PB83-233270
Evaluation of Motor Vehicle and Other Combustion
Emissions Using Short-Term Genetic Bioassays
(U.S.) Health Effects Research Lab.
Research Triangle Park, NC
Jul 83
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TECHNICAL REPORT DATA
(Please rrotl Instructions an the rcrrrsc be/ore rumplcnnxl
I. REPORT NO.
l:PA-(jOO/U-83-078
3. RI:CJ(yf NT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Evaluation of Motor Vehicle and Other Cuubustion
Emissions Using Short-term Genetic Bioassays
5. REPORT DATE
1933
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Joellen Lewtas
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Office of Research & Development
Health Effects Research Laboratory
US Environmental Protection Agency
Research Triangle Park, NC 27711
12. SPONSORING AGENCY NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
A9XA1C
1 1. CONTRACT/GRANT NO.
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Short-term genetic bioassays have been useful in evaluating uregulated organic com-
bustion emissions from motor vehicles. Identification of inutagens and carcinogens
in complex exhaust emissions has been greatly facilitated by the use of bioassay-
directed fractionation and characterization methods. It has also been possible to .
evaluate the effect of fuels, engine types, and control technologies on the rates of
mutagenic emissions from motor vehicles. Greater differences in the rate of muta-
genic emissions have been observed between different engines (e.q.,diesel vs. gaso-
.line) and control technologies (e.g., with and without catalyst) than between dif-
ferent fuels. A comparative evaluation of various combustion sources indicates that
motor-vehicle emissions make a major contribution to the mutagenicity observed in
air.
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Release to I'ublix
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Unclassified
21. NO. OF PAGES
_
20. S tCURITY~inv
22. PRICE
EPA Form 2220-1 (Rov. 4-77) pnr.vious EDITION is OBSOLETE
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PB83-233270
EPA-600/D-83-078
July 1983
EVALUATION OF MOTOR VEHICLE AND OTHER COMBUSTION. EMISSIONS
USING SHORT-TERM .GENETIC BICASSAYS
by
Joel!en Lewtas
Health Effects Research Laboratory
U.S. Environmental Protection Aqency
Research Triangle Park, NC 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE; PARK, NC 27711
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NOTICE
This "document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
11
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EVALUATION OF MOTOR VEHICLE AND OTHER COMBUSTION EMISSIONS USING
SHORT-TERM GENETIC BIOASSAYS .
Joellen Lewtas
Genetic Bioassay Branch, Genetic Toxicology Division,
Health Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
U.S.A. i
ABSTRACT
• - Short-term genetic bioassays have been useful in evaluating
unregulated organic combustion emissions froa motor, vehicles.
Identification of mutagens and carcinogans in complex exhaust
emissions has been greatly facilitated by the use of bioassay-
directed fractionation and characterization methods. It has also
been possible to evaluate the effect of fuels, engine types, and
control technologies on the rates of mutager.ic emissions from
motor vehicles. Greater differences in the rate of rautagenic
emissions have been observed between different engines (e.j.,
diesel vs. gasoline) and control technologies (2.9., with and
without catalyst) than between different fuels. A comparative
evaluation of '/arious combustion sources indicates that motor-
vehicle emissions make a major contribution to the mutagenicity
observed in ambient air.
INTRODUCTION
Combustion emissions from both motor vehicles and stationary
sources contain a complex mixture of organics. Chemical
characterization of these organics shows that they contain
carcinogenic polycyclic aromatic hydrocarbons (PAH), such as
benzo(a)pyrene. Recently, chemical characterization studies of
motor-vehicle emissions have identified the presence of
methylated PAHs (e.g., methylphenanthrenes) (1), nitrated PAHs
(e.g., nitropyrene) (2), oxidized PAHs (e.g., 4 oxapyrene-5-one)
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(3), and a variety of other polycyclic organic compounds not yet
evaluated in animal cancer bioassays.
The development of . nort-term genetic bioassays has provided
relatively simple/ sensitive, and rapid bioassays for mutagenic
and potential carcinogenic activity. Short-term genetic
bioassays have been particularly useful in evaluating organic
combustion emissions. This paper summarizes the results of
studies where short-term genetic bioassays have been used in the
following areas:
(1) identification of mutagens and carcinogens in complex
exhaust emissions,
(2) evaluation of the effect of fuels, engine types, and
control technologies on the mutagenic activity of the emissions,
rnd
(3) comparative assessment of mutagenicity and
carcinogenicity of various combustion sources and their
contributions to the mutagenic activity of ambient air.
IDENTIFICATION OF MUTAGENS AND CARCINOGENS IN COMPLEX EXHAUST
EMISSIONS
Bioassay-directed fractionation and characterization closely
coupled to chemical characterization has been shown to be the
most efficient and effective approach to identifying the specific
chemical compounds in a complex mixture that exhibit a particular
biological activity (4). This approach has been used to identify
tumor initiators and tumor promoters in cigarette-smoke
condensates (5), automotive exhaust emissions (6), and urban-air
particles (7). More recently, this approach has been coupled
with short-term genetic bioassays, including both microbial and
mammalian-cell mutation assays, to identify mutagens and
potential carcinogens in complex mixtures (8). We first employed
this method to identify the chemical classes and specific
components associated with diesel particulate emissions that were
mutagenic in the Ames Salmonella typhimurium mutagenosis
assay (9).
Diesel particles collected by the dilution-tunnel method
(10) were Soxhlet-extracted with dichloromethane and solvent-
partitioned into organic acids, bases, and neutral components.
The neutral components were further fractionated into paraffins
(hexane), aromatics (1% ether/hexane), transitional compounds
(1% ether/hexane, yellow fluorescence), and oxygenated compounds
(50% acetone/methanol). The mutagenic activity of each fraction
was determined using the Ames Salmonella typhimurium/microsome
assay in TA1535, TA1537, TA1538, TA98, and TA100 (9). The
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distributions of the mass of each fraction and of its mutagenic
activity in TA98 are shown in Figure 1 and Table 1 for the
four-stroke V-8 Caterpillar 3208 engine used in urban service
vehicles. The moderately and highly polar neutral compounds in
the transitional (TRN) and oxygenated (OXY) fractions account for
89-94% of the mutagenic activity of the extractable organics and
only 32% of the mass. Conventional gas chromatography/mass
spectroscopy identified many nonmutagenic fluorenones and
methylated fluorenones as major constituents of these fractions.
None of these or other identified constituents account for the
direct-acting frameshift mutagenic activity observed. Mutagenic
Diesel
Particles
Extractable
Organics
J
Particles
24.3% Extractable Mass
Solvent
Partitioning
Organic
Acids
Organic
Bases
%Mass 14.9
% Mutagenicity
• MA 4.9
* MA 9.5
0.29
0.02
0.10
JL
Neutrals
Ether
Insoluble*
3.9
0.36
0.80
Silica Gel
Chromatography
1
1 Paraffins 1
% Mass 36.7
% Mutagenicity
• MA 0
+MA 0
1
Aromatics
6.9
0.60
C.54
!
Transitional 1
5.0
64.9
33.5
I
1 Oxygenated
26.9
29.2
55.4
Figure 1. Distribution from diesel particles of
mass and outagenic activity in Salmonella
typhimurium TA98.
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Table 1. Distribution of the mass and mutagenic activity of fractionated diesel particle organicsa
Mass
Fraction (%)
Organic acids 14.9
Organic bases 0.3
Ether insolubles 3.9
Paraffins 36.7
Aromatics 6.9
Transitionals 5.0
Oxyyenates 26.9
Mutagenic Activityb
(rev/mg)
-MA
193
43.8
53.9
Neg.
49.5
7520
629
+MA
248
132
80.9
Neg.
30.1
2620
798
Weighted
Mutagenic Activity0
(rev/mg)
-MA
28.8
0.13
2.1
0.0
3.42
376
169
+MA
37.0
0.40
3.2
0.0
2.1
131
215
Distribution of
Mutagenic Activity
-MA
4.9
0.02
0.36
0.0
0.60
64.9
29.2
+MA
9.5
0.10
0.80
0.0
0.54
33.5
55.4
a-MA = without metabolic activation; +MA = with metabolic activation.
Aslope determined from linear regression analysis of the initial portion of the dose-reponse curve.
Determined by multiplying the rautagenic activity by the percent mass.
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activity is significantly less in nitroreductase-deficient
strains of Salmonella typhimurium, suggesting that nitrated
polycyclic compounds are present (11). Nitrated polycyclic
aromatic hydrocarbons (N02-PAHs) are potent direct-acting
fraraeshift mutagens detected in xerographic toners (12).
Identification and quantification of a series of N02~PAHs in
diesel extracts allow us to estimate their contribution to the
mutagenic activity of diesel particulate emissions (13).
Particulate emissions from catalyst-equipped gasoline-engine
vehicles using unleaded fuel contain significantly less of these
N02-PAHs. The mutagenic activity of both leaded- and unleaded-
gasoline emissions is substantially increased with the addition
of an exogenous metabolic activation (MA) system, suggesting that
the classical PAHs may play a more important role than do
NOj-pAHs in the mutagenicity and carcinogenicity of gasoline
emissions (14). .
EVALUATION OF THE EFFECTS OF VARIOUS FUELS, ENGINES, AND CONTROL
TECHNOLOGIES
Short-term bioassays have proven useful in evaluating the
effects of various engines, fuels, and control technologies on
the mutagenicity of emissions. To draw meaningful conclusions
from such comparisons, however, Claxton and Kohan (15) studied
the normal variations in the.emissions and in bioassay results
for emissions due to sampling, preparation, and storage for one
engine under standard conditions. For this study, an Oldsmobile
350 diesel vehicle was run on repeated Highway Fuel Economy Test
(HWFET) cycles during one day and on separate days. The
coefficients of variation (CV) for these parameters, as shown in
Table 2, ranged from 0.07 to 0.11. A computerized statistical
method recently developed by Stead et al. (1C) for analysis of
dose-response data from the Ames Salmonella typ';imurium bioassay
greatly facilitated these contparisons. An example of this
Table 2. Coefficients of variation of assay parameters for
standard operation of one diesel vehicle
Range of
Parameters All Days (CV) Separate Days (CV)
Particle emission rate 0.07 0.02 ~ 0.05
% Organic extractables 0.09 j 0.07 - 0.08
I
Mutagenicity slope 0.11 j 0.01-0.11
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\
analysis is shown in Figures 2a and 2b. The average non-linear-
model slope, shown with 95% confidence limits, is then used in
the comparisons.
To compare the mutagenicity of particle emissions from
different sources or fuels, the percent of organics extractable
from the particles and the vehicle's particle emission rate must
also be included in the analysis. The final rate of mutagenic
emissions is determined from all three parameters (Table 3). The
significantly lower rate of mutaginic emissions from the
catalyst-equipped, gasoline-engine vehicle is due primarily to
its much lower particle emission rate (0.0033 g/km), compared
with the leaded gasoline and diesel emissions. The significantly
lower rate of mutagenic emissions from the GM bus (Table 3) than
from the diesel truck and car, however, is due to the
substantially lower mutagenic activity of the organics.
The mutagenic and carcinogenic activities of the extractable
organics from a series of diesel and gasoline particle emissions
have been compared in a battery of short-term bioassays (14).
The bioassays that provided the best quantitative and
reproducible dose-response data were (1) the Ames Salmonella
typhimurium mutagenesis assay, (2) the mouse lymphoma mutagenesis
assay, and (3) the Chinese hamster ovary-cell sister-chromatid-
exchange assay. The results of short-term genetic bioassays were
compared with those of a skin-tumorigenesis assay in SENCAR mice
(17). Within the diesel and gasoline vehicle emission samples
examined, a very high correlation was observed among the results
of these three genetic bio^.isays and also with the mouse skin
tumor initiation assay (14).
The influence of fuel on the mutagenicity of emissions was
initially examined for five fuels, including four No. 2 diesel
fuels and one No. 1 jet fuel, in the two light-duty diesel
vehicles (a Volkswagen Diesel Rabbit and a Mercedes 240D) (9;.
In the VW Rabbit, the emissions from the minimum-quality fuel,
with a higher aromatic and nitrogen content, were significantly
(approximately 5 times) more mutagenic per milligram particulate
emission than were the other fuels. These fuels did not differ
significantly in mutagenicity when they were burned in the
Mercedes (9).
Similar studies were recently conducted (Table 4) for five
fuels in the three heavy-duty vehicles:
(1) GM city bus with a Detroit DD8V-71 engine;
(2) Mack truck with a Mack ENDT 676 engine; and
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Table 3. Comparison of the rates of mutagsnic emissions from motor vehicles
Mutagenicity Extractable
of Organics Organics
Source (rev/yg)« (%)
Diesel fuel
Car (?1erced68)b
Truck (Mack)C
Bus (GM)C
Gasoline fuel
Non-catalyst (Ford Van)d
Catalyst (Mustang II)«
aSalmonella typhimurium TA98
12.0
2.3
0.1
32.0
3.5
with metabolic
8
11
17
19
43
activation;
Particle
Emission Rate
(g/km)
0.24
1.3
2.1
0.03
0.0033
non-linear-model
Hut a genie
Emission Rate
(rev/km)
240,000
320,000
37,000
180,000
5,000
slope analyzed
by the method of Stead et al. (16),
^Mercedes 300D, 1977 model, operated on the HWFET cycle using Ho. 2 diescl fuel obtained
from Union 76.
cHack ENDT 676 diesel engine in a dual-drive tandem-axle truck and GH bun with p Detroit
diasel DD8V-71 engine were operated with the same average Ho. 2 dle&el fuel (EM-239-r)
on the 1983 transient heavy-duty cycle.
°Vord Van, 1970, in-line 6-cylinder engine, operated with leaded gasoline (Premium A) on
the HWFET.
ei?ord Mustang 11-302, 1977, operated with unleaded gasoline on the HWFET.
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SAMPLE ID: MSER-81-0015 LAB: HSHC ACTIUftTIOH: -
STRAIN: TA98 DATE: 06/10^81 TECHHICIAH: ecu
DOSE UNITS PLATE COUNTS
MEAH S.D.
oo
.00 UGS 17 19
3.00* UGS 289 308 382
5.00 UGS 112 108 81
10.00 UGS 208 191 175
30.00 UGS 532 459 522
50.00 UGS 735 677 725
190.00 UGS 1220 1219 1191
300.00 UGS 1748 1769 1588
B<0> B(l> B(2> B(3)
ESTS. 17.513 2.9114 .9851 .09365 2000
TEST CHI -SQUARE DF P LOGL
POISSON 30.62 13 .8038 -92.5840
ADEQUACY 5.f6 3 .1294 -95.4141
TOXICITY 382.76 1 .0088 -286.7941
I1UTAGENICITY14924.67 2 .8000 -7557.7505
IflRfl
AVERAGE SLOPE (HONLIH. MODEL) = 16.880 * °"
95* CONF. LIMITS =< 14.056, 20.272)
AUEKAGE SLOPE (LINEAR REGR.) =• 5.316
95'x. COHF. LIMITS = < 4.255, 6.378)
fl '
v ^»*
2a. Example of a computerized statistical method for
from Ames Salmonella typhimurium without netabol
ORS
/
/
/
{
' 1 1 1 1
\ I
the anal
ic activa
18
299
108
191
584
712
1218
1699
1. EXP
/
/
/
T
\
III
to
ysis o
tion.
.0
.6
.3
.3
.3
.3
.8
.0
US
^
1
2
f «
9 1.
7 9.
3 «6.
3 16.
3 39.
3 31.
8 16.
0 103.
DOSE
_^_~I^^M^^V
^-
1 1 1
8
tose-re
41
71
86
58
5£
01
46
5S
,
I
3
Bf
1
1
I
18
>or
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SAMPLE ID: MSER-81-8813 LAB: NSNC
STRAIN: TA98 DATE: ee/ie/ei
DOSE UNITS PLATE COUNTS
.88 UGS 25 26 26
.58* UGS 539 509 526
5.88 UGS 81 76 65
18.09 UGS 124 111 126
38.08 UGS 448 483 469
58.80 UGS 697 657 716
180.88 UGS 1301 1326 1228
300.ee UGS 1792 1890 1999
ACTIUATIOH: +
TECHNICIAN: BCN
MEAN
25.67
524.67
74.88
120.33
464.00
690.00
1282.33
1893.67
S.D.
.58
15.04
8.19
8.14
21.93
30.12
55.41
183.55
B(8) B(l> B<2> B(3>
ESTS. 24.497 1.9236 1.2412 .08487 2009
TEST CHI -SQUARE DF P LOGL
POISsON 23.75 14 .0490 -90.7998
ADEQUACY 19.86 3 .0003 -100.3287
TOXICITY 616.38 1 .0088 -488.5193 -
HUTAGENICITY28855.64 2 .8000-18528.1465 ,Oflfl
1OOD ""-
AUERAGE SLOPE (NONLIH. MODEL) o 27.090
95* COHF. LIMITS = < 23.835, 38.798)
•»
AUERAGE SLOPE (LINEAR REGR.) * 6.196
95'x. CONF. LIMITS = ( 5.138, 7.254)
WORKING: 3 PARAMETER MODEL DID NOT CONVERGE
0_;
OBS
ft
4
' ' ' 'i
t EXP US
/
V
-
iii.
8 2
DOSE
" '
i i i i
e 3
Figure 2b. Example of a computerized statistical method for the analysis of dose-response data
from Ames Salmonella typhimurium with metabolic activation.
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Table 4. Effect of fuel quality on rate of mutagenic emissions
Fuel Characteristics Mutagenic Activitya/km x 10s
Fuel
Jet
Diesel fl
Diesel 92
Premium
Average
Minimum
% % Cetane
Aromatics Nitrogen No. • Ford/Cftt Mack Truck
2.7 0.025 56.0
10.5 0.006 49.0
16.9 0.046 52.1 7.3 4.2
21.3 0.040 48.0 6.8 5.6
35.8 0.61 42.0 14.0 3.7
GM Bus
0.54
0.88
0.22
0.79
1.1
aSalmonella typhimurium TA98 without metabolic activation.
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(3) Ford Bob Tail Van (LN 7000) with a Caterpillar 3208
engine.
The five fuels ranged in aromatic content from 2.7% to 35.8% and
in nitrogen content from 0.006% to 0.61%. The minimum-quality
fuel produced a higher rate of mutagenic emissions in the Ford/
Cat Van and the GM bus than did the higher-quality fuels. This
difference was not observed for the Mack Truck (Table 4). The
fuels with the lowest aromatic and nitrogen content, jet and
diesel No. 1, did not produce less mutagenic emissions in the ®1
bus than did the diesel No. 2 premium fuel. However, the
mutagenic activity (revertants per microgram) for the extractable
organics from the bus particle emissions was much lower than that
for the emissions from the other two vehicles.
These studies suggest that although poorer-quality fuel,
with relatively high aromatic and nitrogen content, can increase
the rate of mutagenic emissions in certain vehicles, such
differences are not observed in all vehicles. In general, the
changes in mutagenic activity of emissions as a function of fuel
quality are much smaller than the differances between different
types-of engines (e.g., diesel vs. gasoline) and control
technologies (catalyst-equipped vs. non-catalyst-equ _.>ped
vehicles).
COMPARATIVE ASSESSMENT OF VARIOUS COMBUSTION SOURCES
The extractable organics from diluted and cooled combustion
particle emissions from both stationary and mobile sources were
tested in a battery of mutagenesis and carcinogenesis bioassays
(Table 5). The mutagenic or carcinogenic activity per microgram
of extractable organics was generally within two orJers of
magnitude (102). The
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Table S. Comparative nutagenicity and carcinogeniclty of extractable organics
N>
Mouse Skin Tumor
Initiation*
(papilloraaa/
Source mouse/tag)
Mercedes
Nissan
Volkswagen Rabbit
Oldsraobile
Caterpillar
Gasoline catalyst
Mustang II
Caoollno non-catalyst
Chevrolet 366
Ford Van
0.37
0.59
0.24
0.31
Neg.
0.17
-
Mutation in L5178Y
Mouse Lytnphoma Cells
(TJC mutants/
10^ surviving
cells/ug/mli
-MA
0.03
4.2
0.9S
1.2
0.25
0.38
1.2
2.1
+MA
1.5
2.9
0.72
1.3
0.063
1,1
4.9
5.7
SCE in CHO Cellsb
(SCE/cell/ug/ml)
-MA
0.09
0.30
0.075
Neg.
0.011
0.076
0.72
0.62
+MA
0.16
0.071
0.030
0.017
Neg.
-
0.22
0.47
Ames
Salmonella
TA98C
(rev/ug)
-MA
10.0
11.0
3.8
2.2
0.38
1.6
2.9
17.0
+MA
12.0
13.0
3.0
1.5
0.31
3.5
6.2
32.0
(continued)
aBased on papilloma multiplicity data in SENCAR mice (17),
D-MA was 21.5 h exposure and +MA was a 2 h exposure.
cDetertalned .from simple linear regression analysis.
• In progress.
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Table 5, continued
House Skin Tumor
Source
Residential heaters
Oil
Wood
Coal
Utility power plants
Coal, conventional
Coal, FBC
Oil
Initiation*
(paplllotnas/
mouse/rag)
0.12
-
- .
'
-
Mutation in L5178Y
Mouse Lymphoma Cells
(TK mutants/
10 6 surviving
cells/ug/ml)
-MA +MA
1.2 2.6
- -
-
IP*1 IP
IP IP
SCB in
CHO Cellsb
(SCE/cell/ug/ml)
-MA
0.06
-
-
IP
IP
•KMA
0.04
. -
-
IP
IP
Ames
Salmonella
TA98C
(rev/ug)
-MA +MA
1.3 2.1
0.15 0.93
ipd IP
3.1 -
9.4 5.2
IP IP
aBased on papillotna imiltiplicity data in SENCAR mice (17).
b-MA was 21.5 h exposure and +MA was a 2 h exposure.
cDetermined from simple linear regression analysis.
» In progress.
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Table 6. Rate of mutagenic emissions from stationary sources
Source Fuel
Residential heaters
Woods tove Pine
Oak
Residential oil
furnace 81 No. 2 fuel
furnace 92 No. 2 fuel
Utility power
plants (coal)
Mutagenicity3
of Organica
(rev/pg)
1.3
0.9
oil 2.0
oil 5.1
3.1
Organic
Emission Rate
(mg/kg fuel) (ng/J)
8940 508.0
3096 187.0
21 0.5
70 1.5
0.01
Mutagenic Emission Rate
(rev/kg fuel) (rev x 10-3/J)
; i
12,000,000 660.0
2,800,000 163.0 j
40,000 1.0
360,000 7.6
i
0.03,1
aSalmonella typhimuriuai TA98 with metabolic activation.
-------
mutagenic activity observed from respirable'air particles
collected in urban and suburban street-level locations. The
relative contributions from diesel, leaded-gasoline, and
unleaded-gasoline vtM:les depends on their distribution at the
particular location.
REFERENCES
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pp. 591-954.
2. Schuetzle, D., Lee, F.S.-C., Prater, T.J., and Tejada, r.B.:
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T.S., Mejia, V., Schuler, J., Scorziell, G.M., and
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i
6. Grimmer, G., Naujack, K.-W., Dettborn, G. , Bruhe, H.,
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Sandhu, and L. Claxton, eds., Plenum Press, New York,
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Zweidinger, R., Tejada, S., Bumgarner, J., Duffield, F.,
Waters, M., Simmon, V.F., Hare, C., Rodriguez, C., and Snow,
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