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Mutagenic activity detected by the Ames test in river water under the influence of petrochemical industries
Mutation Research, 319 (1993) 31-45 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
31
MUTGEN 01912
Mutagenic activity detected by the Ames test in river water under the influence of petrochemical industries V . M . F . V a r g a s a, V . E . P . M o t t a + a n d J . A . P . H e n r i q u e s b a Funda~o Estadual de Prote~o Ambiental and Curso de P6s-Graduaq~o era Gen~tica, and b Departaraento de Bioffsica and Laborat6rio de Genotoxicidade, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
(Received 6 January 1993) (Revision received 31 March 1993) (Accepted 1 April 1993)
Keywords: Mutagenic activity; Ames test; River water; Industrial wastes; Complex mixtures
Summary The present study was carried out on the waters of the Cal River (Rio Grande do Sul, Brazil) in an area under the influence of a petrochemical industrial complex, as the continuation of a study in which the mutagenic activity of water samples was evaluated in the internal area of this complex. In the previous study, the release of inducing substances was detected, revealing the need for a full analysis of the real ecological impact of the industrial complex on the river. Water samples from different sites along the Cal River were subjected to the Ames test during a study of 20 months duration for the detection of possible mutagens. Strains TA100 and TA98 were used for initial sample screening in the presence and absence of the $9 mix at a standard dose of 2000/.d/plate. When positive activity (values equal to twice the spontaneous mutation rate) a n d / o r cytotoxic activity (cell survival below 60%) was detected, the dose-response relationship was studied. Thirty-four percent of the samples tested were mutagenic, with different values according to collection site. Of the total number of positive responses, 6% were obtained for samples collected at the blank site upstream from the area studied, 82% at sites closest to the industrial complex, and 12% in downstream areas. Strain TA98 was the most sensitive in assays with no metabolic activation. A low frequency of induction (2%) was observed for strain TA102. Application of the Ames test permitted the delimitation of three areas of influence of the petrochemical industrial complex, and the test proved to be adequate for the detection of contaminants from the petrochemical industry.
The contamination of surface waters is an important chapter in the study of the genotoxicity of complex mixtures. Rivers and lakes are the major Correspondence: Dr. V.M.F. Vargas, Funda~o Estadual de Prote~o Ambientat, Av. A.J. Renner, 10, 90245-000 Porto Alegre, RS, Brazil. + Deceased.
reservoirs of water as a source of food, as drinking water, and for recreational or agricultural purposes. Many studies of surface waters using different approaches for genotoxic evaluation have reported contamination of these reservoirs by substances inducing genetic damage (for reviews, see Loper, 1980; Meier, 1988; Stahl, 1991; Houk, 1992; Valent et al., 1992).
32
The Ames test (Salmonella/microsome) has been extensively used for rapid evaluation of the action of chemical substances on the DNA molecule (Ames et al., 1973, 1975). Several genetically modified strains have been produced which combine a low level of spontaneous mutation with an excellent response to mutagenesis induced by different classes of agents (Levin et al., 1982a,b; Maron and Ames, 1983). This test has been used to evaluate the mutagenicity of complex mixtures in the air, in rivers, lakes, sediments, industrial effluents, and in drinking water (for reviews, see McGeorge et al., 1983; Nestmann, 1985; Van Kreijl and Slooff, 1985; Kado et al., 1986; Vargas et al., 1988; Stahl, 1991; Houk, 1992; Valent et al., 1992). The origin of
genotoxicity in water is related to industrial and domestic wastes, to contamination by agricultural products or even to potentially reactive natural products (Forstner and Wittmann, 1981). The use of the Ames test has been recommended for environmental studies by international organizations (Canada, 1986; McGeorge et al., 1983) and is recognized as a primary test for the evaluation of genotoxicity related to carcinogenic potential by the Federal Register, USA (1989). In a previous study on water samples collected in the area of influence of the petrochemical industrial complex of Rio Grande do Sul (Vargas et al., 1988), we detected direct and indirect mutagenicity of the frameshift error and base pair substitution types both in samples of the
oji
{,
i i /
(/
/
\
r.~
t;
~'24.1
Fig. 1. Sampling sites along the Cal River in the area of influence of the industrial complex in Rio Grande do Sul.
33 industrial effluents and in water of the Cal River at sites under the influence of the wastes of this industrial park. This pilot study disclosed the importance of a more complete evaluation of the real ecological impact of this river in terms of the presence of mutagens a n d / o r carcinogens in its waters. Thus, the present study was undertaken to determine the possible presence of mutagenic substances in the waters of the Cal River using the Salmonella/ microsome test for a period of 20 months. Material and methods
Sample Samples of Cal River waters were obtained from the area of influence of the petrochemical industrial complex of Rio Grande do Sul. After standard treatment, the waters of the Cal River are used for drinking purposes, for the protection of aquatic communities, for primary contact recreation and for irrigation (Brazil, 1986). This industrial complex is located upstream from the municipality of Porto Alegre, 30 km from the urban center. The samples were collected according to recommended methods (Standard Methods for the Examination of Water and Wastewater, 1985) for the examination of water. No sodium thiosulfate was added in order to prevent the elimination of possible organochlorated compounds present in the samples. Samples were stored at 4°C for 4 days and then divided into aliquots and stored in a freezer at -20°C. Samples were sterized using a Sartorius filter with 0.22 /zm pores (Carl Zeiss do Brasil). Sampling sites Collection sites were selected on the basis of the physicochemical monitoring data provided by FEPAM for this region, and of mutagenicity analyses previously carried out on the waters of the Cal River (Vargas et al., 1988). Five sites were tested over a period of 20 months. The codes refer to the distance, in km, from the estuary (Fig. 1). Cal 24.1 - blank point located upstream from the area of influence of the industrial complex; Cai 18.6 - site close to the final disposal of
the liquid effluent generated by the industrial complex and to the estuary of the Bom Jardim stream (this stream drains a large area devoted .to agricultural use); Car 13.6 and Cai 13.3 - sites close to the area of disposal of accumulation and safety basins (to retain rainwater that may be accidentally contaminated); Cal 10.6 - a site downstream from the area influenced by the industrial complex.
Ames test Mutagenicity was assayed by the preincubation procedure proposed by Maron and Ames (1983), modified. Different sample volumes were incubated with 100 ~1 of tester bacterial cultures (1-2 x 109 cells/ml) in the presence or in the absence of $9 mix and incubated for 25 min in the dark at 37°C, without shaking. 1-3 ml soft agar (supplemented with histidine and biotin) containing different agar and salt concentrations was then added to the test tube, as described by Vargas et al. (1988). After brief shaking, the material was immediately plated onto minimal medium. Cytotoxic analysis was made by plating 100-200 cells with and without water samples in nutrient agar plates (Maron and Ames, 1983). In assays employing $9 fraction (16.3 m g/ m l protein) 500/zl of $9 mix was added. The $9 fraction was prepared from livers of Sprague-Dawley rats pretreated with polychlorinated biphenyl mixture (Aroclor 1254) as described by Maron and Ames (1983). All assys were carried out in triplicate. Plates were incubated in the dark at 37°C for 48 h, after which revertants and surviving colonies were counted. A negative control (sterile distilled water a n d / o r DMSO solvent) and positive controis (5 ~g sodium azide, AZS, 0.5/zg 4-oxide-1nitroquinoline, 4NQO, UV irradiation for 2 s per plate for strains TA100, TA98 and TA102) were added to each test. For the metabolic activation assay, aflatoxin B 1 (AFB1, 0.5 /xg/plate) was added for TA100 and TA98 or 2-aminofluorene (2AF, 10 /xg/plate) was added for TA102 as positive control. To ensure that Salmonella colonies growing on the incubated Ames plates are true (his ÷) revertants and did not arise due to the presence of excess histidine in the water samples or due to the trace amount of this amino acid released in the toxic samples, 10 colonies
+ -
date
5/86 7/86 8/86 9/86 11/86 4/87 5/87 6/87 7/87 9/87 11/87 1/88 2/88 3/88 4/88 5/88 6/88 7/88 8/88 9/88
250-t- 5 154+29 189+ 1 135+14 1155:7 1485:18 2195:45 1685:16 396 + 43 1735:41 1555:30 151+17 915:6 2065:47 1735:44 2295:23 1235:1 2355:1 199+13 1185:16
R/pl c
100 87 60 100 100 100 73 64 100 89 100 93 85 78 47 100 92 96 100 100
S% d + + +
M
2.3 0.7 1.4 0.9 1.1 1.6 0.8 0.8 0.8 0.8 1.3 7.2
15.5 1.1 0.8 0.8 0.7 0.9 0.6
I
C a l 18.6
2260+28 160+28 148+ 6 155+ 5 1425:9 1605:24 955:11 DNU e 425 5:86 131-1-10 1865:28 146+21 1195:23 3265:39 1915:52 174:1:19 1475:19 2225:41 216+14 9155:99
R/pl
89 70 100 80 80 100 73 100 100 100 87 100
100 100 100 80 100 61 100
S% + + + + + +
13.9 1.8 1.2 0.6 0.7 0.8 1.1 0.7 4.1 1.0 2.6 1.0 1.0 1.1 01.9 3.0 1.0 1.0 3.4 10.0
I
C a l 13.6 M
¢ Number of his+/plate. 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 c o n t r o l . e Data not usable.
/zl/plate, mean 179+45). a +, mutagenic; -, non-mutagenic. b M u t a g e n e s i s i n d e x = N o . o f his + i n d u c e d in t h e s a m p l e / N o ,
100 100 100 80 100 100 100 73 70 78 100 100 100 100 64 91 80 100 100 100
S% + + -
M
1.0 0.7 0.6 0.9 1.0 1.2 1.2
1.2 1.0 0.9 0.8 0.7 0.8 0.7 0.8 3.0 0.8 2.4 0.8
I
C a l 13.3
170-t- 7 1 4 3 + 12 1 6 8 + 15 137+ 5 1325: 1 705:11 1155:16 1605: 8 563 5 : 1 2 5 1595: 2 3115:40 1315:33 DNU 1 9 3 5 : 78 1505:12 1385: 4 1655:14 2615:50 2045:71 1465:15
R/pl
100 38 60 75 45 100 100
75 100 100 84 100 100 100 100 100 76 93 100
% + + -
M
1.9 1.1 0.6 0.6 0.6 0.9 7.6 3.2 1.9 0.9 1.4 0.7 1.3 1.2 0.6 0.9 0.9 1.0 1.0 1.2
I
C a i 10.6
281+33 163+37 109+ 5 127+13 127+ 6 1635:18 12425:82 5265:20 355 5:41 1755:37 1805:31 1185:20 1445:7 230+ 1 1485:16 2015:89 1585:29 2695:52 1735:16 1445:46
R/pl
100 100 64 67 100 68 100 82 66 100 81 77 88 100 73 100 77 I00 82 100
S%
o f s p o n t a n e o u s his + in t h e n e g a t i v e c o n t r o l .
m e a n 1534 + 224) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0
2 0 3 1 + 22 2 6 3 + 27 2 2 0 + 18 1 1 4 + 14 1315:13 1485:18 1715:44 1 4 2 + 16 777 5 : 7 3 1915:10 3435:41 163+ 7 1115:13 2 2 5 5 : 33 2085: 7 5 6 3 5 : 32 1735:19 2655:25 5715:129 12565:292
R/pl
T h e results a r e r e p o r t e d as m e a n s + S D . T h e s a m p l e d o s e u s e d w a s 2 0 0 0 / ~ l / p l a t e . T h e positive c o n t r o l w a s s o d i u m a z i d e (5 ~ g / p l a t e ,
1.5 1.1 1.0 0.7 0.6 0.8 1.3 0.9 2.1 0.9 1.2 0.9 0.8 1.0 0.8 1.0 0.7 0.9 1.2 1.0
I b
C a l 24.1
M a
Analysis
ACTIVATION
S C R E E N I N G O F M U T A G E N I C I T Y I N T H E C A I R I V E R I N T H E A R E A O F I N F L U E N C E O F T H E III P E T R O C H E M I C A L COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 1 0 0 I N T H E A B S E N C E O F M E T A B O L I C
TABLE 1
4~
0.9 1.0
-
2/88
0.9 0.9 0.9
1.4 1.0 0.9 1.1
-
-
1564- 8 1305:1 1225:31 1775:3
1325:6 1735:1 1765:20
1375:10 185 5:20
84 100 100 100
50 100 69
83 100
-
-
+
-
-
-
M
1.8 1.4 0.9 1.3
0.9 1.9 1.2
0.8 2.9
1.0 1.1 0.7
1.4 0.9 0.9 1.3 1.1
0.9 1.2
I
C a l 18.6
1995:29 1845: 1 1275:19 1404- 52
12642 3545:146 224-1- 30
1 1 6 + 10 395 5: 21
1105:12 2404- 11 1654- 52
249-t- 13 1225: 8 1215:15 16046 1435: 2 DNU
182:t: 21 149-t- 14
R/pl
73 100 91 23
100 100 80
100 92
78 93 90
61 100 80 100 100
61 54
S%
-
-
-
-
-
-
M
1.4 1.1 1.0 0.7
0.8 1.0 1.0
1.0 1.5
0.8 1.1 1.0
1.0 0.8 0.8 1.1 1.3 0.9
0.5 0.6
I
C a l 13.6
157+ 3 1525:6 1344- 2 1125:4
1134- 4 1945:11 2004-11
1425:24 273 4- 18
905:10 2325:10 2325:11
1935:5 100:1:5 1045:4 1335:7 161:1:42 1915:36
106+38 1085:8
R/pl
100 93 86 81
100 100 100
30 100
100 100 92
71 100 100 100 100 87
46 80
S%
-
-
-
-
-
-
M
1.0 1.1 1.0 1.2
0.9 1.0 0.9
1.0
0.8 0.9 1.0
0.6 0.9 0.8 1.5 1.1 0.9
0.3 0.5
I
C a l 13.3
1055:1 1505:18 1305:6 192-t- 6
1324- 3 1785:14 1634-11
1494-15 DNU
90+10 1905:19 2374- 8
102-1- 5 1165:15 1004- 1 1894-11 1335:11 1915:35
695:7 85-t- 8
R/pi
92 100 59 100
100 100 100
57
100 96 100
54 100 100 100 100 100
25 50
S%
-
-
-
-
-
-
M
1.9 1.0 1.1 0.8
0.8 1.2 1.5
0.8 1.3
0.8 0.7 1.3
0.9 1.2 1.0 0.8 1.4 0.7
0.4 0.5
I
C a l 10.6
2065:6 1405:1 1455:1 1205:6
123+ 4 2205:16 2734-10
1114-10 235 5:27
925:6 1604-33 2874-21
1125:18 1514- 9 1314- 1 955:10 1725:37 142+11
89+14 945:12
R/pl
F o r a definition o f t h e symbols, see T a b l e 1. T h e positive control w a s aflatoxin B 1 ( 0 . 5 / . ~ g / p l a t e , m e a n 748 + 118) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0 / ~ l / p l a t e , m e a n 157 + 41).
9/88
5/88 6/88 7/88 8/88
4/88
3/88
1/88
100 100
-
6/87 7/87 9/87 11/87
5/87
4/87
DNU 2105:10 1604-28
1.0 0.7
-
8/86 9/86 11/86
53 60 69 62 100 100
0.4 0.9 0.9 0.9 1.2 0.7
-
49 100
S%
745:7 1115:20 1195:16 110+19 1535:12 1465:23
147+18 161-1-30
-
R/pl
5/86 7/86
I
M
date
0.7 0.9
Ca124.1
Analysis
100 100 100 86
97 83 100
94 100
100 100 100
53 65 70 100 100 100
57 71
S%
SCREENING OF MUTAGENICITY IN THE CAi RIVER IN THE AREA OF INFLUENCE OF THE III PETROCHEMICAL COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 1 0 0 W I T H M I C R O S O M E A C T I V A T I O N
TABLE 2
-
0.8 1.4 1.0 0.7 0.7 0.8 0.8 1.3 0.6 0.9 1.0 0.8 0.9 0.9 1.1 0.7 0.7 0.9 1.0 1.9
25+ 1 58+ 9 34-+ 4 18_+ 3 20+ 2 21 + 2 29+ 5 32-+ 8 29+ 1 33+ 4 32+10 31 -+ 3 24_+ 3 33_+ 5 35+ 7 28 -+ 13 27-+ 4 33 _+ 5 41 _+ 5 47+ 3
71 97 100 78 79 64 100 64 100 70 100 93 85 78 47 100 92 96 100 100
+ + + + + 0.7 2.1 0.7 3.0 1.0 0.9 0.6 0.5 1.2 1.3 6.7 18.0
110.6 0.8 0.8 0.8 0.9 1.2 0.7
I
M
S%
C a l 18.6
R/pl
M
I
C a l 24.1
3760+70 33+ 2 28_+ 3 19_+ 1 25 + 2 30+ 6 24+ 2 DNU 31 + 4 76 -+ 16 20+ 7 119-+ 4 28-+ 9 35 + 3 27+ 0 20+ 5 44+ 5 5 2 + 13 266 + 44 445_+ 7
R/pl
100 96 100 80 80 100 73 100 100 100 87 100
100 76 100 100 69 61 100
S%
+ + + + + +
+ + + +
M 148.9 29.0 3.1 1.2 0.9 0.7 3.0 1.3 3.6 2.6 2.1 0.9 0.8 0.7 0.5 6.8 0.8 1.8 4.0 8.4
I
C a l 13.6
5063+194 871+138 110+ 8 29+ 3 25+ 4 17+ 1 106__+ 8 3 4 + 12 1 6 7 + 41 69_+ 3 65+ 5 37_+ 9 23+ 1 28:t: 2 21-+ 4 1 8 5 + 50 31+ 3 72_+ 8 161+ 4 2 1 8 + 73
R/pl 100 65 100 94 49 100 100 60 100 78 100 100 100 100 64 91 80 100 100 100
S% 0.7 1.0 0.9 0.8 0.8 0.9 0.6 4.0 3.0 0.8 0.7 0.7 2.2 1.2 0.6 0.7 1.1 1.3 0.9
+ -
I
+ + -
M
C a l 13.3
25+ 1 42+ 8 30+ 4 21+ 4 22+ 2 23+ 3 22+ 4 91+36 127+ 2 28+11 20+ 2 28_+10 DNU 83+ 9 50+ 1 26+ 3 27+ 8 42_+ 5 51+10 22+ 7
R/pl
100 38 60 75 45 100 100
82 70 86 100 100 100 77 76 100 100 93 100
S%
F o r a d e f i n i t i o n o f t h e symbols, see T a b l e 1. T h e positive c o n t r o l w a s 4 - n i t r o q u i n o l i n e ( 0 . 5 / ~ g / p l a t e , m e a n 253 _+31) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0 / ~ l / p l a t e ,
5/86 7/86 8/86 9/86 11/86 4/87 5/87 6/87 7/87 9/87 11/87 1/88 2/88 3/88 4/88 5/88 6/88 7/88 8/88 9/88
Analysis date
4.1 1.2 0.9 1.0 1.0 1.0 2.7 1.3 0.7 1.5 0.8 1.0 0.9 1.4 0.7 1.3 0.7 1.4 1.3 4.5
140+ 7 51+ 4 30+ 2 25+ 5 26+ I 25+ 4 93_+ 11 34+ 6 34_+11 59+ 9 26-+ 2 38+ 3 26+ 1 52-+ 8 28+ 4 56-+20 27+ 5 54_+ 1 53+ 4 117+ 6
m e a n 34 _+ 10).
+ + +
C a l 10.6
100 80 90 94 77 92 100 100 100 74 81 77 88 100 73 100 77 100 82 100
S C R E E N I N G O F M U T A G E N I C I T Y I N T H E C A d R I V E R I N T H E A R E A O F I N F L U E N C E O F T H E III P E T R O C H E M I C A L COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 9 8 I N T H E A B S E N C E O F M I C R O S O M E ACTIVATION
TABLE 3
1.3 2.4 0.9 1.4 0.6 0.9 1.6 1.2 1.0 1.3 0.9
+ -
R/pl
97 100 83 100 50 100 69 84 100 100 100
100 56 100 91 100 100 100 100
S% 2.4 2.0 1.0 0.9 1.0 0.9 1.1 0.7 1.2 0.5 2.6 1.9 1.0 1.3 1.2 1.4 0.9 1.8 1.1
+ + + -
I
M
1.9 0.7 0.9 1.1 1.2 1.3 0.7 1.9
-
88+ 6 37+ 1 45 + 5 55+ 2 58+ 2 46-t- 3 22+ 1 80+10 DNU 565:11 855:5 325:2 515:16 20:t: 4 335:4 615:25 445: 1 425: 0 535:14 405: 3
M
I
C a l 18.6
C a r 24.1
F o r a d e f i n i t i o n o f t h e symbols, see T a b l e 1. T h e positive c o n t r o l w a s a f l a t o x i n B 1 ( 0 . 5 / z g / p l a t e ,
5/86 7/86 8/86 9/86 11/86 4/87 5/87 6/87 7/87 9/87 11/87 1/88 2/88 3/88 4/88 5/88 6/88 7/88 8/88 9/88
Analysis date
100 100 90 100 92 100 100 80 73 100 91 20
67 100 100 100 80 100 73
S%
+ + -
+ + -
M
2.6 0.9 1.0 0.8 0.5 1.2 0.9 1.4 2.2 1.4 1.6
1.0 0.6 2.3 1.1 1.3 1.3 3.5 1.3
I 42+ 4 36+ 4 89 + 2 52+ 6 62+ 4 43+ 2 121+ 6 56+ 7 DNU 1175:5 40+ 3 345:0 295:7 195:1 465:13 335:1 505: 3 865:10 555:4 675:14
R/pl
100 92 30 100 100 100 100 100 93 86 81
67 54 33 100 100 100 100 100
S%
1.1 1.1 0.9 4.2 0.7 1.6 1.3
+ -
I 4.0 0.7 0.9 0.9 1.2 1.2 1.3 1.4 0.6 1.7 3.4 0.9
+ + -
M
C a r 13.3
184+ 8 40+ 4 45 + 2 46+ 6 59-1- 5 41+ 4 43+ 5 60+ 8 34+ 6 715:16 1535:9 325:6 DNU 405:1 395:5 355:4 1125: 4 305:12 515:8 535: 4
R/pl
100 100 100 100 100 59 100
41 40 96 100 100 100 100 100 100 96 100 57
S% -
M 1.1 0.4 1.5 0.7 1.1 0.8 0.9 1.0 0.8 1.4 1.5 0.9 1.0 1.0 1.0 1.2 0.6 1.1 1.3 0.9
I
C a l 10.6
m e a n 567 + 68) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0 / z l / p l a t e , m e a n 40_+ 9).
110+20 110+ 7 38 + 2 48-t- 4 49+ 7 30+ 1 35+ 4 DNU 38+ 4 495:5 235:1 935:16 715:15 365:7 485:9 435:8 525: 3 395: 1 775:10 495:11
R/pl
C a t 13.6
47+3 21+1 74 + 1 36+1 54+9 26+3 31+1 40+5 40+1 585:3 665:7 325:9 375:1 36+7 385:0 465:3 225:0 485:1 505:4 385:3
R/pl
100 49 40 65 70 100 100 100 100 100 100 94 100 97 83 100 100 100 100 86
S%
S C R E E N I N G O F M U T A G E N I C I T Y I N T H E C A J R I V E R I N T H E A R E A O F I N F L U E N C E O F T H E III P E T R O C H E M I C A L COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 9 8 W I T H M I C R O S O M E A C T I V A T I O N
TABLE 4
38 from one duplicate assay plate w e r e streaked over histidine-free Ames plates with a sterile inoculating loop and then incubated for 48 h at 37°C. Only his ÷ organisms grow on the plates. The area study was conducted in two steps, an initial screening in 2000/zl of sample and a study of the dose-response curve for the sample found to have a mutagenic a n d / o r cytotoxic effect in the initial evaluation. The final criterion used to interpret and consider the results of the Ames test as positive was similar to that employed by McGeorge et al. (1983) and Valent et al. (1992): the number of revertants double that of the spontaneous rate and a reproducible dose-response curve. When one of these two criteria was not satisfied, the sample was considered to present signs of mutagenicity. Samples were considered to be cytotoxic when less than 60% cell survival was obtained in the cell viability test, and to be negative when mutagenicity of less than twice the spontaneous rate was detected in assays with more than 60% cell viability. The dose-response curves were evaluated by the SALMONEL program elaborated and kindly provided by Dr. Myers (Environmental Monitoring System Laboratory, EPA-Software version 2.3, April 1988) and described by Myers et al. (1991). This software predicts that the dose-response relationship of these assays can fit at least four regression models: CONSTANT v = b; LINEAR v = bx + a, LINTOX1 (linear attenuated by simple exponential toxicity) v = ( b x + a ) l X X ; LINTOX2 (linear attenuated by exponential toxicity elevated to the second power) y = (ax + b ) f "rx. The software also considers the Bernstein model (Bernstein et al., 1982) which consists of removing one or more doses from the analysis, using only results compatible with the linear model.
Screening analysis The initial screening of mutagenic samples was performed by the Ames test using strains TA100 and TA98 of S. typhimurium in the presence and absence of microsome fraction $9 for a 2000 /zl sample volume. Tables 1-4 summarize the results obtained in each assay for the different samples. It can be seen that the mutagenic index of 15 samples was much lower than the spontaneous rate (0.5, 0.6) with high cell survival (70% or more), and 14 samples had a low mutagenesis index associated with less than 70% cell survival in the viability test, distributed among the four assays. In general, there was a larger number of samples with mutagenicity in the direct test and a larger number of cytotoxic samples after metabolic activation. Tables 1 and 2 show that, for strain TA100, 14 samples were mutagenic and 3 were cytotoxic in the direct test (Table 1), and 1 was mutagenic and 14 were cytotoxic in the presence of $9 mix (Table 2). All mutagenic samples lost their activity in the presence of $9 mix. Table 3 summarizes the results obtained for strain TA98 in tests without metabolic activation. Twenty-one samples had positive mutagenic activity and 4 samples had a cytotoxic effect. Again, an increase in the number of cytotoxic samples (12) and a decrease in the number of samples with a mutagenic effect (11) were observed in the presence of $9 mix (Table 4). Pearson correlation analysis between the various mutagenesis tests showed a significant correlation between the mutagenesis rates detected for strains TA100 and TA98 in the absence of $9 (r = 0.567, p < 0.001; n = 94). However, in the presence of the $9 mix, the correlation was nonsignificant (r = 0.138, p -- 0.179; n = 96), indicating that these tests measure independent effects.
Results
Analysis of the dose-response curve The samples that showed mutagenic a n d / o r cytotoxic activity in the screening test were analyzed in smaller volumes (1000, 500, 200, and 100 /.d/plate) until the previously detected effect was no longer present. The dose-response curves for these samples were analyzed statistically using the SALMONEL software and have been described by Vargas (1992).
From May 1986 to September 1988 we studied 20 samplings from five collection sites in the Cai River previously analyzed and selected as basic for the study of contamination by mutagenic substances generated by the chemical wastes of this industrial complex of Rio Grande do Sul (Vargas et al., 1988).
39
16 +/-
0
TAtOO-S9mix
~
TA100+ S9mix
T , -t-
Revertonts/2ml Positive
-
Negative
+/-
14
- 2400 I I I 1 I I I I
Indicative
--
B
Bernstein
L
Linear
2100 I ! I I I I I !
L 2 Lintox 2
WM Without Model T
P 1800
12.
Z010E N
:S
T
,J <1
I I !
r~ > C3 Z 14. 0
8"
+/-
i I I I I I I I I I I I I I I I I I I I
m
6,
X W Z +
T
,-
4-/-
I , i
+/_
+
+
'
I
+
4-
+
'
+
I
t
~
I"
+T , I i I' I
i
I
iI ,i I I
;
I, i' m I
'
,','
'!
r1200
l-Z I-" n" Ld n"
"60o
t
,I-300 f
I
+1-
II 'i ! !
1' :
T
il
"1'
:
,"
,
T
:: "
Piooe 1,'.e 1~.8 ,~.e ~.6 ~.8 ~4., ,0.8,3.6 rs~ ,3.8 ,~.~ l~6 ,~.e , ~ Month Year
5 86
.5 86
7 86
5 87
6 87
7 87
7 87
7 87
7 87
1, 87
11 87
Model
B
L
L
WM
B
L
L
L
g
L
B
1 88
L2
2 88
5 88
L
L
! I I I I I I !
;~ o
,~.6,8.8 ,3.6 8 88
9 88
9 88
WM
B
L
Fig. 2. Mutagenic induction for the TA100 strain of S. typhimurium in the presence and absence of metabolic activation after treatment with different water samples from the Cal River.
40
pling. Fig. 2 illustrates the response obtained for strain TA100, with 11 samples classified as positive and 6 as indicative of mutagenicity (signs of mutagenicity) and the more elevated mutagenic peaks at collection sites 13.6 and 18.6. Fig. 3 shows the responses obtained for strain TA98, with the presence of 21 positive samples (12 in the direct test and 9 after metabolization), and 14 indicative of mutagenic induction (9 with and 5 without $9 mix) and the elevated mutagenic peaks at collection sites 24.1, 18.6, 13.6 and 13.3. Table 5 summarizes the responses to the Ames test obtained for each sampling point and in each mutagenesis assay at the different collection sites. The incidence of mutagenic samples was higher in the region located in front of the industrial complex, at collection sites Cai 18.6, Cai 13.6 and
The most frequent regression model for all tests was the linear one with a positive direction in the mutagenic samples (also the Bernstein model) and a negative direction in the cytotoxic samples. We also observed the presence of dose-response curves of the LINTOX2 regression model related to the cytotoxic effect and to combined cytotoxic effect and mutagenic induction. There were also cytotoxic samples with mutagenic activity with a positive (or Bernstein) linear dose-response curve. Figs. 2 and 3 summarize the results of mutagenic induction obtained for each test. The figures illustrate the mutagenesis rates for each sample (2000 tzl/plate), the number of revertants/2 ml for the cases in which it was possible to calculate it, and the place and date of sam-
l(a) 148 1
+/-
['7 TA98- Sgmix TI~ TA98÷Sgmix ~"
Rever tante/2ml
I
÷/-f
+-t-/B L L2 WM
100
Z 0 ~
+/_ --'1 ~
!
Positive Negative Indicative
Bcrnsteln Linear Lint°x2 Without Model
28 N
-J
:3 £3 ->
6
-180
£3 Z _
"r +/-- +
LL
+
U.I Z
o
+
i
2-
n.W > ILl
+
x
T
+
!.-...!
I
+/-
~./_
+T +/I
~
+
+/-
r F ofl +
:....-! +
:!
,;
0 Month Year Model
5 86 8
5 5 5 5 7 86 86 86 86 86 L WM WM B L
7 86 B
8 8 8 86 86 86 L L2 L
5 5 5 87 87 87 B L 8
(/) I-Z
6 7 7 9 87 87 87 87 L WM WM L
9 9 11 87 87 87 L WM 8
~60 i I
i ,
i i ,,/J o
11 11 87 87 L L
Fig. 3a. Mutagenic induction for the TA98 strain of S. typhimurium in the presence (A) and absence (B) of metabolic activation after treatment with different water samples from the Cai River.
41
(b)
i I I I~P420
14-
I
~oo
10"
8
g Z
Z
+/-
6
.I+/~'120 +1-
~
:
I:
2
i :: i:i i:
o
+
T
i
I
i
1 1
'1 i
I
i
I I
+
:
, '
,, i
il'.,,_I o
Fig. 3b. Continued.
Cal 13.3. Forty-two percent of all positive samples analyzed were from collection site Cal 13.6. The blank point in relation to the industrial complex, Cal 24.1, had the smallest number of mutagenic samples (6%) in the entire set of data. At Cal 10.6, located downstream, the percentage was 12%. The direct tests yielded 67% of all positive responses. Strain TA98 was the most sensitive for the samples both in tests carried out with and in those without the $9 metabolizing fraction (27 and 40%, respectively). In summary, the effects detected in the different tests corresponded to 53 responses o f mutagenic activity or indicative of this action, 35 of them in direct tests and 18 in tests after metabolization. Table 6 presents a general picture of the 20 samplings in terms of the presence of induced mutation, classifying the sample as positive, negative, with signs of mutagenicity a n d / o r cytoxicity per sampling point (when the effect was present
in at least one test). It can be seen that the number of positive samples increased from the blank point (Cal 24.1) to the site of greatest influence (Cal 13.6), decreasing again downstream from the area under study. The cytoxicity was already present at the blank point at values of the same order of magnitude as those for the Cal 13.6 site (5 cases). The site with the largest number of cytotoxic samples was Cal 13.3 (7 cases). The results of 5 analyses using strain TA102 (this strain detects a variety of oxidative mutagens, active forms of oxygen and alkylating agents, and has an intact excision repair pathway) (Levin et al., 1982b) in the presence and absence of $9 mix (data not shown) showed the presence of three samples with low cell survival (sites 24.1, 18.6 and 13.6) and one with weak mutagenicity (site 13.6) in tests without metabolic activation. The dose-response curve for the positive sample presented a linear regression model with the estimate of 112 rev/ml. Discussion
The criterion used for the initial screening of induced mutagenesis (doubling of the spontaneous rate) is classically applied in studies for the diagnosis of mutagenicity. However, the most important premise for defining the presence of induction is an increase in the number of mutant colonies that follows a dose-response curve (for reviews, see Bernstein et al., 1982; Maron and Ames, 1983; McCann et al., 1984; Claxton et al., 1987). The shape of the dose-response curve basically depends on the dynamics between mutagenicity and cytoxicity (for review Bernstein et al., 1982; Van der Hoeven et al., 1990). However, in some samples the concentrations tested did not show a dose-response relationship despite the fact that the number of revertants was more than double the spontaneous rate. This fact was observed in 27.7% of the positive responses detected in the screening tests for strains TA100 (6.4%) and TA98 (21.3%). These responses obtained for strain TA98, which do not fit the regression models considered, presented a characteristic response pattern, i.e., a peak of mutagenic induction at the dose of 2000 /~1 (Fig.
42 TABLE 5 SUMMARY OF THE RESPONSES TO THE AMES TEST OBTAINED FOR SAMPLING POINT AND MUTAGENESIS ASSAY Samples
Mutagenic c
Cal 24.1
+ -
18.6
+ -
13.6
+ -
13.3
+ -
10.6
+ -
PT e GT f %
+ + -
T+ g
Mutagenesis assay
Total
TA100
TA100S
TA98
TA98S
1 19 3 (1) 16 6 (1) 14 2 17 2 (2) 18
0 19 2 (1) 17 1 (1) 19 0 19 0 20
0 20 5 (3) 14 10 (4) 10 3 (1) 16 3 (1) 17
2 (1) a 17 3 16 5 (1) 14 3 (2) 16 1 (1) 19
14 84 98 14 86 27%
3 94 97 3 97 6%
21 77 98 21 79 40%
14 82 96 15 85 27%
Partial 3 75 13 63 22 57 8 68 6 74 52 337 389
General
%a
78
4 96 17 83 28 72 11 89 8 92
76 79 76 80
%T + b 6% 25% 42% 15% 12%
13 87
a Percentage of positive and negative results. b %T +, percentage calculated of all positive samples. c +, mutagenic; - , non-mutagenic. d Indicative responses added in all positive samples. e Partial total. f General total. g Percentage of positive tests.
3A, B). T h i s o c c u r r e n c e was also m o r e f r e q u e n t for d i r e c t l y a c t i n g m u t a g e n s . T h e s e o b s e r v a t i o n s suggest t h a t s o m e specific f a c t o r m a y b e a c t i n g o n the chemical interaction with the test organism. T h e m o s t f r e q u e n t d o s e - r e s p o n s e curve m o d e l was l i n e a r r e g r e s s i o n . T h i s r e s p o n s e a g r e e s w i t h t h e results o b t a i n e d b y V a l e n t (1992) in a study o n w a t e r b o d i e s in t h e s t a t e o f S~o P a u l o using a similar m e t h o d o l o g y for statistical e v a l u a t i o n . T h e p r e s e n c e o f a cytotoxic a c t i o n r e s u l t e d in a n e g a tive m o d e l o f l i n e a r r e g r e s s i o n , L I N T O X 2 , o r even a positive l i n e a r m o d e l , i n d i c a t i n g t h e p r e s e n c e o f signs o f m u t a g e n i c i t y . D u r i n g t h e 20 m o n t h s o f s a m p l i n g p e r f o r m e d in t h e p r e s e n t study, only two p e r i o d s d i d n o t p r e s e n t a m u t a g e n i c o r toxic effect. C o n s i d e r i n g t h e r e s p o n s e to b e positive w h e n m u t a g e n i c i t y o r cytoxicity was p r e s e n t in at l e a s t o n e assay ( T a b l e 6), w e o b t a i n e d 34 m u t a g e n i c s a m p l e s a n d 22 cytotoxic s a m p l e s . S e v e r a l s t u d i e s have r e p o r t e d t h a t t h e p r e s e n c e o f cytoxicity c a n b e d e t e c t e d by
a fall in i n d u c t i o n b e l o w e x p e c t e d s p o n t a n e o u s levels. I n this s t u d y w e d e t e c t e d 15 s a m p l e s w h o s e m u t a g e n e s i s i n d e x was m u c h l o w e r t h a n t h e s p o n t a n e o u s r a t e ( M I = 0.5 a n d 0.6), with elev a t e d ( m o r e t h a n 7 0 % ) cell survival in t h e f o u r assays p e r f o r m e d . Similarly, l o w e r i n d i c e s w e r e o b s e r v e d in 14 s a m p l e s , f o l l o w e d by l o w e r cell survival a n d t h e r e f o r e c h a r a c t e r i z i n g a cytoxic effect. O v e r a l l analysis o f t h e d a t a ( T a b l e 5) s h o w e d a l a r g e r n u m b e r o f s a m p l e s with m u t a genicity in d i r e c t tests a n d a l a r g e r n u m b e r o f s a m p l e s w i t h low cell survival in tests c a r r i e d o u t in t h e p r e s e n c e o f $9 mix (25 o f 32 = 78%). T h e s t r a i n m o s t sensitive was T A 9 8 with a n d w i t h o u t $9 mix. T h e u s e o f m e t a b o l i c activation elimin a t e d t h e m u t a g e n i c effect o f c h e m i c a l subs t a n c e s which d a m a g e d D N A by b a s e p a i r substit u t i o n in all s a m p l e s t h a t w e r e positive in t h e d i r e c t test. H o w e v e r , t h e m u t a g e n i c s u b s t a n c e s that caused frameshift damage presented mutag e n i e i n d u c t i o n , o r at l e a s t signs o f this action,
43
TABLE
6
GENERAL DUCED
OF
MUTATION
BILITY LEAST
PICTURE
WHEN ONE
Date
THE
AND
THE
PRESENCE
DECREASE
EFFECT
WAS
OF
IN CELL
PRESENT
INVIA-
IN
AT
TEST
C a l 24.1 C a l 18.6 C a l 13.6 C a l 13.3 C a l 10.6
1986 May
-T
+
+T
±T
+T
Jul.
-T
+T
±T
-T
-T
Aug.
-T
-
+T
-T
+T
Sept.
-
.
Nov.
-
-
-
-
Apr.
-
.
May
-
-
+
-
__.
Jun.
-
DNU
-
+
+
Jul.
+
+
+
+
-
Sept.
-
+
+
-
-
Nov.
+
+
+
.
.
.
- T
1987 .
-
.
.
-
1988 Jan.
-
+
+ T
- T
-
Feb.
-
+
-
DNU
-
Mar.
+ T
-
-
+
-
Apr.
-T
-
-
-T
-
May
-
-
+
-
-
Jun.
-
-
-
+_
-
Jul.
-
-
+
- T
-
Aug.
-
5:
_+
- T
-
Sept.
-
+ T
+
-
+
3
8
12
6
5
17
11
8
13
15
+/± Total % T +/+,
20
19
20
19
20
+
15%
42%
60%
32%
25%
-
85%
58%
40%
68%
75%
5
2
5
7
3
mutagenic/or
indicative of mutagenicity;
34 98
22 -,
non-
m u t a g e n i c ; T, d e c r e a s e in cell viability; D N U , d a t a n o t usable.
with a considerably variable pattern. Metabolization led to a loss of mutagenic effect in 62% of cases, whereas the action persisted in 24% and toxicity was present in 14% of the samples that were positive in the direct test. It was also possible to detect samples with mutagenicity after metabolization in 33% of the positive tests, and 29% of these gave a positive response also in tests carried out in the absence of the $9 microsome fraction. This variable pattern of response is due to the mixture of substances which are part of environmental samples and represents addi-
tional evidence for the fact that the mutagenic and toxic activity detected in these assays is not the sum of the individual effects of each substance. In addition to synergism and antagonism among chemical products, other complications may occur due to the interactions between the mutagenic and antimutagenic agents present. These factors contribute to the ambiguity of the results of the tests (Saxena, 1984) and may also be related to a decrease in spontaneous mutation rates. The higher incidence of direct mutagens which cause frameshift DNA damage has proved to be considerably constant in river samples (Pelon et al., 1977; Grabow et al., 1980; Maruoka and Yamanaka, 1980, 1982; Van Kreijl et al., 1980; Martins et al., 1982; Valent, 1992). As to analysis after metabolic activation, data in the literature have shown that a decrease, or even inactivation of mutagenicity may occur in the presence of the $9 mix fraction (Saxena and Schwartz, 1979; Flanagan and Allen, 1981; Grimm-Kibalo et al., 1981; Valent, 1992). However, some investigators have observed elevation of mutagenicity in rivers after metabolization (for a review, see Meier, 1988). Houk (1992) also emphasized the frequency of directly acting mutagens which cause frameshift DNA damage in petrochemical industry wastewaters. Courtois et al. (1992) have argued that the $9 mix-dependent decrease in mutagenicity of environmental mixtures might not be due only to enzymatic mechanisms. Passive interactions of chemicals with proteins are probably responsible for the decrease leading to the non-expression of the mutagenicity of some direct and indirect mutagens. In mechanistic terms, such interactions seem to be a result of competition at the enzymatic sites rather than of conversion of compounds into non-functional metabolites. It should also be emphasized that complex water samples obtained from water basins are a sum of the chemical, physical and biological interactions of this ecosystem. Thus, the contaminants may already be present in a form differing from that which they had when first dumped into the river. The greater direct action of genotoxic substances in this type of sample may be due to the activity of reactive electrophilic groups generated by organisms present in the water.
44
The selection of sampling sites with the specific objective of evaluating the influence of wastes released by an industrial park led to the observation of sites with more or less constant induced mutagenesis responses. Analysis by collection site showed a lower incidence of positive samples at the blank site, and a clear elevation in number in the zone of influence of the industrial complex, with the Cal 13.6 site presenting the highest frequency of such samples. However, an important decrease in this action was observed in the downstream direction. Sites Cal 18.6, 13.6 and 13.3 were the most heavily involved. These sampling points are located close to the areas of wastewater disposal and to the rainwater accumulation and safety basins. A previous study by our group (Vargas et al., 1988) in this internal area of the industrial complex showed that these sites were contaminated with substances which generated base pair substitutions a n d / o r frameshift errors. The presence of cytotoxic activity at the blank site at levels similar to those detected at Cal 13.6 indicates contamination by biologically active substances. However, a higher percentage of cytotoxicity was observed at the collection site Cal 13.3, also considerably contaminated with mutagens. This toxicity was mainly observed in tests using microsome fraction $9, showing the activity of the metabolites of chemical substances present in the river. However, on the basis of the responses obtained in the present study, we may infer that the methodology used permitted the delimitation of the area of influence of the complex and the release of mutagens that were active and sensitive to the strains employed.
Acknowledgements We are grateful to the students who participated in the experimental part of this study as Scientific Initiation fellows, S. Bresolin and R.R. Guidobono in particular. We are also indebted to the sampling team of FEPAM, especially L.R. Gemelli and C.A. Peixoto, for their dedication. This research was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecno16gico (CNPq), Funda~fio de Amparo h Pesquisa
do Estado do Rio Grande do Sul (FAPERGS), and Financiadora de Projetos (FINEP).
References Ames, B.N., F.D Lee and W.E. Durston (1973a) An improved bacterial test system for detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. USA, 70, 782-786. Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973b) Carcinogens are mutagens: A simple test system combining liver homogenates for activation, bacteria for detection, Proc. Natl. Acad. Sci. USA, 70, 2281-2285. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Bernstein, L., J. Kaidor, J. McCann and M.C. Pike (1982) An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test, Mutation Res., 97, 267-281. Brazil (1986) Conselho Nacional do Meio Ambiente, resolu~o n. 20, de 18 de junho de 1986. Classifica as figuas do Territ6rio National. Difirio Oficial da Repfibliea Federativa do Brasil, Brasflia, 30 de julho. Canada (1986) Minister of National Health and Welfare and Minister of the Environment. Advisory Committee on Mutagenesis. Guidelines on the Use of Mutagenicity Tests in the Toxicological Evaluation of Chemicals, Ottawa, 84 pp. Claxton, L.D., J. Allen, A. Auletta, K. Mortelmans, E. Nestmann and E. Zeiger (1987) Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutation Res., 189, 83-91. Courtois, Y.A., M.L. Pesle and B. Festy (1992) Activation of pro-mutagens in complex mixtures by rat liver $9 systems, Mutation Res., 276, 133-137. Federal Register (1989) Guidelines establishing test procedures for the analysis of pollutants under the clean water act, Federal Register, Part III: Environmental Protection Agency, December 4, 1989, 54 (231), 50216-50223. Flanagan, E.P., and H.E. Allen (1981) Effect of water treatment on mutagenic potential, Bull. Environ. Contain. Toxicoi., 27, 765-772. Forstner, U., and G.T.W. Wittmann (1981) Metal Pollution in the Aquatic Environment, Springer, New York, 473 pp. Grabow, W.O.K., R. Denkhaus and P.G. Van Rossum (1980) Detection of mutagens in wastewater, a polluted river and drinking water by means of the Ames Salmonella-microsome assay, South Afr. J. Sci., 76, 118-123. Grimm-Kibalo, S.M., B.A. Glatz and J.S. Fritz (1981) Seasonal variation of mutagenic activity in drinking water, Bull. Environ. Contam. Toxicol., 26, 188-195. Houk, V.S. (1992) The genotoxicity of industrial wastes and effluents, Mutation Res., 277, 91-138. Kado, N.Y., G.N. Guirguis, C.P. Flessel, R.C. Chan, K. Chang and J.W. Wesolowski (1986) Mutagenicity of fine ( < 2.5 /zm) airborne particles: Diurnal variation in community air
45 determined by a Salmonella micro preincubation (microsuspension) procedure, Environ. Mut., 8, 53-66. Levin, D.E., E. Yamasaki and B.N. Ames (1982a) A new Salmonella tester strain (TA97), for the detection of frameshift mutagens, Mutation Res., 94, 315-330. Levin, D.E., M. Hollstein, M.F. Christman, E.A. Schwiers and B.N. Ames (1982b) A new Salmonella tester strain (TA102) with A.T base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. USA, 79, 7445-7449. Loper, J.C. (1980) Mutagenic effects of organic compounds in drinking water, Mutation Res., 76, 241-268. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Martins, M.T., P.S. Sanchez, M.I.Z. Sato and S. Gaglianone (1982) D e t e c ~ o de subst~ncias mutag~nicas em figuas resultados preliminares, in: VI Simp6sio Anual da ACIESP, S~o Paulo, Anais, vol. 3, ACIESP, S~o Pauio, pp. 114-119. Maruoka, S., and S. Yamanaka (1980) Production of mutagenic substances by chlorination of waters, Mutation Res., 79, 381-386. Maruoka, S., and S. Yamanaka (1982) Mutagenicity in Salmonella typhimurium tester strains of XAD-2-ether extract, recovered from Katsura River water in Kyoto City, and its fractions, Mutation Res., 102, 13-26. McCann, J.L.H., and J. Kaldor (1984) An evaluation of Salmonella (Ames) test data in the published literature: Application of statistical procedures and analysis of mutagenic potency, Mutation Res., 134, 1-47. McGeorge, L.J., J.B. Louis, T.B. Atherholt and G.J. McGarrit (1983) Mutagenicity analyses of industrial effluents: background, results to date, Report of the New Jersey Department of Environmental Protection, Trenton, NJ. Meier, J.R. (1988) Genotoxic activity of organic chemicals in drinking water, Mutation Res., 196, 211-245. Myers, L., N. Adams, L. Kier, T.K. Rao, B. Shaw and L. Williams (1991) Microcomputer software for data management and statistical anlyses of the Ames/Salmonella test, in: D. Krewski (Ed.), Statistical Methods in Toxicological Research, Gordon and Brech, New York, pp. 265-279. Nestmann, E.R. (1985) Detection of genetic activity in effluents from pulp, paper mills: mutagenicity in S. cerevisiae,
in: Mutagenicity Testing in Environmental Pollution Control, Wiley, New York, pp. 118-125. Pelon, W., B.F. Whitman and T.W. Beasley (1977) Reversion of histidine-dependent mutant strains of Salmonella typhimurium by Mississippi River water samples, Environ. Sci. Technol., 11, 619-623. Saxena, J. (1984) In vitro test systems for mutagenicity screening of environmental chemicals, Rev. Microb., 15, 1-16. Saxena, J., and D.J. Schwartz (1979) Mutagens in wastewaters renovated by advanced wastewater treatment, Bull. Environ. Contam. Toxicol., 22, 319-326. Stahl, R.G. Jr. (1991) Genetic toxicology of organic compounds in natural waters and wastewaters, Ecotoxicol. Environ. Safety, 22, 94-125. Standard Methods for the Examination of Water and Wastewater (1985) American Public Health Assoc., American Water Works Assoc. and Water Pollution Control Federation. 16th edn., M.A.H. Franzon, Washington, DC, pp. 856-858. Valent, G.V., M.I. Sato, M.C. Coelho and C.A. Coimbrfio (1992) Monitoring Silo Paulo state river in Brazil for mutagenic activity using the Ames test, Environ. Toxicol. Water Quality, in press. Van der Hoeven, N., S.A.L.M. Kooijman and W.K. Raat (1990) Salmonella test: Relation between mutagenicity and number of revertant colonies, Mutation Res., 234, 289-302. Van Kreijl, C.F., and W. Slooff (1985) Mutagenic activity in Dutch river water, its biological significance for fish, in: Mutagenicity Testing in Environmental Pollution Control, Wiley, New York, pp. 86-103. Van Kreijl, C.F., H.J. Kool, M. De Vries, H.J. Van Kranen and E. De Greef (1980) Mutagenic activity in the rivers Rhine and Meuse in the Netherlands, Sci. Total Environ., 15, 137-147. Vargas, V.M.F. (1992) Avalia~o de testes para triagem e diagn6stico de agentes genot6xicos ambientais, Doctoral Thesis, Instituto de Bioci~ncias, Universidade Federal do Rio Grande do Sul, 237 pp. Vargas, V.M.F., V.E.P. Motta and J.A.P. Henriques (1988) Analysis of mutagenicity of waters under the influence of petrochemical industrial complexes by the Ames test (Salmonella/microsome), Rev. Bras. Gen6t., 11, 505-518.
31
MUTGEN 01912
Mutagenic activity detected by the Ames test in river water under the influence of petrochemical industries V . M . F . V a r g a s a, V . E . P . M o t t a + a n d J . A . P . H e n r i q u e s b a Funda~o Estadual de Prote~o Ambiental and Curso de P6s-Graduaq~o era Gen~tica, and b Departaraento de Bioffsica and Laborat6rio de Genotoxicidade, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
(Received 6 January 1993) (Revision received 31 March 1993) (Accepted 1 April 1993)
Keywords: Mutagenic activity; Ames test; River water; Industrial wastes; Complex mixtures
Summary The present study was carried out on the waters of the Cal River (Rio Grande do Sul, Brazil) in an area under the influence of a petrochemical industrial complex, as the continuation of a study in which the mutagenic activity of water samples was evaluated in the internal area of this complex. In the previous study, the release of inducing substances was detected, revealing the need for a full analysis of the real ecological impact of the industrial complex on the river. Water samples from different sites along the Cal River were subjected to the Ames test during a study of 20 months duration for the detection of possible mutagens. Strains TA100 and TA98 were used for initial sample screening in the presence and absence of the $9 mix at a standard dose of 2000/.d/plate. When positive activity (values equal to twice the spontaneous mutation rate) a n d / o r cytotoxic activity (cell survival below 60%) was detected, the dose-response relationship was studied. Thirty-four percent of the samples tested were mutagenic, with different values according to collection site. Of the total number of positive responses, 6% were obtained for samples collected at the blank site upstream from the area studied, 82% at sites closest to the industrial complex, and 12% in downstream areas. Strain TA98 was the most sensitive in assays with no metabolic activation. A low frequency of induction (2%) was observed for strain TA102. Application of the Ames test permitted the delimitation of three areas of influence of the petrochemical industrial complex, and the test proved to be adequate for the detection of contaminants from the petrochemical industry.
The contamination of surface waters is an important chapter in the study of the genotoxicity of complex mixtures. Rivers and lakes are the major Correspondence: Dr. V.M.F. Vargas, Funda~o Estadual de Prote~o Ambientat, Av. A.J. Renner, 10, 90245-000 Porto Alegre, RS, Brazil. + Deceased.
reservoirs of water as a source of food, as drinking water, and for recreational or agricultural purposes. Many studies of surface waters using different approaches for genotoxic evaluation have reported contamination of these reservoirs by substances inducing genetic damage (for reviews, see Loper, 1980; Meier, 1988; Stahl, 1991; Houk, 1992; Valent et al., 1992).
32
The Ames test (Salmonella/microsome) has been extensively used for rapid evaluation of the action of chemical substances on the DNA molecule (Ames et al., 1973, 1975). Several genetically modified strains have been produced which combine a low level of spontaneous mutation with an excellent response to mutagenesis induced by different classes of agents (Levin et al., 1982a,b; Maron and Ames, 1983). This test has been used to evaluate the mutagenicity of complex mixtures in the air, in rivers, lakes, sediments, industrial effluents, and in drinking water (for reviews, see McGeorge et al., 1983; Nestmann, 1985; Van Kreijl and Slooff, 1985; Kado et al., 1986; Vargas et al., 1988; Stahl, 1991; Houk, 1992; Valent et al., 1992). The origin of
genotoxicity in water is related to industrial and domestic wastes, to contamination by agricultural products or even to potentially reactive natural products (Forstner and Wittmann, 1981). The use of the Ames test has been recommended for environmental studies by international organizations (Canada, 1986; McGeorge et al., 1983) and is recognized as a primary test for the evaluation of genotoxicity related to carcinogenic potential by the Federal Register, USA (1989). In a previous study on water samples collected in the area of influence of the petrochemical industrial complex of Rio Grande do Sul (Vargas et al., 1988), we detected direct and indirect mutagenicity of the frameshift error and base pair substitution types both in samples of the
oji
{,
i i /
(/
/
\
r.~
t;
~'24.1
Fig. 1. Sampling sites along the Cal River in the area of influence of the industrial complex in Rio Grande do Sul.
33 industrial effluents and in water of the Cal River at sites under the influence of the wastes of this industrial park. This pilot study disclosed the importance of a more complete evaluation of the real ecological impact of this river in terms of the presence of mutagens a n d / o r carcinogens in its waters. Thus, the present study was undertaken to determine the possible presence of mutagenic substances in the waters of the Cal River using the Salmonella/ microsome test for a period of 20 months. Material and methods
Sample Samples of Cal River waters were obtained from the area of influence of the petrochemical industrial complex of Rio Grande do Sul. After standard treatment, the waters of the Cal River are used for drinking purposes, for the protection of aquatic communities, for primary contact recreation and for irrigation (Brazil, 1986). This industrial complex is located upstream from the municipality of Porto Alegre, 30 km from the urban center. The samples were collected according to recommended methods (Standard Methods for the Examination of Water and Wastewater, 1985) for the examination of water. No sodium thiosulfate was added in order to prevent the elimination of possible organochlorated compounds present in the samples. Samples were stored at 4°C for 4 days and then divided into aliquots and stored in a freezer at -20°C. Samples were sterized using a Sartorius filter with 0.22 /zm pores (Carl Zeiss do Brasil). Sampling sites Collection sites were selected on the basis of the physicochemical monitoring data provided by FEPAM for this region, and of mutagenicity analyses previously carried out on the waters of the Cal River (Vargas et al., 1988). Five sites were tested over a period of 20 months. The codes refer to the distance, in km, from the estuary (Fig. 1). Cal 24.1 - blank point located upstream from the area of influence of the industrial complex; Cai 18.6 - site close to the final disposal of
the liquid effluent generated by the industrial complex and to the estuary of the Bom Jardim stream (this stream drains a large area devoted .to agricultural use); Car 13.6 and Cai 13.3 - sites close to the area of disposal of accumulation and safety basins (to retain rainwater that may be accidentally contaminated); Cal 10.6 - a site downstream from the area influenced by the industrial complex.
Ames test Mutagenicity was assayed by the preincubation procedure proposed by Maron and Ames (1983), modified. Different sample volumes were incubated with 100 ~1 of tester bacterial cultures (1-2 x 109 cells/ml) in the presence or in the absence of $9 mix and incubated for 25 min in the dark at 37°C, without shaking. 1-3 ml soft agar (supplemented with histidine and biotin) containing different agar and salt concentrations was then added to the test tube, as described by Vargas et al. (1988). After brief shaking, the material was immediately plated onto minimal medium. Cytotoxic analysis was made by plating 100-200 cells with and without water samples in nutrient agar plates (Maron and Ames, 1983). In assays employing $9 fraction (16.3 m g/ m l protein) 500/zl of $9 mix was added. The $9 fraction was prepared from livers of Sprague-Dawley rats pretreated with polychlorinated biphenyl mixture (Aroclor 1254) as described by Maron and Ames (1983). All assys were carried out in triplicate. Plates were incubated in the dark at 37°C for 48 h, after which revertants and surviving colonies were counted. A negative control (sterile distilled water a n d / o r DMSO solvent) and positive controis (5 ~g sodium azide, AZS, 0.5/zg 4-oxide-1nitroquinoline, 4NQO, UV irradiation for 2 s per plate for strains TA100, TA98 and TA102) were added to each test. For the metabolic activation assay, aflatoxin B 1 (AFB1, 0.5 /xg/plate) was added for TA100 and TA98 or 2-aminofluorene (2AF, 10 /xg/plate) was added for TA102 as positive control. To ensure that Salmonella colonies growing on the incubated Ames plates are true (his ÷) revertants and did not arise due to the presence of excess histidine in the water samples or due to the trace amount of this amino acid released in the toxic samples, 10 colonies
+ -
date
5/86 7/86 8/86 9/86 11/86 4/87 5/87 6/87 7/87 9/87 11/87 1/88 2/88 3/88 4/88 5/88 6/88 7/88 8/88 9/88
250-t- 5 154+29 189+ 1 135+14 1155:7 1485:18 2195:45 1685:16 396 + 43 1735:41 1555:30 151+17 915:6 2065:47 1735:44 2295:23 1235:1 2355:1 199+13 1185:16
R/pl c
100 87 60 100 100 100 73 64 100 89 100 93 85 78 47 100 92 96 100 100
S% d + + +
M
2.3 0.7 1.4 0.9 1.1 1.6 0.8 0.8 0.8 0.8 1.3 7.2
15.5 1.1 0.8 0.8 0.7 0.9 0.6
I
C a l 18.6
2260+28 160+28 148+ 6 155+ 5 1425:9 1605:24 955:11 DNU e 425 5:86 131-1-10 1865:28 146+21 1195:23 3265:39 1915:52 174:1:19 1475:19 2225:41 216+14 9155:99
R/pl
89 70 100 80 80 100 73 100 100 100 87 100
100 100 100 80 100 61 100
S% + + + + + +
13.9 1.8 1.2 0.6 0.7 0.8 1.1 0.7 4.1 1.0 2.6 1.0 1.0 1.1 01.9 3.0 1.0 1.0 3.4 10.0
I
C a l 13.6 M
¢ Number of his+/plate. 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 c o n t r o l . e Data not usable.
/zl/plate, mean 179+45). a +, mutagenic; -, non-mutagenic. b M u t a g e n e s i s i n d e x = N o . o f his + i n d u c e d in t h e s a m p l e / N o ,
100 100 100 80 100 100 100 73 70 78 100 100 100 100 64 91 80 100 100 100
S% + + -
M
1.0 0.7 0.6 0.9 1.0 1.2 1.2
1.2 1.0 0.9 0.8 0.7 0.8 0.7 0.8 3.0 0.8 2.4 0.8
I
C a l 13.3
170-t- 7 1 4 3 + 12 1 6 8 + 15 137+ 5 1325: 1 705:11 1155:16 1605: 8 563 5 : 1 2 5 1595: 2 3115:40 1315:33 DNU 1 9 3 5 : 78 1505:12 1385: 4 1655:14 2615:50 2045:71 1465:15
R/pl
100 38 60 75 45 100 100
75 100 100 84 100 100 100 100 100 76 93 100
% + + -
M
1.9 1.1 0.6 0.6 0.6 0.9 7.6 3.2 1.9 0.9 1.4 0.7 1.3 1.2 0.6 0.9 0.9 1.0 1.0 1.2
I
C a i 10.6
281+33 163+37 109+ 5 127+13 127+ 6 1635:18 12425:82 5265:20 355 5:41 1755:37 1805:31 1185:20 1445:7 230+ 1 1485:16 2015:89 1585:29 2695:52 1735:16 1445:46
R/pl
100 100 64 67 100 68 100 82 66 100 81 77 88 100 73 100 77 I00 82 100
S%
o f s p o n t a n e o u s his + in t h e n e g a t i v e c o n t r o l .
m e a n 1534 + 224) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0
2 0 3 1 + 22 2 6 3 + 27 2 2 0 + 18 1 1 4 + 14 1315:13 1485:18 1715:44 1 4 2 + 16 777 5 : 7 3 1915:10 3435:41 163+ 7 1115:13 2 2 5 5 : 33 2085: 7 5 6 3 5 : 32 1735:19 2655:25 5715:129 12565:292
R/pl
T h e results a r e r e p o r t e d as m e a n s + S D . T h e s a m p l e d o s e u s e d w a s 2 0 0 0 / ~ l / p l a t e . T h e positive c o n t r o l w a s s o d i u m a z i d e (5 ~ g / p l a t e ,
1.5 1.1 1.0 0.7 0.6 0.8 1.3 0.9 2.1 0.9 1.2 0.9 0.8 1.0 0.8 1.0 0.7 0.9 1.2 1.0
I b
C a l 24.1
M a
Analysis
ACTIVATION
S C R E E N I N G O F M U T A G E N I C I T Y I N T H E C A I R I V E R I N T H E A R E A O F I N F L U E N C E O F T H E III P E T R O C H E M I C A L COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 1 0 0 I N T H E A B S E N C E O F M E T A B O L I C
TABLE 1
4~
0.9 1.0
-
2/88
0.9 0.9 0.9
1.4 1.0 0.9 1.1
-
-
1564- 8 1305:1 1225:31 1775:3
1325:6 1735:1 1765:20
1375:10 185 5:20
84 100 100 100
50 100 69
83 100
-
-
+
-
-
-
M
1.8 1.4 0.9 1.3
0.9 1.9 1.2
0.8 2.9
1.0 1.1 0.7
1.4 0.9 0.9 1.3 1.1
0.9 1.2
I
C a l 18.6
1995:29 1845: 1 1275:19 1404- 52
12642 3545:146 224-1- 30
1 1 6 + 10 395 5: 21
1105:12 2404- 11 1654- 52
249-t- 13 1225: 8 1215:15 16046 1435: 2 DNU
182:t: 21 149-t- 14
R/pl
73 100 91 23
100 100 80
100 92
78 93 90
61 100 80 100 100
61 54
S%
-
-
-
-
-
-
M
1.4 1.1 1.0 0.7
0.8 1.0 1.0
1.0 1.5
0.8 1.1 1.0
1.0 0.8 0.8 1.1 1.3 0.9
0.5 0.6
I
C a l 13.6
157+ 3 1525:6 1344- 2 1125:4
1134- 4 1945:11 2004-11
1425:24 273 4- 18
905:10 2325:10 2325:11
1935:5 100:1:5 1045:4 1335:7 161:1:42 1915:36
106+38 1085:8
R/pl
100 93 86 81
100 100 100
30 100
100 100 92
71 100 100 100 100 87
46 80
S%
-
-
-
-
-
-
M
1.0 1.1 1.0 1.2
0.9 1.0 0.9
1.0
0.8 0.9 1.0
0.6 0.9 0.8 1.5 1.1 0.9
0.3 0.5
I
C a l 13.3
1055:1 1505:18 1305:6 192-t- 6
1324- 3 1785:14 1634-11
1494-15 DNU
90+10 1905:19 2374- 8
102-1- 5 1165:15 1004- 1 1894-11 1335:11 1915:35
695:7 85-t- 8
R/pi
92 100 59 100
100 100 100
57
100 96 100
54 100 100 100 100 100
25 50
S%
-
-
-
-
-
-
M
1.9 1.0 1.1 0.8
0.8 1.2 1.5
0.8 1.3
0.8 0.7 1.3
0.9 1.2 1.0 0.8 1.4 0.7
0.4 0.5
I
C a l 10.6
2065:6 1405:1 1455:1 1205:6
123+ 4 2205:16 2734-10
1114-10 235 5:27
925:6 1604-33 2874-21
1125:18 1514- 9 1314- 1 955:10 1725:37 142+11
89+14 945:12
R/pl
F o r a definition o f t h e symbols, see T a b l e 1. T h e positive control w a s aflatoxin B 1 ( 0 . 5 / . ~ g / p l a t e , m e a n 748 + 118) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0 / ~ l / p l a t e , m e a n 157 + 41).
9/88
5/88 6/88 7/88 8/88
4/88
3/88
1/88
100 100
-
6/87 7/87 9/87 11/87
5/87
4/87
DNU 2105:10 1604-28
1.0 0.7
-
8/86 9/86 11/86
53 60 69 62 100 100
0.4 0.9 0.9 0.9 1.2 0.7
-
49 100
S%
745:7 1115:20 1195:16 110+19 1535:12 1465:23
147+18 161-1-30
-
R/pl
5/86 7/86
I
M
date
0.7 0.9
Ca124.1
Analysis
100 100 100 86
97 83 100
94 100
100 100 100
53 65 70 100 100 100
57 71
S%
SCREENING OF MUTAGENICITY IN THE CAi RIVER IN THE AREA OF INFLUENCE OF THE III PETROCHEMICAL COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 1 0 0 W I T H M I C R O S O M E A C T I V A T I O N
TABLE 2
-
0.8 1.4 1.0 0.7 0.7 0.8 0.8 1.3 0.6 0.9 1.0 0.8 0.9 0.9 1.1 0.7 0.7 0.9 1.0 1.9
25+ 1 58+ 9 34-+ 4 18_+ 3 20+ 2 21 + 2 29+ 5 32-+ 8 29+ 1 33+ 4 32+10 31 -+ 3 24_+ 3 33_+ 5 35+ 7 28 -+ 13 27-+ 4 33 _+ 5 41 _+ 5 47+ 3
71 97 100 78 79 64 100 64 100 70 100 93 85 78 47 100 92 96 100 100
+ + + + + 0.7 2.1 0.7 3.0 1.0 0.9 0.6 0.5 1.2 1.3 6.7 18.0
110.6 0.8 0.8 0.8 0.9 1.2 0.7
I
M
S%
C a l 18.6
R/pl
M
I
C a l 24.1
3760+70 33+ 2 28_+ 3 19_+ 1 25 + 2 30+ 6 24+ 2 DNU 31 + 4 76 -+ 16 20+ 7 119-+ 4 28-+ 9 35 + 3 27+ 0 20+ 5 44+ 5 5 2 + 13 266 + 44 445_+ 7
R/pl
100 96 100 80 80 100 73 100 100 100 87 100
100 76 100 100 69 61 100
S%
+ + + + + +
+ + + +
M 148.9 29.0 3.1 1.2 0.9 0.7 3.0 1.3 3.6 2.6 2.1 0.9 0.8 0.7 0.5 6.8 0.8 1.8 4.0 8.4
I
C a l 13.6
5063+194 871+138 110+ 8 29+ 3 25+ 4 17+ 1 106__+ 8 3 4 + 12 1 6 7 + 41 69_+ 3 65+ 5 37_+ 9 23+ 1 28:t: 2 21-+ 4 1 8 5 + 50 31+ 3 72_+ 8 161+ 4 2 1 8 + 73
R/pl 100 65 100 94 49 100 100 60 100 78 100 100 100 100 64 91 80 100 100 100
S% 0.7 1.0 0.9 0.8 0.8 0.9 0.6 4.0 3.0 0.8 0.7 0.7 2.2 1.2 0.6 0.7 1.1 1.3 0.9
+ -
I
+ + -
M
C a l 13.3
25+ 1 42+ 8 30+ 4 21+ 4 22+ 2 23+ 3 22+ 4 91+36 127+ 2 28+11 20+ 2 28_+10 DNU 83+ 9 50+ 1 26+ 3 27+ 8 42_+ 5 51+10 22+ 7
R/pl
100 38 60 75 45 100 100
82 70 86 100 100 100 77 76 100 100 93 100
S%
F o r a d e f i n i t i o n o f t h e symbols, see T a b l e 1. T h e positive c o n t r o l w a s 4 - n i t r o q u i n o l i n e ( 0 . 5 / ~ g / p l a t e , m e a n 253 _+31) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0 / ~ l / p l a t e ,
5/86 7/86 8/86 9/86 11/86 4/87 5/87 6/87 7/87 9/87 11/87 1/88 2/88 3/88 4/88 5/88 6/88 7/88 8/88 9/88
Analysis date
4.1 1.2 0.9 1.0 1.0 1.0 2.7 1.3 0.7 1.5 0.8 1.0 0.9 1.4 0.7 1.3 0.7 1.4 1.3 4.5
140+ 7 51+ 4 30+ 2 25+ 5 26+ I 25+ 4 93_+ 11 34+ 6 34_+11 59+ 9 26-+ 2 38+ 3 26+ 1 52-+ 8 28+ 4 56-+20 27+ 5 54_+ 1 53+ 4 117+ 6
m e a n 34 _+ 10).
+ + +
C a l 10.6
100 80 90 94 77 92 100 100 100 74 81 77 88 100 73 100 77 100 82 100
S C R E E N I N G O F M U T A G E N I C I T Y I N T H E C A d R I V E R I N T H E A R E A O F I N F L U E N C E O F T H E III P E T R O C H E M I C A L COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 9 8 I N T H E A B S E N C E O F M I C R O S O M E ACTIVATION
TABLE 3
1.3 2.4 0.9 1.4 0.6 0.9 1.6 1.2 1.0 1.3 0.9
+ -
R/pl
97 100 83 100 50 100 69 84 100 100 100
100 56 100 91 100 100 100 100
S% 2.4 2.0 1.0 0.9 1.0 0.9 1.1 0.7 1.2 0.5 2.6 1.9 1.0 1.3 1.2 1.4 0.9 1.8 1.1
+ + + -
I
M
1.9 0.7 0.9 1.1 1.2 1.3 0.7 1.9
-
88+ 6 37+ 1 45 + 5 55+ 2 58+ 2 46-t- 3 22+ 1 80+10 DNU 565:11 855:5 325:2 515:16 20:t: 4 335:4 615:25 445: 1 425: 0 535:14 405: 3
M
I
C a l 18.6
C a r 24.1
F o r a d e f i n i t i o n o f t h e symbols, see T a b l e 1. T h e positive c o n t r o l w a s a f l a t o x i n B 1 ( 0 . 5 / z g / p l a t e ,
5/86 7/86 8/86 9/86 11/86 4/87 5/87 6/87 7/87 9/87 11/87 1/88 2/88 3/88 4/88 5/88 6/88 7/88 8/88 9/88
Analysis date
100 100 90 100 92 100 100 80 73 100 91 20
67 100 100 100 80 100 73
S%
+ + -
+ + -
M
2.6 0.9 1.0 0.8 0.5 1.2 0.9 1.4 2.2 1.4 1.6
1.0 0.6 2.3 1.1 1.3 1.3 3.5 1.3
I 42+ 4 36+ 4 89 + 2 52+ 6 62+ 4 43+ 2 121+ 6 56+ 7 DNU 1175:5 40+ 3 345:0 295:7 195:1 465:13 335:1 505: 3 865:10 555:4 675:14
R/pl
100 92 30 100 100 100 100 100 93 86 81
67 54 33 100 100 100 100 100
S%
1.1 1.1 0.9 4.2 0.7 1.6 1.3
+ -
I 4.0 0.7 0.9 0.9 1.2 1.2 1.3 1.4 0.6 1.7 3.4 0.9
+ + -
M
C a r 13.3
184+ 8 40+ 4 45 + 2 46+ 6 59-1- 5 41+ 4 43+ 5 60+ 8 34+ 6 715:16 1535:9 325:6 DNU 405:1 395:5 355:4 1125: 4 305:12 515:8 535: 4
R/pl
100 100 100 100 100 59 100
41 40 96 100 100 100 100 100 100 96 100 57
S% -
M 1.1 0.4 1.5 0.7 1.1 0.8 0.9 1.0 0.8 1.4 1.5 0.9 1.0 1.0 1.0 1.2 0.6 1.1 1.3 0.9
I
C a l 10.6
m e a n 567 + 68) a n d t h e n e g a t i v e c o n t r o l w a s sterile distilled w a t e r ( 2 0 0 / z l / p l a t e , m e a n 40_+ 9).
110+20 110+ 7 38 + 2 48-t- 4 49+ 7 30+ 1 35+ 4 DNU 38+ 4 495:5 235:1 935:16 715:15 365:7 485:9 435:8 525: 3 395: 1 775:10 495:11
R/pl
C a t 13.6
47+3 21+1 74 + 1 36+1 54+9 26+3 31+1 40+5 40+1 585:3 665:7 325:9 375:1 36+7 385:0 465:3 225:0 485:1 505:4 385:3
R/pl
100 49 40 65 70 100 100 100 100 100 100 94 100 97 83 100 100 100 100 86
S%
S C R E E N I N G O F M U T A G E N I C I T Y I N T H E C A J R I V E R I N T H E A R E A O F I N F L U E N C E O F T H E III P E T R O C H E M I C A L COMPLEX OF RIO GRANDE DO S U L D U R I N G T H E P E R I O D F R O M M A Y 1986 T O S E P T E M B E R 1988 B Y T H E A M E S T E S T U S I N G S T R A I N T A 9 8 W I T H M I C R O S O M E A C T I V A T I O N
TABLE 4
38 from one duplicate assay plate w e r e streaked over histidine-free Ames plates with a sterile inoculating loop and then incubated for 48 h at 37°C. Only his ÷ organisms grow on the plates. The area study was conducted in two steps, an initial screening in 2000/zl of sample and a study of the dose-response curve for the sample found to have a mutagenic a n d / o r cytotoxic effect in the initial evaluation. The final criterion used to interpret and consider the results of the Ames test as positive was similar to that employed by McGeorge et al. (1983) and Valent et al. (1992): the number of revertants double that of the spontaneous rate and a reproducible dose-response curve. When one of these two criteria was not satisfied, the sample was considered to present signs of mutagenicity. Samples were considered to be cytotoxic when less than 60% cell survival was obtained in the cell viability test, and to be negative when mutagenicity of less than twice the spontaneous rate was detected in assays with more than 60% cell viability. The dose-response curves were evaluated by the SALMONEL program elaborated and kindly provided by Dr. Myers (Environmental Monitoring System Laboratory, EPA-Software version 2.3, April 1988) and described by Myers et al. (1991). This software predicts that the dose-response relationship of these assays can fit at least four regression models: CONSTANT v = b; LINEAR v = bx + a, LINTOX1 (linear attenuated by simple exponential toxicity) v = ( b x + a ) l X X ; LINTOX2 (linear attenuated by exponential toxicity elevated to the second power) y = (ax + b ) f "rx. The software also considers the Bernstein model (Bernstein et al., 1982) which consists of removing one or more doses from the analysis, using only results compatible with the linear model.
Screening analysis The initial screening of mutagenic samples was performed by the Ames test using strains TA100 and TA98 of S. typhimurium in the presence and absence of microsome fraction $9 for a 2000 /zl sample volume. Tables 1-4 summarize the results obtained in each assay for the different samples. It can be seen that the mutagenic index of 15 samples was much lower than the spontaneous rate (0.5, 0.6) with high cell survival (70% or more), and 14 samples had a low mutagenesis index associated with less than 70% cell survival in the viability test, distributed among the four assays. In general, there was a larger number of samples with mutagenicity in the direct test and a larger number of cytotoxic samples after metabolic activation. Tables 1 and 2 show that, for strain TA100, 14 samples were mutagenic and 3 were cytotoxic in the direct test (Table 1), and 1 was mutagenic and 14 were cytotoxic in the presence of $9 mix (Table 2). All mutagenic samples lost their activity in the presence of $9 mix. Table 3 summarizes the results obtained for strain TA98 in tests without metabolic activation. Twenty-one samples had positive mutagenic activity and 4 samples had a cytotoxic effect. Again, an increase in the number of cytotoxic samples (12) and a decrease in the number of samples with a mutagenic effect (11) were observed in the presence of $9 mix (Table 4). Pearson correlation analysis between the various mutagenesis tests showed a significant correlation between the mutagenesis rates detected for strains TA100 and TA98 in the absence of $9 (r = 0.567, p < 0.001; n = 94). However, in the presence of the $9 mix, the correlation was nonsignificant (r = 0.138, p -- 0.179; n = 96), indicating that these tests measure independent effects.
Results
Analysis of the dose-response curve The samples that showed mutagenic a n d / o r cytotoxic activity in the screening test were analyzed in smaller volumes (1000, 500, 200, and 100 /.d/plate) until the previously detected effect was no longer present. The dose-response curves for these samples were analyzed statistically using the SALMONEL software and have been described by Vargas (1992).
From May 1986 to September 1988 we studied 20 samplings from five collection sites in the Cai River previously analyzed and selected as basic for the study of contamination by mutagenic substances generated by the chemical wastes of this industrial complex of Rio Grande do Sul (Vargas et al., 1988).
39
16 +/-
0
TAtOO-S9mix
~
TA100+ S9mix
T , -t-
Revertonts/2ml Positive
-
Negative
+/-
14
- 2400 I I I 1 I I I I
Indicative
--
B
Bernstein
L
Linear
2100 I ! I I I I I !
L 2 Lintox 2
WM Without Model T
P 1800
12.
Z010E N
:S
T
,J <1
I I !
r~ > C3 Z 14. 0
8"
+/-
i I I I I I I I I I I I I I I I I I I I
m
6,
X W Z +
T
,-
4-/-
I , i
+/_
+
+
'
I
+
4-
+
'
+
I
t
~
I"
+T , I i I' I
i
I
iI ,i I I
;
I, i' m I
'
,','
'!
r1200
l-Z I-" n" Ld n"
"60o
t
,I-300 f
I
+1-
II 'i ! !
1' :
T
il
"1'
:
,"
,
T
:: "
Piooe 1,'.e 1~.8 ,~.e ~.6 ~.8 ~4., ,0.8,3.6 rs~ ,3.8 ,~.~ l~6 ,~.e , ~ Month Year
5 86
.5 86
7 86
5 87
6 87
7 87
7 87
7 87
7 87
1, 87
11 87
Model
B
L
L
WM
B
L
L
L
g
L
B
1 88
L2
2 88
5 88
L
L
! I I I I I I !
;~ o
,~.6,8.8 ,3.6 8 88
9 88
9 88
WM
B
L
Fig. 2. Mutagenic induction for the TA100 strain of S. typhimurium in the presence and absence of metabolic activation after treatment with different water samples from the Cal River.
40
pling. Fig. 2 illustrates the response obtained for strain TA100, with 11 samples classified as positive and 6 as indicative of mutagenicity (signs of mutagenicity) and the more elevated mutagenic peaks at collection sites 13.6 and 18.6. Fig. 3 shows the responses obtained for strain TA98, with the presence of 21 positive samples (12 in the direct test and 9 after metabolization), and 14 indicative of mutagenic induction (9 with and 5 without $9 mix) and the elevated mutagenic peaks at collection sites 24.1, 18.6, 13.6 and 13.3. Table 5 summarizes the responses to the Ames test obtained for each sampling point and in each mutagenesis assay at the different collection sites. The incidence of mutagenic samples was higher in the region located in front of the industrial complex, at collection sites Cai 18.6, Cai 13.6 and
The most frequent regression model for all tests was the linear one with a positive direction in the mutagenic samples (also the Bernstein model) and a negative direction in the cytotoxic samples. We also observed the presence of dose-response curves of the LINTOX2 regression model related to the cytotoxic effect and to combined cytotoxic effect and mutagenic induction. There were also cytotoxic samples with mutagenic activity with a positive (or Bernstein) linear dose-response curve. Figs. 2 and 3 summarize the results of mutagenic induction obtained for each test. The figures illustrate the mutagenesis rates for each sample (2000 tzl/plate), the number of revertants/2 ml for the cases in which it was possible to calculate it, and the place and date of sam-
l(a) 148 1
+/-
['7 TA98- Sgmix TI~ TA98÷Sgmix ~"
Rever tante/2ml
I
÷/-f
+-t-/B L L2 WM
100
Z 0 ~
+/_ --'1 ~
!
Positive Negative Indicative
Bcrnsteln Linear Lint°x2 Without Model
28 N
-J
:3 £3 ->
6
-180
£3 Z _
"r +/-- +
LL
+
U.I Z
o
+
i
2-
n.W > ILl
+
x
T
+
!.-...!
I
+/-
~./_
+T +/I
~
+
+/-
r F ofl +
:....-! +
:!
,;
0 Month Year Model
5 86 8
5 5 5 5 7 86 86 86 86 86 L WM WM B L
7 86 B
8 8 8 86 86 86 L L2 L
5 5 5 87 87 87 B L 8
(/) I-Z
6 7 7 9 87 87 87 87 L WM WM L
9 9 11 87 87 87 L WM 8
~60 i I
i ,
i i ,,/J o
11 11 87 87 L L
Fig. 3a. Mutagenic induction for the TA98 strain of S. typhimurium in the presence (A) and absence (B) of metabolic activation after treatment with different water samples from the Cai River.
41
(b)
i I I I~P420
14-
I
~oo
10"
8
g Z
Z
+/-
6
.I+/~'120 +1-
~
:
I:
2
i :: i:i i:
o
+
T
i
I
i
1 1
'1 i
I
i
I I
+
:
, '
,, i
il'.,,_I o
Fig. 3b. Continued.
Cal 13.3. Forty-two percent of all positive samples analyzed were from collection site Cal 13.6. The blank point in relation to the industrial complex, Cal 24.1, had the smallest number of mutagenic samples (6%) in the entire set of data. At Cal 10.6, located downstream, the percentage was 12%. The direct tests yielded 67% of all positive responses. Strain TA98 was the most sensitive for the samples both in tests carried out with and in those without the $9 metabolizing fraction (27 and 40%, respectively). In summary, the effects detected in the different tests corresponded to 53 responses o f mutagenic activity or indicative of this action, 35 of them in direct tests and 18 in tests after metabolization. Table 6 presents a general picture of the 20 samplings in terms of the presence of induced mutation, classifying the sample as positive, negative, with signs of mutagenicity a n d / o r cytoxicity per sampling point (when the effect was present
in at least one test). It can be seen that the number of positive samples increased from the blank point (Cal 24.1) to the site of greatest influence (Cal 13.6), decreasing again downstream from the area under study. The cytoxicity was already present at the blank point at values of the same order of magnitude as those for the Cal 13.6 site (5 cases). The site with the largest number of cytotoxic samples was Cal 13.3 (7 cases). The results of 5 analyses using strain TA102 (this strain detects a variety of oxidative mutagens, active forms of oxygen and alkylating agents, and has an intact excision repair pathway) (Levin et al., 1982b) in the presence and absence of $9 mix (data not shown) showed the presence of three samples with low cell survival (sites 24.1, 18.6 and 13.6) and one with weak mutagenicity (site 13.6) in tests without metabolic activation. The dose-response curve for the positive sample presented a linear regression model with the estimate of 112 rev/ml. Discussion
The criterion used for the initial screening of induced mutagenesis (doubling of the spontaneous rate) is classically applied in studies for the diagnosis of mutagenicity. However, the most important premise for defining the presence of induction is an increase in the number of mutant colonies that follows a dose-response curve (for reviews, see Bernstein et al., 1982; Maron and Ames, 1983; McCann et al., 1984; Claxton et al., 1987). The shape of the dose-response curve basically depends on the dynamics between mutagenicity and cytoxicity (for review Bernstein et al., 1982; Van der Hoeven et al., 1990). However, in some samples the concentrations tested did not show a dose-response relationship despite the fact that the number of revertants was more than double the spontaneous rate. This fact was observed in 27.7% of the positive responses detected in the screening tests for strains TA100 (6.4%) and TA98 (21.3%). These responses obtained for strain TA98, which do not fit the regression models considered, presented a characteristic response pattern, i.e., a peak of mutagenic induction at the dose of 2000 /~1 (Fig.
42 TABLE 5 SUMMARY OF THE RESPONSES TO THE AMES TEST OBTAINED FOR SAMPLING POINT AND MUTAGENESIS ASSAY Samples
Mutagenic c
Cal 24.1
+ -
18.6
+ -
13.6
+ -
13.3
+ -
10.6
+ -
PT e GT f %
+ + -
T+ g
Mutagenesis assay
Total
TA100
TA100S
TA98
TA98S
1 19 3 (1) 16 6 (1) 14 2 17 2 (2) 18
0 19 2 (1) 17 1 (1) 19 0 19 0 20
0 20 5 (3) 14 10 (4) 10 3 (1) 16 3 (1) 17
2 (1) a 17 3 16 5 (1) 14 3 (2) 16 1 (1) 19
14 84 98 14 86 27%
3 94 97 3 97 6%
21 77 98 21 79 40%
14 82 96 15 85 27%
Partial 3 75 13 63 22 57 8 68 6 74 52 337 389
General
%a
78
4 96 17 83 28 72 11 89 8 92
76 79 76 80
%T + b 6% 25% 42% 15% 12%
13 87
a Percentage of positive and negative results. b %T +, percentage calculated of all positive samples. c +, mutagenic; - , non-mutagenic. d Indicative responses added in all positive samples. e Partial total. f General total. g Percentage of positive tests.
3A, B). T h i s o c c u r r e n c e was also m o r e f r e q u e n t for d i r e c t l y a c t i n g m u t a g e n s . T h e s e o b s e r v a t i o n s suggest t h a t s o m e specific f a c t o r m a y b e a c t i n g o n the chemical interaction with the test organism. T h e m o s t f r e q u e n t d o s e - r e s p o n s e curve m o d e l was l i n e a r r e g r e s s i o n . T h i s r e s p o n s e a g r e e s w i t h t h e results o b t a i n e d b y V a l e n t (1992) in a study o n w a t e r b o d i e s in t h e s t a t e o f S~o P a u l o using a similar m e t h o d o l o g y for statistical e v a l u a t i o n . T h e p r e s e n c e o f a cytotoxic a c t i o n r e s u l t e d in a n e g a tive m o d e l o f l i n e a r r e g r e s s i o n , L I N T O X 2 , o r even a positive l i n e a r m o d e l , i n d i c a t i n g t h e p r e s e n c e o f signs o f m u t a g e n i c i t y . D u r i n g t h e 20 m o n t h s o f s a m p l i n g p e r f o r m e d in t h e p r e s e n t study, only two p e r i o d s d i d n o t p r e s e n t a m u t a g e n i c o r toxic effect. C o n s i d e r i n g t h e r e s p o n s e to b e positive w h e n m u t a g e n i c i t y o r cytoxicity was p r e s e n t in at l e a s t o n e assay ( T a b l e 6), w e o b t a i n e d 34 m u t a g e n i c s a m p l e s a n d 22 cytotoxic s a m p l e s . S e v e r a l s t u d i e s have r e p o r t e d t h a t t h e p r e s e n c e o f cytoxicity c a n b e d e t e c t e d by
a fall in i n d u c t i o n b e l o w e x p e c t e d s p o n t a n e o u s levels. I n this s t u d y w e d e t e c t e d 15 s a m p l e s w h o s e m u t a g e n e s i s i n d e x was m u c h l o w e r t h a n t h e s p o n t a n e o u s r a t e ( M I = 0.5 a n d 0.6), with elev a t e d ( m o r e t h a n 7 0 % ) cell survival in t h e f o u r assays p e r f o r m e d . Similarly, l o w e r i n d i c e s w e r e o b s e r v e d in 14 s a m p l e s , f o l l o w e d by l o w e r cell survival a n d t h e r e f o r e c h a r a c t e r i z i n g a cytoxic effect. O v e r a l l analysis o f t h e d a t a ( T a b l e 5) s h o w e d a l a r g e r n u m b e r o f s a m p l e s with m u t a genicity in d i r e c t tests a n d a l a r g e r n u m b e r o f s a m p l e s w i t h low cell survival in tests c a r r i e d o u t in t h e p r e s e n c e o f $9 mix (25 o f 32 = 78%). T h e s t r a i n m o s t sensitive was T A 9 8 with a n d w i t h o u t $9 mix. T h e u s e o f m e t a b o l i c activation elimin a t e d t h e m u t a g e n i c effect o f c h e m i c a l subs t a n c e s which d a m a g e d D N A by b a s e p a i r substit u t i o n in all s a m p l e s t h a t w e r e positive in t h e d i r e c t test. H o w e v e r , t h e m u t a g e n i c s u b s t a n c e s that caused frameshift damage presented mutag e n i e i n d u c t i o n , o r at l e a s t signs o f this action,
43
TABLE
6
GENERAL DUCED
OF
MUTATION
BILITY LEAST
PICTURE
WHEN ONE
Date
THE
AND
THE
PRESENCE
DECREASE
EFFECT
WAS
OF
IN CELL
PRESENT
INVIA-
IN
AT
TEST
C a l 24.1 C a l 18.6 C a l 13.6 C a l 13.3 C a l 10.6
1986 May
-T
+
+T
±T
+T
Jul.
-T
+T
±T
-T
-T
Aug.
-T
-
+T
-T
+T
Sept.
-
.
Nov.
-
-
-
-
Apr.
-
.
May
-
-
+
-
__.
Jun.
-
DNU
-
+
+
Jul.
+
+
+
+
-
Sept.
-
+
+
-
-
Nov.
+
+
+
.
.
.
- T
1987 .
-
.
.
-
1988 Jan.
-
+
+ T
- T
-
Feb.
-
+
-
DNU
-
Mar.
+ T
-
-
+
-
Apr.
-T
-
-
-T
-
May
-
-
+
-
-
Jun.
-
-
-
+_
-
Jul.
-
-
+
- T
-
Aug.
-
5:
_+
- T
-
Sept.
-
+ T
+
-
+
3
8
12
6
5
17
11
8
13
15
+/± Total % T +/+,
20
19
20
19
20
+
15%
42%
60%
32%
25%
-
85%
58%
40%
68%
75%
5
2
5
7
3
mutagenic/or
indicative of mutagenicity;
34 98
22 -,
non-
m u t a g e n i c ; T, d e c r e a s e in cell viability; D N U , d a t a n o t usable.
with a considerably variable pattern. Metabolization led to a loss of mutagenic effect in 62% of cases, whereas the action persisted in 24% and toxicity was present in 14% of the samples that were positive in the direct test. It was also possible to detect samples with mutagenicity after metabolization in 33% of the positive tests, and 29% of these gave a positive response also in tests carried out in the absence of the $9 microsome fraction. This variable pattern of response is due to the mixture of substances which are part of environmental samples and represents addi-
tional evidence for the fact that the mutagenic and toxic activity detected in these assays is not the sum of the individual effects of each substance. In addition to synergism and antagonism among chemical products, other complications may occur due to the interactions between the mutagenic and antimutagenic agents present. These factors contribute to the ambiguity of the results of the tests (Saxena, 1984) and may also be related to a decrease in spontaneous mutation rates. The higher incidence of direct mutagens which cause frameshift DNA damage has proved to be considerably constant in river samples (Pelon et al., 1977; Grabow et al., 1980; Maruoka and Yamanaka, 1980, 1982; Van Kreijl et al., 1980; Martins et al., 1982; Valent, 1992). As to analysis after metabolic activation, data in the literature have shown that a decrease, or even inactivation of mutagenicity may occur in the presence of the $9 mix fraction (Saxena and Schwartz, 1979; Flanagan and Allen, 1981; Grimm-Kibalo et al., 1981; Valent, 1992). However, some investigators have observed elevation of mutagenicity in rivers after metabolization (for a review, see Meier, 1988). Houk (1992) also emphasized the frequency of directly acting mutagens which cause frameshift DNA damage in petrochemical industry wastewaters. Courtois et al. (1992) have argued that the $9 mix-dependent decrease in mutagenicity of environmental mixtures might not be due only to enzymatic mechanisms. Passive interactions of chemicals with proteins are probably responsible for the decrease leading to the non-expression of the mutagenicity of some direct and indirect mutagens. In mechanistic terms, such interactions seem to be a result of competition at the enzymatic sites rather than of conversion of compounds into non-functional metabolites. It should also be emphasized that complex water samples obtained from water basins are a sum of the chemical, physical and biological interactions of this ecosystem. Thus, the contaminants may already be present in a form differing from that which they had when first dumped into the river. The greater direct action of genotoxic substances in this type of sample may be due to the activity of reactive electrophilic groups generated by organisms present in the water.
44
The selection of sampling sites with the specific objective of evaluating the influence of wastes released by an industrial park led to the observation of sites with more or less constant induced mutagenesis responses. Analysis by collection site showed a lower incidence of positive samples at the blank site, and a clear elevation in number in the zone of influence of the industrial complex, with the Cal 13.6 site presenting the highest frequency of such samples. However, an important decrease in this action was observed in the downstream direction. Sites Cal 18.6, 13.6 and 13.3 were the most heavily involved. These sampling points are located close to the areas of wastewater disposal and to the rainwater accumulation and safety basins. A previous study by our group (Vargas et al., 1988) in this internal area of the industrial complex showed that these sites were contaminated with substances which generated base pair substitutions a n d / o r frameshift errors. The presence of cytotoxic activity at the blank site at levels similar to those detected at Cal 13.6 indicates contamination by biologically active substances. However, a higher percentage of cytotoxicity was observed at the collection site Cal 13.3, also considerably contaminated with mutagens. This toxicity was mainly observed in tests using microsome fraction $9, showing the activity of the metabolites of chemical substances present in the river. However, on the basis of the responses obtained in the present study, we may infer that the methodology used permitted the delimitation of the area of influence of the complex and the release of mutagens that were active and sensitive to the strains employed.
Acknowledgements We are grateful to the students who participated in the experimental part of this study as Scientific Initiation fellows, S. Bresolin and R.R. Guidobono in particular. We are also indebted to the sampling team of FEPAM, especially L.R. Gemelli and C.A. Peixoto, for their dedication. This research was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecno16gico (CNPq), Funda~fio de Amparo h Pesquisa
do Estado do Rio Grande do Sul (FAPERGS), and Financiadora de Projetos (FINEP).
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