Environ. Sci. Technol. 1904, 18, 338-348
Mutagenicity and Chemical Characterization of Organic Constituents in Rice Straw Smoke Particulate Matter Terry1 J. Mast,+ Dennis P. H. Hsleh, and James N. Seiber” Department of Environmental Toxicology, University of California, Davis, California 956 16
Whole extracts of rice straw smoke particulate matter (RSS-PM) collected with high volume air samplers from the field and from a specialized burning tower were active in the Salmonella/microsomal mutagenicity assay with bacterial strain TA98. All field samples required S9 activation and were comparable in magnitude to that of barley straw smoke PM. Samples from the burning tower were more active than those from the field and, in one case, contained direct-acting mutagens; these results may have been due to the higher temperature in the tower and environmental conditions at the tower site. Exposure of field RSS-PM to simulated sunlight led to a reduction in mutagenicity-a preliminary indication of the effect of weathering. Extracts of RSS-PM were fractionated by HPLC. GC-MS showed fraction I1 to contain alkylated phenanthrenes which are apparent contributors to the mutagenic activity. HPLC fraction I11 made the largest contribution to whole extract mutagenicity; primarily phenols and related compounds, which are not known mutagens, were identified in this fraction. Introduction Large quantities of agricultural wastes such as sugar cane, orchard prunings, and grain straws are typically disposed of by open burning. In California alone 2-3 million metric tons of rice, wheat, or barley straw are generated each year. Nearly all of the rice straw and approximately half of the wheat and barley straw are burned in the field. Because this burning often occurs seasonally during pruning or harvesting operations, localized deterioration of air quality may result for short periods of time. Rice straw burning, which accounts for 0.8% of the total particulate matter (PM) emissions for California and 4.1% of the PM emissions for the Sacramento Valley (I), may cause visibility reduction in the Sacramento Valley during the months of October and November and again in the early spring. Earlier studies, employing a burning tower at University of California, Riverside, yielded emission factors for rice straw smoke (particulate matter, -4 kg/ton (2);COz, 1045 kg/ton; CO, 36 kg/ton; low molecular weight hydrocarbons, 5 kg/ton (3, 4 ) ) . However, little work has been done to further characterize the organic constituents of the particulate matter. The long-term health effects to populations exposed to smoke PM from agricultural burning are unknown but are of concern since smoke PM from other wood and vegetative sources has been shown to contain polynuclear aromatic hydrocarbons and other biologically active compounds. Examples include wood stove smoke PM (51, forest fire smoke (6), and, more recently, cereal straw smoke PM sampled from an incinerator (7). The organic extracts of the PM from these sources also contain either direct- or indirect-acting mutagens as determined by the Salmonella/microsomal mutagenicity test (6, 8). ‘Present address: California Primate Research Center, University of California, Davis, CA 95616. 338
Environ. Sci. Technol., Vol. 18, No. 5, 1984
Our own preliminary studies have shown that rice straw smoke PM (RSS-PM) collected in the field and from the UCR burning tower is active in the mutagenicity test. We also investigated methods for fractionating and chemically characterizing the extractable organic matter of the RSSP M (9); a method based upon high-performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS) of the fractions was most successful (10).
Little is known concerning the environmental fate of smoke PM in the atmosphere; however, this is an important consideration as the bulk of the populations exposed to smoke from agricultural burning live some distance downwind from the sources. Part of the purpose of this study was to ascertain the short-term effects of weathering on the RSS-PM by collecting samples downwind of a burning field and by exposing filters containing field RSS-PM to simulated sunlight in the laboratory. Further goals of this study were to more completely develop the analytical methodology and chemically characterize the extractable organic matter of the RSS-PM, compare the mutagenic activity of extracts of field samples to those collected a t the specialized burn facility, and compare the mutagenic activity of RSS-PM to barley straw smoke PM and diesel soot. Experimental Section The general strategy employed in this study was as follows: Rice straw smoke particulate matter (RSS-PM) collected from an experimental burning tower and from the field on fiber filters was extracted and fractionated by HPLC into seven fractions, and the whole extracts and HPLC fractions were tested for mutagenic activity. HPLC fractions I-IV were characterized by cap-GC-FID and cap-GC-MS. The mutagenic activity of the whole RSS-PM extracts was compared to whole extracts of barley straw smoke PM and a diesel soot extract. To ascertain shortterm “weathering” effects on RSS-PM, the smoke was sampled 375 m downwind from a burning field, and three field sample filters were exposed to simulated sunlight prior to extraction, fractionation, and mutagenicity testing. Sampling. Rice straw smoke particulate matter (RSS-PM) was collected under two types of experimental conditions: (1)from within the stack of an experimental burning tower located a t the Statewide Air Pollution Control Research Facility, University of California, Riverside (UCR) (see ref 12 for a complete description) and (2) adjacent to, or directly downwind from, a burning rice field. Particulate matter from barley straw smoke was also collected in the field by using the same sampling equipment. Smoke particulate matter from the burning rice or barley straw was collected by using high volume (HiVol) air sampling techniques employing filtration through quartz or Teflon-coated fiber filters (QFF (2500QAO) or TFF (T60A20); Pallflex, Putnam, CT). As our primary concern was to trap only respirable particles, cyclone separators with a 50% ECD (effectivecutoff diameter) of 2.5 pm were placed in line prior to the filters. Only QFF were used in sampling the UCR burning tower, but TFF were used in
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smoke Partioutate h t t e r Sample Quartz or Teflon-coated fiber filter ( 2 0
1
2 5 Cm)
1) Extraction (sonifier horn) 250 mL 1:l benzene-methanol 2 ) Filter; concentrate to 30 mL
(rotary evaporator) 3 ) Filter (0.7 um millipore)
4) Centrifuge 5 ) Concentrate supernatant
under dry N2
Whole Extract of Smoke PM
CC-FID
Mutagenicity Assay
XPLC Semi-preparative CN column 1) Elute with hexane to acetone gradient 2)
collect seven fractions
3)
Concentrate fractions
Fractions GC-US
Mutagenicity Aesay
Figure 1. Analytical methodology scheme Used for smoke particulate matter.
addition to QFF for the collection of field samples. Field samples of smoke particulate matter were collected directly adjacent to the downwind side of back-fired fields of rice or barley straw. RSS-PM samples were obtained from field burns of overwintered rice straw in April 1982, and again in May 1983 in Yolo County, CA, by placing the samplers in the bed of a truck which operated along the south side of the field and was continually maneuvered into the thickest portion of the smoke. Sampler intakes were placed on the upwind side of the truck in order to minimize contamination from exhaust emissions and from dust stirred up by the vehicle. In an attempt to determine short-term effects of air and light on RSS-PM, a stationary sampler was operated simultaneously ca. 375 m downwind of the burning field. During the April 1982 run, wind was from the north at 8-16 km/h, the temperature was 18-21 "C, and visibility was ca. 160 km upwind of the field throughout the sampling period. The conditions for May 1983 were similar except that the wind was out of the south. Sampling times were kept at a minimum to reduce the potential for artifact formation on, and volatilization losses from, the filters: 30-60 min for the TFF, 60 rnin for the QFF, and ca. 2 h for the 375 m downwind sample (QFF). Burning barley straw fields of low moisture content, also located in Yolo County, were sampled in July 1982;winds were 0-5 mph from the south, the temperature was 29 "C, and visibility was ca. 160 km prior to the burn. The sampling time was 50 min. Because the smoke plume rose rapidly, no downwind sample was collected. All samples were stored in the dark at 5OC
a Present in both fractions I1 and 111. Not quantitated. Estimate. Percent recovery.
for the mixture was run on cap-GC-FID under the conditions described for RSS-PM and its HPLC fractions, except the temperature program rate was increased to 8 "C/min to decrease the analysis time while still achieving base-line separation for the individual compounds. Standard curves for each of the 17-compoundsquantified had linear correlation coefficients >0.96. Cap-GC-MSwas used to confirm peak identity and to screen for possible degradation products. Salmonella Mutagenicity Tests. Salmonella mutagenicity tests were carried out as described by Ames et al. (13) by using Salmonella typhimurium strain TA98. The S9 microsomal fraction was from the livers of Arochlor 1254 induced Sprague-Dawleyrats, either prepared by us or purchased from Litton Bionetics, Inc., Kensington, MD. Benzo[a]pyrene (BaP) was used as a positive control for indirect-acting mutagenicity (+S9), and 2-nitrofluorene (2NF) was used as a positive control for direct-acting mutagenicity (-S9). All samples were taken to dryness under N2 and redissolved in dimethyl sulfoxide prior to testing. Mutagenicity results are expressed as revlpg (revertants per microgram) of sample tested, calculated from the slope of an appropriate linear segment of the dose-response curve. Blank filters, GFF, QFF, and TFF, were extracted, concentrated, and tested in the same manner as the smoke PM extracts; no activity was found. The average activity for BaP in the presence of S9 for all tests was 191.3 f 71 rev/pg and, for 2NF in the absence of S9, was 133.2 i 51 rev/pLg. Light Exposure. As an indication of the effect of sunlight on RSS-PM collected in the field, two samples (T1 and T17), Teflon-coated filters, were placed (separately) in a closed, air-filled bell jar (30 cm diameter X 45 cm height) and exposed for 24 h to a xenon lamp (Varian, EIMAC Division, Walnut Creek, CA) placed just above the bell jar. A Pyrex plate covered the opening of the bell jar and supported a Pyrex concave lens used to disperse the light over the entire area of the filter. The lamp emitted a spectral distribution close to that of sunlight, and the 340
Environ. Sci. Technol., Vol. 18, No. 5, 1984
light intensits at the filter surface amroximated that of the summer noonday sun in the Sacramento Valley. Dark controls were achieved by placing replicate filters (T2 and T18), separately, in an identical chamber and protecting them from light by wrapping the chamber with aluminum foil. In order to eliminate possible variation between filters, one filter, field sample Q4, was cut in half; half (Q4L) was exposed to light, and the other half (Q4D) was used as a dark control. The ambient temperature was held constant at 28 "C. Extraction and HPLC fractionation, if done, were completed within 48 h of removal from the chamber.
Results and Discussion Sampling. The use of Teflon-coated fiber filters in addition to the quartz fiber filters for sample collection in the field was prompted by results which demonstrated that the degradation rate of benzo[a]pyrene and benz[alanthracene, applied directly to the filter surface, was slower on TFF than on QFF (15).However, Clark et al. (16) found no difference between diesel soot collected on TFF and QFF with respect to the percent of extractable organic matter (% EOM) or the mutagenic activity. Our results tend to support those of Clark et al. (16) in that there was no significant difference between QFF field samples Q6 and Q4D and TFF field sample T2 (replicates except for filter type) in terms of mutagenic activity of the whole extract and, probably, no significant difference in percent extractable organic matter (% EOM) (Table 11). Field samples T17 and T18, taken a year later and from a different field, gave values for % EOM comparable to those of Q6, T1, and T2. Whole Extracts. The extractable organic matter recovered from field RSS-PM by sonification in benzenemethanol (1:l) averaged 80 f 90% weight of the PM collected on the filters (Table 11). The downwind field samples, Q1 and Q2, were excluded from this average. The RSS-PM % EOM values are higher than those reported for wood burning stove PM (55% EOM (17))and forest fire smoke PM (53% EOM (6)). The higher percent weight recovery of EOM from RSS-PM does not appear to be due to extraction by sonification as a relatively high % EOM, 78, was also recovered by using Soxhlet extraction with benzene-methanol (18). The RSS-PM from the UCR burning tower had a lower percent EOM than samples collected from the field (58.5% EOM vs. 80% EOM), again excluding the downwind field samples from the average. This difference could result from variations in combustion and postcombustion conditions between the burning tower and the field. In the field the temperature decreases rapidly with height and, in fact, can reach ambient within as little as 1m above the ground for short rice stubble (2).Thus, volatilized, uncombusted and/or partially combusted plant constituents such as cuticle waxes might distribute more favorable to the smoke PM in the field. For the burning tower the temperature is as high as 280 "C near the apex of the cone, ca. 3.5 m above the straw, and may be near 150 'T at the sampler intake (4,12). These conditions should favor secondary or more complete combustion and distribution of organic chemicals to the vapor phase. The presence of relatively greater amounts of long-chain alkane/alkene hydrocarbons and fatty acid esters typical of plant waxes in field RSS-PM HPLC fractions I, 111, and IV than in the same HPLC fractions for tower samples supports this argument (see Recovery through HPLC Fractionation). Ramdahl and Becher (7)reported that alkyl esters dominated the moderately polar fraction of barley straw smoke PM emitted from incinerators. They also found that these
indirect mutagenicity (t S9)
direct mutagenicity ( 4 9 )
_ _ _ _ _ -
sample (date sampled)
sample, % wt, mg EOM
QFF (Q17) (July 21,1980)
312.2
QFF (Q3) (July 21, 1980)
202.0
QFF (Q6) (April 28, 1982) QFF ( Q l ) downwind (April 2 8 , 1 9 8 2 ) QFF (Q2) downwind (April 2 8 , 1 9 8 2 ) TFF (T2) (April 2 8 , 1 9 8 3 ) TFF (T2) (April 28,1983) TFF (T18) (June 8 , 1 9 8 3 ) QFF (Q4D) (April 2 8 , 1 9 8 3 )
TFF ( T l ) (April 2 8 , 1 9 8 3 ) TFF ( T l ) (April 2 8 , 1 9 8 3 ) TFF (T17) (June 8 , 1 9 8 3 ) QFF (Q4L) (April 2 8 , 1 9 8 3 )
rev/& of EOM
UCR Tower 59.3 3.70 (0.94) 58.0 3.39 (0.99)
Rice Field 0.62 77.0 (0.98) 0.53 113.9 43.0 (0.97) 0.43 95.7 60.0 (0.96) 0.86 170.0 86.1 (0.99) 1.96 replicate assay (0.95) 114.3 87.3 0.74 (0.90) 103.4 61.0 1.14 (0.99) 141.0
rev/pg of BaP 168.6 (0.99)b 234.0 (0.95) 127.3 (0.98) 127.3 (0.98) 212.0 (0.99) 173.7 (0.99) SO93 (0.99) 192.0 (0.98) 212.0 (0.99)
Rice Field: Light Exposed 173.7 180.0 85.6 0.40 (0.98) (0.99) replicate assay 0.86 309.8 (0.96) (0.99) 0.42 192.0 114.7 84.7 (0.92) (0.98) 0.82 212.0 103.4 62.0 (0.99) (0.99)
QFF (Q9) (July 7 , 1 9 8 2 )
120.0
QFF (Q10) (July 7 , 1 9 8 2 )
142.7
Barley Field 92.0 0.86 (0.99) 60.0 1.71 (0.94)
127.3 (0.98) 176.0 (0.98)
%
BaP a 2.20 1.45
rev/wg of EOM 0 0.51 (0.99)
0.49
0
0.42
0
0.20
0
0.50
0
0.63
0
0.39
0
0.54
0
0.23
0
0.28
0
0.21
0
0.39
0
0.68
0
0.97
0
rev/wg of2NF
%
2NFa
155.6
0
192.2 (0.99)
0.27
115.9 (0.98) 115.9 (0.98) 167.0 (0.98) 158.9 (0.99) 184.9 (0.99) 194.6 (0.98) 167.0 (0.98)
0
158.9 (0.99) 184.9 (0.99) 194.6 (0.98) 167.0 (0.98)
0
115.9 (0.98) 159.0 (0.99)
0 0
0 0 0 0
0 0
G
0 0
a % BaP = [revlpg of sample (+Sg)]/[rev/pgof BaP ( + S 9 ) ] X 100; % 2NF = [rev/pg of sample (-SS)]/[rev/pg of 2NF (--S9)] x 100. b Linear correlation coefficient for dose-response curve, 0-pg dose included.
compounds were absent in the same fraction for wood smoke P M from the incinerator, lending support to the belief that compounds of this type originate from volatilization of plant leaf and stem waxes. The lower percent EOM obtained for the downwind field samples (Ql, 43% EOM; Q2,60% EOM) may be due to (1)oxidation and/or volatilization losses of compounds initially present on the P M and/or (2) collection of fine dust stirred up by the sampling vehicle. Similar results were reported for ryegrass smoke PM where polycylic organic matter (POM) levels were very low or below detectable limits for PM collected downwind from burning ryegrass fields in the Willamette Valley, OR (19). Mutagenic Activity: Whole Extracts. The mutagenic activities for the whole extracts of smoke PM sampled in this study (Table 11) are calculated on the basis of rev/pg of EOM. They were not converted to rev/m3 air sampled because to obtain an adequate amount of smoke P M on the field filters the samplers were continually maneuvered into the thickest portion of the smoke and thus are not representative of average air concentrations. In addition to rev/pg of EOM, the results are also reported as % BaP control for indirect-acting mutagenicity (+S9)and as % 2NF for directiacting mutagenicity (-S9). This calculation permits correction of the results for day to day variations in the sensitivity of the assay that may arise primarily from the use of different batches of S9 microsomal fractions and/or slight differences in bacterial
concentrations and growth phase. Mutagenic activities in terms of % BaP or % 2NF were calculated as follows: rev/pg of sample
x 100 rev/pg of BaP rev/pg of sample % 2NF = x 100 rev/pg of 2NF % BaP =
,. \
(1)
For example, the specific mutagenic activities for replicated assays of a RSS-PM field sample (Tl, Table 11) run on 7/21 and 7/27 were 0.40 and 0.86 rev/pg, respectively. These results are more comparable when expressed as the percent of BaP control, 0.23 and 0.28 respectively. The increased consistency of data when using the % BaP or % 2NF control activity as an internal standard has proven reproducible in our laboratory when assays involving comparisons of samples could not be run on the same day. Whole extracts of RSS-PM from the UCR burning tower contained more mutagenic activity on a weight EOM basis than did RSS-PM samples from the field burns. The average of two tower samples (Q3 and Q17, Table 11)was 1.83% BaP as compared to an average of 0.48 f 0.05% BaP for four field samples (Q6, Q4D, T2, and T18, Table 11). Differing conditions between the tower and the field, especially the higher temperature in the former, may result in the formation of greater levels of PAH (see HPLC fractionation results). Envlron. Scl. Technol., Vol. 18, No. 5, 1984
341
+s 9 I
sample RSS-PM UCR tower (average) RSS-PM field (average) RSS-PM field downwind RSS-PM field light exposed barley straw smoke PM ambient airborne PM Oslo, heavy traffic Oslo, industrial Lexington, KY Los Angeles Basin forest fire smoke PM 100 mi downwind, Lexington, KY woodstove smoke PMe pine oak oil furnace 1e oil furnace 2 e diesel soot a diesel sootg spark ignition engine
rev/mg of PM 2100 64 2 242 465 822
%
BaP
-s9 rev/mg of PM
1.07 0.50 0.31 0.28 0.83
29 6 0 0
10-60 30-500 14.ad 110-610
b b b b b
1300 936 1904 5143 29 1 182-286 400-1200
b b b b b b b
sad
%
2NF
ref
0.15 0
a
a
0 0 0
a
10-105' 30-350 ' 45 58-360 151
b b b b b
22 22 6 24 6
indirect mutagenicity for most samples; overall activity was highest for cold, clear winter days. When these values are considered on a rev/pg of EOM basis, they are more comparable, 0.46 rev/pg for smoky air (53% EOM) vs. 0.33 rev/pg for ambient air (5% EOM). e rev/rng of EOM, % EOM not given. 0.55 rev/pg of EOM x 0.62 x l o 3 pg of EOM/mg of PM = 341 rev/mg of PM. g Values for CH,Cl, extract, May/ June sampling, Alleghany Tunnel (26% EOM). Values for CH,Cl, extract, May/June sampling, Alleghany Tunnel (40% EOM).
Direct-acting mutagenic activity was found in one of the RSS-PM UCR samples shown in Table 11, as well as in other UCR samples tested by us, but not reported in Table 11, and in the diesel soot extract (Table 111). In the case of the UCR samples the level of direct-acting mutagenicity-a small fraction of the indirect activitymay have been an artifact of the environmetal conditions during the run since no direct-acting mutagenicity was detected in any of the field samples. The atmospheric conditions which existed during the UCR burning tower experiments-a stage 2 smog alert with an oxidant level >0.2 ppm and an ambient temperature of 40 OC-may have affected the chemical nature of the RSS-PM leading to formation of direct-acting mutagens on the filter itself. Such a phenomenon was reported by Pitts et al. (20) when benzo[a]pyreneand pyrene deposited on GFF and exposed to gaseous pollutants present in photochemical smog formed direct-acting mutagenic derivatives. The mutagenic activity (rev/pg) of three RSS-PM field samples which had been exposed to simulated sunlight for 24 h was reduced to an average of 58% of the activity found for the corresponding dark controls (Table 11). For the sample set T2 and T1, the decrease was from an average of 0.57% BaP (T2)to an average of 0.26% BaP (Tl). Field samples taken a year later from a different field, T18 and T17, exhibited nearly the same percent reduction in mutagenic activity of the whole extract upon exposure to light (from 0.39 to 0.21% BaP). This apparent decrease in activity upon exposure to sunlight may be significant in terms of human health effects because the bulk of urban populations are exposed to smoke weathered over a distance of 50 miles or more during which time very pronounced changes could take place in chemical composition and mutagenicity. Further support was provided by the downwind RSS-PM filters which contained less activity than samples adjacent to the burning field; however, the possible dust contamination of the downwind sample mentioned above provides an alternate explanation. The mutagenicity of RSS-PM was compared with that of barley straw smoke PM (BSS-PM) and with literature 342
Environ. Sci. Technol., Vol. 18, No. 5, 1984
values for other combustion-generatedPM (Tables I1 and 111). For barley straw smoke mutagenic activity averaged 0.83% BaP control, higher than that found for the RSSPM field samples (Table 11). However, as only one field was sampled, no conclusions should be drawn regarding the relative mutagenicity of the PM from these two sources. Comparisons of the mutagenic activity of RSS-PM with other smoke PM are shown in Table 111. Since much of the literature data is presented on a rev/mg of PM basis, sample averages for RSS-PM samples were recalculated in this manner to facilitate Table I11 comparisons. The indirect (+S9) activity for the RSS-PM field samples (910 rev/mg of PM) is comparable to that obtained for wood smoke (ca. 1000 rev/mg of PM (21)), but somewhat higher than downwind forest fire smoke PM (238 rev/mg of PM (6)). Direct-acting mutagenicity levels comparable to that of the UCR tower sample have been found in other combustion P M samples (6, 16,21,22), as well as in ambient urban air PM (23,24). It was not possible to make these comparisons on a % BaP or 70 2NF control basis as the dose-response curves for the control compounds were not stated in the references. Due to this factor and the normal interlaboratory variation in the Salmonella/microsomal test sensitivity, comparisons of data with those reported in the literature are qualitative at best. Recovery through HPLC Fractionation. The HPLC procedure used to fractionate the RSS-PM EOM, described in detail elsewhere (IO), was characterized more fully by fractionating a mixture of 24 standards to ascertain their elution volumes and percent recoveries (Table I). Nineteen of the 24 compounds fractionated on the semipreparative CN HPLC column were recovered with efficiencies >50%. For the 17 compounds that were accurately quantified the average recovery was 80 f 15%. The lack of quantitative recovery values for 1,2:5,6-dibenzanthracene (fraction 11), P-phenylcinnamaldehyde (fraction IV), (fraction 111), 5-amino-2-methoxypyridine 1-methyl-2-pyridone (fraction VII), and 2-hydroxy-6methylpyridine (fraction VII) was due to inefficient GC
_ 1 _ _ _ _ _ _ _ _ _ ~ 1 _ _ _ _ _ _ _ _ _ _ 1 _ _ 1 _ 1 _ _ _ _
Table IV. Indirect (+ S9) Mutagenicity of Rice Straw Smoke Particulate Matter (RSS-PM) HPLC Fractions RSS-PM field (T17) light exposed RSS-PM field (T18) RSS-PM UCR (Q17) sample % sample % sample % fraction wt, mg rev/gg BaPa wt, wig rev/pg BaPb wt, mg rev/pg BaP b
I _ -
-l____l-_____ I _ _ _ _ _ I
whole
185.00
I I1
4.20 44.80
I11
53.90
IV
24.80
V
37.30
VI
4.40
VI1
5.90
3.70 (0.94) 0 .oo 2.20 (0.99) 5.08 (0.99) 4.74 (0.99) 1.48 (0.99) 7.20 (0.97) 5.77 (0.99)
2.20
62.40
0.00 1.31
3.55 1.05
3.02
10.55
2.82
16.35
0.88
19.80
4.28
1.oo
3.44
1.00
of these compounds, which led to broad and tailing peaks unsuitable for quantitation. Visual estimation indicated >50% recovery for the latter two compounds. Two compounds, dihydroxyphenanthrene and 5methyl-1,lO-phenanthroline, did not elute at all from the fused silica DB-5 capillary column employed in the GCdid elute from the MS. 5-Methyl-1,lO-phenanthroline 30-m SE-54 column in the GC-FID when a standard solution was first prepared, but chromatograms obtained from the same solution 1 week later showed no peak for this compound. Therefore, it was not possible to determine the recovery of these two compounds from the HPLC. The five PAH standards and the three aromatic sulfur heterocycles eluted in fraction 11. Fraction I1 also contained anisole, 2-methylnaphth[l,2-d]oxazole,and thioxanthen-9-one;the latter two were also recovered partially in fraction 111. Fraction I11 contained compounds of greater polarity than those present in fraction 11, a trend which continued for fractions IV, V, and VII. Fractions V and VI contained no compounds present in the initial recovery mix, but fraction V contained a significant amount of a compound having a mass spectrum identical with that of 2,3-dihydro-4H-l-benzothiopyran-4-one, a potential degradation product of thiochroman-Cone. In general, the elution volume appeared to be more strongly governed by polarity than by molecular weight, a situation which was advantageous for a separation of complex mixtures into compound classes. Recovery through HPLC fractionation and subsequent concentration of the eluent was acceptable for the majority of the compounds tested. Mutagenic and Chemical Characterization of HPLC Fractions of RSS-PM. Whole extracts of RSSPM collected from the UCR burning tower (Q17), burning fields (T2 and T18), and field samples exposed to simulated sunlight (T1 and T17) were fractionated by semipreparative HPLC and the fractions tested for mutagenic activity. The results for filters Q17, T18, and T17 are shown in Table IV; because of anomalously low mass recoveries through HPLC fractionation, the results for T1 and T2 are not included. All of the mutagenic activity found in the HPLC fractions was indirect, i.e., required S9 microsomal activation. An RSS-PM sample from the UCR burning tower, Q14, was fractionated (88% recovery of the EOM), and the fractions were characterized by cap-GC-MS; compounds tentatively identified in HPLC fractions 11-IV are presented in Tables VI-VIII.
0.74 (0.90) 0 -00 2.98 (0.90) 1.98 (0.99) 0.99 (0.94) 0.00 4.00 (0.95) 3.80 (0.98)
0.39
61 .OO
0.00 1.50
1.15 1.00
1.03
9.00
0.52
8.75
