Environ. Sci. Technol. 1996, 30, 897-907
Comparative Evaluation of Four Bacterial Assays for the Detection of Genotoxic Effects in the Dissolved Water Phases of Aqueous Matrices C H R I S T O P H H E L M A , * ,† VOLKER MERSCH-SUNDERMANN,‡ VIRGINIA S. HOUK,§ URSULA GLASBRENNER,‡ CARMEN KLEIN,‡ LU WENQUING,| FEKADU KASSIE,† ROLF SCHULTE-HERMANN,† AND SIEGFRIED KNASMU ¨ LLER† Institute for Tumor Biology-Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria, Institut for Medical Microbiology and Hygiene, Faculty of Clinical Medicine, Mannheim, University Heidelberg, P.O. Box 100023, D-68167 Mannheim, Germany, U.S. Environmental Protection Agency, Genetic Toxicology Division, Research Triangle Park, North Carolina 27711, and Department of Environmental Hygiene, Tongij Medical University Wuhan, Wuhan, People’s Republic of China
The aim of this study was to evaluate the perfomance of four bacterial short-term genotoxicity assays (Salmonella/microsome assay, SOS Chromotest, Microscreen phage-induction assay, differential DNA repair test) that are widely used and/or have a promising potential for the genotoxicity testing of water samples. Twenty-three samples of different origins (drinking and bathing water, surface water, municipal and industrial wastewater, pulp mill effluents, groundwater, and landfill leachates) were tested in these assays. In total, 20 samples were genotoxic: 13 in the Salmonella/microsome assay, 13 in the SOS Chromotest, 8 in the Microscreen phage-induction assay, and 19 in the differential DNA repair test. Although the differential DNA repair test was the most sensitive system, positive results were obtained also with some of the negative control samples, and it had the least power to detect different genotoxic potencies. The Microscreen assay was the least sensitive system due to nonlinear results and sample toxicity. The Salmonella/microsome assay and the SOS Chromotest were of equal sensitivity, but the variance of the results was higher in the Salmonella/microsome assay. As the Salmonella/microsome assay also lacks toxicity correction for routine applications and ordinarily utilizes two strains, the SOS Chromotest appears to be the most promising test system for routine screening of water samples. Based on the present experiments, the investigated water samples were
0013-936X/96/0930-0897$12.00/0
1996 American Chemical Society
ranked according to their genotoxic potency as follows: landfill leachates > effluents from pulp production > wastewater > surface water > contaminated groundwater ≈ drinking and bathing water > control samples. The rankings obtained with the individual test systems were generally in good agreement. In addition, we present data on the impact of water treatment methods (activated sludge treatment, UV disinfection) and of alternative technologies (ozone vs ClO2 pulp bleaching) on the genotoxicity of water samples.
Introduction Water is the most important medium in natural and socioeconomic systems. In urban and rural areas, 90% of the total material flows are aqueous (1, 2). As a consequence of the strongly increased anthropogenic material flows and the extensive use of synthetic chemicals, concern about the presence of carcinogenic compounds in water has been raised. In some epidemiological studies, significant correlations between the presence of xenobiotic substances including disinfection byproducts in drinking water and enhanced incidences of neoplastic diseases haves been found (3-6). The presence of genotoxic compounds in the environment can also have adverse effects on ecosystems like enhanced incidences of neoplastic diseases (e.g., in fish and shellfish) (7, 8) and reduction of reproductive success (9, 10). It is well-documented that mutations play an important role in tumor development and that most carcinogens cause DNA damage (11-13). Short-term genotoxicity tests can be used to identify possible carcinogenic hazards posed by environmental samples. For the genotoxicity testing of water samples, bacterial short-term assays, which have been developed for genotoxicity screening of chemicals, can be applied with minor modifications. In contrast to chemical analyses, it is possible to detect the combined effects of all compounds present in a sample, including unknown substances, degradation products, metabolites, and synergistic or antagonistic effects. Although more than 50 genotoxicity assays can be used for water testing (14), the question of which short-term tests are most useful for the routine evaluation of water samples has not been fully adressed. Our aim was to study the perfomance of bacterial genotoxicity assays that are suited for the routine screening of large sample numbers. We selected four bacterial assays with different genotoxic end points (Salmonella/microsome assay, SOS Chromotest, Microscreen phage-induction assay, differential DNA repair test) that are widely used and/or have a promising potential for environmental applications. These assays include the three genotoxic end points detectable in bacteria: point * Corresponding author fax: +43-1-406-07-90; e-mail address:
[email protected]. † University of Vienna. ‡ University of Heidelberg. § U.S. Environmental Protection Agency. | Tongij Medical University Wuhan.
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mutations (Salmonella/microsome assay), induction of DNA repair-specific proteins (SOS Chromotest and Microscreen phage-induction assay), and detection of repairable DNA damage (differential DNA repair test). According to our knowledge, these four assays have not been directly compared before. Testing a variety of water samples from different sources also offered the possibility to obtain a survey of the present contamination with genotoxic compounds and to study the impact of water treatment processes. The most intensively applied assay for genotoxicity testing of water samples (>80% of all applications) (14, 15) is the Salmonella/microsome assay developed by Ames et al. (11). This test is based on the detection of histidineindependent (his+) revertants in selected Salmonella strains after exposure to mutagens. In most studies of water samples, only strains TA98 and TA100 are used (15, 16). Despite its widespread application, the Salmonella/microsome assay has some shortcomings that have led to discussions about its applicability for environmental monitoring. For example, in contrast to the other systems investigated, bacteriotoxic effects, which are very common in environmental samples, are not routinely measured in the Ames test. In addition, it is well-known that the Ames test does not readily detect genotoxic compounds in some important classes of environmental pollutants like heavy metals, chlorinated hydrocarbons, and nitrosamines (17, 18). Therefore, Houk and DeMarini (18) have suggested the additional use of a complementary test system (e.g., Microscreen phage-induction assay) for the testing of complex environmental samples. The SOS Chromotest was developed by Quillardet et al. (19) as an alternative to the Ames test for genotoxicity screening. An Escherichia coli strain PQ37 containing a fusion gene of a β-galactosidase (β-Gal) gene (lacZ) with an SOS response gene (sfiA) is used in this assay. Activation of the SOS repair system by genotoxic agents is measured by photometric determination of the β-Gal enzyme activitiy. Bacteriotoxic effects are indicated by a reduction in the activity of alkaline phosphatase (ap). Only one tester strain is needed, and the results are available within 4-5 h. SOS Chromotest and Salmonella/microsome results are in good agreement (80-90%) (20, 21) for single compounds. The SOS Chromotest has been used successfully to determine the genotoxicity of water samples (22). The Microscreen phage-induction assay with E. coli [WP2s λ] was developed by Rossman et al. (23). Activation of the SOS repair system by genotoxic compounds results in the release of lytic phages, which are detected following infection of indicator bacteria (E. coli TH-008). Several findings indicate that the sensitivity of this assay toward heavy metals, chlorinated hydrocarbons, and radicals is greater than that of the Ames test (18, 23-26). Bacterial toxicity is corrected by sampling phages only from wells with bacterial growth. As the assay is conducted in microtiter plates, very small sample volumes (50 µL) are sufficient. The Microscreen assay has been used for the evaluation of environmental samples such as airborne particulate organic matter (27, 28) and industrial wastes (29). In the latter study, Houk and DeMarini (29) demonstrated that prophage induction is a more sensitive end point for hazardous wastes than point mutations in Salmonella. The DNA repair assay with E. coli strains 343/753 and 343/765 enables the detection of (repairable) DNA damage
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by comparison of the differential survival of strains differing in their DNA repair capacity (uvrB/recA vs uvrB+/recA+). It has been postulated on the basis of a large survey carried out within the framework of the U.S. Gene-Tox program that bacterial DNA repair assays are ideal complements to gene mutation assays (30). More recently, it has been shown that the overall sensitivity of E. coli repair assays is equivalent to that of the Salmonella/microsome assay, but certain classes of genotoxins (e.g., nitrosamines) are detected with higher sensitivity by the DNA repair assays (31-32). Because the E. coli strains used in the present investigation have an intact polysaccharide barrier, they are rather insensitive toward larger molecules. To overcome this problem, indicator cells were made permeable by EDTA treatment (33). This technique was optimized in respect to time and concentration (33). We are unaware of any investigations using DNA repair assays with E. coli for water samples.
Experimental Section Chemicals. Positive controls were 2,4,7-trinitro-9-fluorenone (Salmonella/microsome assay TA98 and TA100 without metabolic activation), methyl methanesulfonate (Salmonella/microsome assay TA100 without metabolic activation), 2-aminofluorene (Salmonella/microsome assay TA98 and TA100 with metabolic activation), 4-nitroquinoline N-oxide (SOS Chromotest without metabolic activation), benzo[a]pyrene (SOS Chromotest with metabolic activation), 2-nitrofluorene (Microscreen assay and differential DNA repair test without metabolic activation), 2-aminoanthracene (Microscreen assay with metabolic activation), and Aflatoxin B1 (differential DNA repair test with metabolic activation). All compounds were obtained from from Sigma (St. Louis, MO). Bacto tryptone and bacto agar were from Difco (Detroit, MI), Nutrient broth No. 2 used for the overnight cultures was from Oxoid (Basingstoke, U.K.). Aroclor 1254-induced (500 mg/kg body weight) rat liver S9 was obtained from Organon Teknika (protein concentration 32 mg/mL, Durham, NC). Acetone (p.A.) used as solvent for concentrated water samples was from Merck (Darmstadt, Germany). Bacterial Strains. Salmonella typhimurium TA98 and TA100 were obtained from B. N. Ames (Berkeley, CA), E. coli PQ37 used for the SOS Chromotest came from P. Quillardet (Paris, France), E. coli WP2s λ and E. coli TH-008 used for the Microscreen phage-induction assay were gifts from D. DeMarini (Research Triangle Park, NC), and E. coli strains 343/765 and 765/753 used for the DNA repair assay were obtained from G. R. Mohn (Leiden, The Netherlands). Sample Sources. The origin of the water samples and available information about quality parameters are summarized in Table 1. Samples were transported to the laboratory in 25-L polyethylene containers and processed immediately after arrival. Comparative investigations with parallel samples transported in glass bottles showed that the container material did not affect the outcome of genotoxicity tests (Table 1). Concentration Procedure. Concentration of water samples was performed according to the procedure recommended by the U. S. EPA (34) with minor modifications: XAD resins (prefabricated Amberlite XAD-2 and XAD-7 columns (20 g/ 60 mL) from Applied Separations, Lehigh Valley, PA) were activated with 200 mL of acetone followed by 200 mL of bidistilled water (Haraeus Destamat).
TABLE 1
Sample Sources and Abbreviations code
origina
C-R1
XAD control, batch 1
C-R2 C-W
XAD control, batch 2 bidistilled water
D-D1
drinking water Vienna (Austria)
D-D2 D-B
drinking water Vienna (Austria) chlorinated swimming pool water
S-1 S-2 S-3
river Wilga (Poland) river Wilga (Poland) river Schwechat (Austria)
S-4
channel Badner Mu¨ hlbach (Austria)
W-1
influent of the wastewater treatment plant Krako´ w (Poland) W-1 after oxygen enrichment W-2 after sedimentation
W-2 W-3 W-4
W-3 after activated sludge treatment
P-C1
untreated effluents from pulp production effluents from pulp production after activated sludge treatment influent of an industrial wastewater treatment plant
P-C2 P-O1
P-O2
P-O1 after activated sludge treatment
G-U
G-T2
groundwater contaminated by hazardous waste landfill groundwater contaminated by hazardous waste landfill purified groundwater
L-1
landfill leachate
L-2
landfill leachate
G-T1
remarks Control Samples XAD-2 and XAD-7 columns were activated as described in Concentration Procedure and eluted with 200 mL of acetone; solvent was evaporated, and residue redissolved in 20 mL of acetone prepared like C-R1 tap water (Vienna) bidistilled with Haraeus Destamat Drinking and Bathing Water low organic content (TOC 1 mg/L), slightly chlorinated ( 0) are indicated in boldface. Abbreviations: CI, confidence interval. b Genotoxic potencies (k) and bacterial toxicities (r) were calculated by fitting the results from individual dose levels (5 doses/sample) to the linear-exponential dose-response model proposed by Leroux and Krewski (41). c Units for the resin control samples are revertants/mL for genotoxicity k and mL-2 for bacterial toxicity r. Note that the concentration factor for the solvent is only 10 compared to 500-10 000 for water samples.
classified as negative. The present findings indicate that the 2-fold rule might be too conservative, especially in the presence of bacteriotoxic effects (see also ref 42). (b) SOS Chromotest. Based on results from the SOS Chromotest, 13 samples were classified as genotoxic. Results from the SOS Chromotest were in good agreement with those from the Ames test (Figure 2). Differences were observed only with three samples. One sample (D-D2) was detected only by the Ames test, and one sample (P-O2) was detected only by the SOS Chromotest. Both samples were of marginal genotoxicity in the corresponding test system (Tables 2 and 3). One sample (W-1) was not mutagenic in the Ames test, and W-3 was not tested in the SOS Chromotest. These findings are in agreement with previous findings with pure chemicals demonstrating a good agreement (>90%) between Salmonella and SOS Chromotest results (20, 21, 37). It was however unexpected that the SOS Chromotest was slightly more sensitive than the Ames test in the present experiments. Quillardet and Hofnung (20) reported after an analysis of a database of 452 pure compounds that the Salmonella/microsome assay detects
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more compounds as genotoxic than the SOS Chromotest. Mersch-Sundermann et al. (21) found a quantitative correlation between the responses of both tests, but the Ames test was able to detect genotoxins at lower doses than the SOS Chromotest. So presently no explanation for the higher sensitivity of the SOS Chromotest for water samples can be given. Although it was necessary to subtract the solvent baseline, due to a reduction of ap activities by acetone, the SOS Chromotest gave very linear results with low variances compared to other assays (Tables 2-5). In addition, the lowest frequency of lost data (e.g., due to infections) was encountered with the SOS Chromotest (data not shown). These features facilitate statistical analysis and enhance the sensitivity of this test system. Our results indicate that quantitative correction of bacterial toxicity (measurement of ap activities) leads to linear doseresponse relationships (data not shown) and that systematic variances can be reduced by physicochemical end point detection. (c) Microscreen Phage-Induction Assay. The Microscreen assay was the least sensitive test in our investigation. Only
TABLE 3
TABLE 4
Genotoxic Potencies of Water Samples in the SOS Chromotesta
Genotoxic Potencies of Water Samples in the Microscreen Phage-Induction Assaya
without S9 genotoxicityb (L-1) sample code
k
C-R1c C-R2c C-W
1.2 0.9 0.7
D-D1 D-D2 D-B
0.8 -1.4 10.8
S-1 S-2 S-3 S-4
54.8 18.9 8.4 -1.6
W-1 W-2 W-3 W-4
53.2 nt nt 32.1
P-Cl P-C2 P-O1 P-O2 G-U G-T1 G-T2 L-1 L-2
95% CI (lower limit)
with S9 genotoxicityb (L-1)
without S9 genotoxicityb (PFU/L)
95% CI (lower limit)
sample code
k
0.4 0.2 -0.5
-1.7 -1.5 -1.7
C-R1c C-R2c C-W
-59 188 108
Drinking and Bathing Water -1.2 1.5 -21.7 -3.6 8.4 4.8
-0.1 -14.6 -2.4
D-D1 D-D2 D-B
Control Samples -1.5 -1.1 -1.7
Surface Water 44.4 -0.2 0.9 -4.5
k
21.7 10.2 8.0 1.8
18.8 4.2 4.1 -1.0
S-1 S-2 S-3 S-4
Wastewater Treatment 21.1 38.8 nt nt nt nt 21.8 13.1
24.1 nt nt 9.1
W-1 W-2 W-3 W-4
Effluents from Pulp Production 654.0 596.7 520.7 44.8 33.8 13.8 156.6 113.3 6.9 31.9 6.5 58.7
410.2 8.0 -25.6 35.9
P-Cl P-C2 P-O1 P-O2
-1.9 3.4 1.5
Contaminated Groundwater -6.9 -0.5 1.3 1.4 -0.4 0.4
-2.9 0.2 -1.0
G-U G-T1 G-T2
298.6 1757.4
Landfill Leachates 193.2 -22.9 594.7 -249.9
-45.2 -644.6
L-1 L-2
95% CI (lower limit) Control Samples -322 -17 -224
with S9 genotoxicityb (PFU/L)
k
95% CI (lower limit)
-245 121 -152
-523 -346 -364
-760.8 nt 13212
Drinking and Bathing Water -520 -192 nt nt 1076 2820
-560 nt 84
5688 4420 5000 2140
Surface Water 1488 -18048 173 -4452 24 2684 -1876 -248
-2840 -19568 576 -8828
40288 -4492 108 -3228
Wastewater Treatment 15896 -29784 -9392 2484 -8592 -1492 -9740 -2936
-48664 -1948 -11068 -17632
Effluents from Pulp Production 467208 46328 921957 2796 -5704 4212 93556 31132 39984 -532 -10240 -9864
868900 -26800 21980 -23072
756 -68 4362
Contaminated Groundwater -124 320 -204 -108 116 292
-122 -356 -624
3908 208176
Landfill Leachates -19556 -3500 -364704 -196868
-12464 -465992
a Assays were carried out according to the procedure given by Mersch-Sundermann et al. (39). For metabolic activation, S9 mix composed according to Mersch-Sundermann et al. (39) was used. Ratios of β-Gal and ap activities (R0) for the negative control were 0.154 ( 0.015 without metabolic activation and 0.630 ( 0.046 with metabolic activation (mean ( standard deviation). Induction factors of the solvent control (acetone 20 µL/assay) were 1.250 ( 0.009 without metabolic activation and 1.138 ( 0.046 with metabolic activation. From each dose level IFD, corresponding IFacetone values were subtracted to obtain sample-induced IFD values that were used for statistical analysis. Positive controls were 4-nitroquinoline N-oxide (0.5 nmol/assay without metabolic activation: IF 22.1 ( 7.4) and benzo[a]pyrene (5 nmol/assay with metabolic activation: IF 7.2 ( 0.4). Statistically significant results (p < 0.05, lower limit of 95% CI > 0) are indicated in boldface. Abbreviations: CI, confidence interval, nt, not tested. b Genotoxic potencies (k) were calculated by linear-regression analysis of the results from individual dose levels (5 doses/sample). c The unit for the genotoxicity parameter k used for resin control samples is mL-1. Note that the concentration factor for the solvent is only 10 compared to 500-10 000 for water samples.
a Assays were carried out according to the procedure given by DeMarini et al. (25). S9 mix was composed as described in ref 25. Negative controls (MST) induced 32 ( 17 PFU/plate without S9 and 39 ( 18 PFU/plate with S9 (mean ( standard deviation). Acetone (25 µL/ assay) gave 43 ( 22 PFU/plate without S9 and 51 ( 9 PFU/plate with S9. From each dose level, PFUD, corresponding PFUacetone values were subtracted to obtain sample-induced PFUD values, which were used for statistical analysis. Positive controls were 2-nitrofluorene (37.5 µg/ assay without metabolic activation: 510 ( 94 PFU/plate) and 2-aminoanthracene (0.25 µg/assay with metabolic activation: 773 ( 191 PFU/ plate). Statistically significant results (p < 0.05, lower limit of 95% CI > 0) are indicated in boldface. Abbreviations: CI, confidence interval, nt, not tested. b Genotoxic potencies (k) were calculated by linearregression analysis of the results from individual dose levels (4 nontoxic doses/sample). c The unit for the genotoxicity parameter k used for resin control samples is PFU/mL. Note that the concentration factor for the solvent is only 10 compared to 500-10 000 for water samples.
eight samples gave positive results (Figure 2). Two of these samples (D-B and G-T2) were not detected by the Salmonella/microsome assay, and one sample (G-T2) was not detected by the SOS Chromotest. Additional use of the Microscreen assay to supplement the Ames test as proposed by Houk and DeMarini (18) would have yielded only minor additional information (detection of two samples with relatively low genotoxicity). However, genotoxicity was detected in chlorinated and ozonated samples (P-C1, P-O1, and D-B) with high sensitivity, which is in accordance with previous findings (18, 25, 26, 29). The power of the phageinduction assay to discriminate between samples of different genotoxicity (e.g., P-C1:P-O1 ratio ≈4.5) was much
The main reason for the low sensitivity of the Microscreen assay was nonlinearities of the dose-response relationships of some samples, an observation which has been made also by Houk and DeMarini (29) for two hazardous wastes. Thus, the use of a linear regression model reduces the number of samples classified as positive in relation to the 3-fold rule proposed by DeMarini et al. (25). Using the 3-fold criteria, samples P-C2 and S-4 would also rate as positive, and the sensitivity would be, with the exception of landfill leachates and the slightly mutagenic samples W-3 and W-4, equal to that of the Salmonella/ microsome assay. As a consequence, we are currently analyzing historical results from the phage-induction assay
higher than the resolution of the Ames test (P-C1:P-O1 ratio ≈2).
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TABLE 5
Genotoxic Potencies of Water Samples in the Differential DNA Repair Testa without S9 genotoxicityb (L-1) 95% CI (lower limit)
with S9 genotoxicityb (L-1) 95% CI (lower limit)
sample code
k
C-R1c C-R2c C-W
-0.14 -0.2 -0.05
Control Samples 0.10 0.02 0.06 -0.5 0.2 -1.2
0.2 -0.3 -0.5
D-D1 D-D2 D-B
-0.2 nt -1.4
Drinking and Bathing Water 0.07 0.0 nt nt -0.9 -0.6
0.2 nt 0.1
S-1 S-2 S-3 S-4
-2.5 -1.3 -1.4 -0.7
Surface Water -1.3 1.9 -0.8 -0.3
W-1 W-2 W-3 W-4
-3.5 -1.9 -2.7 -2.0
P-Cl P-C2 P-O1 P-O2 G-U G-T1 G-T2 L-1 L-2
k
-0.2 -2.1 -0.9 -0.2
0.9 -0.5 -0.2 -0.3
Wastewater Treatment -2.5 -3.8 -0.9 -0.06 -1.2 -1.1 -0.1 -1.3
-1.9 0.9 0.07 -0.4
Effluents from Pulp Production -12.8 -5.6 -6.0 -6.4 -1.8 -3.1 -7.4 0.9 -6.1 -5.9 -2.2 -4.2
-2.3 -1.3 -4.3 -2.9
nt -0.5 -0.3
Contaminated Groundwater nt nt -0.2 -0.6 -0.01 -0.02
nt -0.5 0.09
-5.5 -11.6
Landfill Leachates -2.8 -18.9 -3.3 -17.5
-15.0 -6.2
a Assays were carried out according to the procedure given by Knasmu¨ ller et al. (33). For metabolic activation, standard S9 mix (4%) (35) was used. Survival rates (S0) for the repair-deficient strain (negative control) were 0.74 ( 0.05 without S9 and 2.36 ( 0.35 with S9 (mean ( standard deviation). Differential survival rates for acetone (0.1 mL/ assay) were 1.02 ( 0.08 without S9 and 1.07 ( 0.13 with S9. Positive controls were 2-nitrofluorene (0.24 µg/assay without metabolic activation: differential survival rate 0.13 ( 0.04). Note that genotoxicity is indicated by a reduction of differential survival rates with rising doses, thus giving negative values for the genotoxicity parameter k. Statistically significant results (p < 0.05, upper limit of 95% CI < 0) are indicated in boldface. Abbreviations: CI, confidence interval, nt, not tested. b Measurements were performed within the linear region of the differential survival rates. Therefore, genotoxic potencies (k) were calculated by linear regression analysis of the results from individual dose levels (4 doses/sample). c The unit for the genotoxicity parameter k used for resin control samples is mL-1. Note that the concentration factor for the solvent is only 10 compared to 500-10 000 for water samples.
to develop an appropriate statistical method for data evaluation for this test system. Several features of the Microscreen assay may account for the observed deviations from linearity. The end point detection (infection of indicator bacteria with lytic phages) involves two different organisms and, consequently, depends on more complex biological processes than the end point measurements of the other test systems. Additional sources of error may be introduced by the dilution of phages prior to plating and the subtraction of the acetone baseline. Although bacterial toxicity is compensated by sampling only turbid wells (see Experimental Section), toxic effects on phages may reduce the ability to detect genotoxicity (DeMarini, personal communication). Lower PFUs/plate
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than in control samples are an indication of viral toxicity. This effect was very pronounced in the landfill leachates L-2 and L-1 (less PFUs than in the negative control at all doses, data not shown) and probably prevented the detection of genotoxicity in these samples. (d) Differential DNA Repair Test. The differential DNA repair test with E. coli was the most sensitive test system in our investigation. Nineteen of 21 samples were classified as positive. However, two of three negative controls (CR2, resin control batch2; C-W, bidistilled water) also gave positive results (comparable to surface water) with metabolic activation (Figure 2 and Table 5). As the pure solvent control (acetone) and the resin control from the first resin batch (C-R1) did not cause genotoxic effects, it is possible that impurities were eluted from the second resin batch used in our experiments. Although the formation of mutagenic compounds after reaction of free chlorine with XAD-2 resins was previously reported (43), we are not aware of studies addressing the elution of genotoxic impurities from XAD resins. The differential DNA repair test had the lowest power to detect differences between the genotoxic potencies of different samples (e.g., P-C1:P-O1 ratio ≈1, in contrast to ≈2 in the Ames test). The high proportion of samples detected as positive was also unexpected, because in former studies with pure compounds (31), lower sensitivities than for the other assays used in the present investigation were obtained. As the differential DNA repair test is based on the comparison of the survival curves of two different strains, it is possible that the calculation of differential survival rates (33, eq 4) is not an appropriate method to represent the results from this assay. Currently, we are attempting to optimize the statistical treatment of differential DNA repair data; results from this study will be published separately. Genotoxicity of Water Samples. Control Samples (CR1, C-R2, and C-W). Control samples (resin blanks and bidistilled water) were negative with the exception of the differential DNA repair test with metabolic activation (see above). Drinking and Bathing Water (D-D1, D-D2, and D-B). Tap water from Vienna (D-D1) was negative in all four assays. Several studies have shown that many drinking waters disinfected with chlorine are genotoxic (16). The lack of genotoxicity of D-D1 can be explained with low levels of precursor compounds (TOC 1 mg/L) present in this sample and mild disinfection conditions (
