Energy & Fuels 1995,9, 1051-1057
1051
Preliminary Characterization of Bioreactivity of Novel Carbonaceous Pitches Extracted in N-Methylpyrrolidone C. S. Cohen,?P. G. Stansberry,S A. H. Stiller,#G . Hobbs,g K. Vavro,? and M. R. Miller"?? Department of Biochemistry, Group W Bench, R.C. Byrd Health Sciences Center, West Virginia University, P.O. Box 9142, Morgantown, West Virginia 26506-9142; Department of Chemical Engineering, West Virginia University, P.O. Box 6102, Morgantown, West Virginia 26506-6102; and Department of Community Medicine, R.C. Byrd Health Sciences Center, West Virginia University, P.O. Box 9145, Morgantown, West Virginia 26506-9145 Received March 3, 1995. Revised Manuscript Received August 18, 1995@
Novel pitches produced by N-methylpyrrolidone (NMP) extraction of coal were evaluated for their biological reactivity in the following bioassays: Salmonella reversion or mutation assay, V79 cell micronucleus assay, medaka larval survival assay, and cytochrome P4501A1 induction, as reflected by ethoxyresorufin 0-deethylase activity. The potential health hazards of the NMPderived extracts were evaluated using a wide spectrum of organisms and assays to determine if fundamental toxicity transcended various species and biological endpoints. The bioassays were also used to characterize partially the chemical nature of biologically reactive compounds in the extracts. The samples tested were unprocessed coal, NMP extracted coal, and NMP-extracted coal following hydrogenation in tetralin a t 350, 400, or 450 "C; conventional Ravenswood coal tar pitch and Koppers coal tar pitch were also evaluated for comparison with the pitches produced by NMP extraction. These samples were pulverized with a glass mortar and pestle and extracted in dimethyl sulfoxide, and the dimethyl sulfoxide extracts were evaluated by bioassay. Elemental analysis was performed on samples, and selected samples were analyzed by gel permeation chromatography and Fourier-transform infrared spectroscopy. Unprocessed and NMP-extracted coal exhibited little, if any, biological reactivity in any of the bioassays. Material hydrogenated at 350 "C was weakly reactive, 400 "C material was moderately reactive in all assays, and 450 "C material was highly reactive in all bioassays, indicating a correlation between biological reactivity and temperature of hydrogenation. In most assays the NMP extracts derived from 450 "C hydrogenated coal exhibited biological reactivity comparable t o that of Ravenswood coal tar pitch. Hydrogenation was associated with reduction of number average molecular weight and increased aromaticity of materials. Toxicity of samples correlated well with their ability to induce ethoxyresorufin 0-deethylase activity, suggesting polyaromatic hydrocarbons contributed significantly to the biologically reactive chemicals in the extracts. Furthermore, Salmonella reversion assays using tester strains with normal, elevated, or deficient levels of O-acetyltransferase or nitroreductase indicated the mutagenic compounds in N M P extracts of coal hydrogenated at 450 "C were primarily attributed to aromatic amines and that lower amounts of these mutagens were present in samples hydrogenated at or below 400 "C. These findings suggest that NMPbased processing of coal hydrogenated at high temperatures is likely to produce hazardous materials, whereas processing based on hydrogenation at lower temperatures greatly reduces the production of hazardous materials. These findings must be considered in the design of safe pilot or commercial plants.
Phone: (304)293-2494.
collected, condensed, and fed to a distillation unit for fractionation into distillate cuts. The heavy, nondistillable portion is called coal-tar pitch (CTP). CTP is a valuable byproduct in that it is the preferred substance in the making of bulk carbons, in which CTP functions as a binder. Currently, there is some concern over the availability and quality of CTP. For example, methods used to produce CTP generate considerable environmental pollution and in turn present a serious health concern. Coking processes have been shown t o generate carcinogenidtoxic materia1s.l These important factors may
Department of Biochemistry. Department of Chemical Engineering. 6 Department of Community Medicine. @Abstractpublished in Advance ACS Abstracts, October 1, 1995.
(1)Hubis, W. Health effects in coal-conversion processes: worker health experience. In Toxicology of Coal Conversion Processing; Gray, R. H., Drucker, H., Massey, M. J., Eds.; John Wiley & Sons, Inc.: New York, 1988;pp 553-583.
Introduction Carbonaceous pitches are indispensable materials used extensively by the carbon industries. Tens of millions of tons of these materials are utilized annually in the United States primarily in the fabrication of anodes, electrodes, and artificial graphites. Traditional coal pitches are obtained during the conversion of coal to metallurgical coke. During the process of coal coking volatile organic materials are released. They are then ~
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* Address correspondence to this author.
Fax: (304)293-6846.
*
+
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0887-0624/95/2509-1051$09.00/00 1995 American Chemical Society
Cohen et al.
1052 Energy & Fuels, Vol. 9, No. 6, 1995
indeed force future procedures to resort to nonrecovery methods, in which all the volatile matter is combusted, thereby eliminating the production of CTP. As CTPs are primarily byproducts, there is some advantage in terms of consistency and quality to seek alternative pitch and coke precursors which are in themselves targeted as primary products. In this regard, nontraditional pitches from coal could be a viable substitute for CTP because, similar to them, coal is chemically aromatic. Nevertheless, the major drawback in using coal directly is that it is a solid, containing up to several weight percent mineral matter. The mineral matter in coal is particularly troublesome because the inorganic species are not simple diluents. They affect all aspects of coal utilization. Researchers at West Virginia University have developed a solvent extraction technique in which coal is treated with a class of liquids known as dipolar, aprotic solvents.2 The salient feature of this solvent family is the ability to produce from raw coal low-ash, pitch materials in fairly high yield and under mild conditions. In particular, the solvent N-methylpyrrolidone (NMP) has been shown to be the most effective. Further, it is known that hydrogenation of coal prior to solvent extraction increases dramatically the yield of soluble product over that of the raw coal alone.3 The coal pitches of NMP processing are likely to be different from those produced in the past which relied on other methods of pitch separation and isolation. Although the NMP solvent extraction process2appears t o be a promising alternative for the production of CTP, the potential health risks associated with this process must be determined prior to pilot plant or full scale development. Short-term tests are used to assess initially potential health hazards of materials because they can be conducted relatively quickly and inexpensively. For example, four short-term genotoxicity tests (Ames salmonella mutagenesis assay, chromosome aberration assay, sister chromatid exchange assay, and mouse lymphoma mutagenesis assay) have been shown not to exhibit significant differences in individual concordance with long-term rodent carcinogenicity tests;4the concordance of each test was approximately 60%. Although no single short-term assay can conclusively predict positive or negative toxicity or carcinogenicity, utilization of several short-term tests can provide better information of the potential health hazards of test sample^.^ The goal of these studies was to assess the toxicity of different NMP-derived coal samples using different short-term bioassays. The bioassays chosen utilized a wide spectrum of species and biological endpoints. The working hypothesis of this study was that compounds which are clearly hazardous will be toxic across a spectrum of species with different biological endpoints. The following in vitro assays were therefore used to investigate the potential health risks associated with NMP-derived coal samples, as well as t o characterize partially the nature of biologically reactive components. (2) Renganathan, K.; Zonlow, J. W.; Mintz, E. A.; Kneisl, P.; Stiller, A. H. Fuel Process. Technol. 1988, 18, 273-278. (3) Neavel, R. Fuel 1976, 55, 237-242. (4) Tennant, R. W.; Margolin, B. H.; Shelby, M. D.; Zeiger, E.; Haseman, J . K.; Spalding, J . ; Caspary, W.; Resnick, M.; Stasiewicz, S.; Anderson, B.; Minor, R. Science 1987,236, 933-941. (5) Ashby, J.; Tennant, R. W.; Zeiger, E.; Stasiewicz, S. Mutat. Res. 1989,223, 73-103. (6) Maron, D. M.; Ames, B. N. Mutat. Res. 1983, 113, 173-215.
Table 1. Elemental Analysis of Samples NMP extract H450 Ravenswood CTP c 84.90 85.5 93.8 H 5.3 5.8 3.7 N 2.0 1.7 0.9 S 0.7 0.4 0.6 0 7.0 3.4 1.0 CMb 1.33 1.26 2.11
sample
a
*
Weight percent. Atomic ratio
(1) The Ames6 assay provides a convenient, reliable method of estimating the mutagenic potential of substances in bacteria. Furthermore, bacteria tester strains with elevated or deficient levels of 0-acetyltransferase or nitroreductase have been d e v e l ~ p e d . ~ These , ~ strains exhibit altered sensitivity to particular types of compounds and can be used t o increase sensitivity of detecting mutagens and to characterize partially the chemical nature of mutagens in complex mixture^.^ (2) Micronucleus assays in cultured V79 cellslO determine the ability of samples to induce chromosomal damage in mammalian cells. Like the Ames assay, this assay indicates the genotoxicity of compounds; however, effects are observed in mammalian rather than bacterial cells. (3) General toxicity in vertebrates can be estimated by determining survival of Japanese medaka larvael1J2 following exposure to samples. In contrast to the Ames or micronucleus assay, this assay measures overt toxicity of compounds on complex vertebrates. (4) Cytochrome P4501A1 induction can be employed to assess relative levels of polyaromatic hydrocarbon (PAH)type compounds in different ~amp1es.l~ Cytochrome P4501A1 is induced by PAHs and polychlorinated hydrocarbons14-16 which bind an intracellular arylhydrocarbon receptor; the PAH-receptor complex binds xenobiotic responsive elements of the CYPlAl gene, which increases trans~ription.l'-~~ Because isolated liver cells are being used as a model for hepatotoxicity studies,20 and for P4501A1 induction,21*22P4501A1 induction in isolated teleost liver cells and in medaka liver in vivo were directly compared to determine which system was most sensitive for detecting PAH induction (7) Watanabe, M.; Ishidate, M., Jr.; Mohair, T. Mutat. Res. 1990, 234,337-348. (8) McCoy, E. C.; Anders, M.; Rosenkranz, H. S. Mutat. Res. 1983, 121, 17-23. (9) Stamm, S.C.; Zhong, B.-2.; Whong, W.Z.; Ong, T. Mutat. Res. 1994,321, 253-264. (10)Krishna, G.; Kropko, M. L.; Theiss, J. C. Mutat. Res. 1989,222, 63-69. (11) Hatanaka, J.; Doke, N.; Harada, T.; Aikawa, T.; Enomoto, M. Jpn. J . Exp. Med. 1982,52, 243-253.
(12)Cooper, K. R.; McGeorge, L. Aquatic Toxicology and Risk Assessment. In Fourteenth Volume, ASTM STP 1124, American Society for Testing and Materials; Mayes, M. A., Barron, M. G., Eds.; ASTM: Philadelphia, 1991; pp 67-83. (13) Cohen, C.; Stiller, A.; Miller, M. R. Enu. Cont. Toxicol. 1994, 27, 400-405. (14) Nebert, D. W.; Gonzalez, F. J. Annu. Reu. Biochem. 1987,56, 945-993. (XiEberhart, J.; Coffing, S. L.; Anderson, J. N.; Marcus, C.; Kalogeris, T. J.;Baird, W. M.; Park, S. S.; Gelboin, H. V. Carcinogenesis 1992, 13f2), 297-301. (16) Guengerich, F. Cancer Res. 1988,48, 2946-2954. (17) Okey, A. B. Pharmacol. Ther. 1990,45, 241-298.
(18)Heilmann, L. J.; Sheen, Y.-Y.; Bigelow, S. W.; Nebert, D. W. DNA 1988,7,379-387. (19) Stegeman, J. J.; Woodin, B. R.; Smolowitz, R. M. Biochem. SOC. Trans. 1990, 18, 19-21. (20) Baksi, S.M.; Frazier, J. M. Aquat. Toxicol. 1990,16,229-236. (21) Xu, L.-C.; Bresnick, E. Biochem. Pharmacol. 1990,40, 13991403. (22) Rodman, L. E.; Shedlofsky, S. I.; Swin, A. T.; Robertson, L. W. Arch. Biochem. Biophys. 1989,275, 252-262.
Energy & Fuels, Vol. 9, No. 6, 1995 1053
Bioreactivity of Novel Carbonaceous Pitches
n Ib
1 I
5001
400 300 200 100 0
:
1
'
'
""",
'
10
'
'
"""I
100
Micrograms Xenobiotic
Z-+L 3mo
300
a 5 0 0
WAVENUMBER
Figure 1. Ratio of aromatic (&,I to aliphatic (A,1) hydrogen determined by FTIR: (a) N M P (b) H350; (c) H400; (d) H450.
by complex coal-derived samples. The results of these in vitro bioassays indicate that different NMP-derived coal samples exhibit a wide range of biological properties. Furthermore, there is a correlation between temperature of hydrogenation of the samples and their biological reactivity, which transcends a spectrum of species and biological end points.
o3
Table 2. Ames Assay of Different Concentrations of H450 with Different Salmonella Tester Strains
tester strainn TA98
P&?
+S9
-S9
300 100 10
226 168 99 64 1239 1588 1185 865 78 28 18
393 173
1
YG1024
300 100
(23) Kado, N. Y.; Langley, D.; Eisenstadt, E. Mutat. Res. 1983,121, 25-32.
1
Figure 2. Mutagenicity of coal-derived samples in bacterial mutation assays. Various concentrations (1-330 pg/assay) of test samples were assayed in triplicate for mutagenic potential using tester strain TA98 and bioactivating S9 fraction: H450 (n),Ravenswood CTP (O), H400 (O), Koppers CTP (O), H350 (+I, NMP (W). Mutagenicity is expressed as the number of revertantdplate after exposure to the samples; standard deviations were '5%. Number of revertantdplate in DMSO control was 20.
10
Results Chemical Characterizations. Elemental analysis of coal-derived pitches NMP, H450, and Ravenswood CTP (Table 1) indicate that they consist primarily of carbon. The C/H atomic ratio indicates the CTP (2.11) is highly aromatic; however, this ratio is lower for both NMP extract (1.33) and for H450 (1.26),indicating these materials are less aromatic in nature. Sulfur content is generally low for all the pitches, but nitrogen content is comparatively high for those pitches derived by NMP processing, possibly reflecting a small amount of retained NMP. Gel permeation chromatography (GPC) was used to determine number average molecular weight (MWn). It was not possible to determine M W n for the pitches from the unhydrogenated coals because of their limited solubility in the GPC carrier solvent. Nonetheless, a reduction in molecular weight is evident upon increasing the severity of hydrogenation from 400 "C (-600 MW,) to 450 "C (-500 M W n ) (not shown). Samples NMP, H350, H400, and H450 were examined by Fourier-transform infrared spectroscopy (FTIR). Simple integration of the spectral regions associated and aliphatic hydrogen with the aromatic hydrogenI&( (Le) stretching modes was used to estimate aromaticity. As shown in Figure 1,the most dramatic effect entailed a shiR in the aliphatic and aromatic hydrogen distribution; i.e., hydrogenation increased the aromatic nature of the pitch over that of unhydrogenated coal. Genotoxicity Assays. Potential genotoxicities of the test compounds were initially screened using a bacterial mutation assay.23 Using Salmonella tester strain TA98 in the presence of bioactivating S9 fraction, there was a significant difference in the ability of samples to
"""rJ
1
DNP
300 100
10 NR
1
20
300 100
239 132 88 52
10 1
21
16 329 123 56 16 276 143 17 16 140 95 13 12
Salmonella tester strains are described in the Experimental Section. Amounts of H450 in pg. Assays conducted in the presence (+) of absence (-) of rat liver S9; values are number of revertant bacteridplate as described in Figure 2. Control (DMSO) values were 19 for +S9 and 13 for -S9 for TA98.
induce bacterial mutations (Figure 2), and all positive samples exhibited a dose-dependent increase in mutation frequency. Figure 2 indicates the rank order of mutagenicity of the samples: H450 > Ravenswood CTP >> H400 1 Koppers CTP > H350 = NMP. The mutagenic activities of NMP, coal, and DMSO solvent were not significantly different (not shown). The genotoxicity of the positive control, 2-aminoanthracene (not shown) was similar t o that of the H450 sample. Sample H450 was much more mutagenic than any other NMP-derived coal sample and was therefore further investigated using tester strains TA98, YG1024, DNP, and NR in the presence and absence of liver S9 bioactivating fraction (Table 2). The mutagenicity of H450 was sigmficantly increased in tester strain YG1024 (elevated 0-acetyltransferase), relative to strain TA98, in the presence of S9 fraction. In addition, H450 mutagenicity was dramatically decreased in strain DNP (0-acetyltransferase deficient) in the presence of S9, relative to strains TA98 and YG1024. Nitroreductase deficiency (strain NR) did not alter mutagenicity of H450 in the presence of S9, relative to TA98, but did result in somewhat reduced mutagenicity in the absence of s9.
Cohen et al.
1054 Energy & Fuels, Vol. 9, No. 6, 1995
Table 3. Viability Assay of Coal Samples on Medaka Fry
a,
sample
7,
PPm'
0.1% DMSO 0.01% DMSO
coal coal NMP NMP 350 350 350 400 400 400
350
NMP
COAL
Raven
Koppers
Figure 3. Induction of chromosomal damage in mammalian V79 cells by coal-derived samples, assessed by micronucleus assay. Various concentrations in ppm (solid, 0.1; gray shaded, 0.03;lined, 0.01) of samples were assayed for induction of micronuclei: H450 (450),H400 (400),H350 (350),Ravenswood CTP (R), and Koppers CTP (K) and unprocessed coal (coal). The frequency of micronucleus induction (number of micronuclei/500 cells) was scored 24 h following a 3 h period of exposure to samples. Parameters for scoring micronuclei are those described by Krishna et a1.I0 Background level of micronuclei was -1/500 cells.
The coal-derived samples were examined for genotoxic effects in mammalian cells using the micronucleus assay.1° The micronucleus assay assesses chemicallyinduced genetic damage by quantitating acentromeric chromosome fragments in mononucleated interphase cells. Figure 3 demonstrates that the samples exhibited differences in their abilities to induce micronuclei. The rank order of test compounds to induce micronuclei was Ravenswood CTP > H450 =- H400 = Koppers CTP = H350 > NMP = COAL. log-linear analysis of the micronuclei data (Figure 3) indicates significant effects for the different treatment groups (p < 0.0001);however, a concentration-response relationship was not found. Vertebrate Toxicity Assay. The toxicity of the various coal-derived samples on complex vertebrates was determined by assessing the effects of exposure to the samples on medaka fry survival. Table 3 shows that at the concentrations tested, samples H450, Ravenswood CTP, and Koppers CTP were highly toxic. Other samples did not elicit a toxic response in this assay (Table 3). Cytochrome P4501A1 Induction. The ability of test samples to induce cytochrome P4501A1 in vivo was determined by measuring EROD activity in livers of medaka following a single dose (0.001-1 ppm) of the coal-derived samples added directly to aquaria water.13 Figure 4 indicates that the samples displayed different abilities to induce EROD activity, and all positive samples exhibited a dose-dependent effect. Analysis of variance for the EROD data showed significant effects due to treatment (p < O.OOOl), concentrations ( p < O.OOOl), and the interaction of treatment and concentrations ( p = 0.0294). Overall, the rank order of EROD induction in medaka liver for samples at 0.1 ppm was Ravenswood CTP > H450 > Koppers CTP > H400 > H350 = NMP = coal. The same relative pattern of EROD induction was observed with samples at 1ppm, except H400 was as effective as H450. The ability of samples to induce EROD activity likely reflects the relative amounts of PAH-type compounds present which bind the arylhydrocarbon receptor.
0 0 0 0 0 0 0 0 0 0 0 1 0
100 10 100 10 100 10 1 100 10 1 0.1 100 10 1 0.1 100 10 1 100 10
400 450
toxicityb
0 0
400 450 450 450
450 Ravenswood Ravenswood Ravenswood Koppers Koppers
23 (100%) 21 2 2
23 18 1
23 1
a Concentration (ppm) of coal samples in aquaria water. Toxicity is expressed as number of dead fry following 3 days exposure to coal samples or DMSO solvent. Total fry per exposure group was 23; 23 dead fry is 100%toxicity.
600
1
1
E
d 300 1
,
I
0
/
-F 0.001
Y
y
0.01
0.1
-1
ppm (parts per million)
Figure 4. Induction of medaka liver EROD activity by different concentrations of coal-derived samples added to aquaria water. Medaka were exposed to concentrations of Ravenswood CTP (hourglass symbol), H450 (O), H400 (v), H350 (v),Koppers CTP (W), NMP (crossed square), and coal (shaded circle) ranging from 0.001 to 1 ppm or to 1 ppm DMSO solvent (not pictured) for two days. Animals were then sacrificed and liver EROD activities were determined. The data depicted represents a composite of three individual assays.
To the best of our knowledge, no studies have directly compared the sensitivity of EROD induction in isolated liver cells with that of livers from exposed teleosts. This comparison was undertaken to determine whether in vivo or in vitro EROD induction would be most useful for detecting P a - t y p e compounds in complex samples. In these studies, medaka liver cells were used for direct comparison to in vivo induction; trout liver cells were also utilized because large numbers of liver cells can be obtained from a single animal. Liver cells were isolated from both medaka and trout, placed in culture, and exposed to the same concentrations of H450 used in in vivo exposures (Figure 5 ) for 2 days; cells were then collected and assayed for EROD activity. Previous studies demonstrated maximal EROD induction 2 days after exposing cultured teleost liver cells to P4501A1 inducer^.^^,^^ Figure 5A shows that with medaka liver (24) Miller, M. R.; Saito, N.; Blair, J. B.; Hinton, D. E. Exp. Mol. Path. 1993,58,127-138.
Bioreactivity of Novel Carbonaceous Pitches
Energy & Fuels, Vol. 9, No. 6, 1995 1055
>
.01
0
.1
1
.1
1
P?-
k *O01
f
p
100-
1
%
01
.Ol
0
PP-
Figure 5. Induction of EROD activity in cultured medaka and trout liver cells. Medaka (panel A) and trout (panel B) liver cells were prepared and cultured in 35 and 100 mm dishes, respectively. Various concentrations (0.01-1ppm) of H450 or DMSO solvent (control, 0 ) were added to cultures. Two days later cells were collected and sonicated and EROD specific activities were determined. Both medaka and trout liver cells were assayed in triplicate; results indicate mean and standard deviations.
assay Ames
Table 4. Rank Order of Potency Ravenswood KO pers H450 H400 H350 CTP E)TP NMP coal 10
mutaaenicitv 2 micronucleus
3
4
2
3
5
5
3 3 5
4 5 5
1 1
415 3 2
5 5 5
5 5 5
I
EROD viability
2 1
1
subjective reactivity of samples in bioassays: 1 = high reactivity; 5 = noAow reactivity.
cells EROD induction was detected at 1ppm H450,but not a t the lower doses tested. Figure 5B indicates that with trout liver cells EROD induction is detected a t 0.1 ppm, with further induction at 1ppm. Although EROD specific activities in liver cells from medaka and trout are different (Figure 51, the extent of EROD induction above controls for 1 ppm H450 is similar for medaka cells (-&fold) and trout cells (-&fold).
Discussion The goal of these studies was to use short-term bioassays as a means of assessing potential health hazards of novel pitches produced by extracting coal with NMP and to test the working hypothesis that compounds which are clearly hazardous will be toxic across a spectrum of species with different biological endpoints. The various bioassays measured genotoxicity in bacteria (Figure 2) and in mammalian cells (Figure 3), lethal toxicity to fish fry (Table 3) and cytochrome P4501A1 induction in medaka (Figures 4 and 5). Table 4 summarizes, in a subjective manner, the relative reactivity of the samples in the bioassays tested. In all bioassays performed, H450 and Raven-
swood CTP were highly reactive, indicating their potential t o be hazardous is high. For example, the H450 sample has a similar mutagenic potential as the Ravenswood CTP (Figure 2), which was generated from traditional coking plants and is associated with health hazards, including cancer.1p25-27 In addition, there appeared to be a correlation between temperature of hydrogenation and bioreactivity of "-derived samples; sample H450 was highly reactive in all assays, sample H400 exhibited moderate reactivity in all assays, sample H350 was less reactive, and unhydrogenated NMP extracts (NMP) were essentially unreactive (Table 4). Therefore, NMP extracts of coal samples hydrogenated a t lower temperatures are likely to be less hazardous than those hydrogenated a t higher temperatures. It is not yet clear why bioreactivity is correlated with temperature of hydrogenation of NMP-extracted coal (Table 3). One explanation is that different types of compounds (more reactive) are formed when samples are hydrogenated at higher temperatures. On the other hand, higher temperatures may produce smaller toxic compounds which can more readily enter cells or organisms. The decrease in number average molecular weight when samples are hydrogenated at 400 "C (600 MW,) compared to 450 "C (500 MW,) is consistent with this possibility. Additional studies are in progress t o differentiate these possibilities. Although the different assays measured a range of biological end points in different species, each sample elicited a similar response in the different bioassays (Table 41,with the exception of Koppers CTP. Koppers CTP was toxic in the medaka viability assay, was moderately reactive in the Ames and EROD assays and was weakly reactive in the micronucleus assay. Generally, the use of multiple bioassays increases the specificity of identifying toxins but reduces sensitivity, i.e., the higher the likelihood of a reactive sample producing a negative r e ~ u l t .However, ~ all samples except Koppers CTP elicited consistent biological responses in the specific battery of bioassays utilized herein. This consistency increases confidence in predicting the biological reactivity of the NMP-derived coal samples and supports the hypothesis that compounds which are clearly hazardous will be toxic across a spectrum of species with different biological endpoints. Although extensive chemical separation and analysis will be required t o identify the specific toxic compounds in the NMP-derived extracts, bioassays were used to gain insight to the nature of reactive compounds in the complex NMP-derived coal pitches. The ability of different samples to induce EROD activity in medaka (Figure 4) indicated relative levels of compounds which bind the arylhydrocarbon receptor. EROD induction correlated well with the genotoxic (Figures 2 and 3)and toxic (Table 3) response of the different NMP-derived samples. This finding indicates that PAH-type compounds in NMP extracts may be responsible for the genotoxic and toxic properties of these pitches. Consistent with this hypothesis is FTIR analysis (Figure l),which indicated the aromatic nature of coal samples increased with hydrogenation. The nature of genotoxic (25) Tebbens, B. D.; Thomas, J. F.; Mukai, M. Arch. Ind. Health 1968,17,152-165. (26) Tebbens, B. D.; Thomas, J. F.; Mukai,M . Arch. Ind. Health 1956, 14, 112-120. (27) Palmer, A. J. Occup. Med. 1979,21, 41-44.
1056 Energy & Fuels, Vol. 9, No. 6,1995
Cohen et al.
compounds in the most reactive NMP-derived sample, H450 (Figure 2 and 31, was further investigated using Ames assays with normal tester strain (TA981, strains with elevated (YG1024) or deficient (DNP) O-acetyltransferase or a strain with deficient nitroreductase (NR). Mutagenic nitroarenes are bioactivated by Salmonella nitroreductase-these compounds do not require liver S9 fraction, and mutagenicity would be reduced in strain NR. In contrast, aromatic amines display enhanced mutagenicity in strain YG1024 which overproduces 0-acetyltransferase (the rate-limiting activity in conversion of aromatic amines to ultimate mutagens) and they exhibit reduced mutagenicity in strain DNP which is deficient in this enzyme. Sample H450 displayed greatly enhanced mutagenicity with YG1024 and reduced mutagenicity with strain DNP, relative to TA98, in the presence of S9; mutagenicity with strain NR was only moderately reduced in the absence of S9. These findings indicate aromatic amines are major mutagens in H450. On the other hand, sample H400 displayed 5 50%stimulation in mutagenicity with strain YG1024 and no decreased mutagenicity with strain DNP (not shown), indicating much lower concentrations of mutagenic aromatic amines in this hydrogenated sample or possibly the presence of less biologically potent aromatic amines in this sample. Because both in vivo and cultured liver cell (in vitro) systems are being used for toxicity studies, the sensitivity of detecting EROD induction in medaka liver and in cultured medaka and trout liver cells was directly compared. EROD induction via in vivo exposure of medaka was somewhat more sensitive than in vitro exposure of medaka liver cells and was similar to in vitro exposure of trout liver cells (Figures 4 and 5). It is possible that culture conditions for medaka liver cells are not optimal for cytochrome P4501A1 induction or that these cells are less sensitive than trout liver cells to P4501A1 induction. The in vivo and in vitro EROD induction studies indicate that dosing medaka in aquaria water provides a sensitive bioassay for detecting PAHtype compounds when sufficient quantities of test samples are available; however, isolated trout liver cells provide an appropriate system when amounts of test sample are very small or limit in vivo exposure. Results of short-term bioassay testing has indicated that some aspects of NMP-based processing of coal are likely t o produce hazardous materials. The correlation between temperature of hydrogenation and toxicity (Table 4) indicates that less hazardous materials are produced by processes utilizing lower temperatures of hydrogenation. These findings must be considered in the design of safe pilot or commercial plants. In addition, these studies employed sample preparation for bioassay using traditional DMSO extraction. Because humans will not be exposed to DMSO extracts of NMPderived coal samples, more appropriate methods of presentation in bioassays will be required t o better determine the relevant health hazards to humans.
indicated: vitrinite, 71.4; exinite, 5.5; inertinite, 21.7. The coals were vacuum dried and ground t o
