Environ. Sci. Technol. 1987, Z f , 57-64
(16) Bailey, P. S. Ozonation in Organic Chemistry;Academic: New York, 1978 (Vol. 1)) 1982 (Vol. 2). (17) Herron, J. T.; Huie, R. E. J. Am. Chem. Soc. 1977, 99, 5430-5435. (18) Atkinson, R.; Aschmann, S. M.; Carter, W. P. L.; Pitts, J. N., Jr. Int. J. Chem. Kinet. 1983, 15, 721-731. (19) Atkinson, R. Chem. Rev. 1986,86, 69-201.
Hahn, J. Ann. N.Y. Acad. Sei. 1980,338,359-376. Hatakeyama, S.; Tanonaka, T.; Weng, J.; Bandow, H.; Takagi, H.; Akimoto, H. Environ. Sei. Technol. 1985,19, 935-942.
Niki, H.; Marker, P. D.; Savage, C. M.; Breitenbach,L. P. Environ. Sci. Technol. 1983, 17, 312A-322A. English, J., Jr.; Barber, G. W. J . Am. Chem. SOC.1949, 71, 3310-3313.
Schreiber, S. L.; Claus, R. E.; Reagan, J. Tetrahedron Lett. 1982,23,3867-3870.
Benson, S. W. Thermochemical Kinetics, 2nd ed.; Wiley: New York, 1976.
Received for review April 7, 1986. Accepted July 22,1986. This work was partially supported by Grant-in-Aid for Scientific Research 59740224 from the Japanese Ministry of Education, Science and Culture.
Mutagenic Activity of Two Soils Amended with a Wood-Preserving Waste Klrby C. Donnelly,' Phebe Davol, K. W. Brown, M. Estlrl, and J. C. Thomas Soil and Crop Sciences Department, Texas A&M University, College Station, Texas 77843
Organic compounds extracted from soils amended with a wood-preserving bottom sediment induced a mutagenic response in bioassays using Salmonella typhimurium and Aspergillus nidulans. The maximum level of mutagenic activity was observed in the base fraction from the waste-amended Bastrop soil. In the bioassay using S. typhimurium, the base fraction collected immediately after application from the Bastrop soil induced 77 net revertants/mg of extract, while the base fraction collected 540 days after waste application induced 1561 net revertants/mg of extract. Since the amount of extractable material decreased greatly over this period, degradation appears to have reduced the weighted activity of woodpreserving waste added to soil. The weighted activity, as measured with S. typhimurium strain TA98, of the neutral fraction from 1 g of waste-amended Norwood soil was reduced from 7322 net revertants immediately after application to 1541net revertants 1200 days after application. Major residual organic constituents in the soil were tentatively identified by GC/MS/DS and included pentachlorophenol, trimethylnaphthalene, acenaphthylene, fluoranthene, pyrene, and cyclopentaphenanthrene. Introduction
The application of hazardous waste to soil is restricted by regulations to include only those wastes that will be rendered less hazardous or nonhazardous by chemical or biological reactions in the soil (1). These regulations assume that degradation, immobilization, and transformation will serve to reduce the hazardous characteristics of soilapplied waste constituents. Degradation in soil is generally assumed to be mostly biological. Hamaker (2) states that biological degradation normally accounts for approximately 80% of degradation in the soil. However, in a soil contaminated by toxic and resistant compounds from a hazardous waste, chemical and photochemical degradation will also play an important role. Degradation may not result in the complete mineralization of a hazardous waste but may render waste constituents less hazardous or nonhazardous by removing substituted groups, by breaking an aromatic ring, or by substitutions that produce a less reactive product. Soil degradation may not, however, always result in detoxification of a hazardous waste (3). The major factors influencing the mutagenic potential of residual organic compounds in the soil at a land treatment facility include the number of different compounds present and their concentrations, toxic effects, and interactions. Degradation will influence the mutagenic 0013-936X/87/0921-0057$01.50/0
potential of a waste-amended soil by reducing the concentration of certain compounds and altering the reactivity of others. Oxidation or substitution at specific sites has been shown to increase the reactivity of polycyclic aromatic hydrocarbons (4-7). Thus, techniques are needed to evaluate the influence of degradation on the mutagenic activity of hazardous waste amended soil. The technique currently being used to monitor land treatment and spill sites employs chemical analysis to define the various reaction products that may occur in the soil. Chemical analysis is a valuable tool for defining the types and concentrations of contaminants in the soil. However, chemical analysis alone may fail to account for the synergistic, antagonistic, or additive interactions between soil and waste components and the affect of degradation on these interactions. In addition, the results from a chemical analysis must be extrapolated to estimate the toxicological end point in a biological system. The use of biological analysis alone may fail to account for artifacts generated in the collection or extraction process or the presence of toxic residues bound to the soil in an unextractable form. However, the use of bioassay-directed chemical analysis provides more accurate information than either technique alone from which to obtain a risk assessment. This technique employs a battery of short-term bioassays to define the mutagenic potential of a sample and is followed by chemical analysis of selected samples to identify the chemicals present in the mutagenic samples. The objective of this study was to employ bioassay-directed chemical analysis to evaluate changes in the mutagenic activity of the organic constituents from soil-applied wood-preserving bottom sediment. Information relevant to the environmental interactions of pentachlorophenol, one of the primary ingredients of wood-preserving waste, is pertinent because pentachlorophenol is the most frequently identified pesticide at National Priorities List sites (8). Materials and Methods Two 190-L samples of wood-preserving waste were
collected from a sediment pond at a plant using both pentachlorophenol and creosote. A 1-L subsample of this waste was stored at 0 "C. Mutagenic characteristics of the waste and soils are summarized in Table I. The relatively high mutagenicity of the unamended Bastrop soil may have been related to a previous application of 2,4-dichlorophenol and 2,4,5-trichlorophenol (9). This was not deemed to be a serious problem because the presence of
0 1986 American Chemical Society
Environ. Sci. Technol., Vol.
21, No. 1, 1987 57
Table I. Selected Properties of Hazardous Wastes and Soils Used in the Degradation Study
sample Norwood soil Bastrop soil wood-preserving waste
fraction crude extract crude extract crude extract acidc base neutral
solvent-extractable, organics, mg/g 0.057 0.229 270 23 24 223
net revertants/mg, mean f SD"
weighted activity, rev/gb
35 f 16 2 434 f 96 99 130 f 17 35 100 70 f 17 1610 126 f 11 3 024 90 f 36 20 070 Net TA 98 his+ revertants per milligram. Calculated from dose level of 1 mg/plate using strain TA98 with metabolic activation. Measured from duplicate plates in two independent experiments. Weighted activity = revertants per gram of material extracted; calculated by multiplying revertants/mg times mg/g of extractable hydrocarbons. Acid fraction toxic at 1 mg/pt; value represents mutagenic response a t 500 pg/pt.
these compounds should favor a microbial population that could act on similar compounds likely to be present in the wood-preserving waste. Chemical analysis indicated that the concentration of chlorinated herbicides in the control soil was less than 20 pg/g (9). Previous research has demonstrated that soils with a past history of exposure to oil (10, 11) or polycyclic aromatic hydrocarbons (12, 13) contained relatively high populations of organic degrading microbes. Chemical analysis of the control soils detected primarily short-chain alkanes (Cl2-Cz0),and benzenedicarboxylic acid was additionally detected in the Norwood soil (9). The waste contained 27 % solvent-extractable organic compounds and was applied at a loading rate of 3.1% to two soils selected to exhibit a probable range in soil textures. These soils were Norwood sandy clay (Typic Udifluvent) and Bastrop clay (Udic Paleustalf). Sifted air-dried soil was packed in wooden boxes (45 cm X 57 cm X 20 cm) in 5-cm lifts to a depth of 17 cm. Three boxes of each soil received waste, while three of each served as controls. Waste was incorporated into the soil by removing approximately half of the soil from the box and applying the waste to the remaining soil in the box. The removed soil was returned to the box and mixed with a small trowel until a uniform consistency was achieved. Soil samples were collected before, immediately after, and 180, 360, 540, and 1200 days after waste application. Soil samples were composites of 6-10 randomly selected plugs, each representing 0-17 cm in depth. Samples were stored at 0 OC until analyzed. Following the collection of soil samples, the boxes were subjected to 1 h of simulated rainfall at an intensity of 8.9 cm/h with a rotating disc rainfall simulator, as described by Morin et al. (14). Additional rainwater was periodically added with a sprinkler can to maintain a moisture content near field capacity. Soil and waste samples were extracted with dichloromethane by the procedures of Brown and Donnelly (15). The extracts were taken to dryness on a Brinkman-Bucci rotary evaporator, and the residue was partitioned into acid, base, and neutral fractions following the procedures of Donnelly et al. (16). Compounds present in the acid, base, and neutral fractions of selected samples were tentatively identified with a Finnigan OWA automated gas chromatograph/mass spectrometer (GC/MS/DS) equipped with a J&W Scientific (Orangeville, CA) fused silica capillary column DB5-30W. The DB-5-30W column had a liquid phase that was bonded 1% vinyl/5 % phenylmethyl polysiloxane. One-microliter aliquots were used with a helium carrier gas flow of 36 cm/s. The gas chromatograph (GC) oven temperature program was 60 "C for 1 min and then increased at 6 "C/min intervals to 260 "C with a hold time of 12 min. The OWA unit had a splitless mode injector. The software had a mass spectra library of 31 331 organic compounds. 58
Environ. Sci. Technol., Vol. 21, No. 1 , 1987
Table 11. Characteristics of Bacterial and Fungal Strains Used for Mutagenicity Testing
characteristics mutation detected negative controls none MezSO 8-MOP positive controls B [ ~ I PB ~ I+ P 8-MOP (plus NUV)
S. typhimurium TA98 TAlOO
A . nidulans meth G1 biAl
reverse
reverse
forward
30 f 8 26 f 6 NT
127 f 24 120 f 38 NT
NT 0.7 f 0.68 1.4 f 0.98
131 f 21 28 f 7 609 f 152 707 f 130 NT NT
NT 40.0 f 20.0 245.0 f 45.0
a Controls: All values represent historical mean f SD. Negative controls: none = no additions to top agar; MezSO = 100 pL of dimethyl sulfoxide; 8-MOP = 8-methoxypsoralen. Positive controls: B[a]p - and B[a]p + = 5 pg/plate (Salmonella) or 11 bg/ plate (Aspergillus) benzo[a]pyrene without and with metabolic activation; 8-MOP (plus NUV) = 50 pg/plate 8-methoxypsoralen plus near-UV light. Salmonella data are expressed as total mutation frequency; Aspergillus data are expressed as induced mutants per lo6 survivors. NT = not tested.
Biological Analysis. The SaZmonella/microsome assay of Ames et al. (17)was used to monitor the mutagenic activity of concentrated soil extracts (strains courtesy of Dr. Bruce N. Ames, University of California, Berkeley, CA), with the same procedures as Maron and Ames (18). Extracts were tested on duplicate plates in two independent experiments in the standard plate incorporation assay at a minimum of four sample dose levels with and without metabolic activation (0.3 mL of rat liver/mL of S9 mix) by using strains TA98 and TA100. Aroclor 1254 induced rat liver was obtained from Litton Bionetics (Charleston, SC). Solvent and positive controls were used to verify proper functioning of the microbial strains in each experiment (17). The historical mean and standard deviation of solvent and positive controls for the duration of this project are provided in Table 11, while a more detailed discussion of the variability of the bioassay is provided in Brown et al. (19). (1) Statistics. Data from the Salmonella bioassay were analyzed with two different procedures. First, the data for each sample were analyzed by the modified two-fold rule as described by Chu et al. (20). With this procedure, a response is considered positive mutagenic if the induced mutation frequency is at least twice the solvent control for two consecutive dose levels. Second, a split-plot design, as described by Montgomery (21),was used to evaluate the data with respect to replicate analysis, replicate treatments, and time. The subplot error was found by pooling the three-way interaction between time, treatment, and rep-
Table 111. Mutagenic Activity of Fractions of Solvent-Extractable Organic Compounds from Wood-Preserving Waste-Amended Soils As Measured in the Salmonella Assay
sample waste Norwood
Bastrop
fraction
dose/ plate, rg
acid base neutral acid
500 1000 1000 500
base
1000
neutral
1000
acid
500
base
1000
neutral
1000
net revertants/plate, mean f SD" TAlOO TA9gd +s9 -s9 +s9
day
-s9
0 180 360 540 1200 0 180 360 540 1200 0 180 360 540 1200 0 180 360 540 1200 0 180 360 540 1200 0 180 360 540 1200
0 0 I f 8 ND (a) 7 f 19 (a) 9 f 5 (a) 9 f 6 (a) 74 f 36 (c) 3 f 6 (a) 33 f 33 (a) 82 i 15 (b) 294 i 158 (b) 443 f 52 (c) 1 f 7 (a) 178 f 155 (c) 22 f 6 (a) 23 f 12 (a) 23 f 18 (a) ND (a) 89 f 46 (a) 84 f 141 (a) 153 f 124 (b) 14 f 20 (a) ND (a) 14 f 6 (a) 143 f 93 (b) 1363 f 1242 (c) 52 f 7 (b) 3 f 8 (a) 29 f 22 (a) 0 f 6 (a) 21 f 12 (a) 11 f 8 (a)
I
70 f 17 126 f 11 90 f 36 104 i 118 (a) 108 f 61 (a) 120 f 19 (a) 72 f 13 (a) 225 f 66 (c) 90 f 12 (a) 146 f 138 (a) 501 f 24 (c) 341 f 133 (b) 962 f 170 (c) 65 f 8 (a) 245 f 78 (c) 148 f 24 (b) 105 f 45 (b) 128 f 22 (a) 52 f 11 (a) 93 f 4 1 (a) 221 f 432 (a) 608 f 422 (b) 45 f 27 (a) mm f 24(a) 178 f 48 (b) 409 f 169 (c) 1561 f 410 (c) 110 f 41 (a) 97 f 17 (a) 154 f 8 (a) 93 f 45 (a) 177 f 113 (a) 128 f 22 (a)
0 0 0 ND ND ND ND
264 f 34 276 f 43 195 f 66 186 f 17 200e 152 f 157 109 f 31
ND ND 16 f 11 576 f 91
164 f 22 11 f 53 366 f 99 406 f 53
ND 25 f 39 2 f 29 19 f 9
17 f 45 228 f 152 255 f 23 163 f 51
ND ND ND ND
ND ND 261 f 122 136 f 75
ND ND 10 f 40 230b
121 f 38 139 f 39 296 f 146 306b
ND ND 3 f 19 63 f 75
170 f 30 123 f 95 244 f 121 362 f 325
%
weighted activity, rev/g of soilb
total activity
1610 3024 20070 1456 (a) 1037 (b) 360 (b) 230 (b) 478 (b) 522 (a) 394 (a) 401 (a) 102 (a) 404 (a) 7943 (a) 10315 (b) 5076 (a) 2194 (b) 1541 (b) 1144 (a) 986 (a) 2077 (a) 3770 (a) 342 (b) 308 (a) 552 (a) 450 (a) 624 (a) 165 (a) 12028 (a) 9332 (a) 5134 (b) 7841 (b) 1536 (b)
100 71 25 16 33 100 75 77 20 77 100 129 64 28 19 100 86 182 330 30 100 179 146 203 54 100 78 43 65 13
Mean represents average of three test samples tested on duplicate plates in two independent experiments unless otherwise indicated ( N = 12). ND = none detected (less than background). bNet TA98 his+ revertants per gram of soil (measured with metabolic activation). Calculated by multiplying specific activity (net rev/mg) times residual weight (mg/g). e Mean represents average of only two samples; standard deviation not provided. dData analyzed by t test on four replicates from each of three boxes: (a) = at least one of the three boxes was not significant (p > 0.05); (b) = all three boxes were significant at (p < 0.05 or less; (c) = all three boxes were significant at p < 0.01.
lication and the two-way interaction between time and replication. The results of analysis of variance (ANOVA) for the wood-preservingwaste-amended soils indicated that there was a significant difference between replicate treatments Cp < 0.01), probably due to the heterogeneity of the waste-amended soil. Thus, the statistical analysis of bioassay data were based on a comparison of each of three replicate treatments. To determine if there was a significant change in the number of net revertants induced by a soil extract, a t test was performed on each of three replicate treatments with the data collected 180,360,540, and 1200 days after waste application and compared to the data collected immediately after waste application. (2) Eukaryotic Bioassay. Soil extracts were also tested with Aspergillus nidulans. Samples were tested at one dose level, usually the weight of extract equivalent to 200 mg of soil, and one exposure time (selected to yield approximately 50% survival) on five plates in two independent experiments with and without metabolic activation. The procedures were the same as those in Scott et al. (22),except the cells were exposed in a 13 X 100 mm screw-capped culture tube. The induced mutation frequency for each sample was determined by subtracting the spontaneous mutation frequency from the total mutation frequency. A sample was considered positive if the induced mutation frequency was more than twice the spontaneous mutation frequency. Positive controls included 8-meth-
oxypsoralen plus near-ultraviolet light without activation and benzo[a]pyrene with metabolic activation (Table 11). Results
The results presented in Table I11 indicate that the maximum level of mutagenic activity in the wasteamended soils was detected by using strain TA98 with metabolic activation. All soil extracts induced a positive response in strain TA98 when evaluated with the modified two-fold rule (20). Degradation produced a significant increase in both the total and direct-acting mutagenicity of at least one fraction of the residual organic compounds extracted from both waste-amended soils (Table 111). Degradation in the context of this report is intended to include chemical and photochemical degradation, as well as biodegradation. In the waste-amended Norwood soil, there was a significant increase (p < 0.01) in the directacting (without metabolic activation) mutagenicity of the acid, base, and neutral fractions and the indirect activity (with metabolic activation) of the base and neutral fractions (Table 111). The maximum level of mutagenic activity for the acid, base, and neutral fractions of the waste-amended Norwood soil was observed 1200,1200,and 180 days after application, respectively. In the wasteamended Bastrop soil, there was a significant increase in both direct and indirect mutagenicity of the acid (p < 0.05) and base (p < 0.01) fractions but no significant change in Envlron. Scl. Technol., Vol. 21, No. 1, 1987
59
- S!
+
S!
1200
1200
8oo
800
400
400
v)
c
5 t
+.In t a3
9 c a
g
* 200
200
.*-
100
0
/ / I -
,'-
100
0.1
0.5
1.o
Dose/Plate (mg) Figure 1. Mutagenlcity of base fraction extracted from wood-preserving waste (m) or waste-amended Bastrop soil on day 0 (0),180 (a),360 (O), 540
(A),or 1200 (*), as measured with S . typhmurlom strain TA98.
the mutagenicity of the neutral fraction. The maximum level of mutagenic activity for the acid, base, and neutral fractions of the waste-amended Bastrop soil was observed 540 days after application (Table 111). The most dramatic increase was observed in the base fraction from the Bastrop soil in which the direct and indirect mutagenicity of the sample collected 540 days after application was appreciably greater than the mutagenicity of the sample collected on day 360, although the response induced by the sample collected 1200 days after waste application was approximately equal to that induced by the base fraction collected immediately after waste application (Figure 1). These results indicate that while degradation reduced the quantity of organic compounds residual in the soil, the mutagenicity per unit weight of the residual compounds was increased. Previous studies have also observed increased mutagenicity in soils incubated with polycyclic aromatic hydrocarbons (23) or sewage sludge (24). Results from the biological analysis of the wood-preserving waste-amended soil were also compared by equivalent weights of soil (Table 111). The weighted activity was obtained by multiplying the specific activity (net revertants/mg) by the residue weight (mg/g) for each fraction. The accuracy of these calculations is limited, because it is difficult to ascertain if a reduction in solvent-extractable organics in the soil had a corresponding effect on biological activity. In addition, these calculations can be used to provide a description of the overall effect of soil application on the mutagenic potential of wasteamended soil. Results from the Salmonella bioassay indicate that degradation eventually reduced the mutagenic potential of equivalent weights of waste-amended Norwood soil. The activity of the acid, base, and neutral fractions collected from the Norwood soil 1200 days after waste application was 33, 77,and 19%, respectively, of the ac60
Environ. Scl. Technol., Vol. 21, No. 1, 1987
tivity of the fraction collected immediately after waste application (Table 111). In the waste-amended Bastrop soil, the weighted activity of the acid and base fractions was increased appreciably 540 days after waste application, while the weighted activity of the neutral fraction remained relatively unchanged (Table 111). However, the weighted activity of the acid, base, and neutral fraction collected 1200 days after waste application was 30, 54, and 13%, respectively, of the activity of the corresponding fractions collected immediately after waste application. Thus, the weighted activity of the acid and neutral fractions from the waste-amended Norwood and Bastrop soils was significantly reduced over time (Table 111). In the Aspergillus bioassay, the sample exposure concentration was adjusted to test each sample at a single dose level extracted from an approximately equal weight of soil. The exposure concentrations used were much less than those used in the Salmonella assay. The Aspergillus assay was sensitive to the lower concentrations because, as a forward mutation assay, it is sensitive to a broader range of chemicals (25) and because the liquid exposure assay is generally more sensitive than the plate incorporation assay (26). The data from the Aspergillus assay also displayed a high degree of variability (Table IV). The variability of these data may have been influenced by the complex nature and heterogeneity of the soil extracts. Similar variations were observed in some of the data from the Salmonella assay (Table 111). The overall trend of the weighted activity of the soil extracts in Aspergillus was similar to the trend observed in the Salmonella assay. In all three fractions from both soils, the maximum level of mutagenic activity, with and without metabolic activation, was detected in the sample collected 180 days after waste application (Table IV). In the sample collected 360 days after application from the
Table IV. Total Mutants per Survivor Induced by Fractions of Solvent-Extractable Organic Compounds from Wood-Preserving Waste-Amended Soils As Measured in the Aspergillus Assay
sample waste Norwood
fraction acid base neutral acid base neutral
Bastrop
acid base neutral
dose/ plate, Pg 125 63 125 80 160 15 60 60 16 120 80 15 100
150 50 40 120 15 240 100 11
gram equiv 0.005 0.003 0.0006 1 2 1.5 1 2 1.6 0.2 0.2 0.2 1 2 5 1 2 2 0.2 0.2 1.1
day
0 180 360 0
180 380 0 180 360 0
180 360 0 180 380 0
180 360
induced mutation frequency per IO6 survivors, mean f SEM -s9 +s9 7f4 9f6 5f3 11 & 10 15 f 10 715 918 14 f 11 3f2 10 f 10
36 f 27 4f3 11 f 8 21 f 15 3f4 13 f 9 27 f 21 4f3 20 f 15 93 f 73 3f2
14 f 8 14 f 9 14 f 8 15 f 14 28 f 20 2fl 14 f 14 50 f 36 6f4 9f8 48 f 35 7f5 12 f 9 51 f 37 7f5 17 f 12 40 f 29 4f3 29 f 21 73 f 62 4f3
weighted activity, rev/g of soil" 2800 4667 23333 15 14 1 14 25 4 45 240 35 12 26 1.4 17 20 2 145 365 3.6
%
total activity
100 93 7 100 179 29 100 533 78 100 217 12 100
118 12 100 252 2
" Revertants per gram of waste = induced mutation frequency divided by gram equivalents. Norwood soil, the weighted activity of the acid, base, and neutral fraction was 7, 29, and 78%, respectively, of the weighted activity of the sample collected immediately after waste application (Table IV). While in the bastrop soils, the weighted activity of the sample collected 360 days after application was 12,12, and 2%, respectively, of the activity of the sample collected immediately after waste application. Higher concentrations of the soil extract were not tested in the Aspergillus assay. As a result, there is no additional information to describe the highly mutagenic and persistent residue that was detected with the Salmonella assay. However, the results of the Aspergillus assay do indicate an increase in both direct and indirect mutagenicity 180 days after waste application, with an appreciable reduction in the weighted activity of the waste-amended soil 360 days following waste application. The mutagenic response induced by all three fractions from both the waste-amended Norwood and Bastrop soils 360 days following waste application, as measured with A. nidulans, was near or below that which would be considered nonmutagenic (Table IV). A large number of chemicals were identified in the acid, base, and neutral fractions from the wood-preserving waste-amended Norwood and Bastrop soils (Table V). Due to the large number of compounds present in the crude extract, incomplete separation was obtained in the acid, base, and neutral fractions. Prediction of genotoxic effects from chemical analysis data alone would be difficult if not impossible. A number of alkanes were identified in the base and neutral fractions of the unamended soil extracts. Promoting agents included dodecane, tetradecane, and octadecane, cocarcinogens included octadecane and eicosane, and one inhibitor, hexadecane, were also identified (27, 28). The majority of the alkanes detected in waste-amended soil extracts were also present in control soil extracts (9). Chemical analysis of wood-preserving waste-amended soils tentatively identified more than 14 polycyclic aromatic hydrocarbons (PAH) (Table V). Two-ring aromatic hydrocarbons tentatively identified in the soil extracts
included methylnaphthalene, dimethylnaphthalene, trimethylnaphthalene, and acenaphthylene. Of these, only methylnaphthalene and acenaphthylene have been found to be mutagenic in a foreward mutation assay (29). Nonmutagenic PAH with three or more aromatic rings were identified in the soil-waste extracts and included anthracene, penanthrene, and cyclopentaphenanthrene (29, 30). Of these compounds, only anthracene has been tested in a whole animal bioassay and found to be noncarcinogenic (31). Identified mutagenic PAH included methylphenanthrene and dimethylphenanthrene (4). Pyrene and fluoranthene, which are not carcinogens (32),have, however, displayed cocarcinogenic activity (33). A study by La Voie et al. ( 4 ) found that methylphenanthrene and dimethylphenanthrene were .mutagenic toward Salmonella, although only dimethylphenanthrene acted as a tumor initiator on mouse skin. Two polycyclic aromatic sulfur heterocycles, methyldibenzothiophene and dibenzothiophene, were identified in the soil-waste extracts. Both of these compounds have been tested in the Salmonella assay, and neither was mutagenic (34). The only chlorinated hydrocarbon identified in the waste-soil extracts were pentachlorophenol. Pentachlorophenol is not mutagenic in the Salmonella assay (35) but has been found to induce mitotic gene conversion in Saccharomyces cereuisiae (36). Discussion
For land application of waste to be an environmentally sound disposal alternative, hazardous constituents must be transformed, degraded, or immobilized in the surface layer of soil (I). Since the boxes used in this study were 20 cm deep and the bottoms were sealed, leaching did not occur; however, approximately 14 L of runoff water was collected on five separate occasions from both soils containing a total of 1.59 and 2.29 g of solvent-extractable organics from the Norwood and Bastrop soils, respectively. The quantity of solvent-extractable organics collected in the runoff water accounted for less than 0.1% of the solvent-extractable organics applied with the waste. Since Environ. Sci. Technol., Vol. 21, No. 1. 1987
61
Table V. List of Compounds Tetatively Identified in Wood-Preserving Waste-Amended Soil fraction sample Norwood, day 0
acid, compound (peak n0.p not determined
base, compound (peak noJn methylnaphthalene (249); M1, CO dimethylnaphthalene (289); MO acenaphthylene (314); M1 hexene (332) dimethylbutane (358) unknown (377) (epoxymethy1)pentane (397) unknown (435,461) trifluoromethane (472) unknown (496,523,541,570, 590, 616, 646, 676)
Norwood, day 360
unknown (267) dimethylnonane (303) methylpropylpentanol (358) unknown (377) ethylhexanol (399) pentachlorophenol (419); MO, C1 dimethylundecane (437) trimethyloctane (469) benzenedicarboxylic acid (492); MO fluoranthene (522); M1, CO; CC pyrene (538); M1, CO, CC unknown (662,591,620,639, 657)
unknown (277,303) ethylheptane (318) unknown (339) dimethylnonane (359) unknown (378) dimethylhexane (399) trimethylheptane (469) propylaziridine (492) fluoranthene (523); M1, CO, CC pyrene (539); M1, CO, CC unknown (586,619, 650, 681)
Bastrop, day 0
methylntphthalene (239); M1, CO unknown (246) dimethylnaphthalene (282, 292); MO acenaphthylene (319); M1 alkane (326,367, 407) dibenzofuran (332) trimethyl naphthalene (346); MO phenalene (353); M1 pentachlorophenol (429); MO, C1 unknown (481,515) fluoranthene (535); M1, CO, CC pyrene (553); M1, CO, CC unknown (635,691,728,770,807,846,884)
methylnaphthalene (240); M1, CO dimethylnaphthalene (285, 299); MO acenaphthylene (317); M1 methylethylnaphthalene (326) dibenzofuran (331) unknown (343) phenalene (352); M1 trimethylnaphthalene (363); MO unknown (365) alkane (405) dibenzothiophene (424); MO methyldibenzothiophene (461); M1 methylphenanthrene (478); M1 phenylnaphthalene (504) fluoranthene (533) pyrene (550) unknown (627,679, 730, 775, 822, 876)
Bastrop, day 360
unknown (267) ethylmethylpentanol (277) unknown (303) acenaphthylene (323); M1 trimethylnaphthalene (345); MO dodecane (358); P, CC dimethyloctane (376); P dimethylundecane (398) pentachlorophenol (422); C1 methylpropylnonane (432, 467, 498) phenylnaphthalene (452) cyclopentaphenanthrene (473); MO unknown (510) fluoranthene (519); M1, CO, CC pyrene (634); M1, CO, CC unknown (558)
dimethylhexane (277) unknown (303) acenaphthylene (321); M1 unknown (343, 361) trimethyloctane (397) unknown (456) cyclopentaphenanthrene (476); MO unknown (502) heptadecane (429) dimethylphenanthrene (514); M1 fluoranthene (521); M1, CO, CC pyrene (537); MI, CO, CC
neutral, compound (peak no.)# unknown (238) methylnaphthalene (251, 257), MI, CO biphenyl (281); MO, C1 dimethylnaphthalene (291, 295); MO acenaphthylene (321); M1 dibenzofuran (334) fluorene (359); MO, CO unknown (384,406) dibenzothiophene (424); MO phenanthrene (432); MO, CO unknown (462,478, 503) fluoranthene (531); M1, CO, CC pyrene (547); M1, CO, CC unknown (589, 615, 637) unknown (237, 271, 281) alkane (308) acenaphthylene (324); M1 unknown (334) trimethylnaphthalene (347); MO unknown (375) dimethyloctane (384) dimethylbiphenyl (407) unknown (421) methylpropylpentanol (445) unknown (462) trimethyloctane (476) cyclopentaphenanthrene (481); MO unknown (499) dimethylundecane (508) fluoranthene (529); M1, CO, CC pyrene (544); M1, CO, CC unknown (568) unknown (220) methylnaphthalene (244, 250); M1, CO unknown (277) dimethylnaphthalene (287, 293); MO acenaphthylene (319); M1 alkane (323) dibenzofuran (332) trimethylnaphthalene (337, 343); MO phenalene (358) alkane (364) unknown (378,392) methylfluorene (405) dibenzothiophene (422); MO unknown (438) methyldibenzothiophene (467); MO methylphenanthrene (477); MO cyclopentaphenanthrene (482); MO phenylnaphthalene (501) unknown (511) fluoranthene (531); M1, CO pyrene (548); MI, CO unknown (561, 578) unknown (276) tetradecane (287); P unknown (314) acenaphthylene (335); M1 trimethylnaphthalene (359); MO unknown (385) hexadecane (400); A trimethyloctane (417, 447) octadecane (450); P, CC unknown (467) nonadecane (479) cyclopentaphenanthrene (487); MO eicosane (510); CC fluoranthene (531); M1, CO, CC heneicosane (541) pyrene (547); M1, CO, CC docosane (571) unknown (601,630)
‘Potential genetic toxicity: I = initiator; P = promotor; A = antagonist; CC = cocarcinogen (synergist); MO = nonmutagenic; M1 = mutagenic; CO = noncarcinogen; C1 = carcinogen; all others = no information. The references used in determining these factors include McCann et al. (30),Kaden et al. (29), Goldschmidt (28),and DHEW (31).
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the maximum level of mutagenic activity observed in the runoff water was 475 revertants/mg, loss due to runoff accounted for an absolute maximum of 0.5%of the applied mutagenic activity (29). Loss of mutagens due to volatilization should be insignificant because the wood-preserving bottom sediment contains primarily nonvolatile constituents (19) and because the standard plate incorporation assay is relatively insensitive to volatile compounds (17). Immobilization or binding of organic compounds to the humic fraction of the soil is included as a treatment mechanism by the EPA ( I ) . As a result, transformation of mutagens to compounds bound to the soil humic fraction and thus insoluble in methylene chloride has not been measured in this study. However, since humic acid polymers are relatively resistant to degradation (33, and since organics bound to humic acids would be expected to be less available to further transformation, it is unlikely that future releases of bound mutagens will be significant. Bioassay-directed chemical analysis was employed in this research as an effective means of obtaining a comprehensive view of the mutagenic potential of hazardous waste amended soil. In our opinion, even if the funds and procedures had been available to quantify each chemical constituent of the flaste-amended soil, the results demonstrate that it would have been difficult if not impossible to predict the interactions of soil and waste constituents or the toxicity of degradation products from chemical analysis alone. In addition, biological analysis alone could fail to account for artifacts generated in the collection or extraction process. However, the combined testing protocol employed here provides a description of the mutagenic potential of various mixtures of chemicals, as well as a tentative identification of major organic constituents in the active fractions. Thus, bioassay-directed chemical analysis provides more accurate information from which to make a risk assessment than either method alone. Conclusions
The results of this research indicate that the quantity of solvent-extractable organic compounds in wood-preserving waste-amended soils was significantly reduced with time. In the absence of more detailed results, it is difficult to determine if these losses are a result of degradation, transformation, or volatilization. In addition, it is difficult to determine if the reduction in solvent-extractable organic is an indication of a corresponding reduction in toxicity. Chemical analysis tentatively identified a variety of organic chemicals in the soil 360 days after waste application including pentachlorophenol, fluoranthene, and pyrene. The results from biological analysis indicate that degradation and/or transformation may have reduced the mutagenic potential of equivalent weights of wasteamended soils. However, the combined results from biological and chemical analysis have detected the presence of a highly mutagenic residue in the soil 540 days after waste application, which contains a variety of organic chemicals including mutagens, carcinogens,promotors, and inhibitors. Acknowledgments
Special thanks go to R. Saltarelli for technical assistance, B. R. Scott for conducting the A. n i d u l a n s assay, D. Kampbell of USEPA for chemical analysis, and R. S. Stafford and the staff of the Environmental Mutagen Information Center for the literature review. Registry No. CF&, 75-46-7; (C02H)2CeH4,29010-86-4; Me( C H 2 h M e , 112-40-3; Me(CH&Me, 629-78-7; Me(CH,),,Me,
629-59-4; Me(CH2)16Me,593-45-3; Me(CH2)17Me,629-92-5; Me(CH2)18Me, 112-95-8; Me(CH2)19Me,629-94-7; Me(CH2)20Me, 629-97-0; pentachlorophenol, 87-86-5; trimethylnaphthalene, 28652-77-9; acenaphthylene, 208-96-8; fluoranthene, 206-44-0; pyrene, 129-00-0;cyclopentaphenanthrene, 80455-52-3;methylnaphthalene, 1321-94-4;dimethylnaphthalene, 28804-88-8;hexene, 25264-93-1; dimethylbutane, 38719-68-5; biphenyl, 92-52-4; dibenzofuran, 132-64-9;fluorene, 86-73-7; dibenzothiophene, 13265-0; phenanthrene, 85-01-8; dimethylnonane, 91572-57-5; methylpropylpentanol, 88448-42-4; ethylhexanol, 75737-89-2; dimethylundecane, 79004-83-4; trimethyloctane, 98060-52-7; ethylheptane, 73507-01-4; trimethylheptane, 79004-86-7; dimethylhexane, 28777-67-5; propylaziridine, 104549-74-8; dimethyloctane, 63335-88-6; dimethylbiphenyl, 28013-11-8; phenalene, 203-80-5; methylethylnaphthalene, 31391-42-1;methyldibenzothiophene, 30995-64-3; methylfluorene, 26914-17-0; methylphenanthrene, 31711-53-2;phenylnaphthalene, 35465-71-5.
Literature Cited (1) EPA Fed. Regist. 1982, 47(143), 32274. (2) Hamaker, J. W. In Organic Chemicals in the Soil Enuironment; Goring, C. A. I.; Hamaker, J. W., Eds.; Dekker: New York, 1971; Vol. 2, pp 253-434. (3) Alexander, M. Science (Washington, D.C.) 1981,211,132. (4) La Voie, E. J.; Tulley-Freiler, L.; Bedenko, V.; Hoffmann, D. Cancer Res. 1981,41, 3441. ( 5 ) Huberman, E.; Aspiras, L.; Heidelberger, C.; Grover, P. L.; Sims, P. Proc. Natl. Acad. Sci. U.S.A. 1971,68(12), 3195. (6) Hg,C.-H.; Clark, B. R.; Guerin, M. R.; Barkenbus, B. D.; Rao, T. K.; Epler, J. L. Mutat. Res. 1981, 85, 335. (7) Tikkanen, L.; Matsushima, T.; Natori, S. Mutat. Res. 1983, 116, 297. (8) Hazard. Waste News 1985, Dec. 24, 1984, 415. (9) Brown, K. W.; Donnelly, K. C.; Thomas, J. C.; Davol, P. Sci. Total Enuiron. 1985,41, 173-186. (10) Copper, R. E.; Hedrick, H. G. Soil Sci. 1976,122,331-338. (11) Brown, K. W.; Donnelly, K. C.; Deuel, L. E., Jr. Microb. E d . 1983, 9, 363-373. (12) Shabad, L. M.; Cohan, Y. L.; Ilnitsky, A. P.; Khesina, A. Ya.; Shcherback, N. P.; Smirnov, G. A. J. Natl. Cancer Inst. 1971,47, 1179-1191. (13) Khesina, A. Ya.; Shcherback, N. P.; Shabad, L. M.; Vostrov, I. S. Byull. Eksp. Biol. Med. 1969, 68, 70. (14) Morin, J.; Goldberg, D.; Seginer, I. Trans. ASAE 1967, 74-77. (15) Brown, K. W.; Donnelly, K. C. Enuiron. Pollut., Ser. B 1983, 6(2), 119. (16) Donnelly, K. C.; Brown, K. W.; Scott, B. In TheApplication of Short Term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures; Waters, M. D.;
Sandhu, S. S.; Lewtas, J.; Claxton, L.; Chernoff, N.; Nesnow, S., Eds.; Plenum: New York, 1983; pp 58-78. (17) Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975,
31, 347. (18) Maron, D. M.; Ames, B. N. Mutat. Res. 1983, 113, 173. (19) Brown, K. W.; Donnelly, K. C.; Thomas, J. C. U.S.EPA Report 600/S2-84-135; Environmental Protection Agency: Washington, DC, 1984. (20) Chu, K. C.; Patel, K. M.; Lin, A. H.; Tarone, R. E.; Linhart, M. S.; Dunkel, V. C. Mutat. Res. 1981,85, 119-132. (21) Morrison, D. C. Design and Analysis of Experiments; Wiley: New York, 1984. (22) Scott, B. R.; Sparrow, A. H.: Lamm. S. S.: Schairer. L. Mutat. Res. 19%3,49, 203. Sims, R. C.; Overcash, M. R. In “Proceedings of ASCE National Conference on Environmental Engineering”, Boulder, CO, July 68,1983; Medine, A.; Anderson, A,, Eds.; ASCE: St. Joseph, MI, 1983; pp 1-8. Angle, J. S.; Baulder, D. M. J. Enuiron. Qual. 1984,13(1), 143-146. Scott, B. R.; Dorn, G. L.; Kafer, E.; Stafford, R. Mutat. Res. 1982.98, 49-94. Matsushima, T.; Sugimura, T.; Nagao, M.; Yahagi, T.; Shirai, A.; Sawamura, M. In Short-Term Test Systems for Detecting Carcinogens;Norpoth, K. H.; Garner, R. C., Eds.; Springer: Berlin, 1980; pp 273-285. ?
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(27) Lankas, G. R.; Baxter, C. S.; Christian, R. T. J. Toxicol. Environ. Health 1978, 4, 37. (28) Goldschmidt, B. M. In Carcinogens in Industry and the Environment; Sontag,J. M., Ed.; Dekker: New York, 1981; pp 283-343. (29) Kaden, D. A.; Hites, R. A.; Thilly, W. G. Cancer Res. 1979, 39, 4152.
(30) McCann, J.;Choi, R.; Yamasaki, E.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 1975, 72(12), 5135. (31) Department of Health, Education and Welfare “Survey of Compounds Which Have Been Tested for Carcinogenic Activity”;US. Public Health Service Publication No. 149, Supplement 2; U.S. Government Printing Office: Washington, D.C., 1969. (32) La Voie, E. J.; Tulley-Freiler,L.; Bedenko, V.; Hoffman, D. Mutat. Res. 1983, 116, 91. (33) Hoffman, D.; La Voie, E. J.; Hecht, S. S. In Polynuclear Aromatic Hydrocarbons: Physical and Biological Chemical;
Cooke, M.; Dennis, A. J.; Fisher, G. L., Eds.; SpringerVerlag: New York, 1982; pp 1-9. (34) Pelroy, R. A.; Stewart, D. L.; Tominaga, Y.; Iwao, M.; Castle, R. N.; Lee, M. L. Mutat. Res. 1983, 117, 31. (35) Anderson, K. J.; Leighty, E. G.; Takahashi, M. T. J. Agric. Food Chem. 1972, 20(3), 649. (36) Fahrig, R. In Chemical Carcinogenesis Assays; Montesano; Tomatis, L., Eds.; International Agency for Research on Cancer: Lyon, France, 1974; Vol. 10, pp 161-168. (37) Martin, J. P.; Haider, J. Appl. Environ. Microbiol. 1979, 38(2),283-289. Received for review December 10, 1984. Revised manuscript received March 28,1986. Accepted August 22,1986. Contribution of Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas. This work was funded in part by EPA Cooperative Grant CR-807701-01,
Atmospheric Chemistry of Aniline, N,N-Dimethylaniline, Pyridine, 1,3,5-Triazine, and Nitrobenzene Roger Atklnson,” Ernest0 C. Tuazon, Timothy J. Walllngton, Sara M. Aschmann, Janet Arey, Arthur M. Wlner, and James N. Pltts, Jr.
Statewide Air Pollution Research Center, University of California, Riverside, California 9252 1 The atmospherically important reactions of aniline, N,N-dimethylaniline, pyridine, 1,3,5-triazine7and nitrobenzene, chosen as model compounds for a series of industrially and agriculturally important chemicals, have been studied with the OH radical, 03,and gaseous HN03 by a variety of experimental techniques. At room temperature, the following rate constants (in cm3 molecule-l s-l) were obtained for these gas-phase reactions: (OH radical reactions) aniline, (1.18 f 0.11) X 10-lo;N,N-dipyridine, (4.9 f 0.4) methylaniline, (1.48 f 0.11) X X 10-13; 173,5-triazine,(1.5 f 0.3) X nitrobenzene,
