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Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Mutagenicity of trinitrotoluene and its metabolites formed during composting a
b
b
E. L. Tan , C. H. Ho , W. H. Griest & R. L. Tyndall
c
a
Chemistry Department, University of Tennessee, Knoxville, Tennessee b
Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee c
Health and Safety Research Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, Tennessee, 37831 Published online: 20 Oct 2009.
To cite this article: E. L. Tan , C. H. Ho , W. H. Griest & R. L. Tyndall (1992): Mutagenicity of trinitrotoluene and its metabolites formed during composting, Journal of Toxicology and Environmental Health: Current Issues, 36:3, 165-175 To link to this article: http://dx.doi.org/10.1080/15287399209531632
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
MUTAGENICITY OF TRINITROTOLUENE AND ITS METABOLITES FORMED DURING COMPOSTING
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
E.L.Tan University of Tennessee, Chemistry Department, Knoxville, Tennessee C. H. Ho, W. H. Griest Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee R. L. Tyndall Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
TNT was mutagenic for Salmonella typhimurium without the need of a rat liver metabolic activation system (S9). The mutagenic potency of TNT decreased in proportion to the number of nitro groups that were reduced to the amino form. The presence of a nitro group on the 4 position of the diamino congener is necessary for mutagenicity. Among the active congeners, mutagenicity was generally greater for TA100 than TA98, except that for the 4-amino congener the reverse was true. In cases when S9 was included in the assay, there was always a decrease in the number of mutants induced as compared with those without S9. Tetryl behaved like TNT, except that it was approximately three times more potent. RDX and HMX were not mutagenic under the conditions of the assay. When TNT was composted, the major metabolites identified in organic extracts of compost samples were the 2-amino and 4-amino congeners. An acetonitrile extract of compost was tested and found to be more mutagenic for TA98 than TA100, much like the authentic 4-amino congener, but the amount of this congener in the extract did not account for the degree of mutagenicity.
INTRODUCTION Trinitrotoluene (TNT), Tetryl (N-methyI-A/-2,4,6-tetranitroaniline), RDX (1,3,5-trinitrohexahydro-1,3,5-triazine), and HMX (1,3,5,7-tetranitrooctahydro-1,3,5,7-tetrazocine) are explosives commonly used by the U.S. Research sponsored by the U.S. Army Biomedical Research and Development Laboratory (Dr. W. D. Burrows, COR), Interagency Agreement 1016-B123-A1, under U.S. Department of Energy contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. The authors gratefully acknowledge the expert technical assistance of Elicia Bowman and Kevin Ironside. Requests for reprints should be sent to R. L. Tyndall, Health and Safety Research Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37831. 165 Journal of Toxicology and Environmental Health, 36:165-175, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
166
E. TANETAL.
Army, but the mutagenicity of these compounds has not been studied extensively. TNT is probably the best studied and has been shown to be mutagenic in a number of in vitro test systems (Spanggord et al., 1982; Styles and Cross, 1983; Whong and Edwards, 1984; Won et al., 1976). Owing to widespread contamination of these explosives in the environment of ammunition plants, a need for remedial action became apparent. A cost-effective method to achieve this action was desirable, and composting showed promise as a viable process (Williams et al., 1988; Williams, 1991). That environmental microorganisms can break down organic compounds is well documented and has led to attempts to use composting as a technique to degrade explosives (Sisk et al., 1989). A laboratory study using organisms isolated from areas contaminated with TNT showed that TNT was completely "dissimilated" within 24 h into a variety of organic compounds (Won et al., 1974). Another study showed that in soil "aged" with radiolabeled TNT, the major components detected by HPLC analysis were TNT, 2-amino-4,6-dinitrotoluene, and 4amino-2,6-dinitrotoluene. Thus the purpose of this study was to compare the mutagenicity of TNT transformation products with TNT, Tetryl, HMX, and RDX preparatory to studies on the effects of composting these explosives. Preliminary studies of composted explosive-contaminated soil were also initiated. MATERIALS AND METHODS
Mutagenicity assays were carried out according to the Salmonella/ mammalian microsome plate incorporation method described by Ames et al. (1975) on strains TA98 and TA100, both of which were obtained from Dr. B. N. Ames (University of California, Berkeley). The stability of the test strains was verified periodically by demonstrating their sensitivity to ultraviolet light and crystal violet and their resistance to ampicillin. Rat liver homogenate S9 was prepared from Aroclor 1254-induced male SpragueDawley rats and was purchased from Litton Bionetics (Charleston. S.C.). The homogenate was used at a concentration of 1 mg protein per plate. The 2-amino-4,6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT), 2,4-diamino-6-nitrotoluene (2,4A6NT), 2,6-diamino-4nitrotoluene (2,6A4NT), 2NF (2-nitrofluorene), and 2AAF (2acetylaminofluorene) were obtained from Aldrich Chemical Co (Milwaukee, Wis.). The purity of TNT and its metabolites was greater than 99% as determined by high-performance liquid chromatography (HPLC) methods, and they were used without further purification. TNT, tetryl, RDX, and HMX were obtained from the U.S. Army Toxic and Hazardous Material Agency (Edgewood, Md.). They were of Standard Analytical Reference Materials quality and were used without further purification. The composts tested in this study were generated at the Umatilla
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
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167
Army Depot Activity (UMDA) at Umatilla, Ore., by Roy F. Weston, Inc. The field composting is reported in detail elsewhere (Williams, 1991). Dried and homogenized aliquots of the composts were shipped to Oak Ridge National Laboratory (ORNL), where they were stored in the dark at 4°C. For analyses of explosives and TNT metabolites, 1 g compost was extracted with 4 ml acetonitrile for 18 h in an ultrasonic bath with cooling, and the supernatant was recovered after the solids settled out. Explosives and TNT metabolites were determined using the mixed-mode, anion exchange/reverse-phase HPLC method described in a previous report (Griest et al., 1990). This method has received a USATHAMA Level IB Certification (USATHAMA QA Program, 1987). For mutagenicity testing, 4 g compost was extracted with 20 ml acetonitrile, and 10 ml of the supernatant was evaporated to dryness in a rotary evaporator. RESULTS Authentic standards of TNT and its two major metabolites were assayed for mutagenicity in the presence or absence of a rat liver metabolic activation system (S9). Figure 1 shows that S9 decreased the mutagenicity of TNT and that TNT also caused more revertants in TA100 than TA98. Also, S9 caused a decrease in mutagenicity of the TNT metabolites 2ADNT and 4ADNT. In comparing the number of revertants induced in the two bacterial strains, 4ADNT caused more revertants in TA98 than in TA100, while the opposite was true for 2ADNT (Fig. 2 and Table 1). The reduction of the nitro groups of TNT to the amino products caused a decrease in the mutagenic activity of the compounds, and this decreased in proportion to the number of amino groups formed (Fig. 3 and Table 1). It appears that when a single nitro group is present, it needs to be in the 4 position to elicit a substantial mutagenic response. Tetryl was about three times more mutagenic than TNT (Table 1), but like TNT, tetryl induced more mutants in strain TA100 than TA98, and S9 decreased the number of induced revertants (Fig. 4). The two explosives HMX and RDX, when tested up to concentrations of 1 mg/plate, did not induce revertants in either bacterial strain (results not shown). The acetonitrile extracts of various compost samples yielded considerable mutagenic activity (Table 2). Analysis showed a marked reduction in mutagenic activity over the 90-d composting period. The 10% and 20% (volume percent) soil compost samples showed over an 80% reduction in mutagenic activity. As was seen in compost samples from a different location (Griest et al., 1990) and in TNT per se, the presence of the S9 activation system reduced the ability to detect mutagenic activity with TA98 and TA100 Salmonella. Data presented here are only for experiments without S9. The mutagenic activity of zero time samples was more pronounced with the TA-100 test strain, while the reverse was true with
168
E. TAN ET AL.
1400
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1200 —
1000 —
iu
"5 800 —
600 — LJJ
400 —
200
-
100
150
200
ng/PLATE FIGURE 1. Mutagenicity of TNT in S. typhimurium: TA98 with (O) and without (•) S9; TA100 with (A) and without (A) S9.
the 90-d samples. Concomitant with decreases in mutagenic activity over the 90-d composting period, a marked decrease in concentration of TNT also was observed (Table 3). An attempt to estimate the mutagenic activity of the acetonitrile extract based on the known components in the extract is shown in Table 4. While a greater percentage of mutagenicity is accounted for by TNT and its metabolites at 0 d compared with 90 d, in both cases it represents a minority of the total mutagenicity.
MUTAGENICITY OF TNT AND METABOLITES
169
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500
100
200 Hg/PLATE
300
400
(a) FIGURE 2. Mutagenicity of 2ADNT and 4ADNT in S. typhimurium TA98 and TA100. Circles represent the 2-amino compound and triangles represent the 4-amino compound. Open symbols are in the presence of S9 and closed symbols in the absence of S9. (a) TA98.
DISCUSSION
The four possible mono- and diamino metabolites of TNT studied in this work were all less mutagenic than the parent compound (Table 1). Tetryl was approximately three times more mutagenic than TNT and was similar to TNT in that it induced more revertants in TA100 than TA98 and inclusion of S9 in the assay system decreased its mutagenicity. This is consistent with earlier work (Whong et al., 1980) except that we also found that Tetryl was mutagenic to TA98. We also confirmed that RDX and HMX are not mutagenic to either TA98 or TA100. Of all the metabolites tested, only 4ADNT was more mutagenic to TA98 relative to TA100. Many nitro compounds have been shown to be genotoxic when converted to their ultimate form by nitroreductases (Blumer et al., 1980; McCoy et al., 1981; Rosenkrantz and Mermelstein, 1983; Sugimura et al., 1966). Similarly, TNT has been shown to be dependent on nitroreductase
E. TAN ET AL.
170
700
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600 —
500
-
400
-
LLJ
£
300 —
200 —
100
200
100
300
400
ug/PLATE (b) FIGURE 2. Mutagenicity of 2ADNT and 4ADNT in S. typhimurium TA98 and TA100 [Continued). Circles represent the 2-amino compound and triangles represent the 4-amino compound. Open symbols are in the presence of S9 and closed symbols in the absence of S9. (6) TA100.
activity in order to exert its mutagenic effect on Salmonella typhimurium (Spanggord et al., 1982; Whong and Edwards, 1984). Both groups of investigators have found that TNT is more active toward strain TA100 than TA98 by approximately a factor of two. Spanggord et al. (1982) spectulated that this might relate to the twofold increase in nitroreductase activity of TA100 relative to that of TA98 as reported by Blumer et al. (1980).
MUTAGENICITY OF TNT AND METABOLITES
171
TABLE 1. Mutagenic Potencies of Explosives and Their Metabolites in Salmonella Mutagenic potencies, revertants/plate/mg compounc]
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
TA98 Compound
-S9
TNT 2ADNT 4ADNT 2,4DA6NT 2,6DA4NT Tetryl
5400 500 1000 0 325 8200
TA100 + S9 380 125 275 NTa NT 700
-S9
+ S9
6600 2300 500 0 750 23,000
2000 1233 200 NT NT 3750
a
NT, not tested.
300
50
100
150
200
250
jig/ PLATE FIGURE 3. Mutagenicity of 2,6DA4NT and 2,4DA6NT in S. typhimurium. Circles represent 2,6DA4NT and triangles represent 2,4DA4NT. Open symbols represent the absence of S9 and closed symbols indicate the presence of S9.
E. TAN ET AL.
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600
40
80
120
160
200
ug/PLATE FIGURE 4. Mutagenicity of Tetryl in 5. typhimurium. TA98 with (A) and without (A) S9; TA100 with (O) and without (•) S9.
Our results show the same magnitude of two to three times more revertants in TA100 than TA98. When calculations were made from a concentration response curve (Fig. 2 and Table 1), the decrease in activity due to S9 was 86% for TA98 and 76% for TA100. We find this trend in all our experiments involving TNT and rat liver S9. Other investigators have reported that S9 either TABLE 2. Specific Mutagenicity for Acetonitrile Extracts of Test Composts Specific mutagenicity, revertants/g, average ± SDa Volume percent soil in compost
Days of composting
TA98 without S9
TA100 without S9
10%
0 90 0 90
87,200 14,300 310,000 21,600
100,000 12,800 546,000 14,200
20%
determined on a compost wet weight basis.
± ± ± ±
5390 530 30,700 360
± ± ± ±
2750 1140 25,200 1100
MUTAGENICITY OF TNT AND METABOLITES
173
TABLE 3. Explosives and TNT Metabolites Analyses of Compost Piles Concentration,3 average ± SD, mg/kg
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Sample 10% Contaminated soil, d 0 10% Contaminated soil, d 90 20% Contaminated soil, d 0 20% Contaminated soil, d 90
2,6,DA4NT
2,4,DA6NT
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Mutagenicity of trinitrotoluene and its metabolites formed during composting a
b
b
E. L. Tan , C. H. Ho , W. H. Griest & R. L. Tyndall
c
a
Chemistry Department, University of Tennessee, Knoxville, Tennessee b
Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee c
Health and Safety Research Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, Tennessee, 37831 Published online: 20 Oct 2009.
To cite this article: E. L. Tan , C. H. Ho , W. H. Griest & R. L. Tyndall (1992): Mutagenicity of trinitrotoluene and its metabolites formed during composting, Journal of Toxicology and Environmental Health: Current Issues, 36:3, 165-175 To link to this article: http://dx.doi.org/10.1080/15287399209531632
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
MUTAGENICITY OF TRINITROTOLUENE AND ITS METABOLITES FORMED DURING COMPOSTING
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
E.L.Tan University of Tennessee, Chemistry Department, Knoxville, Tennessee C. H. Ho, W. H. Griest Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee R. L. Tyndall Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
TNT was mutagenic for Salmonella typhimurium without the need of a rat liver metabolic activation system (S9). The mutagenic potency of TNT decreased in proportion to the number of nitro groups that were reduced to the amino form. The presence of a nitro group on the 4 position of the diamino congener is necessary for mutagenicity. Among the active congeners, mutagenicity was generally greater for TA100 than TA98, except that for the 4-amino congener the reverse was true. In cases when S9 was included in the assay, there was always a decrease in the number of mutants induced as compared with those without S9. Tetryl behaved like TNT, except that it was approximately three times more potent. RDX and HMX were not mutagenic under the conditions of the assay. When TNT was composted, the major metabolites identified in organic extracts of compost samples were the 2-amino and 4-amino congeners. An acetonitrile extract of compost was tested and found to be more mutagenic for TA98 than TA100, much like the authentic 4-amino congener, but the amount of this congener in the extract did not account for the degree of mutagenicity.
INTRODUCTION Trinitrotoluene (TNT), Tetryl (N-methyI-A/-2,4,6-tetranitroaniline), RDX (1,3,5-trinitrohexahydro-1,3,5-triazine), and HMX (1,3,5,7-tetranitrooctahydro-1,3,5,7-tetrazocine) are explosives commonly used by the U.S. Research sponsored by the U.S. Army Biomedical Research and Development Laboratory (Dr. W. D. Burrows, COR), Interagency Agreement 1016-B123-A1, under U.S. Department of Energy contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. The authors gratefully acknowledge the expert technical assistance of Elicia Bowman and Kevin Ironside. Requests for reprints should be sent to R. L. Tyndall, Health and Safety Research Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37831. 165 Journal of Toxicology and Environmental Health, 36:165-175, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
166
E. TANETAL.
Army, but the mutagenicity of these compounds has not been studied extensively. TNT is probably the best studied and has been shown to be mutagenic in a number of in vitro test systems (Spanggord et al., 1982; Styles and Cross, 1983; Whong and Edwards, 1984; Won et al., 1976). Owing to widespread contamination of these explosives in the environment of ammunition plants, a need for remedial action became apparent. A cost-effective method to achieve this action was desirable, and composting showed promise as a viable process (Williams et al., 1988; Williams, 1991). That environmental microorganisms can break down organic compounds is well documented and has led to attempts to use composting as a technique to degrade explosives (Sisk et al., 1989). A laboratory study using organisms isolated from areas contaminated with TNT showed that TNT was completely "dissimilated" within 24 h into a variety of organic compounds (Won et al., 1974). Another study showed that in soil "aged" with radiolabeled TNT, the major components detected by HPLC analysis were TNT, 2-amino-4,6-dinitrotoluene, and 4amino-2,6-dinitrotoluene. Thus the purpose of this study was to compare the mutagenicity of TNT transformation products with TNT, Tetryl, HMX, and RDX preparatory to studies on the effects of composting these explosives. Preliminary studies of composted explosive-contaminated soil were also initiated. MATERIALS AND METHODS
Mutagenicity assays were carried out according to the Salmonella/ mammalian microsome plate incorporation method described by Ames et al. (1975) on strains TA98 and TA100, both of which were obtained from Dr. B. N. Ames (University of California, Berkeley). The stability of the test strains was verified periodically by demonstrating their sensitivity to ultraviolet light and crystal violet and their resistance to ampicillin. Rat liver homogenate S9 was prepared from Aroclor 1254-induced male SpragueDawley rats and was purchased from Litton Bionetics (Charleston. S.C.). The homogenate was used at a concentration of 1 mg protein per plate. The 2-amino-4,6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT), 2,4-diamino-6-nitrotoluene (2,4A6NT), 2,6-diamino-4nitrotoluene (2,6A4NT), 2NF (2-nitrofluorene), and 2AAF (2acetylaminofluorene) were obtained from Aldrich Chemical Co (Milwaukee, Wis.). The purity of TNT and its metabolites was greater than 99% as determined by high-performance liquid chromatography (HPLC) methods, and they were used without further purification. TNT, tetryl, RDX, and HMX were obtained from the U.S. Army Toxic and Hazardous Material Agency (Edgewood, Md.). They were of Standard Analytical Reference Materials quality and were used without further purification. The composts tested in this study were generated at the Umatilla
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
MUTAGENICITY OF TNT AND METABOLITES
167
Army Depot Activity (UMDA) at Umatilla, Ore., by Roy F. Weston, Inc. The field composting is reported in detail elsewhere (Williams, 1991). Dried and homogenized aliquots of the composts were shipped to Oak Ridge National Laboratory (ORNL), where they were stored in the dark at 4°C. For analyses of explosives and TNT metabolites, 1 g compost was extracted with 4 ml acetonitrile for 18 h in an ultrasonic bath with cooling, and the supernatant was recovered after the solids settled out. Explosives and TNT metabolites were determined using the mixed-mode, anion exchange/reverse-phase HPLC method described in a previous report (Griest et al., 1990). This method has received a USATHAMA Level IB Certification (USATHAMA QA Program, 1987). For mutagenicity testing, 4 g compost was extracted with 20 ml acetonitrile, and 10 ml of the supernatant was evaporated to dryness in a rotary evaporator. RESULTS Authentic standards of TNT and its two major metabolites were assayed for mutagenicity in the presence or absence of a rat liver metabolic activation system (S9). Figure 1 shows that S9 decreased the mutagenicity of TNT and that TNT also caused more revertants in TA100 than TA98. Also, S9 caused a decrease in mutagenicity of the TNT metabolites 2ADNT and 4ADNT. In comparing the number of revertants induced in the two bacterial strains, 4ADNT caused more revertants in TA98 than in TA100, while the opposite was true for 2ADNT (Fig. 2 and Table 1). The reduction of the nitro groups of TNT to the amino products caused a decrease in the mutagenic activity of the compounds, and this decreased in proportion to the number of amino groups formed (Fig. 3 and Table 1). It appears that when a single nitro group is present, it needs to be in the 4 position to elicit a substantial mutagenic response. Tetryl was about three times more mutagenic than TNT (Table 1), but like TNT, tetryl induced more mutants in strain TA100 than TA98, and S9 decreased the number of induced revertants (Fig. 4). The two explosives HMX and RDX, when tested up to concentrations of 1 mg/plate, did not induce revertants in either bacterial strain (results not shown). The acetonitrile extracts of various compost samples yielded considerable mutagenic activity (Table 2). Analysis showed a marked reduction in mutagenic activity over the 90-d composting period. The 10% and 20% (volume percent) soil compost samples showed over an 80% reduction in mutagenic activity. As was seen in compost samples from a different location (Griest et al., 1990) and in TNT per se, the presence of the S9 activation system reduced the ability to detect mutagenic activity with TA98 and TA100 Salmonella. Data presented here are only for experiments without S9. The mutagenic activity of zero time samples was more pronounced with the TA-100 test strain, while the reverse was true with
168
E. TAN ET AL.
1400
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
1200 —
1000 —
iu
"5 800 —
600 — LJJ
400 —
200
-
100
150
200
ng/PLATE FIGURE 1. Mutagenicity of TNT in S. typhimurium: TA98 with (O) and without (•) S9; TA100 with (A) and without (A) S9.
the 90-d samples. Concomitant with decreases in mutagenic activity over the 90-d composting period, a marked decrease in concentration of TNT also was observed (Table 3). An attempt to estimate the mutagenic activity of the acetonitrile extract based on the known components in the extract is shown in Table 4. While a greater percentage of mutagenicity is accounted for by TNT and its metabolites at 0 d compared with 90 d, in both cases it represents a minority of the total mutagenicity.
MUTAGENICITY OF TNT AND METABOLITES
169
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500
100
200 Hg/PLATE
300
400
(a) FIGURE 2. Mutagenicity of 2ADNT and 4ADNT in S. typhimurium TA98 and TA100. Circles represent the 2-amino compound and triangles represent the 4-amino compound. Open symbols are in the presence of S9 and closed symbols in the absence of S9. (a) TA98.
DISCUSSION
The four possible mono- and diamino metabolites of TNT studied in this work were all less mutagenic than the parent compound (Table 1). Tetryl was approximately three times more mutagenic than TNT and was similar to TNT in that it induced more revertants in TA100 than TA98 and inclusion of S9 in the assay system decreased its mutagenicity. This is consistent with earlier work (Whong et al., 1980) except that we also found that Tetryl was mutagenic to TA98. We also confirmed that RDX and HMX are not mutagenic to either TA98 or TA100. Of all the metabolites tested, only 4ADNT was more mutagenic to TA98 relative to TA100. Many nitro compounds have been shown to be genotoxic when converted to their ultimate form by nitroreductases (Blumer et al., 1980; McCoy et al., 1981; Rosenkrantz and Mermelstein, 1983; Sugimura et al., 1966). Similarly, TNT has been shown to be dependent on nitroreductase
E. TAN ET AL.
170
700
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600 —
500
-
400
-
LLJ
£
300 —
200 —
100
200
100
300
400
ug/PLATE (b) FIGURE 2. Mutagenicity of 2ADNT and 4ADNT in S. typhimurium TA98 and TA100 [Continued). Circles represent the 2-amino compound and triangles represent the 4-amino compound. Open symbols are in the presence of S9 and closed symbols in the absence of S9. (6) TA100.
activity in order to exert its mutagenic effect on Salmonella typhimurium (Spanggord et al., 1982; Whong and Edwards, 1984). Both groups of investigators have found that TNT is more active toward strain TA100 than TA98 by approximately a factor of two. Spanggord et al. (1982) spectulated that this might relate to the twofold increase in nitroreductase activity of TA100 relative to that of TA98 as reported by Blumer et al. (1980).
MUTAGENICITY OF TNT AND METABOLITES
171
TABLE 1. Mutagenic Potencies of Explosives and Their Metabolites in Salmonella Mutagenic potencies, revertants/plate/mg compounc]
Downloaded by [University of Illinois at Urbana-Champaign] at 20:22 05 June 2013
TA98 Compound
-S9
TNT 2ADNT 4ADNT 2,4DA6NT 2,6DA4NT Tetryl
5400 500 1000 0 325 8200
TA100 + S9 380 125 275 NTa NT 700
-S9
+ S9
6600 2300 500 0 750 23,000
2000 1233 200 NT NT 3750
a
NT, not tested.
300
50
100
150
200
250
jig/ PLATE FIGURE 3. Mutagenicity of 2,6DA4NT and 2,4DA6NT in S. typhimurium. Circles represent 2,6DA4NT and triangles represent 2,4DA4NT. Open symbols represent the absence of S9 and closed symbols indicate the presence of S9.
E. TAN ET AL.
172
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600
40
80
120
160
200
ug/PLATE FIGURE 4. Mutagenicity of Tetryl in 5. typhimurium. TA98 with (A) and without (A) S9; TA100 with (O) and without (•) S9.
Our results show the same magnitude of two to three times more revertants in TA100 than TA98. When calculations were made from a concentration response curve (Fig. 2 and Table 1), the decrease in activity due to S9 was 86% for TA98 and 76% for TA100. We find this trend in all our experiments involving TNT and rat liver S9. Other investigators have reported that S9 either TABLE 2. Specific Mutagenicity for Acetonitrile Extracts of Test Composts Specific mutagenicity, revertants/g, average ± SDa Volume percent soil in compost
Days of composting
TA98 without S9
TA100 without S9
10%
0 90 0 90
87,200 14,300 310,000 21,600
100,000 12,800 546,000 14,200
20%
determined on a compost wet weight basis.
± ± ± ±
5390 530 30,700 360
± ± ± ±
2750 1140 25,200 1100
MUTAGENICITY OF TNT AND METABOLITES
173
TABLE 3. Explosives and TNT Metabolites Analyses of Compost Piles Concentration,3 average ± SD, mg/kg
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Sample 10% Contaminated soil, d 0 10% Contaminated soil, d 90 20% Contaminated soil, d 0 20% Contaminated soil, d 90
2,6,DA4NT
2,4,DA6NT
