- Email: [email protected]
Variability over time in the mutagenicity of ashes from municipal solid-waste incinerators
Mutation Research, 301 (1993) 39-43 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00
39
MUTLET 00744
Variability over time in the mutagenicity of ashes from municipal solid-waste incinerators B.S. S h a n e a, W . H . G u t e n m a n n
b and D.J. Lisk b
a Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803, USA and b Toxic Chemicals Laboratory, New York State College of Agriculture and Life Sciences, Cornell University, Ithaca, N Y 14853, USA (Received 29 June 1992) (Revision received 10 August 1992) (Accepted 18 August 1992)
Keywords: Ashes, mutagenicity of; Municipal incinerator ash; Solid-waste incinerators
Summary Incineration of municipal solid waste as an alternative to its disposal in landfills has advantages such as volume reduction and generation of energy. However, both air emissions and the residual ash may pose environmental and human health hazards. The Ames mutagenicity assay was used to determine the mutagenicity of fly and bottom ash from two incinerators over time. This assay is an alternative to costly and time-consuming chemical analyses and is more realistic for the assessment of the best disposition of the ash i.e. whether it could pose a risk to handlers of the ash, whether it can be used in cement or as a fertilizer or whether it should be relegated to a landfill. The mutagenic potency of fly and bottom ash on a per g weight basis of material is similar. Furthermore, the variability over time in mutagenicity indicates that constant monitoring of incineration products and byproducts is essential.
Throughout the US, disposal of solid waste is becoming a major concern due to the extensive areas required for landfills, popular opposition to the siting of these landfills in residential areas, the resulting increase in transportation costs, and the possibility of leaching of toxic components from landfills into groundwater. Thus attention has turned to the feasibility and use of incineration for the destruction of municipal solid waste (MSW). MSW is typically incinerated alone but at Correspondence: Barbara S. Shane, Ph.D., Institute for Environmental Studies, 42 Atkinson Hall, Louisiana State University, Baton Rouge, LA 70803, USA. Tel. (504) 388-4302; Fax (504) 388-4286.
some facilities is burnt with coal. Incineration has a number of advantages but also some disadvantages. The major advantage is the decrease in volume of the waste and the possibility of using the waste as a fuel source. However, discharge of toxic air pollutants by these facilities may pose a risk to inhabitants in the area and the fly and bottom ash so generated, although about one tenth in volume of the original waste, has become enriched with toxic metals and organic compounds. When these ashes are stored in landfills these concentrated toxic components could leach into groundwater. One suggested use for MSW and coal fly ashes is their inclusion in cement for road building, but
40
concern has been expressed regarding the leaching of metals or organics from these roads into the environment. A second possible use is as a fertilizer but the uptake of certain metals such as cadmium from MSW ash amended soil (Bache and Lisk, 1990) and selenium and mutagens from coal fly ash (Shane et al., 1988) into edible plants and thus into the human food supply often precludes the use of fly ash in this manner. For these reasons, most of the fly and bottom ashes generated from municipal incinerators are disposed of in landfills. A major factor relating to the use of fly and bottom ashes is their variability in composition in time and location. Variation in the elemental (Mumma et al., 1990) and organic (Shane et al., 1990) composition of ashes from different municipal solid-waste incinerators has been reported. Also, variation in the elemental composition of ashes from one incinerator sampled at monthly
intervals has been documented (Mumma et al., 1991). It is therefore difficult to determine whether ash from a particular municipal incinerator can be used in road building for example, or whether it should be disposed of in a landfill. One method that can be helpful in evaluating the potential risk an ash may pose is the determination of its biological activity using a mutagenicity assay such as the Ames test. To test the sensitivity and usefulness of this assay for this application, the mutagenicity of fly and bottom ash collected from two incinerators over time was evaluated. Materials and methods
Collection of samples About 15 kg of both fly and bottom ash was collected on three successive dates from two incinerators. Three samples of fly and bottom ash
TABLE 1 M U T A G E N I C I T Y O F I N C I N E R A T O R ASH C O L L E C T E D AT M O N T H L Y O R BI-WEEKLY INTERVALS F R O M T W O M U N I C I P A L SOLID-WASTE I N C I N E R A T O R S Sample
TA98
date
No $9
TA100 rev/gMSC c
+ $9
rev/gMSW
No $9
rev/gMSW
+ $9
rev/gMSW
Incinerator A Fly ash 12/19/89 1/31/90 2/28/90
36+ 6 , a 21 5 : 2 54 5:10 *
17.0 27.6 338.0
305:5 * 26 _+ 1 565:12 *
67.8 62.4 354/7
1045:14 109 5 : 8 171+ 8 *
18.6 41.2 92.6
965:16 87 + 45 1505:16 *
- d 418.9
1735:11 * 182+41 * 28+ 5 *
124.8 96.6 1.1
1985:21 * 2275:33 * 365:6 *
143.2 115.6 2.0
1595:9 * 2385:12 * 1185:3
46.4 76.1 2.0
318+37 * 2705:12 * 1325:12 *
171.2 92.3 3.3
19+ 2 285:10 * 20+ 9
35.3 57.2 109.0
21+ 5 325:7 * 10+ 3
35.0 67~0 -
112+ 9 1005:4 1105:4
19.6 32.8
137+33" 110+10 875:2
58.7 31.2
Bottom ash 12/19/89 1/31/90 2/28/90
Incinerator B Fly ash 5/20/90 6/5/90 6/12/90 " - " control "+"control
175:3 b 372+31 b
195:5 555 5:36 b
1015:15 1063 5:47 b
104+23 259 + 10 b
a Mean + S.D. of at least 2 Expts. performed in duplicate. b DMSO was used as the negative control; 5.0/~g 2-nitrofluorene and 10/.~g sodium azide were used as the positive control in the absence of $9 with TA98 and TA100 respectively, while 10/,Lg 2-aminofluorene was used with both strains in the presence of $9. c R e v e r t a n t s / g municipal solid waste. ,t R e v e r t a n t s / g minicipal solid waste could not be calculated as the number of revertants from the control plate was greater than the number from the experimental plate. * Significantly different from the corresponding control ( P < 0.05).
41 were collected approximately one month apart from the first incinerator (A), and at bi-weekly intervals from the second incinerator (B). The description of the incinerators from which these ashes were collected is outlined in Shane et al. (1990). The incinerators identified as A and B in the previous study refer to A and B in this study.
fidence interval was determined for each ash collected on the three sampling dates. These values were compared with their corresponding controis. The values for the ashes were considered to be significantly different from the control when the 95% confidence interval did not coincide. The data shown in Table 1 is the mean + SD from two experiments performed in triplicate.
Preparation of samples Fly-ash samples were air-dried at room temperature and then mixed by tumbling. In the case of bottom ash, large objects such as stones, glass or ceramic pieces were removed before pulverizing the ash to pass through a 5-mesh (4-mm opening screen). The material was then reduced to a powder in a hammer mill and the powder mixed by tumbling. Two 25-g subsamples of the bottom and fly ashes were extracted in a Soxhlet apparatus for 24 h with 250 ml dichloromethane (DCM). The DCM extracts were pooled and evaporated to dryness under a slow stream of nitrogen. The residue was weighed and reconstituted in 1 ml DMSO.
Ames mutagenicity assay Salmonella typhimurium TA98 and TA100 were kindly supplied by Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley, CA. The plate-incorporation assay as described by Maron and Ames (1983) was utilized throughout the study. Each extract was tested for mutagenicity using Salmonella thyphimurium TA98 and TA100 in the presence and absence of a hepatic microsomal enzyme preparation from rats pretreated with Aroclor 1254. The final protein concentration of $9 was 1 mg/plate. With each experiment negative control plates containing 50 /zl of dimethyl sulfoxide in the presence and absence of $9 were included. Respectively, 5 ~g of 2-nitrofluorene and 10/xg of sodium azide were used as the positive controls for TA98 and TA100 in the absence of $9 while 10 /xg of 2-aminofluorene was used in the presence of $9 with both strains.
Statistical analysis The data were analyzed using Stat View 512. The mean mutation frequency and the 95% con-
Results and discussion
Not unexpectedly the weight of the organic material extracted from the bottom-ash samples (40 _+ 0.8 mg) was greater than that obtained from the two fly ashes (8.3 + 0.8 mg and 4.0 + 1.1 mg) respectively, from incinerator A and B. Although the samples of fly and bottom ash were collected on different days, the weight of extractable organic matter was similar on the three days from the same incinerator. The weight of organic matter obtained per gram of fly ash from the MSW incinerators were 3.32 _+ 0.33 x 10 -4 g and 1.6 + 0.45 x 10 -4 g respectively, from the A and B incinerators while that of bottom ash was 1.6 + 0.87 x 10 -3 g. These values are within the same order of magnitude as that reported by Silkowski et al. (1992). In their case, however, they extracted a mixture of fly and bottom ash, of unknown relative proportions. Mutagenic compounds were detected in most of the samples of both fly and bottom ash from incinerator A but from only a few samples of fly ash from incinerator B (Table 1). Direct-acting frameshift mutagens were found in the first and third sample of fly ash and in all the samples of bottom ash from incinerator A. Direct-acting base-pair mutagens were detected in the third sample of fly ash and in the first two samples of bottom ash. Frameshift mutagens were found in the second sample of fly ash from incinerator B. Base-pair promutagens were only found in the first sample of bottom ash from incinerator A. It was interesting to note that the second bottom ash sample ( 1 / 3 0 / 9 0 ) from incinerator A which gave the highest mutagenic response contained the second highest concentration of organic matter (1.5 mg/50/~1 sample). Without knowing the chemical composition of these bottom-ash samples, it is difficult to assess the concentration of
42 potentially mutagenic compounds that may have been present in the second sample but absent in the third. The mean induced number of revertants/g of fly and bottom ash is shown in Table 1. These values were calculated by subtracting the mean number of revertants from the negative control plates (spontaneous mutagenic frequency) from each experimental plate with both bacterial strains. Normalization of the mutagenic response by computing the net number of revertants per g MSW extracted showed that the activity of the fly ash from incinerator B was lower than that from incinerator A. Per g MSW the net mutagenic response from the bottom ash was lower than that of the fly ash. These normalized mutagenic values (Table 1) are within the same order of magnitude but lower than those obtained by Silkowski et al. (1992) who reported 103.48 and 247.5 revertants/g MSW for TA98 and TA100, respectively. The difference in the results of two studies is probably due to the mixed composition of the MSW evaluated by Silkowski et al. (1992), which contained both fly and bottom ash in unknown proportions. In evaluating the mutagenicity of these ash samples, it was found that a mutagenic response was obtained within a very narrow range of concentrations of the ash extract. At higher concentrations to those reported in this study, the extracts were toxic and at lower concentrations the mutagenic response was above the backgound values but not statistically significant. This phenomenon which may be due to the presence of numerous compounds in the extracts is problematical and interferes with the determination of a dose-response curve. One of the major differences between the type of waste burned in incinerator A and B (Table 1) was the co-combustion of coal in a proportion of 85:15 of coal to MSW in incinerator B while incinerator A only burned MSW. Also, incinerator B is a more modern facility and has two ash-pollution control devices, a baghouse and electrostatic precipitators, while incinerator A only has the latter pollution control device. This probably explains the finding of the much lower concentration of organic compounds in fly ash from incinerator B than incinerator A and the corresponding lower mutagenicity of the fly ash
from incinerator B. In a previous study in which fly-ash samples were collected from these same incinerators, it was found that the concentration of polycyclic aromatic hydrocarbons, chlorobenzenes, phthalates and substituted naphthalenes was 1-2 orders of magnitude higher in the fly-ash samples from incinerator A than B (Shane et al., 1990). Although the samples evaluated in the present study were collected more than a year later than those reported in the previous study (Shane et al., 1990) the percent MSW burned in the respective incinerators has not changed markedly, nor has the configuration of the burner. In the above-mentioned study (Shane et al., 1990), in which fly and bottom ashes from 18 MSW incinerators were evaluated for mutagens, it was noted that in general, the ashes with the highest concentration of organic matter invariably gave the highest mutagenic response. It was also observed at that time that the bottom ash from incinerator A contained both frameshift and base-pair promutagens. Although ashes from only two incinerators have been sampled over time in this study, it is suggested that the weight of extractable organic matter could be used as a crude measure of the likelihood that a particular ash may contain mutagens and thus pose a risk to man or the environment. Mutagens have been found in fly and bottom ash from MSW incinerators in Italy (Rossi et al., 1991), Japan (Kamiya and Ose, 1987a), Sweden (Victorin et al., 1988) and the United States (Shane et al., 1990; Silkowski et al., 1992). The mutagenicity of fly ash collected from a MSW incinerator in Parma, Italy showed a similar variation in response over time (Rossi et al., 1991) to that reported here. Although an equivalent weight of extract was evaluated in their experiments compared to ours, Rossi et al. (1991) found that in some cases their extract was toxic and killed the bacteria. These authors did not dilute these toxic extracts to determine if they would be mutagenic at lower concentrations. In a study in Japan on the mutagenicity of fly and bottom ash from two MSW incinerators, Kamiya et al. (1990) found that neither fly ash nor bottom ash from a modern incinerator in which MSW was burned at 900°C was mutagenic. However, mutagens were detected in the emission gas, as well as fly and
43 bottom ash from a discontinuous batch-type incinerator operated at temperatures between 600 and 900°C. These authors also showed that 90% of the mutagens emitted from this second incinerator were in the gaseous phase and 10% were recovered in fly ash. Only base-pair promutagens were detected in the fly and bottom ash with the activity being higher in the former sample. Although we were unable to sample the gaseous emissions from the incinerators studied, because of technical difficulties and the reticence on the part of incinerator operators to permit this sampling in the US, it appears from both our study and those of Kamiya and co-workers (1987a, b, 1990) and Silkowski et al. (1992) that the risk of exposure to mutagenic compounds from incinerators is dependent on numerous factors. These include the mode of operation of the facility and the apparatus used for environmental control, as well as the waste burned. According to the calculations of Kamiya and Ose (1987b), the mutagenicity of the emission gas from a batch-type incinerator is three orders of magnitude higher than that of light- or heavy-duty trucks, while that from a more modern continuously operated incinerator, in which the temperature of the burner is maintained at 900°C, is much less and may be comparable to a heavy-duty truck. In their study Victorin et al. (1988) showed that the mutagenic potency of emissions from MSW facilities correlated positively with the emission of CO and PAIl. CO production was in turn correlated with the temperature of the furnace. In order to protect human and environmental health and reduce the concentration of mutagens produced during combustion, in concert with the ability to incinerate the enormous volume of MSW produced in the US, a thrust to modernize antiquated facilities must be made. If this goal can be accomplished and the public so informed, opposition to the concept of incineration of MSW in the future is likely to diminish.
Acknowledgements The authors appreciate the technical assistance of Ms. Millie Williams, the editorial contri-
bution of Dr. Maud Walsh and gratefully acknowledge the cooperation of the plant managers of the two incineration facilities.
References Bache, C.A., and D.J. Lisk (1990) Heavy-metal absorption by perennial ryegrass and Swiss chard grown in potted soils amended with ashes from 18 municipal refuse incinerators, J. Agr. Fd. Chem., 38, 190-194. Kamiya, A., and Y. Ose (1987a) Mutagenic activity and PAH analysis in municipal incinerators, Sci. Total Environ., 61, 37-49. Kamiya, A., and Y. Ose (1987b) Study of the behaviour of mutagens in wastewater and emission gas from a municipal incinerator evaluated by means of the Ames assay, Sci. Total Environ., 65, 109-120. Kamiya, A., Y. Ose and T. Sato (1990) Study on behavior of mutagens from municipal incinerators by means of Ames assay, Mutation Environ., Part E, 31-40. Mumma, R.O., D.C. Raupach, K. Sahadewan, C.G. Manos, M. Rutzke, H.T. Kuntz, C.A. Bache and D.J. Lisk (1990) National survey of elements and radioactivityin municipal incinerator ashes, Arch. Environ. Contam. Toxicol., 19, 399-404. Mumma, R.O., D.C. Raupach, K. Sahadewan, B.S. Shane, M. Rutzke, C.A. Bache, W.H. Gutenmann and D.J. Lisk (1991) Variation in the elemental composition of municipal refuse incinerator ashes with time of sampling, Chemosphere, 23, 391-395. Rossi C., P. Poll, A. Buschini, F. Cassoni, A. Galli, R. Vellosi and R. del Carratore (1991) Genetic activity of sfimples collected from a waste incinerator and its neighboring area, Toxicol. Environ. Chem., 30, 51-61. Shane, B.S., C.B. Littman, L.A. Essick, W.H. Gutenmann, G.J. Doss and D.J. Lisk (1988) Uptake of selenium and mutagens by vegetables grown in fly ash containing greenhouse media, J. Agric. Fd. Chem., 36, 328-333. Shane, B.S., C.B. Henry, J.H. Hotchkiss, K.A. Klausner, W.H. Gutenmann and D.J. Lisk (1990) Organic toxicants and mutagens in ashes from eighteen municipal refuse incinerators, Arch. Environ. Contam. Toxicol., 19, 665-673. Silkowski, M.A., S.R. Smith and M.J. Plewa (1992) Analysis of the genotoxicityof municipal solid waste incinerator ash, Sci. Total Environ., 111, 109-124. Victorin, K., M. Stahlberg and U.G. Ahlborg (1988) Emission of mutagenic substances from waste incineration plants, Waste Management and Res., 6, 149-161.
Communicated by J.M. Gentile
39
MUTLET 00744
Variability over time in the mutagenicity of ashes from municipal solid-waste incinerators B.S. S h a n e a, W . H . G u t e n m a n n
b and D.J. Lisk b
a Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803, USA and b Toxic Chemicals Laboratory, New York State College of Agriculture and Life Sciences, Cornell University, Ithaca, N Y 14853, USA (Received 29 June 1992) (Revision received 10 August 1992) (Accepted 18 August 1992)
Keywords: Ashes, mutagenicity of; Municipal incinerator ash; Solid-waste incinerators
Summary Incineration of municipal solid waste as an alternative to its disposal in landfills has advantages such as volume reduction and generation of energy. However, both air emissions and the residual ash may pose environmental and human health hazards. The Ames mutagenicity assay was used to determine the mutagenicity of fly and bottom ash from two incinerators over time. This assay is an alternative to costly and time-consuming chemical analyses and is more realistic for the assessment of the best disposition of the ash i.e. whether it could pose a risk to handlers of the ash, whether it can be used in cement or as a fertilizer or whether it should be relegated to a landfill. The mutagenic potency of fly and bottom ash on a per g weight basis of material is similar. Furthermore, the variability over time in mutagenicity indicates that constant monitoring of incineration products and byproducts is essential.
Throughout the US, disposal of solid waste is becoming a major concern due to the extensive areas required for landfills, popular opposition to the siting of these landfills in residential areas, the resulting increase in transportation costs, and the possibility of leaching of toxic components from landfills into groundwater. Thus attention has turned to the feasibility and use of incineration for the destruction of municipal solid waste (MSW). MSW is typically incinerated alone but at Correspondence: Barbara S. Shane, Ph.D., Institute for Environmental Studies, 42 Atkinson Hall, Louisiana State University, Baton Rouge, LA 70803, USA. Tel. (504) 388-4302; Fax (504) 388-4286.
some facilities is burnt with coal. Incineration has a number of advantages but also some disadvantages. The major advantage is the decrease in volume of the waste and the possibility of using the waste as a fuel source. However, discharge of toxic air pollutants by these facilities may pose a risk to inhabitants in the area and the fly and bottom ash so generated, although about one tenth in volume of the original waste, has become enriched with toxic metals and organic compounds. When these ashes are stored in landfills these concentrated toxic components could leach into groundwater. One suggested use for MSW and coal fly ashes is their inclusion in cement for road building, but
40
concern has been expressed regarding the leaching of metals or organics from these roads into the environment. A second possible use is as a fertilizer but the uptake of certain metals such as cadmium from MSW ash amended soil (Bache and Lisk, 1990) and selenium and mutagens from coal fly ash (Shane et al., 1988) into edible plants and thus into the human food supply often precludes the use of fly ash in this manner. For these reasons, most of the fly and bottom ashes generated from municipal incinerators are disposed of in landfills. A major factor relating to the use of fly and bottom ashes is their variability in composition in time and location. Variation in the elemental (Mumma et al., 1990) and organic (Shane et al., 1990) composition of ashes from different municipal solid-waste incinerators has been reported. Also, variation in the elemental composition of ashes from one incinerator sampled at monthly
intervals has been documented (Mumma et al., 1991). It is therefore difficult to determine whether ash from a particular municipal incinerator can be used in road building for example, or whether it should be disposed of in a landfill. One method that can be helpful in evaluating the potential risk an ash may pose is the determination of its biological activity using a mutagenicity assay such as the Ames test. To test the sensitivity and usefulness of this assay for this application, the mutagenicity of fly and bottom ash collected from two incinerators over time was evaluated. Materials and methods
Collection of samples About 15 kg of both fly and bottom ash was collected on three successive dates from two incinerators. Three samples of fly and bottom ash
TABLE 1 M U T A G E N I C I T Y O F I N C I N E R A T O R ASH C O L L E C T E D AT M O N T H L Y O R BI-WEEKLY INTERVALS F R O M T W O M U N I C I P A L SOLID-WASTE I N C I N E R A T O R S Sample
TA98
date
No $9
TA100 rev/gMSC c
+ $9
rev/gMSW
No $9
rev/gMSW
+ $9
rev/gMSW
Incinerator A Fly ash 12/19/89 1/31/90 2/28/90
36+ 6 , a 21 5 : 2 54 5:10 *
17.0 27.6 338.0
305:5 * 26 _+ 1 565:12 *
67.8 62.4 354/7
1045:14 109 5 : 8 171+ 8 *
18.6 41.2 92.6
965:16 87 + 45 1505:16 *
- d 418.9
1735:11 * 182+41 * 28+ 5 *
124.8 96.6 1.1
1985:21 * 2275:33 * 365:6 *
143.2 115.6 2.0
1595:9 * 2385:12 * 1185:3
46.4 76.1 2.0
318+37 * 2705:12 * 1325:12 *
171.2 92.3 3.3
19+ 2 285:10 * 20+ 9
35.3 57.2 109.0
21+ 5 325:7 * 10+ 3
35.0 67~0 -
112+ 9 1005:4 1105:4
19.6 32.8
137+33" 110+10 875:2
58.7 31.2
Bottom ash 12/19/89 1/31/90 2/28/90
Incinerator B Fly ash 5/20/90 6/5/90 6/12/90 " - " control "+"control
175:3 b 372+31 b
195:5 555 5:36 b
1015:15 1063 5:47 b
104+23 259 + 10 b
a Mean + S.D. of at least 2 Expts. performed in duplicate. b DMSO was used as the negative control; 5.0/~g 2-nitrofluorene and 10/.~g sodium azide were used as the positive control in the absence of $9 with TA98 and TA100 respectively, while 10/,Lg 2-aminofluorene was used with both strains in the presence of $9. c R e v e r t a n t s / g municipal solid waste. ,t R e v e r t a n t s / g minicipal solid waste could not be calculated as the number of revertants from the control plate was greater than the number from the experimental plate. * Significantly different from the corresponding control ( P < 0.05).
41 were collected approximately one month apart from the first incinerator (A), and at bi-weekly intervals from the second incinerator (B). The description of the incinerators from which these ashes were collected is outlined in Shane et al. (1990). The incinerators identified as A and B in the previous study refer to A and B in this study.
fidence interval was determined for each ash collected on the three sampling dates. These values were compared with their corresponding controis. The values for the ashes were considered to be significantly different from the control when the 95% confidence interval did not coincide. The data shown in Table 1 is the mean + SD from two experiments performed in triplicate.
Preparation of samples Fly-ash samples were air-dried at room temperature and then mixed by tumbling. In the case of bottom ash, large objects such as stones, glass or ceramic pieces were removed before pulverizing the ash to pass through a 5-mesh (4-mm opening screen). The material was then reduced to a powder in a hammer mill and the powder mixed by tumbling. Two 25-g subsamples of the bottom and fly ashes were extracted in a Soxhlet apparatus for 24 h with 250 ml dichloromethane (DCM). The DCM extracts were pooled and evaporated to dryness under a slow stream of nitrogen. The residue was weighed and reconstituted in 1 ml DMSO.
Ames mutagenicity assay Salmonella typhimurium TA98 and TA100 were kindly supplied by Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley, CA. The plate-incorporation assay as described by Maron and Ames (1983) was utilized throughout the study. Each extract was tested for mutagenicity using Salmonella thyphimurium TA98 and TA100 in the presence and absence of a hepatic microsomal enzyme preparation from rats pretreated with Aroclor 1254. The final protein concentration of $9 was 1 mg/plate. With each experiment negative control plates containing 50 /zl of dimethyl sulfoxide in the presence and absence of $9 were included. Respectively, 5 ~g of 2-nitrofluorene and 10/xg of sodium azide were used as the positive controls for TA98 and TA100 in the absence of $9 while 10 /xg of 2-aminofluorene was used in the presence of $9 with both strains.
Statistical analysis The data were analyzed using Stat View 512. The mean mutation frequency and the 95% con-
Results and discussion
Not unexpectedly the weight of the organic material extracted from the bottom-ash samples (40 _+ 0.8 mg) was greater than that obtained from the two fly ashes (8.3 + 0.8 mg and 4.0 + 1.1 mg) respectively, from incinerator A and B. Although the samples of fly and bottom ash were collected on different days, the weight of extractable organic matter was similar on the three days from the same incinerator. The weight of organic matter obtained per gram of fly ash from the MSW incinerators were 3.32 _+ 0.33 x 10 -4 g and 1.6 + 0.45 x 10 -4 g respectively, from the A and B incinerators while that of bottom ash was 1.6 + 0.87 x 10 -3 g. These values are within the same order of magnitude as that reported by Silkowski et al. (1992). In their case, however, they extracted a mixture of fly and bottom ash, of unknown relative proportions. Mutagenic compounds were detected in most of the samples of both fly and bottom ash from incinerator A but from only a few samples of fly ash from incinerator B (Table 1). Direct-acting frameshift mutagens were found in the first and third sample of fly ash and in all the samples of bottom ash from incinerator A. Direct-acting base-pair mutagens were detected in the third sample of fly ash and in the first two samples of bottom ash. Frameshift mutagens were found in the second sample of fly ash from incinerator B. Base-pair promutagens were only found in the first sample of bottom ash from incinerator A. It was interesting to note that the second bottom ash sample ( 1 / 3 0 / 9 0 ) from incinerator A which gave the highest mutagenic response contained the second highest concentration of organic matter (1.5 mg/50/~1 sample). Without knowing the chemical composition of these bottom-ash samples, it is difficult to assess the concentration of
42 potentially mutagenic compounds that may have been present in the second sample but absent in the third. The mean induced number of revertants/g of fly and bottom ash is shown in Table 1. These values were calculated by subtracting the mean number of revertants from the negative control plates (spontaneous mutagenic frequency) from each experimental plate with both bacterial strains. Normalization of the mutagenic response by computing the net number of revertants per g MSW extracted showed that the activity of the fly ash from incinerator B was lower than that from incinerator A. Per g MSW the net mutagenic response from the bottom ash was lower than that of the fly ash. These normalized mutagenic values (Table 1) are within the same order of magnitude but lower than those obtained by Silkowski et al. (1992) who reported 103.48 and 247.5 revertants/g MSW for TA98 and TA100, respectively. The difference in the results of two studies is probably due to the mixed composition of the MSW evaluated by Silkowski et al. (1992), which contained both fly and bottom ash in unknown proportions. In evaluating the mutagenicity of these ash samples, it was found that a mutagenic response was obtained within a very narrow range of concentrations of the ash extract. At higher concentrations to those reported in this study, the extracts were toxic and at lower concentrations the mutagenic response was above the backgound values but not statistically significant. This phenomenon which may be due to the presence of numerous compounds in the extracts is problematical and interferes with the determination of a dose-response curve. One of the major differences between the type of waste burned in incinerator A and B (Table 1) was the co-combustion of coal in a proportion of 85:15 of coal to MSW in incinerator B while incinerator A only burned MSW. Also, incinerator B is a more modern facility and has two ash-pollution control devices, a baghouse and electrostatic precipitators, while incinerator A only has the latter pollution control device. This probably explains the finding of the much lower concentration of organic compounds in fly ash from incinerator B than incinerator A and the corresponding lower mutagenicity of the fly ash
from incinerator B. In a previous study in which fly-ash samples were collected from these same incinerators, it was found that the concentration of polycyclic aromatic hydrocarbons, chlorobenzenes, phthalates and substituted naphthalenes was 1-2 orders of magnitude higher in the fly-ash samples from incinerator A than B (Shane et al., 1990). Although the samples evaluated in the present study were collected more than a year later than those reported in the previous study (Shane et al., 1990) the percent MSW burned in the respective incinerators has not changed markedly, nor has the configuration of the burner. In the above-mentioned study (Shane et al., 1990), in which fly and bottom ashes from 18 MSW incinerators were evaluated for mutagens, it was noted that in general, the ashes with the highest concentration of organic matter invariably gave the highest mutagenic response. It was also observed at that time that the bottom ash from incinerator A contained both frameshift and base-pair promutagens. Although ashes from only two incinerators have been sampled over time in this study, it is suggested that the weight of extractable organic matter could be used as a crude measure of the likelihood that a particular ash may contain mutagens and thus pose a risk to man or the environment. Mutagens have been found in fly and bottom ash from MSW incinerators in Italy (Rossi et al., 1991), Japan (Kamiya and Ose, 1987a), Sweden (Victorin et al., 1988) and the United States (Shane et al., 1990; Silkowski et al., 1992). The mutagenicity of fly ash collected from a MSW incinerator in Parma, Italy showed a similar variation in response over time (Rossi et al., 1991) to that reported here. Although an equivalent weight of extract was evaluated in their experiments compared to ours, Rossi et al. (1991) found that in some cases their extract was toxic and killed the bacteria. These authors did not dilute these toxic extracts to determine if they would be mutagenic at lower concentrations. In a study in Japan on the mutagenicity of fly and bottom ash from two MSW incinerators, Kamiya et al. (1990) found that neither fly ash nor bottom ash from a modern incinerator in which MSW was burned at 900°C was mutagenic. However, mutagens were detected in the emission gas, as well as fly and
43 bottom ash from a discontinuous batch-type incinerator operated at temperatures between 600 and 900°C. These authors also showed that 90% of the mutagens emitted from this second incinerator were in the gaseous phase and 10% were recovered in fly ash. Only base-pair promutagens were detected in the fly and bottom ash with the activity being higher in the former sample. Although we were unable to sample the gaseous emissions from the incinerators studied, because of technical difficulties and the reticence on the part of incinerator operators to permit this sampling in the US, it appears from both our study and those of Kamiya and co-workers (1987a, b, 1990) and Silkowski et al. (1992) that the risk of exposure to mutagenic compounds from incinerators is dependent on numerous factors. These include the mode of operation of the facility and the apparatus used for environmental control, as well as the waste burned. According to the calculations of Kamiya and Ose (1987b), the mutagenicity of the emission gas from a batch-type incinerator is three orders of magnitude higher than that of light- or heavy-duty trucks, while that from a more modern continuously operated incinerator, in which the temperature of the burner is maintained at 900°C, is much less and may be comparable to a heavy-duty truck. In their study Victorin et al. (1988) showed that the mutagenic potency of emissions from MSW facilities correlated positively with the emission of CO and PAIl. CO production was in turn correlated with the temperature of the furnace. In order to protect human and environmental health and reduce the concentration of mutagens produced during combustion, in concert with the ability to incinerate the enormous volume of MSW produced in the US, a thrust to modernize antiquated facilities must be made. If this goal can be accomplished and the public so informed, opposition to the concept of incineration of MSW in the future is likely to diminish.
Acknowledgements The authors appreciate the technical assistance of Ms. Millie Williams, the editorial contri-
bution of Dr. Maud Walsh and gratefully acknowledge the cooperation of the plant managers of the two incineration facilities.
References Bache, C.A., and D.J. Lisk (1990) Heavy-metal absorption by perennial ryegrass and Swiss chard grown in potted soils amended with ashes from 18 municipal refuse incinerators, J. Agr. Fd. Chem., 38, 190-194. Kamiya, A., and Y. Ose (1987a) Mutagenic activity and PAH analysis in municipal incinerators, Sci. Total Environ., 61, 37-49. Kamiya, A., and Y. Ose (1987b) Study of the behaviour of mutagens in wastewater and emission gas from a municipal incinerator evaluated by means of the Ames assay, Sci. Total Environ., 65, 109-120. Kamiya, A., Y. Ose and T. Sato (1990) Study on behavior of mutagens from municipal incinerators by means of Ames assay, Mutation Environ., Part E, 31-40. Mumma, R.O., D.C. Raupach, K. Sahadewan, C.G. Manos, M. Rutzke, H.T. Kuntz, C.A. Bache and D.J. Lisk (1990) National survey of elements and radioactivityin municipal incinerator ashes, Arch. Environ. Contam. Toxicol., 19, 399-404. Mumma, R.O., D.C. Raupach, K. Sahadewan, B.S. Shane, M. Rutzke, C.A. Bache, W.H. Gutenmann and D.J. Lisk (1991) Variation in the elemental composition of municipal refuse incinerator ashes with time of sampling, Chemosphere, 23, 391-395. Rossi C., P. Poll, A. Buschini, F. Cassoni, A. Galli, R. Vellosi and R. del Carratore (1991) Genetic activity of sfimples collected from a waste incinerator and its neighboring area, Toxicol. Environ. Chem., 30, 51-61. Shane, B.S., C.B. Littman, L.A. Essick, W.H. Gutenmann, G.J. Doss and D.J. Lisk (1988) Uptake of selenium and mutagens by vegetables grown in fly ash containing greenhouse media, J. Agric. Fd. Chem., 36, 328-333. Shane, B.S., C.B. Henry, J.H. Hotchkiss, K.A. Klausner, W.H. Gutenmann and D.J. Lisk (1990) Organic toxicants and mutagens in ashes from eighteen municipal refuse incinerators, Arch. Environ. Contam. Toxicol., 19, 665-673. Silkowski, M.A., S.R. Smith and M.J. Plewa (1992) Analysis of the genotoxicityof municipal solid waste incinerator ash, Sci. Total Environ., 111, 109-124. Victorin, K., M. Stahlberg and U.G. Ahlborg (1988) Emission of mutagenic substances from waste incineration plants, Waste Management and Res., 6, 149-161.
Communicated by J.M. Gentile