The Science o[the Total Environment, 111 (1992) 109-124 Elsevier Science Publishers B.V., Amsterdam
109
Analysis of the genotoxicity of municipal solid waste incinerator ash Michelle A. Silkowski, S h a n n o n R. S m i t h a n d M i c h a e l J, Plewa* Institute Jbr Environmental Studies, University of Illinois at Urbana-Champaign, Urbana, IL 61801-4723, USA (Received September 20th, 1990; accepted December 31st, 1990)
ABSTRACT Combined bottom and fly ash obtained from a Chicago, IL, municipal solid waste incinerator (MSWI) was extracted with organic solvents, water or acidified water. The mean amounts of organic material isolated from each extraction procedure were 688.2, 91.8 and 167.7/~g/g MSWl ash. These extracts were evaluated for toxicity and mutagenicity in Salmonella typhimurium strains TA98 and TA100. We developed and calibrated a micropreincubation assay to evaluate small concentrations of the organic extracts. No direct-acting mutagens were found, however the acid-treated aqueous extracts were toxic. Materials isolated with methylene chloride methanol were mutagenic after hepatic microsomal activation ($9). The mutagenic potencies of the organic extract normalized to a per gram ash basis was the induction of 103.46 revertants in TA98 and 247.5 revertants in TA100. The aqueous extracts were neither toxic nor mutagenic. However, the acid-treated aqueous extract was mutagenic to TAI00. The organic material isolated from the acidic extract had an induced mutagenic potency of 44.2 revertants/ mg extract. Normalizing these data indicate a mutagenic potency of 7.4 revertants/g MSWI ash leached.
INTRODUCTION M u n i c i p a l solid waste incineration I n c i n e r a t i o n is a widely used m e t h o d o f d i s p o s a l for m u n i c i p a l solid wastes. T h e b u r d e n o f i n c i n e r a t i o n h a s n o t b e e n r i g o r o u s l y assessed in t e r m s o f the p o t e n t i a l d a m a g e to the e n v i r o n m e n t a n d public health. T r e n d s t o w a r d s i n c i n e r a t i o n as a p r e f e r r e d m e t h o d o f w a s t e m a n a g e m e n t m i g h t be altered if risks were a d e q u a t e l y a d d r e s s e d a n d a n a l y z e d (Vining a n d E b r e o , 1990; Vining et al., 1990). M a s s i n c i n e r a t i o n as a n efficient, safe a n d e c o n o m i c m e t h o d o f solid w a s t e d i s p o s a l is b a s e d on the s u p p o s i t i o n t h a t the w a s t e * Corresponding author: Dr Michael J. Plewa, Professor of Genetics, Institute for Environmental Studies, University of Illinois at Urbana-Champaign, i101 West Peabody Dr., Urbana, IL 61801, USA. 0048-9697/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
| ]0
M.A. SILKOWSKI ET AL.
stream can be effectively managed by a sole technique as if it were a homogenous material. Municipal solid waste incinerators (MSWI) are one source of hazardous complex mixtures entering the environment. MSWI produce toxic gas emissions from the stacks and solid residues accumulate in the incinerator grates and combustion chambers. While there is considerable information concerning the constituents of ash residues (Hrudey et al., 1974; Karasek et al., 1987), there is little known about the potential mutagens or promutagens in these complex mixtures which are produced and released into the environment.
Products of incineration Incineration yields three forms of products, energy, gases and solid residues. Energy can be partially recovered and used for electricity generation or steam production. Gas-phase products exit almost solely through the stack. Solid residues (ash) accumulate at two sites, at the bottom on the grates (bottom ash) and at various points in the combustion chamber (fly ash) (Denison and Silbergeld, 1988).
Toxic metals Metals are not destroyed in the incineration process. The concentration of metals found in the waste stream before incineration and in the combined air emissions, bottom and fly ash after incineration are identical (Denison and Silbergeld, 1988). Heavy metals induce a wide range of toxic consequences, including carcinogenic, neurological, hepatic, renal, hematopoietic and other adverse effects (Friberg et al., 1986). Thus, the release of toxic metals from incinerators poses risks to the environment and to public health (Environmental Protection Agency, 1986, 1988; Denison and Silbergeld, 1988).
Toxic organic materials The toxicity of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) is a current subject of concern (Travis et al., 1989). Municipal solid waste incinerators were identified as a source of dioxins in the environment (Tong and Karasek, 1986; Travis and HattemerFrey, 1989). Octachlorinated dioxins were the most abundant isomer detected in residue MSWI ash samples. Smaller amounts of octachlorinated furans were also detected in ash samples (Stern et al., 1989). In a recent paper, Shane et al. (1990) reported P C D D congeners in ash samples from two of 18 MSW incinerators. The concentrations of the P C D D compounds were 57 and 290 ppb.
ANALYSIS OF THE GENOTOXICITY OF MUNICIPAL SOLID WASTE I N C I N E R A T O R ASH
1I I
Polyaromatic hydrocarbons (PAHs) induced toxic and carcinogenic responses under in vivo (Grimmer et al., 1988; Penn and Snyder, 1988; Bonassi et al., 1989) and in vitro conditions (DeFlora et al., 1989). PAHs are found in virtually all media and are generated from combustion processes (Junk and Ford, 1980). Stern et al. (1989) found that the most abundant PAHs in soil samples near a MSWI were pyrene, followed by fluoranthene, phenanthrene, anthracene and benzo[~]anthracene. There is a wide concentration range of aromatic compounds present in incinerator ash. Hrudey et al. (1974) detected 2.5 pg 3-methylphenanthrene/kg ash and 1500 #g di-isobutyl phthalate/kg ash. Recently, Kamiya et al. (1990) reported PAH concentrations in MSWI fly ash ranging from isomers of benzo[a]pyrene at 5 pg/kg ash to phenanthrene or anthracene at 438 #g/kg ash. In a survey of ash from 18 MSWI facilities, relatively high proportions of the organic materials found in MSWI ash were PAHs. There is a relationship between the generation of PAHs and incomplete combustion (Shane et al., 1990). Toxicity and mutagenicity studies on M S W I ash
Gustavsson (1989) found that mortality among workers in a municipal waste incinerator in Stockholm, Sweden, was increased over the general population. The excess deaths were a result of lung cancer and ischemic heart disease. The severity of illnesses was related to occupational exposure. Animal studies on MSWI products have focused primarily on the effects of fly ash. A!arie et al. (1989) found that fly ash inhaled by guinea pigs induced multifocal pneumoconiosis in the pulmonary tissue and elevated heavy metal concentrations in the lungs. Helder et al. (1982) found that fly ash was toxic to fish. Pani et al. (1983) analyzed organic material derived from solvent extractions of airborne dust (not stack emissions) from a MSW incinerator in Trieste, Italy. They found mutagenicity associated with the organic material in the solvent extracts. In a series of studies, mutagens were found to be associated with residue ash, wastewater and stack emissions from a MSW incinerator in Nagoya, Japan (Kamiya and Ose, 1987a,b; Kamiya et al., 1989a,b). After methylene chloride Soxhlet extraction of 18 MSWI ash samples, six extracts contained directacting or promutagenic agents when assayed with S. typhimurium strains TA98 and TA100 (Shane et al., 1990). However, none of the above studies evaluated the mutagenic properties of aqueous extracts of MSWI ash. EXPERIMENTAL PROCEDURES
Municipal solid waste incinerator ash
Municipal solid waste incinerator ash was obtained from the Northwest Waste-To-Energy Facility in Cook County, Illinois, with the cooperation of
l 12
M.A. S I L K O W S K I ET AL.
the Illinois Enviromental Protection Agency, Division of Land Pollution Control. The site inventory number was 0316230004.
Organic extraction of MSW1 ash Erlenmeyer flasks were soaked with N o - C h r o m Mix Acid Wash and washed. Multiple 50-g portions of MSWI ash were placed into the 250-ml flasks. The ash was extracted with 100ml methylene chloride/methanol (1 : 1 v/v). Each flask was sealed and shaken on a wrist-action shaker at a moderate speed for 24h. The solvent was decanted off the ash into 150-ml Corex centrifuge tubes and centrifuged at 2600 g for 20 min. The solvent was decanted into a 1-1 round-bottom flask and the volume reduced to ,-~ 20 ml with rotary evaporation under vacuum from 25 to ,-, 40°C. This residue was stored at - 2 0 ° C . The remaining ash was suspended in 100ml acetone and shaken in the same m a n n e r as above. The acetone was decanted off the ash into 150-ml Corex centrifuge tubes and centrifuged as above. After centrifugation, the supernatant fluid was added to the decanted methylene chloride/ methanol solvent and rotary evaporated under vacuum from 25 to ,--60°C. When dry, the organic residue was removed with 10-20 ml of acetone. The organic residue was then transferred to a tared vial and evaporated with dry argon. The organic residue was weighed, dissolved in a known a m o u n t of dimethylsulfoxide (DMSO) and stored at - 2 0 ° C .
Aqueous extractions of MSWI ash Multiple 50-g portions of MSWI ash were suspended in 100ml of distilled deionized water in 250-ml Erlenmeyer flasks. Samples were then treated by one of two methods. Acid-treated samples underwent weekly additions of sulfuric acid to decrease the pH of the samples to ,-~ 6.0, while unmodified samples had no additions of acid. The samples were shaken in the dark at 150 r.p.m, at 10°C in an environmental chamber for 64-202 days. The pH of both groups was monitored on a weekly basis. Both groups were handled identically after removal from the environmental chamber. The samples were allowed to settle and the liquid was decanted offinto a 1-1 round-bottom flask. The liquid material was frozen at - 70°C and lyophilized to dryness. The flask was removed from the lyophilizer, and 200 ml of methylene chloride/methanol (1 : 1 v/v) was added to the lyophilizate. The samples were then extracted as described above.
Preparation of bacterial cells Salmonella typhimurium strains TA98 and TA100 were provided by Dr Bruce Ames, University of California, Berkeley. The cells were stored as
ANALYSIS OF THE GENOTOXICITY OF MUNICIPAL SOLID WASTE INCINERATOR ASH
1 13
frozen permanent cultures at - 8 0 ° C (Maniatis et al., 1982). Master plates were prepared from the frozen permanent cultures. An overnight culture of S. typhimurium strain TA98 or TA100 was grown from a single colony isolate (Maron and Ames, 1983), washed and concentrated 10-fold in 100mM potassium phosphate buffer, pH 7.4. The titer of each bacterial suspension was determined spectrophotometrically (Plewa et al., 1988). Appropriate amounts of the bacterial suspension and potassium phosphate buffer were combined to yield a final concentration of 1 × 109 cells/ml. These suspensions were placed on ice until used. Hepatic $9 (Aroclor 1254-induced) was obtained from Molecular Toxicology, Inc., College Park, MD, and stored at - 8 0 ° C . The $9 mixture was prepared as a 10% solution (Maron and Ames, 1983). Micropreincubation assay
The micropreincubation assay was developed to measure the mutagenicity and relative toxicity of small quantities of MSWI ash extracts. In general, 100/fl of the stock bacterial suspension (1 x 108cells) was placed in glass reaction tubes. The organic agents extracted from MSWI ash which were dissolved in DMSO were added to the reaction tubes in a range of microliter amounts. For direct mutagenicity tests the total volume of the reaction tubes was brought up to 600pl with potassium phosphate buffer. For tests that included hepatic microsomal activation, a 10% $9 mix was added to the reaction tubes instead of the phosphate buffer. Concurrent negative controls consisted of potassium phosphate buffer with the solvent or $9 mix with the solvent. Concurrent positive controls consisted of 2 5 m M ethylmethanesulfonate or sodium azide (direct tests) or 8.2/~M 2-aminofluorene ($9 activation tests). All reaction tubes were done in triplicate. These components were incubated at 37°C for 30 min with shaking at 200 r.p.m. After incubation, 2 ml of molten VB top agar supplemented with 550 # M histidine and biotin was added to each reaction tube. The top agar was poured onto VB minimal medium plates, incubated for 48 h at 37°C, and revertant his + colonies were counted. The assay was modified to determine the mutagenicity and the relative toxicity of the organic materials derived from the MSWI ash. The reaction tubes were prepared in a similar manner as above except that l l0/A of the bacterial suspension (1.1 × 108cells) was placed in glass reaction tubes. The extracted organic agents dissolved in DMSO were added to the reaction tubes. The total volume of the reaction tubes was adjusted to 610#1 with buffer or $9 mix. The concurrent negative and positive controls were conducted as previously stated. All reaction tubes were done in triplicate. These components were incubated at 37°C for 30 min with shaking at 200 r.p.m.
1 14 TABLE
M.A. SILKOWSKI ET A L
I
organic materials obtained from organic solvent extractions of MSWI ash Sample
g MSWI ash extracted
g organic material obtained
g organic material/ g MSWI ash
6A 7A 2B 5B
200 350 200 200
0.1399 6.995 x 0.1977 5.649 x 0.1377 6.885 x 0.1600 8.000 x Mean _+ SE = 6.882 _+ 0.482 x
10 10 I0 10 10
4 4 4 4 4g
After incubation, 55/d of the suspension was removed and added to a dilution series in phosphate buffer. Approximately 300 cells from each reaction tube were added to molten LB top agar and poured onto LB complete medium plates. The relative percent viable cells was determined after incubation for 24-36 h at 37°C. Molten VB top agar supplemented with 550/~M histidine and biotin was added to the remainder of the cells ( ~ 1 x l0 s) in each reaction tube. The top agar was poured onto VB minimal medium plates. Mutant bacterial colonies were counted after incubation at 37°C for 48 h. Therefore, the relative toxicity and the mutagenicity were determined separately.
Statistics The data were graphed and analyzed using Sigma Plot version 4.0 (Jandel Scientific, Corte Madara, CA). A mean mutation frequency value and the 95% confidence interval was determined for the values obtained for each experimental group. The values were compared with the values determined from their concurrent control. A response was judged to be significantly different from control data when the 95% confidence limits did not coincide. In general, each series of experiments was repeated three times with three VB plates analyzed per concentration. RESULTS
Measurement of the organic materials &olated from organic and aqueous extraction of M S W I ash M S W I ash was extracted with methylene chloride/methanol and with acetone to isolate organic agents. After the organic agents were recovered their weight was determined. The weight of the organic material was divided by the weight of the ash sample that was extracted and was recorded as g organic extract/g M S W I ash. The average amount of organic material isolated from four independent experiments was 688.2/~g/g M S W I ash (Table 1).
ANALYSIS OF FHE GENOTOXICITY OF MUNICIPAL SOLID WASTE INCINERATOR ASH
14
1I5
i
13 3-D-Q---oD-D 121 11 10 212
[] Unmodified Ash O Acid Treated Ash
9 8
7 6
5
0
I
I
1
I
50
100
150
200
TIME OF AQUEOUS EXTRACTION (Doys)
Fig. 1. The pH of the aqueous extract of unmodified (o) and acid-treated (o) MSWI ash as a function of extraction time.
The unmodified aqueous extracts of MSWI ash had a pH range of 12-12.6. The acid-treated aqueous extraction adjusted the range to approximately pH 6-7 (Fig. 1). Analysis of five independent unmodified aqueous extractions resulted in an average of 91.8 #g organic material/g MSWI ash (Table 2). The average amount of organic material from the acid-treated aqueous extracts in three experiments was 167.7/~g/g MSWI ash (Table 3). Extractions of distilled deionized water were conducted as control extractions. The control samples were treated the same as the unmodified aqueous extractions. The analysis of three independent samples resulted in an average of 3.67 ~g organic material/ml distilled deionized water. This material was neither mutagenic nor toxic. TABLE 2
Organic materials obtained from untreated aqueous extractions of MSWI ash Sample
g M S W I ash extracted
g organic material obtained
g organic material/ g MSW! ash
3A 8A 10A 4B 9B
250 250 350 200 250
0.0492 0.0196 0.0111 0.0187 0.0147 Mean ___ SE = 9.184 4-
1.968 7.840 3.170 9.350 5.880 2.820
x x x x x x
10 10 10 10 10 10
4 5 5 5 5 5g
116
MA SILKOWSKIETAL.
TABLE 3 Organic materials obtained from treated aqueous extractions o f MSWI ash Sample
g M S W I ash extracted
g organic material obtained
g organic material/ g MSWI ash
9A 1B 8B
250 250 450
0.0814 0.0210 0.0421 Mean ___ SE = 1.677 _+
3.256 8.400 9.355 0.790
x x × x
l0 4 l0 5 10 10 4g
Calibration of the micropreincubation assay The micropreincubation assay was calibrated with 2-aminofluorene with $9 activation in S. typhimurium strain TA98 and with sodium azide without activation in strain TA100. 2-Aminofluorene in a concentration range from 5 to 75 #M induced a positive concentration-response curve with a significant difference between the control and treatment group at ~> 10/~M (Fig. 2). Sodium azide in a concentration range from 1 to 20#M induced a positive concentration-response curve in TAI00 with a significant induction of revertants at 1/tM (Fig. 3).
Mutagenicity and toxicity of organic' extracts of M S WI ash We determined if direct-acting mutagens were present in agents derived from the organic solvent extractions of MSWI ash. For the mutagenicity and
44
750
IJJ
1303
z
500
I..J n-" I10
250 Z
01
o
25
50
75
2-AMINOFLUORENE (~M) Fig. 2. Calibration of the micropreincubalion assay with 2-aminofluorene with $9 activation.
ANALYSIS OF THE GENOTOXICITY
I
OF MUNICIPAL
I
SOLID WASTE INCINERATOR
I
ASH
] 17
I
-H 150 W
{1O3
~
100
n.kJ
O
o
5o
Z
2~ 0
0
I
I
I
I
5
10
15
20
SODIUM AZIDE (/.t,M)
Fig. 3. Calibration of the micropreincubation assay with sodium azide without $9 activation.
toxicity experiments the concentration range of the organic material was from 120 #g to 1.75 mg. Concurrent positive and negative controls were conducted with each experiment. The results of the mutagenicity and toxicity studies for TA98 and TA100 were negative (Fig. 4). We analyzed the MSWI ash organic extracts for mammalian promutagenic activity by supplementing hepatic microsomes ($9) in the reaction tubes. The 125
I
•~
I
I
~ ° ~ o
10o
loo
~O_ v
P-g
Fr]
75 ° 6 < 75
Z n~
o
50D~
5O
m
nl
z~
O
--
25 o r -
o o.o
0
0.5 1 .o 1.5 MSWl ASH ORGANIC EXTRACT (rng)
2.0
Fig. 4. Genotoxicity (TA98, O; TA I00, I ) and toxicity (TA98, O; TAI00, D) analysis of organic MSWI ash extracts without $9 activation.
I ]8
M.A. SILKOWSKI
300 c~ Lq
+ w
oF o \
EI AL
[]
250
[]
r~
100 --I ~
109
Analysis of the genotoxicity of municipal solid waste incinerator ash Michelle A. Silkowski, S h a n n o n R. S m i t h a n d M i c h a e l J, Plewa* Institute Jbr Environmental Studies, University of Illinois at Urbana-Champaign, Urbana, IL 61801-4723, USA (Received September 20th, 1990; accepted December 31st, 1990)
ABSTRACT Combined bottom and fly ash obtained from a Chicago, IL, municipal solid waste incinerator (MSWI) was extracted with organic solvents, water or acidified water. The mean amounts of organic material isolated from each extraction procedure were 688.2, 91.8 and 167.7/~g/g MSWl ash. These extracts were evaluated for toxicity and mutagenicity in Salmonella typhimurium strains TA98 and TA100. We developed and calibrated a micropreincubation assay to evaluate small concentrations of the organic extracts. No direct-acting mutagens were found, however the acid-treated aqueous extracts were toxic. Materials isolated with methylene chloride methanol were mutagenic after hepatic microsomal activation ($9). The mutagenic potencies of the organic extract normalized to a per gram ash basis was the induction of 103.46 revertants in TA98 and 247.5 revertants in TA100. The aqueous extracts were neither toxic nor mutagenic. However, the acid-treated aqueous extract was mutagenic to TAI00. The organic material isolated from the acidic extract had an induced mutagenic potency of 44.2 revertants/ mg extract. Normalizing these data indicate a mutagenic potency of 7.4 revertants/g MSWI ash leached.
INTRODUCTION M u n i c i p a l solid waste incineration I n c i n e r a t i o n is a widely used m e t h o d o f d i s p o s a l for m u n i c i p a l solid wastes. T h e b u r d e n o f i n c i n e r a t i o n h a s n o t b e e n r i g o r o u s l y assessed in t e r m s o f the p o t e n t i a l d a m a g e to the e n v i r o n m e n t a n d public health. T r e n d s t o w a r d s i n c i n e r a t i o n as a p r e f e r r e d m e t h o d o f w a s t e m a n a g e m e n t m i g h t be altered if risks were a d e q u a t e l y a d d r e s s e d a n d a n a l y z e d (Vining a n d E b r e o , 1990; Vining et al., 1990). M a s s i n c i n e r a t i o n as a n efficient, safe a n d e c o n o m i c m e t h o d o f solid w a s t e d i s p o s a l is b a s e d on the s u p p o s i t i o n t h a t the w a s t e * Corresponding author: Dr Michael J. Plewa, Professor of Genetics, Institute for Environmental Studies, University of Illinois at Urbana-Champaign, i101 West Peabody Dr., Urbana, IL 61801, USA. 0048-9697/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
| ]0
M.A. SILKOWSKI ET AL.
stream can be effectively managed by a sole technique as if it were a homogenous material. Municipal solid waste incinerators (MSWI) are one source of hazardous complex mixtures entering the environment. MSWI produce toxic gas emissions from the stacks and solid residues accumulate in the incinerator grates and combustion chambers. While there is considerable information concerning the constituents of ash residues (Hrudey et al., 1974; Karasek et al., 1987), there is little known about the potential mutagens or promutagens in these complex mixtures which are produced and released into the environment.
Products of incineration Incineration yields three forms of products, energy, gases and solid residues. Energy can be partially recovered and used for electricity generation or steam production. Gas-phase products exit almost solely through the stack. Solid residues (ash) accumulate at two sites, at the bottom on the grates (bottom ash) and at various points in the combustion chamber (fly ash) (Denison and Silbergeld, 1988).
Toxic metals Metals are not destroyed in the incineration process. The concentration of metals found in the waste stream before incineration and in the combined air emissions, bottom and fly ash after incineration are identical (Denison and Silbergeld, 1988). Heavy metals induce a wide range of toxic consequences, including carcinogenic, neurological, hepatic, renal, hematopoietic and other adverse effects (Friberg et al., 1986). Thus, the release of toxic metals from incinerators poses risks to the environment and to public health (Environmental Protection Agency, 1986, 1988; Denison and Silbergeld, 1988).
Toxic organic materials The toxicity of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) is a current subject of concern (Travis et al., 1989). Municipal solid waste incinerators were identified as a source of dioxins in the environment (Tong and Karasek, 1986; Travis and HattemerFrey, 1989). Octachlorinated dioxins were the most abundant isomer detected in residue MSWI ash samples. Smaller amounts of octachlorinated furans were also detected in ash samples (Stern et al., 1989). In a recent paper, Shane et al. (1990) reported P C D D congeners in ash samples from two of 18 MSW incinerators. The concentrations of the P C D D compounds were 57 and 290 ppb.
ANALYSIS OF THE GENOTOXICITY OF MUNICIPAL SOLID WASTE I N C I N E R A T O R ASH
1I I
Polyaromatic hydrocarbons (PAHs) induced toxic and carcinogenic responses under in vivo (Grimmer et al., 1988; Penn and Snyder, 1988; Bonassi et al., 1989) and in vitro conditions (DeFlora et al., 1989). PAHs are found in virtually all media and are generated from combustion processes (Junk and Ford, 1980). Stern et al. (1989) found that the most abundant PAHs in soil samples near a MSWI were pyrene, followed by fluoranthene, phenanthrene, anthracene and benzo[~]anthracene. There is a wide concentration range of aromatic compounds present in incinerator ash. Hrudey et al. (1974) detected 2.5 pg 3-methylphenanthrene/kg ash and 1500 #g di-isobutyl phthalate/kg ash. Recently, Kamiya et al. (1990) reported PAH concentrations in MSWI fly ash ranging from isomers of benzo[a]pyrene at 5 pg/kg ash to phenanthrene or anthracene at 438 #g/kg ash. In a survey of ash from 18 MSWI facilities, relatively high proportions of the organic materials found in MSWI ash were PAHs. There is a relationship between the generation of PAHs and incomplete combustion (Shane et al., 1990). Toxicity and mutagenicity studies on M S W I ash
Gustavsson (1989) found that mortality among workers in a municipal waste incinerator in Stockholm, Sweden, was increased over the general population. The excess deaths were a result of lung cancer and ischemic heart disease. The severity of illnesses was related to occupational exposure. Animal studies on MSWI products have focused primarily on the effects of fly ash. A!arie et al. (1989) found that fly ash inhaled by guinea pigs induced multifocal pneumoconiosis in the pulmonary tissue and elevated heavy metal concentrations in the lungs. Helder et al. (1982) found that fly ash was toxic to fish. Pani et al. (1983) analyzed organic material derived from solvent extractions of airborne dust (not stack emissions) from a MSW incinerator in Trieste, Italy. They found mutagenicity associated with the organic material in the solvent extracts. In a series of studies, mutagens were found to be associated with residue ash, wastewater and stack emissions from a MSW incinerator in Nagoya, Japan (Kamiya and Ose, 1987a,b; Kamiya et al., 1989a,b). After methylene chloride Soxhlet extraction of 18 MSWI ash samples, six extracts contained directacting or promutagenic agents when assayed with S. typhimurium strains TA98 and TA100 (Shane et al., 1990). However, none of the above studies evaluated the mutagenic properties of aqueous extracts of MSWI ash. EXPERIMENTAL PROCEDURES
Municipal solid waste incinerator ash
Municipal solid waste incinerator ash was obtained from the Northwest Waste-To-Energy Facility in Cook County, Illinois, with the cooperation of
l 12
M.A. S I L K O W S K I ET AL.
the Illinois Enviromental Protection Agency, Division of Land Pollution Control. The site inventory number was 0316230004.
Organic extraction of MSW1 ash Erlenmeyer flasks were soaked with N o - C h r o m Mix Acid Wash and washed. Multiple 50-g portions of MSWI ash were placed into the 250-ml flasks. The ash was extracted with 100ml methylene chloride/methanol (1 : 1 v/v). Each flask was sealed and shaken on a wrist-action shaker at a moderate speed for 24h. The solvent was decanted off the ash into 150-ml Corex centrifuge tubes and centrifuged at 2600 g for 20 min. The solvent was decanted into a 1-1 round-bottom flask and the volume reduced to ,-~ 20 ml with rotary evaporation under vacuum from 25 to ,-, 40°C. This residue was stored at - 2 0 ° C . The remaining ash was suspended in 100ml acetone and shaken in the same m a n n e r as above. The acetone was decanted off the ash into 150-ml Corex centrifuge tubes and centrifuged as above. After centrifugation, the supernatant fluid was added to the decanted methylene chloride/ methanol solvent and rotary evaporated under vacuum from 25 to ,--60°C. When dry, the organic residue was removed with 10-20 ml of acetone. The organic residue was then transferred to a tared vial and evaporated with dry argon. The organic residue was weighed, dissolved in a known a m o u n t of dimethylsulfoxide (DMSO) and stored at - 2 0 ° C .
Aqueous extractions of MSWI ash Multiple 50-g portions of MSWI ash were suspended in 100ml of distilled deionized water in 250-ml Erlenmeyer flasks. Samples were then treated by one of two methods. Acid-treated samples underwent weekly additions of sulfuric acid to decrease the pH of the samples to ,-~ 6.0, while unmodified samples had no additions of acid. The samples were shaken in the dark at 150 r.p.m, at 10°C in an environmental chamber for 64-202 days. The pH of both groups was monitored on a weekly basis. Both groups were handled identically after removal from the environmental chamber. The samples were allowed to settle and the liquid was decanted offinto a 1-1 round-bottom flask. The liquid material was frozen at - 70°C and lyophilized to dryness. The flask was removed from the lyophilizer, and 200 ml of methylene chloride/methanol (1 : 1 v/v) was added to the lyophilizate. The samples were then extracted as described above.
Preparation of bacterial cells Salmonella typhimurium strains TA98 and TA100 were provided by Dr Bruce Ames, University of California, Berkeley. The cells were stored as
ANALYSIS OF THE GENOTOXICITY OF MUNICIPAL SOLID WASTE INCINERATOR ASH
1 13
frozen permanent cultures at - 8 0 ° C (Maniatis et al., 1982). Master plates were prepared from the frozen permanent cultures. An overnight culture of S. typhimurium strain TA98 or TA100 was grown from a single colony isolate (Maron and Ames, 1983), washed and concentrated 10-fold in 100mM potassium phosphate buffer, pH 7.4. The titer of each bacterial suspension was determined spectrophotometrically (Plewa et al., 1988). Appropriate amounts of the bacterial suspension and potassium phosphate buffer were combined to yield a final concentration of 1 × 109 cells/ml. These suspensions were placed on ice until used. Hepatic $9 (Aroclor 1254-induced) was obtained from Molecular Toxicology, Inc., College Park, MD, and stored at - 8 0 ° C . The $9 mixture was prepared as a 10% solution (Maron and Ames, 1983). Micropreincubation assay
The micropreincubation assay was developed to measure the mutagenicity and relative toxicity of small quantities of MSWI ash extracts. In general, 100/fl of the stock bacterial suspension (1 x 108cells) was placed in glass reaction tubes. The organic agents extracted from MSWI ash which were dissolved in DMSO were added to the reaction tubes in a range of microliter amounts. For direct mutagenicity tests the total volume of the reaction tubes was brought up to 600pl with potassium phosphate buffer. For tests that included hepatic microsomal activation, a 10% $9 mix was added to the reaction tubes instead of the phosphate buffer. Concurrent negative controls consisted of potassium phosphate buffer with the solvent or $9 mix with the solvent. Concurrent positive controls consisted of 2 5 m M ethylmethanesulfonate or sodium azide (direct tests) or 8.2/~M 2-aminofluorene ($9 activation tests). All reaction tubes were done in triplicate. These components were incubated at 37°C for 30 min with shaking at 200 r.p.m. After incubation, 2 ml of molten VB top agar supplemented with 550 # M histidine and biotin was added to each reaction tube. The top agar was poured onto VB minimal medium plates, incubated for 48 h at 37°C, and revertant his + colonies were counted. The assay was modified to determine the mutagenicity and the relative toxicity of the organic materials derived from the MSWI ash. The reaction tubes were prepared in a similar manner as above except that l l0/A of the bacterial suspension (1.1 × 108cells) was placed in glass reaction tubes. The extracted organic agents dissolved in DMSO were added to the reaction tubes. The total volume of the reaction tubes was adjusted to 610#1 with buffer or $9 mix. The concurrent negative and positive controls were conducted as previously stated. All reaction tubes were done in triplicate. These components were incubated at 37°C for 30 min with shaking at 200 r.p.m.
1 14 TABLE
M.A. SILKOWSKI ET A L
I
organic materials obtained from organic solvent extractions of MSWI ash Sample
g MSWI ash extracted
g organic material obtained
g organic material/ g MSWI ash
6A 7A 2B 5B
200 350 200 200
0.1399 6.995 x 0.1977 5.649 x 0.1377 6.885 x 0.1600 8.000 x Mean _+ SE = 6.882 _+ 0.482 x
10 10 I0 10 10
4 4 4 4 4g
After incubation, 55/d of the suspension was removed and added to a dilution series in phosphate buffer. Approximately 300 cells from each reaction tube were added to molten LB top agar and poured onto LB complete medium plates. The relative percent viable cells was determined after incubation for 24-36 h at 37°C. Molten VB top agar supplemented with 550/~M histidine and biotin was added to the remainder of the cells ( ~ 1 x l0 s) in each reaction tube. The top agar was poured onto VB minimal medium plates. Mutant bacterial colonies were counted after incubation at 37°C for 48 h. Therefore, the relative toxicity and the mutagenicity were determined separately.
Statistics The data were graphed and analyzed using Sigma Plot version 4.0 (Jandel Scientific, Corte Madara, CA). A mean mutation frequency value and the 95% confidence interval was determined for the values obtained for each experimental group. The values were compared with the values determined from their concurrent control. A response was judged to be significantly different from control data when the 95% confidence limits did not coincide. In general, each series of experiments was repeated three times with three VB plates analyzed per concentration. RESULTS
Measurement of the organic materials &olated from organic and aqueous extraction of M S W I ash M S W I ash was extracted with methylene chloride/methanol and with acetone to isolate organic agents. After the organic agents were recovered their weight was determined. The weight of the organic material was divided by the weight of the ash sample that was extracted and was recorded as g organic extract/g M S W I ash. The average amount of organic material isolated from four independent experiments was 688.2/~g/g M S W I ash (Table 1).
ANALYSIS OF FHE GENOTOXICITY OF MUNICIPAL SOLID WASTE INCINERATOR ASH
14
1I5
i
13 3-D-Q---oD-D 121 11 10 212
[] Unmodified Ash O Acid Treated Ash
9 8
7 6
5
0
I
I
1
I
50
100
150
200
TIME OF AQUEOUS EXTRACTION (Doys)
Fig. 1. The pH of the aqueous extract of unmodified (o) and acid-treated (o) MSWI ash as a function of extraction time.
The unmodified aqueous extracts of MSWI ash had a pH range of 12-12.6. The acid-treated aqueous extraction adjusted the range to approximately pH 6-7 (Fig. 1). Analysis of five independent unmodified aqueous extractions resulted in an average of 91.8 #g organic material/g MSWI ash (Table 2). The average amount of organic material from the acid-treated aqueous extracts in three experiments was 167.7/~g/g MSWI ash (Table 3). Extractions of distilled deionized water were conducted as control extractions. The control samples were treated the same as the unmodified aqueous extractions. The analysis of three independent samples resulted in an average of 3.67 ~g organic material/ml distilled deionized water. This material was neither mutagenic nor toxic. TABLE 2
Organic materials obtained from untreated aqueous extractions of MSWI ash Sample
g M S W I ash extracted
g organic material obtained
g organic material/ g MSW! ash
3A 8A 10A 4B 9B
250 250 350 200 250
0.0492 0.0196 0.0111 0.0187 0.0147 Mean ___ SE = 9.184 4-
1.968 7.840 3.170 9.350 5.880 2.820
x x x x x x
10 10 10 10 10 10
4 5 5 5 5 5g
116
MA SILKOWSKIETAL.
TABLE 3 Organic materials obtained from treated aqueous extractions o f MSWI ash Sample
g M S W I ash extracted
g organic material obtained
g organic material/ g MSWI ash
9A 1B 8B
250 250 450
0.0814 0.0210 0.0421 Mean ___ SE = 1.677 _+
3.256 8.400 9.355 0.790
x x × x
l0 4 l0 5 10 10 4g
Calibration of the micropreincubation assay The micropreincubation assay was calibrated with 2-aminofluorene with $9 activation in S. typhimurium strain TA98 and with sodium azide without activation in strain TA100. 2-Aminofluorene in a concentration range from 5 to 75 #M induced a positive concentration-response curve with a significant difference between the control and treatment group at ~> 10/~M (Fig. 2). Sodium azide in a concentration range from 1 to 20#M induced a positive concentration-response curve in TAI00 with a significant induction of revertants at 1/tM (Fig. 3).
Mutagenicity and toxicity of organic' extracts of M S WI ash We determined if direct-acting mutagens were present in agents derived from the organic solvent extractions of MSWI ash. For the mutagenicity and
44
750
IJJ
1303
z
500
I..J n-" I10
250 Z
01
o
25
50
75
2-AMINOFLUORENE (~M) Fig. 2. Calibration of the micropreincubalion assay with 2-aminofluorene with $9 activation.
ANALYSIS OF THE GENOTOXICITY
I
OF MUNICIPAL
I
SOLID WASTE INCINERATOR
I
ASH
] 17
I
-H 150 W
{1O3
~
100
n.kJ
O
o
5o
Z
2~ 0
0
I
I
I
I
5
10
15
20
SODIUM AZIDE (/.t,M)
Fig. 3. Calibration of the micropreincubation assay with sodium azide without $9 activation.
toxicity experiments the concentration range of the organic material was from 120 #g to 1.75 mg. Concurrent positive and negative controls were conducted with each experiment. The results of the mutagenicity and toxicity studies for TA98 and TA100 were negative (Fig. 4). We analyzed the MSWI ash organic extracts for mammalian promutagenic activity by supplementing hepatic microsomes ($9) in the reaction tubes. The 125
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~ ° ~ o
10o
loo
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P-g
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75 ° 6 < 75
Z n~
o
50D~
5O
m
nl
z~
O
--
25 o r -
o o.o
0
0.5 1 .o 1.5 MSWl ASH ORGANIC EXTRACT (rng)
2.0
Fig. 4. Genotoxicity (TA98, O; TA I00, I ) and toxicity (TA98, O; TAI00, D) analysis of organic MSWI ash extracts without $9 activation.
I ]8
M.A. SILKOWSKI
300 c~ Lq
+ w
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250
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r~
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