Bioassay-directed fractionation of mutagenic PAH atmospheric photooxidation products and ambient particulate extracts

Mutation Research, 281 (1992) 67-76 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

67

MUTLET 00611

Bioassay-directed fractionation of mutagenic P A H atmospheric photooxidation products and ambient particulate extracts Janet Arey

a,b, William P. Harger a, Detlev Helmig a and Roger Atkinson a,b

" Statewide Air Pollution Research Center, Unit~ersity of CaliJbrnia, Riverside, CA 92521 (U.S.A.) and t, Department of Soil and Em:ironmental Sciences, Unit,ersity of California, Ricerside, CA 92521 (U.S.A.) (Received 22 May 1991) (Revision received 4 September 1991) (Accepted 13 September 1991)

Keywords: Bioassay-directed chemical analysis; Chemical analysis, bioassay-directed; PAH photooxidation products; Atmospheric mutagens; Nitrofluorenes; Nitrophenanthrene lactones

Summary Simulated atmospheric gas-phase reactions of naphthalene, fluorene and phenanthrene have been carried out in an environmental chamber with bioassay-directed chemical analysis of the reaction products. Nitro-PAH were found to be the most significant mutagens formed from the reactions of naphthalene and fluorene. The mutagram (bar graph of mutagenic activity versus HPLC fraction) of the phenanthrene reaction products closely resembled that of an ambient air particulate extract with the most mutagenic activity being in a fraction more polar than that in which the nitro-PAH elute. Nitrophenanthrene lactones (nitro-6H-dibenzo[b,d]pyran-6-ones) were found to account for the observed activity of this polar fraction of the phenanthrene reaction products. It has been shown that the utilization of an environmental chamber with a known PAH-starting material and the ability to produce sufficient product for isomer-specific identifications of mutagens is a promising complement to bioassaydirected fractionation of ambient air particulate extracts.

Ambient atmospheric particles contain adsorbed organics which are mutagenic and carcinogenic (Leiter et at., 1942; Tokiwa and Ohnishi, 1986, and refs. therein), and these include carcinogens such as the polycyclic aromatic hydrocarbons [PAH] (IARC, 1983) and their nitro-de-

Correspondence: Dr. Janet Arey, Statewide Air Pollution Research Center, University of California, Riverside, CA 92521 (U.S.A.).

rivatives [nitro-PAH] (IARC, 1984; King, 1988). Using bioassay-directed fractionation, investigators found nitro-PAH to be important mutagenic species in diesel-exhaust extracts (Schuetzle et al., 1981; Nishioka et al., 1982), and the technique has been applied to the identification of mutagenic species from such diverse sources as xerographic toners (Rosenkranz et al., 1980), products of the reaction of benzo[a]pyrene with ozone (Pitts et al., 1980), cigarette smoke (Kier et al., 1974), ambient atmospheric particles (Nishio-

68 ka et al., 1988; Lewtas et al., 1990a) and the metabolites of 1-nitropyrene (Lewtas et al., 1990b). The majority of the nitro-PAH observed in ambient air, both gas-phase and particle-associated, are believed to be the products of the gas-phase atmospheric reactions of the parent PAH (Arey et at., 1986, 1987, 1989a, 1990; Zielinska et at., 1989a,b; Atkinson et at., 1990a). For example, 1-nitropyrene is emitted into the atmosphere in diesel exhaust and other direct emissions (Schuetzle et at., 1981; Gibson, 1982; Harris et al., 1984) yet 2-nitrofluoranthene, which is formed from the hydroxyl (ON) radical- and nitrate (NO 3) radical-initiated reactions of gasphase fluoranthene (Arey et at., 1986; Atkinson et at., 1990a,b), is more abundant in ambient particles. Although nitro-PAH make a significant contribution to the direct-acting mutagenicity of diesel-exhaust extracts (Schuetzle, 1983), the nitrofluoranthenes and nitropyrenes, which are among the most prevalent nitro-PAH and generally those found in highest concentrations in ambient air-particle extracts (Nielsen et al., 1984; Ramdahl et al., 1986; Nielsen and Ramdahl, 1986; Atkinson et al., 1988; Nishioka et al., 1988; Zielinska et al., 1989a; Arey et al., 1990), have been reported to contribute less than 10% of the overall direct-acting mutagenicity of ambient air extracts toward Salmonella typhimurium strain TA98 (Arey et al., 1988). In ambient air-particulate extracts, the major mutagenic fractions contain compounds more polar than the nitro-PAH (Nishioka et al., 1988; Lewtas et al., 1990a). Evidence which indicates that these polar mutagenic compounds may be PAH-derivatives, and that these derivatives are formed from the atmospheric reactions of PAH includes the following. Comparing the mutagenicity profiles from high-performance liquid chromatographic (HPLC) fractions of ambient particulate extracts with those of diesel particles, the profile for the ambient extracts is shifted toward more polar mutagenic fractions (Schuetzle and Lewtas, 1986), consistent with the occurrence of chemical transformations of the compounds emitted into the atmosphere during transport from emission source to downwind sites. Nishioka and coworkers (1988), using bioassay-directed frac-

tionation of ambient particle extracts, identified PAH-derivatives in strongly mutagenic polar subfractions. Finally, in a study conducted at seven locations in California chosen to typify different dominant emission sources (Atkinson et al., 1988; Arey et al., 1991a), the direct-acting mutagenic activity of the extracts of particles collected at each site did not correlate with the PAH concentrations themselves, but rather correlated with 2-nitropyrene, a nitro-PAH formed through atmospheric transformations (Arey et al., 1986; Atkinson et al., 1990a). The nitro-PAH account for only a small fraction ( ~ 5%) of the atmospheric reaction products of the PAH with the OH radical, the species responsible for the relatively short lifetimes of the gas-phase PAH in the atmosphere (Arey et al., 1989a,b; 1990; Atkinson et al., 1990a), and the remaining PAH reaction products are expected to include polar compounds which may also be mutagenic. We describe here the application of bioassaydirected chemical analysis to the products of the OH radical-initiated reactions of gas-phase naphthalene, fluorene and phenanthrene. The PAH were reacted in an environmental chamber, with the products being collected and subjected to HPLC fractionation with bioassay using the microsuspension preincubation modification (Kado et al., 1983, 1986) of the Ames assay with Salmonella typhimurium strain TA98. Mutagrams (bar graphs of mutagenic activity versus HPLC fraction) from the reactions of these atmospherically abundant PAH were compared to the mutagrams of ambient air-particulate extracts to look for PAH which produce mutagenic products of the same polarity as the important, but unidentified, mutagens in ambient air. If, as suggested above, the PAH are responsible for a significant portion of the polar direct-acting mutagenicity of ambient air particles through their atmospheric reactions, identification of these mutagenic PAH reaction products would lead to a newly recognized mutagen or class of mutagens whose contribution to ambient mutagenicity could then be assessed. Materials and methods

Ambient samples. Ambient particles were collected on the campus of Harvey Mudd College in

69 Claremont, CA during the South Coast Air Quality Study (Lawson, 1990) in August, 1987. The particulate matter was collected on Pallflex T60A20 Teflon-impregnated glass fiber filters by a high-volume sampler equipped with a 10-micron size-selective inlet and operated at a measured flow rate of 38.2 SCFM (1.1 m 3 rain l). The three samples used for this study were obtained during daytime hours (0600-1800 PDT) on three successive days (August 27, 28 and 29, 1987) using the same sampler. Each filter was Soxhlet extracted for 24 h using CHzC12 and the extracts were concentrated and then fractionated as described below.

Enuironmental chamber reactions. As described in more detail elsewhere (Arey et al., 1986; Atkinson et al., 1987; Atkinson and Aschmann, 1988), reactions of P A H with the O H radical in the presence of N O x were conducted in a 6400 1 all-Teflon environmental chamber equipped with two parallel banks of blacklamps (Sylvania F40/350BL). Hydroxyl radicals were generated by the photolysis at 100% light intensity of methyl nitrite in air at wavelengths > 300 nm. The reaction products were sampled from the chamber by pulling the chamber volume though polyurethane foam (PUF) plugs for 2 min at ~ 1000 1 min-1. The P U F plugs were Soxhlet extracted with CH2CI 2 for 4 h, and the extracts concentrated and separated by H P L C as described below. Naphthalene (initial concentration ~ 910 ppb) and fluorene (initial concentration ~ 90 ppb) were added to the chamber by flowing pure nitrogen though Pyrex tubes packed with the pure PAH. Phenanthrene (initial concentration ~ 160 ppb) was added by spraying a methanol solution into the chamber as a fine mist using a glass atomizer and with the chamber mixing fan on, thereby spreading the phenanthrene over the chamber surfaces. The chamber was flushed with pure air for ~ 15 min to remove most of the methanol. The volatilization of the phenanthrene from the surfaces established the gas-phase phenanthrene concentration in the chamber. The naphthalene and phenanthrene reactions employed 2 parts-per-million (ppm) of methyl nitrite and 1 p p m N O with irradiation for 10 min. Fluo-

rene, with 10 p p m each of methyl nitrite and NO, was irradiated for 5 min. In general, the concentration of hydroxyl radicals in the irradiations increases proportionately with the methyl n i t r i t e / NO concentration ratio and decays effectively instantaneously when the lights are turned off. Blank chamber runs in which C H 3 O N O and N O were photolyzed in the absence of the P A H were also sampled, extracted, fractionated and assayed for mutagenic activity.

HPLC fractionation. The dichloromethane extracts of the ambient filters or P U F plugs from the chamber were filtered (0.45 p.m Teflon filters, Acrodisc, Gelman Sciences) and fractionated by normal-phase HPLC. The H P L C column was a semipreparative Regis Spherisorb S5W silica (5 micron) column, 25 cm × 10 mm. The H P L C instrumentation consisted of a Spectra-Physics Model 8100 gradient liquid chromatograph with a Model 8400 U V / V i s detector (A = 254 nm) and an ISCO fraction collector. The solvent program (at a flow rate of 3 ml min i) was: initially 100% hexane for 10 rain, followed by a 5-min linear gradient to 95% hexane and 5% CH2C12. The solvent was programmed over the next 25 min to 100% CH2CI 2 where it was held for 10 min, then p r o g r a m m e d to 100% acetonitrile over 10 min, held isocratic for 10 min and then p r o g r a m m e d back to the initial conditions. In general, beginning after one minute, 9 nine-minute fractions of increasing polarity were collected during the separation for bioassay and chemical analysis. Bioassay. Salmonella typhimurium, strain TA98, without $9 activation was used to assay the H P L C fractions from ambient air extracts and from the chamber P A H exposures. To enhance sensitivity, a microsuspension preincubation modification of the Ames assay (Kado et al., 1983, 1986) which employs a 90-min preincubation of the test substance with elevated test cell densities in a small-volume buffered-saline suspension was employed. The H P L C fractions were dissolved in CH2C12 and solvent-exchanged into D M S O (Mallinckrodt SpectrAR grade, 5 /zl), combined with the bacteria (100/~1, ~ 1.1 × 101° cells ml -~) and incubated for 90 min at 37°C with vigorous shaking (180 rpm). Following the addition of 2.0

70

ml of soft agar and mixing, each sample was overlayed onto minimal glucose plates which were incubated at 37°C for 63 h and scored by means of an automatic colony counter. On each test day, 2-nitrofluorene was tested as a positive control. To obtain net revertants, the average number of spontaneous revertants (from 6 replicates) was subtracted from all counts and the net revertants were used to calculate the dose-response curve. In order to reserve 50% of each H P L C fraction for chemical analysis, mutagenicity testing was done on single plates using 0.05%, 0.45%, 4.5% and 45% of the sample per plate. The initial linear portion of the dose-response curve was determined by the criterion that a least-squares analysis yielded an intercept within four standard deviations of the spontaneous background revertants; if not, the highest dose point(s) was eliminated until this criterion was met. The slope from this initial linear portion of the dose-response curve was used to calculate the activity.

Chemical analysis. A Hewlett-Packard 5890 G C interfaced to a 5970 Mass Selective Detector was used for G C / M S analysis of aliquots from selected H P L C fractions. More complete characterizations of the reaction products of fluorene (Helmig and Arey, 1991a; Helmig et al., 1991a) and phenanthrene (Helmig and Arey, 1991b) are provided elsewhere. Results and discussion

The ambient sample mutagenicity data given in Table 1 is a tabulation of the revertants in each H P L C fraction from extracts of ambient particle samples collected on three consecutive days in August in Claremont, CA. An averaged mutagram for the ambient samples is given in Fig. 1. The mutagrams from all 3 days looked remarkably similar with the majority of the activity being in H P L C fractions Nos. 6 and 7 (note that the nitro-PAH eluted in fraction No. 4 for these H P L C conditions). Similar mutagrams, that is, with more activity in the later more-polar fractions have been observed for samples collected in El Monte, CA [assayed with the standard Ames test] (Pitts et al., 1984) and more recently in Riverside, CA (Atkinson et al., 1991), and as

TABLE 1 M U T A G E N I C I T Y OF HPLC FRACTIONS OF AMBIENT PARTICULATE O R G A N I C MATTER COLLECTED lN CLAREMONT, CA, August 27-29, 1987 (0600-1800 PDT); MICROSUSPENSION ASSAY; TA98, - $ 9 HPLC Fraction No. 1

2 3 4 5 6 7 8 9 Sum Rev m- 3

Net revertants per fraction ~' 8/27/87

8/28/87

() b

0 b

0 b

0h 280 7 600 12 000 56 000 47 000 1 800 260

0h 130 5 800 8 300 39 000 25 000 1 100 190

110 140 5 800 12 000 57 000 44 000 1 700 310

8/29/87

121 060

124 940

79 520

160

160

100

" In the case of highly mutagenic fractions, to obtain revertant counts in the linear region of the dose-response curve, each fraction was tested over a dose range spanning three orders of magnitude (see text for additional discussion). The 2-nitrofluorene activity was 3100+ 100 revertants nmoles t + the standard error of the slope. b The net revertants were less than four standard deviations above the spontaneous response, i.e., < 20 revertants above the background.

noted above, these findings are consistent with those of other investigators (Nishioka et al., 1988; Lewtas et al., 1990a). In a previous study at 7 sites within California chosen, as noted above, to typify different dominant emission sources, gas-phase and particle-associated P A H and nitro-PAH and ambient particle mutagenicity were measured (Atkinson et al., 1988; Arey et al., 1991a,b). The abundances of some common P A H in these samples were as follows: naphthalene > fluorene ~ phenanthrene > fluoranthene ~ pyrene > benzo[a]pyrene, with the average naphthalene concentration reaching 3600 ng m -3 at Glendora, CA. On the basis of their high ambient concentrations, and since they are expected to be fully in the gas-phase (Coutant et al., 1988), naphthalene, fluorene and phenanthrene were chosen for bioassay-directed chemical analysis of their gas-phase O H radical-initiated reaction products. Significant daytime reaction of these P A H with the O H radical has

71 AMBIENT AIR 80 60

40

W PHENANTHRENE + OH

W

> n o W n/

80

GO

>-

7

40

H

F-- 20 0 <£ 0 z W L3 80 <£ F-Z
FLUORENE + OH

GO

40

20

Lu O

NAPHTHALENE + OH

80 60 40

I

1

2

3

,4

--,5

6

I7

8

g

FRACTInN

Fig. 1. H P L C m u t a g r a m s from the gas-phase O H radical-initiated reactions of phenanthrene, fluorene and naphthalene compared to the m u t a g r a m of Claremont, CA ambient airparticle extracts. The plotted mutagenicity values have been normalized to the sum of the individual fractions. To obtain the ambient sample mutagram, the activity for each fraction of the three samples (Table 1) was s u m m e d and is shown as a percentage of the total activity recovered (sum of all individual fractions). The total revertants recovered from the phenanthrene, fluorene and naphthalene chamber reactions were 341000, 150000 and 97000, respectively.

been observed under ambient conditions (Arey et al., 1989b), and this dominant reaction has been calculated to lead to atmospheric lifetimes of ~ 9 h; ~ 13 h and ~ 6 h for naphthalene, fluorene, and phenanthrene, respectively (Arey et al., 1989a,b). The mutagrams of the products from the O H radical-initiated reactions of these three PAH are shown in Fig. 1. It should be noted that, by rapidly sampling (at ~ 1000 liters min - I ) the chamber contents onto PUF plugs, compounds produced in the gas-phase are sampled, including those which under ambient conditions will become particle-associated to an extent determined by their volatility. Reaction products eluting in fraction No. 4 were responsible for the majority of the mutagenic activity for both naphthalene and fluorene, while HPLC fraction No. 6 of the phenanthrene reaction was highly mutagenic. The activity measured in the chamber blank runs was insignificant when compared with the activity of the PAH reaction products.

Naphthalene. Both 1- and 2-nitronaphthalene and 1-hydroxy-2-nitronaphthalene were identified by G C / M S analysis of fraction No. 4 from the naphthalene reaction. 1-Hydroxy-2-nitronaphthalene has been tested in the standard Ames assay and the Kado microsuspension assay and found to be inactive (Atkinson et al., 1991). Although 1- and 2-nitronaphthalene were reported to be only very weak mutagens toward TA98 in the standard Ames assay, both these compounds showed significant activity when tested with the microsuspension modification (Table 2). By quantifying the nitronaphthalenes, it was estimated that ~ 90% of the activity of fraction No. 4 can be ascribed to the nitronaphthalenes, with the majority of the activity being from 2-nitronaphthalene. The high activity of the nitronaphthalenes in the microsuspension assay, in particular of the 2-isomer, is consistent with the recent work of Kado et al. (1991a) and suggests that the standard plate incorporation assay may underestimate the activity of volatile mutagens (Kado et al., 1991b). It is important to recognize that the ambient particle-phase mutagenic activity, as shown in Fig. 1, would include only a small fraction of the contribution from the nitronaphthalenes, since they were present mainly in the

72 gas-phase (Arey et al., 1987, 1989b). However, it should be n o t e d that the a m b i e n t c o n c e n t r a t i o n s of the volatile n i t r o - P A H , including the nitron a p h t h a l e n e s , m e t h y l n i t r o n a p h t h a l e n e s a n d 3nitrobiphenyl, have b e e n r e p o r t e d to be at least an order of m a g n i t u d e greater than the particleassociated n i t r o f l u o r a n t h e n e s and n i t r o p y r e n e s (Arey et al., 1987). T h e initial n a p h t h a l e n e c o n c e n t r a t i o n in the c h a m b e r reaction for which the p r o d u c t m u t a gram is shown in Fig. 1 was nearly 1 ppm, a n d approximately 5 a n d 10 times the initial p h e n a n t h r e n e a n d f l u o r e n e c o n c e n t r a t i o n s , respectively. In a m b i e n t samples the ratio of n a p h t h a l e n e / p h e n a n t h r e n e has b e e n observed to vary from ~ 20 to ~ 200 (Arey et al., 1987; A t k i n s o n et al., 1988). T h e n a p h t h a l e n e derivatives responsible for the activity in fraction No. 7 of the n a p h t h a lene reaction may thus c o n t r i b u t e significantly to a m b i e n t particle mutagenicity, d e p e n d i n g on the a m b i e n t n a p h t h a l e n e c o n c e n t r a t i o n s and the g a s / p a r t i c l e phase distribution of the mutagen(s). Fluorene. A typical m u t a g r a m from the products of the gas-phase O H radical-initiated reaction of f l u o r e n e is shown in Fig. 1. All four

n i t r o f l u o r e n e isomers (1-, 2-, 3- and 4-nitrofluorene) were identified by G C / M S analysis in H P L C fraction No. 4 of the f l u o r e n e c h a m b e r reaction. A full discussion of the characterization of the n i t r o f l u o r e n e isomers has b e e n r e p o r t e d (Helmig and Arey, 1991a), and a discussion of the n i t r o f l u o r e n e yields and other products formed in this reaction will be p r e s e n t e d elsewhere (Helmig et al., 1991a). The activities of the n i t r o f l u o r e n e isomers in both the s t a n d a r d plate i n c o r p o r a t i o n test and the m i c r o s u s p e n s i o n modification of the A m e s assay are listed in T a b l e 2. In the s t a n d a r d plate i n c o r p o r a t i o n test, 2 - n i t r o f l u o r e n e was the most m u t a g e n i c isomer, followed by 3-nitrofluorene, which is in a g r e e m e n t with a previous report ( W a t a n a b e et al., 1986). 2 - N i t r o f l u o r e n e is predicted to be the most active isomer according to the "para principle" (Arcos a n d Argus, 1974), and 2- and 3 - n i t r o f l u o r e n e would be expected to be the most active according to the general rule that n i t r o - P A H isomers with the N O 2 group orie n t e d along the long axis of the p a r e n t P A H are generally more active (Vance and Levin, 1984), as are isomers that do not have the N O 2 group alpha to a point of ring fusion (Later et al., 1984;

TABLE 2 MUTAGENIC1TY ~ OF NITRONAPHTHALENES, NITROFLUORENES AND NITRODIBENZOPYRANONES Compound

1-nitronaphthalene h 2-nitronaphthalene h 1-nitrofluorene d 2-nitrofluorene d 3-nitrofluorene d 4-nitrofluorene d

Kado microsuspension assay (TA98; $9) Revertants nmoles ~ 48+ 3 890+ 40 860--+ 90 4 100 + 200 5500+300 34+ 2

Plate incorporation assay (TA98; - $9) Revertants nmoles 0.05 c, 0.4 c 0.2 '%0.9 c 4.2 + 1.1 93 + 3 41 -+2 0.40+0.03

2-nitro-6H-dibenzo[b,d ]pyran-6-one ¢

58600 -+700

g

4-nitro-6H-dibenzo[b,d]pyran-6-one ~

480+ 7(1

g

~ For all compounds tested in this laboratory, a minimum of 8 sample doses with 3 replicates at each dose were used and the initial linear portion of the dose-response curve was used to calculate the activity by a simple linear regression. The activity is expressed as revertants nmoles t _+the standard error of the slope. b The nitronaphthalenes were tested on the same day; the 2-nitrofluorene activity in the microsuspension assay was 4900_+200, ~ Data taken from Rosenkranz and Mermelstein (1983). d The four nitrofluorene isomers were tested on the same day. The 2-nitrofluorene activity in the microsuspension assay was 6500+300. The 2-nitrofluorene activity in the microsuspension assay was 5900_+200. No data available.

73 Vance and Levin, 1984). There are small differences in the relative activities between the standard plate incorporation and microsuspension tests, but 2- and 3-nitrofluorene may be said to be comparably strong mutagens and 1- and 4-nitrofluorene to be less potent. 3-Nitrofluorene, the isomer formed in the highest yield, accounted for most of the mutagenic activity of fraction No. 4. Nearly 75% of the activity of fraction No. 4 could be assigned to the nitrofluorenes quantified in this fraction. Since 3-nitrofluorene is the nitro-isomer formed in highest yield in the O H radical-initiated reaction of fluorene and given the carcinogenicity of 2nitrofluorene (Morris et al., 1950) and the comparable mutagenicities of 2- and 3-nitrofluorene, 3-nitrofluorene should also be considered a potential environmental carcinogen.

Phenanthrene. G C / M S analysis of HPLC fraction No. 6 of the phenanthrene chamber reaction s h o w e d a p h e n a n t h r e n e lactone (6H-dibenzo-[b,d]-pyran-6-one) to be the major component of this fraction. Two nitro-derivatives of this lactone (2-nitro and 4-nitro-6H-dibenzo[b,d]pyran-6-one) were also found in fraction No. 6 and these compounds accounted for the majority of the mutagenicity of this fraction, with most of the activity being contributed by the 2-nitroisomer (see also Table 2). The nitrophenanthrene lactones responsible for the activity of fraction No. 6 from the phenanthrene reaction are expected to also be present in fraction No. 6 of the ambient extracts and to contribute to the observed activity. The high activity we observe for the chamber reaction, together with the relative abundance of phenanthrene in ambient atmospheres, suggest that these mutagenic phenanthrene atmospheric transformation products will be significant contributors to ambient mutagenicity. Full chemical characterization of these mutagenic nitrophenanthrene lactones and evidence for their presence in ambient particle extracts will be presented elsewhere (Helmig et- al., 1991b; Helmig and Arey, 1991b). It may be speculated that the nitro-PAH lactones are significant contributors to ambient particulate mutagenicity. Fluoranthene and pyrene are among the additional PAH which will be

studied using the bioassay-directed fractionation described here. Although the ambient concentrations of fluoranthene and pyrene are lower than the PAH reported on here, as noted above, the nitro-derivatives produced from their gas-phase OH radical-initiated reactions are generally the most abundant nitro-PAH observed in ambient particle extracts. Furthermore, two nitropyrene lactone isomers have been found to be approximately an order of magnitude more mutagenic than 1-nitropyrene (E1-Bayoumy and Hecht, 1986).

Conclusions Initial screening of 3 abundant PAH for polar mutagenic reaction products has led to the identification of nitrophenanthrene lactones, which can now be quantified in ambient air and their contribution to ambient mutagenicity assessed. Furthermore, the importance of volatile mutagens (see, for example, Kleindienst et al., 1986, 1990; Shepson et al., 1986, 1987; Dumdei et al., 1988; Lofti et al., 1990) which have been largely overlooked by the analysis of only particle extracts is again e m p h a s i z e d by the activity of 2nitronaphthalene formed from the O H radicalinitiated reaction of naphthalene. Finally, it is concluded that the utilization of an environmental chamber with a known PAH-starting material and the ability to produce sufficient product for isomer-specific identification is a promising complement to bioassay-directed fractionation of ambient particulate extracts. The atmospheric transformation products of the PAH may well be responsible for the direct-acting mutagenic activity of ambient particles and the technique described here has led to the identification of a new compound class of atmospheric mutagens, the nitro-PAH lactones, whose importance to ambient mutagenic activity needs to be assessed.

Acknowledgements We thank Sara M. Aschmann and Patricia A. McElroy for able technical assistance on this work. We acknowledge the financial support of the California Air Resources Board through Con-

74 tract No. A732-154; R a l p h P r o p p e r , Project M a n ager.

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