nishitakase River, one of the tributaries and the micronucleus test, respectively, are suitable for of the Yodo River system, Japan. Since the structure of this purpose. These bioassay techniques are highly these two mutagens was determined to be a 2- sensitive and can measure the cumulative genetic phenylbenzotriazole (PBTA) compound, they were toxicity caused by all the genotoxic pollutants to named PBTA-1 and PBTA-2. Since one gram blue which organisms are exposed in the aquatic environ- rayon hung in the river is capable of collecting about ment. However, field studies examining indigenous 20-l equivalent of river water [24,105], it was aquatic organisms can be hampered by the mobility of estimated that the volume of river water equivalent the sentinel organisms or by the absence of suitable to 27 kg of blue cotton is equal to 540 cubic meters. indigenous animals. Transplantation of aquatic organ- Seven PBTA-type mutagens have by now been isms for monitoring exposures to a polluted water identified from the blue cotton adsorbate samples body, e.g. using an in situ cage study, can preclude collected at sites downstream from sewage plants in these problems and also presents some advantages. By geographically different areas in Japan using transplantation studies, interindividual varia- [61,64,66,71,74,75]. According to the same method, bility can be reduced because aquatic organisms with a new direct-acting 4-amino-3,30-dichloro-5,40-dini- the same life history and a common genetic back- trobiphenyl was identified at the site below chemical ground can be used at similar developmental stages. plants treating polymers and dye intermediate [77]. Moreover, the data obtained after transplantation These reports demonstrates that the combination of could more specifically reflect the geographical and blue cotton/blue rayon as an effective adsorbent and a temporal conditions of exposure because the site and new Salmonella tester strain possessing elevated O- length of exposure can be precisely controlled. acetyltransferase levels as a sensitive bioassay led to However transplantation studies have only recently the identification of novel mutagens in aqueous been utilized, and additional studies are needed to environments. establish this methodology. The efforts on the identification of putative mutagens in surface waters by bioassay-directed 5.2. Suspected or identified mutagens/genotoxins in chemical analysis should be further extended for surface waters better understanding of the risk of adverse effect for humans and indigenous biota. Many mutagenic/genotoxic chemicals, such as heavy metals, PAHs, PCBs, pesticides, were detected in surface waters. However, most of these chemicals 6. Conclusion were not correlated with the observed mutagenicity/ genotoxicity of the surface waters and accounted for Mutagenicity/genotoxicity test of complex mix- quite limited mutagenic/genotoxic activities. tures such as surface waters using variety of bioassays Depledge also described that only limited evidence demonstrates that these environmental mixtures is available to suggest that chemical genotoxins act as contain many unidentified and unregulated toxicants 142 T. Ohe et al. / Mutation Research 567 (2004) 109–149 which may have carcinogenicity and a risk of mutagens/genotoxins formed unintentionally through unknown magnitude. It can be concluded that the industrial process will be identified in the future. The analysis of surface waters proved to be an essential effort on chemical identification of unknown muta- stage of the study to identify areas potentially gens accounting for a substantial portion of the surface contaminated by genotoxic compounds from the water mutagenicity/genotoxicity using bioassay- different sources. In literatures analyzed, some rivers directed chemical analysis should be further expended in Europe, Asia and South America show extreme for better understanding of the post-emission beha- potency (more than 5000 revertants per liter) towards vior, the mechanisms of DNA damage and the risk of Salmonella strains TA98 and TA100 and they are adverse effect of the identified toxicants in humans contaminated with potent direct and S9-activated and indigenous biota. Hopefully, international colla- frameshift-type and base substitution-type mutagens, borative monitoring studies in this area of science although their major mutagens/genotoxins have not should be expected for preservation of the aquatic been clarified. 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