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Method for evaluating mutagenicity of water II. Conditions for applying new sample preparation method to the Ames test
The Science of the Total Environment, 74 (1988) 191 198 Elsevier Science Publishers B.V., Amsterdam Printed in The Netherlands
191
M E T H O D FOR E V A L U A T I N G M U T A G E N I C I T Y OF W A T E R II. C O N D I T I O N S FOR A P P L Y I N G N E W S A M P L E P R E P A R A T I O N M E T H O D TO THE A M E S T E S T
KOHEI URANO, NOBUYUKI HAGA and FUDEKO EMOTO Yokohama National University, 156 Tokiwadai, Hodogaya-ku, Yokohama.City, 240 (Japan) (Received November 18th, 1987; accepted January 21st, 1988)
ABSTRACT Conditions for the application of a new sample preparation method developed in a previous study using the small particle size and highly porous polystyrene and anion-exchange resins CSP800 and CHPA25 were investigated. The desorbing solvents DMSO and 4 N NaNO 3 (0.5 ml), corresponding to i 1 of water sample, could be dosed on a plate for the mutagenicity test, the Ames test. It was confirmed that trace impurities from the resins were not mutagenic. The mutagenicity of several drinking water samples was determined by this new method and evaluated quantitatively by converting to the concentrations of the standard mutagens, 4-nitroquinoline-l-oxide and 2-aminoanthracene, without $9 and with $9 respectively. Furthermore, the mutagenicity of the nonionic pollutants adsorbed on CSP800 and that of the anionic pollutants adsorbed on CHPA25 could be evaluated separately. INTRODUCTION Since various organic compounds have been detected in surface water, g r o u n d w a t e r a n d t a p w a t e r , t h e s a f e t y o f w a t e r is n o w a t t r a c t i n g p u b l i c attention. A convenient method for testing the quantitative mutagenicity of w a t e r is n e c e s s a r y t o e v a l u a t e i t s s a f e t y ( L o p e r , 1980). U n t i l r e c e n t l y , t h e r e w a s no convenient and reliable sample preparation method for testing quantitative m u t a g e n i c i t y ( F o r s t e r a n d W i l s o n , 1981). H o w e v e r , a n e w s a m p l e p r e p a r a t i o n method which can recover various organic substances with high efficiency and a t a h i g h c o n c e n t r a t i o n f a c t o r h a s n o w b e e n d e v e l o p e d ( U r a n o e t al., 1988). I n the present study, the optimum application conditions for this preparative method for the Ames mutagenicity test were investigated and confirmed for several tap water samples. MATERIALS AND METHODS Sample preparation T a p w a t e r s a m p l e s f r o m five r e g i o n s o f J a p a n a n d a m o d e l t a p w a t e r , w h i c h was prepared, as described in the previous paper, from a warm water extract of
0048-9697/88/$03.50
© 1988 Elsevier Science Publishers B.V.
192 TABLE 1 Organic content of the samples Sample No.
Sampling region
THM (ttg 1-1)
TOX (#g 1 1)
TOC (mg 1-1)
A~0 (-/50mm)
1 2 3 4 5 6 7 Model tap water
Tokyo Tokyo Kanagawa Chiba Chiba Ibaragi Hyogo
16 16 11 41 53 21 23 110
81 80 70 135 190 140 185 560
1.2 1.6 0.8 2.2 2.0 1.8 2.0 4.5
0.07 0.04 0.08 0.13 0.08 0.10 0.18 0.25
leaf mold by addition of hypochlorite, were used as the water samples in this study. The organic content of these water samples is given in Table 1. New, highly porous, polystyrene and anion-exchange resins of small particle size, CSP800 and CHPA25, prepared by Mitsubishi Chemical Co., were used for sample preparation after refinement by ethanol and pure water. Five (or 10) millilitres of CSP800 and 2 (or 4) ml of CHPA25 were packed into 6-mm diameter glass columns connected in series. Ten (or 20) litres of water was passed Adsorption System
Desorpt ion System
Fig. 1. Systems for adsorption and desorption.
193 t h r o u g h t h e c o l u m n s at a flow r a t e of 550 (or 1100) ml h 1, up-flow, u s i n g the s y s t e m s h o w n in Fig. 1. T h e c o m p o u n d s a d s o r b e d on CSP800 were d e s o r b e d by d i m e t h y l s u l f o x i d e (DMSO) at a flow r a t e of 10 (or 20) ml h 1, down-flow, and the c o m p o u n d s a d s o r b e d on CHPA25 w e r e d e s o r b e d by 4 N sodium n i t r a t e at a flow r a t e of 10 (or 20) ml h 1, down-flow, u s i n g the s y s t e m s h o w n in Fig. 1. A f t e r a l l o w i n g for the void volumes, D M S O at the s a m e v o l u m e as CSP800 and 4 N sodium n i t r a t e at 2.5 times the v o l u m e of CHPA25 w e r e collected. F i v e (or 10) millilitres of e a c h of the s o l v e n t s c o n t a i n i n g the o r g a n i c s u b s t a n c e s in 10 (or 20) 1 of w a t e r s a m p l e w a s obtained. M i c r o b e s in t h e s e s o l v e n t s w e r e r e m o v e d by filtration t h r o u g h a m e m b r a n e and s u b j e c t e d to the A m e s tests. All chemicals, e t h a n o l , D M S O a n d sodium n i t r a t e , were r e a g e n t g r a d e ( W a k o C h e m i c a l Co.). Details of the o t h e r c o n d i t i o n s and p r o c e d u r e s are the s a m e as r e p o r t e d in o u r p r e v i o u s p a p e r ( U r a n o et al., 1988, this issue).
Mutagenicity test T h e A m e s test was e m p l o y e d in this s t u d y b e c a u s e it is the m o s t widely used m e t h o d for d e t e r m i n i n g m u t a g e n i c i t y . Salmonella s t r a i n s TA98 a n d TA100, the v a r i o u s c h a r a c t e r i s t i c s of w h i c h h a d b e e n c h e c k e d by the N a t i o n a l I n s t i t u t e of Public H e a l t h , J a p a n , were used. T h e s e Salmonella s t r a i n s a n d a n $9 f r a c t i o n o b t a i n e d f r o m the O r i e n t a l Y e a s t Co. were p r e s e r v e d at - 80°C in a freezer. 4 - N i t r o q u i n o l i n e - l - o x i d e ( 4 N Q O ) and 2 - a m i n o a n t h r a c e n e (2AA), the p o s i t i v e s t a n d a r d c o m p o u n d s , were p r e s e r v e d at 10°C in a freezer. $9 cofactor, o b t a i n e d from the O r i e n t a l Y e a s t Co., and o t h e r r e a g e n t s w e r e p r e s e r v e d at 5°C in a r e f r i g e r a t o r . N e g a t i v e r e f e r e n c e tests for o b t a i n i n g the n u m b e r of s p o n t a n e o u s r e v e r t a n t s w e r e c a r r i e d out by a d d i t i o n of D M S O a n d / o r 4 N sodium n i t r a t e solution. T h e positive r e f e r e n c e tests for o b t a i n i n g the a c t i v i t y of the Salmonella s t r a i n s w e r e c a r r i e d out by addition of 4 N Q O or 2 AA a n d the S-9 m i x t u r e . Since the o t h e r o p e r a t i n g p r o c e d u r e s were the s a m e as the g e n e r a l m e t h o d of the Ames test, details are o m i t t e d in this paper. RESULTS AND DISCUSSION
Influence of solvents I f l a r g e a m o u n t s of s o l v e n t s w e r e added to a p l a t e of the A m e s test, g r o w t h of the Salmonella s t r a i n s was inhibited. T h e p o s i t i v e r e f e r e n c e tests of 4 N Q O or 2 AA were c a r r i e d out w i t h a d d i t i o n of different a m o u n t s of D M S O or 4 N sodium n i t r a t e ; the r e s u l t s a r e s h o w n in T a b l e 2. W h e n l m l of D M S O or 4 N sodium n i t r a t e was added, the n u m b e r of r e v e r t a n t colonies was f e w e r t h a n w i t h the o t h e r solvents. B u t w h e n 1 ml of a m i x t u r e of D M S O and 4 N sodium n i t r a t e was added, the n u m b e r of r e v e r t a n t colonies was s i m i l a r to the n u m b e r o b t a i n e d w i t h p u r e water. C o n s e q u e n t l y , 0.5 ml of D M S O a n d / o r 0.5 ml of 4 N
194 TABLE 2 Influence of solvent on revertant colonies of positive references Added solvent
TA98
Pure water (1.0 ml) DMSO (0.5 ml) DMSO (1.0 ml) 4 N NaNO 3 (0.5 ml) 4N NaNO 3 (1.0ml) DMSO * 4 N NaNQ (0.5 ml) DMSO ÷ 4N NaNQ (1.0ml)
TA100
- $9
+ $9
280 320 170 280 190 290 290
210 190 200 210 220 190 220
$9
220 210 240 220 230 210 220
* $9
208 190 210 220 210 190 210
sodium nitrate could be added to an Ames test plate without inhibition. This amount of solvent corresponds to 1 1 of water sample, because each solvent was o b t a i n e d f r o m 1/2000 v o l u m e o f t h e w a t e r b y t h e n e w s a m p l e p r e p a r a t i o n m e t h o d ; i t w a s c o n f i r m e d t h a t t h e o r g a n i c c o m p o u n d s c o n t a i n e d i n 11 o f w a t e r c o l d be d o s e d o n t o a n A m e s t e s t p l a t e .
Influence of impurities eluted from resins The resins were refined with ethanol and pure water before use in sample preparation according to the method described in the previous paper. If the trace impurities from the resins were mutagenic, the test results were not r e l i a b l e . T h e s o l v e n t s , D M S O a n d 4 N s o d i u m n i t r a t e , w e r e fed r e s p e c t i v e l y t o the columns containing CSP800 and CHPA25. These solvents, containing the trace substances from the resins, were then tested for mutagenicity; the results a r e s h o w n i n T a b l e 3, w h e r e t h e y a r e c o m p a r e d w i t h t h e r e s u l t s o f t h e n e g a t i v e and positive reference tests. It was confirmed that the trace impurities from the refined resins were not mutagenic, because the number of revertant colonies per plate for the solvents after treatment with the resins was not different from t h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t c o l o n i e s for t h e n e g a t i v e r e f e r e n c e o f t h e pure solvents.
TABLE 3 Number of colonies produced by trace substances eluted from the resins Sample
Eluted solvent Negative reference Positive reference
TA98
TA100
- S9
+ $9
- $9
+ S9
15 18 288
21 20 192
69 63 220
77 70 224
195
Evaluation of mutagenicity In conventional studies, mutagenicity is usually discussed only qualitatively as positive or negative, because the efficiency with which the organic substances are recovered is very low (2(~40%). Furthermore, the data are presented in the form of the absolute number of r e v e r t a n t colonies per plate or the mutation ratio (MR), which is the ratio of the number of r e v e r t a n t colonies for the sample to the number of spontaneous r e v e r t a n t colonies for the negative reference. But the number of spontaneous r e v e r t a n t colonies and the reaction of the Salmonella strains to the mutagens change during preservation of the strains and differ from one researcher to another. Therefore, the data obtained by one researcher cannot be compared with data of other researchers. Now, however, since the newly developed sample preparation method has been available, with a recovery efficiency for various organic substances in water of 70r 80%, mutagenicity can be discussed quantitatively. The mean number of spontaneous r e v e r t a n t colonies for the solvents, the mean number of r e v e r t a n t colonies for positive reference dosing of 0.5 or 0.05 pg 4 NQO to TA98 strain and TA100 strain, respectively, without S-9 mixture, and 0.2 pg of 2 AA with S-9 mixture to both strains are shown in Table 4. When the number of spontaneous r e v e r t a n t colonies was > 40 for TA98, and > 80 for TA100, and the number of r e v e r t a n t colonies for the standard mutagens 4NQO and 2AA decreased to 70% of the above means, these Salmonella strains were exchanged for new sound strains. The relative activity, X (number of colonies pg 1), for the positive mutagens, 4 NQO and 2 AA, was obtained as follows: the difference between the number of r e v e r t a n t colonies for the standard mutagen, Np, and the number of spontaneous revertants for the pure solvents, Nn, was divided by the dosed amount, A (pg), of the standard mutagen. The mutagenicity of the water sample could then be converted to concentration, C (pg 1 ~), of standard mutagen, 4 NQO or 2 AA, as follows: the difference between the number of r e v e r t a n t colonies for the water sample, Ns, and the number of spontaneous revertants, Nn, was divided by the relative activity, X, and the dosed amount converted to water volume V (1). These procedures can be expressed by the following equation: TABLE 4 M e a n n u m b e r of r e v e r t a n t colonies for negative and positive references (seven replicates) TA98 $9 Negative Positive a
23 302
TA100 + $9 25 199
$9 58 223
+ $9 63 204
0.5 and 0.05 ttg 4 NQO were dosed to TA98 and TA100 in the tests w i t h o u t $9, and 0.2 pg 2 AA was dosed to TA98 and TA100 in the tests with $9.
196
+
© <
Z
o
0 +
>
0
(D
197 30O
O ~T O A + S 90I • ~TA98-$9
~D o
/
TAI0O-S9 A TA98+S9
2OC
I00
1
i
I
0,2
i
,
0.4
i
,
i
0.6
i
0.8
1.0
Dosed omount (woter-l/plote)
Fig. 2. Examples of dose-response relationships for tap water.
C
:
(Ns -
Nn)/XV
:
A(N~
-
Nn)/(Np
Nn) V
The value of V should be in the range from 0.3 to 1.01 so that the linear dose~response relationship is valid. Examples of test results for tap water are shown in Fig. 2. The number of r e v e r t a n t colonies was roughly proportional to the dosed amount; the linear dose~response relationships were obtained in the range from < 0.3 to ~ 1.01 for tap water samples. Consequently, the number of r e v e r t a n t colonies could be converted to the concentrations of standard mutagens by this method, and the difference between the activities of the S a l m o n e l l a strains cancelled, so t hat the data from different researchers can be compared quantitatively. The mutagenicity could also be converted to the c onc e nt r at i on of other standard mutagens. For example, the mutagenicity of the tap water samples whose organic contents are shown in Table 1 is shown in Table 5. The results for the two samples prepared according to the conventional sample preparation method using XAD4/8 are also shown in Table 5 for comparison. The data show t hat the mutagenicity obtained using the new preparative method is two or three times greater tha n t hat obtained using the conventional method. The reason for this difference is that the efficiency with which organic substances are recovered from a water sample by the new sample preparation method is much higher than by conventional methods. Furthermore, these results show t hat the anionic substances not adsorbed by the nonionic resin but by the newly adopted anion-exchange resin have mutagenicities as strong as the substances adsorbed by the nonionic resin. The mutagenicity among the tap water samples differed by as much as a factor of 11. Sample No. 5 showed high mutagenicity, and the anionic substances in this sample showed especially high mutagenicity. CONCLUSIONS The conditions for the application of the newly developed sample preparation method using superior resins and the method for the quantitative
198 e v a l u a t i o n of the m u t a g e n i c i t y of w a t e r were i n v e s t i g a t e d , and the following c o n c l u s i o n s were drawn: (i) T h e solvents, 0.5 ml d i m e t h y l s u l f o x i d e (DMSO) or 4 N s o d i u m n i t r a t e , or a 1 ml m i x t u r e , c a n be added d i r e c t l y to a n A m e s t e s t p l a t e w i t h o u t i n h i b i t i n g g r o w t h of the Salmonella strains. T h e s e a m o u n t s of s o l v e n t s c o r r e s p o n d to 1 1 of w a t e r sample. (ii) T r a c e i m p u r i t i e s f r o m the refined resins w e r e n o t m u t a g e n i c . (iii) T h e m u t a g e n i c i t y of w a t e r c a n be e v a l u a t e d a n d c o m p a r e d q u a n t i t a t i v e ly by c o n v e r t i n g to the c o n c e n t r a t i o n s of s t a n d a r d m u t a g e n s s u c h as 4 - n i t r o q u i n o l i n e - l - o x i d e (4 NQO) in cases w h e r e no $9 m i x t u r e is added, a n d 2 - a m i n o a n t h r a c e n e (2 AA) in cases w h e r e $9 m i x t u r e is added. (iv) It was found t h a t the m u t a g e n i c i t y of w a t e r d e t e r m i n e d by the new s a m p l e p r e p a r a t i o n m e t h o d was m u c h h i g h e r t h a n w h e n u s i n g the c o n v e n t i o n a l method, b e c a u s e the efficiency w i t h w h i c h o r g a n i c s u b s t a n c e s w e r e r e c o v e r e d by the new m e t h o d was m u c h h i g h e r t h a n by the c o n v e n t i o n a l method. (v) U s i n g the n e w method, the m u t a g e n i c i t y of w a t e r s a m p l e s c a n be e v a l u a t e d s e p a r a t e l y for n o n i o n i c and a n i o n i c s u b s t a n c e s . C o n s e q u e n t l y , by u s i n g the new s a m p l e p r e p a r a t i o n m e t h o d d e v e l o p e d in the p r e v i o u s study, the m u t a g e n i c i t y of w a t e r c a n be e v a l u a t e d c o n v e n i e n t l y and q u a n t i t a t i v e l y , a n d the s a f e t y of w a t e r c a n be discussed in m o r e precise terms. REFERENCES Forster, R. and I. Wilson, 1981. The application of mutagenicity test to drinking water. J. Inst. Water Eng. Sci., 35: 259-274. Loper, J.C., 1980. Mutagenic effects of organic compounds in drinking water. Mutat. Res., 76: 241 268. Urano, K., N. Haga, F. Emoto and T. Shinome, 1988. Method for evaluating mutagenicity of water. I. A new method of preparing samples for mutagenicity test. Sci. Total Environ., 74:177 189.
191
M E T H O D FOR E V A L U A T I N G M U T A G E N I C I T Y OF W A T E R II. C O N D I T I O N S FOR A P P L Y I N G N E W S A M P L E P R E P A R A T I O N M E T H O D TO THE A M E S T E S T
KOHEI URANO, NOBUYUKI HAGA and FUDEKO EMOTO Yokohama National University, 156 Tokiwadai, Hodogaya-ku, Yokohama.City, 240 (Japan) (Received November 18th, 1987; accepted January 21st, 1988)
ABSTRACT Conditions for the application of a new sample preparation method developed in a previous study using the small particle size and highly porous polystyrene and anion-exchange resins CSP800 and CHPA25 were investigated. The desorbing solvents DMSO and 4 N NaNO 3 (0.5 ml), corresponding to i 1 of water sample, could be dosed on a plate for the mutagenicity test, the Ames test. It was confirmed that trace impurities from the resins were not mutagenic. The mutagenicity of several drinking water samples was determined by this new method and evaluated quantitatively by converting to the concentrations of the standard mutagens, 4-nitroquinoline-l-oxide and 2-aminoanthracene, without $9 and with $9 respectively. Furthermore, the mutagenicity of the nonionic pollutants adsorbed on CSP800 and that of the anionic pollutants adsorbed on CHPA25 could be evaluated separately. INTRODUCTION Since various organic compounds have been detected in surface water, g r o u n d w a t e r a n d t a p w a t e r , t h e s a f e t y o f w a t e r is n o w a t t r a c t i n g p u b l i c attention. A convenient method for testing the quantitative mutagenicity of w a t e r is n e c e s s a r y t o e v a l u a t e i t s s a f e t y ( L o p e r , 1980). U n t i l r e c e n t l y , t h e r e w a s no convenient and reliable sample preparation method for testing quantitative m u t a g e n i c i t y ( F o r s t e r a n d W i l s o n , 1981). H o w e v e r , a n e w s a m p l e p r e p a r a t i o n method which can recover various organic substances with high efficiency and a t a h i g h c o n c e n t r a t i o n f a c t o r h a s n o w b e e n d e v e l o p e d ( U r a n o e t al., 1988). I n the present study, the optimum application conditions for this preparative method for the Ames mutagenicity test were investigated and confirmed for several tap water samples. MATERIALS AND METHODS Sample preparation T a p w a t e r s a m p l e s f r o m five r e g i o n s o f J a p a n a n d a m o d e l t a p w a t e r , w h i c h was prepared, as described in the previous paper, from a warm water extract of
0048-9697/88/$03.50
© 1988 Elsevier Science Publishers B.V.
192 TABLE 1 Organic content of the samples Sample No.
Sampling region
THM (ttg 1-1)
TOX (#g 1 1)
TOC (mg 1-1)
A~0 (-/50mm)
1 2 3 4 5 6 7 Model tap water
Tokyo Tokyo Kanagawa Chiba Chiba Ibaragi Hyogo
16 16 11 41 53 21 23 110
81 80 70 135 190 140 185 560
1.2 1.6 0.8 2.2 2.0 1.8 2.0 4.5
0.07 0.04 0.08 0.13 0.08 0.10 0.18 0.25
leaf mold by addition of hypochlorite, were used as the water samples in this study. The organic content of these water samples is given in Table 1. New, highly porous, polystyrene and anion-exchange resins of small particle size, CSP800 and CHPA25, prepared by Mitsubishi Chemical Co., were used for sample preparation after refinement by ethanol and pure water. Five (or 10) millilitres of CSP800 and 2 (or 4) ml of CHPA25 were packed into 6-mm diameter glass columns connected in series. Ten (or 20) litres of water was passed Adsorption System
Desorpt ion System
Fig. 1. Systems for adsorption and desorption.
193 t h r o u g h t h e c o l u m n s at a flow r a t e of 550 (or 1100) ml h 1, up-flow, u s i n g the s y s t e m s h o w n in Fig. 1. T h e c o m p o u n d s a d s o r b e d on CSP800 were d e s o r b e d by d i m e t h y l s u l f o x i d e (DMSO) at a flow r a t e of 10 (or 20) ml h 1, down-flow, and the c o m p o u n d s a d s o r b e d on CHPA25 w e r e d e s o r b e d by 4 N sodium n i t r a t e at a flow r a t e of 10 (or 20) ml h 1, down-flow, u s i n g the s y s t e m s h o w n in Fig. 1. A f t e r a l l o w i n g for the void volumes, D M S O at the s a m e v o l u m e as CSP800 and 4 N sodium n i t r a t e at 2.5 times the v o l u m e of CHPA25 w e r e collected. F i v e (or 10) millilitres of e a c h of the s o l v e n t s c o n t a i n i n g the o r g a n i c s u b s t a n c e s in 10 (or 20) 1 of w a t e r s a m p l e w a s obtained. M i c r o b e s in t h e s e s o l v e n t s w e r e r e m o v e d by filtration t h r o u g h a m e m b r a n e and s u b j e c t e d to the A m e s tests. All chemicals, e t h a n o l , D M S O a n d sodium n i t r a t e , were r e a g e n t g r a d e ( W a k o C h e m i c a l Co.). Details of the o t h e r c o n d i t i o n s and p r o c e d u r e s are the s a m e as r e p o r t e d in o u r p r e v i o u s p a p e r ( U r a n o et al., 1988, this issue).
Mutagenicity test T h e A m e s test was e m p l o y e d in this s t u d y b e c a u s e it is the m o s t widely used m e t h o d for d e t e r m i n i n g m u t a g e n i c i t y . Salmonella s t r a i n s TA98 a n d TA100, the v a r i o u s c h a r a c t e r i s t i c s of w h i c h h a d b e e n c h e c k e d by the N a t i o n a l I n s t i t u t e of Public H e a l t h , J a p a n , were used. T h e s e Salmonella s t r a i n s a n d a n $9 f r a c t i o n o b t a i n e d f r o m the O r i e n t a l Y e a s t Co. were p r e s e r v e d at - 80°C in a freezer. 4 - N i t r o q u i n o l i n e - l - o x i d e ( 4 N Q O ) and 2 - a m i n o a n t h r a c e n e (2AA), the p o s i t i v e s t a n d a r d c o m p o u n d s , were p r e s e r v e d at 10°C in a freezer. $9 cofactor, o b t a i n e d from the O r i e n t a l Y e a s t Co., and o t h e r r e a g e n t s w e r e p r e s e r v e d at 5°C in a r e f r i g e r a t o r . N e g a t i v e r e f e r e n c e tests for o b t a i n i n g the n u m b e r of s p o n t a n e o u s r e v e r t a n t s w e r e c a r r i e d out by a d d i t i o n of D M S O a n d / o r 4 N sodium n i t r a t e solution. T h e positive r e f e r e n c e tests for o b t a i n i n g the a c t i v i t y of the Salmonella s t r a i n s w e r e c a r r i e d out by addition of 4 N Q O or 2 AA a n d the S-9 m i x t u r e . Since the o t h e r o p e r a t i n g p r o c e d u r e s were the s a m e as the g e n e r a l m e t h o d of the Ames test, details are o m i t t e d in this paper. RESULTS AND DISCUSSION
Influence of solvents I f l a r g e a m o u n t s of s o l v e n t s w e r e added to a p l a t e of the A m e s test, g r o w t h of the Salmonella s t r a i n s was inhibited. T h e p o s i t i v e r e f e r e n c e tests of 4 N Q O or 2 AA were c a r r i e d out w i t h a d d i t i o n of different a m o u n t s of D M S O or 4 N sodium n i t r a t e ; the r e s u l t s a r e s h o w n in T a b l e 2. W h e n l m l of D M S O or 4 N sodium n i t r a t e was added, the n u m b e r of r e v e r t a n t colonies was f e w e r t h a n w i t h the o t h e r solvents. B u t w h e n 1 ml of a m i x t u r e of D M S O and 4 N sodium n i t r a t e was added, the n u m b e r of r e v e r t a n t colonies was s i m i l a r to the n u m b e r o b t a i n e d w i t h p u r e water. C o n s e q u e n t l y , 0.5 ml of D M S O a n d / o r 0.5 ml of 4 N
194 TABLE 2 Influence of solvent on revertant colonies of positive references Added solvent
TA98
Pure water (1.0 ml) DMSO (0.5 ml) DMSO (1.0 ml) 4 N NaNO 3 (0.5 ml) 4N NaNO 3 (1.0ml) DMSO * 4 N NaNQ (0.5 ml) DMSO ÷ 4N NaNQ (1.0ml)
TA100
- $9
+ $9
280 320 170 280 190 290 290
210 190 200 210 220 190 220
$9
220 210 240 220 230 210 220
* $9
208 190 210 220 210 190 210
sodium nitrate could be added to an Ames test plate without inhibition. This amount of solvent corresponds to 1 1 of water sample, because each solvent was o b t a i n e d f r o m 1/2000 v o l u m e o f t h e w a t e r b y t h e n e w s a m p l e p r e p a r a t i o n m e t h o d ; i t w a s c o n f i r m e d t h a t t h e o r g a n i c c o m p o u n d s c o n t a i n e d i n 11 o f w a t e r c o l d be d o s e d o n t o a n A m e s t e s t p l a t e .
Influence of impurities eluted from resins The resins were refined with ethanol and pure water before use in sample preparation according to the method described in the previous paper. If the trace impurities from the resins were mutagenic, the test results were not r e l i a b l e . T h e s o l v e n t s , D M S O a n d 4 N s o d i u m n i t r a t e , w e r e fed r e s p e c t i v e l y t o the columns containing CSP800 and CHPA25. These solvents, containing the trace substances from the resins, were then tested for mutagenicity; the results a r e s h o w n i n T a b l e 3, w h e r e t h e y a r e c o m p a r e d w i t h t h e r e s u l t s o f t h e n e g a t i v e and positive reference tests. It was confirmed that the trace impurities from the refined resins were not mutagenic, because the number of revertant colonies per plate for the solvents after treatment with the resins was not different from t h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t c o l o n i e s for t h e n e g a t i v e r e f e r e n c e o f t h e pure solvents.
TABLE 3 Number of colonies produced by trace substances eluted from the resins Sample
Eluted solvent Negative reference Positive reference
TA98
TA100
- S9
+ $9
- $9
+ S9
15 18 288
21 20 192
69 63 220
77 70 224
195
Evaluation of mutagenicity In conventional studies, mutagenicity is usually discussed only qualitatively as positive or negative, because the efficiency with which the organic substances are recovered is very low (2(~40%). Furthermore, the data are presented in the form of the absolute number of r e v e r t a n t colonies per plate or the mutation ratio (MR), which is the ratio of the number of r e v e r t a n t colonies for the sample to the number of spontaneous r e v e r t a n t colonies for the negative reference. But the number of spontaneous r e v e r t a n t colonies and the reaction of the Salmonella strains to the mutagens change during preservation of the strains and differ from one researcher to another. Therefore, the data obtained by one researcher cannot be compared with data of other researchers. Now, however, since the newly developed sample preparation method has been available, with a recovery efficiency for various organic substances in water of 70r 80%, mutagenicity can be discussed quantitatively. The mean number of spontaneous r e v e r t a n t colonies for the solvents, the mean number of r e v e r t a n t colonies for positive reference dosing of 0.5 or 0.05 pg 4 NQO to TA98 strain and TA100 strain, respectively, without S-9 mixture, and 0.2 pg of 2 AA with S-9 mixture to both strains are shown in Table 4. When the number of spontaneous r e v e r t a n t colonies was > 40 for TA98, and > 80 for TA100, and the number of r e v e r t a n t colonies for the standard mutagens 4NQO and 2AA decreased to 70% of the above means, these Salmonella strains were exchanged for new sound strains. The relative activity, X (number of colonies pg 1), for the positive mutagens, 4 NQO and 2 AA, was obtained as follows: the difference between the number of r e v e r t a n t colonies for the standard mutagen, Np, and the number of spontaneous revertants for the pure solvents, Nn, was divided by the dosed amount, A (pg), of the standard mutagen. The mutagenicity of the water sample could then be converted to concentration, C (pg 1 ~), of standard mutagen, 4 NQO or 2 AA, as follows: the difference between the number of r e v e r t a n t colonies for the water sample, Ns, and the number of spontaneous revertants, Nn, was divided by the relative activity, X, and the dosed amount converted to water volume V (1). These procedures can be expressed by the following equation: TABLE 4 M e a n n u m b e r of r e v e r t a n t colonies for negative and positive references (seven replicates) TA98 $9 Negative Positive a
23 302
TA100 + $9 25 199
$9 58 223
+ $9 63 204
0.5 and 0.05 ttg 4 NQO were dosed to TA98 and TA100 in the tests w i t h o u t $9, and 0.2 pg 2 AA was dosed to TA98 and TA100 in the tests with $9.
196
+
© <
Z
o
0 +
>
0
(D
197 30O
O ~T O A + S 90I • ~TA98-$9
~D o
/
TAI0O-S9 A TA98+S9
2OC
I00
1
i
I
0,2
i
,
0.4
i
,
i
0.6
i
0.8
1.0
Dosed omount (woter-l/plote)
Fig. 2. Examples of dose-response relationships for tap water.
C
:
(Ns -
Nn)/XV
:
A(N~
-
Nn)/(Np
Nn) V
The value of V should be in the range from 0.3 to 1.01 so that the linear dose~response relationship is valid. Examples of test results for tap water are shown in Fig. 2. The number of r e v e r t a n t colonies was roughly proportional to the dosed amount; the linear dose~response relationships were obtained in the range from < 0.3 to ~ 1.01 for tap water samples. Consequently, the number of r e v e r t a n t colonies could be converted to the concentrations of standard mutagens by this method, and the difference between the activities of the S a l m o n e l l a strains cancelled, so t hat the data from different researchers can be compared quantitatively. The mutagenicity could also be converted to the c onc e nt r at i on of other standard mutagens. For example, the mutagenicity of the tap water samples whose organic contents are shown in Table 1 is shown in Table 5. The results for the two samples prepared according to the conventional sample preparation method using XAD4/8 are also shown in Table 5 for comparison. The data show t hat the mutagenicity obtained using the new preparative method is two or three times greater tha n t hat obtained using the conventional method. The reason for this difference is that the efficiency with which organic substances are recovered from a water sample by the new sample preparation method is much higher than by conventional methods. Furthermore, these results show t hat the anionic substances not adsorbed by the nonionic resin but by the newly adopted anion-exchange resin have mutagenicities as strong as the substances adsorbed by the nonionic resin. The mutagenicity among the tap water samples differed by as much as a factor of 11. Sample No. 5 showed high mutagenicity, and the anionic substances in this sample showed especially high mutagenicity. CONCLUSIONS The conditions for the application of the newly developed sample preparation method using superior resins and the method for the quantitative
198 e v a l u a t i o n of the m u t a g e n i c i t y of w a t e r were i n v e s t i g a t e d , and the following c o n c l u s i o n s were drawn: (i) T h e solvents, 0.5 ml d i m e t h y l s u l f o x i d e (DMSO) or 4 N s o d i u m n i t r a t e , or a 1 ml m i x t u r e , c a n be added d i r e c t l y to a n A m e s t e s t p l a t e w i t h o u t i n h i b i t i n g g r o w t h of the Salmonella strains. T h e s e a m o u n t s of s o l v e n t s c o r r e s p o n d to 1 1 of w a t e r sample. (ii) T r a c e i m p u r i t i e s f r o m the refined resins w e r e n o t m u t a g e n i c . (iii) T h e m u t a g e n i c i t y of w a t e r c a n be e v a l u a t e d a n d c o m p a r e d q u a n t i t a t i v e ly by c o n v e r t i n g to the c o n c e n t r a t i o n s of s t a n d a r d m u t a g e n s s u c h as 4 - n i t r o q u i n o l i n e - l - o x i d e (4 NQO) in cases w h e r e no $9 m i x t u r e is added, a n d 2 - a m i n o a n t h r a c e n e (2 AA) in cases w h e r e $9 m i x t u r e is added. (iv) It was found t h a t the m u t a g e n i c i t y of w a t e r d e t e r m i n e d by the new s a m p l e p r e p a r a t i o n m e t h o d was m u c h h i g h e r t h a n w h e n u s i n g the c o n v e n t i o n a l method, b e c a u s e the efficiency w i t h w h i c h o r g a n i c s u b s t a n c e s w e r e r e c o v e r e d by the new m e t h o d was m u c h h i g h e r t h a n by the c o n v e n t i o n a l method. (v) U s i n g the n e w method, the m u t a g e n i c i t y of w a t e r s a m p l e s c a n be e v a l u a t e d s e p a r a t e l y for n o n i o n i c and a n i o n i c s u b s t a n c e s . C o n s e q u e n t l y , by u s i n g the new s a m p l e p r e p a r a t i o n m e t h o d d e v e l o p e d in the p r e v i o u s study, the m u t a g e n i c i t y of w a t e r c a n be e v a l u a t e d c o n v e n i e n t l y and q u a n t i t a t i v e l y , a n d the s a f e t y of w a t e r c a n be discussed in m o r e precise terms. REFERENCES Forster, R. and I. Wilson, 1981. The application of mutagenicity test to drinking water. J. Inst. Water Eng. Sci., 35: 259-274. Loper, J.C., 1980. Mutagenic effects of organic compounds in drinking water. Mutat. Res., 76: 241 268. Urano, K., N. Haga, F. Emoto and T. Shinome, 1988. Method for evaluating mutagenicity of water. I. A new method of preparing samples for mutagenicity test. Sci. Total Environ., 74:177 189.