composition is different from that of the core, this being detected by photoelectron spectroscopy; heterogeneities of samples trapped on filters (localization and thickness). It is implicitly assumed in most work published that a piece of filter has the same composition as the mean composition of the whole filter. This does not at all correspond to what we have been able to observe in cases where the density is of the order of. 1 pg-cm-*. Errors of 30-40% relative to the mean composition are possible. For this reason, the technique of cutting filters must be used the least, contiguous and simultaneous samples having a better chance of being comparable.
L i t e r a t u r e Cited (1) Saltzman, B. E., Cuddeback, J. E., Anal. Chem., 47,lR (1975). (2) Finlayson, B. J., Pitts, J. N., Science, 192,111 (1976). (3) Novakov, T., Chang, S. G., Dod, R. L., Contemp. Top. Anal. Clin. Chem., 1,249 (1977). (4) Brion, D., Escard, J., J . Microsc. Spectrosc. Electron, 1, 227 (1976). (5) Linton, R. W., Loh, A., Natusch, D. F. S., Evans, C. A,, Williams, P., Science, 191,852 (1976). (6) “Fallout Measurements by the Deposition Plaquette Method”, Standard NF X 43-007, Dec 1973. (7) Carlson, T. A., Carter, W. J., Schweitzer, G. K., J. Electron. Spectrosc., 5,827 (1974). (8) Barbaray, B., Contour, J. P., Mouvier, G., Analusis, 5, 413 (1977).
Acknowledgments
The authors are grateful to Mr. A. Salesse (Centre de Spectrochimie du Goupement Regional de Mesures) for technical assistance in XPS.
Received for review M a y 31, 1978. Resubmitted March 13, 1979. Accepted August 17,1979. One of the authors (B.Barbaray) thanks the Electricit4 de France for financial support.
Cyanoarenes in Soot: Synthesis and Mutagenicity of Cyanoacenaphthylenes S. Krishnan, Debra A. Kaden, William G. Thilly, and Ronald A. Hites’” Massachusetts Institute of Technology, Cambridge, Mass. 02 139
w Two cyanoacenaphthylene isomers were synthesized and used to verify the presence of these compounds in soots generated by the combustion of aromatic hydrocarbon fuels doped with pyridine. Charge exchange-chemical ionization mass spectrometry (CE-CI MS) was utilized in differentiating between the other two cyanoacenaphthylene isomers. The mutagenic activity of the synthesized compounds was measured using a forward bacterial assay. Several polycyclic aromatic hydrocarbons (PAHs) have been identified as carcinogenic compounds associated with soot (1). Nitrogen-containing aromatic compounds (azaarenes) are also present in soot (21, and some are known to be carcinogenic ( 3 ) . However, since azaarenes are much less abundant than PAHs, they have received less attention, and their environmental abundances and biological activities are not well known. This is unfortunate since the environmental occurrence of azaarenes is likely to increase as fuels higher in organic nitrogen content are burned. Dubay and Hites ( 4 ) have recently burned a model fuel containing 1-6% nitrogen and have identified the major azaarenes associated with this soot. The procedure involved Soxhlet extraction of the compounds from soot with CH2C12, the separation of the azaarenes from the bulk of the PAH by column chromatography, and the separation and identification of the individual azaarenes using gas chromatography (GC) and gas chromatographic mass spectrometry (GCMS) . Figure 1 shows the gas chromatogram of the azaarene fraction obtained with a nitrogen-specific GC detector. The major nitrogen-containing compounds in the soot were identified as 1- and 2-cyanonaphthalene (peaks 1 and 2, respectively) and as the four isomers of cyanoacenaphthylene (peaks 3-6, isomers unspecified). The cyanonaphthalenes were identified by comparison of their mass spectra and exact gas chromatographic retention times with those of available standard materials. Cyanoacenaphthylene standards were not Present address, School of Public and Environmental Affairs, Indiana University, Bloomington, Ind. 47405. 1532
Environmental Science & Technology
available; therefore, the identification of these compounds was considered tentative ( 4 ) . The purpose of the present study was to verify the identification of peaks 3-6 as cyanoacenaphthylenes, to specify which peak is which isomer, and to assess the mutagenic activity of these compounds. This has been done by a combination of chemical synthesis of reference compounds and charge exchange-chemical ionization mass spectrometry. The synthesized compounds were tested by a quantitative, forward mutation assay using 8-azaguanine resistance in Salmonella t y p h i m u r i u m . For the reader’s reference, the numbering system of acenaphthylene is shown here:
Experimental
Instrumentation. A Hewlett-Packard 5730A gas chromatograph equipped with dual nitrogen-phosphorus flame ionization detectors was used for GC analyses. A HewlettPackard 5982A mass spectrometer interfaced to a 5933A data system was used for gas chromatographic mass spectrometry. Melting points were measured using an Electrothermal melting point apparatus and are uncorrected. NMR spectra were run on a Varian T-60 instrument. Clones arising on the mutation plates were counted with an Artek Systems automatic colony counter. Synthesis of 1-Cyanoacenaphthylene. Acenaphthylene (1 g) was added to a stirred suspension of 5 g of anhydrous aluminum chloride in 150 mL of hexane; this was followed by 1 mL of bromine dropwise over a 5-min period. The mixture was stirred at room temperature for 1 h under anhydrous conditions (CaC12 drying tube). A t the end of this period, a 0.2-mL aliquot of the reaction mixture was quenched with water and extracted with 1 mL of hexane. Gas chromatographic analysis of the hexane extract showed the absence of acenaphthylene, and GC-MS analysis showed the major
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typhimurium were incubated for 2 h a t 37 "C with several concentrations of each compound dissolved in dimethyl sulfoxide. To allow for the possible metabolic activation of the compounds, a drug metabolizing system derived from Aroclor 1254 induced rat livers ( 7 ) was added to the cultures. Following the incubation, bacteria were centrifuged (2000 rpm x 15 min), resuspended in phosphate-buffered saline, and plated under selective (50 yg/mL 8-azaguanine) and permissive conditions. Colonies were counted after 2 days' growth a t 37 "C, and the mutant fraction for each culture was calculated (the number of colonies observed under selective conditions divided by the number of colonies observed under permissive conditions times the appropriate dilution factors).
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Figure 1. Gas chromatogram of azaarene mixture produced by combustion of 32% pyridine in o-xylene. GC conditions: 1.8 m X 2 mm i.d. glass column packed with 3% OV-17 on 80-100 mesh Supelcoport, programmed from 100 to 300 "C at 8 'C/min, nitrogen-specific FID. Peak identities as established in this study (see below): 1, l-cyanonaphthalene; 2, 2-cyanonaphthalene; 3, 4-cyanoacenaphthylene; 4, 5-cyanoacenaphthylene; 5, 3-cyanoacenaphthylene; 6 , l-cyanoacenaphthylene; 7, isomers of cyanophenanthrene
product to be 1-bromoacenaphthylene [m/e (relative abundance) 233 (14), 232 (97), 231 (14), 230 (94), 151 (loo), 150 (51)]. The reaction mixture was poured into 300 mL of ice-cold water in a separatory funnel and extracted with three 100-mL portions of hexane. The hexane extracts were combined, dried (MgS04),and evaporated in vacuo to remove the solvent. The residue, which contained the 1-bromoacenaphthylene, was treated with CuCN (1g) in refluxing DMF (25 mL) for 14 h ( 5 )t o form 1-cyanoacenaphthylene.Final purification of the product was achieved by alumina column chromatography using hexane as the eluent. The yield was 250 mg (overall yield 22%): mp 91-95 "C dec; GC retention index, 2138; m/e (relative abundance) 178 (12), 177 (loo), 176 (6), 151 (51, 150 (15). Synthesis of 5-Cyanoacenaphthylene. 5-Bromoacenaphthene (4 g), available from Aldrich, was converted into 5-cyanoacenaphthene [2.5 g; 82% yield; mp 109-110 "C; NMR 6 3.39 (s, 4 H), 7.0-8.0 (m, 5 H); m/e (relative abundance) 181 (l),180 (14), 179 (97), 178 (loo), 177 (21), 153 (41,152 (14), 151 (34), 150 (17)] by treatment with CuCN (2.5 g) in refluxing DMF (50 mL) for 6 h ( 5 ) . Dehydrogenation of 5-cyanoacenaphthene (0.15 g) was carried out in refluxing chlorobenzene (10 mL) with 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ, 0.8 g) for 6 h under nitrogen. After this period, the mixture was cooled to room temperature and filtered through a small column of A1203 (25 g). The column was washed with 200 mL of 1:l benzene-petroleum ether. The filtrate and washings were collected together and concentrated in vacuo. Chlorobenzene was removed from the residue by stream distillation, and the 5-cyanoacenaphthylene solid was recrystallized from methanol-water. The yield was 95 mg (63%):mp 82-83 "C; GC retention index, 2083; m/e (relative abundance) 178 (15), 177 (loo), 176 (7), 151 (6), 150 (14). Bacterial Mutation Assay. The compounds were assayed individually for their ability to induce mutations to 8-azaguanine resistance in Salmonella typhimurium as described by Skopek et al. (6, 7 ) . Exponentially growing cultures of S .
Results and Discussion The four cyanoacenaphthylenes may be divided into two classes: (i) bridge substituted and (ii) ring substituted. The two compounds synthesized represent an example from each category. Peaks 4 and 6 were identified as 5- and l-cyanoacenaphthylene isomers, respectively, by coincidence of their exact GC retention times (through co-injection) and of their electron-impact mass spectra. The 1-cyanoacenaphthylene isomer decomposes on melting. In GC experiments, solutions containing equal concentrations of 1- and 5-cyanoacenaphthylene gave a lower response for the 1 isomer, probably due to its partial decomposition in the injection port (300 "C). This factor must be taken into account in the quantitation of 1-cyanoacenaphthylene in environmental samples using gas chromatography. Peaks 3 and 5 (Figure 1) represent either 3- or 4-cyanoacenaphthylene. I t is possible to differentiate between these isomers based on the ( M 1)/M ratios obtained from charge exchange-chemical ionization mass spectrometry (8, 9). This method involves the creation of a plasma containing CzH5+ and Ar+ by using 5-10% methane in argon as a reagent gas. A molecule reacts with the strongly acidic C2H5+ to give the protonated molecular ion (M 1)+or with an Ar+ ion to give the molecular ion (M)+.The intensity ratio of these two ions depends on their relative rates of formation. A high, positive correlation has been observed between the ionization potential of the molecule and the (M 1)/M ratio derived from CE-CI mass spectrometry (8). An examination of data previously reported from this laboratory ( 4 )shows that the (M 1)/M ratios are: 2.33 (peak 3), 2.10 (peak 4), 2.49 (peak 5), and 2.27 (peak 6). With the current knowledge of the identity of peaks 4 and 6 as 5- and 1-cyanoacenaphthylene, respectively, a prediction of the relative ionization potentials of the other two isomers would lead, through the correlation with the (M 1)/M ratios from CE-CI MS, to the assignment of identities for peaks 3 and 5. Charge exchange induced ionization by Ar+ is an electrophilic reaction. Based on reactions of other electrophilic reagents, ionization of simple acenaphthylenes may be expected to occur a t the ethylene bridge. Electron-withdrawing substituents on the aromatic ring would act to decrease the loss of an electron by this process, thereby increasing the ionization potential of the molecule. Such an inductive effect would decrease as the substituent is moved farther away from the reaction site (10). On this basis, one would predict that the ionization potentials would increase in the order &cyanoacenaphthylene < 4-cyanoacenaphthylene < 3-cyanoacenaphthylene. Experimentally observed CE-CI MS values of (M 1)/M increase in the order peak 4 < peak 3 < peak 5. Based on the expected positive correlation between these two sets of parameters, it is predicted that peaks 3 and 5 are 4- and 3-cyanoacenaphthylene, respectively. Acenaphthene, 1-cyanoacenaphthylene, 5-cyanoacenaph-
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murium at concentrations as low as 1000,2800,1100, and 1250 pM, respectively. Acenaphthylene and 1- and 2-cyanonaphthalene did not induce significant mutation a t concentrations up to 1300pM, their limit of solubility under the assay conditions. Benzo[a]pyrene, run as a positive control in each mutation assay, consistently induced mutation a t concentrations as low as 4 pM. Although our results indicate that the active compounds have less than 1%of the activity of benzo[a]pyrene on a molar basis, the significant amounts of cyanoarenes produced by the combustion of nitrogen-containing fuels imply a need for concern. On the other hand, it is not known whether the cyanoarenes are environmentally persistent. Air particulate samples have been shown to contain the cyano functionality (11), but specific cyanoarenes have not been detected in the environment.
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Acknowledgment The authors thank Joanne Donovan for the exploratory work concerning 5-cyanoacenaphthylene.
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Literature Cited
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Figure 2. Dose-dependent mutagenicity (induced mutant fraction vs. concentration) and toxicity (relative surviving fraction vs. concentration) of acenaphthene ( O ) ,acenaphthylene(0),1-cyanonaphthalene (O), 2-cyanonaphthalene ( W), 1-cyanoacenaphthylene (V),5-cyanoacenaphthene (A),and 5-cyanoacenaphthylene (A). All compounds were assayed in the presence of Aroclor-induced postmitochondrial supernatant. Each point represents the average of two independent determinations
(1) Hoffman, D., Wynder, E. L., ACS Monogr., No. 173, 324-65 (1976). ( 2 ) Sawicki, E., McPherson, S.P., Stanley, T. W., Meeker, J., Elbert, W. C., Int. J . Air Water Pollut., 9,515 (1965). (31 DiDDle. A.. ACS Monom.. No. 173.245-314 (1976). (4) DGday; G: R., Hites, R . ’ A., Envhon. Sci. ‘Tecinol., 12, 965 (1978). (5) Friedman, L., Shechter, H., J. Org. Chem., 26,2522 (1961). (6) Skopek, T. R., Liber, H. L., Krolewski, J. J., Thilly, W. G., Proc. Natl. Acad. Sci. U.S.A., 75,410 (1978). (7) Skopek, T. R., Liber, H. L., Kaden, D. A., Thilly, W. G., Proc. Natl. Acad. Sci. U.S.A., 75,4465 (1978). (8) Lee, M. L., Hites, R. A., J . Am. Chem. SOC.,99,2008 (1977). (9) Hites, R. A., Dubay, G. R., in “Carcinogenesis”, Jones, P. W., Ed., Vol. 111, Raven Press, New York, 1978, pp 85-7. (10) Ingold, C. K., in “Structure and Mechanism in Organic Chemistry”, Cornel1University Press, Ithaca and London, 1969,pp 71-83, 280-1. (11) Chang, S. G., Novakov, T., Atmos. Environ., 9,494 (1975).
thene, and 5-cyanoacenaphthylene induced significant mutation (Figure 2) to 8-azaguanine resistance in S. typhi-
Received for review June 18,1979. Accepted August 20,1979. Work supported by the Department of Energy (Grant No. EE-77-S-024267).
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An Estimate of the NO, Removal Rate in an Urban Atmosphere Tai Yup Chang‘, Joseph M. Norbeck, and Bernard Weinstock Engineering and Research Staff, Research, Ford Motor Company, Dearborn, Mich. 481 21
A formulation for estimating the NO, removal rate by chemical reaction in an urban area is presented. Using the (NO,)/(CO) ratio from data obtained in the Los Angeles (LA) Basin, a lower bound for the yearly average removal rate of NO, during daylight hours is found to be 4% h-l. Using the (PAN HNO:J/(NO NO2 PAN “03) ratio from data obtained during the summer months a t West Covina (LA Hasin) and St. Louis by Spicer et al., the average NO, removal rate during the daytime is also found to be 4% h-l. From this analysis, the average residence time for NO, in the LA Basin (all year) and St. Louis (summer) is estimated to be less than 12 days.
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Nitrogen oxides (NO, E NO NOz) are involved in virtually every aspect of photochemical smog formation and tropospheric chemistry. Smog chamber studies and field monitoring reveal that the NO, removal rate depends primarily on 1534
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the degree of photochemical activity. A quantitative understanding of the NO, removal rate is of some importance since the longer the residence time the greater the impact urban NO, has on downwind areas. Despite recent studies, the NO, removal rate in an urban atmosphere is not well understood quantitatively ( I ) . The purpose of this note is to obtain an cstimate of the NO, removal rate in an urban atmosphere using available aerometric data. Tracer and Reactive Species The concentrations of the reactive species in the polluted atmosphere change with time as a result of chemical reaction, changes in pollutant sources, and dispersion/dilution. One tnay isolate somewhat the effects of chemical reaction and disy)ersion/dilution by using the concentration of some relatively inert compound as a normalizing factor. If the concentrations of a reactive species and an inert tracer in an air mass
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