extraction procedures

Genetic Toxicology

ELSEVIER

Mutation Research 343 (1995) 31-52

Use of two short-term tests to evaluate the genotoxicity of river water treated with different concentration/extraction procedures Vera Maria F. Vargas a,b,* R6gis Rolim G u i d o b o n o a, Cleusa Jordao a, Joao A n t o n i o P. H e n r i q u e s c a Funda~do Estadual de Proteqdo Ambiental, Av. A.J. Renner, 10, 90245-000, Porto Alegre, RS Brazil b Curso de P6s- Graduaqdo em Gendtica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Departamento de Bioflsica e Centro de Biotecnologia, UniL,ersidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

Received 4 May 1994; revised 16 November 1994; accepted 28 December 1994

Abstract

The genotoxicity of river water samples was evaluated by the Salmonella mutagenicity assay and by the microscreen phage-induction assay. Different processes of sample treatment were compared using the following assays: different volumes of a non-concentrated sample (direct method); concentrated sample fractionated into portions with acid, basic and neutral activity (liquid-liquid extraction method); sample submitted to extraction of volatile substances (volatile extraction method). Samples that were positive to the Salmonella assay by the direct concentration method lost this activity after liquid-liquid extraction. This difference was related to the loss of substances that volatilize during the extraction process. The study of volatile product concentrates confirmed the role of these compounds in inducing activity present in some samples. The microscreen phage-induction assay proved to be a good screening assay for genotoxic compounds present in small concentration in environmental samples. We conclude that, whenever possible, samples should be treated by the direct method in different volumes to prevent the loss of genotoxic substances. Keywords: Mutagenicity; Genotoxicity; River water; Ames test; Liquid-liquid extraction; Microscreen

1. Introduction

The identification of specific chemical substances with genotoxic activity in drinking water (Meier, 1988), in u n t r e a t e d waters or even in industrial effluents is quite difficult because few c o m p o u n d s are present at high concentrations. T h e most a b u n d a n t substances rarely cause geno-

* Corresponding author.

toxic activity (Stahl, 1991). M a n y times genotoxic activity cannot be attributed to specific comp o u n d s in the mixture but rather to a set of properties and chemical interactions of the sample as a whole ( M c G e o r g e et al., 1983). A relevant aspect is that, although knowing the classes of c o m p o u n d s present in these complex mixture is important for the choice of an appropriate m e t h o d (Claxton et al., 1988; Ashby and T e n n a n t , 1991), this identification is not always possible. T h e study of each substance separately

0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-1218(95)00005-4

32

V..M.F. Vargas et al. /Mutation Research 343 (1995) 31-52

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V.M.F. Vargaset al. /Mutation Research 343 (1995) 31-52

would entail cumbersome laboratory work, with the possibility of not identifying all the compounds present in the mixture and of overlooking the synergism of biological and chemical interactions (Houk, 1992). An ideal method for the evaluation of genotoxicity in water is the use of non-concentrated samples. Although this procedure avoids the loss or alteration of organic substances, the assay may not detect low level of genotoxicants or may be impaired by excessive toxicity. Thus, the development of an appropriate methodology for the fractionation or concentration of sample constituents becomes very important genotoxicity tests (Meier, 1988; Stahl, 1991; Houk, 1992). The methods commonly used to concentrate water samples (liquid-liquid extraction and elution with resins) are equally efficient for the extraction of non-volatile mutagenic compounds. The extraction of volatile compounds and the biological assays for their evaluation require special methods (McGeorge et al., 1983; Kado et al., 1986; Meier, 1988; Houk, 1992). Generally water samples require some process of concentration or fractionation to facilitate the screening of compounds with genotoxic activity, which are usually present in small amounts. The methods most commonly employed are based on the use of XAD resins or on fractionation with organic solvents by the chemical process of liquid-liquid extraction. Non-concentrated samples usually present a low level of mutation induction. However, the process of sample concentration may interfere with the genotoxic response, leading to its increase or even to its loss as a consequence of certain steps in the method itself (Dutka and Switzer-House, 1978; Schwartz et al., 1979). Stahl (1991) emphasized the need for an internationally standardized protocol both for sample collection and processing and for existing genotoxicity tests. The author also points out that these assays need to be properly selected, with a careful review of treatment and interpretation of the results. In a recent study carried out on the waters of the Cai River (Rio Grande do Sul, Brazil) in an area under the influence of a petrochemical industrial complex, we detected direct and indirect mutagenicity as measured by both frameshift er-

33

ror and base pair substitution types. Thirty-four percent of the sample tested were mutagenic, with different values according to collection site (Vargas et al., 1993). In the present study, we evaluated the genotoxic activity of water samples from a Cai river using the Salmonella assay and microscreen phage-induction assay. We have chosen the phage-induction assay to complete the analysis because this assay is considered to be especially appropriate for the detection of halogenated compounds, organochlorine compounds and metals, substances with a low level of detection by the Salmonella assay (Rossman et al., 1984; Houk and DeMarini, 1988; Houk, 1992; Rossman et al., 1991). In this work a comparison was made on the sensitivity of genotoxic assays and different samples treatment: liquid-liquid extraction, direct method and extraction of volatile substances.

2. Materials and methods 2.1. Sample

The sampling and storing methods, as well as the location of the collection sites have been described by Vargas et al. (1988, 1993). Five sampling sites were tested over a period of eight sampling campaigns ( A - H ) and coded as follows (the numbers refer to the distance in km from the estuary): Cai 24.1, a site located upstream from the area of influence of the petrochemical industrial complex of Rio Grande do Sul; Ca~ 18.6, Cai 13.6 and Cai 13.3, located in front of the industrial complex and receiving different types and amounts of industrial wastes; Cai 10.6, a site downstream from the area influenced by the industrial complex (Fig. 1). 2.2. Direct method

This method follows the protocol elaborated by the Institute for Medical Research (1983) (Department of Microbiology, Camden, New Jersey) and described by Vargas et al. (1988), Vargas et al. (1993). This is a modification of the Salmonella assay for the analysis of increasing amounts of

I/.M.F. Vargas et al. /Mutation Research 343 (1995) 31-52

34

Table 1 Preparation of surface agar as a function of sample amount Surface agar/tube

Concentration ~ (%)

Amount/tube (ml)

Amount of s a m p l e / t u b e (ml)

1.0 x

Agar NaCI Agar NaCI Agar NaCI Agar NaCI

3.0

0.1-0.2

2.5

0.5

2.0

1.0

1.0

2.0

1.5 x 2.0 x 3.0 x

0.60 0.50 0.72 0.60 0.72 0.60 1.80 1.50

" After sample addition, the volume and concentration of agar and salts was identical in all tubes.

liquid samples (200, 500, 1000 and 2000 /xl) exposed to a standard number of bacteria (approximately 1.10 s cells). The amounts of agar and NaC1 used on the surface agar at plating time are modified so that each tube will have the same final concentration of NaCI and agar (Table 1).

2.3. Liquid-liquid extraction Water samples were concentrated according to an international guideline (International Organization I S O / T C 147/W67, 1980). Starting from a sample of 800 or 1800 ml of water, three extrac-

tion steps were performed using the solvent methylene chloride (pesticide grade Merck, Darmstadt, Germany) resulting in sample fractionation into extracts of compounds having affinity for neutral, basic and acid pH. After washing with solvent, the extracts were evaporated dry on a water bath under a nitrogen gas flow. The solvent dimethylsulfoxide (DMSO, 0.5 ml) (dimethylsulfoxid-spectranal, Riede-de Hainag, Hannover) was added to each final concentrate, followed by an additional 0.5 ml volume of sterile distilled water after homogenization. Each /xl of the final concentrate corresponded to 0.8 or 1.8 ml of the initial water volume. CONCENTRATING TUBES WITH GLASS CONNECTIONS

ACETONE ROOM TEMPERATURE

S A M P L E ----/

Fig. 2. Schematic drawing of the system for the extraction of volatile substances from water samples.

V.M.F. Vargaset al. / Mutation Research 343 (1995) 31-52 2.4. Extraction of volatile substances As shown in Fig. 2, volatile substances were extracted in a glass system consisting of three collector tubes (20 cm in length and 1 cm in diameter) connected to each other and sealed with ground glass caps. This ensemble was conn e c t e d to a h e a t - c o n t r o l l e d w a t e r b a t h (Cryothermostat WK5- Colora) equipped with a water shaking device. The circuit was connected to a nitrogen gas terminal of adjustable flow. The volatile substances released by gradually heating the sample were mobilized by nitrogen towards the collector tubes. The first tube was left at room temperature, receiving and condensing water vapor. The second and third tubes were placed in a dry ice bath with acetone for condensation of the volatile substances at low temperatures. A 300 ml sample volume was placed in an Erlenmeyer flask sealed with a ground glass cap and submitted to gradual heating in a water bath with gentle shaking for 3 h. The process of volatile extraction was started at 40°C and t e m p e r a t u r e was increased by 10°C at hourly intervals up to 60°C. Nitrogen flow was adjusted according to temperature, starting at 5 l/rain, decreasing to 3 1/min and ending at 2 1/min. After extraction, the tubes were placed in an ice bath and 0.5 ml of the solvent dimethylsulfoxide (DMSO) plus 0.5 ml of ice-cold sterile deionized water was added to each of the two tubes for the collection of volatile substances. The frozen extracts in the collecting tubes were transferred to a storage tube in a dry ice bath and 1 ml of sterile deionized water was added. The storage tube was then sealed and kept at - 2 0 ° C until the time for the mutagenicity test. 2.5. The Salmonella mutagenicity assay The test was carried out by the incubation procedure proposed by Maron and Ames (1983) and Levin et al. (1982,1984) using S. typhimurium strains TA100, TA98 and TA102. The test for cytotoxic evaluation and the analysis of true revertant (his + ) colonies that accompany each assay were carried out as described by Vargas et al. (1988,1993). A modification of the Salmonella

35

assay (Kado et al., 1986) was used for analysis of volatile substances in the water samples. The modification consisted of adding 10 times more bacteria (109 cells/incubation tube) and 5 times less metabolic enzymes in the preactivation test. The battery of tests was prepared in ice baths using sealed tubes and than incubated at 37°C for 90 min. The tubes were then placed in ice baths again and inoculated with surface agar in the usual manner (Maron and Ames, 1983). Positive controls were used at 10-fold lower concentrations. The criterion used to classify the results as positive was similar to that used by Vargas et al. (1993), i.e., a number of revertants double the spontaneous yields accompanied by a reproducible dose-response curve whose significance was evaluated by the S A L M O N E L software (Myers et al., 1991). When one of these criteria was not satisfied, the samples was considered to present signs of positivity. The sample was classified as cytotoxic when a cell survival of less than 60% was observed in the cell viability test. 2.6. Microscreen phage-induction assay The assay was carried out by the methods of Rossman et al. (1984, 1991); H o u k and DeMarini (1988) and as described in Vargas et al. (1990). Decreasing volumes of the test compound diluted in L-medium (1% bacto tryptone, 0.5% Bacto yeast extract and 1% NaCI) was incubated on a sterile microtiter plate at 37°C for 15-16 h in the presence of 100/zl of the lysogenic culture of E. coli B / r WP2s(A) + (trpE, uvr A) in the exponential growth phase (diluted 1:10). The volume of the test fluid ranged from 50 to 300 ~1, for a total of 300 t~l/well. In the test carried out in the presence of microsome activation, the total volume was maintained, the amount of strain added was reduced from 100 to 75 ~1, and 25 /~1 of $9 mix prepared by the method of Maron and Ames (1983) was added. For phage titration, 10 ~ l / w e l l of this mixture was removed and diluted in 2 ml M9 buffer. A 100 p~l amount of this solution was then mixed with an equal volume of the indicator strain (RJF013, spontaneous ampicillin-resistant mutant

V.M.F. Vargas et al. /Mutation Research 343 (1995) 31-52

36

Liquid - Liquid Extraction

Direct Method Sampling A Cai 13.6 TA100 z,~:~

NC~ 1 (b) Acid 1

(a)

50091 1,8 1000911 NT 200091 3

Basici 1 Neutral (25~I) I Neutral (50~i) ~ 1,3

TA98

NC~

.c,

Acid~ 1,2 Basic.Ii~l'I Neutral (2591)10,5 Neutral (5091) ~ 1,1

1

50091~T Z000pl~ 200091

2,t ~

6,8

Sampling B Cai 13.3 TA98 + S9

NC~ I 5009liNT Z 0 0 0 9 l ~ 1,4 2000~1

Aoid~0,9 Basic~i~,

4,1

Neutral (50~1)i~ 1,t Sampling C Cai 18.6

TA98

NC~ 1 50091] NT 1000911 NT 20001~1~ 1,3

Aoid~ 1,4 BasicS1,4 Neutral (5091)~ 2 Cai 13.6

TA98 + S9

NC I

NC I~J I

50091LN T 100091 200091

Ac±d i 1,5 Basic 1,,2

2,3

Neutral (5091)~

1,4

Cai 10.6 TA98

NC , .c !ii~ii!'

50091 N~ I00091 200091

1,3

Acid ~

Basic~ Neutral (5091)~

1,3 1,7

1,9

V.M.F. Vargas et al. /Mutation Research 343 (1995) 31-52

of SR714 strain, trpE, uvrD3-1og-phase culture) in a test tube containing soft agar. This mixture was then plated onto GT-medium plates (0.5% NaC1, 0.8% bacto tryptone, and 0.0001% ampicillin) and incubated at 37°C for 15-16 h. A

37

negative control (sterile distilled water) and a positive control (4NQO, 0.30 g/well in assays without $9 mix, or 2 Aminofluoreno, 2 g/well in assays with $9 mix) were added to each test. The criterion for data interpretation and eval-

Sampling O Cai 18.6 TA100 NC

500pl I000~i 2000~1

Auid~

1,t

Basic ~

1

Neutral (501;1) ~ 0 , 8

7,2

TA98

Aaid ~0,6 Basio~ 0,9 17,8

Neutral (50pl)~0,8

Cai 13,6 TA100

Baaic Neutral 150pil

10

0,8

TA98 .c ~i!ili~,

AaidI 0,7 Basia I~ 0,8 Neutral (50111)~] 1,2

8,4

Cai 10.6 TA98

NC~ ,r,

1000.iz'l

1

:.~ :~ :~ 2,6 Ne~Ccal (501~1)~ ] 1

Fig. 3. Evaluation by Salmonell/microsome assay of four different samplings at seven collection sites in the Ca~ river with positive responses to different methods of sample treatment: concentration by direct method and by liquid-liquid extraction. Left page, mutagenic index = No. of his + induced in the s a m p l e / N o , of spontaneous his + in the negative control (NC). Right page, concentrates: 50 p,I and 25 p,l correspond to 40 ml and 20 ml of water in the initial sample. NT, not tested. For all data see Table 2 in the Appendix.

V.M.F. Vargas et al. / Mutation Research 343 (1995) 31-52

38

DIRECT METHOD TA98

TA100 Cal 18.8 (E)

Nc ~ N T

l(a)

1000~1

20001~.~

Cai 18.6 (E) 500p3. lo OOpl i 2000pl ]

1,2

1,1 ;:

:

2,6

'

5,4 Cal 16.6 (F)

Cat 18.6 (F)

~cL

~1

Nc~l lO00~zl i NT 2000pl

8,2

1,2 Cai 13.6 (G) Cai 13.6 (G)

N C

1

500~. ~

1,2

lOOO~l 2000~zl~

2,4

~

LIQUID-LIQUID EXTRACTION: TA100 Cai 18.6 (E) N c 251al

2,4 I 2,7

TA98 Ca[ 18.6 (E)

Acid

Acid

Basic

Basic

1

5~

,t

~=I 25p3.

1

1

50pl

1,3 Neutral

Neutral ~C ~ 25!~ 50~.

25pl ! • • I 0,9

0,9

50~Lz

1,3

]

1

1,1

Car 18.6 (F)

Cai 18.6 (F)

Acid

Acid 1,3

Basic

Basic

. c 50p.1

I 1,3

. c

1

50~.1~

0,8 Neutral

Neutral 50~C .......... I 1 1,5

50~.1~

] 1,1

Car 13.6 (G)

Ca113.6 (G)

Acid

Ac_i __d_d

501xi

1,3 Basic

Basic

N c 50g3-

i

50~i~ 0,8

1,4

Neutral

Neutral 50~

1,8

50~I~

I

V.M.F. Vargas et aL /Mutation Research 343 (1995) 31-52

uation was based on H o u k and DeMarini (1988) and Rossman et al. (1991). Samples were scored positive when a reproducible, dose-related ratio of greater than 3.0 was seen. If a sample reached or exceeded this value at only one dose, the result was scored as weak. If a given response was not reproducible, the result was scored as inconclusive.

3. Results

3.1. Salmonella mutagenicity assay We evaluated samples from 4 different samplings ( A - D ) carried out at the collection sites Cai 24.1, 18.6, 13.6, 13.3 and 10.6, submitted to the direct concentration method and to fractionation by the liquid-liquid extraction process (acid, basic and neutral portions) using strains TA100 and TA98 in the presence and absence of the $9 mierosome fraction. Fig. 3 shows the responses expressed as mutagenic index obtained for the 11 assays that presented a positive response to at least one of the treatment methods used. It can be seen that 9 of these assays gave positive responses when the direct concentration method was used. The results obtained after liquid-liquid extraction indicated a weak mutagenic induction for strain TA98 in samples from sites Cal 18.6 and Cai 10.6 (neutral portion; double the spontaneous yields, set C) in the absence of metabolic activation. A decrease in cell viability of up to 50% was observed for 3 samples from the collection sites Cai 24.1 (basic portion, set A) and Cai 13.6 (neutral and acid portions, sets A and B) for assays carried out in the absence of metabolic activation (data not shown). The remaining samples analyzed in these four campaigns were negative for both treatments used (data not shown). Fig. 4 presents the results of the mutagenic

39

activity for the three samples from sites Ca~ 18.6 and 13.6 in assays with strains TA100 and TA98 in the absence of $9 mix (samplings E, F and G). These samples were examined after concentration by liquid-liquid extraction (a 1800 ml water volume) and by the direct method. Mutagenic activity of the frameshift error type (TA98) was observed in the three samples and associated with pair base substitution damage (TA100) in the sample from the Cal 13.6 site (G) in an assay using direct concentration. The concentrates did not induce a positive response in their acid, basic or neutral portion. A negative response was also obtained in the assay carried out to evaluate the sample as a combination of the three extracts (20 /xl of the acid portion + 20/xl of the basic portion + 20 /xl of the neutral portion - see Table 3 in the Appendix). A decrease in the cell viability test was observed for the analysis of collection site Cal 18.6 (E) after a chemical extraction process (percent survival 20% for the acid and basic portions and 50% for the neutral portion). The assays with strain TA102 were negative for both concentration methods used (see Table 4 in the Appendix).

3.2. Salmonella assay and microscreen phage-induction assay The mutagenic data obtained for the D set were compared to the phage-induction responses. Most of the responses presented an increase in number of P F U / p l a t e ranged from 2.1 to 2.3 times the spontaneous index. Fig. 5 shows the results of the microscreen phage-induction assay and the Salmonella assay in the absence of metabolic activation for samples concentrated by the direct method. The phage induction was significant (Cai 24.1) or weakly positive (Ca~ 13.6) for two sampling sites. It is interesting to notice that for all sampling sites a significant regression coeficient was observed (see

Fig. 4. Analysis of mutagenicity by the Salmonella assay using strains TA100 and TA98 in the absence of metabolic activation of water samples from the Cai river concentrated by direct method and by liquid-liquid extraction using initial volume of 1800 ml of water. Sampling E, F and G. (a) Mutagenic index = No. of his + induced in the s a m p l e / N o , of spontaneous his + in the negative control (NC). NT, not tested. For all data see Table 3 in the Appendix.

V.M.F. Vargas et al. / Mutation Research 343 (1995) 31-52

40

MICROSCREEN PHAGE-INDUCTION

SALMONELLA ASSAY TAt00

TA98

Cai 24.1

Cai 24.t

cai 24.1 Rc

IgC~ 1

BC

(a)

lOOp1

1,5

~

500pl

1

SOOpZ

200~11

3

1000p1

0,S

1000p1

300pl

2,g

2000~1

0,9

2000;;1 ~ 1 , 8

1,5

Cai 18.6

Cai t8.6

cai 18.6 lC 100p1

200;;1

D,

1 i

500pl HC

O,G

5oo;;1

1000pl

1~

2000pl

zoOOpl

3,7 i~

4,9

?,9

?,3 Cai 13.6

Cai 13.6 Cai 13.6 Ic

we 1 1o0;;1

200;;1

1,3

200;;1

~ 1,4 45

500p1 ~

2,3

300;;1

1000pl 5,3

2oo;;1

1 0,9

500]=1 8,0

5,5

lOOO;;1

5,g

2000pl

Cai 13.3

Cai 1:~-~ Ca t3.3 1

MC BC

BC

1

100pl

1,5

200!~.

2,3

300pl

2,3

500pl

1,3

500~1

1000pl

1,1

1000pX

2000pl

1,1

2000F1 ~0,8

Ca[ 10.6

Cai 10.6

Cai 10.6 ~c

lOOpl

i

300pl

2,T

Mc

t,1

500pI

lOOO~1

1,2

ZOO0pZ

2ooopl

1,1

2ooopl

500pl

200pl

1

1 t,5 2,7 4,5

Fig. 5. Genotoxic evaluation of D sampling by the microscreen phage induction assay and Salmonella assay in the absence of metabolic activation. (a) Induction factor = No. PFU per sample p l a t e / N o . PFU per NC plate. (b) Mutagenic index = No. of his + induced in the s a m p l e / N o , of spontaneous his + in the negative control (NC). For all data see Table 5 in the Appendix.

V.M.F. Vargas et al. / Mutation Research 343 (1995) 31-52

MICROSCREEN PHAGEdNDUCTION

SALMONELLAASSAY TA100

Cai24,! nc~ ioop1 200pl

~

300p1

1,1

100091

1,2

1,2

2000p1

1,6

Cai 24.1

1 (b)

500pl

1,1

TA98 Cai 24.1

•C

(@

i

41

2000p3.

t,6

]

1,3

200pl

1,7

300p1 ~

NC

1

500pl

1

,~oo~

1

1ooop1

I

1ooopl

0,9

lOOpl

2000p1 ~

1,3

1

1,1

Cai 18.6

pc

no

Cai 1.3,6

0,g

Cai 18.6

Cai 10.6

lOOpl

[ - -

.o 1

1,1

Cai 13.6

Cai 13.6

500p1

1

soo~

200pl ~

1,1

1000pl

o,g

1000pl

300~1

1,1

2000p1

1

2000p1

1,3 1,8 l

cai 13.3

]ic

lOOpl

1

200pl

1,1

300pl ~

1

Cai 13.3

Cai 13.3

Cal 10.6

Cai 10.6

++++ ~L +++ ;....+ i

lOOOpZ . . . . pl I

Cai 10.6 1

NC

100111 2001:4

~1,i

NT

WC i000pi

0,7

~]2,T

1 1,1

WC i000pi

1 I1

o,o

Fig. 6. Genotoxic evaluation of D sampling by the microscreen phage induction assay and Salmonella assay in the presence of metabolic activation. (a) Induction factor = No. PFU per sample plate/No. P F U / N C plate. (b) Mutagenic index = No. of his + induced in the s a m p l e / N o , of spontaneous his + in the negative control (NC). NT, not tested. For all data see Table 6 in the Appendix.

42

HM.F. Vargas et al. / Mutation Research 343 (1995) 31-52 TA100

-

TA100 + $9

$9

Cai 24.1

Cai 24.1

,,,c

.c Ilk\ ] l(a)

+0.,I21 I 1,1

1oo.,~+~+~+ 11..

2..., ::?.,8

2o8., ~i i )1o,8

501J' ++;

+ 1,5

Cai 18.6

Cai 18.6

NCI:Ki] 1

NC

60.1~.~

- I 1,1

lOO.,

1,2

+,I+:, i,

108.,

1.6

208.,

1,4 Cai 13.6

Cai 13.6

.cl::i] 1 100.1 ~

108.1 ~ NT

NT

u 1,1 200pl++:+++++l

2oo.

TA98 + $9

TA98 - $9

Cai 24.1

Cai 24.1

.c 50.,

.c .o., I

1.2

1,2 lOO,.,,I+ 6%: + 1.2

lOO., i+ 2

200.1 ~++:+++ +:+ ++++14,

200p +:++++ + ++ 13

:i] 1,6

Cai 18.6

Cai 18.6 NC

:

.c Ii 11 50pi I NT lO8.,[~ 11'1

.11

lO6o.O.,t~%~ , 1,21,3 2oo., [::£+I 1

12

2OOpl

Cai 13.6

Cai 13.6

.clk:ll 6o.,

8.6

lOO.,

200.1

',' iI ~ - - - ~

:

:++~ ~ - ~ r

- ~

:

7 ++:

6 6

'

V.M.F. Vargas et al. /Mutation Research 343 (1995) 31-52

(a)

43

(b)

~.

140

140

120

120

100

100 80

80

• Before

.~

After

60

• Before

c

60

After

1

e,,

40

40

20

20

;;i~!!;ii;!!!i

0 500 pl 1000 pl 2000 pl

1000 pl

2000 pl

Fig. 8. Mutagenicity for the Ca[ 13.6 sample before and after extraction of volatile substances, evaluated by Salmonella assay strain TA98 in absence (a) and presence (b) of $9 microsome fraction. Negative control: 200/xl sterile distilled water, (a) = 22 + 1 his + revertant; (b) = 24 + 1, 3 his + revertant. Positive control: (a) 4NQO(0,05/xg/pl) = 157 +_ 6, (b) aflatoxin Bl(0,05/xg/pl) = 595 _+ 21.

Table 5 in the Appendix). Mutagenicity was positive for samples from collection sites Cai 18.6 (TA98, 189 rev/ml; TA100, 291 rev/ml), 13.6 (TA98, 104 rev/ml; TA100, 652 rev/ml) and 10.6 (TA98, 42 rev/ml). A loss of genotoxicity was observed in all positive assays after metabolic activation (Fig. 6 and Table 6 in the Appendix).

3.3. Mutagenicity of L~olatile substances We evaluated the mutagenicity of extracts of volatile substances obtained from three samples

from collection sites Cal 24.1, 18.6 and 13.6 (H sampling). When the samples were evaluated for mutagenicity (strains TA100 and TA98) before being submitted to the extraction process, the sample from site Car 13.6 was mutagenic for TA98 in the assay without metabolic activation. In the statistical analysis carried out using the SALMONEL software the dose-response curve presented a linear regression model with an estimate of 55 rev/ml. Fig. 7 presents the results of the Salmonella assay for the concentrates of volatile substances

Fig. 7. Mutagenicity of volatile substance extracts from the Cal river evaluated by the Salmonella assay, strains TAI00 and TA98 in the absence and presence of $9 microsome fraction. (a) Mutagenic index = No. of his + induced in the s a m p l e / N o , of spontaneous his + in the negative control (NC); NT, not tested. For all data see Table 7 in the Appendix.

44

V.M.F. Vargas et al. / Mutation Research 343 (1995) 31-52

extracted from samples from the Cai 24.1, 18.6 and 13.6 sites. Mutagenicity was observed in assays with the TA98 strain in the presence and absence of metabolic activation for the concentrate of volatile substances extracted from sample Cai 13.6. Two extractions of the same collection were performed to confirm the response obtained and the results are the mean of the two experiments. Statistical analysis with the S A L M O N E L software produced the Lintoxl regression model in assays with the absence of $9 mix and the Lintox2 model after metabolic activation, with respective estimates of 1243 and 1320 r e v / m l extract. After the extraction process, the sample showed a loss of mutagenicity, presenting spontaneous mutation yields (Fig. 8).

4. Discussion

A comparative study of the methods for sample concentration - direct method and liquidliquid extraction - demonstrated a loss of mutagenic substances after the liquid-liquid procedure with a clear toxic action. This fact was observed for compounds that damage the DNA molecule both by base pair substitution and by frameshift error. Schwartz et al. (1979) also observed a loss of mutagenicity detected by the direct method (strain TA100) in the analysis of organic mixtures in drinking water extracted on polyurethane columns. Rappaport et al. (1979) also observed a difference in mutagenic response in the analysis of water concentrates fractionated or not. These observations constitute cumulative evidence that the ideal condition for evaluating genotoxicity in water samples is the use of unchanged samples (Meier, 1988; Stahl, 1991; Houk, 1992). No method of concentration recovers all the organic compounds present and the results obtained correspond to a small portion of the matrix that does not reflect the true genotoxicity of the sample as a whole (Loper, 1980; Stahl, 1991). Evaluation of the mutagenicity of volatile substance concentrates by the Salmonella assay clearly indicates that these compounds are responsible for the inducing activity detected in the Cal 13.6 sample (Figs. 7 and 8). The samples concentrated by

liquid-liquid extraction lost volatile substances (McGeorge et al., 1983; Meier, 1988) during the processing of sample evaporation (Cheh et al., 1980). Dutka and Switzer-House (1978) observed a loss of mutagenicity due to base pair substitution in samples submitted to evaporation under vacuum at 44°C and related this fact to the presence of substances that volatilize or suffer degradation at this temperature. This treatment is also ineffective for the extraction of polar genotoxins and the substances utilized during extraction may introduce modifications in sample composition, generating some genotoxicity (Stahl, 1991). However, it has the advantage of providing a qualitative estimate of the classes of organic compounds responsible for genotoxic activity, by analysis of the sample fractionated into substances with acid, basic and neutral affinity (Dutka et al., 1981; Grabow et al., 1981; Stahl, 1991). These results agree with the evidence observed in the present study that the loss of sample mutagenicity after liquid-liquid extraction may be related to the elimination of these chemical products during treatment (Cheh et al., 1980; McGeorge et al., 1983; Meier, 1988). The concentrates of volatile substances were gradually isolated at an initial temperature of 40°C and a final temperature of 60°C. The negative response detected after this process does not rule out the possibility of the presence of non-volatile but temperature sensitive mutagenic substances. In addition, one should consider the inability of the liquid-liquid extraction method to preserve polar substances (Stahl, 1991). However, the results obtained with these concentrates permit us to show that at least part of the product mixture responsible for the mutagenicity of the area under study is caused by volatile substances. When comparing the genotoxicity results obtained by Salmonella assay and microscreen phage-induction assays (Fig. 5) in samples tested by the direct method it was observed that these assays differ in the ability to determine the substances present in the various samples. When the Salmonella assay was used, mutagenicity was observed at sites Cai 13.6 and 10.6. In the microscreen assay, significant results (weak or positive induction) were observed in the sampling sites

V.M.F. Vargas et al. / Mutation Research 343 (1995) 31-52

Cal 13.6 and Cal 24.1. After metabolic activation there was loss of genotoxicity measured in the two assays. As to the number of P F U / m l in relation to the number of revertants/ml, the Cai 13.6 sample, which was positive in the two assays, induced 2290 P F U / m l (Table 5 in the Appendix) and 652 or 104 HIS + revertants/ml for strains TA100 and TA98, respectively. Furthermore, the microscreen test detected higher genotoxicity at site Car 24.1, which was negative in terms of mutagenicity, with 1160 P F U / m l values. These observations lead us to infer some aspects: the two methods of genotoxic evaluation are being sensitive to different substances; the microscreen phage-induction assay also detects compounds that are positive in the mutagenicity assay; the microscreen assay can detect other DNA damage (Rossman et al., 1991) in addition to different types of mutation. Thus, the microscreen assay is, probably, a good screening assay of genotoxic compounds present in small concentrations in environmental samples. However, in the present study, the microscreen phage-induction assay did not permit the delimitation of the area influenced by the Petrochemical industrial complex as detected in a previous study using Salmonella assay (Vargas et al., 1993) The positive induction in the blank point (Car 24.1) might indicate the presence of another biological active substances such as halogenated compounds, organochlorine compounds and metals, substances with a low level of detection by the Salmonella assay (Rossman et al., 1984; Houk and DeMarini, 1988; Houk, 1992; Rossman et al., 1991.). Stahl (1991) r e p o r t e d t h a t the

45

Salmonella/microsome test has been used as a method of choice for environmental studies on the basis of its proven efficiency for the detection of genotoxicity of isolated organic compounds. However, the efficacy of this method needs to be better evaluated for safe use in complex mixtures, without the risk of loss of information. In contrast, Houk (1992) considers this method to be accurate for the diagnosis of complex samples. This evidence seems to confirm the considerations extensively discussed in the introduction of the present paper about the use of the Salmonella assay for the evaluation of isolated chemical products (Ashby and Tennant, 1991; Claxton et al., 1988). Similarly, a precise diagnosis of complex mixtures basically depends on the class of compounds involved and on the sample treatment procedure used.

Acknowledgements We are grateful to R.B. Abarkeli for suggesting the system for the extraction of volatile substances. We thank V. Fraga and A.F. Ibias from the F E P A M staff for manufacturing and preparing that system. We are also indebted to A.G. Silva and R.C. Horn for expert technical assistance and to the sampling team of FEPAM, especially L.R. Gemelli and C.A. Peixoto. This research was supported by Conselho Nacional de D e s e n v o l v i m e n t o Cientffico e Tecnol6gico (CNPq), Fundaqfio de Amparo ?a Pesquisa do Rio Grande do Sul (FAPERGS) and Financiadora de Projetos (FINEP).

V.M.F. Vargas et al. / Mutation Research 343 (1995) 31-52

46

Appendix 1 Table 2 E v a l u a t i o n by S a l m o n e l l a a s s a y o f f o u r d i f f e r e n t s a m p l i n g s at s e v e n collection sites in t h e C a i r i v e r with positive r e s p o n s e s to d i f f e r e n t m e t h o d s o f s a m p l e t r e a t m e n t : c o n c e n t r a t i o n by a d i r e c t m e t h o d a n d by liquid-liquid e x t r a c t i o n Sampling Sampling A

Sampling B

C a i 13.6 TA100 M a

TA98 R/pl

bM

R/pl

Sampling C

Sampling D

Ca[ 13.3

C a i 18.6

Cal 13.6

Cal 10.6 C a i 18.6

TA98S9

TA98

TA98S9

TA98

M

M

M

R/pl

R/pl

R/pl M

CM 13.6

TAI00

R/pl

M

TA98 R/pl

M

- 127_+25

-

C a i 10.6

TA100 R/pl

M

TA98 R/pl

M

25_+3

126_+29

-

122_+8+

561-+117+

TA98 R/pl

M

R/pl

Direct method NC c

-187_+24

500

- 241_+2

1000 2000

27_+5

-

27_+2

40_+3

-38_+12

-40_+3

NT

NT

NT

NT

NT d

NT + +563_+32

57_+ 13

-

+185_+50+

37+2

NT

112_+4

- 41_+ 1

- 233_+17+

NT +

-52_+13+86_+10

4 6 8 _ + 4 + 197_+24 +

-54_+I+

915_+99+

26_+3

- 26_+3

142_+23

- 40_+7

1085_+ 13 + 153_+93 + 69_+ 12

445_+7+1256_+292+218_+73+117_+6

Liquid-liquid extraction NC

-

138_+6

33_+8

-

30+2

19_+1

- 27_+1

-19_+1

-139_+15

- 37_+12

-

139_+15

37_+12

-37_+12

Acide50

-

143_+9

41_+5

-

27+8

26_+8

- 42_+1

-24_+0

-

-

-

143_+13

25_+1

-38_+23

- 38_+11

-

31_+9

26+9

- 33_+9

-33-+8

-137-+10

- 32_+11

138_+31

30_+8

-34-+16

-

33_+8

38_+8

- 39_+3+37_+1

-114_+28

-

109_+1

45_+1

- 36_+3

NT

NT

NT

NT

NT

Basic50

-142-+32

Neutral25 e 50

145_+8

16+t

178 _+ 1

37 _+ 1

NT

147_+7

NT

24_+0 30_+6

NT

-

NT

a

+ , m u t a g e n i c ; -, n o n - m u t a g e n i c . b N u m b e r o f his + / p l a t e . c N C , n e g a t i v e control. D i r e c t m e t h o d : 200 p,l sterile distilled w a t e r ; liquid-liquid e x t r a c t i o n : 50 ,ul D M S O / 5 0 % ,l N T , not tested. e C o n c e n t r a t e s : 25 pA a n d 50 /xl c o r r e s p o n d azide/plate:

water.

to 20 ml a n d 40 ml o f w a t e r in t h e initial s a m p l e . Positive control: T A 1 0 0 : 5

1634 _+ 222; T A 9 8 , 0.5 p,g 4 N Q O / p l a t e :

k~g s o d i u m

264 _+ 14.

Table 3 A n a l y s i s o f m u t a g e n i c i t y by t h e S a l m o n e l l a a s s a y u s i n g s t r a i n s T A 1 0 0 a n d T A 9 8 in t h e a b s e n c e o f m e t a b o l i c a c t i v a t i o n o f w a t e r s a m p l e s f r o m t h e cai r i v e r c o n c e n t r a t e d by t h e d i r e c t m e t h o d ( D M ) a n d by liquid-liquid e x t r a c t i o n ( L E ) u s i n g initial v o l u m e o f 1800 ml o f w a t e r D.M

L.E.

TA100

/~l/pl pA/pl

TA98

C a i 18.6 ( E l M a I b R/pl

N.C e

C a i 18.6 (F) c

S% d M I

196 _+ 10 100 NT f

500 1000

NT

2000

1.2 2 3 8 - + 6

Cal 18.6 ( E l S% M I

149-+3 1.4 2 0 9 - + 9

20

100 + 2.6 7 4 _ + 4

100

B a s i c (B) 25

1.0 141 _+ 0

50 N e u t r a l ( N ) 25

1.3 179_+ 25 20 0.9 1 2 1 + 0

50

1.3236_+13 1.4 2 4 9 _ + 2

1.3 179-+ 24 50

NT 1.5 265 _+52 1 0 0 -

1.8324_+1776

1.4 259-+ 75 100

1.4 259 _+3

0.7 130_+3

100 -

C a i 13.6 ( G )

M 1

M I

R/pl 39_+5

39_+5 1.2 4 5 + 1 1

+ 2.7 104_+ 13 + 2.4 9 3 _ + 9 + 8.2 3 2 0 + 9 9 29 + 6

1.2 33 + 14

NT

0.925_+13 1.3 36 _+ 4

NT

100 -

1).4 1 2 _ + 0

0.8 2 4 _ + 0

NT 100

R/pl

1.1 4 2 _ + 0

100-

NT

NT 1.3 239 _+35 100 -

100

+ 5.4 1 5 7 + 5 1 28 _+ 5

NT -

C a l 18.6 ( F )

1.2 3 5 _ + 3

NT 1.1 210 + 2 8 8 7

100

1.1 148-+l)

R/pl 29+6

NT

NT

1.0 134_+ 0

i

200 -+5 NT

R/pl

1.2 245 _+13 llX) + 2.4 3 5 5 _ + 5 0 80 183 + 23 183 + 23

A c i d 25 g ( A ) 50 h

A+B+N

C a l 13.6 ( G ) S% M 1

100

139 + 15

NC

R/pl

-

1.3 3 8 - + 6

0.9 24 + 0

NT

1.1 3 1 _ + 3

1.1 3 3 - + 1

NT

1.1 3 1 _ + 2

+ 2.7 106_+22 42_+ 5 NT 1.249+11 NT 0.8 3 2 - + 3 NT 1.042-+5 NT

a

+ , m u t a g e n i c ; -, n o n - m u t a g e n i c . b M u t a g e n e s i s index. c N u m b e r o f his + / p l a t e . d P e r c e n t survival c a l c u l a t e d in r e l a t i o n to t h e n e g a t i v e control.: < 6 0 % t h e toxic s a m p l e s . e N C , n e g a t i v e control: D M , 200 p.l sterile distilled w a t e r ; L E , 50 p,l D M S O / 5 0 % w a t e r . f N T , not t e s t e d . g,h C o n c e n t r a t e s : 25 /zl a n d 50 p.1 c o r r e s p o n d to 45 ml a n d 90 ml o f w a t e r in the initial s a m p l e . i T w e n t y / x l o f e a c h extract. Positive control, T A 1 0 0 , s o d i u m azide, 5 # . g / p l a t e : 1634 + 222; T A 9 8 - 4 N Q O , 0.5 / x g / p l a t e : 264 + 14.

V.M.F. Vargas et al. /Mutation Research 343 (1995) 31-52

47

Table 4 Analysis of mutagenicity by the Salmonella assay using strains TA102 in the absence of metabolic activation of water samples from the CM river concentrated by the direct method (DM) and by liquid-liquid extraction (LE) using initial volume of 1800 ml of water D.M /zl/pl

L.E. /zl/pl

TA102 Cal 18.6 (12/88) M a

N.C o 2000

I

b

0,9 NC Acid 50 f Basic 50 Neutral 50

CM 18.6 (6/89) R/pl c

M

CM 13.6 (5/89)

I

R/pl

I

R/pl

250 + 83 233 ± 27 NT ~

1.0

326 _+ 6 324 + 32 339 4- 7

1.1

289 _+ 6 315 _+ 116 249 + 21

NT

0.9

313 _+ 27

1.0

243 + 26

NT

0.9

288 +_ 52

1.1

271 ± 27

NT

0.9

306 +_ 42

1.3

324 + 17

a "k-, mutagenic; -, non-mutagenic. b Mutagenesis index. c N u m b e r of his + / p l a t e . d NC, negative control: DM, 200/xl sterile distilled water; FFE, 50 ~1 D M S O / 5 0 % water. c NT, not tested. f Concentrates: 50/zl correspond to 90 ml of water in the initial sample.

M

Table 5 Genotoxic evaluation of the D sampling by the microscreen phage-induction assay and Salmonella assay in the absence of metabolic activation Sample

Dose

Microscreen phage-induction assay Salmonella assay TA100 1

NC ~ Sodium azide 4NQO Cai 24.1

50 ~1 200 ~1 5 ~g 0.015 ~g 0.5 ~g 100 ~1 200 ~1 300 ~1 500 ~1 1000 ~1 2000 ~1

a

Induction TA98

R/pl

b

I

100 #1 200/xl 300/xl 500/xl 1000/zl 2000/xl

100 ~1 200/xl 300/zl 500/zl 1000/~1 2000/zl

26_+3 1701 ± 83

1.0 0.8 0.9

1.8 3.7 7.3

1.4 4.5 8.6 10.0

Response Cal 13.3

100 txl 200 p,l 300 pA 500 Izl 1000/xl 2000/xl

b + sb b + sb ,i (t;n)

163 ± 7 126 ± 29 1380 ± 193

Response Cal 13.6

c

R/pl

NT NT NT 125 101 118

f

+ 13 -+ 9 ± 16

1.0 1.5 1.8

248 ± 34 NT NT NT 25_+6 37±5 47_+3

1.5 3.0 2.9

Response g Cat 18.6 (NC:TA98: 25 ± 3)

PFU/pl

factor c

NT NT NT 233 _+ 17 468 ± 4 915 ± 99 + [291rev/ml] NT 180 ± 59 NT 561 _+ 117 1 0 8 5 ± 13 1252 _+ 292 +

1.3 1.1 1.1

[652rev/ml] NT NT NT 164 ± 4 140 _+ 8 146 -t- 15

1.1 1.2 1.1

NT NT NT 146 + 22 151 _+ 32 144 ± 46

4.9 7.9 17.8

0.9 5.5 5.9 8.4

0.9 1.1 0.8

NT NT NT 122 ± 8 197 _+ 24 445 _+ 7 + [189rev/ml] NT 23 ± 7 NT 142± 23 153 _+ 93 218 ± 73 + [104rev/ml] NT NT NT 24 ± 3 25 ± 4 22 _4-7

0.6 1.2 1.6

1.3 2.3 5.3

1.5 2.3 2.3

249 _+ 30 493 ± 28 467 ± 65 NT NT NT + [1160PFU/ml] 94_+ 18 1 9 1 _+ 15 257 ± 41 NT NT NT

207 + 29 375 + 25 859 ± 68 NT NT NT +/[2290PFU/ml] 244 + 13 380 + 6 370 ± 38 NT NT NT

1.16 ± 0.18 (6.435 * * * ;10)

0.29 ± 0.08 (3.625 * * ;9)

2.29 ± 0.35 (6.542 * * *;11)

0.74 ± 0.10 (7.400 * * *;11)

Response Cai 10.6

Response

100/zl 200/zl 300/xl 500 ~zl 1000/xl 2000 pA

1.5 2.7 4.5

NT NT NT 40 ± 7 69 ± 13 117 ± 7 + [42rev/ml]

1.0 1.4 2.7

[740PFU/ml] 166 _+ 6 224 _+ 28 432 ± 16 NT NT NT [850PFU/ml]

a Mutagenesis index. b Number of his + / p l a t e . c Plaque forming unit (lysis)/plate. Induction factor = No. P F U / s a m p l e plateNo. P F U / N C plate. d Regression coefficient + SD (t test and no. of cases). ° Negative control (sterile distilled water) f NT, not tested. g-, non-genotoxic; ± indicative; + , genotoxic. * * P < 0 . 0 1 ; * * * P < 0.001.

0.85 + 0.16 (5.346 * * * ;11)

V.M.F. Vargas et al. /Mutation Research 343 (1995) 31-52

49

Table 6 Genotoxic evaluation of the D sampling by the microscreen phage-induction assay and Salmonella assay in the presence of metabolic activation Sample

Dose

Salmonella assay TA100

NC d AFB1 Cal 24.1

Response f CM 18.6

Response Cal 13.6

Response Cal 13.3

Response CM 10.6

50 /zl 200 ~t 1 0.5/xg 100/xl 20O ~1 300/zl 500/xl 1000/xl 2000 ~tl 100/xl 200/~ 1 300 ~1 500 Ixl 1000/xl 2000/xl 100/xl 200 tz I 300/xl 500 izl 1000/xl 2000 Ixl 100 ~1 20O/zl 300/xl 5O0/zl 1000/zl 2000/~1 100/xl 200 Ixl 300/zl 500 tzl 1000 pA 2000 Ixl

Microscreen phage-induction assay TA98

I a

R/pl b

1.1 1.2 1.6

110 _+ 6 761 + 64 NT e NT NT 121 _+ 10 132 _+ 6 177 _+ 3

1.0 1.0 1.3

NT NT NT 112 + 13 115 +_ 11 140 + 52

1.0 0.9 1.0

NT NT NT 108 _+ 5 99 _+ 12 112 + 4

1.4 1.7

NT NT NT NT 154 + 3 192 + 6

1.1 1.1

NT NT NT NT 116 __+8 120 + 6

Induction factor c

PFU/pl c

I

R/pl

1.0 0.9 0.9

43 _+ 14 563 + 66 NT NT NT 42 + 6 39 +_ 5 40 + 3

1.3 1.7 0.9

1.0 0.9 1.1

NT NT NT 40 +_ 7 37 + 14 49 _+ 14

429 _+ 0 538 + 54 306 + 32 NT NT NT

1.1 1.1 1.1

1.0 1.3 1.6

NT NT NT 41 _+ 12 56 + 10 67 + 14

347 + 25 360 + 46 347 _+ 21 NT NT NT

1.0 1.1 1.0

1.1 1.2

NT NT NT NT 46 + 7 53_+4

310 +_ 124 334 _+ 85 319 + 83 NT NT NT

0.7 2.7

1.1 0.9

NT NT NT NT 45_+6 38+3

225 + 92 303 + 23 NT NT NT

324 + 8

1.1 1.2 1.6

Response a Mutagenesis index. b N u m b e r of his + / p l a t e . c Plaque forming unit (lysis)/plate. Induction factor = N o . P F U / s a m p l e plate No. P F U / N C plate. d Negative control (sterile distilled water). e NT, not tested. f -, non-genotoxic.

1706 +_ 77 364 + 41 382 + 48 532 + 49 NT NT NT

V.M.F. Vargas et al. / Mutation Research 343 (1995) 31-52

50

Table 7 Mutagenicity of volatile substance extracts from water from the CM river evaluated by the Salmonella assay, strains TA100 and TA98 in the absence and presence ( + $9) of $9 microsome fraction Sample

Dose/plate/xl/pl

TA100

Cal 24.1 NC c 50 100 200 Cal 18.6 NC 50 100 2OO Cal 13.6 NC 50 100 2OO

1.1 1.0 0.8 1.1 1.2 1.0

1.0

TA98

TA100 + $9

M a i b R e v / p l c S% d M I 73 ± 18 83±9 69±3 60 ± 0 73 ± 18 83+4 85 + 17 70 ± 0 141 ± 10 NT NT 140±64

100 1.5 1.7 0.9

100 100

1.2 1.5 1.4

100 74 100

70

1.1

Rev/pl

S%

260 379 432 237 260 312 379 360 155 NT NT 171

100

± + ± ± ± ± ± ± ±

14 45 44 19 14 6 0 0 12

+ 6

M

1.2 1.2 1.4

90 60 100

1.2 1.3 1.0

90 70 100

75

I

+ + +

2.7 3.5 3.6

TA98 + $9 Rev/pl

S%

25 ± 1 30 ± 1 30 ± 2 35 ± 0 25 ± 1 31 + 0 33 ± 5 24 + 10 22 ± 8 60±9 77 ± 44 79+19

100

M

1.2 1.6 1.3

100 100 100 74 100 100 80 60

I

1.1 1.2 + + +

5.5 7.3 6.6

Rev/pl

S%

27 + 5 32±0 44 ± 12 36 + 3 27 ± 5 NT f 32±3 31 ± 8 21 ± 11 116±41 154± 74 139 ± 59

100 90 60 100 90 70 100 90 75 62

+ , mutagenic; -, non-mutagenic. b Mutagenesis index. c Number of his + / p l a t e . J Percent survival calculated in relation to the negative control: < 60% toxic sample. c Negative control: 100/xl DMSO/sterile distilled water. f Not tested. Positive control: TA100-AZS (0.5 / x l / p l ) = 1364 ± 29, TA98-4NQO (0.05 / z g / p l ) = 157 ± 6, aflatoxin B1 (0.05 /xg/pl)-TA100S9 = 763 _+ TA98S9 = 595 ± 21. Table 8 Mutagenicity for the CAi 13.6 sample before and after extraction of volatile substances, evaluated by the Salmonella assay strain TA98 in the absence and presence ( + $9) of $9 microsome fraction Sample ~

Dose/plate/zl/pl

TA98-$9 M b

Before

After

NC 500 1000 2000 NC 2000

+ + +

TA98 + $9 I c 2.4 3.5 5.8 1.4

Rev/pl d

S% c

22 + 1 53 ± 4 78 ± 8 128 + 6 22 _+ 5 31 ± 1

100 100 100 74 100 100

M

I

Rev/pl

S% 100

1.1 1.1

24 ± 13 NT 27 ± 3 26 _+ 2 22 + 5 31 ± 5

1.4

85 60 100 100

a Before or after extraction. +, mutagenic; -, non-mutagenic. c Mutagenesis index. d Number of his + / p l a t e . Percent survival calculated in relation to the negative control. Negative control: 200 /xl sterile distilled water. Positive control: TA98 4NQO (0.05/xg/pl) = 157 ± 6, aflatoxin B1 (0.05 p.g/pl)-TA98S9 = 595 ± 21. b

References

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