Seasonal Variations in the Bacterial Mutagenicity of Airborne Particulate Organic Matter in New York City Joan M. Daisey," Theodore J. Kneip, Irene Hawryluk, and Frank Mukai New York University Medical Center, Institute
of
Environmental Medicine, 550 First Avenue, New York, New York 10016
Seasonal composites of three fractions of particulate organic matter collected from New York City air were tested for direct-acting (no microsomal activation) bacterial mutagenicity by using the Ames bioassay. Winter maxima in the activity per cubic meter of air were found for the polar fraction with Salmonella typhimurium TA-98 and TA-100. The nonpolar and moderately polar fractions exhibited fall-winter rather than distinct winter maxima. These results and a consideration of the principal sources of particulate organic matter indicate that fuel-oil combustion for space heating contributes approximately half of the observed activity per cubic meter in New York City in the winter. Space heating is probably a significant source of these biologically active materials in other northeastern U S . cities as well. Sources which do not exhibit strong seasonal patterns, such as automobiles, must also contribute to the observed activity as biologically active organic matter is present in the aerosol in all seasons. Introduction A number of investigators (1-4) have found particulate organic matter from urban areas to be mutagenic in the Ames bioassay ( 5 ) . Polar as well as nonpolar fractions contained direct-acting (Le., requiring no microsomal activation) mutagens ( 2 ) , indicating the presence of biologically active compounds other than benzo[a]pyrene or unsubstituted polycyclic aromatic hydrocarbons (6).As there is a high correlation between mutagenicity in the Ames test and carcinogenicity (6),these results indicate the presence of unidentified compounds which may pose a health hazard to urban dwellers. Wang and co-workers ( 7 ) have reported evidence that direct-acting mutagens originate from auto exhaust. Mutagenic materials in the aerosol are likely to originate from other combustion sources as well. There are seasonal variations in the composition of aerosols in the northeastern United States. During the winter months increased concentrations of total suspended particulate matter and vanadium are observed due to space heating (8-10). Greater sunlight intensity during the summer months leads to the production of secondary photochemical products, such as ozone, in the atmosphere. Concentrations of still other aerosol species, such as lead, which originates primarily from automotive sources in New York City, show little seasonal variation (10).Thus, variations in the atmospheric concentrations of aerosol species can be indicative of their sources. In view of this, we have investigated the seasonal patterns in the mutagenic activity of particulate organic matter (POM) in New York City. Materials and Methods Weekly samples of respirable (53.5 p m aerodynamic diameter) suspended particulate matter (RSP) were collected on preignited fiberglass filters in New York City from July 1977 to March 1979. The filterhead shielded the samples from all light during collection. A constant-flow sampler (8) was modified by the addition of an Aerotec-2 cyclone as a precollector for particles larger than 3.5 pm. The flow rate through the cyclone was maintained a t 0.45 m3/min in order to get a 3.5-ym "cut" ( 1 1 ) .The sampling station is located on the roof of a residence hall, -60 m above the street, a t the New York University Medical Center. 0013-936X/80/0914-1487$01.00/0
One-half of each RSP sample was sequentially extracted in a Soxhlet apparatus with increasingly polar solvents. Cyclohexane, dichloromethane, and finally acetone were used to extract nonpolar, moderately polar, and polar organic fractions, respectively. Eight-hour extractions were carried out with each of the three solvents, with the samples shielded from the light during the extractions. Extracts were filtered and then reduced to 10 mL in volume with a rotary evaporator equipped with a water bath held a t 40 "C. The samples were then stored in a freezer a t -25 "C. Aliquots of extracts, representing 1000 m3 of sampled air for each week, were composited on a quarterly basis. The months included in each of the seasonal composites were as follows: summer 1977-July, August, September; fall 1977-October, November, December; wintzr 1977-78December, January, February; spring 1978-March, April, May; summer 1978--June, July, August; fall 1978-September, October, November; winter 1978-79-December, January, February. The cyclohexane and dichloromethane composite extracts were evaporated just to dryness, under argon, on a slide warmer ( 3 5 4 0 "C) and redissolved in acetone for testing. Acetone extracts were reduced in volume. The concentrations of the composite samples were determined by weighing duplicate 100-pL aliquots, taken to dryness, on a Cahn Electrobalance. Concentrations of the test solutions were 1-5 mg/mL. The nonpolar (cyclohexane-soluble) fraction of POM is composed of aliphatic hydrocarbons, which constitute 40-60% of the total mass, polycyclic aromatic hydrocarbons (-5%), and other unidentified nonpolar compounds (12).Infrared spectra of the moderately polar and polar fractions indicate that these consist largely of oxidized hydrocarbons. The procedures described by Ames et al. ( 5 ) for bacterial mutagenicity testing were slightly modified. L-Histidine and biotin were incorporated in the bottom rather than the top agar. In brief, 0.1 mL of each sample was incorporated into 2.5 mL of molten soft agar with 0.1 mL of a suspension of the tester strain. This mixture was then poured on bottom agar consisting of 1.5%agar, minimal inorganic salts supplemented with 2% glucose, biotin (0.1 pmol/plate), and histidine (0.1 pmollplate). The number of revertant colonies was scored after 2-day incubation a t 37 "C. Spontaneous reversion for each tester strain was determined by mixing 0.1 mL of bacterial suspension with 2.5 mL of molten soft agar and 0.1 mL of acetone and pouring on bottom agar. Spontaneous revertant colonies on these control plates were -130 (TA-100) and 25 (TA-98). The tester strains were routinely checked for histidine requirements, and TA-100 and TA-98 were checked for the presence of ampicillin resistant R factor ( 5 ) .The presence of toxic effects was checked by routine examination of the background lawn of bacterial growth. If massive cell death has occurred, the background lawn on the test plates will be sparse compared to that on the control plates. In preliminary experiments, extracts were tested for direct-acting mutagens with Salmonella typhimurium strains TA-1537, TA-1535, TA-1538, TA-1975, TA-1978, TA-98, and TA-100. Strains TA-98 and TA-100 were found to be the most sensitive and were used for subsequent studies. Extracts of blank filters were inactive. The three organic particulate fractions were also tested with Arochlor 1254-induced rat liver
@ 1980 American Chemical Society
Volume 14, Number 12, December 1980
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Polar Fraction t
m
1
I
L
Moderately Polar Fraction
Jhm
c c
$ 0
rp’Aatt
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Figure 1. Typical dose-response curves for particulate organic fractions. Figure 1 shows the data for the winter, 1977-78, quarterly composites. Average values of duplicate determinationsare shown.
microsomal fraction (0.3 mL of S-9 mix/plate). Microsomal enzymes reduced the mutagenic activity of all three fractions at the dose tested. The activity of the polar fractions was reduced to the greatest extent (80-100%) while the activity of the nonpolar fractions was reduced 20 and 60%, respectively, with TA-98 and TA-100. In addition, the use of S-9 mix can increase the variability of the response (13).Consequently, the composite samples were tested only for direct-acting mutagens. Each composite extract was tested at four or five doses, in duplicate. All testing was done by the same person (I.H.) to minimize intersample variations. The activity of each extract sample was calculated as net revertant colonies per microgram of organic extract and per cubic meter of sampled air. Net revertants per microgram for a given sample were calculated by a least-squares analysis of the linear portion of the doseresponse curve including the 0,O point. Net revertant colonies per cubic meter of air were calculated as the product of net revertant colonies per microgram times micrograms of extract per cubic meter of air for the tested extract. The slopes of the dose (in m3)-response curves were tested for statistically significant seasonal differences by using a Student’s t test (14):
t = (bz - bl)/u
(1)
where bl and bp are the slopes of the curves and V is the square root of the estimated variance in bz - bl. Only seasons of interest, i.e., seasonal extremes (cf. figures), were tested. In some instances, fall and winter seasons were pooled. All summer vs. winter or fall-winter differences were significantly different at the p 50.05 level or less except for the moderately polar fraction, summer 1978 vs. fall-winter 1978-79 with TA-98. The variability of the mutagenicity testing was also considered independently of the statistical analysis. Such variation, however, would be random in nature. Although this variation is inherent in the data reported here, a strong seasonal pattern is still observed.
Results Typical dose-response curves for the three particulate organic fractions with S. typhimurium TA-98 and TA-100 are shown in Figure 1. The total revertant colonies per plate ranged from -100 to 800 at maximum dose, depending upon the fraction tested. Seasonal variations in the mutagenicity of the three particulate organic fractions, in terms of net revertant colonies per cubic meter of sampled air, are shown in Figures 2 and 3. Winter composites are indicated in the figures with a “W”. In general, all three fractions showed clear fall-winter maxima and spring-summer minima with both S. typhimurium TA-98 1488
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7/77 1/78 6/78 12/78 IOU7 3/78 9/70 3/79
Date Flgure 2. Seasonal variations in net revertant colonies of S. typhimurium TA-98 per cubic meter of air for three fractions of particulate organic
matter collected in New York City.
I2L
Polar Fraction
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Moderately Polar Fraction
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$ 0
t
t
20
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0L
7/77 1/70 6/78 12/78 10/77 3/78 9/18 3/79
Date Figure 3. Seasonal variations in net revertant colonies of S. typhimurium TA-100 per cubic meter of air for three fractions of particulate organic
matter collected in New York City. and TA-100. With the polar fraction, distinct winter maxima were observed. These differences were examined statistically by pairwise comparisons of the slopes of the dose (in m3)response curves (14).The summer vs. fall and/or winter differences were significant at the p 50.05 level, with the exception of the case of the moderately polar fraction for summer 1978 vs. fall/winter 1978-79 with TA-98. This exception is apparent in Figure 2. The activity per cubic meter of air was approximately twice as high for the falllwinter period as for the summer period for both the nonpolar and polar fractions. The difference between the seasons was not as great for the moderately polar fraction. Two factors contribute to the fall/winter maxima in activity per cubic meter observed here. First, the atmospheric con-
Polar Fraction
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Non-polar Fraction 7/77 1/77 6/70 12/70 10/77 3/70 9/70 3/79
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Date
Date Figure 4. Seasonal variations in net revertant colonies of S. typhimurium TA-98 per microgram of particulate organic matter. Atmospheric Concentrations of each fraction of particulate organic matter are shown
Figure 5. Seasonal variations in net revertant colonies of S. typbimurium TA-100 per microgram of particulate organic matter. Atmospheric concentrations of each fraction of particulate organic matter are shown
in the upper curve (right-handscale).
in the upper curve (right-handscale).
centrations of the particulate organic matter tended to reach a maximum during the fall through winter seasons, and a minimum during the summer, particularly for the nonpolar and polar fractions (Figures 4 and 5). Second, the net activity per microgram for the nonpolar and polar fractions was also slightly higher, in general, during winter than during summer, and thus contributed to the observed pattern of fall-winter maxima in activity per cubic meter. The moderately polar fraction showed very little seasonal variation in net activity per microgram with the exception of a summer, 1978, minimum with TA-100 (Figures 4 and 5). This fraction, as was observed in some earlier work in this laboratory ( 3 ) ,was the most active per microgram. However, since this fraction constitutes only 5-10% of the total mass of extractable organic matter, it is comparable to the other two fractions in terms of activity per cubic meter of air.
Fuel-oil consumption for power production and water heating contributes -10% of the total suspended particulate matter in New York City (15, 16) and shows very little seasonal variation (19). Automotive traffic has been estimated to contribute -20% of the T S P ( 1 5 ) .Although traffic may be slightly lower in the summer than in the winter, Kleinman (20) estimated that this results in approximately a 3% winter/summer difference in the automotive contribution to TSP. As the contribution of incineration to TSP is only a few percent of the total (15, 16), even large seasonal differences in emission, which are unlikely, would not result in any significant seasonal difference in the contribution of this source to TSP. The seasonal patterns in source emissions of particulate matter are reflected in the higher concentrations of particulate organic matter which were observed (Figures 4 and 5). Fall, rather than winter, maxima in atmospheric concentrations were observed due to the higher atmospheric dispersion in winter which results in a greater dilution of winter emissions
Discussion The principal anthropogenic sources of airborne particulate matter in New York City are fuel-oil combustion for power production, water heating and space heating, transportation, and incineration (15-1 7). There is no heavy industry in New York City. The single largest energy demand is space heating, which was estimated to be 60% of the total energy demand for 1970 (15).More than 70% of this demand was met by fuel-oil combustion. From approximately November through April, low-sulfur (0.3%)fuel oil is burned for commercial and residential space heating. As December through February are the coldest months of the year, fuel-oil consumption for space heating and emissions of particulate matter peak during these months. We have recently estimated (18)that space heating contributed -40% of the winter levels of total suspended particulate matter (TSP) in 1972-77. Our data (18)from two winter sampling periods in 1977 and 1978 also suggest that 50-70% of the particulate organic matter originates from this source. Other local sources of airborne particulate matter in New York City do not exhibit strong seasonal emission patterns.
(21).
Seasonal and intersite differences in the mutagenic activity of organic particulate fractions also reflect differences in sources. Particulate organic matter collected at rural sampling stations has been found to be less active in the Ames bioassay than that collected in urban areas ( 2 , 2 2 ) .This indicates that anthropogenic activities are the principal sources of organic materials responsible for the observed mutagenic activity. The fall-winter maxima and spring-summer minima in bacterial mutagenicity per cubic meter of air which were observed for New York City clearly indicate that fuel-oil combustion for space heating contributes significantly to the observed activity of particulate organic matter. Space heating appears to contribute to the activity per cubic meter as much as all other nonseasonal activities combined, since winter levels are approximately twice those of summer. Seasonal differences in atmospheric dispersion can influence the atmospheric concentrations of aerosol species. Given a constant emission source, greater atmospheric dispersion Volume 14, Number 12, December 1980 1489
(by 2-3 times) in winter in New York City, due to a higher mixing layer and higher wind speeds, would lead to a greater dilution of emissions in winter than in summer (21).Despite this, fall-winter maxima in the bacterial mutagenicity per cubic meter of air were observed. Although fall-winter maxima were observed in the activity per cubic meter of air for the three organic fractions, biologically active materials were present in the aerosol during all seasons. Thus, emissions from sources which do not show strong seasonal patterns must also contribute to the observed activity. Wang and co-workers (7) reported the presence of directacting mutagens in automobile exhaust and a correlation between TA-98 revertants per cubic meter and the lead content of particulate-matter samples collected in Buffalo, NY, from 1961 to 1963. On the basis of these results, they suggest that the direct-acting mutagens detected originated from automobile exhaust (7). Automobiles are a significant anthropogenic source of total suspended particulate matter in New York City. Since emissions from automotive and other transportation sources are fairly constant throughout the year, such sources are likely to contribute a significant proportion of the mutagenic activity observed throughout the year. The data presented in Figures 4 and 5 also suggest that the nonpolar and polar fractions collected in the winter are slightly more active than those collected in summer in terms of net revertants per unit mass. It may be that organic materials emitted by space-heating sources in winter are inherently more mutagenic. Alternatively, higher ambient concentrations of ozone and greater sunlight in summer could lead to a reduction in the activity of organics emitted during the summer months. There is insufficient experimental data available a t present to distinguish between these possibilities. Summary and Conclusions The mutagenic activity per cubic meter of air of three particulate organic fractions was generally greater during the fall and winter than during the summer in New York City. These results suggest that fuel-oil combustion is an important source of the direct-acting mutagenic materials assayed by the Ames test in New York City, contributing approximately half of the observed activity per cubic meter of air in winter. Sources which do not exhibit strong seasonal patterns, such as automobiles, also contribute to the observed activity, since biologically active materials were observed in the aerosol during summer as well as winter. Acknowledgment We thank Bruce Naumann for the collection and processing of the organic samples and Marie Ann Leyko and Susan Snow for preparation of the composite samples.
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(1) Talcott, R.; Wei, E. J . Natl. Cancer Inst.
j~
Received for review December 12, 1979. Accepted August 13, 1980. This work was supported by Grant No. RP-1,222-1 of the Electric Power Research Institute and by the American Petroleum Institute and is part of a center program supported by the National Institute of Environmental Health Sciences, Grant No. ES00260, a n d the National Cancer Institute, Grant No. CA 13343.
