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Suitability of the modified fluctuation assay for evaluating the mutagenicity of unconcentrated drinking water
Mutation Research, 120 (1983) 97-103
97
Elsevier BiomedicalPress
Suitability of the modified fluctuation assay for evaluating the mutagenicity of unconcentrated drinking water Tina R. Harrington, Earle R. N e s t m a n n and David J. Kowbel Mutagenesis Section, Environmental and Occupational Toxicology Division, Department of National Health and Welfare, Tunney's Pasture, Ottawa, Ont. K1A OL2 (Canada)
(Accepted 27 January 1983)
Summary Filter-sterilized, unconcentrated tap water induced mutagenic responses (p < 0.01) in Salmonella strain TA 100 in fluctuation assays, usually with dose-related increases in positive tubes. Additional experiments were performed to study possible artifacts that could lead to falsely positive results. Determinations of bacterial survival revealed that cell populations in the tubes containing tap water were larger than in the controls. Since spontaneous mutation is a function of cell generation, the increased numbers of bacteria appeared to be responsible for the higher numbers of mutants observed. Therefore, the positive responses must be regarded as artifactual. This study suggests that survival determination should be a routine part of this method, and care should be exercised in the interpretation of positive results.
Bacterial and mammalian assays have shown that mutagens exist in extracts or concentrates of raw and finished waters. Residues from samples of Canadian drinking water (Nestmann et al., 1979a, 1982; Williams et al., 1982) and of water from the Rhine River (Kool et al., 1981), concentrated by XAD resin column chromatography, have been found to be mutagenic in the Salmonella/microsome mutagenicity assay. Dichloromethane liquid-liquid extracts of tap water samples from Pretoria (South Africa), also were mutagenic in Salmonella (Grabow et al., 1980). Residues isolated by reverse osmosis techniques induced genetic activity in Chinese hamster embryonic lung cells (Gruener and Lockwood, 1979) and in both Send correspondenceand reprint requests to Dr. Earle R. Nestmann. 0165-7992/83/0000-0000/$ 03.00 © 1983 Elsevier SciencePublishers
98 Salmonella and BALB/3T3 cells (Loper et al., 1978). Preliminary tests with bacteria have indicated that certain chemicals, usually chlorinated aliphatic hydrocarbons, isolated from finished drinking water, are mutagenic (Simmon et al., 1977; Loper et al., 1982). There are a number of difficulties associated with testing concentrated samples and extracts. For example, during the dichloromethane extraction process, volatile, highly polar or inorganic substances may be lost (Denkhaus et al., 1980). The extremely low recovery (30-40o70) offered by reverse osmosis XAD-2 sorbtion/desorbtion (Loper et al., 1978) indicates that the extract is no longer an accurate representation of the sample. The small yields from XAD-2 resin columns restrict the numbers of assays possible and/or the number of tester strains which can be used (Nestmann et al., 1979a). Water samples concentrated by freeze-drying induce mutations in bacteria (Forster and Wilson, 1981) and in Chinese hamster lung cells (Gruener and Lockwood, 1980). Freeze-drying can give 90°7o recovery of the organic materials in water samples (Gruener and Lockwood, 1980). However, the presence of proportionately large amounts of natural organics (e.g. humic and fulvic acids) contributes to sample insolubility and/or toxicity (Nestmann, 1983). While these techniques have been useful in revealing and comparing the mutagenic potential of drinking water, they involve its conversion into other forms. A more significant estimate of mutagenicity might be obtained by testing water in the form consumed. The Salmonella assay has been used to show weakly positive results with unconcentrated rat water from the Mississippi River (Pelon et al., 1977). However, a more sensitive method, the modified fluctuation assay by Green et al. (1976), has been shown capable of detecting 100-fold lower concentrations of known mutagens compared to conventional plate tests. Since this assay can be adapted to incorporate high proportions of unconcentrated water, the present study has examined its possible suitability in determining mutagenicity of tap water sampled from a single source over several months. An abstract describing these results has been published (Harrington et al., 1982). In preliminary experiments, Salmonella typhimurium strains TA98, TA100 and TA1535 were used. Only strain TA100, which is capable of detecting base-pair substitution mutations, showed a significant mutagenic response (data not shown), and was used in all subsequent experiments. Cultures were stored and grown as described by Ames et al. (1975). Samples were obtained from the same laboratory faucet for all experiments. Previously, water from this source had been shown to be mutagenic after concentration (Nestmann et al., 1979a). The total volume of water to be tested in a single experiment was collected in one large sample after the cold water faucet had run at full force for at least 2 min. This sample was filtersterilized using a Nalgene filter (20 micron) and transferred to a sterile glass bottle. Davis-Mingioli salts (A), as described by Green et al. (1976), were concentrated to 6 times normal to form a solution referred to as 6A, which was autoclaved and
99 TABLE 1 VOLUMES AND PERCENTAGES OF WATER USED IN EXPERIMENTS Flask No.
Volume of tap water (ml)
1
Volume of distilled water (ml)
0
2 3 4 5
5.6 20.8 27.8 81.3
Percentage tap water of maximum (81.3 ml) that could be added
81.3
0
75.7 60.5 53.5 0
6.9 25.6 34.2 100
stored at r o o m temperature. Prior to use, this solution was supplemented with dextrose (2.4070), b r o m o c r e s o l purple (60/~g/ml), histidine (2.5 ttg/ml) and biotin (3.5 /~g/ml). The resulting concentrated solution was then diluted in the following m a n ner: to each o f 5 flasks, 18.7 ml o f supplemented 6A was added. Then, to dilute the salts to 1A (in 100 ml), filter-sterilized tap and distilled water were also added, as shown in Table l, the highest tap water concentrations being limited by the maxi m u m concentration factor o f the salts (6A). 50 #l o f overnight culture was added to each flask, this mixture was dispensed in 1.95-ml aliquots (Nestmann et al., 1979b) into 50 small (13 × 100 m m ) test tubes, and these tubes were incubated at 37°C for 3 days. A turbid or yellow tube indicates that a cell(s) has reverted to histidine independence. This mutant(s) grows in the absence o f histidine, leading to cell turbidity with an a c c u m u l a t i o n o f b y p r o d u c t s with lower p H and can eventually cause a purple to yellow colour change (Green et al., 1976). In this study, the term 'positive tubes' refers to the sum o f yellow and turbid tubes, unless otherwise indicated. 3 negative (clear purple) tubes were saved f r o m each rack for determination o f survival. The cells were diluted (10 -4) in 1A and spread (0.2 ml/plate) o n t o nutrient broth agar plates (1.5°70 agar), with 5 plates per dilution. After 2 days inc u b a t i o n at 37°C, the colonies present were scored. The statistical significance o f results in this study was determined using Chi-Square analysis (Green et al., 1976). TABLE 2 AVERAGE NUMBERS OF ALL 10 TAP-WATER EXPERIMENTS Flask No.
Number of positive tubes
S.D. a
pb
Total number of positive + turbid tubes
S.D. a
1 2 3 4 5
26.7 29.8 32.8 35.8 41.2
6.2 5.2 6.7 5.9 3.8
ns ns ns < 0.05
30.1 33.2 33.6 37.5 42.1
7.6 6.9 7.7 7.2 4.0
aS.D, standard deviation. bns, not significant (o>0.05).
pb
ns ns
ns <0.01
100 TABLE 3 RESULTS OF TAP-WATER EXPERIMENTS Expt. No.
Flask No.
Number of positive tubes
Total number of positive and turbid tubes
1
28
-
2
27 31 39 45
ns ns < 0.05 <0.01
29 27 33 40 45
18 26 20 24 36
ns ns ns <0.01
19 27 21 24 38
3 4 5
36 32 36 39 42 27 33 34 41 44 24 25 37 31 40 10
pa
29 25 26 35 43 21 24 28 28 33
ns ns ns
ns
ns ns
<0.01 <0.01
ns
<0.01 < 0.05 <0.01
38 33 37 41 43 28 35 36 42 44 25 27 38 33 40
<0.01 <0.01
44 46 47 48 48
ns ns ns < 0.05
21 25 29 28 33
ns ns
pa
Averagenumber of cells/ml ( × 107) (where plating done)
ns ns
<0.05 <0.01
ns ns ns
<0.01
as
ns ns ns
ns ns
<0.01 <0.01
ns
<0.01 ns
<0.01
ns
ns ns as
3.20 ns
1.59
ns ns < 0.05
3.88 3.38 1.78
101 T A B L E 3 (continued) Expt. No.
Flask No.
N u m b e r o f pa Positive Tubes
Total n u m b e r of pa positive and turbid tubes
Average number o f cells/ml ( x 107) (where plating done)
1
29
-
30
-
2 3 4 5
31 34 40 44
ns ns <0.05 <0.01
32 37 41 45
ns ns <0.05 <0.01
1
35
-
35
2 3 4 5
36 40 40 43
ns ns ns ns
38 44 40 44
1
30
-
32
2 3 4 5
39 42 38 42
ns <0.01 ns <0.01
40 44 38 44
-
ns <0.05 ns <0.05 -
ns <0.01 ns <0.01
1.66 1.79 2.68 2.83 1.35 1.07 2.07 3.05 2.46 2.41 1.32 1.84 3.39 2.28 2.48
ans, not significant, p > 0 . 0 5 .
Although variable responses were found among the experiments, for convenience, the results of 10 fluctuation tests were averaged. On this basis, a dose-related positive response that was significant (p< 0.01) at the highest dose (Table 2) was observed. 7 of the 10 individual experiments (Table 3) showed significant increases in positives (p<0.01 in 6; p<0.05 in 1). In another experiment (No. 10), the increase was significant at p < 0.01 if turbid tubes were not considered. 2 Expts., Nos. 5 and 7, did not show significant increases of positives (p > 0.05). In 3 of the 4 Expts. which were plated for survival, the average numbers of cells per tube increased proportionately with the numbers of positive tubes scored for each dose (Table 3). Therefore, an elevation in mutation probably was spontaneous (due to cell growth) rather than due to induction by chemical mutagens. One possibility for this excess cell growth could be increasing histidine levels in the tap water. Experiments designed to examine the relationship between histidine levels, numbers of mutants and cell survival showed that the numbers of revertants increased with increasing histidine concentrations. Results of experiments to determine cell numbers were not consistent and appeared not to be the major cause of increasing mutation (data not shown). It has been suggested that under the conditions of the fluctuation test, it is possible that the bacteria may be able to use the available histidine more efficiently (Forster et al., 1982). Experiments were performed to determine whether the variability among the ex-
102 p e r i m e n t s was d u e t o p e r f o r m a n c e o f t h e tests o r to s a m p l e v a r i a b i l i t y . Results o f 2 E x p t s . p e r f o r m e d in p a r a l l e l , using w a t e r f r o m the s a m e s a m p l e , s h o w e d t h a t t h e e x p e r i m e n t a l t e c h n i q u e was consistent. O n the o t h e r h a n d , a large v a r i a t i o n b e t w e e n m o r n i n g a n d a f t e r n o o n s a m p l e s was o b s e r v e d in o n e p a i r o f e x p e r i m e n t s , a n d little v a r i a t i o n was f o u n d in a n o t h e r ( d a t a n o t shown). T h e r e f o r e , it a p p e a r s t h a t the v a r i a t i o n seen a m o n g i n d e p e n d e n t e x p e r i m e n t s was largely d u e to differences in samples rather than experimental performance. P o s i t i v e responses were clearly f o u n d in f l u c t u a t i o n tests with t a p w a t e r , a l t h o u g h these effects m a y n o t h a v e been i n d u c e d b y m u t a g e n s . Results o f p l a t i n g for survival f r o m these e x p e r i m e n t s s h o w e d t h a t increases in b a c t e r i a l c o n c e n t r a t i o n was p a r t i a l ly, if n o t fully, r e s p o n s i b l e for t h e o b s e r v e d d o s e - r e l a t e d positive results. It is n o t k n o w n w h a t o t h e r u n c o n c e n t r a t e d test m a t e r i a l s m i g h t p r o d u c e similar artifacts. H o w e v e r , these findings are consistent with t h o s e r e p o r t e d r e c e n t l y b y F o r s t e r et al. (1982), also with u n c o n c e n t r a t e d d r i n k i n g w a t e r . F o r o t h e r test s u b s t a n c e s , the fluct u a t i o n assay r e m a i n s a simple, sensitive, r a p i d a n d valid test, especially w h e n acc o m p a n i e d b y a p p r o p r i a t e m o n i t o r i n g o f cell survival.
Acknowledgements W e t h a n k Drs. D . H . B l a k e y a n d A . P . H u g e n h o l t z f o r their c o m m e n t s a n d K a t h y N e s b i t t for her usual swift, a c c u r a t e typing.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31, 347-363. Denkhaus, R., W.O.K. Grabow and O.W. Prozesky (1980) Removal of mutagenic compounds in a wastewater reclamation system evaluation by means of the Ames Salmonella/microsome assay, Prog. Wat. Tech., 12, 571-589. Forster, R., and I. Wilson (1981) The application of mutagenicity testing of drinking water, J. Inst. Water Eng. Scient., 35, 259-274. Forster, R., M.H.L. Green, R.D. Gwilliam, A. Priestly and B.A. Bridges (1982) The use of the fluctuation test to detect mutagenic activity in unconcentrated samples of U.K. drinking waters, in: R.L. Jolley, W.A. Brungs and R.B. Cumming (Eds.), Water Chlorination: Environmental Impact and Health Effects, Vol. 4, Ann Arbor Science Publ., in press. Grabow, W.O.K., R. Denkhaus and P.G. van Rossum (1980) Detection of mutagens in wastewater, a polluted river and drinking water by means of the Ames Salmonella/microsome assay, S. Afr. J. Sci., 76, 118-123. Green, M.H.L., W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-42. Gruener, N., and M.P. Lockwood (1979) Mutagenicity and transformation by recycled water, J. Toxicol. Environ. Health, 5, 663-670. Gruener, N., and M.P. Lockwood (1980) Mutagenic activity in drinking water, Am. J. Publ. Health, 70, 276-278.
103 Harrington, T.R., E.R. Nestmann and D.J. Kowbel (1982) Suitability of the modified fluctuation assay for evaluating mutagenicity in bacteria of unconcentrated drinking water, Can. J. Genet. Cytol., 24, in press (abstract). Kool, H.J., C.F. van Kreijl and H.J. van Kranen (1981) The use of XAD-resins for the detection of mutagenic activity in water, II. Studies with drinking water, Chemosphere, 10, 99-108. Loper, J.C., D.R. Lang, R.S. Schoeny, B.B. Richmond, P.M. Gallagher and C.C. Smith (1978) Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity, J. Toxicol. Environ. Health, 4, 919-938. Loper, J.C., M.W. Tabor and S.M. MacDonald (1982) Mutagens from nonvolatile organics of drinking water, Environ. Mutagen., 4, 380 (abstract). Nestmann, E.R. (1983) Mutagenic activity of drinking water, in: H.F. Stich (Ed.), Carcinogens and Mutagens in the Environment, Vol. 2, Naturally Occurring Compounds, CRC Press, Boca Raton, in press. Nestmann, E.R., G.L. LeBel, D.T. Williams and D.J. Kowbel (1979a) Mutagenicity of organic extracts from Canadian drinking water in the Salmonella/mammalian-microsome assay, Environ. Mutagen., 1, 337-345. Nestmann, E.R., T.I. Matula, G.R. Douglas, K.C. Bora and D.J. Kowbel (1979b) Detection of the mutagenic activity of lead chromate using a battery of microbial tests, Mutation Res., 66, 357-365. Nestmann, E.R., R. Otson, G.L. LeBel, D.T. Williams, E.G.-H. Lee and D.C. Biggs (1982) Correlation of water quality parameters with mutagenicity of drinking water extracts, in: R.L. Jolley, W.A. Brungs and R.B. Cumming (Eds.), Water Chlorination: Environmental Impact and Health Effects, Vol. 4, Ann Arbor Science Publ., pp. 1151-1164. Pelon, W.F., B.F. Whitman and T.W. Beasley (1977) Reversion of histidine dependent mutant strains of Salmonella typhimurium by Mississippi River water samples, Environ. Sci. Technol., 2, 619-623. Simmon, V.F., K. Kauhanen and R.G. Tardiff (1977) Mutagenic activity of chemicals identified in drinking water, in: D. Scott, B.A. Bridges and F.H. Sobels (Eds.), Progress in Genetic Toxicology, Elsevier/North-Holland, Amsterdam, pp. 249-258. Williams, D.T., E.R. Nestmann, G.L. LeBel, F.M. Benoit and R. Otson (1982) Determination of mutagenic potential and organic contaminants of Great Lakes drinking water, Chemosphere, 11, 263-276.
97
Elsevier BiomedicalPress
Suitability of the modified fluctuation assay for evaluating the mutagenicity of unconcentrated drinking water Tina R. Harrington, Earle R. N e s t m a n n and David J. Kowbel Mutagenesis Section, Environmental and Occupational Toxicology Division, Department of National Health and Welfare, Tunney's Pasture, Ottawa, Ont. K1A OL2 (Canada)
(Accepted 27 January 1983)
Summary Filter-sterilized, unconcentrated tap water induced mutagenic responses (p < 0.01) in Salmonella strain TA 100 in fluctuation assays, usually with dose-related increases in positive tubes. Additional experiments were performed to study possible artifacts that could lead to falsely positive results. Determinations of bacterial survival revealed that cell populations in the tubes containing tap water were larger than in the controls. Since spontaneous mutation is a function of cell generation, the increased numbers of bacteria appeared to be responsible for the higher numbers of mutants observed. Therefore, the positive responses must be regarded as artifactual. This study suggests that survival determination should be a routine part of this method, and care should be exercised in the interpretation of positive results.
Bacterial and mammalian assays have shown that mutagens exist in extracts or concentrates of raw and finished waters. Residues from samples of Canadian drinking water (Nestmann et al., 1979a, 1982; Williams et al., 1982) and of water from the Rhine River (Kool et al., 1981), concentrated by XAD resin column chromatography, have been found to be mutagenic in the Salmonella/microsome mutagenicity assay. Dichloromethane liquid-liquid extracts of tap water samples from Pretoria (South Africa), also were mutagenic in Salmonella (Grabow et al., 1980). Residues isolated by reverse osmosis techniques induced genetic activity in Chinese hamster embryonic lung cells (Gruener and Lockwood, 1979) and in both Send correspondenceand reprint requests to Dr. Earle R. Nestmann. 0165-7992/83/0000-0000/$ 03.00 © 1983 Elsevier SciencePublishers
98 Salmonella and BALB/3T3 cells (Loper et al., 1978). Preliminary tests with bacteria have indicated that certain chemicals, usually chlorinated aliphatic hydrocarbons, isolated from finished drinking water, are mutagenic (Simmon et al., 1977; Loper et al., 1982). There are a number of difficulties associated with testing concentrated samples and extracts. For example, during the dichloromethane extraction process, volatile, highly polar or inorganic substances may be lost (Denkhaus et al., 1980). The extremely low recovery (30-40o70) offered by reverse osmosis XAD-2 sorbtion/desorbtion (Loper et al., 1978) indicates that the extract is no longer an accurate representation of the sample. The small yields from XAD-2 resin columns restrict the numbers of assays possible and/or the number of tester strains which can be used (Nestmann et al., 1979a). Water samples concentrated by freeze-drying induce mutations in bacteria (Forster and Wilson, 1981) and in Chinese hamster lung cells (Gruener and Lockwood, 1980). Freeze-drying can give 90°7o recovery of the organic materials in water samples (Gruener and Lockwood, 1980). However, the presence of proportionately large amounts of natural organics (e.g. humic and fulvic acids) contributes to sample insolubility and/or toxicity (Nestmann, 1983). While these techniques have been useful in revealing and comparing the mutagenic potential of drinking water, they involve its conversion into other forms. A more significant estimate of mutagenicity might be obtained by testing water in the form consumed. The Salmonella assay has been used to show weakly positive results with unconcentrated rat water from the Mississippi River (Pelon et al., 1977). However, a more sensitive method, the modified fluctuation assay by Green et al. (1976), has been shown capable of detecting 100-fold lower concentrations of known mutagens compared to conventional plate tests. Since this assay can be adapted to incorporate high proportions of unconcentrated water, the present study has examined its possible suitability in determining mutagenicity of tap water sampled from a single source over several months. An abstract describing these results has been published (Harrington et al., 1982). In preliminary experiments, Salmonella typhimurium strains TA98, TA100 and TA1535 were used. Only strain TA100, which is capable of detecting base-pair substitution mutations, showed a significant mutagenic response (data not shown), and was used in all subsequent experiments. Cultures were stored and grown as described by Ames et al. (1975). Samples were obtained from the same laboratory faucet for all experiments. Previously, water from this source had been shown to be mutagenic after concentration (Nestmann et al., 1979a). The total volume of water to be tested in a single experiment was collected in one large sample after the cold water faucet had run at full force for at least 2 min. This sample was filtersterilized using a Nalgene filter (20 micron) and transferred to a sterile glass bottle. Davis-Mingioli salts (A), as described by Green et al. (1976), were concentrated to 6 times normal to form a solution referred to as 6A, which was autoclaved and
99 TABLE 1 VOLUMES AND PERCENTAGES OF WATER USED IN EXPERIMENTS Flask No.
Volume of tap water (ml)
1
Volume of distilled water (ml)
0
2 3 4 5
5.6 20.8 27.8 81.3
Percentage tap water of maximum (81.3 ml) that could be added
81.3
0
75.7 60.5 53.5 0
6.9 25.6 34.2 100
stored at r o o m temperature. Prior to use, this solution was supplemented with dextrose (2.4070), b r o m o c r e s o l purple (60/~g/ml), histidine (2.5 ttg/ml) and biotin (3.5 /~g/ml). The resulting concentrated solution was then diluted in the following m a n ner: to each o f 5 flasks, 18.7 ml o f supplemented 6A was added. Then, to dilute the salts to 1A (in 100 ml), filter-sterilized tap and distilled water were also added, as shown in Table l, the highest tap water concentrations being limited by the maxi m u m concentration factor o f the salts (6A). 50 #l o f overnight culture was added to each flask, this mixture was dispensed in 1.95-ml aliquots (Nestmann et al., 1979b) into 50 small (13 × 100 m m ) test tubes, and these tubes were incubated at 37°C for 3 days. A turbid or yellow tube indicates that a cell(s) has reverted to histidine independence. This mutant(s) grows in the absence o f histidine, leading to cell turbidity with an a c c u m u l a t i o n o f b y p r o d u c t s with lower p H and can eventually cause a purple to yellow colour change (Green et al., 1976). In this study, the term 'positive tubes' refers to the sum o f yellow and turbid tubes, unless otherwise indicated. 3 negative (clear purple) tubes were saved f r o m each rack for determination o f survival. The cells were diluted (10 -4) in 1A and spread (0.2 ml/plate) o n t o nutrient broth agar plates (1.5°70 agar), with 5 plates per dilution. After 2 days inc u b a t i o n at 37°C, the colonies present were scored. The statistical significance o f results in this study was determined using Chi-Square analysis (Green et al., 1976). TABLE 2 AVERAGE NUMBERS OF ALL 10 TAP-WATER EXPERIMENTS Flask No.
Number of positive tubes
S.D. a
pb
Total number of positive + turbid tubes
S.D. a
1 2 3 4 5
26.7 29.8 32.8 35.8 41.2
6.2 5.2 6.7 5.9 3.8
ns ns ns < 0.05
30.1 33.2 33.6 37.5 42.1
7.6 6.9 7.7 7.2 4.0
aS.D, standard deviation. bns, not significant (o>0.05).
pb
ns ns
ns <0.01
100 TABLE 3 RESULTS OF TAP-WATER EXPERIMENTS Expt. No.
Flask No.
Number of positive tubes
Total number of positive and turbid tubes
1
28
-
2
27 31 39 45
ns ns < 0.05 <0.01
29 27 33 40 45
18 26 20 24 36
ns ns ns <0.01
19 27 21 24 38
3 4 5
36 32 36 39 42 27 33 34 41 44 24 25 37 31 40 10
pa
29 25 26 35 43 21 24 28 28 33
ns ns ns
ns
ns ns
<0.01 <0.01
ns
<0.01 < 0.05 <0.01
38 33 37 41 43 28 35 36 42 44 25 27 38 33 40
<0.01 <0.01
44 46 47 48 48
ns ns ns < 0.05
21 25 29 28 33
ns ns
pa
Averagenumber of cells/ml ( × 107) (where plating done)
ns ns
<0.05 <0.01
ns ns ns
<0.01
as
ns ns ns
ns ns
<0.01 <0.01
ns
<0.01 ns
<0.01
ns
ns ns as
3.20 ns
1.59
ns ns < 0.05
3.88 3.38 1.78
101 T A B L E 3 (continued) Expt. No.
Flask No.
N u m b e r o f pa Positive Tubes
Total n u m b e r of pa positive and turbid tubes
Average number o f cells/ml ( x 107) (where plating done)
1
29
-
30
-
2 3 4 5
31 34 40 44
ns ns <0.05 <0.01
32 37 41 45
ns ns <0.05 <0.01
1
35
-
35
2 3 4 5
36 40 40 43
ns ns ns ns
38 44 40 44
1
30
-
32
2 3 4 5
39 42 38 42
ns <0.01 ns <0.01
40 44 38 44
-
ns <0.05 ns <0.05 -
ns <0.01 ns <0.01
1.66 1.79 2.68 2.83 1.35 1.07 2.07 3.05 2.46 2.41 1.32 1.84 3.39 2.28 2.48
ans, not significant, p > 0 . 0 5 .
Although variable responses were found among the experiments, for convenience, the results of 10 fluctuation tests were averaged. On this basis, a dose-related positive response that was significant (p< 0.01) at the highest dose (Table 2) was observed. 7 of the 10 individual experiments (Table 3) showed significant increases in positives (p<0.01 in 6; p<0.05 in 1). In another experiment (No. 10), the increase was significant at p < 0.01 if turbid tubes were not considered. 2 Expts., Nos. 5 and 7, did not show significant increases of positives (p > 0.05). In 3 of the 4 Expts. which were plated for survival, the average numbers of cells per tube increased proportionately with the numbers of positive tubes scored for each dose (Table 3). Therefore, an elevation in mutation probably was spontaneous (due to cell growth) rather than due to induction by chemical mutagens. One possibility for this excess cell growth could be increasing histidine levels in the tap water. Experiments designed to examine the relationship between histidine levels, numbers of mutants and cell survival showed that the numbers of revertants increased with increasing histidine concentrations. Results of experiments to determine cell numbers were not consistent and appeared not to be the major cause of increasing mutation (data not shown). It has been suggested that under the conditions of the fluctuation test, it is possible that the bacteria may be able to use the available histidine more efficiently (Forster et al., 1982). Experiments were performed to determine whether the variability among the ex-
102 p e r i m e n t s was d u e t o p e r f o r m a n c e o f t h e tests o r to s a m p l e v a r i a b i l i t y . Results o f 2 E x p t s . p e r f o r m e d in p a r a l l e l , using w a t e r f r o m the s a m e s a m p l e , s h o w e d t h a t t h e e x p e r i m e n t a l t e c h n i q u e was consistent. O n the o t h e r h a n d , a large v a r i a t i o n b e t w e e n m o r n i n g a n d a f t e r n o o n s a m p l e s was o b s e r v e d in o n e p a i r o f e x p e r i m e n t s , a n d little v a r i a t i o n was f o u n d in a n o t h e r ( d a t a n o t shown). T h e r e f o r e , it a p p e a r s t h a t the v a r i a t i o n seen a m o n g i n d e p e n d e n t e x p e r i m e n t s was largely d u e to differences in samples rather than experimental performance. P o s i t i v e responses were clearly f o u n d in f l u c t u a t i o n tests with t a p w a t e r , a l t h o u g h these effects m a y n o t h a v e been i n d u c e d b y m u t a g e n s . Results o f p l a t i n g for survival f r o m these e x p e r i m e n t s s h o w e d t h a t increases in b a c t e r i a l c o n c e n t r a t i o n was p a r t i a l ly, if n o t fully, r e s p o n s i b l e for t h e o b s e r v e d d o s e - r e l a t e d positive results. It is n o t k n o w n w h a t o t h e r u n c o n c e n t r a t e d test m a t e r i a l s m i g h t p r o d u c e similar artifacts. H o w e v e r , these findings are consistent with t h o s e r e p o r t e d r e c e n t l y b y F o r s t e r et al. (1982), also with u n c o n c e n t r a t e d d r i n k i n g w a t e r . F o r o t h e r test s u b s t a n c e s , the fluct u a t i o n assay r e m a i n s a simple, sensitive, r a p i d a n d valid test, especially w h e n acc o m p a n i e d b y a p p r o p r i a t e m o n i t o r i n g o f cell survival.
Acknowledgements W e t h a n k Drs. D . H . B l a k e y a n d A . P . H u g e n h o l t z f o r their c o m m e n t s a n d K a t h y N e s b i t t for her usual swift, a c c u r a t e typing.
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