4>EPA
United States
Environmental Protection
Agency
Environmental Sciences Research
Laboratory
Research Triangle Park NC 27711
EPA-600 3-79-032
April 1979
ch and Development
Mutagenic
Activity of
Aerosol Size
Fractions
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-79-032
April 1979
MUTAGENIC ACTIVITY OF AEROSOL SIZE FRACTIONS
by
Ronald Talcott and William Harger
College of Natural and Agricultural Sciences
University of California, Riverside
Riverside, California 92521
DA-8-1578J
Project Officer
Lester L. Spiller
Atmospheric Chemistry and Physics Division
Environmental Sciences Research Laboratory
Research Triangle Park, North Carolina 27711
ENVIRONMENTAL SCIENCES RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
-------
DISCLAIMER
This report has been reviewed by the Environmental Sciences Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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ABSTRACT
Several investigators in diverse geographical locations have detected
airborne mutagens with the Ames' Salmonella typhimurium bioassay. Yet to be
established is the aerodynamic diameter of the mutagen-containing particles.
To study this matter, an Andersen 2000 sampler was used to collect size-
fractionated samples in Durham, NC. The samples were extracted and concen-
trated, the masses were determined, and the mutagenic activities were bio-
assayed. Initial studies were designed to establish a protocol suitable for
routine air sampling. A sufficient sample quantity was obtained by operating
- x
four of the Andersen samplers simultaneously over a 5-7 day interval. The
results of 10 weekly samplings indicated that most of the deposited mass,
and most of the mutagenic activity, occurred in the fractions containing the
small (less than 2 ym) particles. Selected extracts, positive for mutageni-
city, were tested for the presence of electrophiles by reaction with nitro-
benzylpyridine (a chemical nucleophile) or by reaction with electrons
provided in the course of dc polarographic analysis. These chemical tests
verified the presence of direct-acting alkylating agents in extracts of
deposited aerosols.
This report was submitted in fulfillment of Contract No. DA-8-1578J by
the University of California, Riverside under the sponsorship of the U.S.
Environmental Protection Agency. This report covers a period from November
23, 1977 to November 29, 1978, and work was completed as of February 1,
1979.
iii
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CONTENTS
Abstract ..... ill
Figures vi
Tables vi
1. Introduction 1
2. Conclusions 2
3. Materials and Methods 3
4. Experimental Procedures 5
5. Discussion 12
References 14
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FIGURES
Number Page
1 Polarographic reduction of air pollution XAD fractions .... 8
2 Nitrobenzyl pyridine reactivity of air pollution extract ... 11
TABLES
1 Mass of Pollutants Deposited and Extracted 9
2 Mass Distribution of Organic Extracts (ug extract/m ) . . . . 9
3 Particle Size Distribution of TA98 Mutagenicity 10
4 Effect on S-9 on TA98 Mutagenicity 10
vi
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SECTION 1
INTRODUCTION
The discharge of combusted organic matter into the air has long been
a process of concern to environmental scientists and others involved in
pollution control. Chemical, physical and biological consequences associated
with the generation, aerosolization, and dispersion of waste matter through-
out the troposphere are subjects that concern scientists in many disciplines.
These challenging problems attract the attentions of diverse workers, leading
to interdisciplinary communication and new problem-solving approaches.
This report is the result of a collaborative effort between our labora-
tory and the Environmental Sciences Research Laboratory. We were charged
with the task of developing a method that could be used to investigate the
particle size distribution of airborne alkylating agents detected as mutagens.
Our strategy was to deposit the size-fractionated aerosol particles on
non-reactive surfaces (coated aluminum foil or Teflon-coated glass fiber
filters) where they could be readily extracted for bioassay.
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SECTION 2
CONCLUSIONS
The Ames1 Salmonella typhimurium bioassay was useful for detecting
airborne alkylating agents in size-fractionated aerosols, provided that
enough deposit was collected on each stage of the sampler. The Ames' TA98
strain was most useful since it was more sensitive than other test strains
to the activities present in the collected samples.
All weekly samplings at a site in Durham, N.C., yielded one or more
fractions that were positive in the Ames1 test. Consistently, fractions
containing particles <2 microns were positive. These fractions contained
about 72% of the total collected mass and 80% of the total mutagenic activity.
The alkylating agents detected as mutagens did not require metabolic
activation by liver enzymes. Samples also contained substances that reacted
positively in a colorimetric test for alkylating agents and appeared to be
reducible by polarographic titration.
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SECTION 3
MATERIALS AND METHODS
SAMPLING SITE
All fractions tested in this study were collected from a site located
on the roof of the Durham, EPA Test Site, Mangum and Cora Streets, Durham,
N.C. This building is located approximately 300 m north of the Durham
expressway and 250 m east of the American Tobacco Company plant.
SAMPLING PROTOCOL
Andersen 2000C samplers (1,2) were used to separate airborne particu-
late matter into five aerodynamic diameter fractions. Stages 1,2,3, and 4
collected particles from 7-11 ym, 4.7-7 ym, 3.3-4.7 pm, and 2.1-3.3 ym,
respectively. The backup filters collected smaller particles.
The initial months of the study were used to establish a sampling pro-
cedure to provide sufficient deposits for mutagenesis testing. Matters of
concern included the quality of air, mass distribution of pollutants, length
of the sampling period, and treatment of filters. From the initial data, we
concluded that approximately 20,000 M of filtered air would deposit enough
mass for the bioassay of each size category. This sample size was attained
by operating four Andersen 2000 samplers in parallel over a 5-7 day period.
Each sampler was loaded with four coated aluminum foil filters on stages
1-4 and a backup filter below stage 4. Samplers were operated at 20
3
ft /min over the sampling period. The filters were then carefully removed
and packaged for shipping to Riverside. Ten samples were collected and
tested between June 15 and November 3, 1978.
Three types of filters were used as backup filters: Gelman glass
fiber filters; Teflon-coated glass fiber filters; and Teflon supported by
polypropylene fibers. The choice of filter did not affect the bioassay
-------
results, but the teflon treated glass fiber filters tended to clog and
were discontinued on October 24.
Originally, the aluminum foil filters were coated with polypropylene
glycol, but this substance proved to be insufficiently adherent; some of the
coating was extracted, precluding the determination of pollutant mass. This
problem was alleviated by replacing polypropylene glycol with propylene
glycol as the coating beginning with the August 22 sampling.
SAMPLE PREPARATION
Aluminum foil filters from corresponding stages of the four samplers
were pooled. Areas of the filters bearing deposit were removed with
scissors and placed in Soxhlet extraction thimbles. Backup filters were
cut into small strips and placed in thimbles. Loaded thimbles were placed
in Soxhlet extraction tubes and extracted for six hours with 100 ml acetone
(Mallinckrodt AR grade). The extracts were then concentrated in vacuo and
their residues were transferred to preweighed vials, dried with nitrogen,
and weighed.
Stock solutions suitable for bioassay were prepared by adding sufficient
dimethyl sulfoxide (DMSO, Aldrich gold label) to make the final concentra-
tions 20,000 "cubic meter equivalents" (erne)/ml in the cases of the foil
extracts and 2,000 cme/ml in the case of the backup filter extracts (one
"cubic meter equivalent" is defined as the quantity of acetone-extractable
3
residue derived from one 1 m of samples air).
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SECTION 4
EXPERIMENTAL PROCEDURES
BIDASSAY
Ames' Salmonella typhimurium bioassay (3) was used to measure the
mutagenic activities in the extracts from foil and backup filters. In the
case of foil filters, sufficient extract was unavailable for testing in
several strains of bacteria. Consequently, initial screenings were executed
to determine the most responsive strains; which proved to be TA98, verifying
previous reports on air pollution mutagenicity testing (4-8).
Four doses of each extract are tested and ^he results are expressed as
net TA98 revertants/500 erne/plate. This mutagenicity test detects the
presence of direct-acting frameshift mutagens in the deposited aerosols in
the various size-fractionated samples.
Extracts obtained from the ten weekly samplings were bioassayed for
TA98 mutagenicity via an Ames1 soft agar test (3). The responsiveness of
the strain was checked with the positive control, benzo(a)pyrene. Each
sample was bioassayed within two weeks of collection. Foil filter extracts
were tested at 250, 500, 1000, and 2000 erne/plate by adding the appropriate
amount of extract (in 0.1 ml DMSO) to tubes containing 2.5 ml top agar and
the tester strain, TA98 (0.1 ml of fully grown culture). Top agar mixtures
were plated and incubated 48 hours prior to the counting of revertant
colonies. The background count of spontaneous revertants was measured in
each set of experiments: For each test plate, net revertants equalled gross
revertants minus spontaneous revertants. For comparison, the results are
expressed in units of net revertant counts/500 erne; 500 erne proved to be
the highest non-toxic dosage.
Backup filter extracts were similarly tested, except that the dosages
used were 25, 50, 100, and 200 erne/plate, and the bioassays were conducted
-------
in the presence and absence of liver S-9. Lower dosages were necessary to
avoid toxicity to the tester strain. The liver S-9 was included as a test
for metabolic requirements. The results, reported in net revertants/500
erne, were calculated by extrapolating from values obtained in the testable
dosage range (i.e., 25-200 cme/plate).
CHEMICAL TESTS
Preussmann et al. demonstrated that a wide variety of alkylating agents
can be detected with nitrobenzylpyridine as the nucleophile (10). The
nitrobenzylpyridine test for alkylating agents was adapted for use in testing
air samples. A backup filter extract from the October 17-24 sampling was
tested at several concentrations using the procedures described below.
The desired amount of sample in 1 ml acetone was mixed with 0.5 ml of
aqueous potassium acid phthalate (0.1 N) and 0.5 ml nitrobenzylpyridine
reagent (5% w/v in acetone); a boiling chip was added and the mixture was
held at 105-110°C for 45 min; allowed to cool to 40-50°C and diluted to
4.5 ml with 50% aqueous acetone. The chromophore was generated by additions
of potassium carbonate (1M, 0.5 ml) and was extracted into 5 ml of methylene
chloride. Absorbance was read from a spectrophotometer at 560 nm.
In another chemical test, an active fraction from the August 22 backup
filter extract was analyzed by differential pulse polarography. This
analytical technique has been used for the determination of metal ions and
electroactive organic molecules, including the nitrosamines. The use of
polarography in the determination of a substance involves either its reduc-
tion at the cathode or its oxidation at the anode. In a typical assay, an
increasing potential is applied to a mercury indicator electrode immersed
in a cell containing the species to be reduced. The current remains nearly
constant until the applied potential is sufficient to reduce the substituent,
at which point a sharp increase in current is sensed by the galvanomenter.
The current continues to increase with voltage until the flow of electrons
is limited by the diffusion of the reducible species to the mercury cathode.
The magnitude of the limiting current, the diffusion current, is proportional
to the concentration of the species.
A preliminary experiment was conducted to examine the feasibility of
polarography in analyzing air pollution extract fractions. XAD-Chromatography
6
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was employed to separate the extract into nonpolar (active) and polar
(inactive) fractions (See fig. 1). A Princeton Applied Research Corporation
Model 174 polarographic analyzer, connected to a Hewlett-Packard XY recorder
and a PARC Model 303 drop former was used to investigate the electroactivity
of the XAD fractions. Titrations were done in a cell containing 10 ml of
acetonitrile, 80 yg of the test substance, and tetrabutylammonium perchlorate
as the supporting electrolyte. The current was recorded over the voltage
range of -0.5V-1.0V.
SUMMARY OF METHODOLOGY
We have combined the technologies of aerosol fractionation and muta-
genicity testing to develop a method to determine the particle size distri-
bution of airborne alkylating agents. Our current procedure is summarized
below.
Four Andersen 2000 samplers are loaded with aluminum foil filters
on stages 1-4 to collect particles 2-11 urn in aerodynamic diameter. For
smaller particles, a backup filter (glat>s fiber or Teflon-supported by
polypropylene fibers) is inserted in each sampler below stage 4.
The four samplers are placed at the sampling site and operated at
20 cfm for 168 hours.
Stage 1 filters are pooled, areas of aerosol deposition are
excised, and total samples is extracted for 6 hours with acetone in
a Soxhlet apparatus. Stage 2,3, and 4 backup filters are similarly
processed.
Acetone extracts are evaporated to dryness under vacuum and the
residues are dissolved in DMSO to make stock solutions of 20,000 cme/ml
(stages 1-4) or 2000 cme/ml (backup filters).
The extracts are tested for mutagenicity with the Ames'
Salmonella typhimurium strain, TA98.
We have now completed the testing of 10 weekly samples collected from
the Durham, N.C., site. The calculations presented below suggest refine-
ments in our methods. These refinements are postulated in section 5.
Calculations
To arrive at an indication of air quality for each weekly sampling, the
quantity of matter deposited on backup filters was divided by the volume of
-------
0.04
10.02
NONPOLAR
FRACTION
(MUTAGENIC)
Figure 1. Polarographic reduction of air pollution XAD fractions.
-------
air sampled. This value averaged 28.4 yg/m and, on the average, 45% of
this mass was extracted by acetone (Table 1).
TABLE 1. MASS OF POLLUTANTS DEPOSITED AND EXTRACTED
Week
6-15
6-28
7-11
8-1
8-22
9-13
9-26
10-5
10-17
10-27
3
yg deposit/m
26
31
21
12
38
29
24
24
45
34
y = 28.4±8.8
3
yg extract/m
9.4
8.1
9.4
9.2
14.5
12.2
12.0
11.2
17.6
15.9
11.9±0.9
yg extract/
yg deposit
.36
.26
.45
.83
.38
.42
.51
.47
.39
.47
0.4510.045
The assessment of airborne particulate mass distribution commenced
with the August 22-29 samples. Results (Table 2) indicate that approximately
72% of the total extractable mass is derived from particles collected on the
backup filters (<2 microns). The distribution of mutagenic activity appeared
to parallel the distribution of mass, as most of the activity was detected
in extracts from the smallest-sized particles (Table 3). The correlation
coefficient (r) for mass distribution versus activity distribution was 0.76.
TABLE 2. MASS DISTRIBUTION OF ORGANIC EXTRACTS (yg extract/m3)
Stage
Week
8-22
9-13
9-26
10-5
10-17
10-27
I
.6
1.1
3.0
0.2
1.1
0.7
y = 1.1210.36
II
0.45
0.75
3.28
0.41
1.2
0.77
1.1410.39
III
0.5
0.8
3.2
0.25
0.46
0.49
0.9510.41
IV
1.5
6.6
2.26
0.06
1.6
0.81
2.1310.86
Backup
14.5
12.2
12.0
11.2
17.6
15.9
13.910.94
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TABLE 3. PARTICLE SIZE DISTRIBUTION OF TA98 MUTAGENICITY
TA98 revertants/500 erne/plate
Week
6-15/6-22
6-28/7-5
7-11/7-18
8-1/8-9
8-22/8-29
9-13/9-19
9-26/10-3
10-5/10-13
10-17/10-24
10-27/11-3
x =
1
(7-lly)
227
160
11
74
124
34
0
0
57
33
72123
2
(4.7-7y)
562
46
1
64
54
54
6
11
91
72
96±50
3
(3.3-4.7p)
59
0
0
14
13
60
4
25
0
59
23.4±8
4
(2.1-3.3y)
62
172
0
50
42
74
6
16
156
101
68±18
Backup
(<2y)
975
655
675
545
703
683
855
462
2677
2887
1111±267
The inclusion of liver S-9 in the bioassay did not enhance the
mutagenicity of the backup filter extracts, except in the case of the June
15-22 sample (Table 4). In most cases, liver S-9 inclusion slightly reduced
the mutagenic activities.
TABLE 4. EFFECT OF S-9 ON TA98 MUTAGENICITY
Week
6-15
6-28
7-11
8-1
8-22
9-13
9-26
10-5
10-17
10-27
TA98 revertants/500
liver S-9 excluded
975
655
675
545
703
683
855
462
2677
2887
11111267
erne/plate
liver S-9 included
3139
601
296
578
612
544
626
398
1937
1697
10421276
One of the extracts (10-17 10-24) was tested for the presence of
alkylating agents that react with 4-nitrobenzylpyridine. A positive
concentration-dependent response was obtained (Figure 2).
10
-------
0.4
0.3
CO
in
E
0.2
0.1
t
4
/
^
^
0
t
/
N02
CHROMOPHORE
N-R
10 15
DOSAGE RANGE, m3 equivalent
20
25
Figure 2. Nitrobenzyl pyridine reactivity of air pollution extract.
11
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SECTION 5
DISCUSSION
Several years ago Bruce Ames and his colleagues recognized the muta-
genicity of complex mixtures of organic matter such as cigarette smoke an4
air pollution extract. Since these initial observations were made, several
reports have been published regarding the mutagenicity of polluted air
collected on various localities in the United States and Europe (4-9). It
appears evident that the production of atmospheric mutagens is not an
atypical phenomenon, but rather a widespread occurrence. Although origins
of the active substances cannot be pinpointed, a striking correlation
between atmospheric lead and mutagenicity has been reported, along with
positive data on the mutagenicity of aufo exhaust (9). These data, of
course, do not rule out the possibility that stationary sources of pollution
also contributed to the atmospheric burden of mutagens.
The first objective of this report was to describe a method that may
prove useful for investigating whether or not mutagens occur on airborne
particles of respirable size. The method involves three phases: a sampling
phase; a sample preparation phase; and a bioassay phase. Sampling utilizes
a system designed to simulate the respiratory system in that larger particles
are deposited on upper stages and smaller particles penetrate to deeper
stages. Collected samples are then extracted with acetone which is removed
in vacuo and the residue is transferred and weighed. DMSO stock solutions
are prepared and bioassayed with the Ames1 test (strain TA98).
The second objective was to present data that have thus far been obtained
via the collection-and-testing system described above. Conclusions concerning
the health effects of air pollution mutagenicity are not to be drawn from
these data, although the results may provide incentive for further research.
12
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A refinement in the bioassay procedure can be made in view of the current
results that establish an effective dosage range (100-500 erne/plate for foil
filters, 25-200 erne/plate for backup filters). Samples should be tested at
several doses within this range and activity reported as the slope of the
best-fit line.
Another modification in the testing phase would be to routinely include
assays for chemical electrophiles. Nitrobenzvlpyridine assay and polaro-
graphic analysis may be the appropriate means. A chemical test for airborne
alkylating agents would be a valuable tool for monitoring air quality.
13
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REFERENCES
1. Andersen, A.A. New Sampler for the Collection, Sizing, and Enumeration
of Viable Airborne Particles, J. Bacteriology, 76:471-484, 1958.
2. Rihm, A. Evaluation of a Particle-Sizing Attachment for High-volume
Samplers. New York State Department of Environmental Conservation
Report No. BTS-3, December 1972.
3. Ames, B.N., J. McCann, and E. Yanasaki. Methods for Detecting Carcino-
gens and Mutagens with the Salmonella/mammalian Microsomes Mutagenicity
Test. Mutation Res. 31:347-364, 1975.
4. Talcott, R. and E. Wei. Airborne Mutagens Bioassayed in Salmonella
Typhimurium. J. National Cancer Institute, 58:449-451, 1977.
5. Tokiwa, H., K. Morita, H. Takeyoshi, T. Katsumi, and Y. Ohnishi.
Detection of Mutagenic Activity of Particulate Air Pollutants. Mutation
Research, 48:237-248, 1977.
6. Pitts, J.Nt, D. Grosjean, T.M. Mischke, V.F. Simmon, and D. Poole.
Mutagenic Activity of Airborne Particulate Organic Pollutants.
Toxicology Letters, 1:65-70, 1977.
7. Dehnen, W., N. Pitz, and R. Tomingas. The Mutagenicity of Airborne
Particulate Pollutants. Cancer Letters, 4:5*-12, 1977.
8. Commoner, B., P. Madyastha, A. Bronsdon, and'A.J. Vithayathel. Environ-
mental Mutagens in Urban Air Particulates. J. Toxicology and Environ-
mental Health. 4:59-77, 1978.
9. Wang, Y. Yi, S.M. Rappaport, R.T. Sawyer, R.E. Talcott, and E.T. Wei.
Direct-acting Mutagens in Automobile Exhaust. Cancer Letters, 5:39-47,
1978. ;
10. Preussmann, V.R., H. Schneider, and F. Epple. The Determination of
Alkylating Agents II. The Detection of Different Classes by Means of
a Modified Color Reaction with 4-(4-nitrobenzyl) pyridine (NBP).
Arzneimittel-Forshung, 19:1059-1073, 1969.
14
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-79-032
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
MUTAGENIC ACTIVITY OF AEROSOL SIZE FRACTIONS
5. REPORT DATE
April 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Ronald Talcott and William Harger
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
College of Natural and Agricultural Sciences
University of California, Riverside
Riverside, California 92521
10. PROGRAM ELEMENT NO.
1AA603A (AC-53) FY79
11. CONTRACT/GRANT NO.
DA-8-1578J
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Sciences Research Laboratory - RTF, NC
Office of Research and Development
U.S. Environmental Protection Agency
Research^ Triangle Park, North Carolina 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final 11/77 to 11/78
14. SPONSORING AGENCY CODE
EPA/600/09
15. SUPPLEMENTARY NOTES
16. ABSTRACT '•
Several investigators in diverse geographical locations have detected airborne
mutagens with the Ames' Salmonella typhimurium bioassay. Yet to be estiablished is
the aerodynamic diameter of the mutagen-containing particles. To study this matter,
an Andersen 2000 sampler was used to collect size-fractionated samples in Durham, NC.
The samples were extracted and concentrated, the masses were determined, and the
mutagenic activities were bioassayed. Initial studies were designed to establish a
protocol suitable for routine air sampling. A sufficient sample quantity was
obtained by operating four of the Andersen samplers simultaneously over a 5-7 day
interval. The results of 10 weekly samplings indicated that most of the deposited
mass, and most of the mutagenic activity, occurred in the fractions containing the
small (less than 2 urn) particles. Selected extracts, positive for mutagenicity,
were tested for the presence of electrophiles by reaction with nitrobenzylpyridine
(a chemical nucleophile) or by reaction with electrons provided in the course of dc
polarographic analysis. These chemical tests verified the presence of direct-
acting alkylating agents in extracts of deposited aerosols.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS |c. COS AT I Field/Group
*Air pollution
*Aerosols
*Mutagens
*Particle size
*Collecting methods
Chemical tests
Ames' tests
Andersen samplers
Durham, NC .
13B
07D
06E
14B
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)"
UNCLASSIFIED
21. NO. OF PAGES
21
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
15
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