Mutagenic activity of swimming-pool water

137

Mutation Research, 78 (1980) 137--144 © Elsevier/North-Holland Biomedical Press

MUTAGENIC ACTIVITY OF SWIMMING-POOL WATER

W.G. HONER *, M.J. ASHWOOD-SMITH** and C. WARBY Environmental Toxicology Research Group, Department of Biology, University of Victoria, Victoria, B.C. (Canada, V8W 2Y2)

(Received 11 September 1979) (Revision received 8 January 1980) (Accepted 28 January 1980)

Summary Swimming-pool water, being chlorinated and exposed to trace organics from use was investigated as a possible source of mutagens using the Salmonella/ mammalian-microsome test. Procedures previously described for the extraction of trace organics from water using XAD-2 macroreticular resin were modified to allow quantitative extraction of mutagens. These procedures were superior to freeze
138

1975). Swimming-pool water, chlorinated to levels as high as 3 ppm, and constantly receiving organics in the form of small amounts of sweat, epithelial cells, urine, menstrual blood, hair and cosmetics would be expected to be a source of chlorinated organics and possible mutagens. As well as reliably detecting over 90% of all organic mutagens tested, the Salmonella/mammalian-microsome test is useful in the detection of mutagenic activity in complex mixtures (Ames et al., 1975; McCann and Ames, 1977; Purchase et al., 1976). This capacity has been demonstrated using cigarettesmoke condensates and urine concentrates from smokers (Kier et al., 1974; Yamasaki and Ames, 1977). We therefore considered it probable that this assay would be capable of detecting organic mutagens after concentration from swimming-pool water. Samples of pool water, like urine, cannot be directly incorporated into the Salmonella mutagenicity assay in amounts larger than 0.1 ml/plate. Various concentration methods can be employed including freezedrying, solvent~extraction using dichloromethane (DCM) and macroreticular chromatography using XAD-2 resin. This particular type of chromatography has been particularly succesful in the extraction of trace organics from water. Techniques described for this prupose (Junk et al., 1974) were modified to allow simple, economical extraction of mutagens from pool water. Using these indirect techniques and the Salmonella/mammalian-microsome test we present in this paper evidence for the existence of several mutagens in swimming-pool water. Materials and methods Preliminary concentration m e t h o d s Swimming-pool water was collected from 3 public indoor pools in the Victoria area (Victoria, B.C., Canada). In early experiments samples were prepared by freeze
139

to sample addition t h e resin was washed briefly with methanol and then with 3 lots (250 ml) of distilled water. A 250-rnl s~mple of pool water (pH 6.5) at room temperature was added to the resin after decanting the final wash. Sample pH could be altered with 0.5% concentrated HC1 or 0.5% concentrated NH4OH. The sample--resin mix was then stirred for 5 min. A distilled water sample was generally extracted first to ensure no previously introduced mutagens remained adsorbed to the resin. After settling the liquid was poured off. A solvent, 250 ml of ether or acetone was added and stirred for 5 rain, then collected for evaporation. The resin was immediately regenerated by the addition of methanol. Ether was evaporated in a water bath at 50°C, the residue then dissolved in DMSO. Acetone was evaporated using a rotary evaporator, also at 50°C. A small amount of water remains in the flask prior to solvent addition. This is immiscible with ether but remains after acetone evaporation. The volume was freeze
Mutagenicity assay The Salmonella/mammalian-microsome mutagenicity assay was performed (Ames et al., 1975). In preliminary experiments Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98 were used, in experiments using XAD-2 concentrates TA100 was used exclusively. Rat-liver microsomal activity was induced with Aroclor 1254, the $9 fraction and $9 mix were prepared as described (Ames et al., 1975). The microsomal mix was used at a concentration of 50 #l/plate (the protein content of $9 = 25 mg/ml); plates were incubated for 48 h. The amount of sample residue dissolved in DMSO was altered to result in different equivalents of pool water per plate, 0.1 ml of concentrate was applied in each case. For freeze
Preliminary experiments Freeze
140

.350

.."

• 300--1-

-'"f"

lso~ lOG "t ~ L' J "

0

"

i

I

I

5 10 15 Equivalents of Pool Water (m|s)

I

20

Fig. 1. M u t a g e n i c i t y o f a c i d - - e t h e r e x t r a c t o f c h l o r i n a t e d s w i m m i n g - p o o l w a t e r . S. t3,phimurfum ( T A 1 0 0 ) in t h e p r e s e n c e o f r a t - l i v e r m i c r o s o m e s , y = 1 1 9 + 1 1 . 5 X ; . . . . . . ,95% confidence limits of regression; b a z is -+ 1 S D o f m e a n .

450

350 .~_300-

~

° -°°" o°.-°°"

,,, 1 5 0 ~ " 100T

i

0

I

I

I

5 10 15 Equivalents of Pool Water (ml$)

I

20

Fig. 2. M u t a g e n i c i t y o f a c e t o n e e x t r a c t o f c h l o r i n a t e d s w i m m i n g - p o o l w a t e r . S. t y p h t m u r i u m ( T A I O 0 ) i n t h e absence o f m i c r o s o m e s , y = 1 3 8 + 1 0 . 5 X ; . . . . . . . 9 5 % c o n f i d e n c e l i m i t s o f r e g r e s s i o n ; b a r is +1 S D of mean.

141

the dose--response relationship was non-linear. However, using freeze-dried concentrates significant activity was observed in all 3 pools tested. DCM concentrates were mutagenic in the same 2 strains at 15 ml equivalent per plate without microsomes. Again toxicity occurred above this level. Residues of evaporated DCM alone were not mutagenic, indicating this known mutagen was not contaminating the sample (Jongen et al., 1978); clearly DCM had been completely removed during the rotary evaporation. Chromatographic analysis indicated a mutagen with R~ of 0.7, significance was achieved in these assays with equivalents of 500--1000 ml/plate. Again, solvent backgrounds were at control levels. The loss of potency could be due to the large number of steps involved in this procedure and the heating necessary for the evaporation of the solvent. X A D - 2 concentrates

Prior to use for pool-water extraction the adsorption and elution capabilities of the XAD-2 resin and various solVents were confirmed using a solution of indole and a spectrophotometric assay. Control responses in the mutagenicity assay were low as expected, with infrequent exception. In the case of 2-aminoanthracene the high response indicates optimal microsomal enzyme activity (Results: Table 1). Initial studies showed little mutagenic activity in unmodified pool water eluted with ether so this possibility was not investigated further. Concentrates of acidified pool water eluted with ether exhibited increased mutagenic activity in the presence of metabolic activation. The response was judged as significant on the basis of being double-control values at 5 ml equivalent per plate and having a linear dose--response relationship well fitted by the regression line (Fig. 1). These criteria are those suggested by Ames et al. (1975). Using an acetone elution neutral pool water was significantly mutagenic in the absence of metabolic activation also at a level of 5 ml equivalent per plate (Fig. 2). In the presence of microsomes the mutagenic response was reduced, possibly due to metabolic detoxification. Acidified pool water eluted with acetone followed a similar pattern (Fig. 3). Significance was apparent in the doubling above control at an equivalent con-

TABLE 1 CONTROL

D A T A W I T H Salmonella typhimurlum ( S T R A I N T A 1 0 0 )

Sample

$9

DMSO

-+

His+ revertants tplate 103 ± 22 98 ± 18

2-Aminoanthraeene

+

Distilled water

-+

107 ± 15 108 ± 22

Tap water (obtained from swimming pool)

-+

98 ± 21 122 ± 22

M e a n s and standard deviations for his + revertantslplate.

~>1200

142 450 j~

400~ 350 --

.~ 3 0 0 e.

o

,52so

200 ~150100

so

I 0

5 Equivalents

of

I

I

10 15 Pool Water (m1$)

20

Fig. 3. M u t a g e n i c i t y o f a c i d - - a c e t o n e e x t r a c t o f c h l o z i n a t e d ( T A 1 0 0 ) i n t h e absence o f m i c r o s o m e s , y = 1 2 1 + 1 0 . 8 X ; . . . . . . b a r i s +1 S D o f m e a n .

s w i m m i n g - p o o l w a t e r . S. t y p h i m u r i u m , 95% confidence Hmits of regression ;

centration of 10 ml/plate and the dose--response relationship was approximately linear. Composite results are shown in Table 2.

Discussion XAD-2 macroreticular resin has been used previously to extract mutagens from another complex mixture, smoker's urine (Yamasaki and Ames, 1977). Concentrates of pool water prepared with similar column-chromatographic

TABLE 2 XAD-2 MACRORETICULAR

pH modification -eluting solvent

$9

RESIN CONCENTRATES

Concentration

OF POOL WATER

of pool water (ml/plate)

1

5

I0

20

Acidified--ether

-+

1 0 7 ± 21 128 ± 49

162 b ± 45 202 b ± 54

161 b ± 28 250 b ± 40

226 b ± 34 335 b ± 37

Neutral--acetone

-+

147 ± 7 126 ± 47

224 b ± 26 153 ± 44

233 b ± 40 163 ± 66

344 b ± 47 215 b ± 46

Acidified--acetone

-+

130 ± 22 119 ± 37

165 a ± 40 143 ± 56

291 b ± 4 5 172 b ± 30

361 b ± 30 229 b ± 34

M e a n s a n d s t a n d a r d d e v i a t i o n s f o r his* r e v e r t a n t s / p l a t e u s i n g s t r a i n T A I O 0 . a Significantly different from appropriate control mean p ~ 0.05. b Significantly different from appropriate control mean p ~ 0.01.

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techniques did not exhibit significant mutagenic activity, although some response was evident. The higher resin-to-sample ratio in our procedures m a y be necessary for the quantitative extraction of these mutagens. This macroreticular resin depends on hydrophobic interactions with organic molecules to separate them from mixtures containing inorganic salts.Acidification of the samples results in the neutralization of any ionised acidic organics, ionisation precludes adsorbtion. The positive results with the unmodified and acidified pool-water concentrates eluted with acetone could be interpreted to indicate the presence of 2 mutagens, one relatively neutral and adsorbable at p H 7.4, the other negatively charged at neutral p H but adsorbable at p H 1. However, the presence of one mutagen capable of being adsorbed at neutral p H and unaltered by the addition of acid would result in a similar response; this seems less likely however. Mutagenic activity in the ether-eluted concentrate is greatly enhanced with metabolic activation and therefore appears to be qualitatively different from that in the acetone~luted concentrates. Failure to detect this activity in the acidified-water acetone-eluted concentrate could indicate inadequacy of acetone as an eluting agent, disruption of microsomal activity by an agent present in the acetone eluate or competition for enzymic activity by an agent in the acetone eluate, diminishing the effectiveness of the necessary activation. The possibility that different patterns of mutagenic activity would result through the use of the microsomes obtained after phenobarbital induction is currently being considered. The evidence thus indicates the presence of at least 3 mutagens in swimmingpool water concentrates. One is acidific, ionised at neutral p H and present in the acidified water-acetone concentrate. A second is acidic, ionised at neutral pH, enhanced by metabolic activation and is present in the acidified waterether concentrate. A third is neutral and present in the unmodified water-acetone concentrate. The reversion of strain T A 1 0 0 indicates all are capable of acting either directly to cause base-pair substitution or indirectly to induce damage due to error-prone recombination repair (Ames et al., 1975; McCann et al., 1975). Without actual isolation and identification of the mutagenic agents present in these concentrates the possible presence of the inorganic mutagens cannot be definitely excluded. Pool water is chlorinated with the direct introduction of chlorine gas or by adding HTH granular-powdered calcium hypochlorite, Ca(OC1)2, which can be of purity as low as 50% in commercial preparations. Impurities include Ca(C104)2, CaC12, CaCO3, Ca(OH)2 and water (10). Sodium hypochlorite and chloramine, a side-product of chlorination, have been shown to be mutagenic (Lu Shih and Ledbrberg, 1976; Shih and Lederberg, 1976; Wlodkowski and Rosencranz, 1975). Direct chlorination of DNA is also possible. However, it is unlikely any one or all of these agents are the sole source of mutagenesis in our concentrates. Inorganic molecules, especially polar hydror philic types are poorly absorbed by the XAD-2 resin. Chloramine with a freezing point of --66°C would nol~ remain ina freeze-dried extract (acetone-elution procedures involve freeze
144

matographic analysis of the concentrates is now in progress to confirm these expectations and indicate the range of possible mutagens present. We have detected mutagenic activity in 3 municipal swimming pools. It must be stressed these pools are well maintained and regulated within clearly defined standards. No attempt on our part has been made at hazard evaluation. However, it seems reasonable that less well controlled residential or motel pools could be sources of higher mutagen concentrations and increased risk. As the mutagenic activity is extractable, alterations in filtration procedures would likely reduce the quantity of mutagens present. Daily analysis of water from a pool over a one-week period or longer, n o w in progress, will indicate if the levels of mutagenic activity are dependent on any particular pattern of use. If so this would explain some of the variation in our results. The clastogenic properties of these concentrates in relation to the induction of sister-chromatid exchanges in Chinese hamster ovary cells is underway. References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for det e c t i ng carcinogens and mut a ge ns w i t h the SalmoneHa/mammalian-microsome m u t a g e n i c i t y test, Mutati on Res., 3 1 , 3 4 7 - - 3 6 4 . Jongen, W.M.F., G.M. Alink and J.H. Koeman (1978) Mutagenic effect of d i c h l o r o m e t h a n e on Salmonella t y p h i m u r i u m , Mutation Res., 56, 245--248. Junk, G.A., J.J. Richard, M.D. Grieser, D. Witiak, J.L. Witiak, M.D. Arguello, R. Vick, H.J. Svec, J.S. Fritz and G.V. Calder (1974) Use of macroreticular resins in the analysis of w a t e r for trace organic contaminants, J. Chromatogr., 99, 745--762. Kier, L.D., E. Yamasak i and B.N. Ames (1974) Detection of muta ge ni c activity in cigarette s moke condensates, Proc. Natl. Acad. Sci. (U.S.A.), 71, 4 1 5 9 - - 4 1 6 3 . Loper, J.C., D.R. Lang, R.S. Schoeny, B.B. R i c h m o n d , P.M. Gallagher and C.C. S mi t h (1978) Residue organic m i x t u r e s from drinking water show in vitro mutageni c and transforming activity, J. ToxicoL Environ. Health, 4 , 9 1 9 - - 9 3 8 . Lu Shin, K., and J. Lederberg (1976) Effects of chloramine on Bacillus subtilis d e o x y r i b o n u c l e i c acid, J. Bacteriol., 125, 934--945. Marx, J.L. (1974) Drinking water: a n o t h e r source of carcinogens? Science, 186, 809--810. McCann, J., and B.N. Ames (1977) The S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test: Predictive value for animal careinogenicity, in: H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of H u m a n Cancer, Cold Spring Laboratory, Cold Spring Harbor, NY, pp. 1431--1450. McCann, J., N.E. Springarn, J. Kobori and B.N. Ames (1975) De t e c t i on of carcinogens as mutagens: Bacterial tester strains with R-factor plasmids, Proc. Natl. Acad. Sci. (U.S.A.), 72, 979--983. Merck Index (1976) Merck, Rahway, NJ, U.S.A. Oliver, B.G. (1 978) Chlorinated non-volatile organics p r o d u c e d by the reaction of chlorine w i t h humi c materials, Can. Res. Develop., 11 (6), 21--22. Page, T., R.H. Harris and S.S. Epstein (1976) Drinking water and cancer m o r t a l i t y in Louisiana, Science, 193, 55--57. Purchase, I.F.H., E. Longstaff, J. Ashby, J.A. Styles, D. Anderson, P.A. Lefewe and F.R. Westwood (1976) E v aluation of six short t e r m tests for detecting organic chemical carcinogens a nd r e c o m m e n d a tions for their use, Nature (London), 264, 624--627. Shih, K.L., and J. Lederberg (1976) Chloramine mutagenesis in Bacillus subtilis, Science, 192, 1141-1143. Wlodkowski, T.J., and H.S. Rosencranz (1975) Mutagenicity of s odi um h y p o c h l o r i t e for Salmonella t y p h i m u r i u m , M u t a t i o n Res., 31, 39--42. Yamasaki, E., and B.N. Ames (1977) C o n c e n t r a t i o n of mutagens from urine by a d s o r p t i o n with the nonpolar resin XAD-2: Cigarette smokers have mutagenic urine, Proc. Natl. Acad. Sci. (U.S.A.), 74, 3555--3559.