The Science of the Total Environment, 116 (1992) 1-13 Elsevier Science Publishers B.V., Amsterdam

1

Generation of mutagenicity by ozonation of humic substances' components Mitsunobu

Hibino a, Hiroaki Matsuda b, Takahiko Sato a, Youki Ose c, Hisamitsu Nagase a and Hideaki Kito a

aDepartment of Public Health, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu City 502, Japan bMie University Hospital, 2-174 Edobashi, Tsu City 514, Japan CGifu City Women's College, 2693 Nagara-Fukumitsu, Gifu City 502, Japan (Received December 6th, 1990; accepted March 4th, 1991)

ABSTRACT Components of humic substances, such as vanillin, syringaldehyde, vanillic acid and di-nbutylphtalate, were ozonated and subjected to the mutagenicity assay using Salmonella typhimurium TA 98 and 100 with and without $9 mix. The strong mutagenic activity was found on all components except di-n-butylphtalate by strain TA 100 with and without $9 mix. Substances with strong mutagenic activity in ozonated vanillin were water-soluble and were slightly extracted with benzene, dichloromethane and ethyl acetate. Following gel chromatography on Sephadex G-10, the strong mutagens generated by ozonation were found with molecular weights > 300.

Key words: Ames assay; mutagenicity; ozonation; vanillin; vanillic acid; syringaldehyde

INTRODUCTION

When drinking water is chlorinated, the organic halides such as trihalomethanes are produced and is of social concern (Rook, 1974, 1976, 1977; US Environmental Protection Agency, 1974). Ozonation is considered the alternative disinfectant to reduce these organic halides. Ozone is now used in > 1000 drinking water plants in Europe (Glaze, 1987). The mutagenicity of ozonated humic substances has been described by various investigators (Gruenner, 1978; Kowbel et al., 1982, 1984; Kamei et al., 1985); however the appearance of mutagenicity was different. The difference is considered to be related to the amount of ozone and the origin of humic substances. It may be important to investigate what components are the precursors of mutagens that are produced by ozonation. Components of humic substances were identified by many researchers (Greene et al., 1962; 0048-9697/92/$05.00

© 1992 Elsevier Science Publishers B.V. All rights reserved

2

M, HIBINO ET AL.

Schnitzer et al., 1972; Martin et al., 1974; Sato et al., 1985; Stephen, 1986), but the mutagenicities of their ozonated components have rarely been investigated. We have selected vanillin, syringaldehyde, vanillic acid and di-nbutylphtalate as components of humic substances and investigated the mutagenicity of their ozonated products. The difference of various author's results may be related to the difference of ozonation methods. Stephen (1986) reported dimethyl acetal production by ozonation of unsaturated chain compounds in anhydrous methanol, and aldehydes and acids production in water. We have investigated the effect of methanol, as it was also used as the solubilizer and the effect of evaporation of separates to dryness. We then investigated the kinds of mutagens which are produced from the components of humic substances; we fractionated mutagenic substances in ozonated products of vanillin with organic solvents extraction (benzene, dichloromethane and ethyl acetate) and investigated the mutagenicity of each fraction. The fractionated ozonated products by gel chromatography on Sephadex G-10, were also investigated. The mutagenicity of each fraction was determined and confirmed the molecular weight of ozonated products from vanillin. MATERIALS AND METHODS

Conditions of ozonation Ozone was produced from dry ultrapure oxygen (99.5%), using an ozone generator Model 0-3-2 (Japan Ozone Co.) at the rate of 36.38 mg O3/min.

Effects of the solvent as the solubilizer The components selected are only slightly soluble in water, hence a need to consider various solvents as solubilizers. Vanillin was ozonated at the weight ratio of vanillin: 03 = 1:2, in 10% methanol (1000 ppm), water (500 ppm) and 10% acetnitrile (1000 ppm), respectively. After ozonation, solutions were aerated for 10 min to eliminate residual ozone, evaporated to dryness at 40°C with a rotary evaporator, dissolved in dimethylsulfoxide (DMSO) and centrifuged (1000 x g, 30 min). Supernatants were applied to Ames assay.

Effect of the evaporation to dryness The influence of concentration methods of ozonated solutions on the mutagenicity was examined. Vanillin (1000 ppm) in 10% methanol was ozonated at the weight ratio of vanillin: 03 -- 1:2, and the mutagenicity was com-

MUTAGENICITY AND OZONATION OF HUMIC SUBSTANCES

3

pared for an unconcentrated aliquot, a x 10 concentrate and a concentrate to dryness. The unconcentrated aliquot and a × 10 concentrate were centrifuged (1000 × g, 30 min), and the supernatants applied to Ames assay. A dried sample was dissolved in DMSO and centrifuged and the supernatant was applied to Ames assay.

Ozonation of components of humic substances Each component of humic substances (vanillin, syringaldehyde, vanillic acid and di-n-butylphtalate) in 10% methanol was ozonated at the weight ratio of 03 to component -- 0, 0.5, 1, 2 and 10. After ozonation, solutions were aerated for 10 min, evaporated to dryness, dissolved in DMSO and centrifuged. The supernatants were applied to Ames test.

Fractionation by organic solvents Ozonated vanillin (900 ml) in 10% methanol was extracted with benzene (90 ml, 5 times), dichloromethane (90 ml, 5 times) and ethyl acetate (180 ml, 10 times), successively. Each extract and water layer was evaporated to dryness, dissolved in DMSO and centrifuged. The supernatants were applied to Ames test.

Fractionation by gel chromatography Ozonated vanillin in 10% methanol (1000 ppm) and ozonated vanillin suspension in water (500 ppm) (vanillin:O3--1:2) were evaporated to dryness. Residues were dissolved in 5 ml of water and fractionated by gel chromatography with Sephadex G-10 (column: 25 x 850 mm, each fraction volume was 10 ml). Each fraction was monitored by measurement of absorbance of 210, 280, 310 nm and collected for each peak. Fifteen fractions obtained from methanol solution, and 14 fractions from water suspension were evaporated to dryness, dissolved in DMSO and centrifuged. The supernatants were applied to Ames assay.

Mutagenicity assay The mutagenicity was investigated using Salmonella typhimurium TA 98 and 100 (Ames et al., 1975) with and without $9 mix and pre-incubation method was adapted (Yahagi et al., 1975).

4

M. H I B I N O ET A L

60

TA 98 (+ $9 mix)

4O

~ ~

30

2ore I00_

0

I I

I

I

l 2.5 5 Ozonated vanillin(mg/pl ate)

TA 98 (- 59 mix) 400

.

.)

g

~

~ 202

300

g

0

>~ 200 ona

d v

i

i (

ate) 5

100

anlll =1

: 1

=1 :2 =1 :I0 Spontaneous revertants

~

0

@

H

II

0

I

I

Fig. 1. Mutagenicity of ozonated vanillin in 10% methanol.

TA 100 (- $9 mix)

800--

v a n i l l i n : 03 = l : 0.5 =l :l H =1:2 = =I :lO H Spontaneous revertants

.: 600-

1=

40@

200-

0

[I 0

1

i 2

l 5 Ozonated v a n i l l i n (mg/plate)

!

1 2.5 5 Ozonated vanil lin(mg/pl ate)

i I0

Fig. 2. Mutagenicity of ozonated vanillin suspension in water.

MUTAGENICITY AND OZONATION OF HUMIC SUBSTANCES

5

RESULTS

Effect of the solvent as the solubilizer Vanillin was ozonated in 10% methanol and the mutagenicity was investigated. The results are shown in Fig. 1. The mutagenicity was observed on TA 100 with and without $9 mix. The mutagenicity was the strongest at the weight ratio of vanillin:O3 = 1:2. The mutagenicity of ozonated vanillin in water and 10% acetnitrile was examined on TA 100 without $9 mix. The results are shown in Figs. 2 and 3. The mutagenicity appeared in both solutions and the strongest mutagenicity was observed at a weight ratio of vanillin:O3 = l:10. As there was no great difference of the mutagenicity among in methanol, water and acetnitrile, we used methanol as the solubilizer because the reproducibility of results in methanol was good.

Effect of the procedures of evaporation to dryness The mutagenicity o f ozonated vanillin with various concentration methods is shown in Fig. 4. The mutagenicity was found with the concentrate to dryness at 1 mg/plate, and the mutagenic activity of 10-times concentrates

5oo-TA lO0 ( -$9 mix ) ~, 400-

/

.,jr.,/F/"

2oo-

\

./

c 300-

ca

~

v a n i l l i n : 03 : 1 : 0

\ k

e

\ \

\

o

u

s\

revertant-s---_

I00-

0

1

0

I

0.5

I

I

1"

2.5 Ozonated v a n i l l i n (mg/pIate)

5

1

0---0

: 1 : o.s :1:1 ,-~ :1:2 = l :1o m--m

Fig. 3. Mutagenicity of ozonated vanillin in 10% acetnitrile.

6

M. H I B I N O ET A L

300

TA 100 ( - $9 mix) c o n c e n t r a t i o n t o drynessO-~O ten times concentration H unconcentration Z~

'-~ 200-

7 tl/

- -- --

=~~o~ L

I

0

I

O.Ol Ozonated

I

I

i

O.l vanillin (mg/pl a t e )

l

Fig. 4. Mutagenicity of ozonated vanillin various concentration methods.

TA 98 (+ S9 mix)

60-

~

400]

TA I00 (+ $9 mix)

340-

!2oi

3o_ 0

1 2.5 5 0zonated syringal dehyde (mg/pl ate )

!-

lO-

TA I00 (- S9 mix)

3oo-I II

0

1 2.5 5 Ozonated syringaldehyde(mg/pl ate)

0oi

~20-

0

0

I

I

/ ,¢~

\\

I

l 2.5 5 Ozonated syringaldehyde(mg/pl ate)

syringaldehyde

: 03 = l =l :I =I =I Spontaneous revertants

: 0 0---0 : 0 . 5 ~ :I H :2 H :I0 H

Fig. 3. Mutagenicity of ozonated syringaldehyde.

I

0

I

I

l-

1

l 2.5 5 Ozonated syringal dehyde(mg/pl ate )

MUTAGEN1CITY AND OZONATION OF HUMIC SUBSTANCES

7

decreased and toxicity appeared. There is a possibility that some toxic substances were lost when aliquots were evaporated to dryness.

Ozonation of components of humic substances Syringaldehyde, vanillic acid and di-n-butylphtalate in 10% methanol were ozonated and the mutagenicities were examined. The results are shown in Figs. 5, 6 and 7. As well as vanillin (Fig. 1), the mutagenicities of ozonated syringaldehyde and vanillic acid were found on TA 100 with and without $9 mix and the mutagenic activities were the strongest when they were ozonated at a weight ratio of component:Oa = 1:2. The mutagenic activity of ozonated di-n-butylphtalate did not appear.

TA 98 (+ $9 mix)

°

400-

TA 100 (+ $9 mix)

~60

~40

o

oo

0 t

-I I I 0 1 2.5 5 0zonated v a n i l l i c acid(mg/plate) l i

0

I I

0

I

l

i

1 2.5 5 0zonated v a n i l l i c acid(mg/plate)

| |

TA 98 (- S9 mix)

II

0

i

400J 7

2 40-i

I

l 2.5 5 0zonated v a n i l l i c acid(mg/plate)

v a n i l l i c acid : 03 = 1 = 1 =l =I = l Spontaneous r e v e r t a n t s F i g . 6. M u t a g e n i c i t y

: 0 : 0.5 :1 :2 : lO

of ozonated

O---O Z~---~ H ~ H

vanillic acid.

TA lO0 (- $9 mix)

~200~

100 0 I

[ I I 1 2.5 5 Ozonated v a n i l l i c acid(mg/plate)

8

M. HIBINO ET AL.

200 --

40q9 8 ~p~l~- -_~_ _ . . ~ ~3o--~w~-

- --~

TA ~

-

(+ $9 mix) ~

- -

~

x)

"~150-(u u~

100

I I l ] I 0 1 2.5 5 0zonated d i - n - b u t y l p h t h a l ate(mg/plate)

II I I I l 2.5 5 0zonated di - n - b u t y l phthal ate(mg/pl ate )

20O~a

~

TA I00 (- $9 mix)

"

.

.

>o

~10--

~ II

I

I

I

0 1 2.5 5 0zonated d i - n - b u t y l p h t h a l a t e ( m g / p l a t e ) di-n-butylphthalate

.

5O-

I I f I 0 1 2.5 5 0zonated d i - n - b u t y l p h t h a l a t e ( m g / p l a t e )

: 03 = l : 0 O----O = 1 : 0.5~---~ =1 : I ~. ~.

= 1 :2 = 1 : 10

H

Spontaneous r e v e r t a n t s

Fig. 7. Mutagenicity of ozonated di-n-butylphtalate.

Extraction of mutagenic substances For each extract of ozonated vanillin by various solvents, the mutagenicity assay was performed, and the results are shown in Fig. 8. The mutagenic activity was rarely extracted with the solvents, such as benzene, dichloromethane and ethylacetate, and the strong mutagenic activity remained in the residual water layer.

Fractionation of mutagenic substances by gel chromatography The gel chromatograms of ozonated vanillin in 10% methanol and in water suspension, and the results of mutagenicity assay of each fraction are shown

9

MUTAGENICITY AND OZONATION OF HUMIC SUBSTANCES

600-

TA 1 ~ ( - S 9 m i x

Q---~Before e~traction O O benzeneextract dichloromethane extract ~ ethyl acetate extract ~ H residual layer ~ Spontaneousrevertants

500 ..: 400g

)

F //~II-------%

~- 300 200100

-

0

II 0

l

I

2.5 0zonated

J

5 vanil]

in

(mg/plate)

l0

Fig. 8. Mutagenicity of the extracts with 3 solvents and residual layer of ozonated vanillin.

in Figs. 9 and 10. In both cases, the molecular weight of products of ozonated vanillin was estimated to be >400 by the eluted positions of vitamin B12 (mol. wt = 1355), Bromocresol Green (mol. wt = 689) and Methyl Orange (mol. wt = 327). The large mutagenic activity was observed in the large molecular weight fractions. DISCUSSION AND CONCLUSION

The appearance of mutagenicity of ozonated humic substances is different between investigators. This difference for the mutagenic assay of ozonated humic substances may be referred to their origins and ozonation conditions. On the ozonated vanillin, syringaldehyde and vanillic acid, the strong mutagenic activities were observed with TA 100 and not with TA 98. The mutagenic activity increased with the increase of ozone dose at the weight ratio of component:O3 = from 1:0 to 1:2, but decreased at 1:10. Van Hooh (1983) and Kool et al. (1986) reported that the substances with the strong mutagenic activity was produced by ozonation, but were decomposed by further ozonation. As the mutagenic activities were found with and without $9 mix, the products with the strong mutagenic activity may be direct mutagens and not be affected by metabolism with the $9 mix. Although we attempted to extract the mutagenic substances with several organic solvents, this was not possible. The oxidized substances with the

7

5

0

r

I

r

/

'

12

13

(MW=3271

Methyl Orange

Nu m b e r I

l ~0

t"

.

~

210 nm

"

.

10

......

.

~

310 nm

280 nm

1 llo0 o n

~.

.

i.

,

Bromocresol Green (MW=689)

(MW=1355)

Vitamin B12

4

23

,5, 6, 7 ,

' 200

14

b

100-

200-

300

0

,,

1

I

•

I 2

•

_2-__---

- ~--- - ~

~

19

--5

I I0

= = ~---'--_

[ 5 Ozonated v a n i l l i n (mg/plate)

S.._-----

L~..~]~'~//~ ~

Fig. 9. Gel c h r o m a t o g r a m with S e p h a d e x G - 1 0 o f o z o n a t e d vanillin in 10% m e t h a n o l ( A ) a n d m u t a g e n i c i t y o f e a c h f r a c t i o n (B).

o

x~

e~

c

~ 6

a

1°

13

o

5

0

3

'

,'

' ~,

/

]

310 nm

(MW:689)

/

'

'

l

t

'

"

I

200

11 i12i 13 I ~. #. ~14

I Methyl Orange (MW=327)

150

21 nOm

~0

Number

" '"

~

g I

100 r a c t i on

. . . .

"~\ "-.

I

280 nm

Vitamin 812 (MW=1355) Bromocresol Green

'],'

1

4

o~ c~

o >

~.J

nm

a~

qJ

b

o

100-

0

200-

300-

~.~

,

1

-

--,--

2

,

Ozonated

5 vanillin

,

"-'~F~-'--~'-'-=-'~'~''~='--

•

~

(mg/plate)

~

~

I

1 ~

10

,

"--'~'~

- ~ _ ~ _ . ~ _ , ~ .

I ~

Fig. 10. Gel chromatogram with Sephadex G-10 of ozonated vanillin suspension in water (A) and mutagenicity of each fraction (B).



?

0 N © Z > ,H

-< > Z

Z

>

l2

M. HIB|NO ET AL.

strong mutagenic activity produced by ozonation were water-soluble. Following fractionation by gel chromatography, substances with the molecular weight >400 were produced by ozonation of vanillin. The molecular weight of vanillin is 152, and they may be produced by interaction of intermediary products by ozonation. It may be very important to identify these produced compounds and make clear the mechanism of their production. The experiments are in progressing. REFERENCES Ames, B.N., J. McCann and E. Yamasaki, 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31: 347-364. Glaze, W.H., 1987. Drinking-water treatment with ozone. Environ. Sci. Technol., 21: 224-230. Greene, G. and C. Steelink, 1962. Structure of soil humic acid. 2. Some copper oxide oxidation products. J. Org. Chem., 27: 170-174. Gruenner, N., 1978. Mutagenicity of ozonated, recycled water. Bull. Environ. Contam. Toxicol., 20: 522-526. Kamei, T., N. Tanpo and S. Tamura, 1985. Environmental mutagenicity of products from humic substances in water by chlorination or ozonation. J. Water Works Assoc. (Japan), 54 (11): 25-33. Kool, H.J. and J. Hrubec, 1986. The influence of an ozone, chlorine and chlorine dioxide treatment on mutagenic activity in (drinking) water. Ozone: Sci. Eng., 8: 217-234. Kowbel, D.J., E.R. Nestmann, M. Malaiyandi and R. Heleur, 1982. Determination of mutagenic activity in Salmonella of residual fulvic acids after ozonation. Water Res., 16: 1537-1538. Kowbel, D.J., M. Malaiyandi, V. Paramasigamani and E.R. Nestmann, 1984. Chlorination of ozonated soil fulvic acid: mutagenicity studies in Salmonella. Sci. Total Environ., 37: 171-176. Martin, J.P., K. Haider and C. Saiz-Jimenez, 1974. Sodium amalgam reductive degradation of fungal and model phenolic polymers, soil humic acids and simple phenolic compounds. Soil Sci. Am. Proc., 38: 760-765. Rook, J.J., 1974. Formation of halohorms during chlorination of natural waters. Water Treat. Exam., 23: 234-243. Rook, J.J., 1976. Haloforms in drinking water. J. Water Works Assoc., 68: 168-172. Rook, J.J., 1977. Chlorination reactions of fulvic acid in natural water. Environ. Sci. Technol., 11: 478-482. Sato, T., M. Mukaida, Y. Ose, H. Nagase and T. lshikawa, 1985. Chlorinated products from structural compounds of soil humic substances. Sci. Total Environ., 43: 127-140. Schnitzer, M. and S.U. Khan, 1972. Humic Substances in the Environment. Marcel Dekker, New York, pp. 137-202. Stephen, D.K., 1986. Action of ozone on methyl ocyadec-9-enoate in polar solvents. Water Res., 20: 167-171. U.S. Environmental Protection Agency, 1974. Lower Mississippi River Facility, New Orleans area water supply study. Analytical Report, 906-10-74-002.

MUTAGENICITY AND OZONATION OF HUMIC SUBSTANCES

13

Van Hooh, F., 1983. Influence of ozonation on direct-acting mutagens formed during drinking water chlorination. In: R.L. Jolley, W.A. Brungs and R.B. Cumming (Eds.), Water Chlorination: Environmental Impact and Health Effects, Vol. 4. Ann Arbor Science, Ann Arbor, MI, pp. 1211-1220. Yahagi, T., M. Nagano, Y. Seino, T. Matsushima, T. Sugimura and M. Okada, 1977. Mutagenicities of N-nitrosamines on Salmonella. Mutat. Res., 48: 121-130.