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Mutation Research, 79 (1980) 193- -202 © Elsevier /Nor th-Hol land Biomedical Press

IDENTIFICATION OF PRIMARY AROMATIC AMINES IN MUTAGENICALLY ACTIVE SUBFRACTIONS FROM COAL LIQUEFACTION MATERIALS

BARY W. WILSON a, RICHARD PELROY b and J.T. CRESTO b

a Physical Sciences Department, b Biology Department, Pacific Northwest Laboratory, Richland, WA 99352 (U.S.A.) (Operated by Battelle Memorial Institute)

(Received 27 November 1979) (Revision received 1 April 1980) (Accepted 9 June 1980)

S, mm~" T

Gas~hromatographic mass spectral (GCMS) analyses were performed on mutagenically active components from the basic, basic tar and neutral tar sub- fractions of a coal<lerived liquid heavy distillate. The latter material is a com- ponent fraction of an experimental oil produced in a solvent refined coal process (SRC II) pilot plant. Mutagenicity was determined with the Ames/Salmonella assay system. Thin-layer chromatography (TLC) was used to separate muta- genically active components of the basic, basic tar and neutral tar fractions from some of the other compounds contained in these complex materials.

For the 3 fractions tested, mutagenic activity was localized in approx, the same TLC regions, with relative mobilities (R~) ranging from 0.1 to 0.3. GCMS analysis of the TLC regions showed that the concentrations of primary aromatic amines, as obtained by measuring peak areas for the (M + 1)* ion as formed in the chemical ionization mode, followed essentially the same distribu- tion as the mutagenic activity. Primary aromatic amines identified in the active regions included aminonaphthalenes, aminoanthracenes, aminophenanthrenes, aminopyrenes and aminochrysenes. With the exception of small amounts of aminonaphthalene, p r imary aromatic amines were not found in TLC regions that lacked mutagenic activity.

The solvent refined coal process II (SRC-II) is a developing technology, currently in the pilot phase, which produces liquid as a major product [6].

Work supported by the U.S. Depa~ment of Energy Under,Contract DE-AC06-76RLO 1830. Abbreviations: AA, aminoanthracenes; AC, amlnochrysenes; AF, aminofluorenes; AN, Rmlnonaph- thalenes; AP, aminopyrenes; APH, aminophenanthrenes.

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Previous studies in this laboratory employing chemical fractionation and thin- layer chromatography, coupled with microbial mutagenesis assays, have shown that SRC-II heavy distillate (HD) is mutagenically active in the Ames/ Salmonella assay system. Heavy distillate is that portion of the coal liquefac- tion product which has a boiling range of approx. 480--850 ° F. These studies also suggested that primary aromatic amines are a major contributor to the mutagenic activity observed in HD. This conclusion is also supported by bio- chemical evidence based on enzyme-activation studies.

The present report concerns the application of GCMS to the specific identifica- tion of chemical constituents in HD, particularly those compounds found in TLC regions exhibiting enhanced mutagenic activity.

The data to be repor ted here are for materials of pilot-plant origin, and should not necessarily be considered representative of SRC liquids which may eventually be produced commercially.

Experimental

Materials Samples of SRC-II HD were obtained from the Fort Lewis, Washington SRC

pilot plant operated by the Pittsburg and Midway Co. The samples were collected on Jan. 29, 1978 during the processing of Pittsburgh Seam coal feed stocks from West Virginia. The solvent extraction procedure used to separate this material into acid, base, neutral, and tar fractions has been described elsewhere [ 9].

Further purification was accomplished by TLC, using a hexane : acetone (9 : 1) mixed solvent system. The TLC regions corresponding to R~ values 0.1-- 0.5 were eluted in 6 or 10 ml of hexane : acetone (1 : 1) and stored in liquid nitrogen. Standards used in GCMS analysis of HD included 2AA, 2AF, 6AC, lAP and 9APH from Aldrich Chemical Corp., and 2AN from Sigma Chemical Corp. All chemicals were of the highest purity available. Prior to GCMS analysis, the sample eluates were divided for concurrent Ames mutagenicity assays.

Gas-chromatographic mass spectrometry Samples for GCMS analysis were concentrated from a volume of 1 ml to 40

pl. Analyses were performed using 2 different GC columns. Preliminary work was done on a 2 m X 4 mm I.D. glass column packed with OV17 stationary phase and programmed from 100 to 320°C and 8 ° C/min. In the electron impact (EI) mode, the helium flow rate was 17 ml/min. In the chemical ionization (CI) mode, methane flow was adjusted to achieve a pressure of about 5 X 10 -1 torr in the ion source.

A capillary column (26 m X 0.25 mm I.D.), coated with SE-52 stationary phase, was programmed from 90--260°C at a rate of 8°C per min at a helium flow rate of approx. 2 ml/min. When used in the CI mode, methane makeup gas was added to achieve the necessary ion source pressure.

Mass spectral analyses were performed on Hewlett Packard 5980 and/or Hewlett Packard 5985 GCMS/Data Systems in both the electron impact (EI) and chemical ionization (CI) modes [7]. In the latter, methane was used both

195

as the GC carrier and the CI reagent gas. Due to its enhanced chromatographic quality, the capillary column was utilized principally to compare GC retention times of various components found in the TLC fractions to those of authentic standards. The CI analyses served to confirm molecular ion (M*) assignments made on the basis of EI spectra.

The methane CI spectra obtained were relatively free from interferences, especially after application of a "background substract" data system routine, and all compounds yielded the characteristic intense (M + 1) ÷ and (M + 29) + ions. Single-ion mass chromatograms were generated and checked to insure that the (M + 29) + ion had the same GC retention time as the (M + 1) ÷ ion in each spectrum. This was done to eliminate consideration of a possible contribution from an (M + 1)* ion formed by a C2 homolog of the compound in question. Such interference could also give rise to an "(M + 29) T M ion. Similar chromato- grams were also generated for the EI spectra to insure the coelution of the molecular and major characteristic fragment ions observed in the mass spectra. In most cases, however, the EI spectra, though informative, did not sufficiently differentiate between the primary aromatic amine and the nitrogen hetero- cyclic methyl homolog of the same elemental composition. It was therefore impossible to positively identify the primary amines on the basis of mass spectra alone, and the relative GC retention times of these 2 types of com- pounds became of primary importance in their identification.

Mu tagenesis assays Agar-plate mutagenicity assays were carried out essentially as described by

Ames et al. [1]. Dimethylsulfoxide (DMSO) was used as solvent for all of the chemicals and complex chemical fractions. To obtain maximum expression of mutagenicity, agar plates were preincubated to a temperature of 37°C before adding the components of the Ames assay to the semisolid agar. A liquid (45°C) top-agar layer was poured onto the 37°C plates and they were incubated in the upright position for several hours to allow hardening of the semisolid top-agars. The assay plates were then inverted and incubated for 24-- 36 h to allow expression of mutant colonies. These Ames assays were carried out with aliquots of the same culture of TA98 and samples from the same batch of liver ($9) homogenates induced with Aroclor 1245 [1]. The same assay conditions were used for all the experiments reported in this paper. Colonies were counted on a New Brunswick Scientific Co. Biotran II auto- mated colony counter.

Assays of the HD basic and tar fractions prior to TLC were done in tripli- cate. The TLC eluates of th¢se materials were done in duplicate. In both cases, the data reported are averages of the mutagenic activity for the various amounts assayed.

Preparation of liver homogenates Aroclor 1254 (Monsanto Chemical Corp.), at a concentration of 250 mg/kg

body weight, was injected intraperitoneally into male Wistar rats. Rats were sacrificed on the 4th day after injection. After sacrifice, the livers were perfused with ice-cold 0.154 M KCI and removed for preparation of $9 homog- enates, following standard procedures [1]. The protein portions used for each of the Ames/Salmonella assays were approx. 870/zg.

196

Thin-layer chromatography Samples were applied, 10/~1 at a time, on a 140-mm line, 30 mm from the

bo t tom and 30 mm from the sides of a 20 × 20 cm silica-gel plate, 0.1 mm in thickness (Eastman Kodak Co.). All samples were added in DMSO. After being allowed to dry for 5 min, chromatograms were placed in tanks containing 100 ml of acetone : hexane (1 : 9). Before beginning TLC separations, the solvent system was allowed to equilibrate for 90 min in the chromatography tanks. When the solvent front reached a line approx. 50 mm from the top of the plates, the chromatograms were removed and allowed to dry at ambient temperature. Fluorescent areas contained .on the chromatograms were located by illumination with a near-UV light source (UVSL-25 mineral light, San Gabriel, CA). The outlines of both the fluorescent and nonfluorescent areas were marked with a pencil, and tracings of these regions were transferred to semitransparent paper for calculation of relative mobilities (Rf values). The TLC plates were then sectioned (using the outl ined areas), cut into small strips, and placed in 6--10 ml of an elution solvent of acetone : hexane (1 : 1). Elu- tions were carried ou t in sealed test tubes (15 × 85 mm) for 24 h at 4°C. Dupli- cate samples of 10 and 20/zl of eluate were added to liquid (45°C) top-agar and analyzed in the Ames mutagenicity assay [ 1 ].

A total to 10 032 #g of basic fraction, or 25 920 #g of basic tar fraction, or 20 232 pg of neutral tar was applied in equal portions to TLC plates, eluted after chromatography and analyzed for mutagenicity as described above.

Results

The basic, basic (acid-insoluble) tax, and neutral (isooctane-insoluble) tar fractions were chemically complex. This is illustrated by the number of components detected after GC of the neutral tar (Fig. 1). These crude fractions contained large concentrat ions of nitrogen heterocyclic compounds, in addition to relatively low concentrations of primary aromatic amines. Primary amines identified in all 3 fractions included aminonaphthalenes (AN), aminoanthra- cenes (AA), and/or aminophenanthrenes (APH), aminobiphenyls, biphenyl amines, aminopyrenes (AP) and aminochrysenes (AC). Compounds having the proper elemental composit ions for aminofluorenes and aminocarbazoles were identified from high-resolution mass-spectral data; however, confirmation of their presence by GCMS was no t achieved due to interference at the appropri- ate m/e ratios and the lack of authentic standards. The capillary GC column used adequate resolution to separate some of the various isomers, as shown in Fig. 1 for the neutral tar. Since authentic standards for many isomers and homologs were not available, specific assigrnents as to configuration have not been made in most cases. The 1- and 2-aminonaphthalenes, 1- and 2-amino- anthracenes, 9-aminophenanthrene, and 6-aminochrysene are compounds for which tentative steric assignments have been made on the basis of retent ion time compared to authentic standards.

TLC of the 3 crude fractions yielded mutagenic subfractions were enriched in primary aromatic amines. This is shown by the data in Figs. 2--4. As can be seen, the mutagenic response exhibited in the Ames assay of TLC regions 1--5

197

mle 24) I ~ N H 2 ~

mle 217 I L ' ~ N H 2

mle 193 . . . . . . . . . . .

mle 143 J ~ ~ - ~ " - ~ - " ~ ' - ~

6 8 10 12 14 16 18 20 22 24 RETENTION TIME IN MINUTES

Fig. I . Single~lon e h r o m a t o g r a m s f o r t h e m/e 1 4 3 (M + f o r AN) , t h e m/e 1 9 3 (M + foz A A a n d A P H ) , t h e m / e 2 1 7 (M + f o r AP) a n d role 2 4 3 (M + f o r AC) s h o w n a b o v e t h e t o t a l i o n c u r r e n t c h r o m a t o g r a m o f a m u t a g e n i c n e u t r a l t a r s u b f r a c t i o n o f HD. G r o u p s o f p e a k s p r e c e d i n g t h e a m i n o c o m p o u n d s ar i se h~om t h e m e t h y l h o m o l o g s o f t h e c o r r e s p o n d i n g n i t r o g e n h e t e z o e y e l i c (e .g . , m e t h y l a c l i d i n e Pzeeedes a m i n o a n t h r a - eene) .

(corresponding to Rf values of 0.1--0.3) is compared with the distribution of several primary aromatic amines. The mutagens (premutagens) in region 2 of the basic subfraction and regions 1--4 of the tar subfractions caused the greatest number of revertan~ colonies in the target cells of S. typhimurium TA98. The relative concentrations of AA, AP, APH and AC were highest in TLC subfractions with the strongest mutagenic activity. Small amounts of

§ io -

I . -

-~ 108o

SRC ii BASIC FRACTION r-"IAC mAA

~ A P r~"~ AF

r'-'-1 APH ~ A N

I 2 3 4 5 (0.08) (0.10) (0.11) (0.17) (0.23)

TLC REGION (Rf)

Fig. 2. T h e re la t ive c o n c e n t r a t i o n o f severa l p r i m a r y a r o m a t i c a m i n e s vs. m u t a g e n i c i t y in T L C s u b f r a c - t i o n s o f t h e H D bas i c f r a c t i o n . M u t a g e n i c l t y d a t a a le f r o m T a b l e 2 foz A m e s assay o f 1 0 #1 o f e lua t e .

198

io §

~ 10001

BASIC TAR II [--]AC i A A ~ A P r'~AN C3APH

r ~

l&2 3 (0.06) (0. 09)

4 5 (0.13) (0.19)

TLC REG I ON (Rf)

1

6 (0.26}

7&8 (0. ~1)

Fig. 3. Th e re lat ive c o n c e n t r a t i o n o f several pr imary a r o m a t i c a m i n e s vs. m u t a g e n i c i t y in a m u t a g e n i c T L C subfrac t ion o f the H D basic tar fract ion . Mutagenic i tY data are f r o m Table 2 for A m e s assay o f 20/~I o f e luate ,

aminonaphthalenes were detected in TLC regions exhibiting little mutagenic activity. Since the aminofluorenes were not identified by GCMS analysis, we have shown their distribution only in the basic fraction, where their presence must be considered tentative.

Data relating to the recovery of mutagenic activity in the HD basic and tar

8

I NEURAL TAR II [] AC IAA AP DAN

~'APH

I 2 3&4 5 (0.06) (0.08) (0.13) (0.2)

TLC REGION (Rf )

I 6&7 (0.2D

Fig. 4 . The relat ive c o n c e n t r a t i o n o f several pr imary a r o m a t i c a m i n e s vs. m u t a g e n i c i t y in m u t a g e n i c TLC subfrac t ions o f the H D neutral tar f rac t ion . Mutagen ic i ty data are f r o m Table 2 for A m e s assay o f 2 0 #1 o f e luate .

199

fractions are presented in Tables 1--3. The mutagenicity of these fractions, expressed in terms of revertant colonies of S. typhimurium TA98 per #g material, ranged from 78 to 198. The number of revertant colonies TA98 expected for Ames assay of the total amounts of these fractions chromato- graphed is shown in column 4 of Table 1.

Ames assay of the TLC eluates from the basic and tar subfractions yielded approx, linear dose--response data (Table 2) which were used to estimate recovery of mutagenic activity in these subfractions as shown in Table 3. Summation of estimated total revertant colonies TA98 expected for the various TLC regions accounted for 70% of the mutagenic activity of basic, 35% of the basic tar and 103% of the mutagenic activity of the neutral tar fractions (Tables 1 and 3).

Some variation was observed between the Rf of chemical mutagens in the basic and neutral tar fractions versus those detected in the basic fraction (Figs. 2 vs. 3 vs. 4). For example, the mutagens in the basic TLC subfractions were

T A B L E 1

M U T A G E N I C I T Y O F B A S I C A N D T A R F R A C T I O N S F R O M H D P R I O R T O T L C

Sample # g (#I) Revertants Mutagenici ty (Y) a fract ion or T A 9 8 ± chemical SE

Tota l revertant equivalents used for T L C b

Basic fract ion

Basic tar fract ion

0 . 8 4 (1) 1 8 3 ± 3 3 1 . 6 7 (2) 3 7 0 ± 61 2 . 5 1 (4) 5 1 0 ± 1 9 3 . 3 4 (6) 6 6 7 ± 1 0 6 4 . 1 8 (8) 8 3 7 ± 4 0

c 8 .36 (10) 351 ± 137

1 . 0 8 (1) 8 9 ± 2 4 2 . 1 6 (2) 1 8 4 ± 9 5 3 . 2 4 (4) 3 3 7 ± 8 8 4 . 3 2 (6) 4 3 3 ± 31 5 . 4 0 (3) 4 1 8 ± 9 1

Y = 1 9 8 x + 7 ¢ = 1.00

Y = 8 8 x + 4 ¢ = 0 . 9

198 revl/~g X 10032 #g = 1.98 X 106 r e v

8 8 rev//~g X 2 5 9 2 0 ~g = 2 . 2 8 X 1 0 6 rev

1 0 . 8 ( 1 0 ) 6 1 4 + 6 3

Neutral tar " 0 . 8 4 (1) 6 4 + 1 5 fract ion 1 . 6 9 (2) 9 9 ± 5 4

2 . 5 3 (4) 2 6 2 ± 5 0 Y = 7 8 x + 1 0 3 .37 (6) 3 7 5 ± 3 2 ¢ = 0 . 8 9 4 . 2 2 (8) 2 3 6 ± 7 2 8 . 4 3 (10) 6 7 4 ± 1 5

2-Aminoanthracene 1 (1) 1 4 0 0 N .A . d N .A . B e n z o [ a ] p y r e n e 5 (5) 4 0 6 N .A . N .A . D M S O o n l y (5) 4 2 ± 1 5 N .A . N . A .

7 8 r e v / # g X 2 0 2 3 2 #g = 1 . 5 8 × 106 rev

a Est imated b y linear regression; y = a x + b w h e r e y , rev co lon ies T A 9 8 ; a , s lope o f regression line in rev co lonies T A 9 8 per g fraction; b, intercept in rev co lonies T A 9 8 at zero samples per plate~ x , #g f r a c t i o n ; ¢ , correlat ion coef f i c ient for x and y .

b Rev co lon ies T A 9 8 / # g fract ion ( ) X # g fract ion chromatosraphed . c Horizonta l line indicates upper concentra t ion l imit (#g per plate) used to est imate mutagenic i ty o f the

crude fract ions . d N .A, , n o t applicable .

2 0 0 ~"

T A B L E 2

M U T A G E N I C I T Y O F T L C S U B F R A C T I O N S F R O M B A S I C A N D T A R F R A C T I O N S O F H D

M a t e r i a l T L C r e g i o n N e t r e v e r t a n t s T A 9 8 a a t Ave rage n u m b e r o f r e v e r t a n t s per/~1

(5) b 1 0 ~1 (10 ) 2 0 ~1 e l u a t e c

Basic 1 1 7 1 4 3 7 39 f r a c t i o n 2 9 5 1 1 7 1 8 1 8 1

3 1 1 9 2 1 2 ' 2 3 4 7 53 - - 5 0 1 2 - -

Basic t a r 1 + 2 1 1 0 2 3 8 1 1 . 5 f r a c t i o n 3 4 1 0 9 0 0 4 3 . 0

4 2 2 1 3 9 0 2 0 . 8 5 59 4 4 4 .1 6 9 1 6 - - 7 + 8 1 6 31 - -

N e u t r a l t a r 1 1 4 9 4 1 1 1 7 . 7 f r a c t i o n 2 5 3 3 1 1 6 2 55 .7

3 ± 4 9 4 1 1 4 7 0 8 3 . 8 5 5 0 1 0 0 5 .0 6 + 7 16 41 - -

a A v e r a g e n u m b e r o f r e v e r t a n t c o l o n i e s T A 9 8 f o r 2 p e t r i p l a t e s m i n u s average b a c k g r o u n d o f 4 2 rever - t a n t s per . p l a t e .

b 5 a n d 1 0 #1 s a m p l e s f o r bas i c f r a c t i o n , 10- a n d 20 -p l s a m p l e s f o r bas i c a n d n e u t r a l t a r f r a c t i o n s . c O b t a i n e d b y a v e r a g i n g n u m b e r o f r e v e r t a n t s T A 9 8 m i n u s b a c k g r o u n d d i v i d e d b y #1 T L C e l u a t e a s s a y e d .

tightly clustered, mostly in TLC region 2 (Rf value approx. 0.11), and only slight activity was observed in regions 3 (Rf 0.08) and 4 (Rf 0.23). No mutagenic activity was associated with any other TLC region. Tar fractions exhibited a more diffuse distribution of mutagenic activity, with significant numbers of revertants recovered from TLC regions with Rf values of 0.06--0.2.

T A B L E 3

R E C O V E R Y O F M U T A G E N I C A C T I V I T Y A F T E R T L C O F B A S I C A N D T A R F R A C T I O N S F R O M H D

M a t e r i a l T L C r e g i o n A v e r a g e n u m b e r V o l u m e o f T L C T o t a l r e c o v e r y o f r e v e r t a n t s p e r e lua t e (#1) m u t a g e n i c a c t i v i t y b

J # l / e lua t e a

Basic 1 3 9 6 0 0 0 2 .3 X 105 f r a c t i o n 2 1 8 1 6 0 0 0 1 . 0 8 X 1 0 6 ~ = 1 .4 ×

3 2 3 6 0 0 0 1 .3 X l 0 s 1 0 6

Basic t a r 1 + 2 1 1 . 5 1 0 0 0 0 1 .2 X 1 0 $ f r a c t i o n 3 4 3 1 0 0 0 0 4 .3 X 105 ~ = 8 ×

4 2 0 . 8 1 0 0 0 0 2 .1 × l 0 s 105 5 4 .1 1 0 0 0 0 4 .1 X 1 0 4

N e u t r a l t a r 1 17 .7 1 0 0 0 0 1 .8 X 105 f r a c t i o n 2 5 5 . 7 1 0 0 0 0 5 .6 X 105 ~ = 1 . 6 3 ×

3 + 4 8 3 . 8 1 0 0 0 0 8 . 4 X 1 0 $ 1 0 6 5 5 . 0 1 0 0 0 0 5 .0 X 1 0 4

a See T a b l e 2. b C o l u m n 3 X c o l u m n 4.

201

Although absolute quantitative data using internal standards have not yet been obtained, computer-generated measurements of mass-spectral peak areas for (M + 1) ÷ ions from the spectra obtained in the CI mode indicate that the highest concentrations of primary amines were found in regions 1--4 of the TLC plates.

Discussion

We have presented evidence that primary aromatic amines are concentrated in the mutagenic TLC subfractions from HD. Although GCMS analyses show them to be low in concentration, the distributions of these primary amines across TLC plates were more similar to the distributions of mutagenic activity in the subfractions than the distributions of any other nitrogen-containing com- pounds detected~ Of special interest was the occurrence of highly active 3- and 4-ring aromatic amines [5] in the most active subfractions. The findings, along with results of previous studies in our laboratory, constitute a growing body of evidence linking aromatic amines to the mutagenicity in HD [8].

Compared to crude petroleums, HD is enriched in nitrogen-containing heter- ocyclics [10], as well as in various polynuclear aromatic hydrocarbons such as benzo[a]pyrene [9], many of which are (or are likely to be) mutagens in the Ames assay [4,5]. However, the mutagens in HD migrated more slowly in the TLC system employed in this study than most of the polynuclear aromatic nitrogen heterocyclic model compounds chromatographed as reference materials. The primary aromatic amines on the other hand, had the same mobility as the mutagens contained in HD. For example carbazoles and quinolines, major compound classes in HD [9,10], peaked in concentration in TLC fractions which displayed decreasing mutagenicity.

Very little evidence is available suggesting primary amines as components in synfuels (such as shale oils) or the SRC liquids studied here. The presence of compounds with primary amino groups has been inferred in both shale oil and coal-derived liquids on the basis of infrared spectroscopy and chemical analysis [4]. With the exception of the evidence presented in this repprt, we are unaware of the direct verification of primary polycyciic amines in SRC liquids. However, primary aromatic amines have received a great deal of attention recently because of evidence linking them to the mutagenicity of pyrolytic pro- ducts from amino acids, peptides [11,12], and cooked meats [2], and an abstract has been published which states that aromatic amines along with aza- arenes, are major constituents of synthetic fuels, including coal<lerived liquids [31.

References

1 Ames, B.N., J, McCann and E. Yamasaki, Methods for detect ing carcinogens ~ad mutagens wi th the Salmonella/manlmalian-mlcrosome mutagenic i ty test, Mutat ion Res., 31 (1975) 347--364.

2 Commoner, B., A. Vithayathli and P. Dolara, Mutageni¢ arudysls of c o m p l e x samples of aqueous effluents, air particulates, and foods, in: M. Waters, S. Nesnow, J. Hulslngh, S. Sandu and L. Clayton (Eds.), Application of Short-Term Bioassays in the Fract ionat ion and Analymds of Complex Eviron- mental Mixtures, EPA-S00/9-Y8-027, NTIS, Springfield, VA, 1978, pp. 529--570.

3 Epler, J., T. Rao, C. Ho and M. Guerin, Identif icat ion of mutagenic c o m p o n e n t s of basic fractions from crude oils, Environ, Mutagen., 1 (1979) 172.

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4 Ho, C., B. Clark, M. Guer in , C. Ma and T. Rao , Aroma t i c n i t rogen c o m p o u n d s in fossil fuels - - a po ten t i a l haza rd , ACS Preprints , Div. Fuel Chem. , 24 (1979) 281 - -291 .

5 McCann, J. , E. Choi, E. Yamasak i a n d B. Ames, De tec t ion of carc inogens as mutagens in the Salmo- ne l l a /mic rosome test: Assay of 300 chemicals , Proe. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5 1 3 5 - - 5 1 3 9 .

6 Moscbi t to , R.D. , Opera t ion of the Ft . Lewis, Washington solvent ref ined coal (SRC) pi lot p lan t in the SRC I a n d SRC II process ing modes , in: Proceedings of the 13 th Energy Convers ion Engineering Conference , Vol. 1, Socie ty o f A u t o mo t i v e Engineers, 400 C o m m o n w e a l t h Drive, Warrendale, PA 1 5 9 0 6 , 1978 , pp. 3 9 5 - - 4 0 1 .

7 Munson , M.0 and F. Field, Chemical ion iza t ion mass s p e c t r o m e t r y I. General i n t roduc t i on , J. Am. Chem. Soc., 88 (1966) 2 6 2 1 - - 2 6 3 0 .

8 Pelroy, R., and A. Gandol f i , Use of a mixed func t ion amine oxidase for metabol ic ac t iva t ion in the Ames /Sa lmone l la assay sys tem, Muta t ion Res., 72 (1980) 3 2 9 - - 3 3 4 .

9 Peterson, M.R., J. F r u c h t e r and J.C. Laul, Charac te r i za t ion of subs tances in p roduc t s , eff luents , and wastes Prom syn the t i c fuel p r o d u c t i o n tests , Q. Rep, U.S. Energy Research a n d Development Admini - s t ra t ion , BNWL-2131 , Battelle, Pacific Nor thwes t L a b o r a t o r y , Rich land , WA 9 9 4 5 3 2 , 1976.

I 0 Schiller, J. , Ni t rogen c o m p o u n d s in coal derived l iquids, Anal. Chem. , 49 (1977) 2 2 9 2 - - 2 2 9 4 . 11 Sugimura , T., T. Kawachi , M. Nagao, T. Yahagi , Y. Seino, T. O k a m o t o , K. Shudo , T. Kosuge, T. Tsuji,

K. Wakabayash i , Y. I i taka and A. Itai, Mutagenic pr inciples in t r y p t o p h a n a n d pheny la l amine py ro ly - sis p roduc t s , Proc. Jpn . Acad. , 53 (1977) 58- -61 .

12 Yoshida, D., and T. Ma t sumoto , Changes in mu t ag en i c i t y of p ro te in py ro lyza t e s b y reac t ion wi th ni t r i te , Muta t ion Res., 58 (1978) 35 - -40 .