, i United States
&~t?\ ~~ Environmental Protection
SEP 1 5 1981 Agency
EPA-600/9-80-057a
November 1980
Reseat ch and Development
Health Effects of
Diesel Engine
Emissions:
Proceedings of an
International
Symposium
Volume 1
Sponsored by:
Health Effects
Research Laboratory
Cincinnati OH 45268
EPA/600/9-80/057a
LIBRARY
LABORATORY
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EPA-600/9-80-057a
November 1980
HEALTH EFFECTS OF
DIESEL ENGINE EMISSIONS
Proceedings of an
International Symposium
December 3-5, 1979
Sponsored by the
Health Effects Research Laboratory
Edited By
W. E. Pepelko, R. M. Danner, N. A. Clarke
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. The views and policies pre-
sented by the individual authors do not necessarily reflect
those of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.
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FOREWORD
The U.S. Environmental Protection Agency was created because
of increasing public and government concern about the dangers
of pollution to the health and welfare of the American people.
Noxious air, foul water, and spoiled land are tragic testimony
to the deterioration of our national environment. The
complexity of that environment and the interplay between its
components require a concentrated and integrated attack on the
problem.
Research and development is that necessary first step in
problem solution and it involves defining the problem, mea-
suring its impact, and searching for solutions. The primary
mission of the Health Effects Research Laboratory in Cin-
cinnati is to provide a sound health effects data base in
support of the regulatory activities of the EPA. To this end,
HERL conducts a research program to identify, characterize,
and quantitate harmful effects of pollutants that may result
from exposure to chemical, physical, or biological agents
found in the environment. In addition to the valuable health
information generated by these activities, new research tech-
niques and methods are being developed that contribute to a
better understanding of human biochemical and physiological
functions, and how these functions are altered by low-level
insults.
Together with the problem posed by air pollution from automo-
tive emissions, the Environmental Protection Agency inherited
a program, designed to define and measure the impact of that
problem, initiated in 1961 within the Public Health Service's
Division of Air Pollution in Cincinnati, Ohio. Part of that
program remains with the Agency's Health Effects Research
Laboratory in Cincinnati. It is therefore appropriate that
the first International Symposium on the Health Effects of
Diesel Emissions should have been held in Cincinnati and that
it should have been sponsored by the Health Effects Research
Laboratory.
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This report of the Proceedings represents an attempt to
assemble current knowledge relevant to an assessment of the
potential impact upon human health and welfare of diesel-
powered light-duty vehicles. With a better understanding of
the health effects, appropriate control measures can be
introduced as necessary.
R. J. Garner
Director
Health Effects Research Laboratory
IV
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PREFACE
The Environmental Protection Agency, under the Clean Air Act,
is charged with the responsibility for regulating emissions
from new motor vehicles. It has been estimated that ten to 25
percent of new U.S. passenger cars could be diesel powered by
1985, since such engines offer about a 25 percent improvement
in fuel savings over comparable vehicles powered by gasoline
engines. The various gases in diesel exhaust are similar to
those emitted by gasoline engines; however, the particles in
diesel exhaust are quite different in composition and quantity
from those in gasoline engine exhaust - even a properly tuned
diesel engine will emit 30 to 100 times more particulate
matter than a comparable gasoline engine with a catalytic
converter. These diesel engine particulates are basically a
carbonaceous material with mainly high molecular weight or-
ganic chemicals adsorbed to them. Additionally these par-
ticulates, because of their small size, are respirable and are
known to penetrate deeply into the lungs. Because of these
facts, there is an obvious need for well designed studies on
the health effects of diesel emissions. These studies will,
hopefully, provide data useful in making risk assessments and
provide the regulators with appropriate criteria for estab-
lishment of scientifically based standards.
The purpose of this Symposium was to bring together scientists
and engineers from the public and private sectors to discuss
their research findings on the health effects of diesel engine
emissions and to conclude with a discussion of health risk
assessment of diesel exhaust.
The Proceedings are organized into eight main sections cor-
responding to the format of the Symposium and addressing
Physical and Chemical Characteristics of Diesel Emissions, In
Vitro Carcinogenic and Mutagenic Effects of Diesel Emissions
and Components, Biochemical and Metabolic Effects, Toxi-
cological Effects of Inhaled Diesel Emissions, Mutagenic and
Carcinogenic Potency of Extracts of Diesel and Related En-
vironmental Emissions, Mutagenicity of Inhaled Diesel Em-
issions, Carcinogenic Effects of Exposure to Diesel Emissions,
Epidemiological Studies, and lastly a panel discussion on
Health Risk Assessment of Diesel Emissions.
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The Proceeding papers, in some cases, are more comprehensive
than the original presentations in order to provide more
thorough coverage of the particular topic. Edited discussions
are included with each paper and wherever possible the
identity of each questioner is indicated. The list of reg-
istrants will enable the reader to contact a speaker for
further information.
Norman A. Clarke
William Pepelko
Robert Danner
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ABSTRACT
The Health Effects Research Laboratory of the U.S. Environ-
mental Protection Agency sponsored an International Sympo-
sium on the Health Effects of Diesel Engine Emissions. It
was held in Cincinnati, Ohio on December 3-5, 1979.
The Symposium brought together scientists, engineers, and
Federal, state, and local public health officials for the
purpose of determining the state-of-knowledge regarding the
physical and chemical characteristics of diesel emissions,
in vitro carcinogenic and mutagenic effects of diesel
emissions and their components, their biochemical and
metabolic effects, the toxicological effects of inhaled
diesel emissions, the mutagenic and carcinogenic potency of
extracts of diesel and related emissions, the mutagenicity
of inhaled diesel emissions, the carcinogenic effects of
exposure to diesel emissions, and the results of epidemio-
logical studies involving human exposure to diesel emis-
sions. The Symposium culminated in a panel discussion on
the health risk assessment of diesel emissions.
The Proceedings consists of 77 manuscripts and associated
discussions, as well as the panel discussion.
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ACKNOWLEDGMENTS
The assistance of the many individuals who contributed to
the success of this Symposium and the timely completion of
the proceedings is gratefully acknowledged. Special appre-
ciation is due to the speakers, for the quality of their
presentations and promptness in submitting their papers, the
session chairmen and the many participants who contributed
to the discussions.
We also wish to acknowledge the assistance of the Center of
Environmental Research Information and in particular the
efforts of Mr. Larry Dempsey, in providing the many services
in connection with arranging the Symposium. We also are
indebted to Verna Til ford and Joan Mattox for assisting with
registration, and to Deborah Dean, Jean Roe and William B.
Peirano who helped in many ways to make the Symposium a
success.
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TABLE OF CONTENTS
Volume I
FOREWORD iii
R. J. Garner
PREFACE v
N. A. Clarke, W. Pepelko, R. Danner
ABSTRACT vii
ACKNOWLEDGEMENTS vlil
KEYNOTE ADDRESS xv
Stephen Gage
SESSION I
PHYSICAL AND CHEMICAL CHARACTERISTICS OF DIESEL
EMISSIONS 1
Chairmen: Ronald Bradow
James N. Pitts
Characterization of Diesel Participate
Exposure 3
Williams, R. L., and D. P. Chock
Characterization of Organic Constituents in
Org
;icul
Diesel Exhaust Participates . 34
Rodriguez, C. F., J. B. Fischer, and D. E.
Johnson
A Rapid Chemical Characterization of Diesel
Particulates by Thermogravimetric Analysis. . . 49
DiLorenzo, A., R. Barbella, G. M. Cornetti,
and G. Biaggini
Preparation and Characterization of Diesel
Exhaust Particles for Biological Experiments. . 82
Graf, J. L.
IX
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TABLE OF CONTENTS
(Continued)
Survey and Analysis of Automotive Particulate
Sampling 93
Duleep, K. G., and R. G. Dulla.
A Particulate Characterization Study of In-Use
Diesel Vehicles 113
Wotzak, G., R. Gibbs, and J. Hyde
Measurement of Unregulated Emissions - Some
Heavy Duty Diesel Engine Results . 138
Perez, J. M., Ph.D.
Polynuclear Aromatic Hydrocarbons in Diesel
Emission Particulates 175
Choudhury, D. R., and B. Bush
Emissions of Inorganic Compounds from Heavy
Duty Diesel Trucks on the Road . 187
Kiyoura, R.
Interactions Between Diesel Emissions and
Gaseous Co-Pollutants in Photochemical Mr
Pollution: Some Health Implications.. . . . . 188
Pitts, J. N., Jr., A. M. Winer, D. M.
Lokensgard, S. D. Shaffer, E. C. Tuazon,
G. W. Harris
Optimizing Diesel Combustion: Improving Fuel
Economy, Engine Life, and Reducing Particulate
and N0y Emissions with Electrostatic Fluid
Processors 210
Gibbons, R. A., and Dr. B. A. Wolf.
SUMMARY DISCUSSION FOLLOWING SESSION 1 225
SESSION II
IN-VITRO CARCINOGENIC AND MUTAGENIC EFFECTS
OF DIESEL EMISSIONS AND DIESEL EMISSION
COMPONENTS 228
Chairmen: Joellen Huisingh
F. Bernard Daniel
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TABLE OF CONTENTS
(Continued)
Page
Diesel Particulate Collection for Biological
Testing: Comparison of Electrostatic Precipi-
tation and FiItration . 230
Chan, T. L., P. S. Lee, and J. S. Siak
Diesel Particulate Extracts in Bacterial Test
Systems 245
Siak, J. S., T. L. Chan, and P. S. Lee
Kutagenic Activity of Diesel Emission Particu-
late Extracts and Isolation of the Mutagenic
Fractions 263
Choudhury, D. R. and C. 0. Uoudney
Mutagenicity Studies on Diesel Particles and
Particulate Extracts. . 276
Loprieno, N., F. DeLorenzo, G. M. Cornetti,
and G. Biaygini.
The Hutagenicity of Diesel Exhaust Exposed to
Smog Chamber Conditions as Shown by Salmonella
Typhimurium 309
Claxton, L., and H. M. Barnes
Salmonella/Microsome hutayenicity Assays of
Exhaust From Diesel and Gasoline Powered Motor
Vehicles 327
Lofroth, G.
Biological Availability of Mutagenic Chemicals
Associated with Diesel Exhaust Particles. . . . 345
Brooks, A. L., R. K. Wolff, R. t. Royer, C.
R. Clark, A. Sanchez, and R. 0. McClellan
Diesel Particulate Matter Chemical and Biolo-
gical Assays 359
Risby, T. H., R. E. Yasbin, and S. S. Lestz
Diesel Particulate Extracts in Cultured Mam-
malian Cells . . 385
Rudd, C. J.
Diesel Soot: Mutation Measurements in Bacter-
ial and Human Cells 404
Liber, H. L., E. M. Andon, R. A. Kites, and
W. G. Thilly
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TABLE OF CONTENTS
(Continued)
Page
Studies on the Effects of Diesel Particulate
on Normal and Xeroderma Pigmentosum Cells . . . 413
McCormick, J. J., R. M. Zator, B. B.
DaGue, and V. h. Maher
Benzo(a)pyrene Alters Lung Collagen Synthesis
in Organ Culture 416
Bhatnagar, R. S., M. Z. Hussain, and
S. D. Lee
Application of a Battery of Short Term Muta-
genesis and Carcinogenesis Bioassays to the
Evaluation of Soluble Organics from Diesel
Particulates 427
Huisingh, J., S. Nesnow, R. Eradow
and M. Waters
A Review of In-Vitro Testing Systems Appli-
cable to Diesel Health Effects Research .... 431
Whitmyre, Gary i\.
The DMA Damage Activity (PDA) Assay and
its Application to River Waters and Diesel
Exhausts 448
Doudney, C. 0., M. A. Franke, and
C. N. Rinaldi
SESSION III
BIOCHEMICAL AND METABOLIC EFFECTS OF DIESEL
EMISSIONS AND DIESEL EMISSION COMPONENTS 463
Chairman: Robert M. Danner
Lung Biochemistry of Rats Chronically Exposed
to Diesel Particulates 465
Misiorowski, R. L., K. A. Strom, J. J.
Vostal , and h. Chvapil
DNA-Binding Studies with Diesel Exhaust Par-
ticle Extract 481
Pederson, Thomas C.
xn
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TABLE OF CONTENTS
(Continued)
The Effect of In Vivo Exposure of Rats to
Diluted Diesel Exhaust on Microsomal Oxi-
dation of Benzo(a)pyrene .". . . . 498
Charboneau, J. and R. McCauley
Benzo(a)pyrene Metabolism in Mice Exposed to
Diesel Exhaust: I. Uptake and Distribution . . 508
Tyrer, H. VJ., E. T. Cantrell, R. Horres,
I. P. Lee, W. B. Peirano, and R. M. Uanner
Benzo(a)pyrene Metabolism in Mice Exposed
to Diesel Exhaust: II. Metabolism and
Excretion 52U
Cdntrell, E. T., H. W. Tyrer, W. B. Peirano,
ana R. Ni. LJanner
Effect of Exposure to Diesel Exhaust on
Pulmonary Prostaglandin Dehydrocjenase (PGDH)
Activity 532
Chaudhari, A., R. G. Farrer and S. Dutta
Effect of Diesel Particulate Exposure on Aden-
ylate and Guanylate Cyclase of Rat and Guinea
Pig Liver and Lung 538
Schneider, D. R. and B. T. Felt
Biochemical Alterations in Lung Connective
Tissue in Rats and Mice Exposed to Diesel
Emissions 557
Bhatnagar, R. S., h. I. Hussain, K.
Sorensen, F. M. Von Dohlen, R. M. Danner,
L. McMillan, and S. D. Lee
xm
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KEYNOTE ADDRESS
Stephen J. Gage
Assistant Administrator for
Research and Development
U.S. Environmental Protection Agency
Washington, DC
I am delighted to be here today addressing this symposium on
the health effects from diesel emissions, because that
subject is a very good example of the problems associated
with environmental regulation based on health effects and
how health effects research can and cannot influence the
regulatory process. The salient points for discussion
are:
1. The effects of concern, principally cancer and muta-
genesis in the diesel case, are chornic and slow to
develop as opposed to acute.
2. The science behind the effects of concern is not well
understood.
3. The effects of concern are expected to be of low
probability for an individual, but the exposed pop-
ulation will be large.
4. The effects of concern are currently believed to have
a possibility of occurring at all exposure levels no
matter how small.
5. The toxic agent is a mixture of literally hundreds of
compounds. Furthermore, the mixture varies in ways
that are not well understood as a function of such
non-precise factors as engine design, operating con-
dition, source of fuel, etc.
xv
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6. The object of possible regulation, in this case the
diesel car, is not the only source of the toxic
pollutant.
7. The diesel engine is seen by many to offer advantages
to society that we need, and need now.
The fact that the effect is slow to develop and chronic, its
science not well understood, and its occurrence of low
individual probabiity means that whatever health effects
research we do will not be related to health effects in
human beings in any tightly argued scientific way. First
of all, this may mean that epidemiology useful for standard
settting probably cannot be done. The expected low proba-
bility of the effect means that we probably won't get
studies showing a positive result, but on the other hand we
won't be able to use the negative results because the power
of the studies will not be strong enough for us to say that
diesel soot is of no concern, especially when the exposed
population is so large. Second, for obvious ethical reasons,
exposure of humans cannot be done which leads us to the now
familiar arguments of the validity of extrapolating from
mouse to man. The subspecies of arguments contained in the
mouse to man argument are many and include variation between
species in pharmacodynamics, routes of exposure, dose
levels, preparation of samples, deposition and clearance,
etc. I am, of course, not say-ing that because of these
perturbing factors and lack of good theory and knowledge as
to the mechanisms of carcinogenesis, that the animal experi-
ments are essentially useless for assessing human health
risk. Such obviously is not the case. I am, however,
pointing out that even in the area where health effects
research is most applicable, the result is not a scientific
answer in the sense that the laws of Newton provide a
scientific answer for landing on the moon, but a much vaguer
argument, elaborate but still basically post hoc ergo
propter hoc. The conclusion that we must draw is that
even where science makes its strongest contribution to
a regulatory health effects question, when it comes to
deciding what regulating to actually do, there remain all
the basic arguments about environmental regulation about how
much is enough and why. I should note here that the Federal
Government's Regulatory Council has generated a cancer
policy on how cancer data is to be used for regulatory
purposes by the Federal Government.
The use of health effects research gets even less scientific
when we with horror discover that for regulation it is not
enough to assert that a substance such as diesel soot is a
carcinogen for regulatory purposes, but that an estimate of
potency is needed as well. This is so because many perceive
xvi
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benefits associated with the generation of such substance
and they are not willing to forego them without at least a
respectable argument made that the asserted adverse health
effects will be in some, and always arguable, sense "signi-
ficant". The argument is further fueled by EPA's policy
(consistent with current science, but not proven by it
either) that there is no threshold exposure effect for
chemical carcinogen. This assumption means, in the current
case, that no matter how small the increased exposure to
diesel soot, we will expect some increased number of cancer
cases. Obviously, whatever the controversy about going from
"mouse to man" for establishing human carcinogenicity, the
controversy over a numerical estimate of a substance's
potency as a human carcinogen is considerably greater. This
is not the place to discuss quantitative cancer risk assess-
ment, but I would like to make on point quite clear. At the
present time, quantitative chemical cancer risk assessment
at environmental exposures is not science, but an analytical
technique consistently using the available health effects
data to fulfill a policy and regulatory need for some
estimate of a substance's human carcinogenic potency.
The fact that diesel soot is a mixture of hundreds of
organic compounds of various types, with the composition of
this mixture varying as an unkown function of such imprecise
thing as engine design, operating conditions, source of
fuel, etc., is another place where the diesel soot regula-
tory issue is similar to other regulatory problems. While
the final word has not been said, some think that the
compound by compound identification, potency testing and
control design for complex mixtures of chornic toxic sub-
stances is not going to be a fruitful approach for regula-
tory purposes. Assuming arguendo the above we see that a
method for estimating carcinogenic potency is needed which
1) can be applied to mixtures, and 2) does not require a
long time to get answers. For example, whole animal cancer
testing is considered in this sense "too slow". In fact,
even when dealing with a pure substance, whole animal cancer
tests produce results on a time scale that is felt awkwardly
slow by the public and regulators. To put it another way,
test procedures are needed to assess relatively quickly
whether or not the mixture produced after changes are made
in how it is generated, is more or less toxic. To take the
present case, if we are to reduce the cancer potency of
diesel soot by engine design, short-term tests will be
needed if the redesign is not to take forever.
Hence, after pointing out above, the arguments engendered
by mouse to man extrapolation, I shall now suggest that
research must be aggressively pursued so that we go from
microbe or cell to man where feasible. The general problem
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needing research to develop methods is then short-term tests
for chronic effects. The chronic effect of interest with
the diesel is cancer, but for general environmental protec-
tion the problem is more broad in that such test systems are
needed in the areas of chronic lung disease, neurological
disorders, immune system impairment, etc.
The Environmental Protection Agency's diesel research
effort, as you will hear, is pursuing such research both in
the Health Effects Research Laboratory here in Cincinnait
and at the one in North Carolina. We currently think that
in developing such test systems, three aspects need empha-
sis. First, the systems should produce very few false
positive results, or to say it another way, the false alarm
probability should be low. Second, the systems should
produce very few false negatives or the detection probably
should be high. Third, the systems need to quantitatively
assess human potency. I believe I have listed these needed
characteristics in order of difficulty, and speaking off the
cuff, I think the end is in sight in the case of cancer for
a low false alarm (i.e., no or few false positives) short-
term test system. At the risk of being slightly parochial,
I can think of no area of health effects research that is
more crucial to EPA's regulatory needs than the development
of such short-term tests for quantitatively and reliably
assessing chronic human effects.
Preliminary estimates have indicated that even if diesel
cars emitting particles at the current level extensively
penetrate the country's automobile fleet that they will not
be the only major contributor of diesel particles in the
air. As the energy crunch continues, we expect more diesels
to show up including such areas as cogeneration of elec-
tricity with space heating and cooling, and heavy and light
duty trucks. The implications are clear, regulation of one
part of a potential diesel problem will affect the regula-
tion of other diesels. This will almost certainly force
health effects work on other sources of diesel particles and
from there to the health effects of carbon containing
particles in the air. Carbon particles (from all sources)
are the second most abundant species in the air after
sulfates nationwide, and indeed are the most abundant
species contributing to fine particles in many western
urban locations.
My last point for discussion, namely that many people feel
that diesel cars have features that we need and need now, is
the one where the contribution of health effects research
runs right into all the other things we think are important
in life besides health. It is in this area where we health
researchers are apt to feel that no one listens to us or if
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they do, they completely misinterpret what we have to say.
All I can say is this is where we as scientists must make
the impact of health effects research felt. We must not
present our data nad arguments as being more scientific than
they are, however, we must not allow ourselves to be pushed
aside in our areas of competence. This requires that we
educate ourselves so that we understand what regulators
think, but furthermore, that we educate the regulators so
they understand what we have to say.
In closing, I would like to stress that in spite of the fact
that there seems to be no prospect that health effects
research will take the controversy out of standard setting
or render having to think about a problem obsolete, health
effects research is one of the major components in standard
setting. It is necessary to keep us from paying an unaccept-
ably high price (in terms of deleterious health effects) for
an otherwise beneficient technology or alternatively to keep
us from foregoing valuable technology from fear of phantom
health effects. This health research must be done and done
well. In your pursuit of this work, we all, and indeed the
country, must wish you Godspeed. Thank you.
xix
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Session I
PHYSICAL AND CHEMICAL CHARACTERISTICS
OF DIESEL EMISSIONS
Chairmen:
Dr. Ronald Bradow
Dr. James N. Pitts
Characterization of Diesel Participate Exposure.
Williams, Ronald L. and David P. Chock.
Characterization of Organic Constituents in Diesel Exhaust
Particulates.
Rodriguez, C. F., J. B. Fischer, and D. E. Johnson.
A Rapid Chemical Characterization of Diesel Particulates by
Therniogravimetric Analysis.
DiLorenzo, A., R. Earbella, G. M. Cornetti, and G.
Biaggini.
Preparation and Characterization of Diesel Exhaust Particles
for Biological Experiments.
Graf, Jean L.
Survey and Analysis of Automotive Particulate Sampling.
Duleep, K. G., and R. G. Dulla
A Particulate Characterization Study of In-Use Diesel
Vehicles.
Wotzak, G., R. Gibbs, and J. Hyde.
Measurement of Unregulated Emissions - Some Heavy Duty
Diesel Engine Results.
Perez, Joseph M., Ph.D.
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Session I
(Continued)
Polynuclear Aromatic Hydrocarbons in Diesel Emission
Participates.
Choudhury, Dilip R., and Brian Bush.
Emissions of Inorganic Compounds from Heavy Duty Diesel
Trucks on the Road.
Kiyoura, Raisaku.
Interactions Between Diesel Emissions and Gaseous Co-Pol1u-
tants in Photochemical Air Pollution: Some Health Implica-
tions.
Pitts, Jarnes N., Jr., Arthur M. Winer, David M.
Lokensgard, Steven D. Shaffer, Ernesto C. Tuazon
and Geoffrey W. Harris.
Optimizing Diesel Combustion: Improving Fuel Economy,
Engine Life, and Reducing Particulate and NQX Emissions
with Electrostatic Fluid Processors.
Gibbons, Robert A., and Dr. B. A. Wolf.
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CHARACTERIZATION OF DIESEL
PARTICULATE EXPOSURE
Ronald L. Williams and David P. Chock
Environmental Science Department
General Motors Research Laboratories
Warren, Michigan 48090
ABSTRACT
This report summarizes our work on the physical and chemi-
cal characterization of diesel exhaust with emphasis on the
extractable and benzo(a)pyrene content of the particulate.
Compared with benzene-ethanol, methylene chloride is an
unsuitable extraction solvent for the measurement of benzo-
(a)pyrene in diesel particulate. Using recently developed
analytical techniques, a series of studies of sampling
parameters shows that the composition of the particulate is
fixed when it leaves the tailpipe and that the composition
does not change with dilution. Further, sampling results
in a dilution tunnel agree quite well with those observed
in open air sampling. Finally, based on various air
quality models and projections of the diesel sales-emission
scenario, we find that the light-duty diesel will be a
contributor to total ambient particulate.
INTRODUCTION
In an era constantly threatened by energy shortages,
efficient usage of our energy resources is of primary
importance. The most attractive feature of diesel engines
is clearly their superior fuel economy, as compared to
gasoline engines. However, there has been concern
expressed in some quarters about the potential health
effects of diesel emissions. Extensive research has been
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conducted for some time at General Motors and elsewhere to
assess the potential health effects due to exposure to
diesel particulate and other diesel emissions. Fundamental
to these efforts is an understanding of the physical and
chemical character of diesel emissions, as well as the
anticipated levels of human exposure. We will discuss the
latest information on the physical and chemical charac-
teristics of diesel emissions and, with that background,
assess the air quality impact of expanded usage of light-
duty diesel vehicles.
CHARACTERIZATION OF DIESEL EMISSIONS
The facilities used to generate and collect diesel emis-
sions and the details of the chemical analyses will not be
described in this paper. Previous publications on these
subjects may be consulted (1-3). Instead, we will begin
with the analysis of filter samples of diesel particulate
and then use those results to examine filter sampling tech-
niques and the potential interactions between the gases and
particles emitted by diesel vehicles.
Analysis of Filter Samples
Extraction of Particulate Matter: Solvent extraction
is a convenient method for dividing diesel exhaust parti-
culate into two components. The composition of the
extraction solvent determines the quantity and composition
of the material extracted. We have elected to use a mix-
ture of benzene and ethanol (4:1) in order to extract both
the relatively nonpolar organic materials, e.g., hydro-
carbons, which are readily soluble in benzene, and the more
polar organic materials, e.g., aldehydes, ketones, perox-
ides, acids, or heterocyclic compounds which tend to be
more soluble in ethanol. This choice of solvent was based
in part on earlier research in the determination of poly-
cyclic aromatic hydrocarbons in vehicle exhaust (U,5).
Each weighed filter was cut into strips approximately 12 mm
x 50 mm and extracted in a 30-mm i.d. Soxhlet apparatus.
In order to reduce both the volume of extraction solvent
required and the syphon-cycle time, a 19-mm diameter by
80-mm-long glass rod was put into the extraction chamber
with the filter. The 125-ml boiling flask with 60-ml
extraction solvent and several Teflon boiling-aid chips was
set directly into steam to nearly one-half the flask
diameter, and the entire assembly was wrapped in a
12-mm-thick glass blanket to reduce heat loss. Syphon
cycle-time (the time between batch extractions) was
approximately three minutes, and the total heating time was
three hours so about 60 syphon-cycles took place. Four
extraction units were usually operated simultaneously.
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The extract was filtered into a 100-ml beaker through a
small, tared glass-fiber filter to remove any carbon
particles which may have washed off the sample filter. The
extract volume was reduced to a few ml by evaporation of
the solvent under a stream of filtered air. The extract
concentrate was then transferred to a small, tared vial,
and the evaporation of solvent was completed in the same
manner. The vial, the extracted exhaust sample-filter, and
the extract clearing-filter were stored a minimum of 16
hours in the controlled atmosphere of a balance room and
then weighed to determine the weights of extractables and
dry soot.
In completing the evaporation of solvent, some of the
diesel fuel constituents which would have been extracted
will also be evaporated. This loss is minimized by
stopping the air flow as soon as the residue appears to be
stabilized, i.e., no further reduction in volume or visual
absence of solvent (6). Such loss of diesel fuel is prob-
ably modest, considering that material balances in the
total extraction procedure have averaged above 93%, as
shown in Table I.
Table I.
Test
Series
a
b
c
d
e
f
Overall Recovery of Particulate Matter
in Extraction of Diesel Soot on Filters
Number of
Filters
12
16
19
13
24
14
Average Percent
Accounted For
94.4
98.2
99.2
93-3
96.5
96.3
± 8.7
± 4.8
± 5.5
± 4'7
i 17.4
+ 8.8
The percentage of exhaust particulate matter soluble in
benzene-ethanol differs among cars, and for individual cars
it differs with the mode of operation. In our experience,
the soluble fraction has ranged from about 0.1 to 0.9.
Only in the case of one car, at low cruise speed, did the
soot contain sufficient liquid to soak into the sample
filter, giving the filter an oily appearance. The extract
was most often a pasty solid, but was sometimes a rela-
tively viscous oil and at other times a dry-appearing solid.
-------
Several other solvents have been used in Soxhlet extrac-
tions to determine the extractable mass compared to that
obtained with benzene ethanol (Table II). A considerable
range in extractable mass was observed. The final choice
of an extraction solvent is dictated by the kind of anal-
ysis intended for the extracts. These strong organic
solvents are well suited for chemical analysis purposes,
but they may be inappropriate in biological studies.
Table II. Potential Solvents for Extracting
Diesel Particulate
Solvent Index of Solubility3
benzene-ethanol 1.00
methylene chloride 0.66
dichloroethane 0.66
cyclohexane-ethanol 0.93
cyclohexane-isopropanol 0.80
chloroform-ethanol 0.99
dichloroethane-ethanol 1.12
methylene chloride-ethanol 0.88
benzene-isopropanol 0.92
dichloroethane-isopropanol 0.85
Extractable mass relative to benzene-ethanol
During the course of this work, the EPA recommended that
methylene chloride (dichloromethane) be used as the
extracting solvent (7). In order to compare the efficiency
of methylene chloride with benzene-ethanol for the extrac-
tion of benzo(a)pyrene (BaP), a series of extractions were
performed using 20-mg portions of diesel exhaust parti-
culate which had been collected in replicate tests. The
bulk particulate was wrapped in a fiberglass filter paper
and extracted for 3-hours (60 cycles) using either benzene-
ethanol (80:20) or methylene chloride. The results are
shown in Table III.
Trials 1, 2, and 3 in Table III show that methylene
chloride extracts only 60 to 70% as much BaP from diesel
exhaust particulate as benzene-ethanol. (Similar experi-
ments using cyclohexane and methanol showed that these
-------
Table III. Comparison of Quantities of BaP
Extracted (ng BaP/mg participate) with
Benzene-ethanol and Methylene Chloride
Benzene Methylene
Trial Extraction ethanol Chloride
1 3-h Soxhlet 8.93 6.28
2 3-h Soxhlet 9.41 5.50
2A Second 3-h Soxhlet 0.75 0.38
with fresh solvent
3 3-h Soxhlet 7-98 4.94
3A Second 3-h Soxhlet with 0.04 3-63
the solvents reversed
solvents extracted only about 40$ as much BaP as benzene-
ethanol.) Trial 2A, Table III, shows that a second 3-hr
extraction yields only a small amount (7-8J) of BaP. In
contrast Trial 3A with the solvents reversed shows that
after one 3-hr extraction with methylene chloride consid-
erable benzene ethanol extractable BaP is left on the
diesel particulate. This demonstrates convincingly that
the low results for methylene chloride extractions are not
due to decomposition of BaP in methylene chloride.
To further test the extraction efficiency, several
experiments were performed in which the diesel exhaust
particulate was "spiked" with a solution of BaP before
extraction. These experiments were performed in parallel
to those shown in Table III, so a "total expected" BaP
value could be calculated from the benzene-ethanol results
in Table III plus the spike. These results are shown in
Table IV.
The benzene-ethanol results in Table IV show the excellent
recovery of the spikes obtained with this solvent. The
methylene chloride results show lower recovery and less
consistency; the BaP recovery varies from 65 to 86% of the
total expected. Finally, Trial 3A again shows that
benzene-ethanol effectively extracts additional BaP after a
methylene chloride extraction, but the reverse is not
true. Since BaP is biologically active and, thus, a rea-
sonable model compound, the extraction results indicate
that benzene-ethanol is an efficient extraction solvent but
that methylene chloride is totally unsuitable as a solvent
for diesel particulate extraction.
-------
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Analyses for Carbon, Hydrogen, Oxygen, and Nitrogen:
Elemental analysis of extracts and of the dry soot
remaining after the extraction has been obtained for
carbon, hydrogen, oxygen, and nitrogen (C, H, 0, N). The
methods employ combustion oxidation/reduction according to
Pregl or Dumas, and utilize a Perkin-Elmer model-240 ele-
mental analyzer. Typical results are illustrated for a
series of seven extracted filters and the extracts of two
of those filters (Table V). The extract and the dry soot
remaining after extraction both contain considerable
oxygen. The principal difference in composition is the
lower hydrogen and the higher carbon content of the dry
soot.
Table V. Elemental Analyses of Diesel
Exhaust Particulate Matter
Benzene-ethanol Extract
iight %
N
Vehicle
Operation
48 km/h
Idle
C
77.1
72.8
Weight %
H 0
10.9 11.3
7.4 19.0
0.70
0.73
Dry Soot (material remaining after
extraction with benzene-ethanol)
Vehicle
Operation
48 km/h
64
80
96
S-7
HFE
Idle
C
81.9
78.8
84.7
83.6
82.6
76.5
75-3
Weight
H
1.4
1.6
1.6
3-6
2.7
2.4
1.7
j
0
16.2
18.9
12.9
11.4
14.1
20.3
22.1
N
0.60
0.70
0.73
1.40
0.59
0.90
0.96
Molecular Weight Distribution by Gel Permeation
Chromatography (GPC): Extracts of soot from several cars
were analyzed for molecular weight distribution by gel
permeation chromatography, also sometimes referred to as
liquid exclusion chromatography. The gel column was one
commonly used for styrene polymers and therefore was
optimized for relatively large molecules. Relatively low
concentrations of substances below a molecular weight of
-------
about 150 could not be measured accurately. A typical GPC
chromatogram for a diesel particulate extract is illus-
trated in Fig. la, along with chromatograms for diesel
fuel, Fig. Ib, and SAE-30 lubricating oil, Fig. Ic. It is
evident that the molecular-weight-distribution curve for
the soot extract is more nearly like the distribution for
the lubricating oil than the distribution for the diesel
fuel. The extract is shown to include more components with
high molecular weights than lubricating oil, but, because
the extract contains more polar oxidation products, the
actual molecular weight distribution may differ from the
calibrations with polystyrene. Because the GPC procedure
doesn't apply thermal stress to the sample, it is more
appropriate than distillation or the GC-simulated distil-
lation procedure for the determination of molecular weights
above about 600.
A summary of average molecular weights for several extracts
is given in Table VI. MN refers to the number-average
molecular weight and My to the weight-average molecular
weight. For the purpose of calculating a mean-molecular
formula, M^ is utilized to show the mean-molecular size
by frequency of occurrence.
Table VI. Molecular Weight of Extracts of Typical
Exhaust Particulate Matter Compared to
Diesel Fuel and Lubricating Oil
Operating Condition M,. My Range
48 cruise 369 476 150-5000
64 cruise 381 552 150-5000
80 cruise 394 64? 200-7600
96 cruise 371 619 200-7600
S-7 cycle 400 607 150-5000
HFE cycle 385 520 150-5000
Idle 384 531 150-5000
Diesel Fuel 199 223 150-550
SAE #30 Lubricating Oil
Fresh 443 584 200-3000
Used 487 595 200-3000
10
-------
(Q)
Extract of
Diesel Particulate
(0
SAE 30
Lubricating Oil
Figure 1. Gel Permeation Chromatograms Indicating
Molecular Weight Distribution Based
on Polystyrene.
Utilizing MN values from Table VI and the elemental com-
positions from Table V, the general molecular formulae for
the extract samples would be:
30 mph
Idle
H39 02.6 NO.18
C23 H29 Oi*.7 N0.21
The mean molecular weight MN of the extract under all
operating conditions is somewhat lower than that of SAE-30
11
-------
lubricating oil (MN = c^ %8^' but the distribution
covers a wider range.
Infrared Absorption Spectroscopy: Infrared absorption
spectroscopy has been utilized to obtain structural infor-
mation on the benzene-ethanol extracts of diesel particu-
late. The method, as applied, is semiquantitative.
Because of the viscous nature of the samples and the small
sample quantities available, the spectra were obtained by
the attenuated total reflectance (ATR) technique. For most
extracts, carbon-hydrogen structures similar to those in
lubricating oil predominated. Aromatic absorption at 6.25
microns was also similar to that in lubricating oil and
diesel fuel. In addition, strong absorptions in the 5-85
micrometer region, indicative of C=0 structure, and in the
2.8-3-1 micrometer region, indicative of OH or NH struc-
tures, were present. Thus, the extracts contained a
substantial fraction of partially oxidized hydrocarbons,
e.g., ketones, aldehydes, or acids. The relatively strong
C=0 and OH structures were supported by the oxygen content,
as determined by elemental analysis.
Volatility by Thermogravimetry: The analytical tech-
nique of thermogravimetry (TGA) determines the change in
sample mass as a function of temperature. Although the
technique can be as simple as periodically weighing a
sample at room temperature or other controlled temperature
(static mode), it often finds its most useful application
in the dynamic mode by recording the sample mass as a
function of continuously increasing temperature. The
sample, in close proximity to a small thermocouple, is
suspended on a sensitive balance in a small furnace through
which gas of controlled composition flows. For all samples
analyzed by TGA, the temperature rise was 20°C per minute
to 700°C, starting at room temperature (about 23°C). Gas
flow was 100 ml per minute. Samples were heated first in
nitrogen to 500°C, then in air to 700°C.
This approach distinguishes between volatile material and
combustible material, as shown in Fig. 2. Using the ther-
mogram, one can replot the mass removed relative to the
total mass removed at 700°C in air. Results for diesel
particulate are illustrated in Fig. 3, along with results
for diesel fuel and SAE-30 lubricating oil. It is evident
that the particulate matter contained components which
overlap the high boiling end of diesel fuel. However, the
majority of the weight loss was associated with material
less volatile than diesel fuel, i.e., mainly in the
volatility range of lubricating oil.
The weight loss (in air) above about 150°C in the TGA
procedure is due mainly to combustion of carbon, but also
12
-------
Region of Volatilization
i 100
z
I 96
I
5 92
Region
of
Combustion
SO 100 150 200 250 300 350 400 450 500 550 600 650 700
Temperature (°C)
Rate of Change in Mass
50 100 150 200 250 300 350 400 450 500 550 600 650 700
Temperature (°C)
Figure 2. Typical Thermogram of Diesel Exhaust
Particulate (On Glass Fiber Filter)
Heated to 500°C in Nitrogen, Then
Reheated to 700°C in Air.
20
BO
100
100
200 300 400
Temperature (°C)
500
6OO
700
Figure 3. Temperature Dependence of Weight Loss of
Diesel Exhaust Particulate (calculated
percent weight loss based on combustion
end point).
13
-------
to the combustion of a smaller amount of high-molecular-
weight organic matter, the ignition of which causes the
composite to burn at a lower temperature. For example, if
a particulate sample (on glass-fiber filter paper) is
heated in air, the onset of the carbon combustion is in the
region of J»75-525°C. If, however, the sample is first
heated in nitrogen to 500°C, the temperature for the onset
of the carbon combustion in air is 600-625°C, an elevation
of about 100°C.
The TGA process is not a vapor/liquid equilibrium process.
The temperature in the TGA apparatus at which 50$ of the
diesel fuel is lost (Fig. 3) is 150°C. At this temper-
ature, the equilibrium vapor pressure of the midpoint
fraction of diesel fuel is about 10 torr (8). Rapid loss
of diesel fuel in the TGA apparatus occurs about 100°C
below the normal boiling point of the midpoint fraction of
diesel fuel. For lubricating oil, the midpoint in the TGA
procedure (Fig. 3) is about 300°C, at which temperature the
vapor pressure of the midpoint fraction is also about
10 torr. In this case, the TGA removal temperature is
about 180°C below the normal boiling point of the midpoint
fraction.
Correlation of Extractables with Volatiles: Solvent
extraction separates the total particulate into two
fractions which can be used in chemical and biological
analysis. Thermogravimetry may be used to determine the
mass of volatile material and the mass of combustible
material in a particulate sample, but it destroys the
sample. However, the relative simplicity of thermo-
gravimetry makes it an attractive measurement tool if it
yields results similar to extraction results. Depending on
the diesel engine involved, we have found various levels of
correlation between these two methods. The results for one
particular diesel vehicle are shown in Fig. JJ. In this
example the solvent extractables and TGA volatiles agree
very well. The least-squares best fit through the origin
has a slope of 1.04, and the correlation coefficient is
0.982. A wide range of driving modes are included in the
data set and the extractables cover nearly the full range
of extractables observed for any diesel. Using other
diesel cars, unexplained deviations from this simple
correlation have been observed, as shown in Fig. 5. Ther-
mogravimetry remains an attractive alternative to solvent
extraction but care must be exercised in its application
and interpretation.
Filter Sampling Techniques
Verification of the Filter Sampling Technique:
Although filter sampling is commonly used to collect
samples of particulate emissions from a variety of sources
14
-------
10
20
30 40 60
Solvent Extr«ct«ble (%)
60
Figure 4. Correlation of TGA Volatiles with Solvent
Extractables for Cyclic and Cruise Driving
of One Diesel Car.
(9,10), very little is known about changes in the particles
which might occur during the collection process, particu-
larly when the source is a diesel-powered automobile. This
part of our report describes a series of experiments which
were designed to examine some of the factors which might
cause changes in diesel particulate during the collection
and analysis of the filter samples. These experiments
include: 1) sampling rate variations, 2) sampling time
variations, 3) additional exposure to diesel exhaust gases,
and t) stability during filter-sample storage.
a) Sampling Rate Variations: Sampling rate is one of
the factors which could change the quantity and composition
of diesel particulate collected on a filter. A set of nine
filters was collected from the dilution tunnel at sampling
rates which ranged from 0.062 to 0.276 m3/min, each with
a sampling period of 10.0 min. The concentration of parti-
culate in the sample stream was calculated from the mass on
the filter and the volume of diluted exhaust sampled.
-------
100
BO
60
«o
20
X
X
X
o/
X
/
20 40 60
Solvent Extractable (%)
80
100
Figure 5- Weak Correlation Between TGA Volatiles and
Solvent Extractables for Two Diesel Vehicles
in Cruise Driving.
The extractable percentage (benzene-ethanol solvent) and
the BaP concentration in the particulate were also deter-
mined. The results of the sampling rate experiment are
shown in Table VII.
If filter efficiency is low, we would expect the efficiency
to increase with loading of particles on the filter. How-
ever, for a filter with high efficiency, the loading rate
on the filter will be proportional to the sampling rate.
For each filter in this study, the mass concentration of
particulate in the stream sampled was calculated (see Table
VII). The mass concentration was 9.89 + 0.16 mg/m3, and
there was no discernible trend with sampling rate, which
confirms the high efficiency of the Dexiglass filters.
For the range of sampling rates studied, the extractable
percentage was 36.2 + 1.8, and the BaP content was 10.2 +
1.1 ng/mg. Neither the extractable percentage, which would
reflect adsorption, nor the BaP concentration, which would
reflect chemical reactions, showed any dependence on
sampling rate.
16
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b) Sampling Time Variations: A set of eight filter
samples was collected from the dilution tunnel at a con-
stant sampling rate of 0.20 m^/min, but the sampling
period was varied from 1 to 25 minutes. The concentration
of particulate in the sample stream, the extractable per-
centage, and the BaP concentration are shown in Table
VIII. The range of sampling times covers the practical
range commonly used in vehicle testing.
Any change in collection efficiency which might occur
during extended sampling periods would lead to changes in
the apparent mass concentration in the diluted exhaust.
The mass concentration was 9-^6 + 0.35 mg/m3) with no
dependence on the duration of the sampling period.
Processes such as adsorption and chemical reaction could
affect the composition of the particulate on the filter
since particles collected in the initial layer of particu-
late are exposed to gaseous emission components transmitted
by particle layers collected later. As can be seen in
Table VIII, the extractable percentage and the BaP concen-
tration were again constant with no dependence on the
duration of the sampling period.
Therefore, variations in flow rate and in the duration of
the sampling period have no effect on the particle mass
concentration, the extractable percentage, or the BaP
concentration. Over the practical range of these two
variables we find no evidence for filter efficiency
changes, adsorption, or chemical reaction.
c) Additional Exposure to Diesel Exhaust Gases: A set
of four identical filter samples were collected from the
dilution tunnel to study the effect of additional exposure
of particulate to diesel exhaust gases. The sampling
period for these filters was 10 minutes during which about
20 mg of particulate were collected. To complete the
experiment, two filter holders were connected in series for
additional sampling. A clean filter was placed in the
first holder to remove the particulate. Two of the pre-
viously loaded filters were placed one at a time in the
second holder for exposure to diluted exhaust gas. One was
exposed for one minute, and a second filter was exposed for
10 minutes. The extractable percentage and the BaP concen-
tration in the particulate were determined on the four
filters (Table IX, Set 1). This experiment was repeated
using lighter loaded filters, collected under different
driving conditions (Table IX, Set 2).
In both sets the additional exposure to diesel exhaust
gases caused no significant change in mass on the filter,
in the extractable percentage, or in the BaP concen-
tration. Again, it appears that interactions between the
18
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diesel exhaust gases and particles in this sampling system
have reached a steady state before the particles are
collected on the Dexiglass filter.
d) Stability During Filter Sample Storage: Eleven
additional replicate filter samples were collected from the
dilution tunnel for a filter storage study. The amount of
particulate collected on each filter was 7.5 ± 0.2 mg.
These filter samples were stored in a room at 24 + 0.5°C
and 45 + 1H relative humidity and with normal fluorescent
light. Periodically, up to 150 days after collection,
filters were randomly selected for analysis. The extract-
able percentage and the BaP concentration are listed in
Table X.
Table X. Effect of Filter Sample Storage
on Diesel Exhaust Particulate
Storage BaP
3
3
t\
5
6
7
8
9
10
11
Period
(days)
9
9
9
9
28
28
28
91
91
147
147
Extractable
Percentage
34.1
32.9
32.2
32.0
38.8
39.2
40.0
44.6
40.1
36.5
37.8
37.1 + 4.0a
Concentration
(ng/mg)
12.1
11.1
11.1
10.9
9.8
9.0
10.1
7.7
6.3
5.5
4.3
One standard deviation.
While the extractable percentage has a standard deviation
of 4.0J, it shows no systematic change with storage time.
The total BaP loss in 150 days was 57? and the BaP concen-
tration decreased linearly with time at a rate of
0.046 ng/mg/day. While long term storage does cause a
decrease in the BaP concentration, the loss in 20 days of
21
-------
storage is less than one standard deviation normally found
for BaP in replicate samples analyzed on the same day. To
avoid excessive loss of BaP by unidentified processes, the
storage conditions should be carefully controlled, and good
practice would dictate minimizing storage time.
Comparison of Dilution Tunnel Sampling with Open Air
Sampling: When diesel exhaust exits the tailpipe, it is
diluted very rapidly. Roadway experiments have shown that
exhaust is diluted at least 1000-fold in the first fraction
of a second after it enters the free atmosphere (11).
This, of course, results in a very rapid decrease in both
the temperature and the concentration of the exhaust con-
stituents. In order to collect samples of sufficient size
without the confounding influence of large quantities of
extraneous ambient particulate and without requiring pro-
hibitively large sampling equipment, certain compromises
must be made in the test procedure. In general, vehicle
sampling has been carried out in dilution tunnels at five-
to twenty-fold dilution.
The question, of course, is to determine what actually
happens in the atmosphere. To this end, we have carried
out a series of experiments to make a direct comparison
between dilution in a tunnel and dilution in the free
atmosphere. A vehicle was allowed to idle in a partially
enclosed courtyard area and samples were taken at different
distances from the tailpipe to provide various dilution
ratios. At the same time, COg measurements were taken at
the sampling point to provide a measure of the dilution.
The range of dilution in these experiments was 2 to 350.
The mass of particulate relative to C02 concentration was
independent of the dilution ratio, as shown in Table XI.
Two measures of particulate composition were examined: the
extractable fraction and the BaP content. The same vehicle
was then tested using a dilution tunnel at two pump speeds
which correspond to dilution ratios of 14 to 1 and 26 to
1. These data points are also included in Table XI, and it
can be seen that the two experiments agree within experi-
mental error. The extractable fraction does not change
with dilution. The fact that the BaP content tends to
increase with dilution is interesting. Our interpretation
of this data is that sampling in a concentrated exhaust
stream causes significant BaP to be converted to other
products, probably by reaction with NC^- These results
may have important implications in the area of biological
testing of diesel particulate material. These experiments
strongly suggest that the hydrocarbon is bound to the
particles as they leave the tailpipe and that the hydro-
carbon remains attached, at least through the initial
dilution process.
22
-------
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23
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Filter sampling is intended to separate an aerosol into its
gaseous and particulate components. Concern has been
expressed about the potential desorption of hydrocarbons
from diesel particles and the potential for such gases to
participate in atmospheric photochemical reactions. To
begin answering these questions, we have carried out a
series of studies in which the weights of filters con-
taining diesel particulate were monitored for as long as
seven months (Table XII). The average weight loss of the
particulate on the filters was only 0.40J. The extractable
portion of the particulate in this set of samples ranged
from 15> to 45$, representative of the wide range of
sampling and operating conditions used to generate these
samples. By way of contrast, an experiment in which diesel
fuel was added to a clean filter resulted in 94$ evapo-
ration of the fuel in 40 days. The lack of desorption of
hydrocarbons from diesel particulate is certainly not
surprising since it is well known that the vapor pressure
of hydrocarbons is dramatically reduced when they are
adsorbed on carbon (12,13).
Table XII. Evaporation of HC from Diesel
Particulate Aged on Filters (room
temperature storage)
Days
After
Collection
80
80
80
80
80
190
190
190
190
190
210
210
210
210
Particulate $ of
Mass on Particulate
Filter (mg) Mass Evaporated
17-7
19.5
18.6
39.2
32.4
52.8
38.7
37.4
42.6
29.1
16.8
17.5
34.8
34.5
Avg. $ Evaporated
0.29
2.05
0.84
0.02
0.19
-0.10
-0.07
-0.05
-0.41
0.97
0.35
0.10
0.80
0.58
0.40
% of
Particulate Mass
Extractable*
28
37
36
16
17
15
17
22
18
41
29
30
42
45
* Determined 24 hours after sample collection on paired
filter.
24
-------
When considering even longer periods of time, it is impor-
tant to remember that the control of exhaust hydrocarbon
emissions is directed toward the reduction of ozone (formed
by a photochemical process). The hydrocarbons attached to
diesel particulates tend to have very high molecular
weights (Hi), i.e., almost all the hydrocarbons are larger
then C±2- High-molecular-weight hydrocarbons such as
these tend to be very unreactive, and when they react the
products tend to be more polar and even less volatile.
These considerations suggest that the heavy hydrocarbons
are deposited on diesel particulates before they enter the
atmosphere and that their reactivity is so low that they
will make a negligible impact on photochemical ozone
formation.
Gaseous Emissions
While the issue of potential health effects of diesel
engine emissions centers on particulate emissions, chemica-
characterization and biological effects studies should not
ignore gaseous emissions which occur concurrently with the
particulate. A list of exhaust emissions from an experi-
mental 5.7 L diesel under Federal Test Procedure conditions
is presented in Table XIII. The raw (undiluted) exhaust
concentration of each component was calculated using an
exhaust flow rate estimated from the average tunnel dilu-
tion ratio. The total particulate concentration in the raw
exhaust was 85 mg/m3, from which the other concentrations
may be scaled in animal exposure studies using diluted
diesel exhaust. Similarly, any interactions which might
occur between the gases and particles emitted from diesels
must occur at these same relative concentrations regardless
of the degree of dilution. Inadequate dilution of the
gaseous emissions might give rise to effects in extended
animal exposures regardless of the character of the
particulate under study.
AIR QUALITY IMPACT
Emission Standards and Diesel Market Share
The Proposed Standards: While the health effects of
diesel particulate have been under intense study for some
time, to date, no adverse health effects have been
observed. The extent of any diesel health effects will
surely depend on the level of exposure. In order to limit
exposure, the Environmental Protection Agency (EPA) pro-
posed on February 1, 1979 a particulate emission standard
for light-duty diesel vehicles: 0.38 g/km (0.6 g/mile) for
the 1981 and 1982 model years, 0.12 g/km (0.2 g/mile) for
the 1983 model year and thereafter (15).
25
-------
Table XIII. Exhaust Emissions from an Experimental
Diesel (FTP data)
Emission
N0x
N02
HC
CO
Sulfur Dioxide
Total Aldehydes
Hydrogen Cyanide
Ammonia
Sulfate
Total Particulate
Benzo(a)pyrene
Mass
Emission Rate
0.77 g/km
O.I'* "
0.24 "
1.06 "
515 mg/km
12 "
1.2 "
0.6 "
8.0 "
380 "
1-9 yg/km
Cone, in
Raw Exhaust
82 ppm
15 ppm
27 ppm
188 ppm
10 ppm
1.3 ppm
0.2 ppm
0.2 ppm
1.8 mg/m3
85 mg/m3
0.43 Pg/rn3
Calculated using exhaust flow rate estimated from
average tunnel dilution ratio.
According to the accompanying EPA documents, this emission
standard was based on the total suspended particulate air
quality standards and on EPA's judgement of technological
feasibility. From both the technology and the air quality
viewpoints, the proposed standard appears to be very strin-
gent, particularly in the initial period. In response to
EPA's proposal, General Motors proposed on April 19, 1979 a
Corporate Average Particulate Standard (CAPS) which repre-
sents an average over the entire fleet of light-duty
vehicles produced by a manufacturer in one model year
(16). In this proposal, an eventual corporate average
particulate standard of 0.031 g/km (0.05 g/mile) was
suggested for the 1987 model year and thereafter. If the
market share of light-duty diesels is expected to stabilize
at about 25% this standard represents an average emission
rate of 0.12 g/km (0.2 g/mile) for the diesel fleet, which
agrees with EPA's proposed diesel particulate emission
standard for the model year 1983 and thereafter.
Diesel Penetration in the Light-Duty Fleet: In the
PEDCo report (17), on which EPA's proposed standard was
based, two diesel sales scenarios were considered: the
26
-------
"best estimate" which assumed an ultimate market share of
10%, reached by model year 1983; and the "maximum estimate"
which assumed an ultimate market share of 25$, reached by
model year 1983. Our estimate, on the other hand, assumed
that an ultimate market share of 25% would not be reached
until model year 1990. For the purpose of estimating the
air quality impact, we assumed that the diesel portion of
the total vehicle-miles traveled by light-duty vehicles
would be 25%' This latter assumption requires the mainte-
nance of a market share of 25% for about 10 years or more.
Based on GM's and EPA's ("maximum") diesel sales projec-
tions, the above assumptions may be realized by the year
2000, and in any case the total particulate from light-duty
diesels prior to 2000 would not exceed the rate for the
year 2000.
Projected Particulate Concentrations due to Light-Duty
Diesels
Concentration Estimates: The primary National Ambient
Air Quality Standards for total suspended particulate are
75 yg/m3 (annual average) and 260 yg/m^ (24-hour
average). Physically, time averaging has the same effects
as spatial averaging. The extent of spatial averaging
depends on the emission-source distribution and the
fluctuations of the wind. For an urban area, because of
the rather diffuse distribution of motor vehicles, the
roadside annual average concentration due to emissions from
motor vehicles should be essentially equivalent to the
regional average over the whole urban area. An upper bound
for this number would be the average observed at or near
the downtown of an urban area. The 24-hour average, on the
other hand, represents a local average for, say, a portion
of a downtown area. From observations (18,19), the maximum
24-hour averages are about 2.5 times higher than the annual
averages. In the existing total-suspended-particulate air
quality standard, however, the ratio of the 24-hour average
to the annual average is about 3.5 which means that the
annual average is a more stringent standard.
In this connection, it should be mentioned that in the
PEDCo study (17), in addition to the regional annual
(geometric) mean, a roadside annual (geometric) mean was
also defined. (There is no compelling reason to favor a
geometric mean or an arithmetic mean. The geometric mean
derives from the assumption that concentrations averaged
over a fixed duration are lognormally distributed if each
of these concentrations are treated as an independent
statistical variate. While lognormality is not always
appropriate, it is not a major concern here since, in
general, the two means are not very far apart typically
within 10% of each other. It can be proven that for
27
-------
positive quantities, such as concentrations, the arithmetic
.mean is greater than or equal to the geometric mean.) The
'roadside annual Bean should be essentially the same as the
regional annual mean, especially when the upper bounds are
considered. Unfortunately, PEDCo arbitrarily multiplied
the regional mean by a factor of 11 to obtain the roadside
mean. The factor of 11 defies common sense. Worse yet,
PEDCo also estimated a regional 24-hour maximum which was
assumed to be about three times the regional annual mean.
This regional 24-hour maximum was then raised by a factor
of 11 to obtain a roadside 24-hour maximum. In other
words, the roadside 24-hour maximum is now about 33 times
the regional annual mean, whereas observations indicate
that the factor is typically 2.5.
To show how much EPA and PEDCo overestimated the roadside
concentration, we will compare their estimate with one of
our own. We use the worst-case meteorological condition
(stable, parallel wind at less than 1 m/s) observed during
the General Motors Sulfate Dispersion Experiment (11) and
by scaling the observed data, estimate the concentration at
3.5 meters above ground and 3.8 meters from the road. The
concentration is 9-2 yg/m3 for a traffic density of 17000
cars per day, with 25 J of the cars being light-duty diesels
emitting 0.6 g/km (1 g/mile) of particulate matter. Since
the worst-case condition cannot be sustained throughout the
day, we estimate the highest 24-hour concentration to be
about one-half of 9.2 yg/mS, or 4.6 yg/m3. EPA, on the
other hand, estimated a value of 52.9 yg/mS, at a
position three meters above ground and 4 meters from the
road. This is about 11 times too high! (The 52.9 yg/m3
estimate is the light-duty diesel portion of the total
mobile-source estimate of 75.6 yg/m3. See EPA (20),
Table IV-5.)
There are many existing models for describing short-term
(up to 24-hour) and long-term (annual) averages. In parti-
cular, the tracer (or surrogate) model is very simple to
use. It relates the unknown concentration with the
observed concentration of a tracer (e.g., lead), by multi-
plying the latter by a scaling factor which is the ratio of
diesel particulate and tracer emission rates. It is quite
reliable for long-term estimates, but is probably less
useful for short-term estimates because short-term averages
are more sensitive to local fluctuations. Yet, comparisons
of the tracer model estimates are consistent with those
from the Simple Line-Source Model (21) for worst-case
short-term concentrations. Therefore, the tracer model
offers a reliable methodology for both long-term-regional
and short-term-maximum concentration estimates. In what
follows, the tracer model will be used in our concentration
estimation. We shall assume a growth rate of 1J per year
28
-------
for the total vehicle miles traveled, a stabilized fleet of
25% light-duty diesels by the year 2000, and a particulate
emission of 0.12 g/km (0.2 g/mile). The estimated contri-
butions from light-duty diesels to particulate concen-
tration in 2000 for selected urban areas are shown in Table
XIV. It can be seen that the contributions from light-duty
diesels are a small fraction of the air quality standard
for total suspended particulate. It should also be pointed
out that the estimated concentrations are for worst cases
in the downtown areas where particulate emissions from
mobile sources are emphasized.
Table XIV. Projected Particulate Concentration Due
to Light-Duty Diesels for Various Cities
in the Year 2000 (Pg/m3j
Regional Roadside
Annual Mean 24-Hour Maximum
Los Angeles 10.5 26.2
Freeways
Los Angeles 6.0 15.0
Downtown
Chicago, New York 3-5 8.8
Downtown
Cincinnati, Denver,
Philadelphia 2.0 5.0
Downtown
Manhattan Taxi Cab Scenario: In the foregoing we
implicitly assumed that light-duty diesels are evenly
distributed throughout the country. It has been suggested
that this might not be the case. For example, all taxis in
Manhattan, New York might be powered by diesels. According
to the New York City Traffic Bureau (22), the traffic
density on major streets is about 400 vehicles per hour per
lane during a typical rush-hour period, with 50% taxis, 26$
passenger cars, 20$ trucks, and 4$ buses. We assumed that
60$ of the vehicles were light-duty diesels. These should
cover the entire taxi fleet and a substantial portion of
the private-passenger cars. We also chose a high emission
rate of 0.4 g/km (0.7 g/mile) for all light-duty diesels.
Then, using the APRAC Street Canyon Model (23) under the
worst meteorological conditions, we estimated the light-
duty diesels contribution to the roadside 24-hour maximum
concentration to be 40 yg/m3. This concentration should
be compared to the 24-hour particulate air- quality standard
of 260 ug/n>3.
29
-------
DISCUSSION
A detailed study of solvents, using BaP as a model
compound, showed that benzene-ethanol is an efficient
solvent for extracting BaP from diesel particulate while
methylene chloride is unsuitable because it gives lower and
less consistent BaP recoveries. Various chemical and
physical analyses showed that diesel particulate extract
resembles slightly oxidized engine lubricating oil.
A series of studies of sampling parameters showed that
Dexiglass filters are highly efficient filters for diesel
particulate, and that the composition of the particulate is
fixed when it leaves the tailpipe. As a result, the
composition is relatively stable in diluted exhaust. The
particulate composition is also quite stable to storage,
but a slow degradation of BaP (50% in 150 days) was
observed. Sampling results in a dilution tunnel agreed
quite well with those observed in open air sampling.
The concentrations of some of the gaseous emissions which
accompany the particulate emissions from diesels were
presented. Inadequate dilution of these gaseous emissions,
which are similar to those from gasoline engines, might
give rise to effects in extended animal exposures regard-
less of the character of the particulate under study.
Based on various air quality models and on our projections
of the diesel sales-emission scenario, it is estimated that
the light-duty diesel will be a minor contributor to
ambient particulate concentrations. These diesel emissions
will maximize in the year 2000 at about 2 yg/m3 (annual
average) for most cities and at less than 10 pg/m3
(annual average) in the worst locations. Roadside 24-hour
maximum concentrations will be typically 2.5 times higher
than the regional annual means, in contrast to EPA's
earlier estimation of a factor of 33.
30
-------
REFERENCES
1. Williams, R. L. and C. R. Begeman (1979), "Character-
ization of Exhaust Participate Matter from Diesel
Automobiles," General Motors Research Laboratories
Publication GMR-2970.
2. Cadle, S. H., G. J. Nebel, and R. L. Williams (1979),
"Measurements of Unregulated Emissions from General
Motors' Light-Duty Vehicles," SAE Paper 790694.
3. Swarin, S. J. and R. L. Williams (1979), "Liquid
Chromatographic Determination of Benzo(a)pyrene in
Diesel Exhaust Particulate: Verification of the
Collection and Analytical Methods," General Motors
Research Laboratories Publication GMR-3127-
4. Begeman, C. R. and J. M. Colucci (1962), "Apparatus
for Determining the Contribution of the Automobile to
the Benzene-Soluble Organic Matter in Air," National
Cancer Institute Monograph No. 9, Washington, D.C.,
p. 17.
5. Begeman, C. R. (1964), "Carcinogenic Aromatic Hydro-
carbons in Automobile Effluents," SAE Technical
Progress Series, Vol. 6, VEHICLE EMISSIONS, Society of
Automotive Engineers, New York, NY.
6. Williams, R. L. and S. J. Swarin (1979), "Benzo(a)-
pyrene Emissions from Gasoline and Diesel Automo-
biles," SAE Paper 790419-
7. Zweidinger, R. B., S. B. Tejada, D. Dropkins,
J. Huisingh, and L. Claxton (1978), paper presented at
Symposium on Diesel Particulate Emissions Measurement
Characterization, Ann Arbor, MI, "Characterization of
Extractable Organics in Diesel Exhaust Particulate."
8. Maxwell, J. B. and L. S. Bonnell (1955), "Vapor
Pressure Charts for Petroleum Hydrocarbons," Esso
Research and Engineering Co.
9. Gelman, C., and T. Meltzer (1979), "Membrane Filters
in Air Analysis," Anal. Chem., 51, p. 22A.
10. Witz, S. and R. MacPhee (1977), "Effect of Different
Types of Glass Filters on Total Suspended Particulates
and Their Chemical Composition," J. Air Pollut.
Contr. Assn., 27, p. 239.
31
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11. Cadle, S. H., D. P. Chock, P. R. Monson, and
J. M. Heuss (1977), "General Motors Sulfate Dispersion
Experiment: Experimental Procedures and Results," J_._
Air Pollut. Control Assoc., 27, p. 33.
12. Pupp, C., R. Lao, J. Murray, and R. Pottie (1974),
"Equilibrium Vapor Concentrations of Some Polycyclic
Aromatic Hydrocarbons, As^Og, and Se02 and the
Collection Efficiencies of These Air Pollutants,"
Atmos. Environ., 8_, p. 915.
13. Commins, B. T. (1962), National Cancer Institute
Monograph No. 9, Washington, D.C., p. 225.
14. Black, F. and L. High (1979), "Methodology for Deter-
mining Particulate and Gaseous Diesel Hydrocarbon
Emissions, SAE Paper no. 790422.
15. EPA (1979), "Control of Air Pollution from New Motor
Vehicles and New Motor Vehicle Engines, Certification
and Test Procedures - Particulate Regulation for
Light-Duty Diesel Vehicles," Federal Register, JK) (23)
6650-6671, February 1.
16. GM (1979), "Response to EPA Notice of Proposed Rule-
making on Particulate Regulation for Light-Duty Diesel
Vehicles," April 19.
17. PEDCo Environmental, Inc., (1978), "Air Quality
Assessment of Particulate Emissions From Diesel-
Powered Vehicles," EPA-450/3-78-038.
18. Colucci, J. M., C. R. Begeman and K. Kumler (1969),
"Lead Concentrations in Detroit, New York, and
Los Angeles Air," J. Air Pollut. Control Assoc., 19,
p. 255.
19. EPA (1977), "Air Quality Criteria for Lead,"
EPA-600/8-77-017, ORD, Washington, D.C., December.
20. EPA (1978), "Draft Regulatory Analysis - Light Duty
Diesel Particulate Regulations," December 22.
21. Chock, D.P. (1978), "A Simple Line-Source Model for
Dispersion near Roadways" Atmos. Environ. 12, p. 823-
22. New York City Traffic Bureau (1974), File No. 6337.
23. Johnson, W. B., W. F. Dabberdt, F. L. Ludwig, and R.
J. Allen (1973), "An Urban Diffusion Simulation Model
for Carbon Monoxide," J. Air Pollut. Control Assoc.,
23, p. 490.
32
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General Discussion
B. PETERS: Since there is some concern that a lot of
the potency could be due to oxygenated polycyclics, do
you have any information on other pure polycyclic com-
pounds besides BaP?
R. WILLIAMS: We have a limited amount of information
on other pure polycyclics. We have no analytical in-
formation on the oxygenated polycyclics derived from the
pure parent compounds.
R. FREEDMAN: Have you tried toluene-ethanol, and if
so, how does it compare to benzene-ethanol?
R. WILLIAMS: Yes, we have tried toluene-ethanol. We
do find comparable results. I think the important in-
gredient here is the combination of an aromatic solvent
and an alcohol when one is comparing mass, and also per-
haps for the penetration of particles for the extraction
of tightly bound materials like BaP. I would say we find
very comparable results, mass-wide and BaP-wise for tol-
uene-ethanol compared to benzene-ethanol. Since I am also
involved with some of the CRC Chemical Characterization
Panel's work, we worry a great deal about benzene. We
need to look at solvents other than benzene, and I think
toluene would be a very, very good substitute material.
A. LAWSON: You said the gas particle conversion of
the tailpipe is status stay. What does that amount to in
terms of dilution ratios in your tunnel?
R. WILLIAMS: The dilution ratios covered in the open
air samples range from five to 350. Our tunnel can be
run at a dilution ratio of either 14 or 26. We have
numerous samples in that low dilution ratio range where
the mass of material is easily gathered. Significantly
less material was collected at 300 to one, but the data
shown was for the entire range. In the paper you will
see the numerical data for all of the samples we col-
lected.
A. LAWSON: Are you saying that in the tunnel, if you
get 14 to one dilution you will be reaching status stay?
R. WILLIAMS: A dilution ratio of 14, and even five to
one, is in good agreement with that value.
33
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CHARACTERIZATION OF ORGANIC CONSTITUENTS
IN DIESEL EXHAUST PARTICULATES
C.F. Rodriguez, J.B. Fischer, and D.E. Johnson
Southwest Research Institute
ABSTRACT
The diesel engine is experiencing increased use outside the
industrial sector, particularly in the area of automotive
propulsion. Concern has arisen over the possibly toxic
organic compounds released to the atmosphere as constitu-
ents of the large amounts of particulate matter present
in diesel exhaust. The objective of the program reported
here was the preparation of large amounts of organics
extracted from diesel exhaust particulates for submission
to biotoxicity testing. Large quantities of particulates
collected on glass fiber filters were extracted in a
Soxhlet apparatus and fractionated into seven groups of
compounds. The fractions were submitted for toxicity
testing by various in-vitro methods which then guided sub-
sequent analytical chemical characterizations. Analytical
determinations included elemental analysis, infrared
spectroscopy, flame photometric gas chromatography, and
gas chromatography/mass spectrometry. The work was of a
preliminary nature and resulted in the tentative identifi-
cation of approximately 40 compounds in three organic
fractions.
The diesel engine has been shown to be more fuel efficient
than most other types of propulsive power plants and it
produces lower emissions of some of the gaseous pollutants.
However, there is still concern about potentially toxic
pollutants in the particulate emissions which form a
significant fraction of diesel exhaust emissions. The
solids are known to contain toxic organic compounds some
of which are carcinogenic, for example benzo(a)pyrene.
34
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Consequently, a great deal of work has been done at many
institutions in attempts to characterize the production
and effects of this compound and others like it.
The Department of Automotive Emissions at Southwest
Research Institute has for a number of years conducted
engineering studies for both industry and the EPA. These
studies have included many types of engines categorized
as both mobile and stationary source emitters. These
studies have been directed at determining the effects of
engine modifications on exhaust emissions and the impact
on the environment. One such program for the EPA involved
two engines, a two-stroke cycle and four-stroke cycle
diesel, which were run on dynamometers on specified duty
cycles for extended time periods.
We in the Department of Environmental Sciences were asked
to provide support for this program in the preparation of
samples for biological, in-vitro, toxicity testing. Also,
the application of various analytical characterization
methods was to guide the preparative efforts and provide
information for preliminary identifications of compounds
or compound groups which might be responsible for any toxic
effects noted.
The objectives of the characterization phase of this study
were to:
1. sample a large amount (200 g) of diesel engine
exhaust particulates,
2. separate organic compounds from the particulate
matter,
3. submit the organics for in-vitro toxicity testing,
in particular the Ames mutagenicity test, and
4. provide analytical information for the identifi-
cation of compounds.
The decision to collect a large amount of particulates
was based on the need to have enough material for the tests
which were to be conducted which included the Ames test and
several other mammalian cell tests. These would require
large amounts of material for each culture which is run at
varous concentration levels. We also knew that the organic
compounds extracted from particulates would be many in
number and complex so we anticipated fractionating into
functional groupings that would simplify the analytical
work and make toxicity testing more meaningful. Material
would also be needed for the analytical characterization
work.
The particulates were deposited onto a fluted 30 cm x 300 cm
glass fiber filter contained in a "backpack" holder through
which the whole raw exhaust passed directly. Organics were
35
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extracted into dichloromethane (DCM) followed by extraction
into acetontrile (ACN). The DCM extracts were then frac-
tionated into acids, bases, and neutrals by liquid-liquid
partitioning. The neutrals were subfractionated by column
liquid chromatography on silica gel adsorbent. All of the
fractions and the two extracts were submitted for biological
testing as enough material became available during the
course of the extractions and fractionations. Analytical
characterization of the various fractions consisted of
evaporating the solvent and weighing the residue; C, H, N
determinations; infrared spectroscopy; and selective detec-
tion gas chromatography.
The two-stroke cycle engine exhaust deposited 3-4 g parti-
culates on each filter and the other engine 2-3 g particu-
lates. In preparation for extraction two filters were
laid lengthwise one over the other and offset 8-10 cm.
The uncovered flap of the lower filter was carefully folded
over the upper one and the two were rolled into a cylinder
12-17 cm in diameter by 30 cm long. The filter was placed
in a glass Soxhlet thimble which had been constructed by a
glassblower since they are not commercially available. It
took practice to roll the filters properly - if the cylinder
was too large it would not fit in the thimble or if it was
too tight the extraction solvent would not make good con-
tact with the particulate sample.
The Giant Soxhlet extractors used are available commercially
from Ace Glass Co. and we had one available. To get better
than three months delivery we had three others made by
Houston Glass Fabricating Co. None of these extractors
would siphon with either DCM or ACN. We effected proper
siphoning action by placing fluorocarbon tubing inside
and pushing it to the top of the siphon tube and then
placing a smaller diameter tube inside it. As with rolling
the filters this operation was an art and it required a
great deal of experimentation to find the proper size
tubing that would provide proper siphoning.
The 12 1 flask was charged with 5-6 1 solvent and run for
24 hours at a cycle time of 70-90 minutes which resulted
in 14-17 cycles/run over about 22 hours with the remaining
time being given to take-down and start-up. When all of
the filters in an engine series had been extracted with
DCM the drained filters were then extracted with ACN.
Extract batches from four filters were concentrated and the
solvent recovered for reuse by distillation. The concen-
trates were then evaporated to dryness by inert gas imping-
ment, and the residue was weighed and taken up in DCM for
storage at -20°C.
36
-------
First attempts to separate the acids, bases and neutrals
were hampered by the formation of emulsions between DCM
and water, and we eventually replaced the DCM with diethyl
ether. The DCM containing 10-15 g of sample was evaporat-
ed; the residue taken up in 250 ml ether was extracted
with 0.1 MOH which was made acidic with ^04 and the
acids were then extracted into ether. The ether solution
was dried, weighed and stored in DCM solution as the ACD
fraction. The remaining ether solution was extracted
with 1 J^ H3P04 and the basic compounds (BAS) were extracted
into ether from the solution made basic with KOH. The com-
pounds remaining in the original ether solution were named
the neutral fraction (NUT). During the partitioning
processes some tars formed and were deposited on the walls
of the separatory funnels. Originally this material was
recovered and stored as fraction INT but we found later
that most of it could be dissolved by successive washings
with the partitioning solvents. In some few cases the
insoluble material from the four-stroke cycle engine would
not completely redissolve.
The neutral group contained most of the extracted organics
and was still quite complex so it was further fractionated
on a column of silica gel primarily to separate the alipha-
tic hydrocarbons present in large quantities but which are
not expected to exhibit any appreciable toxicity. The NUT
solution containing approximately 4 g of the two-stroke
or 2 g of the four-stroke sample was evaporated to near
dryness and mixed with 1 g silica gel.* A hexane slurry
was made and transferred to a 25 mm diameter glass column
packed with 66 g silica gel which had been activated by
heating 18 hours at 600 C. With this treatment the silica
gel could be reused indefinitely without any adverse effects
on the separations.
The aliphatic hydrocarbons (PRF) were eluted as the first
fraction with 225 ml hexane. The end point can be deter-
mined by monitoring the Schlieren patterns in the column
under visible light. Also, material fluorescent under
long wave length UV reaches the bottom of the column at
this point, and the eluant is changed to 1% diethyl ether
in hexane. The aromatic fraction (ARM) is then collected
in about 800 ml eluant, the cut off point being signaled
by a narrow, yellow, nonfluorescent band reaching the
bottom of the column. The yellow band containing the
"transitional" compounds (TRN) is then collected in 400-
450 ml eluant which is then changed to acetone/methanol
(1:1). About 200 ml removes the oxygenated fraction (OXY),
* Bio-Si! A, 100-200 mesh, available from Bio Rad
Laboratories, Richmond, California
37
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a dark orange-brown material which has to be chased with
200 ml methanol to remove the remaining tint from the
column.
The fuel used in these engines was also chromatographed
on silica gel. The fuel did not contain any acidic or
basic compounds measurable by gravimetric determination
so 2 g was chromatographed neat. The results of fractiona-
tion of the fuel and organic-soluble particulates are
shown as Table I. Apparently, most of the compounds com-
prising the fuel are aliphatic and small ring aromatic.
The results are calculated as a percentage of the weight
of total sample taken, exhaust particulates or liquid fuel.
The differences in amounts and types of organics produced
by the two engines are quite obvious in this table and
become even more so in subsequent analytical determinations.
Elemental determinations were made on each fraction and the
results are shown as trends of C/H ratios in Figure 1. The
graph is a good preliminary indicator of the character of
the exhaust organics. Generally, as the C/H ratio increases
substitution of heteroatoms and aromaticity increases. Also,
lower ratios for the two-stroke cycle engine (I) fractions
indicate the greater aliphatic character of the compounds
in these groups.
The infrared spectrum was determined from 1 mg of each
fraction on a KBr disc. A summary of the results is shown
as Table II with interpretive comments alongside the major
absorption bands. These results were expected for the
most part but there is a large degree of aliphatic character
indicated as present but not wholly anticipated in the
transitional and oxygenated fractions. The greater cyclic
and aromatic character of the four-stroke organics indi-
cated by the carbon-hydrogen ratios is emphasized by the
infrared results.
Each of the fractions from the engine exhausts was gas-
chromatographed on 3% OV-17 liquid phase with several
detectors of varying selectivity. The flame ionization
gas chromatograms of the NUT from the two engines are shown
in Figure 2. It can be seen that these two groups of com-
pounds are very different both qualitatively and quanti-
tatively. The chromatogram from each of the engine frac-
tions was essentially identical with the corresponding
whole neutral fraction shown here. Most of the compounds
from Engine I elute early in the lower molecular weight
region between the indexes at (44-024; the Engine II com-
pounds are concentrated in the higher molecular weight
region between 024-640.
Figure 3 shows chromatograms resulting from using a sulfur-
38
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TABLE I. RESULTS OF FRACTIONATION OF DIESEL FUEL
AND ORGANIC - SOLUBLE PARTICULATES
FRACTION
DCM
ACN
ACD
BAS
INT
NUT
Loss P
PRF
ARM
TRN
OXY
Loss F
2-STROKE
63.0
9.0
0.8
0.06
61.3
0.2
37.1 (60
8.8 (14
3.5 (5
9.2 (15
2.6 (4
% TOTAL WEIGHT
4-STROKE
25.0
9.9
2.1
0.2
2.1
20.2
0.1
.5) 8.6 (42.6)
.3) 0.9 (4.4)
.7) 1.2 (5.9)
.0) 7.0 (35.6)
.2) 2.5 (12.4)
FUEL
--
--
--
--
--
--
--
74.0
21.5
0.7
0.3
3.5
39
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TABLE II. INFRARED SPECTROSCOPY OF
SOLUBLE-ORGANIC FRACTIONS
FRACTION
PRF
ACD
BAS
ARM
TRN
OXY
ABSORBANCE, cm'1
2915,2850,1460,1375
3700-2300 broad
1700 broad
1600
1280,1225
3400 very broad
1700 strong, broad
1600 strong
810,750
880,830
2915,2850,1460,1375
1720,1690
1710,1600sh
1790
1600-1790
REMARKS
aliphatic mixtures of pe-
troleum orgin
0-H stretching
C=0
aromatic C=0 stretch
C-0 stretch
hydrogen bonded N-H stretch
possibly amide C=0
C=C stretch
C-H bending v. strong in
4-stroke
additional C-H in 4-stroke
aliphatic, very similar to
PRF
carbonyl , largely aliphatic
in 2-stroke; conjugated,
unsaturated and aromatic
keytones in 4-stroke
very similar to TRN
aromatic ketones
acyclic esters in 2-stroke;
aromatic ketones and
esters, y- and 6-lactones
in 4-stroke
40
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10
9
8
7
6
5
-ENGINE I
o-ENGINEII
PRF NUT DCM TRN BAS OXY ARM ACN INT ACD
FIGURE I. CARBON/HYDROGEN RATIOS OF ORGANIC
SOLUBLE DIESEL EXHAUST PARTICULATE
FRACTIONS.
41
-------
C32 C36\ C40
ENGINE II
FIGURE 2. FLAME IONIZATION GAS CHROMATOGRAMS
OF NEUTRAL FRACTIONS.
42
-------
ENGINE II
ENGINE I
ENGINE II
ENGINE I
FIGURE 3. SULFUR-SELECTIVE FLAME PHOTOMETRIC
GAS CHROMATOGRAMS OF NEUTRAL AND
ALIPHATIC FRACTIONS.
43
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selective Flame Photometric Detector. The chromatograms
of the neutral fractions are not unlike those exhibited
with the flame ionization detector except that they are
shifted approximately four carbon units to longer retention
times. The aliphatic fractions, as expected, indicate no
detectable sulfur content. The transitional and oxygenated
fractions from Engine II had some individual peaks super-
imposed on the envelope shown here for NUT in the region
between C to
At this stage of the program we had enough of each of the
fractions from the DCM extracts to begin submitting them
for biological in-vitro testing. The most important
results were to be those of the Ames mutagenicity tests
conducted by Dr. Vincent Simmon at SRI International.
The results of these tests were to guide the direction
that further analytical characterization was to take.
Other tests run were in-vitro mammalian cell assays by
Dr. Martin Meltz, Southwest Foundation for Research and
Education, unspecified tests, Dr. Leonard Schechtman,
microbiological associates and Drosophila fruit fly
screening, Dr. Ruby Vallencia, University of Wisconsin.
The results of the biological tests were reported to
Drs . Ronald Bradow and Mike Waters, EPA, Research Triangle
Park, North Carolina. The C, H, N determinations were
made by Galbraith Laboratories in Tennessee, HPLC and in-
organic ion chromatography was conducted by Dr. Sylvestre
Tejada at EPA-IERL, North Carolina, and some preliminary
capillary GC/MS and high resolution MS by Dr. Evan Horning,
Institute for Lipid Research, Houston, Texas.
The results of the Ames mutagenicity tests in decreasing
order of activity are, TRN II, OXY II, TRN I, OXY I, ACD II,
ACD I, INT II, BAS II, ARM II, ARM I. The remaining
fractions, PRF I and II, BAS I, and INT I showed no activity.
Activity is apparently concentrated in fractions containing
aromatic/oxygenated functionalities and seems to be greater
in the fractions from the four-stroke cycle engine. The
results from the basic and insoluble tars fractions are
ambiguous because of the very limited amounts which were
available. These test results directed our analytical
efforts to concentrate on the transitional and oxygenated
fractions from the two engines. Gas chroma tographic/mass
spectrometric determinations were then begun on these
fractions and some of the results are shown as Figure 4.
It was apparent that packed column gas chromatography would
not provide very useful mass spectra of the individual
compounds without a great deal of time and effort expended
on computer manipulations of the data. Wet then attempted
further fractionation of these groups of compounds by a
"quick and dirty" procedure which follows:
44
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o
o
CD
O
in
o
o
m
o
in
O
o
o
K
O i±J
& 3>
O
o
m
GO
a: i-
o o
O Z
H- <
LJ
OL
ID
45
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1. soak Sephadex LH-20* in iso-octane, methanol,
chloroform,
2. pack swollen LH-20 gel in a 100 mm (1) x 4 mm
(i.d.) glass column to a height of 40 mm,
3. place 1-1.5 mg sample in 250 yl solvent on the
LH-20 column,
4. elute with the solvent mixture, catch five 1 ml
fractions, and
5. elute fraction 6 with 20 ml methanol.
The glass column in Step 2 is a Pasteur pipet with the tip
cut off at a convenient point. Nitrogen at a very low
pressure (<5 psig) was applied to the head of the column
to hasten elution of the solvent.
GC/MS of the subfractions from the gel chromatography
provided chromatograms that were much more useful than
those from the whole fractions. Some of these chromato-
grams are shown as Figure 5. The peaks are more discretely
separated, but the "envelope" which results from the incom-
plete separation of many similar compounds is still
present. The same characteristics of earlier chromatograms
are again evident, i.e., the two-stroke exhausts are made
up of earlier eluting compounds of low molecular weight.
Useful mass spectra could be extracted from these total
ion chromatograms with more ease than from those of the
whole fractions. Interpretable spectra were generated
by careful choice of the peaks for study and by subtraction
of background taken from both the leading and trailing
sides of the peak. These spectral enhancement manipulations
resulted in the tentative identification of a number of
compounds, as follows:
alkyl naphthalenes (ARM I, II)
anthracene/phenathrene (ARM I, II)
alkyl anthracene/phenanthrenes (ARM I, II)
fluoranthene (ARM I)
pyrene (ARM I)
benzofluorene and/or methylpyrene (ARM I)
o-phenyl anisole (TRN II)
4-methylphenylbenzo(c)cinnoline (TRN II)
nitropyrene (TRN II)
phenylethylketone (OXY I)
chromone (OXY I)
coumarin (OXY I, II)
formylnaphthalene (OXY I, II)
alkyl naphthols (OXY I, II)
*A cross-linked dextran gel available from Pharmacia
Fine Chemicals, Piscataway, New Jersey
46
-------
100
LJ
P 100 i
LU
>
4-STROKE
TRN
100 i
4-STROKE
OXY
2-STROKE
OXY
50 100 150 200 250 300 350 400 450
SPECTRUM NUMBER
FIGURE 5. TOTAL ION CHROMATOGRAMS OF DIESEL
ORGANIC SUBFRACTION 4
47
-------
perinaphthindenone (OXY I, II)
anthroquinone (OXY I, II)
benzanthrone (OXY I, II)
m-hydroxybenzaldehyde (OXY I, II)
methoxybenzaldehyde (OXY I)
naphthol (OXY I, II)
p-phenylphenol (OXY I, II)
dibenzo-p-dioxin (OXY I, II)
naphthoic anhydride (OXY I, II)
o-phenylphenol (OXY I)
methylbenzanthrone (OXY II)
anthrone (OXY II)
The fractions each compound was indicated to be present
in are indicated in parentheses. For example, (OXY I, II)
indicates the spectrum was found in the oxygenated
fraction from both engines.
The analytical characterizations were secondary to the
main purpose of this program; hence, were of preliminary
and supportive nature. The primary objective of providing
a large amount of diesel engine exhaust particulate
organics in useful compound groups for biotoxicity testing
was accomplished. The fractionation methods described
may prove useful for continuing studies of the biological
effects of diesel exhaust constituents and determination
of the compounds responsible for toxic effects. These
studies could include more definitive determinations of
toxicity, determination of the fate of exhaust compounds
as they are emitted to the atmosphere, mammalian exposure
and inhalation experiments, and the development of methods
of eliminating unwanted compounds from diesel exhaust.
This project was sponsored by the EPA, Research Triangle
Park, North Carolina, Dr. Ronald Bradow; Project Officer
48
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A RAPID CHEMICAL CHARACTERIZATION
OF DIESEL PARTICIPATES BY THERMOGRAVIMETRIC ANALYSIS
A. DiLorenzo and R. Barbella
Laboratorio di Ricerche sulla Combustione
Napoli, Italy, 80125
G. M. Cornetti
FIAT - Research Center
Turin, Italy
G. Biaggini
IVECO - Engrg.
Turin, Italy
ABSTRACT
Thermogravimetric analyses, carried out on particulate
produced by an oil flame and by a diesel engine, allow the
determining, in a short time, of the volatile content
associated to the particulate and the distinguishing of the
unreacted fuel and/or lubricant oil from the combustion
products.
Thermogravimetric values, compared to those obtained by
solvent extraction, display that the extraction procedure
usually employed was not complete and in some cases the
extracted organics account for about 50% of the volatile
content of the particulate. These results are confirmed by
thermogravimetric analysis of particulate after solvent
extraction.
49
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The thermogravimetric technique is compared and integrated
by direct gas chromatographic and mass spectrometric analysis
of particulate before and after solvent extraction.
INTRODUCTION
Sampling and collection, chemical and physical characteriza-
tion, health effect evaluation are the three main steps
involved .in diesel particulate emissions. The complexity
of the different problems related to these three steps
requires a very long time to obtain significant experimental
evidences. Thus, the possibility to achieve a first rapid
information about particulate exhausted by diesel engines
would be welcome. This is particularly true for the manu-
facturers who need a quite short assessment in order to test
the influence of technological engine modifications on
quality and quantity of the organics associated to particu-
lates.
The aim of the present paper is to face with the thermo-
gravimetric characterization of the organics in order to
obtain a short answer to the main engine parameter changes.
At the same time the need for particulate collection and the
possibility for the health effect studies following this
rapid chemical characterization are examined.
At first this new method has been verified on the steady-
state pilot burner due to the better possibility of obtain-
ing large amounts of particulate. Then it has been extended
to diesel particulates and preliminary evidences will be
shown.
For the better understanding of the chemical composition of
the organics the new system has been compared and integrated
by direct gas chromatographic and mass spectrometric analy-
sis of particulates.
EXPERIMENTAL
Samples
The measurements have been carried out on two series of
particulates. The first was produced by an oil flame
(described in detail in a previous paper) (1), that pre-
sented properties similar to those that may be found in
residential and industrial burners. The picture of Fig. 1
50
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shows the oil flame. Two particular zones can be distin-
guished along the axis: the first one a spray zone, the
second one a combustion zone. The sampling was made by
means of a sintered bronze filter placed inside a water-
cooled stainless steel probe, that can be moved along three
axes of the flame to investigate on the droplets vaporiza-
tion and combustion and participate formation and evolution
at different stages of the combustion.
The second consists of particulate samples exhausted from a
N/A 2.4 1 displacement 4-cyl IDI engine, with and without
oxidation catalyst, tested under 1975 Federal Test Procedure-
Urban Driving Dynamometer Schedule, and collected according
to EPA proposed procedure (2).
Procedure
Thermogravimetric measurements of particulates were carried
out using a Perkin-Elmer TGS-2 Analyzer (Fig. 2). A detail
of furnace and balance is shown in Fig. 3. The samples, 5 *
10 mg, were put in a pan platinum inside a furnace heated up
to 600° C. The heating rate of the furnace, 10° C/min, was
controlled by a Model UU-1 temperature program control. The
furance atmosphere was controlled by a flow of purified
helium (SIO UPP type, 99.998%, 10 ml/min) at atmospheric
pressure. The first derivative, weight loss versus temper-
ature (dW/dT), was diagrammed by a model FDC-1 derivative
computer. The thermogravimetric method was firstly cali-
brated with known syntehtic mixtures of diesel fuel and/or
lubricant oil on carbonaceous support.
Figs. 4 and 5 show some examples of thermogravimetric curves
of a particulate, the first containing 10.7% diesel fuel
only, and the second 33% diesel fuel and 13% lubricant
oil.
Quantitative values of unreacted fuel and/or lubricant
oil in particulates were obtained measuring the area of
the peaks of the first derivative with a maximum at about
150° C for unreacted fuel, and 300 °C for lubricant oil.
The gas chromatographic analysis of particulates was carried
out by a direct sampling of solid samples, 1*5 mg, in
the injection block of a gas chromatograph (C. Erba mod. GI)
kept at 450° C, by a solid injector apparatus (3). The
volatile products were separated by a stainless steel
column, 2 m x 2 mm, packed with 5% Dexsil-300-GC on Chromo-
sorb W, 80 * 100 mesh. The column temperature was iso-
therm for 5 min and then programmed linearly to 370° C at 6°
C/min; helium was used as carrier gas, at 10 ml/min.
51
-------
Figure 1. Oil flame.
52
-------
Figure 2. Thermogravimetric apparatus.
53
-------
Figure 3. A detail of furnace and balance of the termo-
gravimetric apparatus.
54
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7.1
or
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1000
600
bQQ
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400 o_
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The particulates were also analyzed by direct sampling in a
mass spectrometer with a data system in line (4-5). The
samples, about 0.1 mg, were placed in a quartz capillary at
the tip of a solid probe. The probe was then inserted into
the mass spectrometer and heated up to 500° C, with an
automatically controlled temperature programmer, at about
40° C/min. An El source with electron energy at 70 eV was
used.
The extraction and separation of the organic components
present in both types of particulates were carried out
according to the EPA procedure (6): the particulate,
collected in repeated runs in different filters, was placed
in batches in soxhlet thimbles (previously, extracted for
6 hr with dichloromethane) and extracted for 8 hr with
dichloromethane (DCM).
The particulate after extraction was dried and used for
further analysis by means of mass spectrometry and thermo-
gravimetry. The extract material was evaporated to dryness
and then weighted. This value was compared with that
obtained by thermogravimetric analysis.
Then the dried extract was dissolved in diethyl ether and
subdivided on a silica gel column into paraffinic (PRF),
aromatic (ARM), transitional (TRN) and oxygenated (OXY)
fractions. Samples of all the above fractions were prepared
for in-vitro mutagenicity analysis (7). Part of TRN frac-
tion was dried and analyzed by direct sampling in a mass
spectrometer using a solid probe.
RESULTS AND DISCUSSION
The particulate samples include adsorbed uncombusted fuel
(and/or oil lubricant in those emitted from diesel engine)
and a variety of combustion products including some high
molecular weight organics. The thermogravimetric analysis
of these samples allows the determining in a short time of
the volatile content associated to the particulate by weight
loss versus temperature (T). Furthermore the first deriva-
tive of the TG curves (dW/dT) allows the distinguishing of
the unreacted fuel, the lubricant oil and the combustion
products capable of being vaporized up to 600° C in inert
gas flowing.
Fig. 6 shows, for example, the thermogravimetric curves
related to a soot sample collected immediately to the
downstream spray zone of the oil flame. The total weight
loss up to 600° C was 12.3% of which a large amount was in
the temperature range betweeen 100° and 240° C. The first
56
-------
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r
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moo
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time (minutes)
Figure 6. Thermogravimetric curves related to a soot sample
collected immediately to the downstream spray
zone of the oil flame. Total weight loss=12.3%.
57
-------
derivative illustrates this result better: there is a very
big peak with a maximum at 140° C and another evident peak
has a maximum at about 600° C. The big peak was very
similar to that obtained in calibration curves of diesel
fuel on carbonaceous support and was attributed to the
unreacted fuel; the percentage calculated of diesel fuel was
7.1%. Other analyses of particulate collected inside the
spray zone of the oil flame allowed the tabulation of the
content of unreacted fuel and of combustion products, and
the possibility of having more detailed information on the
structure and properties of the spray combustion of liquid
hydrocarbons. The TG results, moreover, were compared with
those obtained on optical measurements based on laser light
scattering used to distinguish fuel droplets and particulates
in the spary zone (8). From the comparison of the first
results it seems that these two techniques together can give
a more detailed information on particulate formation in
practical combustion devices.
Fig. 7 illustrates the thermogravimetric curve and the first
derivative of soot collected in the combustion zone of an
oil flame. The total volatile content was 3.3%; the peak
related to unburned fuel had completely disappeared, while
that of combustion products was particularly evident.
Fig. 8 illustrates the thermogravimetric curve and the first
derivative of a particulate sample exhausted from a N/A 2.4
1 displacement 4-cyl DI Diesel engine without oxidation
catalyst. The organic content was 39%, of which the part
evaporated up to 250° C accounts for about 50%. The first
derivative shows the peak of the unburned fuel and that of
combustion products, less evident in this analysis was the
peak of lubricant oil, with a maximum at about 300° C.
There is also to note that the first derivative shows, at
low temperature, the presence of compounds produced from
the combustion process, by the cracking of higher molecular
weight materials.
Fig. 9 shows the thermogravimetric curve and the first
derivative of particulate sample exhausted from the same
N.A. Diesel engine of Fig. 8 with oxidation catalyst. The
total volatile content in this soot was less than that of
Fig. 7, 17.6%. The distribution of unreacted and combustion
products is particularly evident from the first derivative:
the peak with a maximum at 60° C gives evidence of the
cracking products from higher molecular weight materials,
the peak at 180° C of unreacted fuel, the peak at 300° C
of lubricant oil, the last peaks of the combustion products.
For a more significative characterization of the TG peak of
unreacted fuel associated to particulates, we have analyzed
soot samples as collected by direct sampling in a gas
chromatograph.
58
-------
i-lnoo
Figure 7. Thermogravimetric curves related to a soot sample
collected in the combustion zone of the oil flame.
Total weight loss=3.3%.
ritmo
2.1
fib
Lime (minutes)
Figure 8. Thermogravimetric curves of a particulate
exhausted from a diesel engine without oxidation
catalyst. Total weight loss=39%.
59
-------
to.1
9.1
4b
1000
-800
-bUD
400
-too
tjme (minutes)
Figure 9. Thermogravimetric curves of a particulate
exhausted from a diesel engine with oxidation
catalyst. Total weight loss=17.6%.
60
-------
Figs. 10 and 11 show the gas chromatographic results for
participate collected inside the spray zone of the oil flame
and of the fuel, respectively. The distribution of peaks
is about the same, this means that the attribution of the TG
peak at about 170° C to the unburned fuel was correct.
Several significant differences were noted, instead, in
the gas chromatogram for particulate collected downstream
of the spray zone, Fig. 12. The first part of the chro-
matogram can be ascribed to unreacted fuel, the second
part shows peaks of combustion products; nevertheless,
also in this soot the content of unburned fuel can be
correctly calculated.
Particulate samples produced in spray oil flame at dif-
ferent heights along the vertical axis (that correspond to
different stages of the combustion) and by Naturally
Aspirated (NA) and Turbocharged (TG) engines, in some
cases with oxidation catalyst (ox. cat.) and/or exhaust
gas recirculation (EGR), were extracted with dichloro-
methane (DCM). Table 1 shows extraction and the volatile
content determined as percentage of weight loss of soot as
collected by thermogravimetric curves (TGI). TGI values
exhibit a volatile content of particulates much higher
than those obtain by DCM extraction. This means that the
extraction procedure was not complete and in some cases
the extracted organics account for about 50% of the
volatile content of the particulate.
In the same Table 1 can also be seen the thermogravimetric
weight loss values of the particulate analyzed after DCM
extraction (TG2). These values confirm that the DCM
extraction was not complete. In the last line of Table 1
are shown the calculated differences between TGI and TG2
(ATG). A good correspondence was observed between these
values and those of DCM organic extract.
An example of thermogravimetric analysis of a particulate
before and after DCM extraction is reported in Fig. 13.
This particulate was collected immediately downstream of
the spray zone of oil flame. The comparison shows that
the higher compound content remains unextracted in soot
particles.
For the characterization of this higher compound content,
we have extensively analyzed the particulate samples,
before and after extraction, by direct probe-mass spectro-
meter interfaced with a data system. In this connection
particular attention has been devoted to the content of
higher polycyclic aromatic hydrocarbons (PCAH).
The following experiments were carried out on soot pro-
duced in oil flame spray.
-61-
-------
Figure 10. Gas chromatogram of a particulate collected
inside the spray zone of the oil flame.
Figure 11. Gas chromatogram of the fuel.
62
-------
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64
-------
4000
ib-
40 ,. , , bO
time (minutes)
lODO
20
time Cminuta)
Figure 13. Thermogravimetric curves of particulate, before
(A) and after DCM extraction (B). This particu-
late was collected immediately downstream of the
spray zone of the oil flame.
65
-------
Fig. 14 illustrates the mass spectrum related to the higher
m/e ratios analyzing soot samples as collected. The spec-
trum shows clearly that compounds with high molecular masses
are associated with soot: the most prominent ions are 350,
374, 398, 400, 424, 448 and 472, corresponding to some
possible aromatic structure with from 8 to 11 rings.
Fig. 15 shows the profiles versus time (and temperature) of
the total ion current (TI) and of single ions, attributed to
PCAH, for soot collected in the combustion zone of oil
flame, similar to that of Fig. 7. Of course, vaporization
carried out under vacuum and a temperature programmed up to
500° C, at 40°/min, does not separate the compounds of the
mixture one by one; nevertheless, the ability of the on line
computer to store and follow the spectra taken during the
analysis can help to give useful information and evalutions.
Moreover, the profile of the total ion current, reproduces
with good approximation the peak of the first derivative
related to the combustion products of Fig. 7.
Fig. 16 shows the mass spectrum of the soot after DCM
extraction. The spectrum confirms the information obtained
by TG analysis about the presence of substances at higher
molecular mass in soot after extraction and that the most
prominent masses correspond to the higher PCAH.
Bearing in mind that the mass spectra of unsubstituted PCAH
consists of a very intense molecular ion accompanied by a
few weak fragment ions, it was possible to obtain a semi-
quantitative determination of PCAH present in soot before
and after DCM extraction.
Figs. 17 and 18 illustrate some examples of the profiles of
molecular ions of different PCAH obtained by direct probe
analysis of soot before and after extraction. The PCAH with
lower molecular masses, such as 202, 226 and 252 (Fig. 17)
present in soot before extraction are absent in soot after
extraction. The result is different in the profiles of
higher PCAH: the area of molecular ions 374 and 398 in soot
after extraction (Fig. 18) represent a significative percen-
tage as compared to that of soot before extraction and this
percentage is higher for the ion 398.
An example of spectrum related to soot after DCM extraction
is reported in Fig. 19. Also in this spectrum the presence
of PCAH with higher molecular masses is evident.
The computer calculated areas under the peaks related to
molecular ions of PCAH for soot before and after extraction
are listed in Table 2, where the calculated area ratio
percentages of ions are also reported.
66
-------
tee
ae
S0
40
20
0
100
80
68
40
20.
e.
ill
II,
llm
l||li;,i,l,li
II
340
Illl
Illl
III
IllllllllHI
li.
see
iilniilll!iiili!i!i
I'MM|IM!'MM|MI
360
5^.0
ll
IliillllllMl
Illllllllllllllll
1
lii
380 40C 420
54
t 560 SB
e
liiii||iii|i|i
llbLi
440 460 480
600 ese £4«
Figure 14. Mass spectrum related to the higher m/e ratios
of soot collected in oil flame.
67
-------
472.3
424.2
398.2
374.0
350.0
300.0
276.0
252.0
226.0
202.0
A=I5248
A=4I864
A=47727
A=79I88
A=73I26
A=I2I849
16
Figure 15. Profiles versus time (and temperature) of the
total ion current (TI) and of single ions,
attributed to PCAH for soot collected in oil
f1ame.
68
-------
00-
50 100 150 200 250 300 350 400 45O 500
Figure 16. Mass spectrum of soot after DCM extraction.
69
-------
IOO-
8O-
60-
40-
20-
300-
200-
100-
0
SOOT AS COLLECTED
.202
30 60 90 120 ISO ISO 210
SOOT AFTER EXTRACTION
340
370
400
430
100-
226
30 60 90 120 ISO ISO 210
SOOT AFTER EXTRACTION
250
340
370
100-
0-
60-
40-
20
200-
100-
SOOT AS COLLECTED
292
SO «0 90 120 ISO 180 210
SOOT AFTER EXTRACTION
r-
'282
220
250 2SO
310
370 400
430
Figure 17. Profiles of molecular ions of PCAH at lower
molecular masses obtained by direct probe mass
spectrometry of soot before and after extraction.
70
-------
600!
500
400-
300-
200-
100-
300
200-
IOO-
30
SOOT AS COLLECTED
374
60 90
120 ISO 15 ISO 210
220
SOOT AFTER EXTRACTION
374
280
4OO 430
200-
396
0 30 60 90 120 ISO ISO 210
SOOT AFTER EXTRACTION
220 250 280 310 340 370 400 430
Figure 18. Profiles of molecular ions of PCAH at higher
molecular masses obtained by direct probe mass
spectrometry of soot before and after extraction.
71
-------
lee ne 120 130 140 ise 160 ina 180 138 zee 210 220 230 240 250 260 270
, 1
II.L ,.i..i.llllil. . .1....
|llll|ll.).ll|llll|IHjUU,UT.₯..l|,ll|)i.|,^.1~r
280 290 306 318 328 336 348 350 360 378 380 390 400 410 429 430 440
450 460 470 480 490 WO 510 32C 530 540 550 560 370 580 590 600 618 620
Figure 19. Mass spectrum related to soot after DCM extrac-
ti on.
72
-------
CQ
4-1 H
0 o
3 O
s-i in
4J
CO CW
O
CJ
H 01
4-1 00
nJ CB
£5 4-1
O C
^J CU
td o
i-l 01
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ex
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cu -H
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ex M
n)
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cu O
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td o
m
-------
These values are almost negligible up to 300 m/e, and
increase very quickly at higher m/e values, showing that
the more incomplete DCM extration was the higher the mole-
cular masses of PCAH were.
This significant lower yield of extraction is better depicted
in Fig. 20. The efficiency in DCM extgraction from soot of
unsubstituted PCAH from seven-ring polycyclic compounds
(coronene) to ten-ring (ovalene) decreases rapidly down to
very low values. In our opinion, it does not fall to zero
at higher masses because these compounds are difficult to
vaporize even when the soot was heated up to 500° C.
A much greater fraction of the high molecular weight PCAH
extracted with DCM was present in the transitional fraction
(TRN). This fraction was dried and then analyzed by direct
probe mass spectrometry. Figs. 21-23 show the resulting
mass spectra at different times of analysis of the fraction.
Molecular ions of higher PCAH can be recognized in the
spectra, such as 350, 374, 376, 398, 400. Figs. 24 and 25
show the profile of some molecular ions of PCAH present in
TRN Fraction, and give an idea of their relative abundance.
There are some problems associated with the diesel particu-
late collection system usually employed. In fact filtration
of diesel exhaust hinders to achieve a sufficient particulate
amounts useful for a their complete chemical characterization
by means of thermogravimetry, gas chromatography and mass
spectrometry. The collection efficiency of filters recom-
mended by EPA as printed out in references (9,10) and the
plugging of filters resulting from the particulates going
inside the filter pores does not allow to remove the parti-
culate from the filter surface.
A collection system based on the thermal gradient effect
appears the most promising in order to collect the very low
size (< 0.2 y) non conductive diesel particles with high
efficient and in the same time to allow scraping the parti-
culate on the thermal precipitator walls.
Preliminary mass spectrometric analyses of diesel particles
have shown that there are a few differences between the
combustion product distribution associated to these par-
ticles and that associated to particles collected in oil
flame spray. The less controlled combustion in diesel
engines generates a greater variety of combustion products,
above all some partially oxidized organics.
However, we are also going to carry out analysis on diesel
particulates, before and after extraction, in thermogravi-
metric apparatus working under high vacuum and interfaced
with mass spectrometer. This combined system may allow to a
74
-------
b"IOO
ce
o
o
50
200
300
400
500
Figure 20. The efficiency in DCM extraction from soot of
PCAH at different molecular masses.
120 140 160 ISO 200 220 240 260 280 300
320 340 360 380 400 420 440 460 480 500
Figure 21. Mass spectrum 68 of the transitional fraction.
75
-------
120 140 160 180 200 220 240 260
.li .-iJIL ..III...
270 290 310 330
350 370 390 410
,XIO
420 440 460 480 500 520 540 560
Figure 22. Mass spectrum 133 of the transitional fraction.
120 140 160 180 200 220 240 260 280 300
L
"ffcf-f-E
320 340 360 380 400 420 440 460 480 500
Figure 23. Mass spectrum 147 of the transitional fraction.
76
-------
TRN
580 610
3000-
2000-
1000-
580
640
670
700 730
380
Figure 24. Profiles of molecular ions of some PCAH present
in the transitional fraction.
77
-------
TRN
'400
600 620 640 660 680 700 720 74O
200
100'
0-1
980
600 620 640 665 680 700 720 740
2000
580 »00 820 84O 66O 68O 700 720 740
Figure 25. Profiles of molecular ions of some PCAH present
in the transitional fraction.
78
-------
direct and better characterization of the thermogravimetric
curves and the first derivative peaks, and to carry out
analysis in a very short time, without manipulations.
CONCLUSIONS
The experimental results presented suggest the following
final considerations:
1) Thermogravimetric analysis of soot samples collected
inside and downstream the spray zone of oil flame allows
the calculation of the unreacted fuel and the combustion
products, giving a more detailed information on the
structure and properties of the spray combustion of
liquid hydrocarbons. The same analysis carried out
on particulate exhausted from a diesel engine shows
cracking products from higher molecular weight mater-
ials, the unreacted fuel, the lubricant oil and com-
bustion product at high molecular mass;
2) The comparison of volatile organics associated to
particulate obtained by TG and the DCM extractable
materials shows that compounds at high molecular mass
remain adsorbed in the particulate, both produced in
oil flame and in a diesel enigne;
3) The direct sampling in mass spectrometer-data system, by
a heated solid probe, of particulates, collected in oil
flame spray, gives a detailed information of the highest
molecular weight compounds adsorbed on particulate. The
same analysis carried out on particulate after DCM
extraction can evidence higher compounds, mainly PCAH
that remain adsorbed in soot. The compared analysis of
soot before and after DCM extraction evidence that the
extraction is more incomplete the higher the molecular
masses of PCAH were;
4) Direct mass spectrometric analysis of transitional
fraction of extracted organic contains PCAH at high
molecular masses;
5) Other diesel particulate collection systems, different
from the mechanical filtration till now employed, have
to be performed in order to obtain sufficient amount of
particulate for its complete chemical characterization
and health effect evaluation.
79
-------
ACKNOWLEDGEMENTS
The authors wish to convey their appreciation to Ron Bradow
by U.S. EPA-Mobile Source Emission Research for his helpful
criticism and Joseph Sturm by U.S. DOT-Transportation System
Center for assistance on regulated and unregulated emission
measurement. Thanks are also extended to Anna Ciajolo for
useful cooperation in experimental work.
REFERENCES
1) Beretta, F., Cavaliere, A., D'Alessio, A., Noviello,
C.: "Visible and U.V. Spectral Emission and Extinction
Measurements in Oil Spray Flame", Combust. Sci. Techn.,
in press.
2) Huising J. et al.: "Application of Bioassay to the
Characterization of Diesel Particulate Emissions",
Symposium on Application of Short-term Bioassay in the
Fractionation and Analysis of Complex Environmental
Mixtures, 1978.
3) DiLorenzo, A.: "Direct Gaschromatographic Analysis of
Polycyclic Aromatic Hydrocarbons in Soot Samples" Chim.
Ind. (Milan), 55_, 573 (1973).
4) DiLorenzo, A.: "Analysis of Organic Air Pollutants
Using the Combination of Thermogravimetry and Mass
Spectrometry", Fourth International Clean Air Congress
of the International Union of Air Pollution Prevention
Association, Tokyo, May 1977.
5) DiLorenzo, A.: "Analysis of Higher Polycyclic Aromatic
Hydrocarbons in Soot Samples by Using Direct Probe Mass
Spectrometry", 8th International Mass Spectrometry
Conference, Oslo, Aug. 1979.
6) Zweidinger, R.B. et al.: "Characterization of Extrac-
table Organics in Diesel Exhausted Particulates",
Symposium on Diesel Particulate Emissions and Measure-
ment Characterizations, May 1978.
7) Loprieno, N., DeLorenzo, F., Cornetti, G. M., Biaggini,
G.: "In-Vitro Mutagenicity Analysis of Diesel Particu-
late Extracts by Different Genetic Systems", This
Symposium.
8) Cavaliere, A., D'Alessio, A., Noviello, C., Venitozzi,
C.: "Laser Light Scattering Measurements in Spray Oil
Flame", XXXIII ATI Congress, Palermo, Oct. 1979.
80
-------
9) FIAT Response to EPA NPRM on "Particulate Regulation for
Light-Duty Diesel Vehicles", March 20, 1979.
10) Bassoli, C., Cornetti, G. M., Biaggini, G., DiLorenzo,
A.: "Exhaust Emissions from a European Light Duty
Turbocharged Diesel", SAE Paper n° 790316, Detroit,
Feb. 1979.
81
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PREPARATION AND CHARACTERIZATION
OF DIESEL EXHAUST PARTICLES
FOR BIOLOGICAL EXPERIMENTS
Jean L. Graf
Fine Particles Research Section
IIT Research Institute
Chicago, Illinois
ABSTRACT
Bioassays of diesel engine exhaust components are being con-
ducted at IITRI to determine toxic and carcinogenic poten-
tials of the exhaust. The bioassay method, intratracheal
instillation of saline suspensions of test materials in ham-
sters, requires preparation of stable suspensions of test
materials.
A method to prepare suspensions of whole particle diesel ex-
haust in saline has been developed. The diesel exhaust par-
ticle material was supplied to IITRI as a dry, loose powder
by the U.S. EPA from a light duty diesel test engine. Pre-
liminary characterizations of the powders indicated aggrega-
tion of exhaust particles had occurred both before and
during capture on collection substrates. Flake-like sheets
and hollow spheres of aggregated particles up to 150 ym in
size were present in the powders. Therefore, the powders
were ball-milled to geometric particle sizes more amenable
to the animal administration technique to be employed.
Grinding, suspension preparation and particle concentration
assaying methods have been developed. Particle (geometric)
size and morphological characterizations have also been per-
formed on the as-received powders and prepared suspensions.
A method to prepare emulsions (liquid-liquid suspensions) of
the dichloromethane extracts of whole particle diesel ex-
haust has also been developed.
82
-------
INTRODUCTION
Under contract to the U.S. EPA Biomedical Research Branch,
IIT Research Institute (IITRI) is conducting studies to de-
termine the acute toxicity of diesel engine exhaust compon-
ents when administered to hamsters by the intratracheal in-
stillation method of Saffiotti (1). The Saffiotti model is
designed to determine if the instilled materials possess
carcinogenic and/or co-carcinogenic activity relative to the
pulmonary epithelium.
Two types of diesel engine exhaust components are being
evaluated: whole particle exhaust which is a carbonaceous
soot with adsorbed liquid and gaseous species; and a dichlor-
omethane extract of the whole particle exhaust. Both types
of materials are being supplied to IITRI by the U.S. EPA
Mobile Source Emissions Resources Branch. The methods of
exhaust generation, capture and characterization have been
described elsewhere (2). Briefly, exhaust generated by a
350 Oldsmobile test engine was diluted and captured on a
20" x 20" teflon-coated glass fiber filter. Continuous op-
eration of the engine resulted in deposition of sufficient
solid exhaust material to allow scraping of the exhaust ma-
terial from the filter. The whole particle diesel exhaust
was supplied to IITRI as a loose powder, packaged in glass
bottles purged with nitrogen and frozen with dry ice.
Extractions of the diesel exhaust-laden filters were per-
formed by the EPA with dichloromethane (3) to prepare the
second type of diesel exhaust material to be bioassayed.
The extracts were supplied as frozen 86 mg/ml solutions in
dichloromethane.
The Saffiotti intratracheal instillation method requires
preparation of these two types of diesel exhaust materials
as stable suspensions in saline. Gelatin is typically used
as a protective colloid for suspension stability purposes.
The Saffiotti method also utilizes inert carrier dusts such
as ferric oxide (Fe203), to which the test materials are at-
tached, or at least are intimately mixed with, in order to
increase the retention time of the test material at the tis-
sue test site. The bioassay test protocol requires prepara-
tion of various concentrations of the two types of diesel
engine exhaust materials as gelatin-saline suspensions.
This paper describes methods of suspension preparation and
characterization.
Whole Particle Diesel Exhaust Suspensions
Microscopical examination of the as-received diesel exhaust
powder revealed no significant contamination by teflon and
glass fibers from the collection substrate, but that severe
agglomeration and aggregation of the submicrometer exhaust
83
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particles had occurred. The presence of large diameter (up
to 70 ym), hollow carbonaceous spheres indicated some aggre-
gation of the individual exhaust particles had occurred in
the exhaust stream, before deposition on the collection fil-
ter. The predominant particle morphologyflakes of aggre-
gated particles ranging in thickness from near 0 to 0.5 ym
and up to 150 ym in lengthecu!d have been formed both be-
fore and after deposition on the filter.
To be suitable for intratracheal suspension, the diesel ex-
haust powder aggregates and agglomerates had to be reduced
in geometric size, preferably to a point where 90% by mass
of the material was below 10 ym in size. Attempts to dis-
perse the powder into its constituent submicrometer parti-
cles by conventional deagglomeration techniques such as ul-
trasonics confirmed the conclusion that the diesel exhaust
particle morphologies observed microscopically were indeed
aggregates rather than simple agglomerates. Further micro-
scale testing demonstrated that the only way in which the
exhaust particulate material could be dispersed into the
individual submicrometer particles was by dissolving the
condensed organic species (extractable materials); these
sorbed species served as a cementing agent that bound the
ultrafine carbon particles in sheets and hollow spheres.
Since microscopical analysis indicated that some of the ag-
gregates had been formed before the exhaust was captured on
the filter, the most logical approach to reducing the parti-
cle size distribution of the powder to a range suitable for
intratracheal instillation was to crush the aggregates.
The low density of the diesel exhaust powder, its wide par-
ticle size range and its electrostatic nature precluded the
possibility of dry grinding the powder before suspension in
gelatin-saline. The hydrophobic nature of the powder and
sterility requirements suggested that size reduction and
suspension in gelatin-saline were best accomplished in one
operation.
Before size reduction-suspension methods could be evaluated,
a suitable wetting agent for the diesel exhaust powder had
to be selected. Although some degree of wetting could be
achieved by lengthy, vigorous agitation of very small quan-
tities of the powder with the gelatin-saline, the quantities
and concentrations of suspension required for intratracheal
instillation ruled out this approach to wetting. Reagent
grade propylene glycol was finally chosen as a suitable wet-
ting agent, based on its relatively low toxicity (4), misci-
bility with saline and non-solubility (at room temperature)
of the extractable components of the diesel exhaust.
Low and high intensity ultrasonic treatments, conventional
vortex mixers and high speed shear blenders were evaluated
84
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as methods of simultaneous exhaust particle size reduction
and suspension in gelatin-saline. None of these techniques
sufficiently dispersed or reduced sizes of the diesel ex-
haust particles. A ball-milling technique, developed at
IITRI for preparation of benzo(a)pyrene-gelatin-saline sus-
pensions (5), was finally selected as the method of suspen-
sion preparation. Milling apparatus consists of wide-mouth,
cylindrical pyrex glass jars with silicon rubber-lined plas-
tic caps; 3-5 mm solid pyrex glass beads; and a variable
speed roller. Milling vessels containing the appropriate
quantities of gelatin and saline were autoclaved before the
propylene glycol-wetted diesel exhaust particles were added.
Three concentration ranges of diesel exhaust particle sus-
pensions were each to be prepared with and without equiva-
lent mass concentrations of Fe203 carrier dust. Table 1
lists the composition of suspensions prepared. Each con-
TABLE 1. COMPOSITION OF WHOLE PARTICLE
DIESEL EXHAUST SUSPENSIONS
Dose Ranges 5, 3 and 1 mg/0.2 ml
Carrier Liquid Saline with 0.5% w/v gelatin
Wetting Agent Propylene glycol -7% by volume
Carrier Dust Fe203, 5, 3 and 1 mg/0.2 ml
centration was prepared individually rather than by dilution
of the highest concentration suspension.
A milling time of 10 days was required to reduce 75% by mass
of the diesel exhaust aggregates below 10 ym. A longer
milling time was required to reach the desired size of 90%
by mass below 10 ym, but significant contamination of sus-
pensions with glass fragments began to occur after 10 days.
Table 2 compares the size distributions of the as-received
and milled diesel exhaust particles.
85
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TABLE 2. PARTICLE SIZE DISTRIBUTIONS OF
WHOLE PARTICLE DIESEL EXHAUST
Geometric
Diameter (vim)
0
1.0
3.0
5.5
8.0
10.5
13.0
15.5
18.0
20.5
Cumulative Number %
Greater Than Size
As Received
100
83.9
54.3
21.9
10.2
5.4
2.3
1.0
0.5
0.2
Milled
100
83.7
48.2
20.5
7.9
2.6
1.0
0.3
0.1
0
Cumulative Mass %
Greater Than Size
As Received
100
99.7
97.2
82.7
63.0
43.6
26.0
14.4
7.3
2.5
Milled
100
99.6
95.7
76.1
48.1
25.3
12.4
4.7
1.5
0
Particles in the suspension quickly agglomerated after mill-
ing halted, but could be readily redispersed by simple
shaking. However, aged suspensions required more vigorous
deagglomeration methods and suspensions stored at room tem-
perature or in a conventional refrigerator for four weeks or
more could not be deagglomerated.
Several different types of assays were developed to deter-
mine the diesel exhaust particle and Fe203 concentrations in
the final suspensions. Aliquots of each suspension were
filtered through tared 0.05 urn pore size membrane filters.
This total suspended particle assay provided direct deter-
mination of diesel exhaust particle concentration in those
suspensions not containing the Fe203 carrier dust. Diesel
exhaust particle concentrations in the suspensions contain-
ing the Fe203 carrier dust were determined by low tempera-
ture ashing the filter containing both particle types (from
the total suspended particle assay). The material remaining
after ashing was the Fe203 plus a predictable (6) quantity
of filter ash. Table 3 presents typical results of the fil-
tration and ashing assays. Reproducibility of the filtra-
tion technique was determined to be ±3%. The two different
assaying methods produced diesel exhaust particle concentra-
tions within ±5% of each other.
86
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Determined diesel exhaust particle concentrations in the pre-
pared suspensions were consistently lower than the theoreti-
cal concentrations by 7 to 10%. The low concentrations can
be attributed to two factors: solubility of some diesel ex-
haust components in saline; and the difficulty in completely
transferring a particle-liquid suspension from one vessel to
another, particularly during separation of the grinding media
from the suspension.
Diesel Exhaust Extract Suspensions (Emulsions)
Upon removal of the dichloromethane carrier solvent under a
slow stream of nitrogen, the diesel exhaust organic extract
was found to be comprised of two phases: a light amber oily
liquid; and a semi-solid, dark brown tarry material.
Neither material was appreciably soluble in or miscible with
saline. Therefore, a wetting agent which, as in the case of
the whole exhaust particles, was relatively non-toxic and
miscible with saline had to be selected before emulsion pre-
paration could proceed. The two-phase nature of the diesel
exhaust extract and the semi-solid nature of the one extract
phase required that the wetting liquid essentially dissolved
the semi-solid phase. Without solvation, there was no prac-
tical method for removing the dichloromethane carrier sol-
vent and transferring the sol vent-free extract to an appro-
priate mixing vessel, or thoroughly mixing the saline with
the solid phase in the vessel in which the solvent removal
had been carried out.
Since propylene glycol had already been introduced into the
bioassay system for the whole diesel exhaust particle sus-
pensions, this was the first solvent tested for use in pre-
paration of the diesel exhaust extract emulsions. At room
temperature, the semi-solid phase of the diesel exhaust
extract was not particularly soluble in the propylene glycol;
however, by gentle heating (50-65°C) the semi-solid diesel
exhaust extract phase became more fluid and essentially dis-
solved in the propylene glycol. The extract oil phase was
not miscible with propylene glycol at any temperature.
With propylene glycol selected as a wetting-solvation agent
for the diesel exhaust extract, mechanical methods for emul-
sifying the extract in gelatin-saline were investigated.
The need of a surface active agent was immediately evident.
Based on conversations with pharmaceutical companies (7) and
U.S. Pharmocopeia data (8), sorbitan monooleate--SPAN-80 (9)--
was selected as a suitable surfactant. Several different
types of conventional mixing emulsifying devices were evalu-
ated for emulsion preparation. Bath-type ultrasonic devices
did not provide enough energy to sufficiently disperse the
extract components in the gelatin saline, nor did vortex-
style mixers. Metal probe-type mixers, including ultrasonic
88
-------
probes and high-speed stirrers, were also found unsuitable
because the extract components preferentially adhered to the
metal surfaces; no amount of ultrasonic energy would prevent
plating out of the diesel exhaust extract on the ultrasonic
probe. High-speed blenders with glass blending vessels
forced deposition of the extract material on the metal
mixing blades, while blenders with metal blending cups re-
sulted in near total loss of the diesel exhaust extract
material on both the vessel walls and mixing blades. Teflon
coated metal mixer components proved to be no better than
the metal mixer surfaces; preferential deposition of signif-
icant quantities of extract material on teflon surfaces
still occurred. Glass appeared to be the most desirable
surface to subject the diesel exhaust extract and gelatin-
saline mixture to; no preferential adhesion of extract com-
ponents occurred. Therefore, glass vessel emulsifying appa-
ratus were evaluated. Standard all-glass Potter-Eljevehm
tissue grinders were found to adequately mix and emulsify
the diesel exhaust extract in gelatin-saline. Advantages of
using such emulsifying apparatus were numerous: mixing ap-
paratus could be sterilized before use; solvent removal from
the as-received exhaust extract could be carried out direct-
ly in the mixing vessel, thereby eliminating transfer losses;
precise weights of extract emulsified with gelatin-saline
could be determined; small volumes of emulsion could be pre-
pared, should emulsion stability be a problem; and glass was
the only possible contaminant which would be introduced into
the system.
Emulsion stability was found to be poor; near complete sepa-
ration of components occurred with an hour of emulsion prep-
aration. Therefore, an additional stabilizing agent, gum
arabic (4), was added to the gelatin-saline before emulsi-
fying with the propylene glycol-diesel exhaust extract
mixture. Table 4 lists the components and their concentra-
tions in the final emulsions. Note that all extract emul-
sions are prepared with Fe203 carrier dust.
TABLE 4. DIESEL EXHAUST EXTRACT EMULSIONS
Dose Ranges 5, 3 and 1 mg/0.2 ml
Carrier Liquid Saline with 0.5% w/v gelatin and 0.25%
w/v gum arabic
Wetting Agents Propylene glycol - 10% by volume and
Sorbitan Monooleate - 0.1% by volume
Carrier Dust Fe203, 5, 3 and 1 mg/0.2 ml
89
-------
Addition of the gum arabic increased emulsion stability to
approximately 12 hours. Although the light oil phase of the
diesel exhaust extract separated within minutes of prepara-
tion, the oil could be redispersed by simple shaking. The
short stability time required that emulsions be prepared
fresh for each instillation. Each extract concentration
range was prepared individually rather than diluted from the
highest concentration emulsion.
At present, no adequate assay method has been developed for
the diesel exhaust extract-saline emulsions because of the
complex natures of both the extracts and the saline carrier
fluid. Assays of the delivered dose of ferric oxide are
performed, however. One concern is the possible loss of
diesel exhaust extract components during heating with pro-
pylene glycol . To minimize this loss, a protective layer of
non-heated saline is added before heating is begun.
FUTURE WORK
Although the toxic and carcinogenic potentials of the vari-
ous non-diesel exhaust components of the suspensions and
emulsions being intratracheally instilled are simultaneously
tested in the bioassay program (control solvents containing
all components except the diesel exhaust material), work is
continuing in whole particle diesel exhaust suspension and
diesel exhaust extract emulsion preparation methodologies.
Other wetting agents, surfactants and protective colloids
which are demonstrated to produce suitable suspensions or
emulsions are being bioassayed. Other mechanical methods of
suspension and emulsion preparation are also being investi-
gated in order to find a mixing device which might allow
elimination of some of the wetting, stabilizing and surface
active agents.
A simple, routinely applicable assay method for the diesel
exhaust extract concentration in the final emulsions is
being developed. In all probability, two assay types will
be developed: one assay will be occasionally performed to
demonstrate that no components of the original diesel ex-
haust extract have been perferentially lost during storage
and handling of the extract; the other assay will be per-
formed on all prepared emulsions to determine diesel exhaust
extract concentration by focusing on one or one group of
diesel exhaust extract components.
CONCLUSIONS
Methods for preparation of stable suspensions of whole par-
ticle diesel exhaust and emulsions of diesel exhaust
(organic) extracts in a saline carrier fluid have been
developed. Suspensions and emulsions are being intratrache-
ally instilled in hamsters to determine toxic and
90
-------
carcinogenic potentials of diesel exhaust components to pul-
monary epithelium.
Methods for assaying concentrations of whole particle diesel
exhaust concentrations in the final suspensions have been
developed. No methods for assaying the concentration of
diesel exhaust extract in prepared emulsions are available;
however, methods are being developed.
ACKNOWLEDGMENTS
This work was performed under U.S. EPA Grant Nos. R806326010
and R806929010.
The author gratefully acknowledges the efforts of Dr. R.
Bradow and Dr. R. Zweidinger of the U.S. EPA in supplying
the diesel exhaust materials for test; Dr. W. Eisenberg,
Mr. K. Brown and Mrs. E. Segers of IITRI for their assis-
tance in characterizing and preparing the suspensions and
emulsions; and Mr. A. Shefner of IITRI for biophysical and
biochemical aspects of the tasks.
REFERENCES
1. Montesano, R., 0. Saffiotti, and P. Shubik. The Role of
Topical and Systematic Factors in Experimental Respira-
tory Carcinogenesis. In: Inhalation Carcinogenesis,
M.G. Hanna, Jr., P. Nettesheim and J.R. Gilbert, eds.
AEC Symposium Ser. 18 U.S. Atomic Energy Commission,
Division of Technical Information, Oak Ridge, TN, 1970.
2. Bradow, R.L. Diesel Total Particle Collection Techniques
at CRC-APRAC Diesel Exhaust Emission Measurement
Symposium, Chicago, IL, April 1977.
3. Private communications with Dr. Roy Zweidinger, U.S. EPA,
Research Triangle Park, NC.
4. Merck Index, Ninth Edition. M. Windholz, S. Budavari ,
L.Y. Stroumtsos and M.N. Fertig, eds. Rahway, NJ, 1976.
5. National Cancer Institute Contract Nos. NIH-69-0275,
NIH-70-2245, N01-CP-33296.
6. Millipore Corp., Catalogue 1978/1979, Bedford, MA.
7. In private communications with a major U.S. pharmaceuti-
cal co., it was learned with European manufactured aero-
sol inhalator type medications (e.g., bronchial asthma
inhalators) already on the U.S. market contain SPAN-80,
and that U.S. manufacturers are preparing similar
medications containing SPAN-80.
91
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8. United States Pharmacopeia, XIX.
9. Atlas Chemical Industries, Inc., Wilmington, DE.
92
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SURVEY AND ANALYSIS OF AUTOMOTIVE.PARTICULATE SAMPLING
K. G. Duleep and R. G. Dulla
Energy and Environmental Analysis, Inc.
1111 North 19th Street
Arlington, Virginia 22209
(703) 528-1900
INTRODUCTION
PARTICULATE EMISSIONS FROM INTERNAL COMBUSTION ENGINES are
viewed by the EPA as being potentially harmful to human
health, and there is much interest in the accurate sampling
and characterization of exhaust particulate. EPA has issued
a Recommended Practice to measure the total weight of parti -
culates emitted by a light-duty vehicle over the Federal Test
Procedure (FTP). Laboratory measurement techniques follow
the general guidelines of the EPA recommended procedure, but
there are many parameters that can be varied or are uncon-
trolled within the scope of the recommended practice. The
objective of this paper is to evaluate the effects of those
parameters that are identified as having a significant impact
on particulate emissions.
The evaluation of these parameters is constrained by the data
available and the errors associated with the data. Compari-
son of data from different sources is sometimes inconclusive
because many parameters are varied simultaneously, making it
difficult to isolate the effects of each parameter. Thus,
the conclusions of the paper reflect the informed judgement
of researchers where the data do not provide enough informa-
tion.
The organization of this paper is as follows: The particu-
late formation process is briefly described and a general
methodology to measure combustion particulate emissions is
93
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outlined. This is done to explain the rationale of the EPA
Recommended Procedure for particulate sampling from automo-
biles. The EPA Practice is reviewed and the differences
possible in the implementation of this procedure are identi-
fied. These differences are grouped into two categories,
namely (a) simulation parameters which affect the amount of
particulate emitted by the automobile and (b) collection
parameters which affect the measured particulate emissions.
The effects of parameters in each group are discussed and the
error introduced by the parameters on measured particulate
emissions are detailed.
PARTICULATE COLLECTION AND RATIONALE
Particulates are defined to be any dispersed matter, both in
the solid and liquid phases, present in dilute exhaust gases
at conditions close to ambient. The lower limit of particu-
late size is not defined clearly, but practical considera-
tions and statistical significance place the lower limit at
about 100 A . This value is associated with some representa-
tive dimension of the particle, such as the diameter for a
spherical particle, or some average dimension for an irregu-
larly shaped particle. Most automotive particulate emissions
are composed of many differently sized particulates; size
descriptions are usually given in statistical distributions.
The size determines the aerodynamic behavior of the particle
and this must be accounted for as well in the measurement.
The following two sections detail the formation process of
exhaust particulates and the rationale for a collection
technique that would duplicate this formation process.
Particulate Formation
Researchers have compared the formation of particulates from
various combustion sources and found that the size of par-
ticulates obtained from natural gas flames, oxyen-acetylene
flames, and diesels is similar. Lipkea and Johnson, (1) who
have surveyed results of particulate emissions studies from
a wide variety of sources, indicate that all combustion pro-
duces primary particles that range in size from 0.01 to 10
microns but vary in composition and physical properties, de-
pending on the type of combustion and fuel used. This find-
ing indicates that similar measurement techniques can be ap-
plicable to the variety of engines currently used for auto-
motive propulsion such as Otto cycle, stratified charge, and
diesel engines.
A simplified diagram of particulate formation in internal
combustion engines is given in Figure 1. Khan, e_t al_. (2)
suggest that the formation of soot particles involves the
pyrolysis of fuel hydrocarbons, both in the gas and liquid
phase. Nucleation, or the formation of embryonic nuclei,
94
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FIGURE 1
PARTICIPATE FORMATION PROCESS IN A DIESEL ENGINE
occures in locally fuel-rich areas where combustion is in-
complete. This process develops because of the extremely
complex turbulent mixing and combustion occuring in the cyl-
inder. Intermediate species are formed in the pre-combustion
zones, and these species have side reactions forming poly-
unsaturated hydrocarbons which lead to the growth of nuclei
into soot particles by aggregation. As the exhaust gas
passes through the manifold, it is cooled somewhat, and the
aggregation processes continue along with the adsorption of
hydrocarbons on the surface of the particulate. Physical
coagulation or "agglomeration" also takes place and, when the
exhaust is released to ambient air, the sudden cooling and
dilution causes the hydrocarbon matter to condense, resulting
in greater agglomeration and absorption. It is at this
diluted level that the particulate matter must be sampled and
measured.
Rationale of Measurement Techniques
It is clear that the exhaust pipe and the atmosphere play a
part in the formation and growth of particulate matter.
Consequently, any laboratory procedure must (a) simulate
engine load and speed conditions occurring in normal use, (b)
simulate flow through the exhaust pipe and dilute the exhaust
emerging from the exhaust pipe with ambient air, and(c)
sample the dilute exhaust in such a way that particulates may
be trapped without interfering with the particulate growth
process or aiding in the formation of artifacts due to mea-
surement. The atmosphere offers the capability of infinite
dilution, a situation that cannot be achieved in any simula-
tion. However, several studies have suggested that particu-
late growth is virtually complete within a few feet of the
tailpipe outlet. Dr. Bradow, in an oral presentation to SAE,
(3) showed that the dilution ratio remains fairly low (around
10:1 to 20:1) in the immediate vicinity of the tailpipe for
an Oldsmobile but rises dramatically (to about 1000:1) as the
95
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air vortex from the automobile roof mixes with exhaust. This
suggests that laboratory simulation can be realistic at
manageable dilution ratios, if particulate formation is
complete near the tailpipe as suggested by the studies.
REVIEW OF SAMPLING TECHNIQUES
EPA Recommended Procedure
The procedure recommended by EPA for particulate sampling and
measurement is an extension of the basic constant volume
sampling (CVS) method used for gaseous pollutant measurement.
It must be noted that the procedure only measures the total
weight of particulates emitted during the Federal Test
Procedure driving cycle. The EPA method involves driving a
car on a chassis dynamometer in accordance with the Federal
Test Procedure test cycle to simulate typical vehicle speeds
and loads. The vehicle tailpipe(s) is connected by a short
length of tubing to a diluton tunnel, where the exhaust gas
is mixed with filtered ambient air at a dilution ratio that
approximates real world conditions. The dilute exhaust is
sampled downstream from the point at which air and exhaust
are mixed together at a distance sufficient to allow the
mixing, hydrocarbon condensation, and agglomeration process
to be essentially complete. The sampling is by means of a
probe and filter arrangement. The net flow through the
tunnel (i.e., the sum of exhaust gas and dilution air) is
kept constant, to provide proportional sampling, by means of
heat exchanger and a positive displacement pump (POP) or a
critical flow venturi (CFV). EPA's entire sampling system is
shown schematically in Figure 2.
Identification of Uncontrolled Parameters
There is wide diversity in the particulate collection schemes
employed, and these differences can exist even within the
scope of the EPA recommended procedure. The differences,
comprised of various uncontrolled parameters, can be divided
into two main categories. The first category classified as
simulation parameters, includes parameters that simulate the
(1) state and heat transfer of the tailpipe; (2) dilution air
properties and dilution ratio: (3) effect of mixing air
and exhaust to maintain time-temperature-concentration
profiles similar to those encountered by the particulate.
The second category, classified as collection parameters,
includes: (1) the tunnel configuration and the effects of
tunnel maintenance procedures; (2) the probe position, type
and length of line to the filter; (3) the type of filter or
collection apparatus used to trap the particulate. All these
uncontrolled parameters can affect the weight, size distri-
bution, organic fraction, and chemical species present on
the particulate.
96
-------
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EFFECTS OF SIMULATION PARAMETERS
As stated previously, simulation parameters affect the
processes that govern particulate formation. This is be-
cause, in simulation, the vehicle is stationary on the
chassis dynamometer and the exhaust is artificially mixed
with a finite amount of dilution air, in contrast to a
moving vehicle discharging exhaust into an infinite sink.
Because of the interaction between the dilution ratio and
mixing rate, these variables are discussed jointly. The
effects of specific simulation parameters on particulate
formation are detailed below.
(1) Tailpipe State and Heat Transfer Simulation
Theory indicates that the temperature gradients in the
tailpipe are sufficiently high to seriously affect the
aggregation, agglomeration, and condensation processes
occuring in the tailpipe. In addition, the tailpipe has
particulate deposits on its walls and produces particulates
due to corrosion and wear. The tailpipe and connecting pipe
roughness also play a part by trapping particulates on its
walls. Simulation of the exhaust pipe state was studied by
Danielson, (4) who showed that particulate formation de-
creases with successive runs at wide open throttle (WOT) (see
Figure 3). In the Federal Test Procedure, after a WOT
preconditioning, diesel emissions decreased 16 percent in an
Oldsmobile diesel but did not decrease in a Mercedes-Benz
300D. This indicates that preconditioning effects are a
function of both the exhaust pipe design and the total par-
ticulate emissions of the vehicle.
The effect of heat transfer on the exhaust pipe is difficult
to examine because airflow under a moving vehicle is diffi-
cult to simulate with a stationary vehicle. However, heat
transfer effects can be estimated in an indirect fashion by
studying the impact of the connecting pipe, which provides
additional surface area for heat transfer. In an unpublished
study, Danielson (5) found that a long connecting pipe
(about 15 ft) with a rough inner surface yielded errors of
+20 percent in particulate emission tests. This error
existed when measuring the total weight of particulates; even
greater errors are possible when measuring soluble organic
matter emissions and individual chemical species. Thus, it
appears that heat transfer, exhaust pipe preconditioning, and
pipe surface roughness could substantially affect particulate
measurement.
(2) Dilution and Mixing of Exhaust
The cooling and dilution of exhaust with filtered ambient air
in the dilution tunnel cause some gaseous hydrocarbons to
98
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PARTICULATE EMISSIONS
CMS/MILE
5.0
4.0 -
3.0 -
2.0 -
1.0 -
OLDSMOBILE
350 D
MERCEDES
300 D
3
TEST SEQUENCE
FIGURE 3
PRECONDITIONING STUDY - WOT PARTICULATE EMISSIONS
condense on the particulate surface. Particulate agglomera-
tion and hydrocarbon adsorption/desorption also occur upon
cooling and dilution. In the EPA method of measurement, the
dilution ratio (or the volumetric ratio of ambient air to
exhaust) is not kept constant during particulate emission
measurement over the prescribed driving cycle. Rather, the
principles of the EPA-specified constant volume sampling
method require that the total flow (exhaust plus dilution
air) through the tunnel be kept constant and, therefore, the
dilution ratio at any instant is inversely proportional to
the exhaust flow. The only requirement for dilution is that
the peak temperature of dilute exhaust not exceed 125 F.
99
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In the dilution tunnel, the dilution ratio simultaneously
determines the final temperature of exhaust, the mixing rate,
and the particle residence time in the tunnel. This is
because, for a fixed tunnel size, increasing the dilution
ratio increases the flow rate through the tunnel which, in
turn, increases the rate of mixing of exhaust and air, and
decreases the time the particle spends in the tunnel before
being sampled. Since the dilution air properties are con-
stant, increasing the dilution ratio lowers the dilute
exhaust temperature and also changes the relative humidity of
dilute exhaust. Most experiments performed on the effect of
dilution ratios do not control for the other variables that
are affected simultaneously. The separation of effects of
the individual variables is not possible from the data.
Several studies have addressed the effect of the dilution
ratio on particulate size distribution. Schreck, e_t al_. (6)
measured size distributions of particulate drawn directly
from the raw exhaust with a jet pump diluter and from the
same exhaust after further transit through the exhaust, 14:1
dilution, and cooling to ambient temperature. They reported
that particulate aerodynamic diameter was approximately
doubled after transit, dilution, and cooling, in comparison
to the former case. Black ahd High (7) measured size dis-
tribution at average dilution ratios varying from 8 to 18 and
found that particulate size distribution shifted towards the
smaller particles with increasing dilution ratios.Laresgoiti,
ejt a^. (8) confirmed this result for dilution ratios varying
from 4 to 12. Their explanation was that increasing the
dilution ratio decreases particle concentration and hence
decreases the coagulation rate. The coagulation rate is
proportional to the square of the concentration of particles
in a given size range, and a lower coagulation rate results
in a larger percentage of smaller particles. Due to the
differences in the sampling train, the results of Schreck, et^
al., are not directly comparable to the other results.
The effect of dilution ratio on the weight of particles
emitted and the soluble organic content of particulate has
been evaluated by several researchers' in comparing the
results; errors have been introduced because of the differ-
ences in sampling and characterization methods used by the
researchers. The differences attributable to the errors
associated with the choice of filters or the choice of sol-
vent for extracting the organics may be greater than the
variable under study, i.e., the dilution ratio. Laresgoiti,
e_t al_. (9)' sampled particulate from a Mercedes-Benz OM616
engine, using a glass fiber filter, and found no difference
in weight of particulate emissions from raw and dilute (8:1)
exhaust. Frisch, Johnson, and Leddy (10) measured particu-
late emissions from a Caterpillar 3208 heavy-duty diesel
engine at various dilution ratios ranging from 0 to 50. They
100
-------
found large increases in total particulate weight with in-
creasing dilution ratios and accounted for this increase in
terms of the total soluble organic fraction (DCM* soluble) of
the collected particulate. The results, illustrated in
Figure 4, also show the effects of mixture temperature and
different filter media on this result. Condensation of
hydrocarbons due to their high initial concentration may
account for these results. Williams and Begeman (11) in-
vestigated a light-duty diesel at various steady state speeds
with two different dilution ratios. At all speeds except
idle, an increase in dilution ratio led to an increase in the
percentage of soluble organic matter (solvent: Benzene+EtOH)
present on the particulate. The effect of dilution on total
weight of particulate was not reported. The results of
Laresgoiti, et^ at_., appear inconsistent, but it may be due to
the fact that their sampling method did not use a dilution
tunnel.
PARTICULATE CONCENTRATION,
MG/STD M3 OF EXHAUST
100 n
60-
40-
3208 Caterpillar Diesel
1700RPM ----- ModeS
BIVIEP - 28 Pounds force 'in? Fuel 2
47mm Fluoropore
O 47mm Glass liber
if Avg insoluble fraction
* 8x 10 in Glass fiber
1
1
30 300
DICHLOROMETHANE
1 1 III!
5
1
200
INSOLUBLE
l l
10
r i
100 90
FRACTION
1
80
50 100
Volume Dilution Ratio
1
70
Mixture Temperature, °F
FIGURE 4
EFFECT OF DILUTION RATIO ON PARTICULATE EMISSIONS
* DCM - Dichloromethane
101
-------
Black and High (12) performed a series of transient tests on
diesel-powered automobiles at different average dilution
ratios ranging from 8 to 20. No significant differences were
found in the particulate emissions or the soluble organic
fraction (DCM soluble) of the particulate. Peak temperature
recorded during the tests was 136 F, but the average temper-
ature was below 125 F. Tests by EPA have extended this
result to very high dilution ratios, up to 275:1. All dilu-
tion values quoted in connection with transient tests are
average values, and instantaneous values can differ by as
much as an order of magnitude between, for example, the idle
mode and high-speed wide-open throttle mode. The results
indicate that the dilution ratio is a less important variable
for transient tests than for steady-state tests.
Cuthbertson, et^ al_. (13) studied the effect of varying
sample temperature alone at constant dilution ratio. The
results, illustrated in Figure 5, are based on driving a car
over the (hot) FTP and show that particulate weight and the
organic content of the particulate are strong functions of
filter temperature. Particulate weight reaches a maximum of
approximately 125 F. Below 125 F, the results showed con-
siderable scatter, possibly due to the capture of light
volatiles on the filter that later are lost or retained,
depending on the pre-weighting process. This result indi-
cates that average filter temperatures may have the greatest
effect on collected particulate weight. The interactions
with the filter are discussed in greater detail in the
following section.
EFFECTS OF COLLECTION PARAMETERS
The collection parameters include the apparatus necessary to
sample the exhaust and collect the particulate material. The
effects of these parameters on particulate emission levels
are detailed below.
(1) Tunnel Type and Preconditioning
In spite of a wide variety of tunnel sizes and lengths used
in measurement tests, particulate emission results can be
affected only if (a) the residence time of exhaust after
dilution changes; (b) particulate matter is lost to tunnel
walls; or (c) particulate matter and/or hydrocarbons are
desorbed from the tunnel wall. Comparisons of emission
results from similar automobiles tested in tunnels having
different residence times indicate no systematic variations
due to tunnel size. Particulate losses to tunnel walls are
generally very small. Daniel son (14) has shown that succes-
sive test results after tunnel cleaning were identical.
Tunnel preconditioning and wall losses are, thus, not an
important factor in particulate emission measurements.
102
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Nevertheless, researchers recommend that for successive
measurements from sources having very different particulate
characteristics (such as gasoline and diesel vehicles), the
tunnel be cleaned to avoid particulate desorption. Similarly,
2 LITRE DIESEL CAR DRIVEN OVER THE FTP
Particulate HC
by TG/HFID
Total Particulates
(Filter Weighing)
..__ Particulate
Solid Carbon
__ HC After Filter
byHFID
TEMPERATURE °C
GRAMS PER TEST
FIGURE 5
VARIATION OF PARTICULATES AND HC WITH FILTER TEMPERATURE
103
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the use of stainless steel walls would preclude desorption of
any organic gases or corrosive particles from the wall,
although there is no evidence that this is a major factor in
measuring participate emissions. EPA's recommendation for an
electrically conductive and grounded tunnel would prevent
electrostatic precipitation of particulates due to charged
tunnel walls.
(2) Sample Probe and Line Configuration
The effects of the sample probe and line can be subdivided
into 1) the need to sample isokinetically and 2) the particu-
late losses occurring in the line connecting the probe to the
filter. There is uniform agreement that, due to the very
small particulate size encountered, there is no necessity to
sample isokinetically. The effects of isokinetic and non-
isokinetic sampling and the effects of sample line length
have been investigated by Black and High. (15) Results
indicate that emission measurements are not sensitive to
probe and line configuration. The effects of probe cleaning
and preconditioning also are not expected to affect measure-
ment results.
(3) Trapping Media and Configuration
Although there has been some investigation of electrostatic
precipitators, the use of filters to trap particulates is far
more common. This later method is useful only for determin-
ing total particulate weight and for chemical characteriza-
tion. Size distribution usually is determined by directly
passing dilute exhaust through an optical or mechanical size
measuring device. The filter technique can miscalculate
factors due to (a) absorption of gaseous hydrocarbons by
filter media; (b) artifact formation due to catalysis of
chemical reactions by filter media. Filter media normally
used are glass fiber, teflon coated glass fiber, and fluoro-
pore membrane filters. Other filters such as quartz fiber
and cellulose ester membrane filter also have been investi-
gated.
Black and High (16) measured the sorption of gaseous organics
with uncoated glass fiber and Teflon coated glass fiber
media. Bench test experiments with gas phase organics alone
indicated that the THC loss across the filter was less than
5% for Teflon coated filters versus 25 +5% of total THC for
the glass fiber filters. Tests with vehicle exhaust showed
that glass fiber filters (GF/AE) measured consistently higher
particulate sample weights than Teflon coated filters; this
difference is shown in Table 1. The difference could be
accounted for by the difference in organic extracts on the
particulate. This appears inconsistent with the results
shown in Figure 3, where higher particulate sample weight was
104
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ters. In a similar study, Cuthbertson, e^t al_. (17) compared
the results of several FTP tests on Diesel cars using Teflon
coated (Pallflex) and glass fiber (Watmans type GF/A) filters
and found both filters gave identical test results. The
reasons for the differences in the results of the three
studies are not obvious but may be due to differences between
different brands of glass fiber filters.
In a more detailed analysis of artifact formation on the fil-
ter, Lee, et_ al_. (18) analyzed various filters with respect
to the sample integrity of B(a)P and other PAH compunds pre-
sent on the particulate. The errors and inefficiency associ-
ated with the extraction and dilution of these compounds are
reflected in the recovery values for B(a)P and PAH compounds
on the filter. The method utilized to study the extent of
degradation of B(a)P was the radiotracer method where ll*C-
B(a)P was used as a model compound and radioactive 11+C
activity was examined. Table 2 illustrates the percentage
recovery of 14C-B(a)P spiked on blank filters after sampling
prefiltered ambient air. It is clear that the occurrence of
B(a)P/gas reactions depends both on the surface characteris-
tics of the filter and the sample flow rate. It is seen that
Tissuequartz and glass fiber filters cause maximum degrada-
tion of the sample. It was further shown that the presence
of diesel particulate reduced the percentage of 14C-B(a)P
recovered from the filter. Many of the PAH in the free
molecular state are reactive and, hence, can be expected to
undergo facile transformations when exposed to gaseous pol-
lutants. The transformations of 1!*C-B(a)P to polar and even
acidic products were found in the presence of diesel particu-
late. The dependence of the transformation on the type of
filter is high but occurs to some extent on all filters. In
the case of Fluoropore filters, the mechanism of 14C-B(a)P
loss is not due to air oxidation of B(a)P, nevertheless the
mechanism is still unknown.
Attempts to compare particulate obtained by filtration with
particulate obtained by other means have led to mixed re-
sults. Researchers at GM (19) compared the deposits built up
over time on the walls of the dilution tunnel with a fresh
filter sample from a diesel vehicle. It was found that the
sample from the tunnel walls had a much lower soluble organic
fraction than the sample on the filter (9% versus 59.6%), but
the solubles were relatively similar in molecular weight dis-
tribution. In a similar study by Cuthbertson, ejt al_. (20),
fresh deposits from tunnel walls showed exactly the same
hydrogen to carbon ratio as the sample on the filter. Since
the results obtained by GM were from tunnel deposits that
were relatively "old," the prospects of desorption of or-
ganics from particulate exists. Tests with stored samples of
particulate (21) on the filter have not shown the effects of
106
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desorption, however. This may be due to the fact that vola-
tile hydrocarbons condense on the filter to form an oily
cross-linked structure that is thermally stable, whereas the
deposits on the tunnel walls are free from such condensation.
SUMMARY AND CONCLUSIONS
The dilution -tunnel and filter method of particulate sampling
affect the sample integrity in a number of ways. This paper
has examined those parameters that were identified as having
affected the weight and character of particulate samples col-
lected; these parameters are dicussed in turn below.
1) The effect of tailpipe heat transfer and preconditioning
on particulate emissions is significant. Particulate buildup
on the walls is caused by wall roughness and the thermal
gradient; the deposited particulate is blown out periodically
especially at non-FTP related driving conditions. The de-
posited particulate may be higher in organics and nitrated
compounds than particulate in the gas stream. Results show
that WOT preconditioning can reduce particulate emissions as
much as 15% on the FTP. (22)
2) The dilution ratio affects several variables simultan-
eously, such as the temperature of the mixture of air and ex-
haust, the rate of mixing, and the residence time of particu-
late in the tunnel. It is difficult to separate the effects
of each of these variables, but it appears that the variable
with the strongest effect is the dilute mixture temperature.
Most researchers show that collected particulate weight dur-
ing steady state tests reaches a maximum at temperatures be-
low 125 F. However, it still is not clear whether this re-
sult is biased due to the filter sampling technique employed.
Transient tests employing variable dilution ratios (and tem-
perature) show particulate emissions to be insensitive to the
average dilution ratio employed, as long as the average
temperature of the sample was below 125 F; steady-state tests
have shown strong effects of varying dilution ratios. This
is an indication that the effects of filtration may be mask-
ing the effects of dilution ratios on transient tests.
3) Sampling diesel particulate on a filter has been found to
lead to a loss of sample integrity due to the continuous
sample flow through the filter. Teflon coated filters have
been found to be superior to other types of filters to
minimize sorption of gaseous organics and catalysis of re-
actions involving organic compounds, but there are some
inconsistent results from glass fiber filters. However,
there is no simple method to evaluate the sorption of or-
ganics by the particulate matter on the filter. The use of
the filtration method leads to the oxidation of PAH compounds
and possible reaction of the PAH with pollutant gases such as
108
-------
NO and sulfates, but the extent of sample degradation shows
a Strong dependence on both sample flow rate and filter type.
The participate sample on the filter also may underestimate
the effect of desorption that normally would occur in atmo-
spheric particulate.
Comparisons of a Teflon coated filter and a glass fiber
filter measuring particulate emissions show a 15% difference
in the total weight of particulate and a 25% difference in
the weight of the soluble organic fraction. (23) B(a)P
recovery on these filters, using 14C-B(a)P spiked filters
that were exposed to a continuous sample of air for 24
hours, was 11% for the glass fiber filter and 71% for the
Teflon coated (Fluoropore) filter. (24)
Table 2 - The Depencence of B(a)P Recovery on Filter
Types and Sampling Conditions (20)
a/
Filter Type Sampling Conditions
Time (hours) 17 8 IS 24 30
Air Volume (m3) 35 80 140 250 145
Amount of B(a)P Spiked
on the Filter (ug)b/ 0.5 0.5 0.5 CLS_ 5_
Glass Fiber 35 - - 11 75
Tissuquartz - - 1.3 - 62
A Micro Glass Fiber
Filter with Teflon
Binder on Fibers - 50 40 86
Fluoropore 60 55 65 71 90
Data listed in the same column represent runs made simultaneously under
identical sampling conditions.
The amount of B(a)P spiked on s
for the first four samples and was ~100ng/cm/ for the last sample.
This run was made separately but undei
those runs listed in the same column.
' The amount of B(a)P spiked on each filter was, ~10ng/(cm filter surface)
This run was made separately but under similar sampling conditions as
109
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REFERENCES
1. ,N.H. Lipkea and J.H. Johnson, "The Physical and Chemical
Character of Diesel Participate Emission," SAE Report
SP-430, 1978.
2. I.M. Khan, et al., "Coagulation and Combustion of Soot
Particles in Diesel Engines," Combustion and Flam, Vol.
17, No. 3, December 1971.
3. R. Bradow, "Sampling Diesel Particles," Oral presentation
at the 1979 SAE Congress, March 2, 1979.
4. E. Danielson, "Particulate Measurement - Vehicle Pre-
conditioning," EPA Technical Report SDSB 79-05.
5. E. Danielson, Oral Communication with the author.
6. R.M. Schreck, et al., "Characterization of Diesel Ex-
haust Particulate under Different Engine Load Conditions,"
APCA Paper 78-33.5, June 25-30, 1978.
7. F. Black and L. High, "Diesel Hydrocarbon Emissions,
Particulate and Gas Phase," presented at Symposium on
Diesel Particulate Emissions Measurement and Character-
ization, 1978.
8. A. Laresgoiti, A.C. Loos, and G.S. Springer, "Particulate
and Smoke Emission from a Light Duty Diesel Engine,"
'Environmental Science and Technology' (11:10) October
1977, p. 973.
9. Ibid.
10. L.E. Frisch, J.H. Johnson, and D.G. Leddy, "Effects of
Fuel and Dilution Ratio on Diesel Particulate Emissions,"
SAE Paper 790 417, 1979.
11. R.L. Williams and C.R. Begeman, "Characterization of
Exhaust Particulate Matter from Diesel Automobiles," GM
Research Publication GMR-2970 ENV #61, 1979.
12. F. Black and L. High, "Methodology for Determining Par-
ticulate and Gaseous Diesel Hydrocarbon Emissions," SAE
Paper 790 422, 1979.
13. R.D. Cuthbertson, A.C. Stinson, and R,W. Wheeler, "The
Use of a Thermogravimetric Analyzer for the Investigation
of Particulates and Hydrocarbons in Diesel Engine Ex-
haust," SAE Paper 790 814, September 1979.
110
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REFERENCES (continued)
14. E. Danielson, "Particulate Measurement - Dilution Tunnel
Stabilization," EPA Technical Report LDTP 78-14, November
1978.
15. F. Black and L. High, "Methodology for Determining
Particulate and Gaseous Diesel Hydrocarbon Emissions,"
SAE Paper 790 422, 1979.
16. Black and High, op. cit.
17. R.W. Wheeler, "The Use of a Thermogravimetric Analyzer
for the Investigation of Particulates and Hydrocarbons
in Diesel Engine Exhaust," SAE Paper 790814, September
1979.
18. F. Lee, et al., "Chemical Analysis of Diesel Particulate
Matter and an Evaluation of Artifact Formation," pre-
sented at Conference on Sampling and Analysis of Toxic
Organics in the Atmosphere, Colorado, August 1979.
19. Schreck, et al., op. cit.
20. Cuthbertson, et al., op. cit.
21. S. Tejada, personal communication with the author.
22. E. Danielson, op. cit.
23. Black and High, op. cit.
24. Lee, et al., op. cit.
General Discussion
R. SCHRECK: Our data is not to be interpreted as saying
that increased dilution produces increased particle size.
What our results have shown is that you can produce dilu-
tion systems that allow for a coagulation effect that will
result in a larger particle size.
K. DULEEP: Certainly there are many differences when
the entrained sulphur being used is compared by other peo-
ple. This is just a comparison of data from various sources.
D. KITTLESON: We have done calculations of coagulation
in different sampling systems and it can be demonstrated
experimentally that an increasing dilution ratio and a
decreasing residence time, acting together tends to result
in smaller particles. If you go beyond a certain limit
however, you freeze out the particle size distribution and
111
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there is no further change. The place where care must be
taken occurs for example, when running something like a ten
to one diluted exhaust, which is typical tunnel dilution,
and having residence times much longer than five or ten
seconds. Than you can start to get significant shifts.
However, if you keep your residence times less than a few
seconds under those conditions you shouldn't have any trouble.
K. SPRINGER: I don't believe that the emissions from a
diesel engine, the particulates, will be the same from hot
filtered raw exhaust as they will be from diluted tunnel
exhaust, and I want to take exception to that.
K. DULEEP: I tend to agree with you. This is just a
review of all the results and some of the contradictions
expressed in the published literature.
112
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A PARTICULATE CHARACTERIZATION STUDY
OF IN-USE DIESEL VEHICLES
G. Wotzak, R. Gibbs, J. Hyde
New York State Department of Environmental Conservation
Automotive Emissions Laboratory
50 Wolf Road
Albany, New York 12233
ABSTRACT
Particulate sampling methods and vehicle test protocols are
described for a study to characterize emissions and fuel
economy from approximately 20 diesel vehicles in consumer use
throughout a two-year mileage accumulation period. At this
time, three diesel cars have been tested by these procedures
to collect large quantities of particulate for chemical
characterization and bioassay studies to proceed in parallel
with testing of in-use vehicles. Primary attention is focused
on particle-bound organics removed by solvent extraction of
filter samples followed by chemical characterization of
extract using GG, HPLC, GC/MS and a new fluorescence mapping
technique identified as total luminescence spectroscopy (TLS).
TLS results are presented for one particulate extract sample
and its sub-fractions to demonstrate the application of this
technique.
INTRODUCTION
In anticipation of increased numbers of diesel automobiles in
future years, New York State, under EPA support, is con-
ducting a study to obtain characterization data from diesel
cars in consumer use. In preparation for repetitive testing
of approximately 20 consumer-use diesel cars over a two-year
mileage accumulation period, bioassay and analytical tools
have been applied to selected samples of diesel particulate,
as described in this and other papers at the current sym-
posium (1-3). These analytical and assay methods will be
subsequently used to characterize particulate samples from
113
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the fleet test portion of the study in which a range of vehi-
cles, fuels, oils, and test conditions will be explored.
Particulate Sampling
The major thrust of characterization effort is aimed at
particulate emissions, although regulated emissions and fuel
economy are routinely measured by the 1979 Federal Test
Procedure for new car certification. Particulate emissions
are measured by the standard dilution tunnel technique as
detailed in the Federal Register Notice of Proposed Rule
Making (4). Some sample-system modifications have been
incorporated, as shown in Figure 1. Mass flow controllers
have been installed in order to regulate particulate sampling
through the 47 mm filters used for determination of emission
rate. A system for collecting large particulate samples has
been added utilizing a 20" x 20" filter holder. Particulate
is collected on the 20" x 20" filter at a nominal 100 cfm
and the total volume filtered in any test segment is measured
as shown in Figure 1. Pallflex T60A20 Teflon-coated glass
fiber filter media are used for both the 47 mm and 20" x 20"
filter systems. Exhaust dilution ratio is adjustable by a
10 HP variable-speed motor driving a positive displacement
pump CVS. With this drive the CVS flow rate is continuously
adjustable from 200-530 cfm, and dilute exhaust temperature
is kept below 52°C for different vehicle types and driving
cycles.
Vehicle Testing
The present testing plan includes three similar one-day phases
to complete each vehicle test. These test phases will employ
different fuel/oil combinations as follows: 1) fuel as
delivered to the Automotive Emissions Laboratory (AEL), oil as
delivered to AEL; 2) AEL control fuel, lubricating oil as
delivered; and 3) AEL control fuel and fresh lubricating oil.
Each day of the vehicle test begins at mid-day with vehicle
preparation and an appropriate fuel/oil switch followed by a
30 min 50 mph cruise to purge fuel lines and precondition
the vehicle. Immediately following preconditioning, the
following cycles are used to determine particulate emission
rates and collect large filter samples: 1) a 30 min 50 mph
cruise, 2) Congested Freeway Driving Schedule (GFDS), 3)
Three successive Highway Fuel Economy Tests (HFET), and 4) a
114
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30 min idle. Gaseous emissions are not measured in this
afternoon sequence. After overnight soak, a morning test
sequence is begun that includes gaseous/particulate emissions
and collection of large filter particulate samples from the
following tests: 1) FTP, 2) GFDS, and 3) Three HFET. Fuel
and/or oil is then switched to begin the next test phase.
Selected 20" x 20" filters from each three-day vehicle test
will be solvent extracted for subsequent chemical characteri-
zation and bioassay, with the remaining filters archived in
freezer storage for future studies.
Figure 1
VCQ
T
DISPLAYS
ACCUMULATION
A NO RATE
VARIABLE
SPEED DRIVE
tO-IOOCFM
CONSTANT VOLUME
SAMPLER POSITIVE
DISPLACEMENT PUMP
SCHEMATIC OF PARTICULATE COLLECTION APPARATUS
115
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Collection of Large Samples
A 20" x 20" filter assembly was used to collect many
filters from a few vehicles to provide large samples for
detailed studies. A Mercedes 300-D was used to accumulate
180 filters for EPA use in evaluating diesel particulate.
The vehicle was operated on a daily schedule that included
one FTP for vehicle warm-up followed by 24 HFET cycles broken
down into blocks of three cycles to yield one filter from the
FTP and eight filters from HFET tests. FTP filters were kept
for project analysis and HFET filters shipped to EPA. After
completion of this 6,000 mile test program, a similar
project of shorter duration was undertaken on the same
vehicle using the AEL control fuel scheduled for use through-
out the in-use vehicle study.
A diesel Rabbit was subsequently operated with one FTP and
24 HFET cycles each day (AEL control fuel) to generate
particulate sample used for detailed characterization
analyses described in this and connected papers at the pre-
sent symposium. A final large sample is now being collected
from an Oldsmobile 350 diesel. Frozen portions of extract
from all of these large samples will serve as reference bio-
assay specimens throughout the in-use vehicle portion of the
study. Background data on the samples from the Mercedes 300-D
and Rabbit are given in Table 1.
Extraction of 20" x 20" Filters
All 20" x 20" Pallflex filters (Type T60A20) used for par-
ticle collection were extracted with dichloromethane in order
to remove the soluble organic fraction. Filters were folded
and placed in 50 mm Soxhlet extractors without thimbles.
Extraction was carried out with 300 ml of dichloromethane
(Burdick and Jackson Laboratories, Distilled in Glass grade)
for 24 hr at three to four cycles per hour.
Extracts were filtered by vacuum through an 0.2 y,m Fluoropore
filter (Millipore FGLP) to remove any particles which may
have been carried over. The extract solution was boiled
before filtering and the filtering apparatus was kept hot
during filtration to prevent precipitation of any material
on the filter.
Solvent was removed from the filtered solutions by heat and/
or vacuum. When a composite of several filter extracts was
being prepared, the volume of each extract was first reduced
and the concentrated extracts were combined. Aliquots of the
composite were transferred to tared vessels and the remaining
116
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solvent removed by vacuum at room temperature. These samples
were desiccated over Drierite and weighed. Extracted
material was fractionated into acidic, basic, and neutral
cuts. The neutral fraction was further fractionated into
seven cuts on a silica gel column with a gradient of hexane
to dichloromethane to ether (2) with partition percentages of
72.9, 6.0, 2.1, 3.8, 4.8, 8.6, and 1.8 for fractions 1
through 7 respectively. Results for the Ames bioassay for
these fractions are given in Table 2. Outpoints for these
fractions were adjusted so that Cut 1 contained parafinic
material and Gut 2 included the unsubstituted polynuclear
aromatic compounds.
Characterization of Organic Extract
In order to characterize organic species adsorbed on carbon-
aceous particles emitted by diesel engines, a combination of
analytical methods and instruments have been employed in a
cooperative effort between the Automotive Emission Laboratory
of the Department of Environmental Conservation and the
Toxicology Laboratory of the Department of Health. The focus
of this effort is twofold. The first program area will be
the identification of patterns and trends of emissions of
adsorbed organic species as a function of engine configura-
tion, fuel, lubricant, and operating conditions. These
results are to be used in conjunction with mutagenic activity
data in order to relate diesel system operation with possible
carcinogenic activity of diesel exhaust. A second task
involves use of analytical "fingerprints" of adsorbed organic
species in order to infer the possible compounds or classes
of compounds which exhibit mutagenic activity. Again, muta-
genic activity data is necessary for a systematic determina-
tion of the identity of compounds with significant mutagenic
impact.
At present, this characterization effort requires four
instrument systems. Capillary gas chromatography is used
for determination of carbon number distributions (5) of
diesel fuel, lubricant, and gross particulate extract. High
pressure liquid chromatography is necessary for fractionation
of the neutral fraction of the diesel extract. Total
luminescence spectroscopy and GC/MS systems are then utilized
in order to obtain "fingerprints" of the composition of these
extract fractions.
Total Luminescence Spectroscopy
As many different techniques as possible will be employed in
an interactive manner in order to explore the many facets of
the qualitative and quantitative problems associated with
characterization of this material. In this paper, we present
some of the capabilities of total luminescent spectroscopy as
118
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an analytical tool for determination of possible compounds
and classes of compounds adsorbed on diesel particulate.
Simple luminescence methods have offered sensitivity and
specificity for analysis of single compounds. For these
methods, use of principle excitation and emission spectrum is
often sufficient for identification of a pure compound. How-
ever, in mixtures of compounds the overlapping of emission
spectra of the individual components often results in the
inability to identify peaks corresponding to individual spe-
cies. Complex mixtures can be analyzed by luminescence
techniques only if total luminescence data is acquired and
analyzed.
Total luminescence data is the observed intensity of lumines-
cence as a function of all accessible excitation and emission
wavelengths. In order to obtain this total picture of
luminescence information, we must acquire a large number of
emission spectra, each taken for a given excitation wave-
length. Contours of equal intensity have been chosen as a
convenient method for display of this voluminous data.
Total Luminescence Spectroscopy (TLS) is the term that has
been chosen for the computer data acquisition, manipulation,
display, and interpretation of total luminescence data. This
method has recently (6-8) been found to be an effective tool
for identifying crude oil spills, as well as toxic and
hazardous materials. Figure 2 illustrates a TLS spectrum
for the neutral fraction of extract from a large pooled sample
produced by a diesel Rabbit. We note that the upper left
region (excitation wavelength > emission wavelength) contains
no luminescence information since no emission can occur in
this region. Although it is not significant in this case,
Rayleigh and Tyndal scattering are centered around the 45°
line where the emission wavelength equals the excitation
wavelength. Even though there is little structure in
Figure 2 because of the superposition of peaks from many
individual species, one characteristic group of peaks does
appear to be present in the region of 320 to 340 nm excita-
tion wavelengths and 340 to 390 nm emission wavelengths.
This structure provides a possible fingerprint of some of the
more dominant compounds in a neutral fraction, which probably
contains thousands of chemical species. Fractionation of
this neutral cut is necessary before more information of the
composition of this sample can be obtained.
TLS spectra for this study were obtained on a Baird Corpora-
tion SFR-100 Ratio Recording Spectrofluorometer. Data
acquisition and scan control for this instrument is locally
provided by a Baird MP-100 microprocessor controller which
is linked to a host Data General Nova 3 Minicomputer system
with 64K words of core and a 10 megabyte disk. Software was
120
-------
11/27/79
Figure 2
350--
250
300
350 400
EMISSION WRVELENGTH (NM)
1450
500
90D05-NEU DIESEL RRBBIT - NEUTRfll IN DICHLOROMETHRNE
MflX.=9.15 X1Q RT 35U.O NM.FM , 30q 0 NM EX.
CONTOUR INTERVRU 5.00 5.00 TO 95.00 PCT OF MHX
121
-------
supplied by Baird Corporation and modified by AEL personnel
with the inclusion of data smoothing algorithems. Contour
plots are produced on a Houston Instruments COMPLOT X-Y
Plotter.
TLS Spectra of Individual Compounds
Some insight into the capabilities of total luminescent
spectroscopy can be provided by consideration of spectra of
individual pure compounds. Spectra for napthalene, pyrene
and fluoranthene in a methylcyclohexane solvent are given in
Figures 3-5 in order to illustrate the wide varieties of
contour structure found for some polynuclear aromatic com-
pounds. An additional indication of the specificity of the
TLS technique is provided by a comparison of spectra obtained
for isomers of the same compound. Figures 6 and 7 illustrate
the great difference between the contour structure of benzo-
(a)pyrene and benzo(e)pyrene, respectively.
An additional dimension to the specificity of the TLS method
is provided by solvent effects on spectra of the same com-
pound. Compound identification during analysis of mixtures
can be aided by taking TLS spectra of the mixture in
different solvents. Solvent effects are minimal for some
compounds but are significant for other species. These
solvent effects can aid in increasing the degree of certainty
for compound identification. For example, there is a signi-
ficant change in the fingerprint for pyrene in dichloromethane
(Figure 8) relative to that found for pyrene in methylcyclo-
hexane (Figure 9). By contrast, benzo(a)pyrene does not show
any marked shifts in spectra with the same solvents (Figures
10 and 11).
TLS Spectra of Diesel Extract Fractions
Organic extracts of exhaust particulate have been obtained
from a 1979 Volkswagen Rabbit Diesel as previously described.
Approximately 200 micrograms of each fraction were dissolved
in 4 milliliters of solvent (methylcyclohexane, dichloro-
methane, or methanol) and run on the TLS system. Results for
these seven fractions with dichloromethane as the solvent are
presented in Figures 12-18.
We note that Cut 1 exhibits a significant fluorescent level.
This behavior is expected since the cutpoint on the separa-
tion scheme was chosen to include as much of the parafinic
material as possible. Therefore, some of the aromatic
portion of the sample was included in this parafinic cut.
A complicated contour structure is noted in the Cut 2 spec-
trum. Identification of compounds by use of the left-hand
side of the contour map is difficult because of the severe
122
-------
11/26/79
350--
o
s
d
300'
250
Figure 3
H 1 1 (-
300
350
400
EMISSION WflVELENGTH
(NM)
450
500
NRP-17. -MCH _NHPTHRLENE -17. MG/L IN METHYLCYCLOHEXflNE
MflX.-1.40 X10~' HT 322.0 NM.FM.. 278 0 NM.FX.
CONTOUR INTERVRL= 10. DO
10.00 TO 90.00 PCT.OF MflX
11/27/79
Figure 4
350--
cr JUU- -
300
EMISSION WHVELENGTH 1NM)
500
PYR-l.O-MCH _P1"RENE - 1.0 MG/L IN METHYLCYCLOHFXflNE
MHX.-4.97 X10 RT 382.0 NM.EM.. 338.0 NM.FX.
CONTOUR INTERVRL= 10 00
:0.00 TO 90 00 PCT.OF MRX
123
-------
.11/27/79
Figure 5
350--
tr 3UU--
300
350 ' 400
EMISSION WRVELENGTH (NM)
450
SOO
FLU-l.O-MCH FLUORRNTHENE - 1.0 MG/L IN METHYLCYCLOHEXRNE
MflX.-8.58 Xio"2 RT 458 0 NM.FM.. 290.0 NM EX
CONTOUR INTERVflL= 10.00
10.00 TO 90.00 PCT OF MRX
11/27/79
Figure 6
350--
E 300-
250
300
350
450
400
FMISSION WRVELENGTH.(NM)
BRP-0 1-MCH BENZOIRIPYRENF - 0 1 MG/L IN MFTHYLC-TCLOHEXRNE
MRX. -4.51 X!0~ RT 402.0 NM.FM.. 300 0 NM.FX.
CONTOUR INTERVRL= 10 00 10 00 TO 90.00 PCT OF MHX.
500
124
-------
11/26/79
Figure 1
350--
E 300--
250
300
350
450
400
EMISSION WPVELENGTH (NMI
BFP-l.O-MCH BENZOIEIPTRENE - 1.0 MG/L IN METHTLCTLOHEXqNE
MRX.-4 C5 X10 RT 386 0 NM EM . 292.0 NM.EX
CONTOUR INTERVPL- 10 DO 10 00 TO 90 00 PfT OF MPX
500
11/27/79
Figure 8
350--
tr 300--
250
300
400
EMISSION WflVELENGTH
450
500
(NM)
PTR-l.O-OCM PYRENE - 1 0 MG/L IN DICHLOROMETHflNE
MHX.-1.39 X10'! HT 390 0 NM.FM.. 340 0 NM.FX.
CONTOUR INTERVflL- 10 00 10 00 TO 90 00 PCT OF MRX.
125
-------
11/27/79
Figure 9
350--
300--
250
300
350 400 45
EMISSION WflVElENGTH (MM)
500
PYR-l.O-MCH ^PYRENE -1.0 MG/L IN METHYLCYCLOHEXRNE
MHX.-I4.97 X10~2 RT 382.0 NM.EM . 338.0 NM.EX
CONTOUR INTERVfll_= 10 00 10.00 TO 90.00 PCT OF MflX
11/27/79
Figure 10
350--
£ 300--
250
300
350
450
400
EMISSION WflVELENGTH (NM)
BRP-O.l-DCM RENZOlfilPYRENE - 0.1 MG/L IN OICHLOROMETHHNE
MRX. -8 13 X)0~ RT 404 0 NM.FM. . 302.0 NM.FX.
CONTOUR INTERVflL= 10.00 10.00 TO 90.00 PCT . OF MHX.
500
126
-------
11/21/79
350--
a 300--
250
300
350
1400
FMISSION WflVELENGTH (NM)
500
BflP-0 1-MCH BENZO(R)pTRENE - 0.1 MG/L IN MFTHYLCYCLOHEXRNE
MflX.-U.51 Xio" RT 402 0 NM EM . 300 0 NM.EX
CONTOUR INTERVRL= 10 00 10 00 TO 90 00 PCT.OF MflX
11/26/79
Figure 12
350--
300--
250
450
500
FMISSION WftVELENDTH (NM)
9000S-C1 DIESEL RflBBIT - CUT 1 IN DICHLOROMETHRNE
MBX.-1.64 X10" m 344.0 NM.EM.. 292.0 NM.FX.
CONTOUR INTERVBL= 5.00 5.00 TO 95.00 PCT.OF MflX.
127
-------
11/21/79
Figure 13
350--
£ 300-r
250
300
350 MOO
FMISSiON UfWELENDTH INK)
450
500
90005-C2
^DIESEL RflBBIT - CUT 2 IN DICHLORGMETHflNE
MBX.-l 32 X10 HT 372 0 NM EM . 340 0 NM.EX.
CONTOUR INTERVflL= 5 00
5.00 TO 95 00 PCT 0F MRX
11/26/79
Figure 14
350--
300
250
350 400
EMISSION WflVELENGTH 1NM)
USD
500
90005-C3 _OIESEL RflBBIT - CUT 3 IN DICHLOROMETHRNE
MRX.-1.53 XIO"' RT 352.0 NM.EM. 292.0 NM EX
CONTOUR INTERVflL= 5.00
5.00 TO 95.00 PCT.OF MflX
128
-------
11/26/79
Figure 15
250
11/26/79
350
WO
EMISSION WfWFLENGTH
450
500
90005-C4-H
(NM)
IN OICHLOROMETHRNF
OIFSEL RflBBIT - GUI <4
MRX -4 55 X10~2 RT 348 0 NM FM. 296 0 NM FX
CONTOUR 1NTERVRL- 5. DO
5 00 TO 95 00 PCT Op MRX
Figure 16
350 +
300
250
300
400 ' 450
EMISSION WflVELENGTH (NM)
9D005-C5-H _DIESEL RflBBIT - CUT 5 IN DICHLOROMETHqNE
MRX.-2.49 X10 RT 350.0 NM.EM. 294.0 NM.FX.
CONTOUR INTERVflL= 5 00 S.OO TO 95 00 PCT.OF MRX.
500
129
-------
11/29/79
11/29/79
Figure 17
250
300
350
400
EMISSION WHVELENGTH (NM)
500
90005-C6-H _DIESEL RRBB1T - CUT 6 IN DICHLOROMETHflNE
MRX.-2.97 X10 flT 348 0 NM.FM. . 294.0 NM.FX.
CONTOUR INTERVRlr 5.00 5.00 TO 95.00 PCT.OF MflX.
Figure 18
250
300
500
EMISSION WflVELENGTH (NM)
90005-C7-H _DIESEL RPBBIT - CUT 7 IN DICHLOROMETHflNE
MflX.-2.20 X10~ flT 352.0 NM.FM.. 290.0 NM.FX.
CONTOUR INTERVflL= 5.00 5.00 TO 95.00 PCT.OF MflX.
130
-------
overlap of spectra in this region. However, the characteris-
tic pattern of benzo(a)pyrene (see Figure 6) is noted in the
upper portion of Figure 13. This benzo(a)pyrene fingerprint
is clearer in Figures 19a and 19b where the same information
as Figure 13 is presented for methylcyclohexane solvent. In
addition, the lower portion of this spectrum is presented
with a fine contour grid. The characteristic ridges of
fluoranthene at 290 and 360 nm (see Figure 5) are also found
in Figure 19b. Gut 2 was also analyzed by TLS in dichloro-
methane as shown in Figure 20. Although the benzo(a)pyrene
and fluoranthene structure is weaker with this solvent, the
pyrene (see Figure 8) spectra is more evident in Figure 20
than in Figure 19a.
Another manifestation of the use of solvent effects in the
compound identification process is illustrated by the con-
tour maps for Gut 5 in methylcyclohexane (Figure 21) and
in dichloromethane (Figure 22). A low overall level of
fluorescence for this fraction results in noisy spectra,
although the smoothed results of Figures 21 and 22 yield a
reasonable amount of contour detail. Although the broad peak
in the area of 285 nm excitation, 450 nm emission is visible
in the methylcyclohexane spectrum, a much clearer picture of
this fluorescent contour structure is found in the TLS
spectrum of Cut 5 in dichloromethane.
Quantitative information can also be obtained from TLS,
Available software enables the plotting of any excitation or
emission scan. For example, the principle excitation and
emission scans for benzo(a)pyrene in methylcyclohexane are
presented in Figures 23 and 24 respectively. Analysis of
TLS results for the 386 nm excitation scan of Cut 2 in
methylcyclohexane coupled with the TLS spectra of pure benzo-
(a)pyrene indicates that benzo(a)pyrene accounts for 0.2
weight percent of Cut 2, which translates to an effective
emission rate for benzo(a)pyrene of 9.0 micrograms per mile.
Before serious extract characterization and compound identi-
fication can be initiated, a library of TLS spectra for
pertinent pure compounds must be produced. The software
library must be modified to incorporate a soon-to-be-released
addition-subtraction program. This program will allow
subtraction of known compounds from the spectra in order to
produce a clearer picture of the remaining contour structure.
In addition, the addition-subtraction routine will allow the
determination of quantitative information for extract
fractions which contain small numbers of fluorescing com-
pounds.
131
-------
11/21/79
Figure 19a
11/28/79
350--
E 300
250
300
400 450
FM1SSION WRVELENGTH (NMI
9005-C2-M DIESEL RRBBIT - CUT 2 IN METHYLCYCLOHEXRNE
MflX.--l.34 X10~! RT 348 0 NM.EM . 300.0 NM.EX
CONTOUR INTERVflL= 5.00 5.00 [0 95.00 PCT OF MflX
500
Figure 19b
300
250
300
yso
EMISSION WflVELENGTH (NM)
9005-C2-M
DIESEL RflBBIT - CUT 2 IN METHTLCTCLOHEXflNE
MHX.-1.84 X10 flT 348.0 NM.EM.. 300.0 NM EX.
CONTOUR INTERVflL= 2.00
500
2.00 TO 40.00 PCT.OF MRX.
132
-------
11/21/79
Fiaure 20
350--
cr 300
250
300
350 MOO ' 45
EMISSION WRVELENGTH 1NM)
90QOS-C2 _D!ESEL RP8BIT - CUT 2 IN DICHLOROMETHRNE
MRX -1 82 X10~' RT 372 0 NM EM . 340 0 NM.EX
CONTOUR INTERVRL- 5 00 5 00 TO 95.00 PCT 0^ MRX
500
11/26/79
Figure 21
350--
E 300
250
300
400
EMISSION WRVELENDTH (NM)
450
500
9QOOS-C5-M DIESEL RRBBIT - CUT 5 IN METHTLCTCLOHEXflNE
MRX.-2.23 X10~ RT 328 0 NM.EM., 288.0 NM.EX
CONTOUR INTERVHL= 5 00
5.00 TO 95.00 PCT.OF MRX.
133
-------
11/26/79
350--
300
250
SO 400 45
EMISSION WRVELENGTH (NM)
90005-C5-H DIESEL RRBBIT - CUT 5 IN DICHLOROMETHRNE
MHX.-2.49 X10 RT 350.0 NM.EM.. 294.0 NM.EX.
CONTOUR INTERVRL= 5.00 5.00 TO 95.00 PCT.OF MflX.
500
11/26/79
5.00*10~2
Figure 23
= 402.0 NM
250
300 350
WflVELENGTH (NM)
400
BflP-O.l-MCH BENZOtfllPYRENE ^0.1 MG/L
IN METHYLCYCLOHEXflNE
134
-------
11/26/79
5. 00*1 CT2
EX= 300.0 NM
Figure 24
300 ' ' ' 350
i 1 1 1 1 1 r
400
WRVELENGTH (NM)
500
BflP-O.l-MCH BENZO(fl)PYRENE - 0.1 MG/L
IN METHYLCYCLDHEXflNE
135
-------
Acknowledgements
Appreciation is directed to Stan Byer in helping with the
arduous procurements necessary for this project, Ben Hill for
help in the computer software developments, and Bob Johnson
and Paul Werner in performing the vehicle testing. This work
was partially supported through EPA research grant R805934010.
REFERENCES
1. Choudhury, D., and B. Bush. 1979. Contribution of
Particulate Diesel Emissions to Composition of Poly-
nuclear Aromatic Hydrocarbons in Air. Symposium on Health
Effects of Diesel Engine Emissions.
2. Choudhury, D., and C. Doudney. 1979. Isolation of
Mutagenic Fractions of Diesel Exhaust Particulate and an
Approach to Identification of the Major Constituents.
Symposium on Health Effects of Diesel Emissions.
3. Doudney, C., M.A. Franke, and C.N. Rinalde. 1979. The
Escherichta Coli Rec Uvr DNA Damage Activity Assay: A
Sensative Measure of Diesel Exhaust Extract Mutagenicity.
Symposium on Health Effects of Diesel Emissions.
4. Particulate Regulation for Light-Duty Diesel Vehicles.
Federal Register. Vol. 44, No. 23, Thursday, February 1,
1979.
5. Black, F., and L. High. 1979. Methodology for Determin-
ing Particulate and Gaseous Diesel Hydrocarbon Emissions.
Society of Automotive Engineers Paper No. 790422.
6. Giering, L., and A. Horning. 1977. Total Luminescence
Spectroscopy: A Powerful Technique for Mixture Analysis.
American Laboratory, Nov. 1977, pp 113-123.
7. Chisholm, B., H. Eldering, L. Giering and A. Horning.
1976. Total Luminescence Contour Spectra of Six Topped
Crude Oils. Bartlesville Energy Research Center Report
BERC/RI - 76/16.
8. Giering, L. 1979. Identification and Quantitation of
Aromatic Pollutant Mixtures Without Physical Separation.
Baird Corporation Report, Bedford, MA.
136
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General Discussion
P. THILLY: Maybe I forget my LCAO modeling, but it
seems to me that in a system such as diesel soot overloaded
with methyls, dimethyls and ethyl substituents, that this
procedure would more or less lump such substituent com-
pounds and not shift their behavior in your system. Yet
such substitutions would be important from a first chemical
characterization and secondly from a biological characteri-
zation. Am I in error in remembering that result?
G. WOTZAK: What you find with simple methyl and ethyl
substitution, there isn't any great shifts in the TLS spec-
tra involved. What you do find when you have substitution
of, let's say, CHOH group or NH2 or something like this, is
that there are significant solvent effects in terms of
shifting over to the right and upward of the whole spectra
involved. You tend to lump some of the simple alcohol
substitutions involved for the PAH's involved here. That
is one problem you have in this, but you can get some fall-
out of any of the oxygenated compounds. Just from what we
have seen here you have the whole thing with rather simple
PAH's and also the oxygenated species. You are going to be
able to see some differences both from just plain TLS spectra
plus also going to high polarity solvents. If you go from
methyl cyclohexane to dichloro methane, going up to methanol,
higher polarity, you notice some shifts of some oxygenated
species. So there is another case where solvent effects
may get a picture of what type of compounds is involved.
137
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MEASUREMENT OF UNREGULATED EMISSIONS -
SOME HEAVY DUTY DIESEL ENGINE RESULTS
Joseph M. Perez, Ph.D.
Research Department TC-E
Caterpillar Tractor Co.
100 N.E. Adams
Peoria, IL 61629
ABSTRACT
Development of analytical capabilities to evaluate unregu-
lated emissions are discussed. The sampling and analysis
methods along with some problem areas are included. Results
obtained on three heavy-duty diesel engines are reported.
Emphasis was placed on analysis of the particulate fraction
including the solvent extractable material. Preliminary
experiments suggest appreciable quantities of organic
extract and BaP pass through the primary filter. Other
species analyzed to obtain a baseline for emission reduction
research included aldehydes, sulfates, sulfur dioxide,
ammonia, hydrogen sulfide and hydrogen cyanide. Of primary
concern is the proper assessment of the results.
INTRODUCTION
The composition of diesel exhaust has challenged investiga-
tors with its complexity. Studies in the late sixties and
early seventies, stimulated by an interest in exhaust
reactivity or diesel odor, did produce some information
on diesel exhaust composition (1,2,3,4).*
''Numbers in () are References at end of text.
138
-------
However, most research on diesel emissions was aimed at
measuring and reducing gaseous emissions (5,6,7,8). The
projected increase in diesels (9) and the issuance of
Advisory Circular-76 coupled with recent advances in chro-
matography and spectroscopy have resulted in increased
efforts to chemically characterize and measure diesel
exhaust constituents. This is evidenced by recent publi-
cations on both light (10) and heavy duty diesel (11,12)
exhaust composition.
This paper discusses some of our current efforts to measure
the constitutents shown in Table 1 and to characterize
diesel exhaust. Caterpillar Tractor Co.'s research program
on the chemical composition of diesel exhaust dates back
some 30 years. Early work by Dr. E. W. Landen on smoke,
nitric oxide and fuel composition effects (13,14) demon-
strated the advantages of physical and chemical diagnostics
on hardware development and resulted in a continuous program
in this area.
Upon receipt of Advisory Circular-76 the decision was made
to build-up our in-house capability to measure unregulated
emissions. This required increasing our commitment in
facilities and people in this area as well as addition of
new equipment. Selection of methods and instrumentation was
facilitated by visits to various laboratories working in the
field. The cooperation of laboratories such as Southwest
Research Institute (SwRI), Department of Energy (DOE)
Bartlesville and EPA-RTP were very helpful.
Use of an outside contract laboratory, Illinois Institute of
Technology Research Institute (IITRI), helped to accelerate
some method developments during the build-up phase of the
program. Personnel also attended instrument suppliers
workshops. These efforts resulted in tested methodology
and trained personnel and within 6 months measurement of
unregulated emissions was started. Our laboratory facility,
Figure 1, has limited access, special lighting and adequate
temperature and humidity control.
ANALYTICAL PROCEDURES
SAMPLING
The sampling system used in this work is shown in Figures 2A
and B. The mini-dilution tunnel shown in Figures 2A and B
is a 17.8 cm x 3.1 meter tunnel of 500 cfm capacity. Dilu-
tion air enters through an Ultra Aire^ filter and is mixed
with raw exhaust by a converging-diverging nozzle and
orifice plate. The raw exhaust flow is controlled by a
139
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TABLE 1 UNREGULATED EMISSIONS
ALDEHYDES
AMMONIA
HYDROGEN CYANIDE
HYDROGEN SULFIDE
METALS
NITROSAMINES
POLYNUCLEAR AROMATIC HYDROCARBONS
BENZO -«- PYRENE
BENZO -«- ANTHRACENE
SOLVENT EXTRACTABLE FRACTION OF PARTICULATES
TOTAL PARTICULATES
SOLUBLE SULFATE
SULFUR DIOXIDE
FIGURE 1 UNREGULATED EMISSIONS LABORATORY
140
-------
FIGURE 2a CATERPILLAR MINI-DILUTION TUNNEL
FIGURE 2b UNREGULATED EMISSIONS SAMPLING SYSTEM
BACKPRESSURE
CONTROL
FOUR 3/8' OD
CHEM SAMPLING
PROBES
CENTRIFIGAL
BLOWER
200 CFM
(500 CFM
CAPACITY)
20' 5/16" I D
HEATED LINE
190°C
SAMPLE PORT
OPTION 2
141
-------
backpressure butterfly valve in the stack and flows into the
tunnel through a 1.5 meter, 0.95 cm O.D. line. Dilution
ratio varied from about 10:1 to 15:1 depending on operating
conditions. Two particulate and four chemical sampling
probes are located near the tunnel outlet. The particulate
filters were attached to the probes and held at 52° C or
less depending on the exhaust flow into the tunnel.
The chemical sampling carts shown in Figures 3A and B are
similar to those used at SwRI (12). Each cart has two
heated Teflon^ sampling lines and four sampling stations.
The sampling lines are 6 meters long and 0.95 cm I.D. The
line temperature is maintained at 52° C and the sampling
rates are varied depending on the species sampled.
In general the samples were taken as recommended by Refer-
ence 15. The temperature of the diluted exhaust to the
glass bubblers or filters was maintained at 52° C. The
bubblers were immersed in an ice bath. The flow rate was 4
liters per minute for 30 minutes and the volume of sample
through each sampler was measured by individual gas meters
down-stream from the pumps. Volumes were corrected to STP
conditions.
During an unregulated emissions engine test, Figure 4, a
total of some 175 to 200 samples are collected over a
five-day period for subsequent chemical analysis.
CHEMICAL METHODS
The chemical speices analyzed and the methods used are shown
in Table 2 and are described briefly in the following para-
graphs. Some of the methods are adopted from Reference 15
which details procedures validated by SwRI for unregulated
emissions in gasoline exhaust.
Ammonia
Ammonia in diesel exhaust can be measured in the protonated
form NH|, after collection in dilute H2S04. The acidifica-
tion is carried out in two glass bublers in series main-
tained at ice bath temperature, each containing 25 ml 0.01 N
H?S04. The sample is analyzed for ammonium ion in the ion
chromatograph and compared to standards of known NH^ concen-
trations.
142
-------
FIGURE 3a CATERPILLAR SAMPLING CART
143
-------
FIGURE 3b SAMPLING CART (SCHEMATIC DIAGRAM)
FILTERS
(AS REQUIRED)
GAS VOLUME
EMERGENCY
POWER
144
-------
FIGURE 4 UNREGULATED EMISSIONS SAMPLING
TABLE 2 CHEMICAL METHODS
UNREGULATED EMISSION
1. AMMONIA
2. ALDEHYDES
3. HYDROCARBONS
A) TOTAL
B) PART. FRAC.
C) PART. FRAC.
D) POST FILTER
4. TOTAL CYANIDE
5. HYDROGEN SULFIDE
6. METALS
7. N-NITROSAMINES
8. ODOR
9. PARTICIPATE
10. SOLVENT EXTRACTABLE
FRACTION
11. PNA (BaP)
12. SOL. SULFATES
13. SULFUR DIOXIDE
ANALYSIS METHOD
ION CHROMATOGRAPHY
HPLC
FID
GC/MS
COAM
COAIW/GC
GC/MS
COLORIMETRIC/UV
(Methylene Blue)
AA//X-RAY FLUOR.
GC/MS
COAM
GRAVIMETRIC BALANCE
GRAVIMETRIC BALANCE
GC/MS//COAM
HPLC//GC/MS
ION CHROMATOGRAPHY
ION CHROMATOGRAPHY
145
-------
Carbonyl Compounds
Initially a 2,4-dinitrophenylhydrazin (DNPH) procedure
utilizing gas chroraatography (19) was tried. Resolution
of peaks and retention repeatability were unsatisfactory.
A variation of the procedure using a modified sample work-
up followed by HPLC analysis using a UV detector was devel-
oped with IITRI. Some nine carbonyl compounds were studied.
Additional work on this method is continuing. An HPLC
trace of some standards is shown in Figure 5.
Total Cyanide
The measurement of hydrogen cyanide and cyanogen in diesel
exhaust is accomplished by collecing exhaust in two glass
bubblers in series maintained at ice bath temperature,
each containing 25 ml 1.0 N KOH. An aliquot of the absorb-
ing solution is treated with potassium phosphate and chlora-
mine-T. In Reference 15 a portion of the resulting cyanogen
chloride is analyzed by a gas chromatograph with an electron
capture detector. We are using the GC/MS in place of the
E.C.D. Our minimum detection limit is 0.1 ppm in solution.
No cyanides were detected in the tests to date.
Hydrogen Sulfide
The measurement of H2S in exhaust gas is accomplished by
collecting exhaust in two glass bubblers in series main-
tained at ice bath temperature. The absorbing solution is
buffered zinc acetate solution which traps the sulfide ion
as zinc sulfide. The absorbing solution is treated with N,N
dimethylparaphenylene diamine sulfate and ferric ammonium
compound, methylene blue. The colored solution is analyzed
by spectrophotometer at 667 nm in a 1 cm pathlength cell.
Our minimum detectable concentration is 0.1 ppm in solution.
Metals
An initial survey of several samples was conducted. A NIOSH
procedure (18) using atomic absorption (AA) detection
methods was used. The samples were collected on 0.8 y
cellulose acetate filters, Millipore Type AA, 44mm, diges-
ted with nitric and perchloric acids and analyzed by AA.
Most metals with the exception of traces of Zn and Ca were
not detected. X-ray fluorescense is currently under study
to verify the results.
146
-------
o
S3
o
PM
W
O
w
Q
i-J
H
W
O
W
S3
o
H
W
O
W
o
Pi
o
147
-------
Nitrosamines
An initial attempt was made to detect the presence of
dimethylnitrosamine (DMNA) and dibutynitrosarnines (DBNA).
Samples were collected at two engine conditions on filters
and TenaxR GC traps. These were eluted or thermally
desorbed and examined at IITRI using SIM GC/MS. No nitro-
samines were detected. Samples of DMNA and DBNA standards
processed similarly showed detected limits in the ppb range.
However, the smapling procedures for nitrosamines were not
considered adequate and further work may be required to
validate our sampling method.
Particulate
Measurement of total particulate was conducted by sampling
both direct and diluted exhaust. Initial procedure develop-
ment involved direct sampling of engine exhaust to expedite
collection of samples.
However, all particulate data reported on the three heavy
duty diesel engines were obtained using diluted exhaust
sampling. Samples were obtained using 47 mm and 70 mm
diameter Teflon coated glass fiber filters.
Solvent Extractable Fraction (SEF)
The diesel particulates in this study were collected on
Teflon coated glass fiber filters. The amount and compo-
sition of the extractables depends on the extraction proce-
dure and the solvent used (16,17). In this study the SEF
refers to material extracted by methylene chloride. Soxhlet
extraction for 6 hours at 6 cycles per hour using 20 r,l of
solvent was normally used. The SEF was further processed
for PNA analysis and in some cases characterized by liquid
chromatography (LC) or gas chromatography-mass spec (GC/MS).
Benzo-oc-Pyrene
Analysis for BaP, a suspected carcinogen, was performed on
the SEF of all engine samples. The procedure was developed
under contract by IITRI and utilizes HPLC with fluorescence
detection. The procedure involves concentration of the SEF,
fractionation on silica gel, concentration of the PNA frac-
tion and a solvent change prior to analysis HPLC. The HPLC
148
-------
analysis is on a DuPont Zorbox ODS reverse-phase column with
an acetonitrile-water mobile phase. Elution is isocractic
and components are detected by flubrorneter. The fluorescent
detector is set at xex = 280 and has an emission cutoff
filter at \esn > 389. Using this procedure fourteen PNA's
were studied, Table 3. The BaP analysis was quantified and
used to measure BaP in the engines.
Soluble Sulfate
Sulfuric acid aerosols and other sulfates in diesel exhaust
are collected on a fluorocarbon membrane filter. The
soluble sulfates are leached from the filter with water and
analyzed by ion chromatography as was sulfur dioxide. The
recommended procedures in Reference 15 converts the soluble
sulfates to ammonium sulfate which is analyzed by a barium
chloranilate procedure using high performance liquid chroma-
tography. Our procedure is reliable and less time is
required for sample work-up.
Sulfur Dioxide
The concentration of S0£ in diesel exhaust is measured as
sulfate using the ion chromatography. Exhaust samples are
collected in two glass bubblers in series maintained at ice
bath temperature, each containing 25 ml of 3% hydorgen
peroxide. The samples are then directly analyzed on the
ion chromatograph and compared to standards of known sulfate
concentrations.
MEASUREMENT AND CHEMICAL CHARACTERIZATION OF SOLVENT
EXTRACTABLE FRACTION (SEF)
Chemical characterization of complex mixtures such as diesel
exhaust can involve identification and quantification of
specific compounds such as the PNA's or it can quantify
fractions or classes of compounds. In the latter case the
fractionation depends on the chemical separation process
employed. In this study a LC technique was used to charac-
terize the SEF. It is a modification of an odor analysis
method (COAM) used by this laboratory. Essentially the
percent aromatic hydrocarbons and percent polar compounds or
"oxy" compounds were determined using the system shown on
Figure 6A. Typical analyses using UV fluorescence at 254
nm are shown on Figure 6B. The first peak in each pair
represents the aromatic content, the second, the "oxy".
149
-------
TABLE 3
RELATIVE RETENTION TIMES OF VARIOUS PNA
RELATIVE TO FLUORANTHENE
PNA Retention Ratio
Acenaphthene - (Acn) 0.70
Phenanthrene - (Ph) 0.74
Anthracene - (An) 0.79
Fluoranthene - (Fla) 1.00
Pyrene - (Py) 1.14
Benz[a]Anthracene - (BaA) 1.39
Chrysene - (Chr) 1.38
Benz[e]pyrene - (BeP) 1.96
Perylene - (Per) 1.96
Benzo[k]fluoranthene - (BkF) 1.96
Benzo[a]pyrene - (BaP) 2.23
Dibenz [a,h]anthracene - (DBahA) 2.52
Benzo[g,h,i]perylene - (BghiP) 3.25
Dibenz[a,i]pyrene - (DBaiP) 4.96
150
-------
FIGURE 6a LIQUID CHROMATOGRAPH
CYCLOHEXANE
2-PROPANOL
ROTARY
VALVE
PULSE DCMCE
DAMPENER yALVE
SEPTUM INJECTOR
LIQUID CHROMATOGRAPHIC
COLUMN
PRESSURE
GAGE
UV RECORDER
DETECTOR
FIGURE 6b TYPICAL LC (COAM) ANALYSES
COLUMN: CORASIL II; 50cm x 2mm
SOLVENTS- HEXANE; IPA
ENGINE B ENGINE C
IPA
IPA
151
-------
The paraffin-naphthene fraction was determined by subtract-
ing the total of the aromatic and oxy fractions from the
SEP. The COAM can also be modified to separate a transition
fraction. Similar separations can be obtained using HPLC
but require considerably more effort. A comparison of the
COAM and HPLC method is shown on Table 4.
In addition to the above procedure the SEF was analyzed by
GC/MS and the molecular weight distribution compared to the
fuel.
ENGINES AND FUEL
Engines
Three engines were used in this study and are described in
Table 5. They are typical production heavy duty diesel
engines. Engines A and B are similar engines. Engin B has
exhaust gas recirculation (EGR) for NOX control. Prior
to an unregulated emission test the engines were run on the
13-Mode Federal Test Cycle to check the emissions and insure
typical performance.
Fuel
The fuel used in the tests was a typical No. 2 diesel fuel.
Since the fuel came from a central system, samples were
taken prior to and at the completion of each test for
comparison by GC/MS. A typical total ion chromatogram of
the fuel trace is shown in Figure 7. Typical physical
properties of the fuel are shown in Table 6. No evident
change in the physical properties of the fuel was noticed
from test to test. Some change in BaP content was noticed.
13-Mode Cycle
All engine samples were obtained at stead-state engine
operating conditions. The individual 13-Mode Federal Test
Cycle engine modes were analyzed and a weighted composite
value calculated.
152
-------
TABLE 4 SEF CHARACTERIZATION
LC VS. HPLC
SAMPLE
HEAVY DUTY
DIESEL
TIME REQUIRED
LC (COAM) , % HPLC-UV, %
METHOD* AROM TRANS OXY AROM TRANS OXY
1 26.7 - 73.2 -
2 26.8 15.2 58.0 -
3 30.8 17.2 51.8 34.8 22.5 42.7
FOR ANALYSIS, MINUTES:
METHOD LC (COAM) HPLC
13
25 90
37 90
*METHOD:
1 - CORASIL-II; NORMAL SOLVENT SEQUENCE FOR LC
2 - CORASIL-II; DELAYED SOLVENT SEQUENCE FOR LC
3 - PORASIL-C; DELAYED SOLVENT SEQUENCE FOR LC
TABLE 5 HEAVY DUTY DIESEL ENGINES
ENGINE
TYPE
NO. CYLINDERS
BORE & STROKE, mm
RATED SPEED, RPM
RATED POWER, Kw
A
DINA
8
114X127
2800
157
B
DI-EGR
8
114X127
2800
149
FEDERAL TEST CYCLE DATA, G/BHP-HR:
CO
HC
HC+NO2
5.92
0.77
8.89
5.37
0.57
5.73
C
DITA
6
137X165
2100
284
2.20
0.27
7.93
Dl = DIRECT INJECTION
NA = NATURALLY ASPIRATED
EGR= EXHAUST GAS RECIRCULATION
T . = TURBOCHARGED
A = AFTERCOOLED
153
-------
FIGURE 7 FILTER EFFICIENCY CHECK
BB243 "IIESEL FUEL :»*PLE COLLECTEJ !M'7 10.1 UL
!»SE 37,321,344 ZERO 238,784 SCBLE 1.89
TQTflL ION CHROHflTOCRfln
OCT 23.1979 7:<3BH
4 8 12 16 28 24 28 32 36 48 44 48 52 5« 68
UllLJLll
» 4 8 12 16 28 24 28 32 36 48 44 48 52 56 68
TABLE 6 TYPICAL FUEL PROPERTIES
DISTILLATION, TEMP °C
API GRAV
% SULFUR
% CARBON
% HYDROGEN
% AROMATICS
% OLEFINS
% PARAFFIN/NAPHTENES
VIS, SSU @ 210°F
35.2 '
I
0.27
I
85.5
I
12.9 ,
34.4 ,
0.8 ,
64.8 |
1.03 |
IBP
5
10
50
90
95
E.Pt.
REC, %
354
394
412
494
583
611
629
98
154
-------
EXPERIMENTAL RESULTS
Particulates
The ability to reproducibly obtain diesel particulate
samples was studied. The variables studied include:
o Type of filter medium,
o Weighing precision and accuracy,
o Filter handling, drying and weighing,
o Water and fuel efefcts, and
o Filter efficiency.
In this study both T60A20 and TX40H120WW filters were used.
They are vey similar Teflon coated glass fiber filters. The
Pallflex TX40H120WW filters appear to be more efficient for
particulates. The filters are compared on Figure 8 and
Table 7. Fluoropore filters were also tried but had the
highest pressure drop limiting teh size of particulate
sample collected. Fluoropore filters are used for sulfate
analysis sampling. Glass fiber filters reportedly (16)
react with collected PMA but have been pretreated and used
effectively (17).
All weighings were conducted on microbalances in the same
laboratory. Humidity in the lab ranged from 35 to 65% over
the period of the study while temperature was maintained at
22 +_ 2°C. Under these conditions there is no problem in
weighing filters after storing them in a desiccator for up
to two weeks before using them. Storage in a desiccator
overnight was sufficient time to equilibrate filters even
when spiked with moisture and raw No. 2 diesel fuel.
Storage of equilibrated samples for up to three weeks did
not significantly affect the results.
Serious consideration was given to normal engineering
tolerances in establishing our analytical procedure. The
resulting protocol used in this study is shown on Figure 9.
Particulate values obtained from the three engines are shown
in Table 8. Both the composite values (G/BHP-HR) and the
range of values for individual modes (G/HR) are shown. The
SEF was obtained using methylene chloride. The particulate
values are similar to those obtained by others studynig
heavy duty diesel engines (11,20). Characterization of the
SEF on two of the engines is discussed later. One item of
concern is how to assess the risk associated with these
levels. In some cases OSHA values or TLV recommendations
may give some guidance. Another approach is to conduct
155
-------
156
-------
TABLE 7 FILTER EFFICIENCY CHECK
TEST CONDITIONS: 2 FILTERS IN SERIES; 70 mm FILTERS; DILUTED EXHAUST.
CONDITION
1*
2**
HC,
ppm
322
208
SMOKE,
% OPACITY
27
<2
% PARTIC.
T60A20
6.3
9.9
ON 2ND FILTER
TX40H120WW
O.I
3.8
* 25 L/MIN FLOW
** 55L/MIN FLOW
FIGURE 9 CHEMICAL CHARACTERIZATION BY LC (COAM)
MG/M3
[329|
114 61 ENGINE B
INTERMEDIATE SPEED
_____ _]B
LOAD LI 0 25 50 75 100 °
RATED SPEED
( ) = % OF TOTAL HC BY FID
PARAFFIN
NAPHTHENE
_ n ]'
LOAD 0 2S 5O 75 100
TABLE 8 PARTICULATES
ENGINE
A
B
C
TOTAL PARTICULATES
G/BHP-HR RANGE, G/HR
0.77 4.6 - 260
1.21 2.7 - 268
0.33 1.5 - 134
SOLVENT EXTRACT FRACTION
G/BHP-HR % RANGE, %
0.19 25 6-92
0.079 6.5 2 - 89
0.037 11 5-61
157
-------
evaluations using established air models and compare the
results with available data. As a first pass an EPA Hiway
Model (21) was used to evaluate the composite and other
selected values. Using an unlikely truck population density
and extreme model conditions the maximum particulate expo-
sure standing next to a highway with bumper to bumper
traffic with idling trucks was < 0.05 yg/M3. Values
calculated for other species and conditions were less.
Sulfur Compounds
The S02 and 864 values are shown on Table 9. As expected
the sulfur values were conssitent with fuel consumption.
Approximately 90% of the fuel sulfur was acdcounted for in
the analyses. This compares to 95% reported for gasoline
vehicles in Reference 15. Less than 1% of the fuel sulfur
was found as soluble sulfate.
Hydrogen sulfide would not be expected to be present at
non-catalyzed, lean operating conditions of the diesels.
Under our conditions, detectability is 0.01 ppm in solution
or about 0.1 ppm in the undiluted exhaust. H£S was not
detected at this level but may be mased by interference
from S02 in the exhaust. The 0.1 level is well below
the recommended 10 ppm TLV level. h^S also has a strong
offensive odor detetable at about a 0.1 ppm level.
Carbonyl sulfide (COS) would be formed under the same
conditions that favor H2S formation. Based on the
results, samples were not taken for COS analysis.
Ammonia
This compound would not be expected in the diesel exhaust
but was detected at levels of 0.77-1.15 mg/BHP-Hr. However,
background levels or interferences with the method produced
positive results of about the same magnitude. The maximum
corrected concentration found in the exhaust, 0.4 mg/M3,
whether real or not is well below allowable recommended OSHA
levels, 35 mg/M3.
PNA
The procedure has the potential for detecting a number of
PNA but to date most of our work has involved BaP and to a
158
-------
TABLE 9 SULFUR COMPOUNDS
ENGINE
A
B
C
SO2
G/BHP-HR
0.88
1.01
0.91
S04
mg/BHP-HR
8.08
10.2
17.2
H2S
ppm
<0.1
<0.1
<0.1
TABLE 10 BaP by HPLC
ENGINE
A
B
C
WBHP-HR
1.08
4.34
0.34
RANGE,
mq/Ka FUEL
0.001-0.031
0.001-0.199
<0.001-0.021
FUEL BaP,
ma/Ka FUEL
4.7
14.6
12.7
TABLE 11 EXHAUST: FUEL BaP
ENGINE A
ENGINE B
Hg BaP/BHP-HR
1.08
4.34
mg BaP/Kg FUEL
4.7
14.6
RATIO*
0.0012
0.0014
*RATIO =
BaP OUT IN EXHAUST
BaP IN FUEL IN
159
-------
lesser extent BaA. The GC/MS has been used to verify BaP
and BaA values. The composite or cycle BaP values in
^g/BHP-Hr for the three engines are shown on Table 10. The
range of BaP values in Mg/Kg fuel burned is shown for the
11-Modes of the 13-Mode cycle. The BaP values are consis-
tent with reported levels (11,22). However, fuel BaP values
appear to be considerably higher than reported values from a
survey of some 20 diesel fuels (22). The engines are
efficient in burning the fuel but some of the exhaust BaP
could have originated in the fuel as indicated by the ratio
in Table 11.
Characterization
The preceeding results are part of our effort to establish a
data base for unregulated emissions. The procedures used
are similar to those used by a number of laboratories and
the engines tested and scheduled for testing are representa-
tive of our various engine families. In addition to this
effort we have also been evaluating other ways of character-
izing the emissions, especially the particulates. We have
looked both at a modification of the Caterpillar LC odor
analysis method (COAM) and GC/MS to evaluate the SEF.
The results of the LC method is shown on Figures 10 and 11
for Engines B and C. The difference between the distribu-
tion of oxy and aromatic fractions is obvious. The transi-
tional fraction referred to by EPA would be part of the
"oxy" fraction. Preliminary results to break this fraction
out are shown in Table 4. The BaP is a minor portion of the
aromatic fraction as shown in Figures 10 and 11.
The same fractions shown in Figures 10 and 11 were character-
ized by GC/MS for molecular weight distribution and compari-
son with diesel fuel. Some typical results are shown in
Figures 12 and 13 and Table 12. The results are in general
agreement with those reported by F. Black of EPA-RTP (9).
The fractions are essentially in the upper molecular weight
range of the fuel with some higher molecular weight material
either formed by polymerization or originating in the
lubricant. The traces for 0-1oad and 25% load shown on
Figure 12 are typical of those containing larger fractions
of higher molecular weight material. BaP on these traces
would elute at about 42 minutes.
Tests were conducted at Mode 11 using a hot (190° C) filter
to obtain a SEF for comparison with a SEF fraction taken at
52° C. The samples were taken from the dilution tunnel,
Figure 14, and the same protocol followed. The SEF from the
hot (190° C) particulate sample is less than a tenth of the
160
-------
FIGURE 10 CHEMICAL CHARACTERIZATION BY LC (COAM)
MG/M3 -
SEF CHEMICAL CHARACTERIZATION
r
-
"
-
-
_
17-5)
.
-
13 51
Pi
I
167)
^
ENGINE C
IN
55
i
TERMEDIATE SPEED
15
) - % OF TOTAL HC BY FID 10
1366)
11 0
I
P
1
PARAFFIN
NAPHTHENE
OXY 5
AROMATIC
fl 1 n
r
-
-
-
_
~
_
r
h
%
1343)
v\^
244
I
ENGINE C
RATED SPEED
266
I
( ) = % OF TOTAL
HYDROCARBONS - FtO
MS 9)
PARAFFIN-
NAPHTHENE
^ ^ °XV
^
^ AROMATIC
n i
_ _ >" = 0 _ _ _ >p
LOAD L1 0 25 50 75 100
LOAD 0 25 50 75 100
FIGURE 11
PROTOCOL FOR CHARACTERIZATION OF PARTICULATES
1 PLACE FILTERS IN DESICCATOR UNTIL READY TO USE, MINIMUM - OVERNITE
2 WEIGH FILTER
3 DESICCATE UNTIL DAY OF USE
4 COLLECT PARTICULATE SAMPLE
5 DESICCATOR OVERMTE AS MINIMUM, KEEP IN DARK OR REDUCED UV LIGHTING
6 WEIGH FILTER + PARTICULATE
7 SOXHLET EXTRACTION, MeClj,, 6-HOURS, 6-CYCLES/HR IF UNABLE TO EXTRACT
SAME DAY. STORE IN DARK IN FREEZER I-23°C)
C
1
I
:ONCENTRATE
SAMPLE TO
10 MLS
1
[ CONC TO ( ML
[ SILICA GEL
CLEANUP
COLLECT
& CON(
* t
LC (coAM)| | IGC/MS] i
AROI
II1. , * , *
/I|-|OXY| |BAP| MOL. WT
DISTRIB'N
1
EVAPORATE
TO DRYNESS
1
WEIGH
1
ADD 1 ML Me
FRACTIONS
:ENTRATE
CI2
nHPLC ANALYSIS
(PNA, BAPI
J
-
| QUANTIFY & RELATE BACK TO EXHAUST |
-------
FIGURE 12 CHEMICAL CHARACTERIZATION BY GC/MS
IOOJ
ENGINE C
INTERMEDIATE
SPEED
0-LOAD
90
10
5 9 13 17 21 29 29 33 37 41 45 49 83 5T 61 65
100
25% LOAD 50
""v
10 14 18 22 26 30 34 36 42 46 50 54 58 62 66 70
100 n
75% LOAD 50
tv» h
I
I
V
10 14 18 22 26 30 34 38 42 46 30 54 58 62 66 70
162
-------
FIGURE 13
100 i
A
10
SEF - ENGINE B
INTERMEDIATE SPEED
25% LOAD
S 9 13 IT 21 25 29 33 37 41 46 49 S3 57 SI 69
IOO
50% LOAD
10
04 12 16 20 24 28 32 36 40 44 46 52 56 60
100
SO
10
JL
100% LOAD
10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70
163
-------
TABLE 12 GC/MS SEF FRACTIONIZATHDN
A% = CnH2n-l-2
FUEL
ENGINE B
MODE 2
3
3*
3**
4
5
6
7
8
9
10
11
12
C12
15.9
0.5
0
0
5.2
0.0
0.2
0.0
1.2
0.5
0
0.2
0.2
0
C14
18.0
0.3
0.2
0.2
32.2
0.1
3.6
1.4
1.2
0.8
1.1
2.2
0.2
0.1
C16
23.3
1.3
2.5
0.6
33.2
0.4
6.4
4.0
1.2
3.4
3.6
8.3
0.7
1.0
C20
30.0
16.8
19.7
3.4
24.3
7.3
33.4
58.3
12.9
40.3
21.4
24.9
16.7
12.2
C40
12.8
81.1
77.8
62.5
5.1
92.2
56.4
35.8
83.5
55.1
73.9
64.4
82.2
88.7
* HOT FILTER
** TRAP AFTER 52°C FILTER
164
-------
FIGURE 14 HOT & COLD PARTICULATE SAMPLING
-FILTER
FLOW METER
CHROMOSORB TRAP
VALVE
EXHAUST
HOT
' PARTICULATE
FILTER
FILTER
PARTICULATE FILTER (52°C
FLOW METER
CHROMOSORB TRAP
VALVE
EXHAUST
165
-------
SEF from the cold (52° C) participate sample, Table 13.
The GC/MS trace, Figure 15, showed loss of the "lighter"
material. This was similar to data reported in 1968 (7) at
which time vaporization of hydrocarbons from the filter of
direct exhaust samples was suggested. This study would tend
to agree that the hot FID analysis for total hydrocarbons
accounts for about 95% of all hydrocarbons in the exhaust.
Recently, Johnson et al. (23) in characterization studies of
particulates indicated appreciable amounts of hydrocarbon
can be adsorbed on the surface of some particulates. In
addition, hydrocarbon aerosols can readily be formed on
cooling. An inability to obtain a hydrocarbon material
balance in the preceeding experiments and our earlier
characterization studies suggested a significant unaccounted
fraction, resulted in two additional experiments at Modes 3
and 11. The tests were conducted using a Chromosorb 102
trap after both hot (190° C) and cold (52° C) filters shown
on Figure 16. It was found, Table 14, that in some tests
more solvent extractable material is passing through the
filter than is collected on it. Of even more significance
the BaP levels on the Chromosorb can exceed that of the SEF.
The molecular weight distribution for the fractions trapped
on the Chromosorb are compared to the SEF on Figure 16. The
SEF trace shown is for the material trapped on the filter
upstream from the Model-11 (25% load, rated speed) chromo-
sorb trap. The traces shown are for the corresponding
Mode-3 and Mode-11 samples shown on Table 14. Attempts to
validate the results and establish the variables affecting
the split are in progress. The work raises some very
significant questions regarding the extensive amount of
risk assessment work underway using the MeClz extractable
particulate fractions. Are we testing the right material?
Are some of the differences between laboratories in short
term tests and animal tests due to differences in particu-
late collection methods?
SUMMARY AND CONCLUSIONS (FIGURE 17)
This work was an attempt to obtain baseline data on unregu-
lated emissions for some heavy duty diesel engines. In
comparison to established OSHA and ACGIH values none of the
substances we have measured to date are present in the
exhaust at concentration levels that could be considered
dangerous at least for short term exposures.
The complexity of the exhaust and the time consuming effort
required to conduct unregulated emission analyses suggest
the need for prioritization with characterization of the
particulate and solvent extractable fraction having high
166
-------
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iH
II
||
H 9
O O
w o
K
167
-------
FIGURE 15 SEF-GC/MS
SO-
HOT (190°C)
PARTICULATE
EXTRACT
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
IOO-
r~\
50
COLD (52°C)
\ PARTICULATE
EXTRACT
04 8 12 16 20 24 28 32 36 40 44 48 52 56 60
168
-------
FIGURE 16 CHEMICAL CHARACTERIZATION OF TRAP PRODUCTS
1001
CHROMOSORB 102
TRAP PRODUCT
INTERMEDIATE SPEED,
25% LOAD
0 4 8 12 16 20 24 28 32 36 40 44 48 92 96 SO
100 i
90-
SEF - COLD (52%°C)
PARTICIPATE
RATED SPEED,
25% LOAD
10 14 18 22 26 30 34 38 42 46 50 54 98 62 66 70
100
90
CHROMOSORB 102
TRAP PRODUCT
RATED SPEED,
25% LOAD
10
'6 4 8 12. 16 20 24 28 K 36 4O 44 4« S2 M »0
169
-------
TABLE 14 CHROMOSORB TRAP PRODUCT
SAMPLING AS SHOWN ON FIGURE 14
COLD FILTER (52 °C)
P ARTICULATES , G/HR
SEF, G/HR
BaP, mg/HR
CHROMOSORB TRAP
SOLVENT EXTRACT, G/HR
BaP, mg/HR
HOT FILTER (190 °C)*
P ARTICULATES , G/HR
SEF, G/HR
BaP, mg/HR
CHROMOSORB TRAP
SOLVENT EXTRACT, G/HR
BaP, mg/HR
MODE 3
17.9
9.67
0.10
18.8
2.2
7.04
0.05
25.0
0.41
MODE 11
107.6
9.59
5.25
25.7
1.9
125.2
0.35
1.51
35.4
0.43
*UNDILUTED EXHAUST
170
-------
FIGURE 17
MEASUREMENT OF UNREGULATED EMISSIONS
SOME HEAVY DUTY DIESEL ENGINE RESULTS
SUMMARY AND CONCLUSIONS
BASELINE UNREGULATED EMISSIONS
DATA ON SOME HEAVY DUTY
DIESEL ENGINES
NO UNUSUAL CONCENTRATION LEVELS
VALIDATION OF METHODS
CHARACTERIZATION OF SEF -
HIGH PRIORITY, IN PARTICULAR:
1) ORIGIN OF CONSTITUENTS
2) SIGNIFICANCE OF MATERIAL
PASSING THROUGH FILTER
BEST JUDGEMENT IN RELATING TO
RISK ASSESSMENT:
1) MEASUREMENT
2) MODELS
3) ENGINEERING TOOLS
171
-------
priority. The immediate concern is to establish the signi-
ficance of the material passing through the participate
filter in the gaseous phase.
Validation of the chemical methods for analysis of a number
of unregulated emissions in diesel exhaust is required. The
ability to measure and characterize diesel emissions remains
a challenge but must be vigorously pursued if a fair assess-
ment of the risk, problem comopunds, and benefits of control
technology is to be made.
ACKNOWLEDGEMENTS
R. V. Bower, Caterpillar Research and M. Poff, Technical
Facilities were instrumental in setting up our facilities.
K. Claar and R. D. McDowell's cooperation on obtaining and
operating the engine test and evaluation facilities for this
study and the technical assistance of R. V. Bower, L. A.
Schoepke, and V. J. Huisenga in the chemical areas are
appreciated.
Dr. W. J. Eisenberg, IITRI, conducted method development
work on PNA and aldehydes under contracts for Caterpillar.
His cooperation on these and other tests was very helpful
in developing our in-house capability.
REFERENCES
1. Landen, E. W. and Perez, J. M., "Some Diesel Exhaust
Reactivity Information Derived by Gas Chromatography",
SAE Paper No. 740530 (June 1974).
2. A. Dravnieks, et al., "Gas Chromatography Study of
Diesel Exhaust Using a Two Column System", ACS Div. of
Water, Air and Waste Chemistry Meeting, Los Angeles,
March 1971.
3. P. L. Levins, "Chemical Analysis of Odor Components in
Diesel Exhaust", CRC-APRAC Symposium, Washington, DC,
March 1973.
4. R. S. Spindt, G. J. Barnes and J. H. Sommers, "The
Characterization of Odor Components in Diesel Exhaust
Gas", SAE Paper No. 710605, June 1971.
5. J. M. Perez and E. W. Landen, "Exhaust Emission Charac-
teristics of Precornbustion Engines", SAE Paper No.
680421 (May 1968).
172
-------
6. R. W. Hurn and W. F. Marshall, "Techniques for Diesel
Emissions Measurement", SAE Paper No. 680418 (1968).
7. Vehicle Emissions Part III, SAE Progress in Technology,
Vol. 14, 1971 (Library of Congress Catalog Card No.
64-16836).
8. H. E. Dietzman, et al., "Diesel Emissions as Predictors
of Observed Diesel Odor", SAE Paper No. 720757.
9. F. Black and L. High, "Methodology for Determining
Particulate and Gaseous Diesel Hydrocarbon Emissions",
SAE Paper No. 790422 (Feb. 1979).
10. S. H. Cadle, G. J. Nebel and R. L. Williams, "Measure-
ments of Unregulated Emissions from General Motors'
Light-Duty Vehicles", SAE Paper No. 790694 (June
1979).
11. C. T, Hare and R. L. Bradow, "Characterization of
Heavy-Duty Diesel Gaseous and Particulate Emissions,
and Effects of Fuel Composition", SAE Paper No. 790490
(Feb. 1979).
12. H. E. Dietzmann, and F. M. Black, "Unregulated Emis-
sions Measurement Methodology", SAE Paper No. 790816
(Sept. 1979).
13. E. W. Landen, "Nitrogen Oxides and Variables in Precom-
bustion Chamber Type Diesel Engines", SAE Paper 630167
(June 1963).
14. E. W. Landen, "Combustion Characteristics of Diesel
Fuels", SAE Quarterly Transactions, Vol. 3, 1949, p.
200.
15. "Analytical Procedures for Characterization of Unregu-
lated Pollutant Emissions from Motor Vehicles", EPA
Report No. 600-2-27-017, Feb. 1979.
16. F. S. Lee, et al., "PAH Transformation During Filter
Collection of Airborne Particulates - A Quantitative
Evaluation", Presented at 4th International Symposium
on Polynuclear Aromatic Hydrocarbons, Battelle Columbus
Laboratories, Oct. 1979.
17. S. J. Swarin and R. L. Williams, "Liquid Chromato-
graphic Determination of BaP in Automobile Exhaust
Particulate: Verification of the Collection and
Analytical Methods", Presented at 4th International
Symposium on Polynuclear Aromatic Hydrocarbons, Bat-
telle Columbus Laboratories, Oct. 1979.
173
-------
18. MIOSH Method - No. P&CAM 173.
19. F. Stumpf, "Oxygenated Compounds in Automobile Exhaust-
Gas Chromatographic Procedure", MSERB-ESRL-EPA, Research
Triangle Park, NC.
20. L. E. Frisch, J. H. Johnson and D. G. Leddy, "Effect
of Fuels and Dilution Ratio on Diesel Particulate
Emissions", SAE Paper No. 790417 (Feb. 1979).
21. J. R. Zimmermann and R. S. Thompson, "User's Guide for
Highway: A Highway Air Pollution Model", EPA Report
No. 650/4-74-008, Feb. 1975.
22. R. S. Spindt, et al., "Polynuclear Aromatic Content
of Hevay Duty Diesel Engine Exhaust Gases", CRC-APRAC
Project CAPE 24-72, Gulf Research and Development
(Dec. 1974) NTIS PB238 688/AS.
23. K. Carpenter and J. H. Johnson, "Analysis and Physical
Characteristics of Diesel Particulate Matter Using
Transmission Electron Microscope Techniques", SAE Paper
Mo. 790815 (Sept. 1979).
174
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POLYNUCLEAR_AROMATIC HYDROCARBONS IN
DIESEL EMISSION PARTICULATES
Dilip R. Choudhury and Brian Bush
Division of Laboratories and Research,
New York State Department of Health, Albany, NY 12201
ABSTRACT
A preliminary characterization of polynuclear aromatic hydro-
carbons (PAH) in dichloromethane extracts of diesel exhaust
particulates is described. Acid-base liquid-liquid partition-
ing followed by adsorption chromatography was used to isolate
the PAH fraction. Compounds were identified primarily by the
mass spectra of high-resolution gas chromatographic effluents.
Three-four-ring PAHs and their alkyl-substituted homologs
were the predominant constituents. In addition, ultraviolet
spectra of four high performance liquid chromatography-sepa-
rated PAHs were superimposable with those of reference com-
pounds leading to their unambiguous identification. These
results show the advantage of using several complementary
techniques for characterization rather than gas chromatogra-
phy-mass spectrometry alone.
INTRODUCTION
Polynuclear aromatic hydrocarbons are products of various
combustion processes. Most of the carcinogenic PAHs in the
ambient air are associated with the particulate matter(1).
The expected increase in the use of diesel powered vehicles
will greatly increase the automotive contribution of partic"
ulate-adsorbed PAHs and other organics in air because these
vehicles emit 30-50 times more particulates than a comparable
gasoline-powered vehicle (2). Consequently it is important to
characterize thoroughly the particulate-adsorbed PAHs in
diesel exhausts.
Several workers have identified PAHs in diesel exhaust par-
ticulate extracts (3,4), these identifications were based
primarily on data from analysis by gas chromatography-mass
spectrometry (GC-MS) or thin layer chromatography and high
performance liquid chromatography. Because several isomeric
PAHs are difficult or impossible to separate by GC, a firm
identification is difficult to achieve by GC-MS alone.
175
-------
In this communication we describe preliminary results of a
comprehensive characterization study of PAHs in diesel emis-
sion particulates. Most of the work was performed on a sam-
ple from a Volkswagen (VW) Rabbit driven in a highway fuel
efficiency test (HFET) mode. Preliminary results from a GC-
MS examination of a sample from a Mercedes 300-D exhaust
(federal test procedure {FTP} driving cycle) are also inclu-
ded.
EXPERIMENTAL
Collection of Particulates and Preparation of the Organic
Extract
Diesel-exhaust particulates were collected by a modification
of the standard dilution-tunnel technique. The Pallflex
T60A20 Teflon-coated glass fiber filters (20" x 20") contain-
ing the particulates were Soxhlet extracted with dichloro-
methane for 24 h. The extract was filtered through a 0.2-ym
Fluoropore filter under vacuum, and the solvent was removed
under vacuum with gentle heating. A composite of several fil-
ter extracts was prepared for chemical characterization. De-
tails of the sampling procedure and extraction are given else-
where in this Proceeding (5).
Isolation of the PAH Fraction
The crude extract is a highly complex mixture. To isolate
the PAHs from interfering non-PAH compounds, the neutral
fraction was first separated by acid-base partitioning. The
PAH fraction was then isolated from the neutral fraction by
silica-gel adsorption chromatography (6). Fluorene and cor-
onene were chromatographed under similar conditions to de-
termine the elution volume of the PAH fraction.
Solvents and Chemicals
Solvents for high performance liquid chromatography (HPLC)
and other uses were glass distilled, ultraviolet grade (Bur-
dick and Jackson Laboratories, Inc., Muskegon, MI). PAH
reference compounds (Aldrich Chemical Co., Eastman Kodak,
and K § K Laboratories) were checked for purity and, if
necessary, were purified further by column chromatography on
silica gel and recrystallization from an appropriate solvent.
Glass Capillary Column for High Resolution Gas
Chromatography
A glass capillary column (45 m x 0.35 mm) was drawn from Py-
rex glass tubing using a Shimadzu capillary drawing machine
(Shimadzu Scientific Instruments, Inc., Columbia, MD). The
column was filled with hydrogen chloride gas, sealed, heated
176
-------
for 4 h at 350°C, and cooled to room temperature. The hy-
drogen chloride was blown but with dry nitrogen, and a solu-
tion of 5% 1,1,1,3,3,3-hexamethyl disilazane in toluene (5 ml)
was passed through the column. The column was washed with dry
toluene, dried by passing dry nitrogen, and then dynamically
coated with a solution of SES4 gum in isooctane under a con-
stant pressure of nitrogen.
GC and GC-MS Analysis
For GC analysis the PAH fraction was dissolved in a small
volume of chloroform, and aliquots of 1-2 yl were injected
without stream splitting into a Hewlett-Packard 5840A gas
chromatograph with microprocessor controls. The injector
temperature was held at 230°; the pressure at the injector
head was 6 Psi. Careful optimization of the injector head
pressure was critical for optimal resolution. The oven temp-
erature was started at 110°C, and 2 min after injection a
multistep temperature program was initiated, reaching a fi-
nal temperature of 290°C. Nitrogen was the carrier and make-
up gas. The analytical conditions were carefully optimized
for best resolution, minimum analysis time, and peak sharp-
ness.
For GC-MS analysis the same column was directly connected to
the source of a Finnigan 4000 mass spectrometer equipped with
an INCOS 2300 data system and operated at 70 eV. Helium was
the carrier gas.
High Performance Liquid Chromatography and Ultraviolet Spec-
troscopy
The HPLC method for analysis of PAHs was developed during our
previous work on airborne PAHs (Choudhury and Bush, manu-
script in preparation). A Zorbax ODS column (4.6 mm x 25 cm;
DuPont Instruments, Wilmington, DE) was used with a solvent
gradient system. Two solvent systems were used: methanol-
water (20-80) and methanol. Elution was started with 84%
methanol-water and continued for 20 min. A gradient to 100%
MeOH over 20 min was then initiated. Ultraviolet spectra
of the HPLC eluates were obtained by interfacing a millisec-
ond-scan vidicon ultraviolet spectrophotometer to the HPLC
instrument. This instrument, capable of providing an in-
stantaneous and complete ultraviolet spectrum of every HPLC
eluate in a continuous mode, will be described elsewhere
(Smith, Aldous, Choudhury and Bush, manuscript in prepara-
tion) .'
RESULTS AND DISCUSSION
Trace analysis of PAHs in environmental samples is a diffi-
cult task. Blumer and Giger observed in 1974 that even when
177
-------
large samples are available, as in petroleum analysis, "a
complete analytical resolution of the PAH fraction exceeds
the capability of any existing combination of analytical
techniques" (7). Computerized GC-MS is the most powerful
technique normally applied to identification of PAHs and
other trace organics in a complex environmental sample.
The two-step clean-up procedure used in this work was highly
effective for isolation of the PAH fraction. No interfer-
ing compounds of other type were detected in this fraction
by mass spectrometry. Compounds were identified primarily
by high resolution GC retention times and mass spectra of
the GC-separated components. Additional evidence for the
identity of certain compounds was obtained from ultraviolet
spectra of the HPLC eluates and HPLC retention times. Low
resolution packed columns were not used, as the superior per-
formance of high resolution capillary columns is well estab-
lished. Glass capillary columns coated with several sili-
cone-based stationary phases (SP2100, SE30, SE52, SE54) were
examined for their suitability for analysis of PAHs in die-
sel emission particulate samples. In our hands the SE54-
coated column gave the best resolution and peak sharpness,
and the analysis time was quite short.
The GC profile of the PAH fraction from a VW Rabbit exhaust
sample is shown in Figure 1. Comparison of the peak reten-
tion times with those of representative parent PAHs indica-
ted that the major components were low molecular weight PAHs.
The minor components could not be compared with a high degree
of confidence. Four parent PAHs were identified: phenan-
threne (peak 1), anthracene (peak 2), fluoranthene (peak 11) ,
and pyrene (peak 12). The retention time of peak 7 corre-
sponded with 2-phenylnapthalene. For more confirmed charac-
terization of these and other constituents the mass spectrum
of each component was determined using the same capillary GC
column.
Some loss of chromatographic resolution was observed in the
GC-MS work, presumably because the injector of the Finnigan
GC-MS is drastically different from that of the Hewlett
Packard gas chromatograph. We have found it virtually im-
possible to maintain maximum chromatographic resolution in
GC-MS work. Chromatographic and mass spectral scan param-
eters were optimized separately for the best possible reso-
lution.
The total ion chromatogram of the PAH fraction is shown in
Figure 2. M/e values of the major components of all observ-
able peaks were determined, and the parent ions were noted.
Mass chromatograms of the parent ion and key fragment ions
were then generated, and clean spectra were obtained by com-
puter-assisted background subtraction. These spectra and the
retention times were compared with those of reference com-
178
-------
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loo.o r
[n] = m/e
600 800 1000 1200 1400 1600 1800 2000 SCAN
10-00 13:20 16:40 20:00 23'-20 26:40 30'00 33:20 TIME
Figure 2. Total ion chromatogram of PAH fraction of diesel
particulate extract (VW Rabbit).
180
-------
pounds, when available. In other cases the background sub-
tracted spectra were compared with the best-fitting spectra
from the computer-stored mass spectral library. In addition
to the compounds whose masses are shown in Figure 2, many
minor components were characterized. A total of 45 spectra
were extracted, and tentative structures were assigned.
Four compounds with parent ions of m/e 192 (also the base
peaks) were present. Key fragment ions were observed at
masses corresponding to Mt-l, Mt-27, and M?+. These spectra
were in excellent agreement with those of methyIphenanthrene/-
anthracene. The peak with the parent ion m/e 204 was simil-
arly identified as 2-phenylnapthalene. Three components
having parent ion of m/e 206 had spectra in agreement with
those of dimethyIphenanthrene/-anthracene and/or ethylphen-
anthrene/-anthracene. At least four compounds with a parent
ion of m/e 220 were detected; their mass spectra agreed with
those of trimethyIphenanthrene/-anthracene or methylethylphen-
anthrene/-anthracene. The m/e 228-parent ion peak was simil-
arly identified as chrysene/benzo(a)anthracene/triphenylene.
Gas chromatographic separation of these three components is
incomplete. The mass spectrum of the peak with a parent ion
of m/e 226 corresponded to that of benzo(ghi)fluoranthene,
and the GC retention index of this compound (391) was in ex-
cellent agreement with that reported (389.6) by Lee et al.
(8).
At least two components having parent ion of m/e 252 were
detected. The first (scan 1725) was characterized as benzo-
(b)fluoranthene/benzo(j)fluoranthene/benzo(k)fluoranthene on
the basis of mass spectrum and retention time. The reten-
tion time and mass spectrum of the second (scan 1775) corre-
sponded to that of benzo(e)pyrene/benzo(a)pyrene. Although
the GC column used can separate the two, no separate peaks
were observed, presumably because of their low concentrations
and loss of resolution of the gc column in GC-MS. Compounds
with a higher ring system could not be detected in this sam-
ple.
Some additional minor components characterized tentatively
by mass spectral data were isomeric methyldibenzothiophenes/
-dibenzodioxins, isomeric dimethyldibenzothiophenes/-di
benzodioxins, benzo(a)fluorene, benzo(b)fluorene, and isomer-
ic methylpyrenes/-fluoranthenes.
Since mass spectrometry cannot differentiate between isomeric
PAHs, additional independent evidence for identification of
individual isomers is desirable. This is particularly impor-
tant because the carcinogenic properties of PAHs are depen-
dent on the isomeric structure. PAHs absorb strongly in the
ultraviolet region, and we took advantage of this distinctive
property to achieve their unambiguous characterization. HPLC
181
-------
retention times could also be used for identification, as
HPLC gives excellent separation of PAHs. Our ultraviolet
spectra were obtained by interfacing a millisecond-scan ultra-
violet spectrophotometer to the HPLC instrument. We have
also used this instrumentation to obtain unambiguous identi-
fication of several PAHs in airborne particulates (Choudhury
and Bush, manuscript in preparation).
The HPLC profile of the PAH fraction of the VW Rabbit sample
is shown in Figure 3. The retention times of components 1,
2, 3, and 4 corresponded with those of phenanthrene, anthra-
cene, fluoranthene, and pyrene respectively. The ultraviolet
spectra of these components were superimposable on those of
authentic standards of phenanthrene, anthracene, fluoranthene
and pyrene respectively (Figure 4), establishing firmly their
presence in the sample. Since PAHs with higher ring systems
were present only in low concentrations, no effort was made
to obtain their ultraviolet spectra. Work is under way to
preconcentrate the higher molecular weight PAHs for firm
characterization.
We have also examined the PAH fraction of the particulate
extract from a Mercedes 300-D. Its GC-MS profile was very
similar to that obtained from the VW Rabbit sample. Its
total ion chromatogram (Figure 5) showed components charac-
terized by GC-MS as phenanthrene, anthracene, methylphenan-
threne/-anthracene, dimethyIphenanthrene/-anthracene, benzo-
(ghi)fluoranthene, chrysene/benzo(a)anthracene/triphenylene,
benzo(b)fluoranthene/benzo(k) fluoranthene,benzo(e)pyrene
benzo(a)pyrene. In this sample fluoranthene appeared as a
shoulder of one of the dimethyIphenanthrene/-anthracene peaks.
The HPLC-ultraviolet data are not yet available.
In contrast to the diesel exhaust results, we have found
that airborne particulates collected from several areas of
New York State using hi-vol samplers contain qualitatively
similar amounts of most parent PAHs containing 3-7 rings
(Choudhury and Bush, manuscript in preparation). Very few
alkyl-substituted PAHs were detected in our samples. The
sample collection methods were different, however, and there-
fore while it is an important observation, a strict compari-
son between airborne PAHs and those in diesel particulates
is inappropriate.
182
-------
40 42
Figure 3. HPLC profile of PAH fraction of diesel particulate
extract (VW Rabbit). HPLC condition: 4.6-mm x 25-cm Zorbax
ODS column. MeCH/H20 (84/16). 1.6 ml/min. Linear gradient
to 100% MeOH (20min.) started after 20 min.
,Peak I
Peak 3x
Peak 2
Peak 4
210
365 210
WAVELENGTH(nm)
365
Figure 4. Ultraviolet spectra of HPLC eluates of PAH fraction
of diesel particulate extract (W Rabbit).
183
-------
100.0
i
o
400 600 800 1000 1200 1400 1600 1800 2000 SCAN
6:40 10=00 13:20 16:40 20:00 23:20 26"-40 30=00 33:20 TIME
Figure 5. Total ion chromatogram of PAH fraction of diesel
particulate extract (Mercedes 300-D).
184
-------
ACKNOWLEDGMENT
Sincere appreciation is extended to Dr. P. Dymerski for his
assistance in mass spectrometry work, to Mr. E. Barnard for
general technical assistance and to Dr. R. Gibbs and his as-
sociates of the N.Y.S. Dept. of Environmental Conservation
for collection and extraction of samples. This research was
partially supported by USEPA grant no. R805934010.
REFERENCES
1. Cautreels, W., Van Cauwenberghe, K. 1978. Experiments on
the Distribution of Organic Pollutants Between Airborne
Particuiate Matter and the Corresponding Gas Phase. Atmos.
Environment, 12, 101.
2. Earth, D.S. and Blacker, S.M. 1978. The EPA Program to
Assess the Public Health Significance of Diesel Emission.
J. Air Pol. Control Assoc., 28_, 760.
3. Karasek, F.W., Smythe, R.J., and Laub, R.J. 1974. A Gas
Chromatographic-Mass Spectrometric Study of Organic Com-
pounds Adsorbed on Particuiate Matter from Diesel Exhaust.
J. Chromatography, 101, 125.
4. Bricklemyer, B.A. and Spindt, R.S. 1978. Measurement of
Polynuclear Aromatic Hydrocarbons in Diesel Exhaust Gases.
SAE technical paper 780115.
5. Wotzak, G., Gibbs, R., and Hyde, J. 1980. A Particuiate
Characterization Study of In-Use Diesel Vehicles. In: Proc.
of the Int. Symp. on Health Effects of Diesel Engine Emis-
sions.U.S. Environmental Protection Agency, this volume.
6. Choudhury, D.R. and Doudney, C.O. 1980. Mutagenic Acti-
vity of Diesel Emission Particulates and Isolation of the
Active Fractions. In: Proc. of the Int. Symp. on Health
Effects of Diesel Engine Emissions. U.S. Environmental Pro-
tection Agency, this volume.
7. Blumer, M. and Giger, W. 1974. Polycyclic Aromatic Hydro-
carbons in the Environment. Anal. Chem., 46, 1633.
8. Lee, M.L., Vassilaros, D.L., White, C.M. and Novotny, M.
1979. Retention Indices for Programmed-Temperature Capillary-
Column Gas Chromatography of Polycyclic Aromatic Hydrocarbons.
Anal. Chem., 51, 768.
185
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General Discussion
A. KOLBER: How were the airborne particles collected?
D. CHOUDHURY: The typical high volume samples were
extracted with benzene. At that time we were using benzene.
Most of the samples were collected about 1975 to 1977.
A. KOLBER: Could you describe the sampling technique
further.
D. CHOUDHURY: This was a typical high volume sample
using glass fiber filters.
J. HORWITZ: I didn't hear whether these were wall-
coated open-tubular capillaries; what kind of capillaries
were employed here?
D. CHOUDHURY: These were wall-coated capillaries, by
cell fractioning.
J. HORWITZ: Were the molecular ions determined with the
CI or the El source?
D. CHOUDHURY: They were El. We have also done CI.
D. HOFFMANN: You are comparing engine emission pol-
lutants collected one way and urban pollutants collected
another. How do you know that you are not collecting any
hydrocarbons, or hardly any, and therefore no methylene.
Isn't this comparison a little weak? Must you not use the
same collection system in order to say that this indicates
some diesel pollution. Is it not correct that you can do
it only when you have the same data collection system.
D. CHOUDHURY: The question is not a fixed comparison
between what is and what isn't there. It is an observation
and you take the data from different sources and they are
all different. Now what one sees in the air is a com-
bination of pollutants that come from all sorts of com-
bustion sources, so you cannot really isolate their source.
Basically the idea is that exhaust contains this type of
agent, whereas, typically the air sample contains that kind
of agent. Gasoline has one type while other combustion
samples would have another type of PH profile. This is
basically an observation, and it is not really strict in
saying that this has that and why do we have that.
186
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EMISSIONS OF INORGANIC COMPOUNDS
FROM HEAVY DUTY DIESEL TRUCKS ON THE ROAD
Raisaku Kiyoura
Chairman, Prof. Emeritus
Research Institute of Environmental Science
4 Kojamachi 5-Chome
Chiyoda - Ku
Tokyo, Japan
ABSTRACT
Experiments have been conducted to measure diesel trucks'
inorganic emissions at the Nihonzaka two-lane northbound
tunnel of 2 km long located 170 km west of Tokyo. Average
traffic of heavy duty diesel trucks was 600 per hour during
the two-day period of measurements. Truck speed was 80 km
per hour. Sulfur content of fuel oil was 0.4-0/0. Measure-
ment procedures are almost similar to the Allegheny Tunnel
study by Pierson. Preliminary study done in 1972, present
study was started in 1978 and will continue to 1981.
Average emissions rates of heavy duty diesel truck were
found to be:
(1) Nitrogen oxides
(2) Sulfur dioxide
(3) Sulphate
(4) Nitrate
(5) Total particulates
7-9 grams/km
1.5 grams/km
50 milligrams/km
3 milligrams/km
0.8-1.0 grams/km.
The overall sulfur dioxide conversion to sulphate of emis-
sions was 3-0/0.
The measurements at the ambient are on the way. Sulfuric
acid particulates of 2-30 microns spheres were observed by
microscope on the thymol blue dye coated films exposed in
the ambient 100 meters distant from the tunnel portal.
The author thanks Dr. Pierson, Dr. Witz, Dr. Perhac and
EPA-RTP Officers for their assistance through valuable
discussions.
187
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INTERACTIONS BETWEEN DIESEL EMISSIONS AND GASEOUS
CO-POLLUTANTS IN PHOTOCHEMICAL AIR POLLUTION;
SOME HEALTH IMPLICATIONS
James N. Pitts, Jr., Arthur M. Winer, David M. Lokensgard,
Steven D. Shaffer, Ernesto C. Tuazon and Geoffrey W. Harris
Statewide Air Pollution Research Center
University of California
Riverside, CA 92521
ABSTRACT
A complete assessment of the health effects of diesel
emissions must take into account the possible chemical and
biological transformations of particulate organic matter
due to reactions with the many gaseous co-pollutants which
have now been unambiguously demonstrated to be present
in atmospheres burdened by photochemical air pollution.
These co-pollutants include the "trace" species, nitric
(HN03> and nitrous (HONO) acids, the nitrate radical (N03>,
formaldehyde (^CO) and formic acid (HCOOH), as well as
the criteria pollutants, ozone (03) and nitrogen dioxide
(N02>. Techniques for establishing the atmospheric
concentrations of the trace pollutants (and their spatial
and temporal variations) are briefly described, and we
present results of investigations into the reactions of
polycyclic aromatic hydrocarbons (PAH) coated on filters and
exposed to ambient concentrations of 03 and N02- Envi-
ronmental health implications of these results are discussed
and include the potential for sampling "artifacts" and their
possible effects on the correlation (or lack thereof)
between ambient PAH levels and urban lung cancer rates, as
well as the problems associated with understanding the
appropriate POM "dose" to be employed in animal testing
and assessments of impacts on human health.
INTRODUCTION
Two major pollutants of concern in diesel exhaust emissions
are gaseous oxides of nitrogen (NOX) and particulate
organic matter (POM). Nitrogen dioxide, a key constituent
188
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of NOX, is a "criteria" pollutant per se, and also repre-
sents, through its photolysis, the only known anthropogenic
source of ozone in photochemical smog. Diesel POM is emitted
in the submicron, respiratory size range and like most com-
bustion-generated particulates, contains a number of promu-
tagenic and carcinogenic polycyclic aromatic hydrocarbons
(PAH) as well as other unidentified mutagens that exhibit
strong direct activity in the Ames Salmonella/mammallian
microsome assay.
A comprehensive assessment of the biological effects of
diesel POM emissions must consider not only their composi-
tion as primary pollutants but also the nature of the ulti-
mate products formed (during dispersion and transport) by
their physical and chemical transformations in the atmos-
phere.
In regions experiencing photochemical air pollution, many
nitrogenous and oxygenated compounds are formed as secondary
pollutants by complex photochemical and thermal reactions.
Some of these compounds are known to exhibit toxic, muta-
genic and/or carcinogenic effects in experimental animals.
Others, as yet untested, may also present health hazards.
Some of these species may be capable of reactions with
components of diesel POM to form potentially hazardous
products.
We believe that a reliable prediction of the future health
impact of greatly increased diesel emissions will require an
understanding of:
Current ambient levels of noncriteria pollutants such as
nitric acid, formaldehyde, nitrous acid, etc., in airsheds
suffering from moderate to heavy photochemical smoginto
which additional NOX and POM from diesels will be intro-
duced , and
The chemical, physical and biological results of inter-
action of diesel POM with these co-pollutants, as well as
with the criteria pollutants 03, N02, S02 and CO, and, in
certain instances, sulfuric acid.
These data are required in order to estimate reliably the
total "dose" of pollutants to which individuals might be
exposed in various types of air pollution episodes involving
significant levels of diesel emissions. Obtaining such
informationthe task of atmospheric scientistsis parti-
cularly difficult in the case of diesel NOjj and POM
emitted into photochemical smog. Not only are homogeneous
gas phase processes involved but heterogeneous reactions may
also be important. These include the well-known formation
of secondary nitrate aerosols and a wide range of possible
189
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reactions on the surfaces of primary organic particulates.
Chemical and physical transformations may occur in the
presence of sunlight, oxygen, water and a spectrum of
co-pollutants during transport of diesel POM and NOX in
the atmosphere over periods of hours, days or even weeks.
Present mixtures of gaseous and particulate pollutants in
urban airsheds are already exceedingly complex, but they may
be expected to become even more so in the future as our
nation utilizes such alternatives as diesel engines, synfuel
technologies, and coal-fired power plants to meet the
economic challenges of the energy crisis. Clearly, the
identification and determination of toxic, mutagenic and/or
carcinogenic gaseous and particulate species in the polluted
atmospheres of the 1980s will require a high level of effort
employing sophisticated instrumentation and analytical
procedures (chemical as well as microbiological). These
data are required to avoid two potentially costly misjudg-
ments; on the one hand, unnecessary economic burdens if
overly stringent controls are mandated, and on the other,
threats to public health if possible adverse biological
impacts are not recognized and resolved in a timely, econo-
mically and environmentally sound manner.
An additional level of complexity associated with this
problem is the question of possible "artifact" effects
occurring during the collection, extraction and analysis of
POM. As illustrated in Figure 1, if artifacts are important
in these procedures, the actual "dose" of the pollutants
inhaled by man may be significantly different qualitatively
and quantitatively from the dose assumed to be administered
to experimental animals calculated on the basis of chemical
or microbiological analyses of, for example, hi-vol samples
collected on glass fiber filters. This point will be
considered later.
In order to best confront the challenging problems presented
by these issues, a joint fundamental-applied research
approach involving both simulated and real atmospheres was
developed at the University of California Statewide Air Pol-
lution Research Center (SAPRC). Certain of our programs have
concentrated on the characterization of the pollutant "dose"
typical of atmospheres containing photochemical oxidants.
More recently, our research has been extended to include
studies of POM emitted from diesel engines and other major
combustion sources.
Towards these ends, we have developed and employed state-
of-the-art smog chamber and spectroscoplc systems to iden-
tify and measure for the first time several gaseous pollut-
ants that have been reported or postulated to exist in pho-
tochemical smog. In the following sections we will briefly
190
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DIESEL AND Sf*RK
IGNITION ENGINE
ARTICULATE
EMISSIONS
TRANSPORT AMD
CHEMICAL AND PHYtKAL
TRANSFORMATIONS INVOLVINC
GASEOUS CO-POLLUTANTS
HUMAN EXPOSURE
U U
HI-VOL COLLECTION
EXTRACTION, SEPARATION
COAL AND OIL-FIRED POWER PLANT
ARTICULATE EMISSIONS
IN-VIVO IN-VITRO
MUTAGENIC AND CARCINOGENIC TESTING
Figure 1. Chemical and physical transformations of PAH on
ambient particulate matter during transport through photo-
chemical air pollution and during collection on hi-vol
filters may result in significantly different "doses" to man
and animals than the POM emitted originally from primary
sources.
illustrate the use of such instruments, and also consider
the chemical and biological effects of interactions of a
representative PAH, benzo(a)pyrene (BaP), with the "criteria"
co-pollutants, N02 and 03. References to original litera-
ture can be found in recent publications (1-8) from this and
other laboratories.
GASEOUS CO-POLLUTANTS WITH DIESEL POM IN PHOTOCHEMICAL SMOG
The use of smog chambers fitted with long path White-cell
type optics interfaced to prism and dispersive infrared
spectrophotometers originated more than two decades ago.
Thus, in the classic studies of Hanst, Stephens, Schuck,
Doyle and their co-workers in the 1950s and early 1960s (8),
a variety of compounds formed in HC-NOx-air-UV systems
were identified and time-concentration profiles established.
In the past decade, two major advances have been achieved
in this field. One is the design of more sophisticated smog
chambers and solar simulators; the other is the use of
191
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Fourier-transform (8) infrared spectrometers. Identifica-
tion and measurement of a number of important labile pol-
lutants in simulated and ambient systems is briefly dis-
cussed in the following sections.
Smog Chamber Studies; Identification of Labile Products in
Propene-NOy-Air Systems
Irradiated mixtures of propene, NO and N0£ in air consti-
tute a useful and well studied model system for the photo-
oxidation of ambient olefinic compounds (9). We have
thoroughly investigated this system using our 5800-liter
evacuable environmental chamber. The temperature of the
chamber is controllable to + 1°C in the temperature range
from -20° to +100°C, and it utilizes a 25 kw solar simu-
lator as a light source (Figure 2).
AMBIENT
AIR
INLET
MAGNETIC/ILLY
COUPLED
STIRRING FANS
PRIMARY MIRROR
CHAMBER IRRADIATION WINDOWS
ULTRAVIOLET GRADE QUARTZ
Figure 2. SAPRC evacuable smog chamber and solar simulator
facility.
An Eocom interferometer, interfaced to an 85 m pathlength
multiple-reflection cell in the evacuable chamber is used to
obtain infrared spectra. The short scan times, high resolu-
tion, and large wave number range per scan afforded by an
interferometer makes long-path FT-IR spectroscopy an ideal
tool for identifying and obtaining time-concentration pro-
files of labile gaseous nitrogenous and oxygenated species
formed in such experiments.
192
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A high-resolution (0.125 cm~l) spectrum from a run in which
a mixture of propene, NO and N02 (at concentrations in the
ppm range) in air at 9«4°C (48°F) was irradiated is shown
in Figure 3. This spectrum permits identification
04
0 3
0 I
00
700
800 900 1000
FREQUENCY (cm'1)
1100
Figure 3. Nitrogenous and oxygenated compounds observed by
FT-IR spectroscopy during irradiation of propene-NOx system
at 48°F in SAPRC 5800-liter evacuable chamber.
and quantification of such species as ^05, HN03, pernitric
acid (H02N02), methyl nitrate (CH3ON02) and PAN (CI^COC^NC^)
Additionally, and of importance when considering possible
health effects of ambient photochemical oxidant, formic acid
and formaldehyde were also observed as products.
The propene system has much in common with the many other
more complex systems we have studied in the evacuable chamber
facility, and spectroscopic identification of the above
compounds in such simulated atmospheric systems suggests
their presence in ambient polluted atmospheres as well. At
least two major differences exist between smog chamber
simulations and real polluted atmospheres; first, meteoro-
logical conditions can be effectively controlled in chambers
and, second, higher concentrations of reactive species (ppm
rather than ppb levels) can be employed. In ambient
measurements of trace pollutants and studies of their inter-
actions the former problem is intractable; however, the
latter can be overcome by exploiting the enhanced sensitivity
afforded by kilometer pathlength optical systems (e.g., 1000
m vs. 40-100 m pathlengths in smog chambers), in conjunction
with either IR or visible-UV methods.
193
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Identification and Measurement of Labile Noncriteria
Pollutants in Ambient Photochemical Air Pollution
Applications of FT-IR Spectroscopy
In collaboration with Dr. P. L. Hanst, of the EPA, who
designed and furnished the original instrument, a very long
pathlength FT-IR facility was established at SAPRC in 1976.
In this system, an FT-IR spectrometer is interfaced to a
multiple-reflection cell consisting of eight gold-coated
mirrors with a 22.5-m base path (Figure 4). This cell has
been routinely operated in urban atmospheres at total
pathlengths of 1 km or more.
Figure 4. Kilometer pathlength multiple reflection infrared
cell and FT-IR spectrometer.
During the summer of 1976, the first spectroscopic detection
of HNC>3 and HCHO in ambient smog was achieved. Addition-
ally, time-concentration profiles were obtained for these
species, as well as for formic acid, ammonia, PAN and ozone
(10). These measurements were carried out at Riverside,
California, a "downwind receptor" site in the South Coast
Air Basin.
In 1978 the system was moved "upwind" to Claremont, a mid-
basin site. Again the diurnal and seasonal variations in
the ambient levels of nitric acid and formaldehyde were
measured concurrently with ozone, PAN, formic acid and
ammonia. A representative set of data obtained in a con-
tinuous 36-hour monitoring effort on October 12 and 13,
1978 is shown in Figure 5.
194
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-OCTOBER 12, 1978-
- OCTOBER 13, 1978-
500
1000 1400 1800 2200 0200 0600 IOOO 1400 1800 2200
IOOO
1400 1800 2200 0200 0600
OCTOBER 12. 1978 "
IOOO
1400 1800 2200
-OCTOBER 13, 1978-
TIME (PDT)
Figure 5. Time-concentration profiles of ozone and other
toxic pollutants present in photochemical smog (i.e.,
photochemical oxidant) determined with 1 kilometer path-
length, Fourier transform infrared spectroscopic system in
Claremont, California during October 1978.
High "noncriteria" pollutant levels were observed during
this severe smog episode. Indeed, one can see from these
plots, as well as from the bar graph, Figure 6, that on
this particular occasion the aggregate of these known or
potentially toxic species represented a very substantial
fraction of the levels of the ozone itself. For example, on
the afternoon of the 13th when the ozone level was 0.12 ppm,
the sum of formaldehyde, formic acid, nitric acid and PAN
was 0.11 ppm. A more complete set of data from these
experiments has recently been published (8).
195
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« 3rd STAGE
EPA HAZARDOUS"
2nd STAGE
1st STAGE
CALIFORNIA AIR QUALITY STANDARD
OZONE FORMALDEHYDE NITRIC
ACID
FORMIC
ACID
TOTAL ASSOCIATED
GASEOUS POLLUTANTS
Figure 6. Maximum concentrations of ozone and "noncriteria"
pollutants determined by kilometer pathlength FT-IR spec-
troscopy during a severe October smog episode in Claremont,
California in 1978.
Applications of Long Pathlength Differential Optical
Absorption Spectroscopy
While kilometer pathlength FT-IR spectroscopy is a powerful
tool for the study of atmospheric systems, it suffers from
certain inherent disadvantages. Such facilities are expen-
sive, complex and not readily mobileI.e., not suitable for
tracking pollutants across an air basin over a time span of
several hours or days. Even more importantly, the IR
extinction coefficients of certain key labile species are
significantly smaller than the corresponding UV coefficients
resulting in a corresponding lower IR sensitivity (for a
given optical path).
For these reasons, and in order to further probe the con-
stituents of polluted atmospheres, we recently assembled
and employed a long path UV-visible spectroscopic instrument
to act as a complement to the km pathlength FT-IR system.
The computer-based UV-visible instrument, called a long
pathlength differential optical absorption spectrometer, was
developed by a group of German scientists from the Institut
fur Chemie, Kernforschungsanlage, Julich, directed by
Professor Dieter Ehalt. These scientists first used the
system (shown in Figure 7) to identify and measure formal-
dehyde in the sub-ppb range in maritime air using a 10 km
pathlength (11). Subsequently, for the first time, they
unequivocally identified nitrous acid (HONO) in a relatively
clean atmosphere (12) at Julich, a small nonindustrial town
in West Germany.
Last summer (1979), in collaboration with Drs. Ulrich Platt
196
-------
COLLECTION MIRROR
/\
\ X
<^
3 (long postulated to
play important roles in atmospheric chemistry) in photochem-
ical smog are briefly discussed below, with special atten-
tion to their possible interactions with POM.
REACTIONS OF BENZO(a)PYRENE WITH NITROGEN DIOXIDE AND OZONE
IN SIMULATED ATMOSPHERES
Our discovery in 1975 of direct mutagenic activity of the
organic extract from samples of ambient aerosol collected
throughout southern California led us to investigate the
reactions of benzo(a)pyrene deposited on glass fiber filters
(used for hi-vol sampling) with (a) the gaseous components
of ambient photochemical smog, and (b) with N02 and 63 at
197
-------
2J
CL
Q.
I
"|- riONO r
N02 f T I
1 T H--^ T
I2r , /^f t1 1 -.- -
en
o
1
o
o
x
n
4- \^ i ! *
/rt \.
v4 * \
i T\
M
LOCAL SUNRISE
AUG 4 1979 | AUG 5 1979 |
1)1)1111111
^
H[
ll 1 1 1
100
_o
CL
Q.
80 ~
O
H-
60 $
40 X
^
CM
20 0
n
21 22 23 0 I 23 456
TIME OF DAY (PDT)
Figure 8. Concentration-time dependence of HONO and N02,
measured by Differential Ultraviolet Visible Spectroscopy
(DUVVS) system at Riverside, California, August 4-5, 1979.
<
c
o
X
200
100-
SEPT 12, 1979
> NOj at 623 nm ]
f NOj at 662 nm
N02
1300
3.
O
-200
o
z
X
00
/ .
4/
1800 1900
i
2000
^;
»^s<^^
1
2100 2200
X
F=-K^__
2300
~+-
2400
-100
O
TIME OF DAY (POT)
Figure 9. Concentration-time dependence of N03, N0£ and
03, measured by Differential Ultraviolet Visibile Spectros-
copy system at Riverside, California, during a photochemical
smog episode on September 12, 1979.
198
-------
simulated ambient levels in the laboratory.
Extraction and fractionation of the reaction mixtures, ini-
tially by TLC and subsequently by HPLC techniques, showed
that a number of derivatives of BaP were readily formed in
both types of experiments. This was interesting since for
many years, despite certain intriguing observations to the
contrary, it was assumed by some scientists that polycyclic
aromatic hydrocarbons were virtually inert in atmospheric
systems.
Furthermore, in contrast to BaP, which requires microsomal
activation to produce mutagenic activity in the Ames assay,
directly* mutagenic mononitro- or oxygenated derivatives
were readily formed upon exposure of the BaP-coated filters
to either 0.25 ppm NC>2 (+ -10 ppb HN03) or to 0.1 ppm 03 in
air, respectively. The values of 0.25 ppm NC>2 and 0.10 ppm
03 for one hour correspond to the California air quality
standards for these pollutants. The N0£ standard is
frequently exceeded in the coastal and downtown regions
of Los Angeles and its immediate environs (e.g., West Los
Angeles and Pasadena) in the winter months. The 03
standard is exceeded over 200 days/year (and at times by as
much as a factor of A) in the mid-basin and "downwind"
regions in the late spring, summer and fall.
It is also interesting that a directly mutagenic mononitro
derivative, 3-nitroperylene, was formed when ppm levels of
NC>2 (+ ~10 ppb HNC>3) in air was passed over perylene
similarly deposited on a glass fiber filter. The parent
PAH, perylene, is an isomer of BaP, but a much weaker
activable mutagen than BaP.
We have hypothesized that such reactions with NC>2 and 03 may,
in part, account for the formation of the compounds (as yet
unidentified) responsible for the "excess" carcinogenicity
found in samples of POM collected from ambient air or
exhaust emissions from LDMV with spark ignition engines.
We use the term "excess carcinogenicity" to describe that
activity which is greater than can be accounted for on the
basis of the known carcinogenic polycyclic aromatic compounds
measured in these samples.
Iwe define "direct mutagen" as mutagenic activity without
the use of a microsomal activation system (S9) under
normal testing procedure. We are aware of the possibility
of increased sensitivity of the intracellular nitrogen
reductase enzymes of the original tester strains and the
potential for false positives.
199
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The half-life of BaP on a glass fiber filter exposed to 0.1
ppm O^ in air is less than one hour; such high reactivity
and the formation of direct mutagens are consistent with our
earlier hypothesis that directly mutagenic and/or carcino-
genic compounds can be formed by "atmospheric activation" of
PAH during transport in regions with moderate to heavy
photochemical air pollution. However, it is important
to recognize that these exposures have been carried out on
glass fiber filters. There well may be a substrate depen-
dency of the rates and products of nitration or ozonolysis
of BaP. Results of such exposure may differ significantly
when carbon, quartz, Teflon fibersor indeed raw diesel
POMserve as the sites of BaP deposition. Studies are
currently underway to explore this issue.
The nitration of BaP in simulated atmospheres appears to be
catalyzed by nitric acidand as our longpath FT-IR studies
have shown, there is ample HN03 in ambient photochemical
smog to serve this function. Moreover, we now have confir-
mation of the presence of HONO and N03 in ambient air. It
is possible that these co-pollutants of POM may also react
with BaP and other reactive PAH.
Furthermore, the reaction of BaP with relatively low levels
of 03 (100 ppb) is so fast on glass fiber filters that
this reaction would appear to be quite feasible in real
atmospheres if the substrate dependence is small. To
determine whether this is really the case, we must develop
an "artifact free" system for collecting ambient POM. We
are currently designing a "diffusive de-nuder" that selec-
tively removes the reactive gases N02, PAN and 03 prior
to collection of the POM. Preliminary results at relatively
low flows have been encouraging, and if substantiated for
higher collection rates, may ultimately lead to an "add-on"
device for particulate samplers that will minimize artifac-
tual problems in the routine collection of ambient POM.
Finally, we point out that in order to reliably establish
correlations between, for example, oxidant and N0£ levels
and the direct mutagenic activity of ambient aerosol
samples, it is necessary to improve the precision and
accuracy of the chemical and microbiological procedures
involved in the complete characterization of an environ-
mental sample of POM. We have refined our analytical and
Ames test procedures with the goal of achieving such
improvements (15). The dose-response data shown in Figure
10, are from four samples of ambient POM collected concur-
rently on the SAPRC "mega sampler". The good agreement
between these curves suggests that overall precision for
sample handling, from collection through extraction and
chemical and microbiological assays, of around +15% can
be achieved if appropriate precautions are taken.
200
-------
LJ
_
CL
1000-
900-
800-
to 700-
I
LOCAT10N. EL MONTE, CA.
DATE: OCT. Z-OCT 3, 1979
COLLECTION TIME- 27 HOURS, 18 MINUTES
OVERALL FLOW RATE- 640 C.F.M.
AMBIENT AIR SAMPLED: 7420 M3 PER FILTER
FILTER MEDIA: TEFLON-IMPREGNATED GLASS FIBER
SPONTANEOUS REVERTANT COLONIESISTRAIN TA98)
-S9 31 REVERTANTS/PLATE
I 20 60 100
10 40 80
150 250
SAMPLE/PLATE
500
Figure 10. Direct mutagenic activity of four samples of
ambient POM with strain TA98 collected concurrently using
the SAPRC "mega sampler." The samples were collected on
Teflon impregnated glass fiber filters at El Monte, Calif-
ornia on October 2-3, 1979. (Spontaneous reversion fre-
quency for TA98 was 31 revertants per plate.)
DISCUSSION OF POTENTIAL HEALTH IMPLICATIONS
We start from the premise that the composition of POM
emitted from the tailpipe of light duty motor vehicles with
diesel or spark ignition engines (or indeed, emitted from a
fossil fueled power plant) may be very different chemically
and in its biological activity from the POM that ultimately
201
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impacts man. Thus, assessments of the health effects of
exhaust emissions from diesel engines must take into account
not only the chemical and physical characteristics of the
primary emissions of particulate organic matter, but also
their chemical and physical transformations which may occur
before or during receptor impact. Such transformations of
POM by gaseous co-pollutants might occur: (1) in primary
exhaust streams, (2) during transport in polluted atmos-
pheres, (3) on filters during the act of collection and (4)
possibly in situ in the lungs.
Transformations of POM in Primary Exhaust Streams
A common assumption is that if PAH levels in exhaust streams
are reduced by certain control techniques (e.g., by a
catalytic converter on a diesel engine operating in a mine)
the health impact of the POM will necessarily be lessened.
Actually, this may or may not be the case, especially for an
aged and inefficient catalyst. Thus, if BaP reacts (and
disappears from the system), new species may be formed which
are more mutagenic and/or carcinogenic than the original
BaP. If this is the case, the health impact may be worse
than before treatment of the POM by an oxidizing catalyst;
if not, genuine gains will be achieved.
^Transformations During Transport in Polluted Atmospheres
Since diesel particulates fall in the respirable, submicron
particle size range, they can remain in the atmosphere and
be transported and transformed over a period of hours (or
possibly even days under the stagnant weather conditions
characteristic of severe smog episodes).
Three general cases should be considered when developing
"impact" or "assessment" documents: injection of diesel
emissions into (a) "clean" air, (b) light-to-moderate air
pollution and (c) severely polluted atmospheres. In addi-
tion, two general types of "smog" that are chemically quite
different should be considered. "London"-type smog contains
carbonaceous particles and oxides of sulfur (SOX) in a
generally reducing atmosphere at relatively low temperatures
and high humidities, while "Los Angeles"-type photochemical
smog is characterized by highly oxidizing atmospheres, high
ambient temperatures, clear skies and lower relative humid-
ities.
Chemistry and health effects, of course, can be very differ-
ent for diesel POM in these two quite different types of
smog. We stress that our comments here are directed primar-
ily to major air basins already suffering from moderate
(-0.20 ppm maximum hourly average several times per year)
to severe (>0.30 ppm 03 hourly average on say, five or
202
-------
more days per year) levels of photochemical oxldant. In
principle, however, the suggested approach to this problem
i.e., research on the possibility of secondary pollutants
interacting in the atmosphere with diesel POMalso seems
appropriate for areas with heavy sulfurous type smog.
Finally, it is clear that when evaluating the chemical and
physical transformation of POM, gaseous co-pollutants to be
considered should include not only the "criteria" pollutants
but also such species as PAN, nitric and nitrous acids,
formaldehyde, formic acid and the nitrate radical (N03>.
We have recently shown (16) that the latter reacts very
rapidly with phenol and the cresol isomers, as well as with
olefins; it also may do so with PAH.
Transformations of POM During Sampling
As noted previously, it is critical to define precisely
what we mean by the "dose of diesel POM" and to be aware
that its properties may be substantially modified by, for
instance, sampling. Thus recent studies in this and other
laboratories show that "filter artifacts" can occur in
which, for example, reactive polycyclic aromatic hydrocar-
bons such as benzo(a)pyrene are transformed on conventional
hi-vol glass fiber filters by gaseous pollutants present in
ambient photochemical smog. They may also react with SOX
species (e.g., S02, 803, ^SO^, etc.), but we have not as
yet examined these systems.
One of the most intriguing preliminary results from our
studies of the interactions of ozone in air at ambient
levels with BaP on glass fiber filters is that we have some
evidence, based on HPLC retention times, mass and fluores-
cence spectra, that a BaP-epoxide may be formed. However,
the analysis of the complex product mixture formed in this
reaction is difficult. We can state that the HPLC fraction
which shows strong direct mutagenic activity contains
predominantly qulnones which are inactive to strain TA98 in
the Ames test. Thus, we suspect that the activities in this
predominantly quinone fraction may be due to small amounts
of a powerful mutagen(s) formed from BaP during its oxida-
tion by ozone. Studies are underway to further resolve this
fraction and to identify this powerful mutagen which we
suspect may be an epoxide of BaP.
Should this mutagen prove to be a BaP epoxide, it is tempt-
ing to speculate that it might also be one of the species
present in raw diesel exhaust and in ambient POMand be
a contributor to the strongly mutagenic activity of the
polar fractions of these materials. While it is speculation
to suggest that BaP-epoxide might be present in raw diesel
exhaust, it is a possibility which should be investigated,
203
-------
since it is well known, for instance, that BaP-4,5-oxide is a
very powerful direct mutagen.
In photochemical smog substantial levels of ozone coexist
with BaP present in ambient POM. If the reactions on glass
fiber filters do indeed occur in ambient air such a species
as the epoxide may well be formed. We are currently inves-
tigating this possibility.
Transformations of POM In Situ in Lungs
If reactive PAHssome of which are known carcinogensreact
on filters with atmospheric pollutants such as nitrogen
dioxide and ozone, they might also react in situ after
deposition in the lung with such gaseous co-pollutants
(possibly dissolved in lung fluids). Hence, even laboratory
animal exposure studies designed to elucidate health effects
arising from emissions of diesel POM to the atmosphere may
be incomplete unless the studies simulate this potential
synergism between the POM and its co-pollutants.
Implications to Epidemiological Studies
The ease with which BaP deposited on glass fiber filters
reacts with ambient levels of N02 and 03 to form direct
mutagens has implications not only for artifacts in analyses,
but also for epidemiological studies in which ambient BaP
(or POM) are correlated with lung cancer. In this context,
the following observation should be considered: it was
reported in 1972 that levels of BaP in ambient air in
California's South Coast Air Basin were much lower than in
most other major cities in the world (5).
From this it could be concluded that (a) primary emissions
of POM (and associated BaP) from industry and motor vehicles
were much lower in the Los Angeles area than in other urban
areas and (b) since BaP levels were lower in Los Angeles,
the risk of lung cancer that may be associated with ambient
BaP was correspondingly less. However, if the rates of
reaction of BaP with such species as 03 or N02 are as
fast in ambient photochemical smog or during the sampling
procedures employed in these studies, as we have shown them
to be in our laboratory simulations, these processes could
convert the BaP into species not detected by the analytical
techniques commonly used in the 1950-1970 period (e.g.,
fluorescence). This alternative explanation could account
for the relatively low levels of BaP reported for Los
Angeles (5).
In this regard, epidemiologists Goldsmith and Friberg (17)
report the following anomaly:
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"If urban pollution by benzo(a)pyrene makes
an important contribution to the urban excess, lung
cancer in the locations most polluted by this material
should be highest, and when the agent decreases, lung
cancer should do so as well. This has not been shown
to occur."
Actually there may be little reason to expect that such a
correlation should exist in areas heavily polluted with
photochemical oxidant. In these regions the BaP may be
efficiently transformed into other compounds in the air or
on the sampling filters. Thus, the ambient levels of BaP as
reported by air monitoring networks and utilized by
epidemiologists and control officials estimating health
effectsmay be seriously misleading.
In conclusion, atmospheric scientists should continue to
attempt to define the complexities inherent in fully char-
acterizing the atmospheric "dose" associated with diesel
emissions. However, of course, the ultimate judgments
concerning possible health effects (i.e., search for and
characterization of the "response") must be made by the
biological and medical communityhopefully in close colla-
boration with the atmospheric scientists.
ACKNOWLEDGMENTS
We gratefully acknowledge support for the research described
here from the National Science Foundation (No. PFR-7801004),
Department of Energy (No. DE-AT03-79EV10048), U. S. Envi-
ronmental Protection Agency (No. 804546) and California Air
Resources Board (No. A7-138-30). We thank Ms. Yvonne
Katzenstein for valuable editorial assistance in preparing
this manuscript and Professor W. Belser, Dr. R. Graham and
P. Hynds, T. Fisher, C. Smith, P. Ripley, A. Thill for their
contributions to this research. We also express our appre-
ciation to Mr. Robert Danner, the Symposium organizer, for
his professional assistance.
REFERENCES
1. Pitts, J. N., Jr. 1980. Atmospheric interactions of
nitrogen oxides. In: Nitrogen Oxides and Effects on
Health (S. D. Lee, ed.), Ann Arbor Science, Ann Arbor,
Michigan.
2. Pitts, J. N., Jr. 1979. Photochemical and biological
implications of the atmospheric reactions of amines and
benzo(a)pyrene. Phil. Trans. R. Soc., 290:551-576.
3« Pitts, J. N., Jr., K. A. Van Cauwenberghe, D. Grosjean,
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J. P. Schmid, D. R. Fitz, W. L. Belser, Jr., G. B.
Rnudson and P. M. Hynds. 1978. Atmospheric reactions
of polycylic aromatic hydrocarbons: facile formation
of mutagenic nitroderivatives. Science, 202:515-519.
4. . Pitts, J. N., Jr., D. Grosjean and T. M. Mischke. 1977.
Mutagenic activity of airborne particulate organic
pollutants. Tox. Lett., 1:65-70.
5. Committee on Biologic Effects of Atmospheric Pollutants.
1972. Particulate Polycylic Organic Matter, National
Academy of Sciences, Washington, B.C.
6. Hoffmann, D. and E. L. Wynder. 1977. Organic particu-
late pollutants - Chemical analysis and bioassays for
carcinogenicity. In; Air Pollution, Volume II (A.
Stern, ed.), Academic Press, Inc., New York, New York.
7. Lane, D. A. and M. Katz. 1977. The photomodification
of benzo(a)pyrene, benzo(b)fluoranthene and benzo(k)-
fluoranthene under simulated atmospheric conditions.
In; Fate of Pollutants in the Air and Water Environ-
ments, Part 2 (I. A. Suffet, ed.), John Wiley and Sons,
Inc., New York, New York.
8. Tuazon, E. C., A. M. Winer, R. A. Graham and J. N.
Pitts, Jr. 1980. Atmospheric measurements of trace
pollutants by kilometer-pathlength FT-IR spectroscopy.
In; Advances in Environmental Science and Technology,
Volume 10 (J. N. Pitts, Jr. and R. L. Metcalf, eds.),
John Wiley and Sons, Inc., New York, New York.
9. Carter, W. P. L., A. C. Lloyd, J. L. Sprung and J. N.
Pitts. 1979. Computer modeling of smog chamber data:
Progress in validation of a detailed mechanism for the
photooxidation of propene and n-butane in photochemical
smog. Int. J. Chem. Kinet.. 11:45-101.
10. Tuazon, E. C., R. A. Graham, A. M. Winer, R. R.
Easton, J. N. Pitts, Jr. and P. L. Hanst. 1978. A
kilometer pathlength Fourier-transform infrared system
for the study of trace pollutants in ambient and syn-
thetic atmospheres. Atmos. Environ., 12:865-875.
11. Platt, U., D. Perner and H. W. Patz. 1979. Simulta-
neous measurement of atmospheric CI^O, 03 and N02
by differential optical absorption. J. Geophys. Res.,
84:6329-6335.
12. Perner, D. and U. Platt. 1979. Detection of nitrous
acid in the atmosphere by differential optical absorp-
tion. Geophys. Res. Lett., 6:917-920.
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13. Platt, U., D. Perner, A. M. Winer, G. W. Harris and
J. N. Pitts, Jr. 1980. Detection of NC>3 in the
polluted troposphere by differential absorption.
Geophys. Res. Lett.. 7:89-92.
14. Platt, U., D. Perner, G. W. Harris, A. M. Winer and
J. N. Pitts, Jr. 1980. Observations of HONO in an
urban atmosphere by differential optical absorption.
Nature, in press.
15. Belser, W. L., Jr., S. D. Shaffer, R. D. Bliss, P. M.
Hynds, L. Yamamoto, J. N. Pitts, Jr. and J. A. Winer.
1980. A standardized procedure for quantification of
the Ames Salmonella/mammalian microsome mutagenicity
test. Environ. Mutat.. submitted for publication.
16. Carter, W. P. L., A. M. Winer and J. N. Pitts, Jr.
1980. Kinetics of the gas phase reactions of the
nitrate radical with phenol and the cresols. J. Phys.
Chem., submitted for publication.
17. Goldsmith, J. R. and L. T. Friberg. 1977. Effects of
air pollution. In; Air Pollution, Volume II (A.
Stern, ed.), Academic Press, New York, New York.
General Discussion
R. KLIMISCH: There is some work that says that BaP
isn't nearly as reactive when it is on soot. It doesn't
react with N02 very readily, and I would suspect that it
won't react with anything else very readily when it is
absorbed on soot. So I really think your experiments are
unrealistic in terms of the reactions of PAH.
J. PITTS: That's a very good point and a very good
question. You recall I stressed that it might very well be
substrate dependent. This may very well be the case. We
also have received, in the last six months, a grant from
the Department of Energy to look at exactly this question.
What are the reactions on glass fiber, on soot, on coal
fired power plants flyash. They will likely be very dif-
ferent. Certainly, I think the photochemistry is dif-
ferent, and I have not even talked about just direct photo-
chemical rearrangements. There is evidence in the lit-
erature that suggests the photochemistry just in clean air
may be very different on, say, flyash as against soot. So
your point is well taken, they may not, but we are going to
continue in a very orderly fashion to try to explore these
different subjects. We have an application in now and
hopefully, we will be working with DOE and NSF to use pho-
toacoustic photoscopy to actually get the absorption spec-
trum of these PAH's and PNA's as they are bound on these
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different substrates. As an old geriatric photochemist who
has been in this game probably longer than he wants to
admit, the first thing we taught our students, and the
first thing I was taught, is before you start any photo-
chemical studies, you have to measure the absorption spec-
trum for the compounds you are looking at. The first law
of photochemistry says if it doesn't absorb, it isn't going
to react photochemically. Well, very little is known about
absorption spectrum of PAH's. So this is going to be a
very exciting field, and it is a tough field, but it is
exciting and it will provide answers to all kind of ques-
tions.
R. KLIMISCH: I think you will find that PAH's are al-
ways associated with particulates and carbon,
J. PITTS: Depending on whether they are sitting on the
flyash inorganic site with a particular service area and
service density, and service properties as against say the
kind of loose soot that are hung together in diesel, they
can be very different.
B. BELINKY: With respect to the nitro derivatries of
PAH, have you actually determined their presence in either
diesel emissions or ambient air, and have you received any
comments about their susceptibility.
0. PITTS: We have not as yet. We have looked for ni-
tros at Riverside but one problem is that N02 levels are
very low at that location. We never exceed the air quality
standard there. We will be looking for nitro derivatives
this winter near the beach in Southern California where NC>2
levels are high. The second problem is that they are pho-
tochemical ly reactive. We have not measured the quantum
levels as we intend to, but they do photo decompose and
ultimately wind up as clinodes. The NOg rearranges to a
nitrate to NO bond, NO rips off in sunlight and then you
oxidize it ultiamtely to a clinode. Here is another point
which should be mentioned. In these transformations, I
want to be very clear about this, we may form compounds
that are more toxic. We may also detoxify the compound. It
is very important to recognize that if you allow ozone
oxidation to proceed long enough, you will have a system
comparable to the S9 oxidation system cells. That is, the
PAH's are oxidized with formation of hydroxyl radicals and
thus might be detoxified also. We have to look at that
path.
J. PATTISON: I presume the nitrate radicals were meas-
ured in Riverside. However, this might have been the time
of day that the LA smog drifted into that area, rather than
say that it is forming at that time of day which was late.
So your interpretation is, if you measured it downtown you
would find it in the day rather than in the early evenings?
J. PITTS: That is a "downwind" phenomena. Most of the
smog comes into Riverside, for example, from LA along the
mountains, Pasadena, Ontario, up through Oregon County. So
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this would basically be it.
J. PATTISON: In other words, it drifted in rather than
formed there.
J. PITTS: It drifted in, but it just sat there. It sat
there lone enough - it was a fairly stable mass.
It had moved in and, in fact, it was an episode that lasted
several days. We never went down to clean air. We didn't
have a transport through to clean it up every night.
This was like that Clairmont episode. It stayed, and it
built up, so the inversional phenomena of the build-up
during the night is a real phenomena.
J. PATTISON: In other words, it would be retained in
the evening?
J. PITTS: No, no, in this case the nitro derivatives
formed and when the sun went down disappeared. So it was
formed-by photochemistry, but it didn't get there sooner
because it hadn't drifted in yet. It had to be actually
made there. It was in the element of the smog mask that
moved in - contained in certain compounds then when the
right NO conditions, particularly concentration and ratio
were reached, it could form which it did. Then with the
addition of NO, NOX reacts with NO, N03 radical plus NO
reacts at collison frequency practically to form NOz- So
it is destroyed. If you have a lot of cars go by the free-
way, you get more NO, and it is gone. It is a very complex
system, but it is there.
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OPTIMIZING DIESEL COMBUSTION: IMPROVING FUEL ECONOMY.
ENGINE LIFE. AND REDUCING PARTICULATE AND NQy
EMISSIONS WITH ELECTROSTATIC FLUID PROCESSORS
Robert A. Gibbons
Diesel Automobile Association USA
and
Dr. B. A. Wolf
Consultant
Clearwater, Florida USA
ABSTRACT
Emission legislation has adversely affected fuel economy of
gasoline engines, and threatens similar effect upon diesel-
powered vehicles. This would be unfortunate, for diesel
automobiles can have a significant fuel conservation impact,
improving fuel economies by as much as 100 percent over
comparable gasoline engines in actual use-modes.
Increasing application of diesel power to autos and light
trucks has been somewhat inhibited by two related emissions
concerns: speculative concern about health effects of
components of diesel particulates, and technical difficulty
of simultaneous achievement of proposed particulate and
nitrogen oxides emission levels.
This paper illustrates the positive potential of Electro-
static Fluid Processing systems (EFP) to substanitally
reduce both particulate and NOX emissions without a fuel
economy penalty, and, with added benefit of extending
engine/component life. Also, as diesel fuels continue to
deteriorate in quality, and as manufacturers are under
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pressure from emissions proposals to develop increasingly
sensitive microprocessor-controlled fuel injection equipment
and/or such wear-inducing techniques as exhaust-gas recir-
culation, the role of EFP is expected to assume critical
importance in diesel power applications.
INTRODUCTION
Relatively neglected areas of inquiry in the concern about
maximizing fuel economy and minimizing engine emissions are
those of fuel composition, fuel quality, contamination of
fuels in processing, handling and distribution, and in fuel
systems, and, related issues regarding interaction of
lubricating oil contamination, engine emissions, engine
life and fuel economy.
Obviously, concern with emissions from fuel burned in an
engine or other combustion system properly includes concern
and inquiry about the incombustible, partially combustible,
and other fuel components and contaminants which inhibit
optimal combustion, and are emitted as exhaust components.
The computer industry truism, "...garbage in...garbage out",
is also true of fuels/engines systems.
Furthermore, in reciprocating piston engines, especially
in diesels, crankcase lube-oil contaminants may plate upon
or even aspirate into combustion chambers, and there inter-
mingle with fuel and fuel contaminants, causing an increase
in emission product.
Such contaminants, in addition to the partially-understood
phenomenon of "carbonizing" (1), dynamically affect combus-
tion in the known and partially known phenomena of wall-
quenching, flame quenching, and fuel droplet-quenching.
Such contaminants may also act as "condensation nuclei" and
chemical reactants within the fluid dynamic of combustion.
It is known conclusively that automotive fuel filters and
lube-oil filters of nominal 5-micron size capacity actually
cannot remove particles smaller than 10 to 13-microns with
consistency. It may be confidently asserted that fuels and
lube oils, as they are used in engines, have contaminants,
residues and detritus which are larger and much smaller than
these sizes. These contaminants contribute to engine wear,
to reduced fuel economy, and to increased emissions. Con-
versely, removal of and/or prevention of the formation of
such particulates as appear in fuels and lube-oils of
diesels will have a salutary and economical effect upon
the parameters of emissions, fuel economy and durability.
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TECHNIQUES OF PARTICULATE REMOVAL FROM HYDROCARBON LIQUIDS
The problem of removing very fine particles from a body of
fluid is not readily solved by mechanical filter alone,
since it becomes necessary to use a filter element having
such fine pores or openings to pass the fluid that they are
easily clogged by removed particles lodged in the pores.
The resistance to flow increases rapidly as the pores become
clogged. Resistance to fluid flow would be intolerably
great even without such clogging when the openings are
sufficiently small to retain particulates of micron size.
Centrifuges (centrifugal separators) remove particles and
particulate contamination to the 10 micron range, and less
consistently to 5+ micron size. It is interesting to note
the effect of even this relatively poor level of diesel
fuel purification upon parameters of contamination and
combustibility:
TABLE 1. BENEFITS OF PURIFICATION
WITH CENTRIFUGE EQUIPMENT (2)
Ash Content
Water Content
Net Calorific
Value, BTU/lbs.
Before Purification
0.09%
1.3 %
17,603
After Purification
none measurable
none measurable
18,081
Particulates of 10 micron are the smallest size visible to
the unaided eye (2), and, according to ASME/ASTM Standard
118, oil purification equipment must remove particulate at
least to 10 micron. Other specifications call for reduction
of particle contaminants to one micron size.
Water-washing, a technique which removes particulate from
fuels and oils to one micron, but not smaller, is highly
unlikely to find application aboard a motor vehicle.
However, since hydrocarbon liquids are dielectric fluids
having relatively high dielectric value at a wide range
of temperatures, an electrostatic field may be employed
to remove particles down to 0.001 microns, (10~9 meter)
(3).
THE ELECTROSTATIC FLUID PROCESSING SYSTEM (EFP)
It is well known, especially in treating dirty gases, that
suspended particles can be charged electrically and then
caused to migrate to and be collected on a surface under the
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influence of an electric field. In a typical electrostatic
precipitator, the force of the electric field on a 0.5
micron (micrometer) particle is several thousand times the
force of gravity on such a particle. When hydrocarbon
liquids are substantially free of water, the force of an
electric field upon particles in such a liquid hydrocarbon
remain approximately the same as that upon particles sus-
pended in a gas.
The EFP system was originally designed for cleansing fluids
for missiles, where there is a definite need for liquid of
the greatest possible freedom from contamination, and the
liquid to be cleaned has a high dielectric value. EFP
systems have been used successfully for many years in such
applications. It is a system that will remove very fine
suspended particles, and includes an effective water-strip-
ping component capable of removing water to 0.005%. The
system is an electrostatic processor capable of removing
sub-micronic particles from a dielectric liquid stream or
bulk storage.
The EFP unit designs offer negligible resistance to fluid
flow. It is flexible in operation, economical to manufac-
ture in various capacities and flow rates, and is simple to
maintain in efficient operating condition. EFP systems
remove macro and sub-micronic particulates from fuels and
lube-oils, and remove liquid contaminants by breaking up
emulsions.
EFP SYSTEM PRINCIPLES DIFFER FROM GASEOUS PRECIPITATORS
While the EFP system uses electrical charging fields,
principles of dielectrics, electrostatics and electro-
magnetics similar to those used in electrostatic gaseous
precipiators, the phenomena of corona discharge used in
gaseous precipitators do not occur in dielectric liquids.
Rather, electrostatic induction, electrophoresis, and/or
electrowinning agglomeration processes are used to remove
all particulate contaminants without destroying any of
the liquid or changing any of its chemical or physical
properties, except that removal of contaminants restores
the original viscosity, improves flamability and the like.
A high voltage electric charge, applied to the fluid causes
an excess of electrons in the fluid. These electrons sepa-
rate covalently bonded materials by filling the void in the
valence of the co-valently bonded atoms. By this means,
dissimilar materials are separated and similar atoms then
ionize, flocking together into larger particles which adhere
to the charged surface of the opposite polarity. Thus,
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elemental as well as chemically complex substances, whether
metallic, non-metallic, organic or inorganic, can be removed
from petroleum liquids and non-petroleum hydrocarbon liquids.
EXPERIMENTAL DETERMINATION OF SUBSTANCES REMOVED
Test conducted by the U.S. Air Force over a period of
several years determined the following substances are
effectively removed by EFP systems processing fuel oils,
hydraulic fluids, turbine oils and lubricating oils (4):
Iron
Aluminum oxide
Red iron oxide
Biotite mica
Limestone
Titanium
Manganese
Lead
Silver
Zinc
Steel
Quartz
Delustered dacron
Brass, bronze
Calcite
Hornblende
Calcium
Silicon
Aluminum
Chromium
Phosphorus
Chlorine
Stainless steel
Magnesium
Limonite
Paper fibers
Carbon
Potassium
Copper
Tin
PROLONGED ELECTROSTATIC PROCESSING EFFECTS ON HYDROCARBON
LIQUIDS
The U.S. Air Force subjected used purging oil to 80 hours of
electrostatic filtration, with the following test results:
New Purging Oil
Used Purging Oil Before Filtration
Used Purging Oil After 80 hrs EFP Filtration
Flash Point
208° F
151° F
162° F
Viscosity
Used Purging Oil Before Filtration
Used Purging Oil After 12 min.
Used Purging Oil After 8 hrs Filtration
Used Purging Oil After 60 hrs Filtration
.0516 centistokes
.0526 centistokes
.0526 centistokes
.0524 centistokes
INFRARED ANALYSIS
Samples of used purging oil were compared with the used
purging oil after it had been electrostatically filtered
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for 80 hours and no change in the composition was detected
(5), other than the removal of contaminants, which tends
to improve viscosity, etc. The Air Force laboratory inves-
tigators concluded:
"We have not encountered any type of contamina-
tion that is not effectively removed by the
electrostatic filters. Contamination commonly
found in hydraulic systems and motor oils is
effectively removed...The fact is, we have not
found any kind of solid material the filter will
not remove, regardless of how small it is. The
electrostatic filter removed particles of 5
microns and smaller very effectively." (Whereas)
..."Mechanical means of filtering using 5-micron
absolute filters, cannot remove this contamina-
tion and the level of this small size, 5-micron
and smaller, would gradually increase, causing
changes in the viscosity of the fluid...The
smaller the particle the more effective is
electrostatic filtration..." (6) (Emphasis in
original).
ON-BOARD ELECTROSTATIC PROCESSING OF DIESEL FUEL AND DIESEL
ENGINE LUBRICATING OIL EXPERIENCE IN THE MEXICAN
NATIONAL RAILROAD
The popular press in the United States has characterized
Mexico as a nation unconcerned with environmental pollution,
and since revelation of the extent of Mexico's petroleum
reserves, as also unconcerned with conservation. As will
be seen, this is unfair to Mexico and is in fact, not the
case.
The Mexican National Railroad and the Mexican government
have expressed their interest in conservation of fossil
fuels and in reducing pollution from combustion by having
sponsored a three-month test of EFP units aboard an Electro-
Motive Division (EMD) diesel locomotive of the Ferrocarriles
Nacionales de Mexico. From the results of this test, the
Mexican government has expressed its interest in installa-
tion of EFP systems on all 1,500 Mexican railroad locomo-
tives.
SUMMARY OF RAILROAD DIESEL TEST RESULTS
An EFP system consisting of two EFP units 27 inches wide
by 46 inches high by 36 inches deep was installed in an
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EMD SD40 3000 HP diesel locomotive for continuous processing
of engine fuel and lubricating oil. Units were use-dedi-
cated, one for diesel fuel and one for lubricating oil, and
were installed aboard Engine #8720 in Mexico City. Another
locomotive, Engine #8709, also an EMD SD40 3000 HP model,
was used as the control or comparison engine. Both engines
had just received major overhaul, and were as close to being
mechanically identical as was possible. The two engines
were coupled together to permit them to receive identical
fuel, lubricants, filters and operators. They were desig-
nated to pull the same load under identical conditions. The
only significant difference between the two engines was the
EFP system aboard #8720, processing the fuel and lube-oil of
#8720's diesel engine.
1. One of the major differences in operation noted was the
disappearance of the majority of the black smoke emitted
by locomotive #8720, which contained the EFP system.
2. Another significant change was the cleanliness of fuel
as it passed through the sight-glass. Upon installa-
tion, the fuel passing the sight-glass was extremely
dark and non-transparent. Within minutes after the
EFP system was turned on, fuel passing the sight-glass
changed to clear and lighter color.
3. Fuel tanks on locomotives apparently have tendency to
build up an iron oxide scaling, extremely small in
micron size, which is continuously carried through to
injectors and engine combustion chambers by the fuel
flow. After the EFP system was operating, the excessive
fuel over engine dema.nd was force-fed back to the fuel
tanks. Iron oxides which were stripped from the inner
tank walls were captured and retained by the system,
preventing these contaminants from being transmitted
to the injectors, combustion chambers, and hence, to
exhaust emissions.
4. With regard to fuel cleanliness, the locomotive tanks
were filled with centrifuged fuel, which purportedly is
free of contamination by particulate matter and water.
A sample of the fuel was taken while the tanks were
being filled. Then the locomotive was started and the
EFP system turned on. At the end of three hours, a
sample of processed fuel was taken from the drain of the
locomotive tank. Both samples were in the same size and
type containers; the difference was so visible that the
centrifuged fuel appeared unprocessed when compared to
that processed by EFP.
5. The incandescent material normally emitted from the
stacks of a diesel locomotive began to visibly disappear
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from the emission of #8720, while it increased in that
of #8709. Within three days, the incandescent emission
from #8720 had been reduced to the extent that it was
completely cooled and no longer visible beyond a two-
foot distance of the stack. The possibility of #8720
starting a roadside fire had been eliminated, and the
total mass particulate emissions had been dramatically
reduced as evidenced by substantial reduction of smoke
and other visible particulate emissions. Most certainly
there was a reduction of particles of iron oxide which
are normally emitted from the locomotive's exhaust
stacks.
6. Upon termination of the test, examination of the cathodes
revealed the EFP units had captured and retained various
contaminants ranging in size from the smallest visible
under a microscope to sizes in excess of 100 microns.
While we have no calculation of the percentage this
removal is of the total fuel mass consumed in the test,
there were 10 collecting plates of approximately 16
inches in diameter that contained a coating in excess
of 1/8-inch thickness of contaminants removed from the
fuel. Further examination of the polyurethane pads of
the EFP unit, and the fuel residue in the bottom of the
EFP housing revealed considerably more of the same sizes
and types of contaminants. A good portion of the
samples were given to Mexican Railroad chemists, there-
fore it was not possible to estimate a ratio of fuel/
contamination or the percentage of contaminants in the
fuel.
7. Within one week of operation (7 days), the engineer com-
plained of the difference in performance between the two
locomotives. He felt the fuel was being restricted in
#8709 and asked that the mechanical filters be changed.
The mechanical filters in both locomotives, #8709 and
#8720 (EFP-equipped), were changed simultaneously. The
weights of the filters prior to and after installation
were recorded:
TABLE 2. WEIGHTS OF
New Filters (dry)
New Filters (wet)
#8709 Wt. on removal
Percent Wt. increase
over wet
#8720 Wt. on removal
Percent Wt. increase
over wet
MECHANICAL FILTERS ON
Primary
2788 g
3895 g
4056 g
3.97 %
3895 g
0.0 %
Secondary #1
230 g
320 g
330 g
3.03 %
320 g
0.0 %
LOCOMOTIVES
Secondary #2
230 g
320 g
326 g
1.84 %
320 g
0.0 %
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Thus, it may be seen in Table 2, that on-board, in-use
application of EFP systems processing of diesel fuel
prevents a significant percentage by weight, of contaminants
from reaching the standard mechanical filters. It may be
confidently asserted, therefore, that EFP systems prevent
a significant percentage of contaminants smaller than 5
microns which would usually pass through conventional
filters, from reaching combustion and from being emitted
as exhaust product.
FUEL ECONOMY IMPROVEMENTS
The first distance run made by the locomotives was a three
hundred (300 km) kilometer round trip. Upon their return,
they were refueled and the quantities recorded:
TABLE 3. FUEL CONSUMED. AMPERAGE DEVELOPED
Locomotive Fuel Consumed
#8709 2069 liters
#8720 (EFP-equipped) 1862 liters
Difference: 207 liters =10 percent
Amperage Developed/Traction Motor Load
#8709 500 Amperes
#8720 500 Amperes
Inches of Rack Indicated for 500 Amp Load
#8709 92 inches
#8720 104 inches
Difference: 12 inches
(Note: The lower the inches of rack the more fuel consumed.)
When two locomotives, coupled back to back, are pulling the
same train and the inches of rack are at different settings
to produce the same amperage loading, the engine with the
lower numerical inches of rack setting is consuming more
fuel. In this case, the non-EFP equipped engine #8709
consumed 10 percent more fuel.
LUBRICATING OIL PROCESSING
The lubricant used in #8720 had no additives and was
continuously processed by the on-board EFP system's lube-
side unit, removing all metallic particles normally caused
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by wear, in addition to all of the carbon and metal-infused
carbon particles, plus other inorganic substances usually
found in the carter (pan) of the locomotive diesel engine.
Simultaneously, the moisture was removed from the lube-oil,
preventing the possible formation of hydrochloric and
sulfuric acids which have a tendency to etch the metal
surfaces of engine and components. (Note: Additives used
in lubricating oils for the detection of water are abrasive,
and although smaller than 5 microns in physical size, cause
internal wear of the engine.)
Through systematic processing of the lubricant, noticeable
increase in lubricity of the fluid, combined with increased
volatility of EFP-cleaned fuel, significantly and noticeably
lowered the noise level of diesel engine #8720 compared to
that of engine #8709. Operator comments were: a) Locomo-
tive #8720 operates more smoothly and has more positive
control, particularly when switching cars in the yard; b)
The diesel engine noise is reduced from what it was before;
c) The black smoke and sparks have almost completely dis-
appeared, compared to the emissions from the sister loco-
motive #8709 which did not have an EFP system installed
(7).
DISCUSSION OF MEXICAN RAILROAD DIESEL TESTS OF EFP SYSTEMS
while the objective results reported are impressive -- a ten
percent fuel economy improvement; substantial reduction of
emissions; evidence of potential extended engine life; and
the fact that EFP systems can operate successfully in harsh
conditions for at least 1120 hours without downtime or
maintenance, the fuel economy data are from one test situ-
ation, therefore the developer does not claim a similar
percent improvement will always prevail. However, the data
indicate that EFP systems can provide emissions control
at some level of improvement, without increasing engine
frictional or thermal loadings, and without a fuel consump-
tion penalty. This is more than an be said of many tech-
nologies being investigated for improvement of diesel
combustion.
Also, while reduction of black smoke and visible particles
in exhaust emission is not conclusive evidence of reduction
of smaller, invisible particulate emission, the fact that
incombustible particulate of sizes down to 0.001 microns
are consistently removed from fuels and lube-oil prior to
combustion is such evidence, and means that these very fine
particulates are prevented from entering combustion, and
hence, are prevented from emission. It can be said that
EFP systems do reduce total particulate emission.
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NITROGEN OXIDE EMISSIONS AND EFP SYSTEMS
It is known that high values of NOX formation occur under
conditions of high smoke formation (8). These conditions
are a function of high temperature, heat transfer due to
carbon formation and proximity to a cold surface (wall-
quenching). While NOX emissions from EFP-fitted diesels
have yet to be measured, it is likely that EFP-caused
reduction in smoke formation conditions is accompanied
by some reduction in NOX.
Also, to the extent that fuel volatility is increased by EFP
fuel processing, combustion temperatures can be lowered, and
duration of combustion may be shortened.
Secondly, EFP removal of incombustible particles from the
fuel probably has the effect of reducing droplet, flame, and
wall-quenching phenomena, which would tend to increase the
critical concentration of combustible fuel droplets during
the mean period of major heat release. If so, this would
help "...win the race against the N0(x) kinetic", in the
words of the distinguished Dr. W. T. Lyn, and would contri-
bute to such victory over NOX as is now being sought by
means of increasing injection pressures and rates (9).
PARTICULATE EMISSIONS AND EFP SYSTEMS
We have seen that EFP-fitted diesels emit fewer large par-
ticles, and we have seen EFP systems prevent much of the
sub-micronic particulate matter from entering combustion or
appearing in exhaust emissions. This improvement occurs in
diesels using fuel of relatively good quality -- a centri-
fuged fuel. To the extent that fuels contain carbonaceous
particles in sizes 10"^ meter and larger, including those
containing the high molecular weight ring compounds, they
are removed. Certainly carbonaceous particles are formed
instantaneously in diesel combustion no matter how pure the
fuel and lube-oil in the engine, and we make no claim EFP
will prevent all of this.
However, it is likely that much of the carbon formation in
high-pressure, high-temperature combustion is stimulated by
presence of contaminants and carbon residue particles of
0.001 microns and larger. Given the lack of a precise
understanding of the nature of such carbon formation, a
hypothesis of "precursor" particles or of "precursor elec-
trical charges" from such particulate as is present in fuels
unprocessed by EFP system should not be dismissed without
empirical investigation.
220
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In any case, EFP systems provide an absolute improvement in
particulate emissions regardless of any other parameter of
engine design, performance, operating conditions or fuel
composition. As certainly, EFP systems will provide some
level of reduction in particulate emissions additional to
any provided by engine design or injection system design
improvements or exhaust after-treatment.
DIESEL FUEL COMPOSITION, PARTICIPATES AND EFP: MYTHS ABOUT
FUEL
ASTM Limiting Specifications for No. 2-D diesel fuel allow
the following amounts of solid and liquid contaminants:
Water and Sediment, % vol. 0.05
Carbon Residue, % 0.35
Ash, % wt. 0.01
Sulfur, % wt. 0.50
Except for ash and sulfur which remain the same, No. 1-D
specifications are about one-half this amount of contami-
nant. Since EFP systems remove water to 0.005%, and con-
sistently remove all of the above solid contaminants to
the 0.001 micron level, these specifications, often not
realized in the 'real world', can be surpassed by appli-
cation of EFP systems.
The vastly complex subject of petroleum fuels production and
refinement is beyond the scope of this paper, nevertheless,
it is useful to note recent trends indicating possible
decline in fuel quality, and to reflect on the ways fuels
become contaminanted.
Generally, 'straight-run' diesel fuels are the highest
quality, i.e., those boiled off at the lowest distillation
temperature for that strata of the barrel of crude. It is
said that European diesel fuel is in greater proportion,
straight-run fuel, and perhaps it is not a coincidence that
actual Cetane Number values in Europe are reported to be 55
CN, whereas in the United States, where more diesel fuel is
derived through thermal cracking and other high-temperature,
high-pressure processes, the CN average is falling to 45 CN
(10).
While refineries take great care to ensure their fuels meet
specifications, it would be interesting to have results of
carbon residue and other contaminant averages in fuels
actually available in the United States. For, it is said
the demand upon refineries to produce unleaded gasoline has
taken much mid-distillate stock for this purpose, requiring
221
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increased cracking of heavier stock to meet demand for
diesel. Despite this, U.S. government analysis of the
quality of diesel fuel is apparently still conducted upon
mainly straight-run fuel samples of the highest purity
available from refiner's diesel stock. If increased crack-
ing processes are used, it would be expected that carbon
residue, ash, sediments and perhaps other contaminants are
increased.
Diesel Automotible Association motorist members report
marked increase in water and sediment contamination of fuels
as a consequence of disruption in the supply system this
year. The appetite of diesel fuel for water is well known,
and diesel vehicles accumulate condensation water in fuel.
Increased tendency of users to store diesel fuel against
threatened shortages of supply increases likely contamina-
tion of water, bacteria and fungi.
In handling, delivery of fuels and filling of vehicles,
there are opportunities for contaminants to enter the
fuel. High-speed flow in fill-lines generates electro-
static charges and frictional heat. Some additives, such
as surfactants, increase susceptibility of fuels to water
attraction, contamination and deterioration. If benefits
from EFP of fuel economy, reduced emissions, reduced wear
were not sufficient to attract manufacturers to EFP, insur-
ing diesel users against further deterioration in fuel
quality ought to be.
OTHER APPLICATIONS OF EFP SYSTEMS IN FUELS/OILS PROCESSING
Today we are concerned with transportation diesel applica-
tions of EFP systems, and our developers are engaged in
production of EFP prototypes suitable in size and manufac-
turability to diesel automotive and automobile applications,
however, there are several others of interest to conserva-
tion of fuels and reduction of atmospheric and environmental
pollution:
Purification of Power Plant Boiler Fuels -- especially
in reduction of particle sulfur and particulate in
residual fuel of declining quality.
t Purification of Combustion Turbine Fuels -- especially
removal of trace metals in particle or compound form,
which, when not removed, cause costly damage to turbine
blading.
Reclamation of Used Automotive Lube Oils/Cutting Oils.
222
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t Processing Coal-Derived Liquid Fuels -- especially
improving stability of coal liquid product.
SUMMARY
In addition to improved fuel economy in diesels, reduction
of emissions, reduction of wear, downtime, and associated
maintenance, all of which are consistent benefits of EFP
systems, the following specific benefits obtain from EFP
application to diesels:
Reduced Fuel Pump/Injector Wear;
Reduced Failure of Seals, Gaskets, 0-Rings, and Poly-
urethane Fuel Injection Parts;
Reduced Wear of Cylinder Liners, Piston Rings, Valves;
Reduced Corrosion, Reduced Acid Formation;
Improved Combustion;
Reduced Noise;
Reduced Carbon Deposition on Exhaust Valves, Combustion
Surfaces;
Easier Cold Starts; --
and the potential for design of EFP systems which completely
eliminate oil drain interval's^
NOTES/REFERENCES
1. Discussion of "Conradson Carbon Residue", Scientific
Encyclopedia, 5th Ed. Van Nostrand, New York 1976.
2. "Centrifuges for Power Generating Plants", DeLaval
Separator Co., Poughkeepsie 1979.
3. "Characteristics of Particles and Particle Dispersoids"
Stanford Research Institute Journal, 3rd Quarter 1961,
Palo Alto.
4. Electrostatic Information Packet, Diesel Automobile
Assoc., Concord, NH and Fort Lee, NJ, July 1979.
5. Ibid.
223
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6. Ibid.
7. "Mexican National Railroad Report", B. A. Wolf, May
1979, contained in Electrostatic Information Packet,
Op. cit.
8. "Optimization of Diesel Combustion Research", W. T. Lyn,
Cummins Engine Co., the Horning Memorial Award Lecture,
Society of Automotive Engineers Paper #SP-433, 1978.
9. Ibid., p. 5.
10. Diesel Automobile Association interviews of DOE and
other U.S. government fuel specialists.
224
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SESSION I
Summary Discussion Following Completion of Session I
225
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General Discussion
A. KOLBER: Is there any information on pollutants that
are produced by diesel which have a high vapor pressure or
vapor phase materials that might be trapped and tested? Is
there any information at all as to what they might be, and
if they exist can they be tested?
J. PITTS: The ones we are most concerned with are N02
and NO. These levels are relatively high, in relation to
the spark-ignition engine versus a diesel system. However,
there are others, including some of those that he had on
his slide, for example, formaldehyde. Who would like to
answer that question? Someone who has actually measured
the gaseous pollutants.
R. BRADOW: I guess in terms of vapor-phase components,
diesel engines in general don't look greatly different from
what we have seen from gasoline engines. There are a num-
ber of papers in the literature over a long period of time
relative to the important gaseous constitutents of both
gasoline and diesel engine exhaust. Organic compounds we
are dealing with are, of course, mainly hydrocarbons. In
general, about ten percent of what we measure as total
hydrocarbon is present as low molecular weight oyxgenates
both in gasoline and diesel engine exhausts, and these are
dominated by formaldehyde. I would estimate for both gaso-
line and diesel engine exhaust, probably, formaldehyde
would exceed all the other aldehydes combined in concen-
tration. There are great differences in emission rates for
oxygenates and hydrocarbon depending on the way the vehicle
or test engine is operated. Some of the problems in making
point-to-point comparisons between heavy duty diesel en-
gines and those for the use of passenger cars, for example,
are centered around the fact that it is hard to get com-
parable cyclic emission tests right now. Possibly when the
coming transient test for diesel engines is run for these
pollutants we can do a little better job in making one-to-
one comparisons between types of engines. I think that so
far, we have come to the conclusion that the organic mate-
rials associated with the particles, oxygenates, and hydro-
carbons have molecular weight aromatic hydrocarbons and
differ markedly from engine to engine. It is a little
difficult to say where the sort of unifying similarities
might be. I think the slides I showed this morning rela-
tive to the HPLC chromatograms for the variety of different
diesel and gasoline engines were sort of indicative of
that. Frankly, I would really like to have somebody else
speak on that issue. Does anyone else believe that the
differences among engines, diesel, and gasoline are bigger
than the similarities?
R. KLIMISCH: There is one difference I would like to
mention. That is the fact that evaporative emissions from
226
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diesel engines were much lower than gasoline engines. That
becomes significant in terms of the total hydrocarbons.
R. BRADOU: I quess that is true. Evaporative loss from
diesel engines is very low. We have seen, though, rela-
tively low evaporative loss in a lot of newer gasoline
engine cars. The important issue here however, is really
tailpipe emissions. Previously when there were a lot of
cars burning leaded gasoline you really had something in
the evaporative loss to talk about, for example, ethylene
dichloride, ethylene dibromide, and perhaps some of the
lead alkyls as possible toxic agents that might be present
in the ambient air to some respectable level. Evaporative
emissions generally do not appear to be such a big problem
any more. Also there are very few cars being made that
burn leaded gasoline. I think in the 1980 model year one
gasoline automobile was certified for leaded gasoline out
of all the makes and models that exist. So, it appears to
me that certianly in terms of oxygen precursors, we still
have a little problem with evaporative loss. But we never
have had the problem with regard to toxic agents that we
have with tailpipe emissions. We have not tested enough
different kinds of cars and do not possess the analytical
techniques that are needed to assess their difference and
similarities. I think these are the major issues that need
to be addressed.
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Session II
IN-VITRO CARCINOGENIC AND MUTAGENIC EFFECTS
OF DIESEL EMISSIONS AND DIESEL EMISSION COMPONENTS
Chairmen:
Dr. Joellen Huisingh
Dr. F. Bernard Daniel
Diesel Participate Collection for Biological Testing:
Comparison of Electrostatic Precipitation and Filtration.
Chan, T. L., P. S. Lee, and J. S. Siak.
Diesel Participate Extracts in Bacterial Test Systems.
Siak, J. S., T. L. Chan, and P. S. Lee.
Mutagenic Activity of Diesel Emission Participate Extracts
and Isolation of the Mutagenlc Fractions.
Choudhury, Dilip R. and Charles 0. Doudney.
Mutagenicity Studies on Diesel Particles and Particulate
Extracts.
Loprieno, N., F. DeLorenzo, G. M. Cornetti, and G.
Biaggini.
The Mutagenicity of Diesel Exhaust Exposed to Smog Chamber
Conditions as Shown by Salmonella Typhimurium.
Claxton, Larry, and H. M. Barnes.
Salmonella/Microsome Mutagenicity Assays of Exhaust From
Diesel and Gasoline Powered Motor Vehicles.
Lofroth, Goran.
228
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Session II
(Continued)
Biological Availability of Mutagenic Chemicals Associated
with Diesel Exhaust Particles.
Brooks, A. L., R. K. Wolff, R. E. Royer, C. R. Clark,
A. Sanchez, and R. 0. McClellan.
Diesel Participate Matter Chemical and Biological Assays.
Risby, T. H., R. E. Yashin, and S. S. Lestz.
Diesel Particulate Extracts in Cultured Mammalian Cells.
Rudd, Colette J.
Diesel Soot: Mutation Measurements in Bacterial and Human
Cells.
Liber, H. L., B. M. Andon, R. A. Hites, and W. G.
Thilly.
Studies on the Effects of Diesel Particulate on Normal and
Xeroderma Pigmentosum Cells.
McCormick, J. Justin, Roselyn M. Zator, Beverly B.
DaGue, and Veronica M. Maher.
Benzo(A)pyrene Alters Lung Collagen Synthesis in Organ
Culture.
Bhatnaga, R. S. and M. Z. Hussain.
Application of a Battery of Short Term Mutagenesis and
Carcinogenesis Bioassays to the Evaluation of Soluble
Organics from Diesel Particulates.
Huisingh, Joellen, Stephen Nesnow, Ronald Bradow and
Michael Waters.
A Review of In-Vitro Testing Systems Applicable to Diesel
Health Effects Research.
Whitmyre, Gary K.
The DNA Damage Activity (PDA) Assay and its Application
to River Waters and Diesel Exhausts.
Doudney, Charles 0., Mary A. Franke, and Charles N.
Rinaldi.
229
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DIESEL PARTICULATE COLLECTION FOR BIOLOGICAL TESTING:
COMPARISON OF ELECTROSTATIC PRECIPITATION AND FILTRATION
T. L. Chan, P. S. Lee and J. S. Siak
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
ABSTRACT
The extent to which a particle collection method can
influence the chemical composition and biological activity
of diesel participate extracts was investigated. Undiluted
diesel particles were collected from the exhaust of a 5.7L
GM diesel engine at specific collection temperatures by
electrostatic precipitation (ESP) and filtration. Parallel
samples were taken with an electrostatic precipitator and
Pallflex filters under the same sampling conditions. The
percent of extractable organic compounds by dichloromethane
for the ESP sample was higher than the filter sample and
was dependent on collection temperature. The extracts were
chemically fractionated into nine components according to a
solvent partitioning scheme. These extracts and their
fractions were used in in vitro biological studies.
INTRODUCTION
The potential health effects of diesel exhaust emissions
should be determined with the expected increased usage of
light duty diesel engines. In addition to chronic exposure
studies with dilute diesel exhaust, the chemical and bio-
logical characteristics of diesel particles and the ex-
tracts would provide relevant information for the assess-
ment of health hazards associated with inhaled diesel
particles. Short term bioassays, such as the Ames test,
have shown that diesel particulate extracts are mutagenic
with varying degrees of biological activity dependent on
230
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engine types and fuels. Most of the biologically active
compounds found on diesel particles are suspected to be
hydrocarbon compounds formed either during the combustion
process or adsorbed and condensed on the particles during
the collection process. Experimental artifacts may exist
if the diesel particles are not collected under proper
conditions. The standard sampling method using appropriate
filter papers may not be satisfactory for diesel particle
collection if the biological activity of the particles is
to be studied. The high pressure drop across the filter
can lead to losses of volatile organic compounds on the
particles on the filter due to the partial vacuum.
Another serious problem associated with filter sampling
may be the chemical conversion of the organic compounds on
the particles by engine exhaust gases, such as nitrogen
oxides and other reactive hydrocarbon compounds. Pitts,
et al [1] reported that filters spiked with benzo[a]pyrene
were found to be partially converted to nitrosubstituted
compounds by 1 ppm nitrogen dioxide in laboratory studies.
These factors may play important roles in the relative
biological potency of diesel particles and their extracts.
In order to avoid some of these artifacts associated with
filter sampling, electrostatic precipitation (ESP) was used
in parallel to examine the role of collection method on the
chemical and biological properties of diesel exhaust
particles. The collection efficiency of ESP is almost as
high as filtration, although the mechanisms of collection
are distinctly different. Electrostatic collection of
particles offers low pressure drop across the collector and
allows the exhaust gases to pass over the collected
particles instead of passing through them. Thus, losses of
volatile compounds due to the partial vacuum and the mass
transfer of gaseous hydrocarbons and nitrogen oxides to the
particles should be reduced since only a small fraction of
the gases is in contact with the diesel particles.
The objective of this study is to determine the effect of
particle collection method on the chemical composition and
biological activity of diesel particulate extracts. In
this study, a 5.7L GM diesel engine was operated at 65 km/hr
road load conditions. Type 2D federal compliance diesel
fuel was used and a regular oil change was performed every
3,000 miles with 30W lubricating oil. Diesel particles
collected by either ESP or filtration were extracted in a
Soxhlet apparatus with dichloromethane. The diesel partic-
ulate extracts were chemically fractionated into nine
components according to a solvent partitioning scheme. The
biological activity associated with the extracts and their
various fractions was studied using the Ames test.
231
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EXPERIMENTAL METHODS
Collection of Diesel Particles
The sampling system for diesel particulate collection is
shown in Fig. 1. A 5.7L GM diesel engine produced by
Oldsmobile was controlled by a water brake dynamometer at
1350 rev/min and 96 N-m, representing 65 km/h road load
cruise conditions. The exhaust from the engine passed through
a normal passenger car exhaust system. A gate valve down-
stream of the muffler controlled the total diesel exhaust
airflow and the collection temperature in the electrostatic
precipitator. Parallel filter samples were obtained from the
filter sampling ports using 47 mm Pall flex filter paper.
Speed and
- Load Controls
Exhaust
3 Way
Valve
Figure 1 Electrostatic and filter collection of undiluted
diesel particles from the exhaust of a 5.7 L GM
diesel engine produced by Oldsmobile.
The basic principle of ESP collection of particles is a two-
stage process: particle charging and particle collection.
Particles entering the precipitator are charged electrically
in a unipolar ion field provided by the corona discharge in
the charging section. The charged particles are then imme-
diately subjected to a high electric potential gradient
and are driven towards the collection plates. A modified
electrostatic precipitator (Trion, Inc., Model #424635)
was used for the diesel particulate collection. The inlet
and outlet of the precipitator were replaced by stainless
steel reducers which provided a more developed flow field
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in the precipitator. Additional modifications included
the isolation of the power supply module from the ESP
mainframe (to prevent thermal degradation of the electrical
and insulating components) and the installation of a
removable front panel to gain access to the collector
section. The collection temperature was usually maintained
at normal tailpipe temperatures of 90-100°C. Direct
readout of temperature was provided by a thermocouple
placed in the ionization section of the precipitator.
Particles deposited on the first 18 cm of the collection
plates were shown to be within ±5°C of this temperature.
Undiluted diesel exhaust particles were collected for
fifteen minutes and the airflow and power to the ESP were
turned off. The collection section was removed from the
ESP and placed in a hood immediately. Particles collected
on the first 18 cm of the collection plates were scraped
off, placed in amber glass vials, sealed, and stored in
a -80°C freezer. The collection and storage of particles
were performed under these defined conditions to reduce
the probability of losses and conversions of the biologically
active compounds in the diesel particles.
Extraction of Diesel Particles
The organic compounds in the diesel exhaust particulates
were extracted by dichloromethane (DCM) in a Soxhlet
apparatus. The frozen samples collected either by ESP or
filtration were allowed to reach room temperature by
placing them in a dark hood for at least an hour. The
cellulose extraction thimbles were also stabilized at 20°C
and 45% relative humidity prior to weighing. Filter
samples were placed vertically and ESP samples loaded
directly in the thimbles. If necessary, a small pyrex rod
was inserted into the extraction chamber to reduce the net
volume and to increase the reflux rate to about 3-5
minutes/cycle. Routine extractions were performed at 40°C
for four hours. When extraction was complete, the thimble
was dried and restabilized at 20°C and 45% relative
humidity to determine the mass lost. The total volume of
the solution in the flask was reduced to about 5 mi by
gentle evaporation with a mild jet of nitrogen. Additional
rinses of the flask with 2 mL of redistilled dichloro-
methane were performed and the solution added to a tared
vial. Finally, the solution was evaporated to dryness
under nitrogen and the weight of extractable fraction
determined.
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Chemical Fractionalion of Diesel Particulate Extracts
The diesel particulate extracts were fractionated into nine
components according to a solvent partitioning scheme
outlined in Fig. 2. The procedures used were based on the
systematic extraction scheme of Novotny et al [2] in studies
related to the identification of polynuclear aromatic com-
pounds. The nine fractions obtained from the solvent
partitioning scheme have been named WATER SOLUBLE, STRONG
ACID, WEAK ACID, ACID SALT, BASE, BASE SALT, NEUTRAL-POLAR,
NEUTRAL-NONPOLAR I and NEUTRAL-NONPOLAR II fractions. The
WATER SOLUBLE fraction was obtained by extracting the diesel
particulate extract in DCM with water. Soluble compounds
such as acids, alcohols, ketones, aldehydes with low
molecular weight, and inorganic salts should be present in
this fraction. The organic layer was extracted with 5%
NaHC03 solution. The aqueous NaHC03 layer was acidified,
back extracted into dichloromethane, and dried in an
evacuated rotary evaporator to yield a STRONG ACID fraction.
Free organic acids such as sulfonic acids (-S03H), carboxylic
acids (-COOH), and sulfinic acids (-S02H) belong to this
category. The organic CH2C12 layer was treated with a strong
base (NaOH) to yield a WEAK ACID fraction. Compounds such as
SOLVENT PARTITION SCHEME FOR DIESEL PARTICULATE EXTRACT
Diesel Particulate Extract
H,0
CH2CI2 Layer
I NaHCO3
Water Layer
(WATER SOLUBLE)
CH2CI2 Layer
I NaOH
NaHCO3 Layer
(STRONG ACID)
CH2CI2 Layer
H2° (ACID SALT)
HCI
NaOH Layer
(WEAK ACID)
CH2D2 Layer
I H2O (BASE SALT)
HCI Layer
(BASE)
Evaporate to Dryness Dissolve in £}
I CH3 OH/H2O
O Layer
I CH3 NO2
CH3OH/H20 Layer
(NEUTRAL-POLAR)
I I
OCH~ NO, Layer
Layer 3 2
(NEUTRAL-NONPOLAR I) (NEUTRAL-NONPOLAR II)
Figure 2 Solvent partition scheme for diesel
particulate extracts.
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phenols (ArOH), primary and secondary nitro compounds, aryl-
sulfonyl derivatives of primary amines (ArS02NHR), unsubsti-
NOH
ii
tuted arylsulfonamides (ArS02NH2), oximes (-C-), thiophenol
0
II
(ArSH), hydroxanic acid (-C-NHOH) and active methylene
0 0
II H
compounds (_-C-CH2-C-) are typical examples in this group.
The ACID SALT fraction was obtained next after the removal
of both the STRONG and WEAK ACID fractions by treating
with water. This fraction should consist of similar
compounds as in the WEAK ACID fraction but with higher
molecular weights. Addition of HC1 to the organic layer
produced the BASE fraction containing mainly amines and N-
heterocyclic compounds. The organic CH2C12 layer was
extracted with water to yield an aqueous layer named the
BASE SALT fraction.
The remaining neutral compounds in the CH2C12 layer were
evaporated to dryness and dissolved in cyclohexane. The
solution was then extracted successively with solvents of
decreasing polarity to yield three neutral fractions named
NEUTRAL-POLAR, NEUTRAL-NONPOLAR I and NEUTRAL-NONPOLAR II
compounds. Extraction with a 4:1 mixture of methanol/water
yielded the NEUTRAL-POLAR fraction consisting of oxy-
compounds such as aldehydes, ketones, alcohols, epoxides,
nitrosamines, etc. Subsequent extraction with nitromethane
gave the final two fractions. The cyclohexane layer
provided the NEUTRAL-NONPOLAR I fraction with aliphatic
and one-ring aromatic compounds. Finally, the nitromethane
layer provided the NEUTRAL-NONPOLAR II fraction which should
contain substituted and unsubstituted polynuclear aromatic
hydrocarbons as well as aromatic nitro compounds.
Biological Activity of the Extracts
The biological activities of diesel particulate extracts
and fractions were studied using a short term bacterial
mutagenicity test system generally known as the Ames test.
Experimental details of the assay are described in a
separate report [5]. Since tester strain TA 98 was proven
to be sensitive and stable in earlier experiments with
diesel particulate extract, it was used to evaluate the
biological activity of the extracts and fractions obtained
from samples collected by ESP and filter samples. The
mutagenic potency of the individual extract fractions
would provide useful information regarding the significance
of sampling methodology. Typically, the mutagenic activity
recovered from all the fractions represented between 75-90%
of the original extract.
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RESULTS AND DISCUSSIONS
Collection of Diesel Particles
We have shown in pilot studies that the collection efficiency
of diesel particles was 92% using an electrostatic sampler.
In this study, a larger electrostatic precipitator was
operated at about 300 L/min and 100°C. Under these
sampling conditions, particle reentrainment and charging
effects reduced the collection efficiency to 85%. It was
possible to determine the collection efficiency only
during the initial sampling period since the section of
the exhaust line between the ESP and the downstream
sampling port was never absolutely free of particles. Some
of these particles can be reentrained to cause a decrease
in the measured collection efficiency.
The most serious problem associated with the filter
sampling of diesel particles was the potential chemical
conversion of organic compounds to nitro-substituted com-
pounds by the nitrogen oxides in the exhaust gases.
Although electrostatic precipitation can effectively
reduce these reactions with the exhaust gases passing over
the collected particles, corona discharge and ozone forma-
tion during the particle charging process could alter the
chemical species on the particles. In order to examine
the role of ozone, a chemiluminescence ozone analyzer
(Monitor Labs, Model #8410) was used to detect ozone at
the outlet of the precipitator. When clean air was drawn
through the ESP, 0.2 ppm of ozone was measured. However,
no ozone was detected at a 5 ppb detection limit in the
presence of undiluted diesel exhaust. Apparently, the 10%
available oxygen in the diesel exhaust, the absorption of
ozone by carbonaceous diesel particles, and the rapid
reactions with reactive nitrogen oxides in the exhaust
caused the dramatic reduction of ozone concentration.
These considerations illustrated that advantages and
limitations are associated with each sampling method.
Thus, a thorough understanding of every particle collection
mechanism and its potential artifacts is necessary in the
interpretation of experimental data.
Extraction of Diesel Particles
Parallel samples obtained from both ESP and filter sampling
at specific collection temperatures were extracted by
dichloromethane. Results showing the percent of extractable
compounds by weight to the collection temperature were
given in Fig. 3. An increase in the extractables was
found for the ESP sample compared to the filter sample at
236
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15
O
CM
O
.O
CD
X
LU
en
(D
10
0
ESP Samples -
Filter Samples
60 70 80 90 100 110 120
Collection Temperature (°C)
Figure 3 Percent of extractable compounds in diesel
particles collected by ESP or filtration.
Soxhlet extraction for four hours at 40°C
was performed with dichloromethane as the
solvent.
the same collection temperature. Both diesel particulate
extracts exhibited similar dependence on collection temper-
ature, i.e., an increase in % extractables at lower
temperatures. This was reasonable since various gaseous
hydrocarbon compounds could easily be adsorbed or condensed
onto the diesel particles.
The characteristics of undiluted diesel particles at
tailpipe conditions were studied since it would allow us to
examine the effects of dilution and cooling of diesel
exhaust. At the normal exhaust temperatures of 100°C at
the tailpipe, 11% was extracted from the ESP sample but
only 6.2% was obtained in the filter sample. Since both
samples were collected simultaneously, the differences
observed must be related to differences in the collection
methodology. Volatile compounds which could be lost in the
filters would be retained in the ESP. Direct acting
mutagenic compounds may be formed by chemical conversion by
the exhaust gases in the filter. Moreover, formation of
new compounds during the particle charging process may be
possible in the presence of excess ions provided by the
corona discharge. Detailed chemical fractionation of the
diesel particulate extracts were therefore necessary to
determine differences in the chemical profiles due to the
collection method.
237
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Chemical Profiles of the Particulate Extracts
Parallel samples collected simultaneously by ESP and
filtration at 90-100°C were fractionated according to the
solvent partitioning scheme described earlier. Only
paired samples were compared to avoid variations due to
other experimental parameters. Results indicated that more
than 60% of the extractables were found in the NEUTRAL-
NONPOLAR I fraction (Fig. 4). This fraction consisted
primarily of aliphatic and one-ring aromatic compounds.
Initial data indicated that the overall chemical profiles
were quite similar with the exception of the ACID SALT
fraction. Significantly greater amounts were found in this
fraction which accounted for most of the increase in
extractables in the ESP sample. Since most of the com-
pounds in this ACID SALT fraction were not expected to be
volatile, the increase must be attributed to some charac-
teristics associated with electrostatic collection. The
corona discharge could provide an abundance of ions in the
charging section of the electrostatic precipitator.
Chemical kinetics studies have shown that free radicals are
formed under field ionization [3,4]. These free radicals
can undergo rapid chemical reactions to form larger mole-
cules. Therefore, it was conceivable to see some conversion
CHEMICAL PROFILE OF DIESEL PARTICULATE EXTRACT
100
Weak
Acid
Strong
Acid
Base
Salt
Neutral- Neutral- Neutral- Acid
Polar Nonpolar II Nonpolar I Salt
Water
Soluble
Figure 4 Chemical fractions of diesel particulate extracts
for both ESP and filter samples. The NEUTRAL
-NONPOLAR I fraction accounted for more than
60% of the extractables.
238
-------
of the WEAK ACID compounds to similar compounds with higher
molecular weights in the ACID SALT fractions in the ESP
sample. This mechanism would also lead to the decrease in
the amount of WEAK ACID and STRONG ACID fractions for the
ESP samples. Additional paired samples collected under the
best reproducible conditions are being evaluated to deter-
mine the variations in the data. With regard to the
formation of nitro-substituted compounds on the filter
samples due to the nitrogen oxides, there was very little
difference in the amount of extract fraction. However, the
specific bioactivity of these extract fractions may be
different and must be examined in in vitro test systems
such as the Ames test.
In Vitro Biological Activity of the Extract Fractions
Although the ESP sample extract exhibited a significant
increase in the amount of extractables in the ACID SALT
fraction, no activity was detected in the Ames test. By
comparison, the same fraction from the filter sample
extract accounted for 1.5% of the total mutagenic activity.
Apparently, charging effects during the electrostatic
collection process enabled the conversion of some of the
compounds in the WEAK ACID and STRONG ACID fractions to
non-mutagenic compounds classified under the ACID SALT
fraction. Thus, increased amounts of extractable mass in
the ESP sample do not give rise to a corresponding increase
in the bioactivity in the diesel particulate extract. The
overall biological activity of the extracts for ESP and
filter samples (expressed as net revertants/mg of particles
basis) was quite similar [5].
The subtle effects of the sampling methodology on the bio-
activity of the extract can be seen by examining the
specific activity of the most active extract fractions
(Fig. 5). Higher specific activities were observed in the
filter samples in the WEAK and STRONG acid fractions.
Significant increases in specific activity were seen in the
filter sample extracts tested under the inactivated system
for both NEUTRAL-POLAR and NEUTRAL NON-POLAR II fractions.
This strongly suggested the existence of more direct acting
mutagens in the filter sample. Work is still in progress
to identify the active compounds in these fractions.
239
-------
ESP Sample (+MT)
ESP Sample (-MT)
Filter Sample (+MT)
Filter Sample (-MT)
Weak Acid
Strong Acid
Neutral-Polar Neutral-Nonpolar II
Figure 5
Specific activity of diesel particulate extract
fractions determined by the Ames test. +MT
indicates metabolic activation with S9, -MT
indicates no activation.
SUMMARY
The amount of extractable compounds from the diesel
exhaust particles depended on collection temperature
and collection method. Under the same collection
temperature, more extractables were found in diesel
particles collected by electrostatic precipitation
(ESP) than filtration.
The chemical profiles of the two particulate extracts
were similar with over 60% of the extractable mass
classified as a NEUTRAL-NONPOLAR I fraction. This
fraction was obtained from a solvent partitioning
scheme and should contain aliphatic and one-ring
aromatic compounds.
Most of the mass increase in the extractables in the
ESP sample was found in an ACID SALT fraction and
could be attributed to charging effects. Although
there was more than a two-fold increase in this
fraction, no bioactivity was observed in the Ames
test.
240
-------
4. Bioactive compounds determined by the Ames test were
found in the NEUTRAL-POLAR and NEUTRAL-NONPOLAR II
fractions. These fractions accounted for more than
70% and 90% of the bioactivity in the filter and ESP
samples, respectively. However, only 10-15% by
weight of the extractables belonged to these frac-
tions.
& CONCLUSIONS
We have examined the effect of sampling methodology on the
chemical composition and biological activity associated
with the diesel particulate extracts. Electrostatic
precipitation gave a larger amount of extractables with
most of the increase in an ACID SALT fraction. This
fraction was subsequently shown to be inactive in the Ames
test. Filter sample extracts do exhibit higher specific
activities in some fractions and may be attributed to the
presence of some direct acting mutagens formed by reaction
with the exhaust gases. On the other hand, charging
effects during electrostatic precipitation may convert
some of the mutagenic compounds to less mutagenic forms.
Additional experiments are required to determine the
dominant mechanism. However, the overall biological
activity of the extracts (expressed as net revertants/mg
of particles basis) was quite similar. These subtle
differences due to the sampling methodology must be
recognized in the assessment of health hazards associated
with inhaled particles. Due to the presence of hydro-
carbon compounds and nitrogen oxides in the gaseous phase
in urban atmospheres [6], routine filter samples drawn for
extended periods of time could create some direct acting
mutagens in the extracts. Therefore, studies involving
short term bioassays of particulate extracts [7,8] would
provide relevant but only relative information concerning
the biological hazards of airborne particles.
ACKNOWLEDGEMENTS
We thank M. Baxter, J. M. Dickman, R. A. Gorski, W. E.
Hering and J. T. Johnson for their technical assistance in
the collection, extraction, fractionation and biological
testing of the diesel particulate samples. The ozone
measurements were performed by Dr. K. A. Strom.
241
-------
REFERENCES
1. Pitts, J. N., Jr., Van Canwenberghe, K.A., Grosjean,
D., Schmid, J.P., Fitz, D. R., Belzer, W. L. Jr.,
Knudson, G. B. and Hynds, P.M., "Atmospheric reactions
of polycyclic aromatic hydrocarbons: facile formation
of mutagenic nitro derivatives." Science, 202:515-
518, 1978.
2. Novotny, M., Lee, M. L. and Bartle, K. D., "The
method for fractionation, analytical separation and
identification of polynuclear aromatic hydrocarbons
in complex mixtures." J. Chromat. Sci. 12:606-612,
1974.
3. Derrick, P. J. and Burlingame, A. L., "Kinetics and
mechanisms of unimolecular gas-phase reactions of
radical cations at times of 10 n to 10"5 seconds
following field ionization." Accounts of Chemical
Research, 7:328-333, 1974.
4. Thomas, C. L., Egloff, G. and Morrell, J.C., "Reactions
of hydrocarbons in electrical discharges." Chem.
Rev., 28:1-70, 1941.
5. Siak, J., Chan, T. L. and Lee, P.S., "Diesel particu-
late extracts in bacterial test systems." Presented
at the Symposium on Health Effects of Diesel Engine
Emissions, USEPA, December 3, 1979 at Cincinnati, OH.
6. Contreels, W. and Van Canwenberghe, "Experiments on
the distribution of organic pollutants between airborne
particulate matter and the corresponding gas phase."
Atmospheric Environment 12:1133-1141, 1978.
7. Dehnen, W., Pitz, N. and Tominges, R., "The mutageni-
city of airborne particulate pollutants." Cancer
Letters, 4:5-12, 1977.
8. Daisey, J.M. and Mukai, F., "Short-Term in vitro bio-
assays: applicability to air monitoring in the coal
conversion and shale oil industries." Am. Ind. Hyg.
Association Journal, 40:823-828, 1979.
General Discussion
W. BAL60RD: You mentioned several types of possible
artifacts with filters; I wonder if you had considered the
possibility of your electrostatic precipitator producing
ozone. Did you measure that?
T. CHAN: Yes, first of all we did some ozone measurements
242
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before we installed the precipitator in the collection
system. In the presence of clean air we can measure up to
0.2 PPM of ozone. However, in the presence of undiluted
diesel exhaust, the amount of ozone was below our detection
limit which is 0.2 PPB. Apparently what happened is the
ozone is taken out by the hydrogen dioxide in the exhaust
gas or could be absorbed by the particles. In this case,
considering that the concentration of the nitrogen oxide
in the exhaust is so high, the 0.2 PPM of ozone would not
be a problem.
A. KOLBER: Can you give us an estimate of what percentage
the polar neutral fraction represents of your total ex-
tractable mass?
T. CHAN: It was 60 percent.
A. KOLBER: It represented 60 percent of the polar?
T. CHAN: Sixty percent of the extractable mass. I
indicate here also that the percent, by mass, of the active
fractions only amounts to 10 - 15 percent of the extractable
mass for diesel particles.
R. OALLEY: Could you tell us why you looked at the raw
exhaust and also what type of filter was used?
T. CHAN: I will answer the second question first.
We used pallflex filters, and we chose to sample from the
undiluted exhaust simply because we wanted it to determine
the activity and the chemical composition at the point of
emission.
R. DALLEY: How is the temperature controlled?
T. CHAN: In the case of a precipitator, we could con-
trol it with the gate valve upstream of the precipitator,
which controls the flow rate into the precipitator. In
case of a filter, we controlled it with a gate valve behind
the filter.
D. KITTELSON: Did you use a positive or a negative
corrider with the percipitator, and did you try changing
the corrider polarity? That would change the sources of
compounds that you might be forming in the corrider and
thus perhaps give rise to artifact formation.
T. CHAN: We used a positive corrider and since the
percipitator was available from this company, we have no
way of switching polarity. We would like to, but we have
no way of doing that. We also considered the possibility
of not using a target section, but we are in the process of
working out the details of that.
J. HUISINGH: We have done some experiments over the
last year with an electrostatic precipitator in ambient air
and have compared charging the corrider two different ways.
We have measured ozone and have found no difference in
mutagenic activity in three different locations, including
on top of a coke oven. The chemistry of the PAH's were
also characterized and we essentially found no major dif-
ferences, so it would appear that, if similar to the diesel
exhaust, ozone would not be a problem with diesel. I have
243
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a question about your fractions. You saw a lot more activ-
ity than we have seen in the nonpolar neutral fractions,
and yet it was direct acting activity. When we tried a
fractionation scheme to this, we saw the same thing and
assumed that we got spill-over of the oxygenated type ma-
terials into the PAH fraction. Do you think that it was
the PAH's that we were seeing?
T. CHAN: I don't think so.
J. HUISINGH: The PAH's would be indirect acting and
your fractions were direct acting.
T. CHAN: Dr. Siak will give the next paper to discuss
details of the biological testing. I think it would be
appropriate for him to answer that question after his talk.
R. BRADOU: We have conducted a similar experiment. This
morning I described a split flow system wherein the diluted
raw exhaust was compared to a filtered exhaust with a filter
held at 100 degrees centigrade. We obtained an electrostatic
precipitator very similar to the one used by General Motors
and have reconducted that same experiment electrostatic
precipitator plate (ESP). Generally speaking, we got about
the same mutagenicity from the Pallflex filter that we got
in those that were obtained from extracts of the (ESP)
plates.
J. HUISINGH: Is that after diluting?
R. BRADOW: No, that was raw exhaust. So our conclusion
was that the diluted sample was very similar, slightly
lower in activity, because at the somewhat lower temp-
erature there was larger retention of polyaromatic hydro-
carbon so the material was less diluted. Pallflex filter
samples and ESP samples collected at that location were
essentially comparable by HPLC analyses as well as by the
Ames Test. So in the raw exhaust, we found no difference
between ESP and the Pallflex filter.
244
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DIESEL PARTICULATE EXTRACTS IN BACTERIAL TEST SYSTEMS
J. S. Siak, T. L. Chan and P. S. Lee
Biomedical Science Department
General Motors Research Laboratories
Warren, Michigan 48090
ABSTRACT
The Ames bacterial mutagenicity test system was used to
evaluate parameters which may affect the mutagenic activity
of diesel participate extracts. The optimal extraction
conditions, extractability of mutagens by simulated bio-
logical fluids and the effect of collection method were
investigated. The role of solvent was examined by extract-
ing diesel particles with methanol, acetone, cyclohexane,
ethyl acetate, n-hexane, dichloromethane, benzene and a
benzene-ethanol mixture. Of these, the dichloromethane
extract exhibited the highest activity in the Ames test,
although methanol yielded the largest extractable mass.
Diesel particles were also extracted by dimethyl sulfoxide
(DMSO) and four other simulated biological fluids for 48
hours at 25°, 37° and 45°C to study the effects of temper-
ature. The mutagenic activity of the DMSO extract began to
decline at temperatures higher than 37°C after 8 hours of
incubation. Fetal calf serum was the only simulated
biological fluid which eluted mutagenic activity from the
particles. No activity was detected in the 0.5% bovine
serum albumin, simulated lung surfactant and saline extracts.
Diesel particles collected by electrostatic precipitation
(ESP) and filtration were studied. The mutagenic activities
of both extracts were- comparable when expressed as rever-
tants per mg of particle. After the extracts were separated
into nine fractions by a solvent partitioning scheme, the
majority of the activity was found in the neutral-nonpolar
II, neutral polar, strong acid and weak acid fractions. The
acid salt fraction from the ESP sample was inactive. These
results demonstrate that differences in the extraction
245
-------
conditions can result in differences in the mutagenic
activity of diesel participate extract. Since the, mutagens
in the extracts are not readily extractable by simulated
biological fluids, the question of bioavailability of
mutagens in diesel particles must be considered in the
final assessment of their potential effects in biological
systems and organisms.
INTRODUCTION
Recent studies have indicated that the organic solvent
extracts of diesel particles exhibited different mutagenic
response in the Ames mutagenicity test. The variability in
the findings may be attributed, in part, to differences in
the composition of the extracted compounds. Although
Huisingh et al [1] found that after extraction of diesel
particles with dichloromethane, the extract was mutagenic
in the Salmonella typhimuriwn mutagenicity test [2],
McGrath et al [3] observed that diesel particles were only
slightly mutagenic when the particles were tested as a
dimethyl sulfoxide suspension. These observations suggest
that the mutagens in diesel particles have different
extractability in different solvents. Similar findings
were observed in fly ash studies [4]. Also, it has
been shown that serum extracts only small quantities of
benzo[a]pyrene from benzo[a]pyrene-enriched carbon black
particles [5]. Moreover, the results obtained in the
studies with organic solvents do not reflect the conditions
in vivo. This study was designed to examine the roles of
various parameters on the Ames mutagenicity test. The
optimal extraction conditions, the extractability of the
mutagens in simulated biological fluids, and the effect of
particulate collection method were investigated.
EXPERIMENTAL METHODS
Collection of Diesel Particles
The diesel particles were collected either by a Pallflex
filter or by an electrostatic precipitator [6] from a GM
5.7 L diesel engine. The engine was operated on a water
brake dynamometer at 1350 rev/min and 96 N-m, representing
65 km/h road-load cruise conditions. The particles were
collected from the undiluted exhaust at the normal tail-
pipe temperature of 100°±5°C. Type 2D federal compliance
diesel fuel was used in this study.
Preparation of Simulated Biological Fluids
Four solutions were prepared to simulate body fluids.
Saline (SLN) was prepared as 0.9% sodium chloride in
deionized water. A 0.5% bovine serum albumin (BSA) in
246
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saline was used to simulate the proteinaceous material in
body fluids. To simulate the surfactant layer of the
alveoli, dipalmitoyl-2-lecithin, palmitic acid, stearic
acid and tripalmitin, and cholesterol were used to generate
simulated lung surfactant (SLS) vesicles according to the
method of Schroit and Pagano [7]. Fetal calf serum (PCS)
was chosen to represent body fluids with complex properties.
Extraction Procedures
Diesel particles (200-300 mg) were extracted with 150 ml
dichloromethane (DCM) or other solvents (Table 1) in a
Soxhlet apparatus for 4 hours accounting for 20-25 solvent-
wash cycles. The volume was reduced to 10-15 ml under
vacuum in a Rotavapor (Buchi) and the remaining solvent was
evaporated under nitrogen to dryness to determine the
extractable mass. The extracts were stored at -80°C and
were subsequently redissolved in DMSO for use in mutageni-
city tests.
The concentration of diesel particles used in the SLN, 0.5%
BSA, PCS, SLS and dimethyl sulfoxide (DMSO), was 5 mg/mL.
The extractions were performed in a shaking water bath at
the desired temperature (25°C, 37°C, or 45°C). Two milli-
liters of the extraction mixture were removed at selected
time intervals and centrifuged at 10 000 g and room temper-
ature for 30 minutes. The samples were frozen at -80°C
until they were examined in the mutagenicity test.
Preliminary experiments indicated that bacterial contamina-
tion may occur in FCS extract after prolonged incubation.
This problem was eliminated either by incubating the extrac-
tion mixtures at 45°C or by adding ampicillin (50 pg/mL)
to the extraction mixture. Ampicillin had no effect on the
mutagenicity of the extract as demonstrated by the negative
controls used in each test.
For the multiple solvent-wash cycle study, 2 grams of
diesel particles were extracted with 50 ml of dichloro-
methane for 10 minutes under constant agitation at room
temperature. After the extract was separated from the
particles by centrifugation, the remaining particles were
re-extracted with the same volume of fresh solvent as
described above. This procedure was repeated ten times.
The extracts were then evaporated to dryness and stored
at -80°C as described earlier.
Bacterial Mutagenicity Test
The bacterial mutagenicity test used in this investigation
was essentially the same as described by Ames et al [2].
Preliminary experiments indicated that extracts of diesel
247
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particles elicited significant mutagenic activity in the
TA1538, TA98 and TA100 strains of Salmonella typhiwupivm.
Of these, TAT538 and TA98 gave the most reproducible
responses. In addition, TA98 is resistant to ampicillin
whereas the TA1538 strain is not. Consequently, TA98
strain was used in this study since ampicillin had to be
used in conjunction with the simulated biological fluids.
Bacteria were grown in nutrient broth (TN) containing 0.9%
sodium chloride, 0.4% tryptone, 0.25% yeast extract, and
0.1% glucose. The cultures were kept at 37°C in a shaker
water bath and grown to an optical density (Ai^s.) of 1.5 to
2.5. The bacteria were harvested by centrifugation at
10 000 g and 25°C. The cell pellets were resuspended in
sterile saline supplemented with 10% TN broth to an optical
density of 2, which represented a cell density of 2-5 x
108/mL. The suspensions were kept on ice during the
experiment to maintain the viability of the bacteria.
The Aroclor 1254-induced rat liver enzyme preparation, S9,
was purchased from Litton Bionetics, Kensington, Maryland.
A reaction mixture was prepared according to Ames_et al [2]
and sterilized by filtration through a Mi 11iporeQvfiHer
(0.45 urn pore size). The final volume applied was 20 pL S9
in 0.5 mL of reaction mixture per plate.
The test compound was first introduced in a tube containing
2 ml molten agar overlay (0.6% agar, 0.05 mM histidine,
0.05 mM biotin, and 0.9% NaCl). Then, 0.1 ml of the
bacterial suspension was added and mixed thoroughly. If
the test required metabolic transformation, 0.5 ml of the
S9 mixture was added before mixing. This mixture was
poured onto Vogel-Bonner E medium and allowed to solidify.
The plates were incubated at 37°C for 48 hours, and the
number of colonies was determined by an automated counter
(Biotran Colony Counter).
Five doses of each extract were used to establish the dose-
response curve, and the activity of each dose was deter-
mined by duplicate plates. The mutagenic activity of the
extracts was expressed as: 1) extract activity (his+
revertant per mg extract for DCM extract or his+ revertant
per ml extract solution for simulated biological fluids and
DMSO extracts), and 2) specific activity (his+ revertant per
mg particle).
248
-------
The extract activity was calculated from the slope of the
linear portion of the dose-response curve: y = a + bx.
1. For DCM extracts:
y is the number of revertants per plate,
x is the concentration of the extract per plate,
(mg extract/plate)
a is the intercept and b is the slope of the
regression line (revertants/mg extract).
2. For simulated biological fluids and DMSO
extracts:
y is the number of revertants per plate,
x is the volume of extract solution per plate
(ml/plate)
a is the intercept and b is the slope of the
regression line (revertants/mL extract
solution).
The specific activity (S.A.) is calculated as follows:
1. For DCM extracts:
S.A. (his revertants/mg particle) =
£(rev/mg) Percentage Extractable Mass (%)
2. For simulated biological fluids and DMSO extracts:
<, A b frev/ml extract solution)
' ' c (mg particle/ml extract solution)
where c is the mass of particles per volume of
extract solution (mg particle/ml extract solution).
RESULTS
Optimal Extraction Conditions
The choice of extraction solvent, extraction time and
extraction temperature were examined experimentally. A
total of eight organic solvents ranging from methanol to a
benzene-ethanol azeotrope were used. Table 1 lists the
solvents used along with their respective dielectric
constants and boiling points. First, the amount of extract-
able mass seemed to correlate with the polarity of the
solvents. More polar solvents extracted more mass than the
less polar solvents as shown in Figure 1. However, the
extractable mass did not correlate with the mutagenic
activity of the extracts. Methanol yielded the most
249
-------
Table 1
Physical
Characteristics of the Solvent
Dielectric Constant
Solvent
Water
Methanol
Acetone
Dichloromethane
Ethyl acetate
Benzene
Cyclohexane
n-Hexane
OJ
-D
to
4->
« ao-
+j
X
LU
£
** 10-
^
^
SY
//
U)
at 25°C
78.54
32.63
20.7
8.895
6.02
2.274
2.015
I
1
I
.882
metHoool \ dK:H(oro-
I
JL i cxc|°
m%
ItAvnnA
ocetote
Figure 1 Percent of
by organic solvents.
1500-
Ol
E
98 His+ Revertant/
Diesel Particle
, § i
i i i
i
Boil ing
Point (°C)
100
64.96
56.2
40
77.06
80.1
80.74
68.95
I
he '
ethonol
extractable mass of diesel particles
I
^
1
i^
^i
-S9
I
Figure 2 Mutagenic activity of diesel participate extracts
expressed as TA98 net revertants/mg of particle without S9
activation.
250
-------
extractable mass from the diesel particles, but the extract
was not the most active in the Ames test. Cyclohexane and
n-hexane extracts were also relatively less active.
Dichloromethane extract was found to be most active,
followed by the benzene and the benzene-ethanol extracts.
Thus, DCM seems to be the best solvent for the extraction
of mutagenic activity from diesel particles, although it
does not remove the maximum amount of extractable organic
matter from the particles (Figure 2).
Secondly, the number of solvent wash cycles required to
' remove most of the mutagenic activity from the diesel
particles was examined from the successive dichloromethane
extracts obtained from the 10 minute washes. Figure 3
shows the cumulative percentage of the extractable mass and
mutagenic activity of the extracts as a function of the
number of successive washes. More than 90% of the extract-
able mass and mutagenic activity were recovered after only
<4 washes. After 10 washes, the mass and activity extracted
from the particles were within 1% of the amount removed by
Soxhlet extraction over 20-25 solvent wash cycles. From
these results, we established that a minimum of 10 solvent
wash cycles was sufficient to elute most of the mutagenic
activity from the diesel particles.
100 n
A EXTRACTABLE MASS
X MUTAGENIC ACTIVITY (S9)
34567
NUMBERS OF SOLVENT WASHES
10
Figure 3 Cumulative percentage of extractable mass and
mutagenic activity from diesel particle by multiple solvent
wash cycles.
251
-------
Thirdly, the effect of extraction temperature was investi-
gated by incubating the diesel particles with dimethyl
sulfoxide at 25°, 37° and 45°C. The mutagenic activities
of these extracts were determined up to 48 hours. The
activity of the extracts at these temperatures reached the
maximal level after 1 to 2 hours of extraction (Figure 4).
At 25°C, the activity remained stable throughout the
extraction duration; whereas the activity of the extracts
at 37° and 45°C began to decline after 6-8 hours of extrac-
tion. These results indicated that some of the mutagenic
activity in the diesel particles was affected at the higher
extraction temperatures, a moderately low 37-45°C. The
combination of heat and oxidation effects may be responsible
for the decline of activity in the extracts.
100
o
<
80 -J
O
z
60-
O
<
20
25° C
x 37° C
D 45° C
TA 98 -S9
0 6 12 18 24 30 36 42
DURATION OF EXTRACTION, h
Figure 4 The effect of extraction temperatures on the
mutagenic activity of the DMSO diesel particulate extracts.
The activity of DCM extract was taken as 100%.
252
-------
Extractability of Mutagens in Simulated Biological Fluids
In order to compare the activity of the extracts from dif-
ferent solvents, the DCM extract activity was used as a
reference to calculate the relative mutagenic activity of
these extracts. Figure 5 shows the relative uiutagenic
activity of the PCS and the DMSO extracts at different
times during one extraction at 45°C. The DMSO extract
reached its maximum level of activity after two hours of
extraction, and the activity began to decline after 8 hours
of exposure to 45°C. On the other hand, the activity of
the FCS extract increased slowly during the entire period
of extraction. On an average, 6 ± 5.2% of the activity was.
eluted by FCS after 48 hours at 45°C in five experiments
and the range was 3.6 to 12.6%.
100
>-
I
>
»
o
o
z
Ul
o 5°-
A DMSO
TA 98 -S9
x FCS
o SLS, BSA ASLN
6 12 18 24 30 36 42
DURATION OF EXTRACTION, h
Figure 5 The relative mutagenic activity of diesel par-
ticle extracts at 45°C versus time. The activity of DCM
extract was taken as 100%.
253
-------
A typical dose-response curve of the mutagenic activity of
the DMSO and PCS extracts is shown in Figure 6. Each point
of the curve was determined from four plates. The dura-
tions of extraction were 2 hours for DMSO and 48 hours for
PCS. Extracts obtained at these durations exhibited the
highest activity. Note that the activity of the PCS
extract was much lower than the DMSO extract, both with and
without metabolic transformation despite the more than
twenty times longer extraction period.
Five experiments were repeated with different batches of
diesel particles, and the results are summarized in Figure
7. The data again indicated that dichloromethane is much
more effective in extracting the mutagens from diesel
particles than dimethylsulfoxide and that the simulated
biological fluids remove little or no mutagens from diesel
particles.
Comparison of Mutagenic Activity of Diesel Particles
Collected by Electrostatic Precipitator and Filtration
The effect of collection method on the chemical composition
and biological activity of diesel particles was described
in a separate report [6]. Although the percentage of
extractable mass from the electrostatically collected
sample (ESP) was greater than that obtained from the filter
sample, the mutagenic activities of the extracts expressed
as net revertants/mg of diesel particles were comparable as
shown in Table 2. Most of the increase in extractable mass
for the ESP sample was found in the acid salt fraction
obtained after chemical fractionation of the extract [6].
Mutagenic activities for all nine extract fractions were
Table 2
TA98 Specific Activity of Diesel Particles
Collected at 100°C by ESP and Filtration
his net revertants/mg particle
Sample
Diesel
Diesel
Particle
Particle
(ESP)
(Pallflex
n*
12
filter) 4
-S9
440±105**
367± 45
+S9
419+1
307±
75
22
* number of experiments
**mean ± standard deviations
Positive Controls (n=6):
2-nitrofluorene (2.5 yg) (-S9) 368±22
2-aminoanthracene (2.5 pg) (+S9) 1047±91
254
-------
150
100-
CU
CO
E-
Z
OS
w
a:
.22 soH
CO
O)
PCS -S9
+39
a DM50 -S9^
DMSO +S9
SO 100 150
MICROLITER/PLATE
200
Figure 6 Dose-response curves of DMSO and PCS extracts of
diesel particles.
150
O
O
z
UJ
o
\
^
2
UJ
>
t-
<
_i
UJ
Q£
Figure 7 Comparison of the mutagenic activities of diesel
particulate extracts DCM (dichloromethane), DMSO (dimethyl
sulfoxide), PCS (fetal calf serum), BSA (0.5% bovine serum
albumin), SLS (simulated lung surfactant), and SLN (saline),
TA 98 -S9
!
1
T
l~
255
-------
determined in the Ames test where the acid salt fraction
from the ESP sample was found to be inactive. More than
90% of the biological activity was accounted for in the
neutral-nonpolar II, neutral polar, weak and strong acid
fractions. The mutagenic activity of each extract fraction
is presented in Figures 8 and 9 as a percent of the total
activity of all the extract fractions.
DISCUSSION
Among the solvents used in this study, DCM was the most
effective solvent to elute the mutagens from diesel parti-
cles. In contrast, DMSO showed only a moderate ability to
extract mutagens from the particles. PCS extracted 6 ±
5.2% (range 3.6 to 12.6%) of the activity found in the DCM
extracts, whereas no activity could be detected in the 0.5%
BSA extracts. The difference in mutagenic activity between
the PCS and the 0.5% BSA extracts may be attributed to the
presence of lipoproteins and phospholipids in the fetal
calf serum and to the fact that the lipids and lipoproteins
in the PCS may extract mutagens from the particles which
are not extractable by proteins alone. Indeed, benzo-
[a]pyrene was found to associate with serum lipoproteins in
vitro [8]. However, the fact that the simulated lung
surfactant (SLS) lacks any mutagenic activity despite the
presence of significant amounts of phospholipids does not
fully support this explanation.
Although earlier studies have reported that diesel partic-
ulate extracts obtained by powerful organic solvents have a
significant mutagenic activity, the results of this study
indicate that the mutagenic activity in diesel particles is
not readily removable by simulated biological fluids. In
addition, 90-95% of the mutagenic activity of the diesel
particulate extract was contained in only two fractions
consisting of polynuclear aromatic compounds. Since the
chemical species in these fractions are expected to be only
slightly soluble in aqueous solutions, the question of
bioavailability of the mutagens in diesel particles must be
considered in the final assessment of any potential effects
of diesel particles in biological systems and organisms.
The effect of the extraction temperature on the mutagenic
activities of DMSO extracts is important in the preparation
of diesel particulate extracts for chemical and biological
analyses. Indeed, the mutagens extracted by DMSO at
45°C for more than 8 hours were shown to be unstable and
may lose a significant amount of their activity after long
exposures to this temperature. Therefore, caution should
be exercised in the extraction of diesel particles for
testing of mutagenic effects if losses of biological
activity are to be minimized. On the other hand, the
256
-------
100
>>
4->
>
o
<=c
!Z
o>
o
S-
ESP Sample (+SS'l
[3 ESP Sample (-S9
Filter Sample <-f S9|
("] Filter Sample (- S91
Weak Acid
Strong Acid
Neutral-Polar Neutral-Nonpolar II
Figure 8 Most of the mutagenic activity of the diesel
particulate extracts was found in four extract fractions.
ESP indicated sample was collected by electrostatic precip-
itation. +S9 and -S9 indicated S9 activation or no activa-
tion in the Ames test using TA98.
2.0
ESP Sample (+ S9)
0 ESP Sample (- $9)
Filter Sample (+S9)
Filter Sample (- S9)
Base
Base Salt
Neutral-
Nonpolar I
Acid Salt
Weak Salt
Figure 9 Contribution of mutagenic activity in the less
active extract fractions of the diesel particulate extracts.
*Below detection limit
257
-------
bioactivity expressed on a particle weight basis is not
affected by the collection methods studied.
CONCLUSIONS
The Salmonella typhimuriim mutagenicity test system is a
useful technique to study the mutagenic properties of diesel
particulate extracts. However, temperature and oxidation
effects can alter the mutagenic activity of the extract.
Caution should be exercised during the extraction and
handling of the diesel particulate extracts in order to
prevent any loss of biological activity. The choice of
solvent in the extraction is also critical. Although
methanol extracted the greatest amount of mass from the
diesel particles, the dichloromethane extract was found to
be most active in the Ames test. The acetone, cyclohexane
and benzene extracts exhibited less activity compared to the
DCM extract. The particulate collection method and the
sampling conditions must also be examined and standardized
to avoid sampling artifacts.
No mutagenic activity could be detected in the 0.5% bovine
serum albumin, simulated lung surfactant, and saline ex-
tracts, even at a particulate concentration of 5-10 mg/mL in
the extraction mixture. Only the fetal calf serum displayed
the ability to extract some mutagenic activity from diesel
particles. These results indicated that the mutagens in
diesel particles would not be readily available in vivo.
Thus, the question regarding the biological fate of inhaled
diesel particles and the metabolic transformation of the
active compounds found in the diesel particulate extracts
should be addressed in future studies.
ACKNOWLEDGEMENT
The authors are grateful to Dr. Kenneth Strom for his assis-
tance in formulation and preparation of the simulated lung
surfactant. Also, we are thankful to Janet Dickman, Jim
D'Arcy and Tom Johnson for their technical assistance.
258
-------
REFERENCES
1. Huisingh, J., Bradow, R., Jungers, R., Claxton, L.,
Zweidinger, R., Tejada, S., Bumgarner, J., Duffield,
F., Water, M., Simmon, V. F., Hare, C., Rodriguez, C.
and Snow, L. Application of Bioassay to the character-
ization of diesel particle emissions. In: Application
of short-term bioassays in the fractionation and
analysis of complex environmental mixtures. EPA 600/9-
78-027, September 1978.
2. Ames, B.N., McCann, J. and Yamasaki, E., 1975. Methods
for detecting carcinogens and mutagens with the
Salmonella mammalian-microsome mutagenicity test.
Mutation Res., 31:347-356.
3, McGrath, J.J., Schreck, R.M. and Siak, J.S., 1978.
Mutagenic screening of diesel particulate material.
Paper No. 78-33.6, 71st Annual Meeting of the Air
Pollution Control Assn., Houston, Texas.
4. Chrisp, C.E., Fisher, G.L. and Lammert, J.E., 1978.
Mutagenicity of filtrates from respirable coal fly ash.
Science, 199:73-75.
5. Falk, H. L., Miller, A. and Kotin, P., 1958. Elution
of 3,4-benzpyrene and related hydrocarbons from soots
by plasma proteins. Science, 127-474-475.
6. Chan, T. L., Lee, P.S. and Siak, J.S., Diesel Particu-
late collection for biological testing: comparison of
electrostatic precipitation and filtration. In:
Proceedings of International Symposium on Health Effects
of Diesel Engine Emissions, USEPA, Cincinnati, OH,
December 3-5, 1979.
7. Schroit, A.J. and Pagano, R.E., 1978. Introduction of
antigenic phospholipids into plasma membrane of mamma-
lian cells: organ and antibody redistribution. Proc. of
National Academy of Science, 75:5529-5533.
S.Shu, H.P. and Nichols, A.V., 1979. Benzo[a]pyrene
uptake by human plasma lipoproteins in vitro, Cancer Res.,
39: 1224-1230.
259
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General Discussion
R. BRADOW: Recently, one piece of evidence has come out
of our work relative to the extraction of the diesel with
protic-solvents. Apparently this happens both with meth-
anol extraction are with extractions with benzene/methane!
or toluene/methanol. The protic solvents seem to have a
tendency to extract fairly well the sulphate and nitrate
ions, inorganic anions. We avoided that approach fearing
some toxicity in the biological testing due to the elevated
levels of electrolytes in the medium. Have you experienced
such effects? Our endochromatography indicates that a
fairly substantial level of sulphate can be obtained.
J. SIAK: Most of the studies in our department have
used BZMA X-rays. We have done some other experiments
where we extracted particles with methanol first, coex-
tracted it, and re-extracted it with dichloride methane.
The dichloride methane extract by the secondary extraction
has brought very high activity in a low percentage re-
covery. They are very active, and I think we have not
looked into the problem. I think the methane cold wash
will remove a lot of salt and then you can clean your sys-
tem quite well by using that two-step procedure.
R. BRADOW: Yes, initially the attack that we made on
that similar problem was to extract with DCM and then fol-
low it subsequently with acetonitrile, but even a nonpro-
tic, highly polar solvent such as acetonitrile was fairly
effective in removing the inorganic anions. Ultimately we
abandoned that procedure because of the problems in dis-
tinguishing between the organic material and the inorganic
substance.
P. NETTESHEIM: What are the conclusions you drew from
the fact that the particles seem to extract so poorly in
simulated body fluids?
J. SIAK: You can look at our chemical analyses. Most
of them are what we call neutral polar, neutral nonpolar -
you don't realize they are not dissoluble in aqueous sol-
utions, to the best of my interpretation.
J. HUISINGH: We started sometime ago looking at ex-
tracts of serum and found similar results; then we went
back and extracted the same particles with DCM. Thus we
knew how much DCM extractable mutagenic activity there
should be on a certain mass of particles. We mixed the
total DCM extract with serum, and the activityent right
down to what it was when we incubated serum with the particles
So it looked as though serum was decreasing the activity,
either through binding or other means. We couldn't measure
it, however, and we had considerable difficulty in releasing
that activity although we have tried with solvents, col-
umns, and proteases. Do you have anything like that and
have you combined extracts with serum?
260
-------
T. CHAN: I haven't done that experiment yet. There is
a paper being released showing that BaP can bind the lipid
protein of the serum fractions. The lipid protein may thus
be a serum fraction that will extract some mutagens out.
But, we have to do the study before we say anything. What
conclusions are you making from the fact that you are hav-
ing such a rapid degradation of the mutagenic activity in
the serum? The interpretation of what you have offered was
that you feel the serum can extract the mutagenic activity.
J. HUISINGH: I think possibly this approach should be
used to look at other test systems where mutagens bound
with serum may be more detectable using cell culture sys-
tems.
MR. 6EEM: Is there also a possibility that there may be
some detoxification forces going on in the serum?
J. SIAK: No, when doing serum culture, using serum
medium, you have serum there already.
L. SCHECHTMAN: In tissue culture systems, such as the
serum hamster embryo system, we have used fetal calf serum
to extract whatever mutagenic or transforming activity
might be found using mutagenic compounds such as benz(a)-
pyrene. We find that with continued incubation of BaP in
fetal calf serum at 37 degrees over a 24 to 48 hour period
one can, in fact, introduce as much as 10 to 30 micrograms
of BaP per ml of pure fetal bovine serum. When that same
serum is used as the serum supplement to growth medium to
incubate the serum hamster embryo cells, one can change
those cells to morphically transformed phenotypes. The
serum serving as a "solubilizer" of the polycyclic hydro-
carbon may be working as a double-edged sword not so much
in terms of inactivating but making the carcinogen more
bioavailable to the target cell system.
J. HUISINGH: I think that is an important point. We
have not yet incubated these particles with serum and then
introduced them to cell culture systems. I don't know
whether General Motors has done that experiment or not, but
I think that would help address the question. My assumption
was that the serum was not inactivating but was simply
binding the mutagens in such a way that they were not avail-
able to the bacteria and that is why we had attempted to
release them or extract them back. I think it is just a
matter of technique; we have not yet found the right meth-
od. We have had trouble with emulsions forming when we do
extractions and proteas, although they might break up the
proteins and create more histidine which interferes with
the assay so we haven't found the right method yet.
0. CHOUDBURY: We observed that in extracting fiberglass
splinters containing airborne particulates, a polar solvent
like methanol extracts a lot more mass. We extracted the
filters with benzene first and if the extraction is carried
out for six hours almost 94-95 percent of it is extracted.
We then took a series of these extracted filters and ex-
tracted them further with acidified and basic methanol and
261
-------
found that these methanol extracts had much more of the
mass than any other solvent, but the mutagenic activity was
very, very low. My assumption is that perhaps there is a
lot of organic material in there, but the TLC plate shows
that there isn't any organic. It is very highly polar.
R. YASBIN: Microbiologists have known for some time
that even things like antibiotics when mixed with serum
have great difficulty interfering with the processes in
gram negative organisms. They are not detoxified; they are
just bound, and I don't think detoxification is an ex-
planation of what is happening with the serum. Probably,
at least relating to what we have learned with antibiotics,
they are bound and not allowed to get through the gram
negative wall or membrane.
J. HUISINGH: I think that is an important point that
probably should be tested. You are suggesting that serum
can bind materials and be less available to bacteria, and
from Dr. Schechtman's statements, it may make them more
available to the mammalian cells. To draw conclusions
about these serum extraction experiments, we really need to
know a little more about what we are extracting.
262
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MUTAGENIC ACTIVITY OF DIESEL EMISSION PARTICULATE
EXTRACTS AND ISOLATION OF THE MJTAGENIC FRACTIONS
Dilip R. Choudhury and Charles 0. Doudney
Division of Laboratories and Research
New York State Department of Health, Albany, N.Y. 12201
ABSTRACT
Mutagenic activity in diesel emission particulate extracts
was detected by the Salmonella typhimurium/microsome assay.
Direct-acting mutagens as well as promutagens requiring
metabolic activation were detected. The extracts were frac-
tionated into acidic, basic, and neutral fractions, and the
neutral fraction was chromatographed into seven subfrac-
tions. Differences in the mutagenic potency of these frac-
tions and subfractions were determined by the Salmonella as-
say. Fractions containing as yet unidentified~compounds,
but not polynuclear aromatic hydrocarbons; were found to
make a major contribution to mutagenic activity of the ex-
tracts.
INTRODUCTION
The carcinogenic activity of gasoline-powered automobile ex-
haust condensates is well known (1). Recently, organic ex-
tracts of diesel exhaust particulates have been shown to
possess carcinogenic (2) and mutagenic (3) activities. How-
ever, the emission characteristics of diesel-powered vehi-
cles are significantly different from those of gasoline-pow-
ered vehicles. A diesel automobile emits 30-50 times more
particulates by mass than a comparable gasoline-powered au-
tomobile (4). Moreover, 90% of the diesel emission particu-
lates are of respirable size, <1 Mm (5), and high molecular
weight organics are adsorbed onto them. The anticipated in-
crease in use of diesel-powered light-duty vehicles will thus
increase the automotive contribution to atmospheric particu-
late pollutants which are of public health concern. These
considerations emphasize the importance of determining the
toxicologic properties of organic compounds adsorbed onto the
diesel exhaust particulates and identifying the constituents
most responsible for the genotoxic effects.
Determination of the carcinogenic potential of an environ-
mental sample by the conventional mouse-skin painting test,
however, is time-consuming, expensive, and difficult. The
263
-------
microbial assay system introduced by Ames and his co-workers
(6) is a fast, inexpensive, sensitive and reliable way to de-
termine the mutagenic activity of a chemical, which has a
strong qualitative correlation with its carcinogenic poten-
tial. Several histidine-requiring strains of Salmonella
typhimurium are used for the assay.
Previous efforts to identify genotoxicants in diesel exhaust
particulates have involved searching for compounds of known
or suspected carcinogenicity, such as polynuclear aromatic
hydrocarbons (PAH) (7), by appropriate analytical techniques
--commonly gas chromatography-mass spectrometry. 'We assumed
that the complex mixture of organic compounds in diesel par-
ticulate extracts is likely to contain mutagenic compounds
other than commonly suspected ones. We therefore used the
Ames test to detect mutagenicity in particulate extracts and
to monitor the distribution of mutagenicity amoung various
fractions of the extract. This communication presents the
preliminary results of these assays, the fractionation of the
extracts, and the isolation of fractions with relatively high
mutagenic activity. Comprehensive chemical characterization
of these fractions is under way.
EXPERIMENTAL
Sample Collection
Emission particulates from a diesel Volkswagen (VW) Rabbit
and a diesel Mercedes 300-D were collected by a modification
of the standard dilution tunnel technique (8) and using Pal-
Iflex T60A20 Teflon-coated glass fiber filters. The filters
were extracted with dichloromethane for 24 h in a Soxhlet ex-
tractor. After filtration under vacuum through a 0.2-um
filter, the solvent was completely removed under vacuum with
gentle heat. Details of the sample collection and extraction
are described elsewhere in this Proceeding (8).
Fractionation of the Diesel Particulate Extracts
The first step in the fractionation was a liquid-liquid par-
titioning to separate the acidic, basic, and neutral com-
pounds (Figure 1A). The sample was partitioned between dich-
loromethane and aqueous 0.1 N HoSC^ and aqueous 0.1 N NaOH
to extract the bases and acids respectively in the aqueous
phases. The basic and acidic compounds were subsequently
back-extracted into dichloromethane after the pH of the
aqueous phase was adjusted. When the acids and bases had
been extracted, the organic layer was washed with water and
dried over anhydrous sodium sulfate, and the solvent was
evaporated to give the neutral fraction.
264
-------
0.1 N H2S04
ORGANIC
AQUEOUS
ORGANIC
0.1 N KOH
AQUEOUS
B
X
o
too
80
60
40
20
5N H2S04
(PHI)
CH2C12
pH 10-11
-CH2CI2-C6H,4-
Et20
567
200 400 600
ELUTION VOLUME (ml)
800
Figure 1. A) Fractionation of diesel emission particulate
extracts. B) Elutant profile for chromatographic fractiona-
tion of neutral diesel particulate extract.
265
-------
For the VW Rabbit sample the neutral fraction comprised ap-
proximately 94% of the extract by mass. It was further frac-
tionated into seven subtractions by adsorption chromatogra-
phy. Silica gel 60 (E. Merck, Germany) was washed with
hexane and dichloromethane, dried and activated at 110°C for
12 h. The sample was then adsorbed onto 1 g of the freshly
activated silica gel, which was added to the top of a chro-
matographic column (28 x 1 cm) packed with 9 g of dry, acti-
vated silica gel. A solvent gradient of hexane to dichloro-
methane to diethyl ether was used for elution. The elution
profile is illustrated graphically in Figure IB. The frac-
tionation was monitored qualitatively by observing the fluo-
rescence with an ultraviolet light.
Mutagenicity Assay
Histidine-requiring strains of Salmonella typhimurium (TA98,
TA100, TA1535, TA1537, TA1538) were obtained from Dr. Bruce
Ames of the university of California at Berkeley. The
strains were routinely checked for histidine auxotrophy,
deep rough character, and the presence of R factor (TA98 and
TA100) as described by Ames et al. (6). The cells were
grown in Difco bovine heart suspension. Liver homogeneate
(S-9) from Aroclor 1254-induced male Sprague Dawley rats was
obtained from Litton Bionetics (Kensington, Md). We found
0.075 ml of this S-9 preparation per ml of S-9 mix to be op-
timum for promoting optimal mutagenesis in TA98 by benzo(a)-
pyrene (BaP) and 2-aminofluorene (2AF). This concentration
was used in all subsequent experiments. Tests of diesel ex-
tract samples with lower or higher concentrations of S-9 did
not increase the mutagenesis.
Each tester strain was routinely checked in the presence and
absence of S-9 for optimal mutagenesis as follows:
TA98: BaP, 5 yg; 2AF, 2.5 yg; and 4-nitroquinoline-N-oxide
(4NQO), 0.5 yg.
TA100: BaP, 5 ug, and 4NQO, 0.5 yg.
TA1535: N-methyl-N-nitro-N-nitrosoguanidine, 10 yg.
TA1537: 9-aminoacridine, 25 yg.
TA1538: 2AF, 2.5 yg.
A diesel particulate extract sample from a Nissan van was
used with every set of experiments to check the consistency
and reproducibility of the mutagenic response of each tester
strain.
Each extract sample was dissolved in dimethyl sulfoxide and
examined in the presence and absence of the metabolic acti-
vation system (S-9) in a dose response fashion (5-8 doses/
tester strain. Revertant colonies were counted with a Bio-
tran II, model Clll automated colony counter (New Brunswick
Scientific Co., New Brunswick, NJ). Duplicate plates were
run except for the subfractions (where the quantity of sam-
266
-------
pie was insufficient), and the count of revertants was aver-
aged. Each experiment was repeated at least once. All data
were corrected for the spontaneous background reversion rate
observed with each tester strain. A blank glass fiber fil-
ter was also extracted, and the extract was assayed for mu-
tagenic response.
Solvents and Reagents
All solvents used for extraction and chemical fractionation
were glass distilled,ultraviolet grade (Burdick and Jackson
Inc., Miskegon, MI). Benzo(a)pyrene (gold label) and 2AF
were obtained from Aldrich Chemical Company (Milwaukee, WI,
4NQO from ICN Pharmaceuticals (Plainview, N.Y.), and 9-
aminoacridine and dimethylsulfoxide from Sigma Chemical
Company (St. Louis, Mo).
RESULTS AND DISCUSSION
Extract of diesel exhaust particulates from the VW Rabbit
showed positive mutagenic response to all five tester
strains. The results with TA98, TA100, and TA1538 are shown
in Figure 2. The responses with TA1535 and TA1537 were
rather low. The highest response was with TA100.
Significant mutagenic activity was observed with all five
strains in the absence of the metabolic activation system
(S-9), indicating the presence of direct-acting mutagens in
the extract. Incorporation of S-9 lowered the mutagenic
activity except in the case of TA1538. Similar deactivation
of some direct-acting mutagens in the presence of S-9 has
been reported before (9). The positive response with all
strains also indicates that the mutagens in the extract pre-
sumably act by both base-pair substitution and frameshift
mutation. An extract of Mercedes 300-D exhaust particulates
also showed a high response with TA98 and TA100 (Fig. 3).
The activity without S-9 was again higher than with S-9.
The crude extracts were tested in duplicate at five concen-
trations in the range of 20-800 ug/plate. The number of re-
vertants/plate (with background correction) was plotted
against the dose (yg/plate), and slope of the linear part of
the induction curve was determined by the least squares
method. This slope was assumed to reflect the specific ac-
tivity (revertants/yg). The results showed that specific
activity of the Mercedes sample was somewhat higher than the
Rabbit sample. In general, we found that TA98 and TA100 are
good general indicators of mutagenicity of these samples.
These two strains were therefore used to screen fractions of
the extracts.
The extract was first fractionated into acidic, basic, and
neutral fractions (Figure IB). Some emulsion was formed dur-
ing extraction with base and acid solutions. Use of dilute
H2S04 and NaOH solutions (0.1 N) in partitioning helped to
267
-------
s
X
0>
CD
4->
03
iI
O
H
f-.
OS
-------
800
600
cr
LU
UJ
cr
400
200
0
QTAIOO
OTA 98
20 50 100 150
EXTRACT (//g)
200
Figure 3. Mutagenic activity of diesel particulate extract
(Mercedes 300-D).
269
-------
reduce this problem. The extracts did not dissolve complete-
ly in ether, which could have minimized the emulsion problem.
The specific activities of the three fractions and the crude
extract are shown in Figure 4. The acidic fraction had the
highest specific activity, which was significantly higher in
the absence than in the presence of S-9. The basic frac-
tion, 0.5% by mass of the total, showed enhanced activity in
the presence of S-9. The neutral fraction showed somewhat
higher mutagenic response in the presence of S-9 than in its
absence; the difference was smaller than for the crude ex-
tract. The results suggest that much of the direct-acting
mutagens in the crude extract are acidic. Since the neutral
fraction was the major fraction (95%) by mass, it was ex-
pected to contain most of the mutagenic compounds, includ-
ing PAHs, We therefore developed an adsorption chromato-
graphic system to subfractionate the neutral fraction.
Since the extracts are not soluble in hexane and since ap-
plication of the sample in a polar solvent such as dichloro-
methane would result in loss of chromatographic resolution,
we decided to apply the sample preadsorbed to a small amount
of silica gel, which could be added to the top of a column
containing dry, activated silica gel. This method of sample
application is simpler than the one described by Rodriguez
(10). The elution volume of the PAH fraction was determined
by the elution volumes of fluorene and coronene, chromato-
graphed under identical conditions.
The elution was started with hexane to carry off the paraf-
finic materials (fraction 1), and 70 ml of eluate was col-
lected. The elutant was changed to 5% dichloromethane in
hexane when fluorescent materials, observed under longwave
UV, reached the bottom of the column. This fluorescent band
was the PAH fraction (fraction 2). The elutant was gradual-
ly changed to 100% dichloromethane in several steps and fi-
nally to 100% diethyl ether (Figure IB). Final elution with
diethyl ether was essential to elute some highly adsorbing
compounds.
Seven fractions were collected. The bulk of the solvent
from each fraction was evaporated on a steam bath. The con-
centrated residue was quantitatively transferred to small,
tared, screw-cap test tubes, dried under a gentle stream of
nitrogen, and weighed. In the VW Rabbit sample the percen-
tage of various fractions by mass was: fraction 1, 72.9%;
fraction 2, 6.0%; fraction 3, 2.1%; fraction 4, 3.8%; frac-
tion 5, 4.8%; fraction 6, 8.6%; and fraction 7, 1.8%.
All subfractions were assayed with strains TA98 and TAIOO.
The tests were run at six to eight concentrations in the
range of 2-100 yg/plate. Direct-acting mutagens were de-
tected in subfractions 3-7. Subfraction 4 had the highest
specific activity (Figure 5), as detected by both TA98 and
TAIOO. The activity of subfractions 3 and 5 was enhanced in
the presence of S-9, particularly with the strain TAIOO.
270
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Crude extract
Neutral
Figure 4. Mutagenicity of diesel particulate extract
and its fractions (VW Rabbit) .
271
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14
12
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Figure 5. Distribution of mutagenicity among subfractions
of neutral diesel particulate extract (VW Rabbit).
272
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The paraffinic subfraction 1 showed no detectable activity,
the PAH subfraction 2 showed marginal activity only in the
presence of S-9. These results demonstrate that the objec-
tive of using the Salmonella assay and chemical fractiona-
tion to identify mutagenic fractions in the extracts has
been achieved.
Since the paraffinic subfraction 1 is probably of little
toxicological significance, we are ignoring it for the time
being in our chemical characterization work. Subfraction 4
and the entire acid fraction will receive high emphasis for
structural characterization; subfractions 3, 5, and 6 will
also be characterized. Some subfractions will be further
fractionated by high performance liquid chromatography for
additional concentration of active components and to facili-
tate the identification of mutagenic compounds. Although
the PAH subfraction 2 showed only marginal activity, many
members of this class are recognized carcinogens and muta-
gens. Consequently we have undertaken a complete character-
ization of this fraction too. Preliminary results are de-
scribed elsewhere in this Proceeding (11). No non-PAH com-
pound was detected in this fraction by mass spectrometry,
indicating clean separation of PAHs from paraffins and other
classes of compounds.
Our results show the feasibility of the Ames test for detec-
ting activity in diesel particulate extracts and various
chemical subfractions of these extracts. Mutagens of var-
ious chemical classes can thus be separated from non-muta-
gens and weak mutagens, thereby facilitating chemical char-
acterization. Also the relative contribution of known clas-
ses of mutagens (e.g. PAHs) compared to unknown ones in a
complex mixture can be evaluated. We have used this test
previously to isolate airborne mutagens (12), and it should
be applicable to other complex environmental samples as well.
ACKNOWLEDGEMENT
We thank Ms. Mary Franke and Mr. E. Barnard for technical
assistance and Dr. R. Gibbs and his associates of the N.Y.
State Department of Environmental Conservation for the col-
lection and extraction of the samples. This research was
partially supported by USEPA grant no. R805934010.
REFERENCES
1. Hoffman, D., Theisz, E., and Wynder, E.L. 1965. Studies
on the Carcinogenicity of Gasoline Exhaust. J. Air Poll.
Contr. Assoc., 1.5_, 162.
2. Kotin, P., Falk, H.L., and Thomas, M. 1955. Aromatic
Hydrocarbons III. Presence in the Particulate Phase of
273
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Diesel Engine Exhausts and the Carcinogenicity of Exhaust
Extracts. AMA .Arch. Ind. Health, LI, 113.
3. Huisingh, J., Bradow, R., Jungers, R., Claxton, L.,
Zweidinger, R., Tejada, S., Bumgarner, J., Duffield, F.,
Waters, M., Simmon, V.F., Hare, C., Rodriguez, C., and Snow
L. 1978. Application of Bioassay to the Characterization of
Diesel Particle Emissions. In: Application of Short-term
Bioassays in the Fractionation and Analysis of Complex Envi-
ronmental Mixtures, USEPA, EPA-600/9-78-027, p. 382.
4. Barth, D.S. and Blacker, S.M. 1978. The EPA Program to
Assess the Public Health Significance of Diesel Emissions.
J. Air Poll. Control Assoc., 28, 760.
5. Lipkea, W.H., Johnson, J.H., Vuk, C.T. 1978. The Physi-
cal and Chemical Characterization of Diesel Particulate emis-
sions --Measurement Techniques and Fundamental Considerations,
SAE special publication 450.
6. Ames, B.N., McCann, J., and Yamasaki, E. 1975. Methods
for Detecting Carcinogens and Mutagens with the Salmonella/
Mammalian-Microsome Mutagenicity Test, Mutation Res., 31_,
347.
7. Karasek, F.W., Smythe, R.J. and Laub, R.J. 1974. A Gas
Chromatographic-Mass Spectrometric Study of Organic Com-
pounds Adsorbed on Particulate Matter from Diesel Exhaust.
J. Chromatography, 101, 125.
8. Wotzak, G., Gibbs, R., and Hyde, J. 1980. A Particulate
Characterization Study of In-Use Diesel Vehicles. In: Pro-
ceedings of the International Symposium on Health EfTects of
Diesel Engine Emissions, USEPA, this volume.
9. DeFlora, S. 1978. Metabolic Deactivation of Mutagens in
the Salmonella-Microsome Test. Nature, 271, 455.
10. Rodriguez, C.F. 1978. Characterization of Organic Con-
stituents in Diesel Exhaust Particulates, South West Research
Institute, Draft Final Report.
11. Choudhury, D.R. and Bush, B. 1980. Polynuclear Aromatic
Hydrocarbons in Diesel Emission Particulates, In: Proceedings
of the International Symposium on Health Effects' of Diesel
Engine Emissions, USEPA, this volume.
12. Choudhury, D.R., Bush, B., Miller, T. and Wolin, M.J.
1978. Separation, Bioassay and Identification of Mutagenic
Organic Compounds in Airborne Particulates, Presented at the
176th National Meeting of the American Chemical Society,
Miami Beach, September 11-17.
274
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General Discussion
D. HOFFMANN: Your slides were the first I had seen of
all the fractionation studies which showed that through
fractionation you lose 20 percent of the whole tar, neu-
tral, basic etc. Did you ever try the freeze-drying water
layer and testing it? We have obtained mutagenicity with
tobacco tar which was very surprising. We found activity
when we took the water layer and freeze-dried it.
D. CHOUDHURY: I have not tried that. I think that 20
percent is primarily because there is a lot of water sol-
uble material which is staying in the water layer and so
far we haven't looked at that. I think your guess is right,
and I tend to agree with that.
I. ALFHEXN: When you say you have a 20 percent loss, is
that of the total extractable material, or is it a 20 per-
cent loss of mutagenicity during the fractionation?
D. CHOUDHURY: That is the material by weight.
I. ALFHEIM: Did you know the recovery of the mutagenic
activity of this fractionation?
D. CHOUDHURY: We haven't tried that, and in fact, I am
not sure if one can actually do that. As you keep on frac-
tionating this crude extract, you do get all kinds of sen-
ergisms and antagonisms coming into play and I am not sure
if you can combine the activity and compare with the total.
Some people have tried to do that and they claim they have
done it quite successfully.
I. ALFHEIM: We do it on ambient air samples. When we
combine the different fractions and test it again, we find
the same as when we add the testing results from separate
fractions.
D. CHOUDHURY: You mean the combining of different frac-
tions together and then testing?
I. ALFHEIM: Yes, but they still seek the percentage
loss during a procedure quite similar to this. That is why
I asked if this was from different samples?
D. CHOUDHURY: We haven't done that, but I don't think
there is actually any artifact during these fractionations.
What you say would indicate that during fractionation,
there is probably some change in the chemical character of
this compound. That would perhaps occur. You lose the
biological activities.
I. ALFHEIM: I was asking if there is any degradation?
D. CHOUDHURY: I believe there is some degradation but
I do not have proof of that so far.
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MUTAGENICITY STUDIES ON
DIESEL PARTICLES AND PARTICULATE EXTRACTS
N. Loprieno
Laboratorio di Genetica of the University, Pisa Italy
F. DeLorenzo
II Cattedra di Chimica Biologica of
The 2nd Medical Faculty of the University, Napoli Italy
G. M. Cornetti
FIAT Research Center, Torino, Italy
G. Biaggini
IVECO Engineering, Torino, Italy
ABSTRACT
Short-term mutagenicity assays by employing both different
genetic end points and organisms treated with several
methodologies have been developed on particles and DCM-
extracted fractions obtained from a naturally aspirated
swirl chamber high speed european diesel engine as a pre-
liminary approach to the definition of the potential nega-
tive health effects on human population exposed to diesel
emissions.
The in vitro methodologies have been based on the
induction of:[i) reverse mutation on S. typhimurium
TA1535, TA1537, TA1538, TA98, and TA100 strains; (ii)
forward mutation on the yeast S. pombe PI strain; (iii)
mitotic gene conversion on the yeast S. cerevisiae D4
strain; (iv) unscheduled DNA synthesis (UDS) on human
heteroploid EUE cell line.
276
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The results so far obtained have shown the direct mutagenic
activity of the particles when tested in vitro on Salmonella
TA98 strain; this activity might be possibly due to the
active fractions TRN and OXY which have resulted mutagenic
on Salmonella.
The OXY fraction has been demonstrated to be able to produce
forward mutation and mitotic gene conversions in two differ-
ent yeast systems. The present in vitro data confirm those
obtained by other AA and complete them, by showing the
direct mutagenic activity of particles and the genotoxicity
of the OXY fraction in other genetic systems not analyzed by
other AA.
Preliminary data in our laboratories have been obtained on
two heavy duty (sample A) and light duty (sample B) diesel
engine extracts obtained from EPA: mutagenicity in vitro
assays on Salmonella have confirmed the EPA previously
demonstrated activity of these extracts. The Sample A
diesel extract has been also tested for the induction of
unscheduled DNA synthesis on human cells: the fraction
has been found negative, but it inhibits the normal ^H-TdR
incorporation of the human cells.
The future plan of this project intends to develop other J_n
vitro mutagenicity assays which analyze the induction of
other genotoxic end points (gene-mutation in hamster cells,
UDS in human cells, chromosome aberrations in human lympho-
cytes) and short term in vivo mutagenicity assays on mice
and/or rats treated by different ways with particles or
active particulate extract fractions (liver and lung tests
with yeast; urine and serum tests with Salmonella; chromo-
some aberrations on bone marrow cells).
INTRODUCTION
Several AA (5,11,12,14) have recently shown that short-term
mutagenicity in vitro assay, such as the Salmonel1a-micro-
some plate test, represents a suitable biological analytical
procedure for the evaluation of the mutagenic activity of
extractable organic compounds obtained from particles
produced by different types of combustions (5,12) including
different types of diesel engines (11): this approach
allows both the identification of those fractions of chemi-
cals produced during the combustions process which possess
mutagenic activity and which could be further selected for
chemical identification studies and for the evaluation of
potential carcinogenic risk of diesel engine emissions due
to the high positive correlation which has been observed
between mutagenic and carcinogenic compounds (20,22,23,24,
25).
277
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These results have stimulated the interest of public author-
ities and of automobile manufacturers in a possible negative
health effect which could be the result of the exposure of
human popoulation to diesel engine exhaust emissions, taking
into account also the most probably future penetration of
the market by diesel vehicles.
This consideration has prompted the development of chemical
and mutagenic/carcinogenic researches on diesel engine
particulate extract fractions with the aim of either iden-
tifying the combustions conditions on which it might depend
the formation of mutagenic compounds, and either to evalu-
ate, by applying the most appropriate short-term in vitro
and in vivo mutagenicity assays, the potential carcinogenic
and genotoxic risk to human population as a consequence of
the exposure to diesel engine exhaust emissions, thus
reducing, and possibly avoiding, a series of long-term
carcinogenic animal assays.
Short-term in vitro mutagenicity assays which employ differ-
ent biological systems (from bacteria to animals) (17), on
which genetic end points of different molecular nature (gene
mutation and chromosomal mutation) and of a different
relevance for the evaluation of the genetic/carcinogenic
risk represent only the possible rationale for identifying
the highly genotoxic and potential carcinogenic fractions of
chemicals present on the surface of the particulate and
produced in a diesel engine combustion chamber.
More relevant however for the present interest is the use
of appropriate in vivo methodologies which make use of
microbial genetic systems and small rodents: these can be
employed to confirm in vivo (in the mice) the mutagenic
activity of the particles per se or of those fractions which
have been identified as mutagenic after several in vitro
experiments on different organisms. These methodologies, of
the type of the host mediated assay (3,9) have been recently
improved for their sensitivity (3): it is, therefore,
possible to assess the presence or the in vivo formation of
active metabolites in the liver, or in other organs (lung),
by a direct assay, or to assess the presence of active
metabolites in organic fluids, as the urines or the serum,
taken from animals treated with diesel particles or their
extracts. Other genetic end points, such as the chromosome
mutation induction, may also be studied in vivo, in the
somatic cells of treated animals (17), or in vitro, on human
lymphocytes (17).
In the present paper we report the preliminary results
obtained by in vitro mutagenicity experiments on the parti-
cles and four particulate DCM-extract fractions obtained
from a naturally aspirated high speed swirl chamber european
278
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diesel engine (4); the assays have been developed on Salmo-
nel1 a five strains (TA1535, TA1537, TA1538, TA98, and TA100)
(2), and two yeasts, namely S. pdmbe (PI strain) (3) and _$._
cerevisiae (D4 strain) (15,16) which have allowed the
evaluation of the potential activity of the samples for the
production of gene-mutation in prokaryotic and eukaryotic
cells and of the gene-conversion in a diploid cell.
Analyses have been made also on two EPA heavy duty (sample
A) and light duty (sample B) diesel engine DCM total ex-
tracts with Salmonella 5 strains, by varying the amount
of S-9 mix and for the induction of UDS (unscheduled DNA
synthesis) on a human cell lines, an in vitro test that we
want to apply to other samples of diesel engine exhausts
emissions.
Further development of this work will make use of several
in vivo methodologies, as those reported in figs. 1 and 2.
MATERIALS AND METHODS
I. Genetic systems: Figs 1 and 2 report the battery of
short-term in vitro and ji_n vivo tests for the evalua-
tion of mutagenic potential of diesel engine particles
and particulate extracts which we intend to use in our
project. In the present paper the results of only the
in vitro test 1, 2, 3 and 4 have been reported.
I.A Salmonella reverse mutation test: The method described
by B. N. Ames et al. (2) has been used; in all experi-
ments 500 nil of S-9 mix per plate was employed. In the
experiments reported in Table 3 and Fig. 3 different
amounts of S-9 mix were incorporated into the plates.
I.B S. pombe forward mutation tests: Yeast cell gene
mutation test with Schizosachargmyces pombe is based on
the treatment of a haploid double mutant strain (PI) of
the genotype Spade 6-60, rad 10-198, h. It allows the
evaluation of the frequency of forward gene mutations
induced by a chemical in five different genetic loci
located on the three chromosomes of this yeast. The
strain employed consists of purple colonies (on com-
plete medium), and the induced mutants consist of white
colonies: they are of the genotype ade 6-60, adex, rad
10-198, and contain a second ade x mutation which has
occurred in one of the following 5 genetic loci, namely,
ade 1, ade 3. ade 4, ade 5. ade 9 (10). Forward muta-
tions induced by chemicals in each one of these 5 loci
279
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have been shown to consist of basepair substitutions
as well as insertion/deletion base-pairs (10): 96
chemicals of different molecular nature have been so
far tested in this mutational assay and 54 of them have
been found positive. These include 38 direct acting
mutagens and 16 promutagens (18).
I.C S. cerevisiae gene conversion test: Yeast cell mitotic
gene-conversion (mitotic intragene-recombination) test
in Saccharomyces cerevisiae is based on the treatment
of a diploid multimutant strain (D4) of the genotype
a/a, gal2/+. ade 2-2/ade 2-1, trp 5-12/trp 5-27, leu
T/+. It allows the evaluation of the frequency of gene
conversions in the ade 2 and trp 5 loci, located on
different chromosomes of the yeast induced by chemi-
cals. Gene-conversions ade 2 and trp 5 have been
shown to be sensitive to hundreds of direct and in-
direct acting mutagenic chemicals (16).
I.D EUE cell line UPS test: EUE human cells have been used
in the DNA repair test which allows the detection of
the ability of a chemical to stimulate the unscheduled
DNA repair syntehsis (1,6,19). DNA repair is not a
direct demonstration of the induction of a genetic
effect; this process, however, is related to mutational
as well to carcinogenic events (6) and the test allows
the detection of many carcinogens (19).
2. Methodologies. The in vitro methodologies employed in
the present study do include the treatment of the cells
in the plates (Salmonella) or in liquid suspensions
(yeast cells and human cells). Yeast cells were
treated for several hours during their growth, and
the general methodology was that used in several other
studies already described (15). EUE human cells were
seeded in each of 50 mm petri dishes, containing 4
coverslips and incubated with Dulbecco's modified
Eagle's Minimal Essential Medium + 8% of calf serum at
37° C for 24 hours. After washing with Hank's balanced
salt solution containing 20 mM HEPES buffer (HBSSH),
the cells were treated with known concentations of
chemicals under analysis, at 37° C for 1 hour, which
were dissolved in small volumes of DMSO and then
diluted with HBSSH. At the end of treatments, cells
were washed 3 times with HBSSH incubated with fresh
medium, or fresh medium +5 mM solution of Hydroxyurea
(HU). After 15 min., all cultures received 10 uCi/ml
^H-TdR (15-25 Ci/mmoles) and were further incubated
for 4 hours. The cultures on coverslips were washed
twice with cold HBSSH and extracted with ice-cold 1 M
280
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HCL for 20 min.; they were then washed twice with
ice-cold ethanol/ethylether (1:1) and twice with
ice-cold ethylether dried and incubated overnight
with toluene 350 (Packard) at room temperature, in
scintillation vials. After addition of 10 ml standard
scintillation mixture, the samples were counted in
a Packard Tri-Carb Spectometer. The in vitro metho-
dologies (Salmonella, S. pombe, S. cerevisiae, EUE
cells) have all made use of S-9 mix obtained from
aroclor treated rats (liver) according to previously
described methods (3).
3. Chemicals: Particles were collected from a naturally
aspirated swirl chamber high speed european diesel
engine,1 according to the methods described by C.
Basso!i et al. (4); particulate extract fractions PRF,
ARM, TRN, and OXY were prepared by A. DiLorenzo et al.
(8). Reference mutagenic chemicals such as methyl-
methane-sulfurate (MMS) and dimethylnitrosamine (DMNA),
employed in the UDS test, and 9-amino acridine, sodium
azide, 2-aminofluorene employed in the Salmonella test
were all of pure analytical grade. Sample A (heavy
duty) and Sample B (light duty) diesel engine parti-
culate DCM total extracts have been provided by U.S.
EPA.
4. Evaluation of data: The evaluation of the induced
genetic effect has been based on the regression analy-
sis according to the equation y_ = a + bx, where (y) is
the induced frequency of the genetic eTTect under
consideration, and (x) is the dose of the mutagen
(rng/plates in the case of Salmonella and mg/ml in the
case of the two yeasts); the coefficients (a) and (b)
are calculated by means of the least squares method
(7). For each set of data the correlation coefficient
r has been also evaluated.
RESULTS
Sample A and Sample B diesel extracts obtained from EPA (TRN
fractions) were tested on the five strains of Salmonella
typhimurium with and without S-9 mix: the same samples have
been distributed in several laboratories. The results,
reported in Tables 1A, IB, 2A, and 2B, are quantitatively
similar to those obtained by other AA (11). The presence of
S-9 mix in the plates reduces the toxic effects of extracts:
U-cyl 2.5 1 displacement Diesel engine tested under 1975
Federal Test Procedure - Urban Driving Dynanometer Schedule.
281
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with the aim to evaluate the possibility that a reduction of
lethal effect might allow more revertant colonies to grow,
an experiment has been performed on the Sample B diesel
extract with different amounts of S-9 mix: the results are
reported in Table 3 (Fig. 3) and they show that S-9 mix does
not influence the mutagenic potency of this sample.
Sample A diesel extract has been also tested for its ability
to induce unscheduled DNA syntehsis in human cells (EUE);
this test informs us about the direct reactivity of a
chemical under analysis towards the DNA, by evaluating the
repair synthesis of DNA which might follow the induction of
some type of damage to the DNA structure.
The results of this assay are reported in Tables 4A, 4B, 4C
(Fig. 4); reference compounds such as MMS (Tab. 5B) and DMNA
(Tab. 5A) have also included in this assay: concentrations
below 1 mg/ml of Sample A extract in absence of S-9 mix do
not stimulate unscheduled DNA synthesis, nor they show toxic
effect (Tab. 4A); when more concentrated solutions have been
tested these have been found highly toxic for normal ^H-TdR
incorporation (Tables 4B, and 4C and Fig. 4); on the contrary
reference mutagenis either direct (MMS) or indirect (DMN)
show a relative lower toxic effect and an increase of the
^HTdR incorporation in the presence of HU (Tables 5A and
5B). By the present analysis it is possible to consider
this diesel extract fraction as able to interfere with DNA
replication, thus resembling the classical DNA inhibitors
FUdR and BUdR (1), but, in any case there is an indication
of a stimulation of an unscheduled DNA synthesis.
PRF, ARM, TRN and OXY particulate extract fractions have
been tested on Salmonella, S. pombe, and S. cerevisiae, by
the in vitro methodology. PRF and ARM fractions resulted
negative in all test systems (Tables 6A, 6B, 7A, 7B, 8A,
88); on the contrary TRN was mutagenic on TA1537, TA1538,
TA98, and TA100 strains of Salmonella (Table 6C): this
fraction resulted to contain direct mutagenic chemicals,
although the presence of the metabolic system showed some
increase of activity (Fig. 5). TRN was not mutagenic for
the yeast S. pombe, and it did not induce mitotic gene
conversions in S. cerevisiae (Tables 7C and 8C).
The OXY fraction was found positive in all three microbial
systems (Tables 6D, 7D, and 8D). On Salmonella strains the
higher tested dose produced a relevant toxicity (2,000
mg/plate); also for this fraction the presence of S-9 mix
Tncreased the mutagenic activity (Fig. 6). On S. pombe the
OXY fraction was mutagenic (Table 7D) without S-9 mix, and
the presence of the metabolizing system inhibited completely
its mutagenic effect (Fig. 7). On S. cerevisiae the OXY
fraction was also found mutagenic in the direct assay (Table
80), but some genotoxic activity was also observed when the
282
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S-9 mix was present (Fig. 8). Particles collected from this
european diesel engine were assayed for their complete
mutagenic activity in vitro, on Salmonella TA98 strain.
In vitro diesel exhaust particles were mutagenic (Table 9)
either in presence and in absence of S-9 mix: its mutagenic
activity has been compared with one of TRN and OXY fractions
on the same strain: the results are reported in Fig. 9.
These two fractions represent 3.16% a.,d 10.81% respectively
of the total amount of diesel particles.
When we compare the specific direct mutagenic activity of
the particles and the TRN and OXY fractions (250 nig/plate)
the following values of revert ants/nig are obtained: parti-
cles = 0.74; TRN = 2.08, and OXY = 6.70. These values
correspond to the concentration ratios between TRN or OXY
and the particles. Further experiments will define better
the quantitative comparison of the mutagenic content of
particles and the DCM-extracted fractions.
Preliminary experiments have been developed on some of the
test assay presented in Figs. 1 and 2 for a definition of
the most appropriate methodological procedures to be used
in the future.
DISCUSSION
The Ames test applied to diesel particulate extracts pre-
pared by EPA has shown that two samples analyzed have
produced in our laboratories comparable values of mutagenic
activity with those obtained by EPA: the increase of S-9
mix amount to the plates does not influence the mutagenic
activity of the Sample B extract. A test has been also
performed for the evaluation of the induction of unscheduled
DNA synthesis in a human heteroploid cell line grown in
culture: the sample has resulted inactive at non toxic
levels (below 1 mg/ml), whereas it is extremely toxic to
cell DNA replication at levels above 1 mg/ml: at concen-
trations higher than 1 mg/ml diesel particulate extract
inhibits the normal DNA replication, thus demonstrating a
direct effect of this fraction on DNA of the type of that
observed for well known DNA inhibitors, such as BUdR and
FUdR (1). In the presence of S-9 mix at 1 mg/ml, which does
not inibit the normal DNA replication, the fraction does not
stimulate the unscheduled DNA synthesis.
In the studies developed on the particles and the four
extracted fractions obtained from a naturally aspirated
swirl chamber high speed euorpean diesel engine, the Salmo-
nella test has demonstrated the mutagenic activity on TA98
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strain of the crude particles when tested directly and of
the TRN and OXY fractions: these results confirm also for
this type of particulate extracts the previous data (11).
If the positive results obtained so far will be confirmed
also by further experiments on other types of diesel engine
particles, it would appear the possibility to operate on
particles instead of particulate extracts.
The OXY fraction resulted the most active in Salmonella in
the absence of a metabolic activation system, which confirms
the presence in the extract of direct acting frameshift
mutagens, active on TA1537, TA1538, TA100, and TA98; this
fraction resulted toxic at a level range of 1,000-2,000
mg/plate.
The OXY fraction has resulted also genotoxic for the yeast
S. pombe PI (forward gene mutation system) and for the yeast
S. cereyisiae D4 (mitotic gene conversion system), thus
supporting the hypothesis that this fraction contains highly
aspecific mutagenic chemicals, as they have been found
positive in several short-term assays (26); the present data
confirm and complete the results obtained recently by the
EPA program.
Other methodologies of the type shown in Fig. 1 are in
progress in our laboratories on components exhausted and on
different fractions from diesel engine emissions, as well
as from particles obtained from industrial and domestic
burners with the same fuel as that supplied to the present
diesel engine: these comparative values are of the extreme
relevance for the interpretation of the quantitative poten-
tial mutagenic/carcinogenic risk of diesel emissions.
At the present it is still impossible to define the poten-
tial genotoxic risk for human generation posed by diesel
engine exhaust emissions, on the base of in vitro mutage-
nicity experiments if there are no sufficient in vivo
experiments; moreover all these studies should produce
results to be used for a comparison with similar risk
posed by other air contaminants present in the human
environment.
ACKNOWLEDGEMENTS
The AA are very grateful to the important contributions
given to the experimental part of this project by Drs.
A. Abbondandolo, R. Barale, L. Zaccaro, and D. Zucconi
(University of Pisa) and by Drs. G. Vricella, A. Belisario,
E. Buonocore, and I. Quinto (University of Napoli), Italy,
and to Dr. R. Cortesi and Dr. J. Huisingh of U.S. EPA and
Dr. J. C. Sturm of U.S. DOT-TSC for useful suggestions and
discussions.
284
-------
IN VITRO
1 Salmonella
Reverse Mutation
2 S pombe
Forward Mutation
3 S cerevisiae
Gene Conversion
4 EUE cell line
UDS
5 Hamster V79 cell
Forward Mutation
6 Human Lymphocytes
Chrom aberrations
Figure 1. Battery of Short-Term in Vitro an Vivo Tests for
the Evaluation of Mutagenic Potential of Diesel
Engine Particles and Particulate Extracts.
IN VIVO (mouse)
1 URINE TEST Salmonella Rev. Mutation
2 SERUM TEST . Salmonella Rev Mutation
3. LIVER TEST : S pombe Forw. Mutation
4. LUNG TEST : S. pombe Forw. Mutation
5 BONE MARROW . Chromosome Mutation
yeast cells
injection
intraperitoneal
treatment
ANALYSIS OF TISSUES
1
Liver Lung
(yeast)
(0-24 hours )
ANALYSIS OF FLUIDS
Bone marrow Serum Urines
(cytogenetic) (.Salmonella)
( 1-7 days )
Figure 2. In Vivo Bioassay of Diesel Engine Particles.
285
-------
450-
-400
£ 350 -
Ul
>
Ul
S.typhlmurlum TA 1538
EPA SAMPLE B DIESEL EXTRACT
(400 jig/plate)
250 5OO 1OOO
S 9 mix >il /plate
Figure 3. Reverse Gene Mutation Assay.
,00(1 364x10
0.01
+ S-9 mix
1 2
mg/ml
Figure 4. Unscheduled DNA Synthesis Assay.
(Sample A) Diesel Extract: EUE Cells
286
-------
1000-
100-
10
K
z
<
a.
Ul
1O-
SWIRL CHAMBER EURO DIESEL
TRN-1 SMlmonell* TA1538
5 50 00 250
500
1000
jjg/plate
Figure 5. Reverse Gene Mutation Assay
*S-9 mix
SWIRL CHAMBER EURO. DIESEL
OXY-1 Smlmonell* TA 1538
5 50 100 250
500
1000
yg/plate
Figure 6. Reverse Gene Mutation Assay.
287
-------
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288
-------
TABLE 1A. MUTAGENICITY OF EPA SAMPLE A DIESEL EXTRACT
DISSOLVED IN DMSO IN ABSENCE OF S-9 MIX ON SALMONELLA
Treatment
DMSO
Sample A
Particulate
Extract
9-Amino
Acridine
Sodium Azide
u9 x
Plate
0.2ml
0
50
100
200
400
800
10
5
TA1535
19.5
19.5
19.0
14.0
12.5
13.5
12.5
_
1290
TA1537
5.5
5.5
11.5
8.5
7.5
10.5
7.0
>1000
_
TA1538
14.5
15.0
14.5
25.5
28.0
42.0
73.5
_
-
TA100
74.5
74.0
98.0
170.0
219.0
335.0
493.0
_
1020
TA98
34.0
36.0
41.0
75.5
117.5
175.5
276.0
^
-
TABLE IB. MUTAGENICITY OF EPA SAMPLE A DIESEL EXTRACT
DISSOLVED IN DMSO IN PRESENCE OF S-9 MIX (500 L S-9
MIX ON SALMONELLA
Treatment
DMSO
Sample A
Diesel Partic.
Extract
2-Amino
Fluorene
ug x
Plate
0.2ml
0
50
100
200
400
800
5
TA1535
19.5
19.5
18.5
19.5
19.0
18.0
21.5
_
TA1537
5.5
5.5
7.5
6.0
7.0
4.0
4.0
_
TA1538
14.5
15.0
18.5
25.5
42.0
69.5
160.0
530.0
TA100
14.5
74.0
95.5
110.0
119.0
147.0
210.0
435.0
TA98
35.0
36.0
35.0
46.5
62.0
94.0
155.0
765.0
289
-------
TABLE 2A. MUTAGENICITY OF EPA SAMPLE B DIESEL EXTRACT
DISSOLVED IN DMSO IN ABSENCE OF S-9 MIX ON SALMONELLA
Treatment
DMSO
Sample B
Diesel
Extract
Plate
0.2ml
0
50
100
200
400
800
TA1535
18
22
14
15
16
22
26
TA1537
5
5
7
12
25
24
30
TA1538
16
15
40
75
108
133
TOXIC
TA100
79
74
293
706
902
1500
2000
TA98
32
35
144
368
620
877
1042
9-Amino
Acridine
Sodium Azide
10
>1000
1290
1020
TABLE 2B. MUTAGENICITY OF EPA SAMPLE B DIESEL EXTRACT
DISSOLVED IN DMSO IN PRESENCE OF S-9 MIX (500 L S-9
MIX) ON SALMONELLA
Treatment
yg x
Plate
TA1535 TA1537 TA1538 TA100 TA98
DMSO
Sample B
Diesel
Extract
2-Ami no
Fluorene
0.2ml
0
50
100
200
400
800
18
17
23
22
23
54
54
7
11
6
7
13
18
16
15
54
121
270
425
634
520
79
72
161
228
408
641
1100
460
32
35
88
133
352
636
850
730
TABLE 3. MUTAGENICITY OF EPA SAMPLE B DIESEL EXTRACT
DISSOLVED IN DMSO IN THE PRESENCE OF DIFFERENT AMOUNT
OF S-9 MIX ON SALMONELLA
Treatment yg x plate S-9 mix 1 x plate
TA1538
Sample B 400
Diesel 400
Extract 400
250
500
1000
395
416
393
290
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295
-------
TABLE 6A. MUTAGENICITY OF PRF-1 FRACTION FROM PARTICIPATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO. DIESEL
ENGINE ON SALMONELLA WITH AND WITHOUT S-9 MIX
S-9
Treatment Mix
DMSO
PRF
-
_
_
-
-
-
DMSO +
PRF +
+
4
+
+
+
4
plate
0.2ml
5
50
100
250
500
1000
5000
0.2ml
5
50
100
250
500
1000
5000
TA1535
9
5
7
6
6
10
7
8
10
8
12
5
9
5
10
7
TA1537
5
5
3
7
3
9
7
10
7
2
3
3
3
9
6
3
TA1538
16
6
12
6
2
3
10
3
12
7
7
6
5
7
9
8
TA100
154
158
147
131
100
101
111
90
137
130
133
134
147
149
172
165
TA98
43
40
41
31
33
33
36
22
30
36
35
34
37
19
14
34
TABLE 6B. MUTAGENICITY OF ARM-1 FRACTION FROM PARTICULATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO, DIESEL
ENGINE ON SALMONELLA WITH AND WITHOUT S-9 MIX
S-9
Treatment Mix
DMSO
PRF
-
-
-
-
-
-
DMSO +
PRF +
+
4
4
4
4
4
yg
plate
0.2ml
5
50
100
250
500
1000
3000
0.2ml
5
50
100
250
500
1000
3000
TA1535
5
5
9
9,.
r
3
7
9
10
7
17
6
6
8
11
13
TA1537
6
4
10
7
11
6
1
5
8
2
8
4
4
9
8
17
TA1538
9
5
4
15
7
7
6
8
9
9
12
9
17
11
28
30
TA100
139
133
144
145
144
139
144
138
122
136
151
152
148
170
195
200
TA98
23
12
26
20
19
18
20
39
22
16
14
30
34
30
34
57
296
-------
TABLE 6C. MUTAGENICITY OF TRN-1 FRACTION FROM PARTICULAR
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO. DIESEL
ENGINE ON SALMONELLA WITH AND WITHOUT S-9 MIX
Treatment
DMSO
TRN
DMSO
TRN
S-9
Mix
-
+
I
plate
(x)
0.2ml
5
50
100
250
500
1000
0.2ml
5
50
100
250
500
1000
Regression Analysis
TA1537
TA1538
TA100
TA98
-S-9
+S-9
-S-9
+S-9
-S-9
+S-9
-S-9
-S-9
y
y
y
y
y
y
y
y
= 13.
= 46.
= 48.
= 152
= 295
= 513
= 125
= 493
TA1535
(y)
5
6
15
15
12
11
17
10
12
36
54
55
19
3
(TOXIC)
410
169
861
.31
.94
.60
.3 H
.95
+
+
+
+
+
+
h 1
+
0.
1.
0.
7.
3.
6.
TA1537
(y)
6
9
40
50
92
170
180
8
20
190
263
450
232
55
(TOXIC)
31662x
7288x
56534x
364x
859x
9407x
.4230x
3.0942x
TA1538
(y)
9
14
110
166
200
300
620
8
54
738
1060
1884
1600
800
(TOXIC)
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
TA100
(y)
139
207
570
843
1385
2124
2420
122
247
1320
1740
1950
2150
1340
(TOXIC)
TA98
(y)
23
50
190
370
520
975
1460
22
60
430
940
2100
2800
3000
99386***
96722***
98320***
97323***
98356***
84305*
98323***
80213*
297
-------
TABLE 6D. MUTAGENICITY OF OXY-1 FRACTION FROM PARTICULATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO. DIESEL
ENGINE ON SALMONELLA WITH AND WITHOUT S-9 MIX
Treatment
DMSO
OXY
S-9
Mix
-
_
-
_
_
-
_
plate
(x)
0.2ml
5
50
100
250
500
1000
TA1535
(y)
9
10
12
24
22
20
17
(TOXIC)
DMSO
OXY
_
+
+
+
+
+
+
+
+
2000
0.2ml
5
50
100
250
500
1000
2000
10
13
18
25
66
63
36
8
(TOXIC)
TA1537
(y)
5
13
62
112
264
541
291
(TOXIC)
7
8
25
60
175
381
443
(TOXIC)
81
(TOXIC)
TA1538 TA100
(y)
16
24
150
330
661
1052
1020
(y)
154
161
431
630
1010
1500
1770
(TOXIC)
12
21
125
300
762
1860
1130
491
685
(TOXIC)
137
134
332
490
880
1284
2020
1820
(TOXIC)
TA98
(y)
43
63
403
830
1680
2120
2350
(TOXIC)
30
41
228
504
1600
3200
4120
3600
(TOXIC)
Regression Analysis
TA1537
TA1538
TA100
TA98
-S-9
+S-9
-S-9
+S-9
-S-9
+S-9
-S-9
+S-9
y = 5.7616
y = 4.7539
y = 56.
084
+ 1.
+ 0.
+ 2.
y = 0.40559 + 3
y = 246
y = 193
y = 430
y = 438
.25
.19
.36
.38
+ 2.
4 2.
+ 2.
+ 1.
0635x
75638X
0956x
.6722x
6613x
3181x
3499x
9597x
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
r = 0.
99980***
99748***
98993***
99548***
98167***
98595***
89859*
96698***
298
-------
TABLE 7A. MUTAGENICITY OF PRF-1 FRACTION FROM PARTICIPATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ENGINE ON A FORWARD MUTATIONAL YEAST ASSAY
(S. POMBE) WITH AND WITHOUT $-9 MIX
Treatment
Control
PRF
Control
PRF
S-9
mg/ml Mix
0
0.47 -
0.94 -
1.88 -
0 +
0.47 +
0.94 +
1.88 +
No. Mutants: Mutations
No. Colonies Frequency xlO"
2: 5500
1:27027
1:15150
2: 4761
1:15870
1:27030
1:13510
1:17240
0.36
0.37
0.66
0.42
0.63
0.37
0.74
0.58
Survival
4 xlO-2
100
71
70
79
100
83
60
76
TABLE 7B. MUTAGENICITY OF ARM-1 FRACTION FROM PARTICULATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ENGINE ON A FORWARD MUTATIONAL YEAST ASSAY
(S. POMBE) WITH AND WITHOUT S-9 MIX
Treatment
Control
ARM
Control
ARM
S-9
mg/ml Mix
0
1.2 -
2.4 -
4.9 -
0 +
1.2 +
2.4 +
4.9 +
No. Mutants: Mutations
No. Colonies Frequency xlO"
4:28200
5:21675
7:23625
3:30750
2:20550
4:30750
6:26700
7:25275
1.41
2.30
2.96
0.97
0.97
1.30
2.24
2.76
Survival
4 xlO-2
100
77
83
100
100
100
100
100
299
-------
TABLE 7C. MUTAGENICITY OF TRN-1 FRACTION FROM PARTICULATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ENGINE ON A FORWARD MUTATIONAL YEAST ASSAY
(5. POMBE) WITH AND WITHOUT S-9 MIX
S-9 No. Mutants: Mutation Survival
Treatment mg/ml Mix No. Colonies Frequency xlO"^
Control
TRN
Control
TRN
0
0.5
1.0
2.0
0
0.5
1.0
2.0
6:27675
7:22725
5:12600
5:32400
2: 7350
3:36675
13:26025
5:37725
8:17640
3:85760
10:28275
8:47100
2:13050
2:18075
5:23475
4:47925
2.62
3.54
1.76
3.15
2.44
2.61
1.27
1.47
100
63
60
89
100
79
41
68
300
-------
TABLE 7D. MUTAGENICITY OF OXY-1 FRACTION FROM PARTICIPATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ENGINE ON A FORWARD MUTATIONAL YEAST ASSAY
(S. POMBE) WITH AND WITHOUT S-9 MIX
Treatment
Control
OXY
Control
OXY
mg/ml S-9
(x) Mix
0
0.5 -
1.0 -
1.5 -
2.0 -
0 +
0.5 +
1.0 +
1.5 +
2.0 +
No. Mutants:
No. Colonies
5:33525
6:27675
10:48300
10:40500
11:12900
2: 5775
2: 3244
TOXIC
2:25425
8:17640
9:61800
11:34950
3:37950
5:18300
6:29550
5:26040
Mutation
Frequency xlO~^
(x)
1.82
2.88
6.00
6.16
2.65
2.29
1.76
1.69
1.92
Survival
xlO-2
100
100
29
10
TOXIC
100
100
92
100
100
Regression Analysis:
y = 2.19 + 0.75x
r2 = 0.99315***
301
-------
TABLE 8A. MUTAGENICITY OF PRF-1 FRACTION FROM PARTICULATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ON A MITOTIC GENE CONVERSION (S. CEREVISIAE: 04)
WITH AND WITHOUT S-9 MIX
Treatment
Control
PRF
Control
PRF
mg/ml
0
1.2
2.4
4.9
0
1.2
2.4
4.9
Mitotic Gene
S-9 Frequency
Mix ADE?
1.75
1.61
1.43
1.19
+ 1.24
+ 1.39
+ 0.82
+ 1.15
Conversion
xlO-5
TRPc,
1.25
1.24
1.40
1.13
0.69
0.99
0.58
1.19
Survival
xlO-2
100
100
100
100
100
82
100
91
TABLE 8B. MUTAGENICITY OF ARM-1 FRACTION FROM PARTICULATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ON A MITOTIC GENE CONVERSION (S. CEREVISIAE: D4)
WITH AND WITHOUT S-9 MIX
Treatment
Control
ARM
Control
ARM
mg/ml
0
0.47
0.94
1.88
0
0.47
0.94
1.88
Mitotic Gene
S-9 Frequency
Mix ADE?
1.75
1.62
1.32
1.38
+ 1.24
+ 1.23
+ 1.31
+ 1.02
Conversion
xlO~5
TRPc;
1.25
0.90
0.94
0.84
0.69
0.80
0.62
0.50
Survival
xlO-2
100
100
100
100
100
86
82
90
302
-------
TABLE 8C. MUTAGENICITY OF TRN-1 FRACTION FROM PARTICIPATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ON A MITOTIC GENE CONVERSION (S. CEREVISIAE: D4)
WITH AND WITHOUT S-9 MIX
Treatment
Control
TRN
Control
TRN
mg/ml
0
0.5
1.0
2.0
0
0.5
1.0
2.0
Mitotic Gene
S-9 Frequency
Mix ADE?
2.51
2.64
1.37
2.11
+ 0.82
+ 2.11
+ 1.32
+ 2.42
Conversion
xlO'5
TRPc;
1.74
1.55
1.47
1.38
0.85
0.93
1.05
1.23
Survival
xlO-2
100
100
100
88
100
70
77
73
TABLE 8D. MUTAGENICITY OF OXY-1 FRACTION FROM PARTICULATE
EXTRACTS OF A NATURALLY ASPIRATED SWIRL CHAMBER EURO.
DIESEL ON A MITOTIC GENE CONVERSION (S. CEREVISIAE: D4)
WITH AND WITHOUT S-9 MIX
Treatment
Control
OXY
Control
OXY
mg/ml S-9
(x) Mix
0
0.5
1.0
1.5
0 +
0.5 +
1.0 +
1.5 +
Mitotic Gene Conversion
Frequency xlO"5
ADE?
2.51
1.76
1.76
3.10
0.82
0.84
1.03
2.67
(y) TRPR
1.74
1.83
3.38
3.45
0.85
0.87
0.93
1.76
Survival
xlO-2
100
83
70
34
100
97
100
56
Regression analysis:
y = 1.5980 + 1.336X
r = 0.91526*** (trp 5)
303
-------
TABLE 9. IN VITRO MUTAGENICITY ON SALMONELLA TA98 OF
PARTICLES COLLECTED FROM A NATURALLY ASPIRATED SWIRL CHAMBER
EURO. DIESEL ENGINE
Dose
mg/plate
0
250
500
1000
2000
No. Revert ants
- S-9 Mix
(y)
28.6 +_ 4.0
186.3 i 13.0
292. 3 _+ 3.2
398.6 +_ 19.3
714.6 ± 27.5
per plate (+S.E.)
-i- S-9 Mix
(y)
45. 3 ^ 7.4
153.6 + 4.9
248.0 _+ 24.8
386.0 i 32.1
698.0 ± 22.1
Regression analysis:
- S-9 y = 82.475 + 0.3223 x r = 0.9882**
-i- S-9 y = 67.325 + 0.3185 x r = 0.99794**
304
-------
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2. Ames, B. N., J. McCann, and E. Yamasaki. Methods for
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mammalian-microsome mutagenicity test. Mutation Res.
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Congress and Exposition, Detroit, Feburary 26-March 2,
1979.
5. Chrisp, C. E., G. L. Fisher, J. E. Lammert. Mutage-
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Science, 199, 73-75, 1978.
6. Cleaver, J. E. Methods for studying excision repair of
DNA damaged by physical and chemical mutagens. In B.
J. Kilbey: Handbook of mutagenicity test procedures.
Elsevier, Amsterdam, 19-48, 1977.
7. Crow, J. F. and M. Kimura. An introduction to popula-
tion genetics theory. Harper & Row, New York, p. 7,
p. 486-493, 1970.
8. DiLorenzo, A., R. Barbella, G. M. Cornetti, G. Biaggini.
A rapid chemical characterization of diesel particulates
by thermogravimetric and mass spectrometric analysis.
EPA, International Symposium on health effects of
diesel engine emissions. Cincinnati, December 3-5,
1979.
9. Fahrig, R. Host mediated mutagenicity tests. Yeast
systems. Recovery of yeast cells out of testes, liver,
lung, and peritoneum of rats. In B. J. Kilbey:
Handbook of mutagenicity test procedures. Elsevier,
Amsterdam, 135-147, 1977.
10. Gutz, H., H. Heslot, U. Leupold, and N. Loprieno.
Schizosaccharomyces pombe. In R. C. King: Handbook of
Genetics. Plenum Press, New York, _!, 395-446, 1974.
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11. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R.
Zweidinger, S. Tejada, J. Bumgarner, F. Duffield, M.
Waters, V. F. Simmon, C. Hare, C. Rodriguez, L. Snow.
Application of Bioassay to the characterization of
diesel particle emissions. In "Application of short-
term bioassays in the fractionation and analysis of
complex environmental mixtures" EPA-600/9-78-027, 32
pp, 1978.
12. Kubitschek, H. E. and Luz Venta. Mutagenicity of coal
fly ash from electric power plant precipitators..
Environmental Mutagenesis JU 79-82, 1979.
13. Legator, M. S., Lantruong, and T. H. Connor. Analysis
of body fluids including alkylation of macromolecules
for detection of mutagenic agents. In A. Hollaender
and F. J. DeSerres: Chemical mutagens. Principles and
methods for their detection. Plenum Press, New York,
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14. Lofroth, G. Genotoxic compounds in the oxidation fuel
cycles. Present status of combustion emissions.
Presented at the "Research coordinated meeting of the
International Atomic Energy's Coordinated Programme on
Comparative Biological Hazards from Low-Level Radiation
and Energy-related Chemical Pollutants. Leiden, 27-29
August 1979.
15. Loprieno, N. The use of yeast cells in the mutagenic
analysis of chemical carcinogens. Colloques interna-
tionaux du C.N.R.S. 256, 315-331, 1977.
16. Loprieno, N. Use of yeast as an assay system for
industrial mutagens. In A. Hollaender and F. J.
DeSerres: Chemical mutagens. Principles and methods
for their detection. Plenum Press, New York, _5, 25-53,
1978.
17. Loprieno, N. Mutagenicity tests for evaluating chemi-
cal substances. ETS, Pisa, 1-148, 1978.
18. Loprieno, N. Screening of coded carcinogenic/noncar-
cinogenic chemicals by a forward mutation system with
the yeast Schizosaccharomyces ppmbe. International
Program for the evaluation of short-term tests for
carcinogenicity (ICI/MRC/NIEHS), 1979, 16 pp.
19. Martin, C. N., A. C. McDermid, and R. C. Garner.
Testing of known carcinogens and non carcinogens for
their ability to induce unscheduled DNA synthesis in
HeLa cells. Cancer Res. 38, 2621-2627, 1978.
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20. McCann, J., E. Choi, E. Yamasaki, and B. N. Ames.
Detection of carcinogens as mutagens in the Salmonella/
microsome test: Assay of 300 chemicals. Part 1.
Proc. Natl. Acad. Sci. (USA) 72, 5135-5139, 1975.
21. Matsushima, T., M. Sawamura, K. Hara, and T. Sugimura.
A safe substitute for polychlorinated biphenyls as an
inducer of metabolic activation system. In F. J.
DeSerres et al.: In vitro metabolic activation in
mutagenesis testing. Elsevier, Amsterdam, 85-88,
1976.
22. Poirier, L. A., and F. J. DeSerres. Initial National
Cancer Institute studies on mutagenesis as a prescreen
for chemical carcinogens: an appraisal. J. Natl.
Cancer Inst. 62, 919-926, 1979.
23. Purchase, I. F. H., E. Longstaff, J. Ashby, J. A.
Styles, D. Anderson, P. A. Lefevre and F. R. Westwood.
An evaluation of 6 short-term tests for detecting
organic chemical carcinogens. Br. J. Cancer, 37,
873-903, 1978. ~~
24. Rinkus, S. J. and M. S. Legator. Chemical characteri-
zation of 465 known or suspected carcinogens and their
correlation with mutagenic activity in the Salmonella.
Cancer Res. 39, 3289-3318, 1979.
25. Sugimura, T., S. Sato, M. Nagao, T. Yahagi, T. Matsu-
shima, Y. Seine, M. Takeuchi, and T. Kawachi. Overlap-
ping of carcinogens and mutagens. In "Fundamentals in
Cancer Prevention". Ed. by P. N. Magee et al., Univer-
sity of Tokio Press, 191-215, 1976.
26. Application of a battery of confirmatory carcinogenesis/
mutagenesis bioassays to the evaluation of mobile
source and related emissions. Accomplishment Report
Sept. 1979. EPA.
General Discussion
A. KOLBER: I notice as I extrapolate your data that
for this fraction you get between two and three revertents
per microgram, which is the kind of mutagenicity quantifi-
cation that a lot of people get. I noticed from Dr. Chan's
talk from General Motors, they obtained as much as 40
revertents per microgram in their neutral polar fraction.
I wonder if either one of you would like to comment about
the quantification. Why is one sample so highly mutagenic
and another rather low? Is there any reason for this dif-
ference in quantification?
307
-------
N. LOPRIENO: Our data shows an induction of mutation.
I showed that one of the fractions was completely different
from the other, but I don't really have an explanation.
J. SIAK: I will reply to the question. We follow all
the activity we put in and then calculate all the activity
from each fraction. We get around 85 to 90 percent of the
activity we put into it before the fractionation scheme.
N. LOPRIENO: We have not completed all the work with
regards to the total extract.
J. SIAK: The discrepancy may be due to the procedures
used in your activation process. We avoid light and we
don't expose to high temperature. We try to evaporate
under pressure, or under vacuum, and try not to use diethyl
ether. So I think for those doing fractionations they
should consider those procedures to avoid the loss of ac-
tivity.
N. LOPRIENO: As I stated, our first aim was to develop
a small data set from a different system on just the one
sample, and then the next step was to do very quantitative
studies in order to correlate to the mutagenic potential
with different test systems. The numerical data you saw
are only that way we use to evaluate the positivity of
themselves by the regulation analysis.
J. HUISINGH: Do you have any data yet on the V79 cell
line?
N. LOPRIENO: No, I do not yet.
308
-------
THE MUTAGENICITY OF DIESEL EXHAUST
EXPOSED TO SMOG CHAMBER CONDITIONS
AS SHOWN BY SALMONELLA TYPHIMURIUM
Larry Claxton
Genetic Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Ayency
Research Triangle Park, North Carolina 27711
H. M. Barnes
Environmental Sciences Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
ABSTRACT
Since previous work demonstrating the mutagenicity of
particle-associated organics was performed using particles
collected with laboratory dilution tunnels, this study
explored the significance of ambient conditions through the
use of a smog chamber. Diesel particles exposed within the
Calspan Smog Chamber Facilities, Ashford, New York, were
collected on Palfex T60A20 glass fiber filters, extracted in
dichloromethane, solvent exchanged to dimethylsulfoxide and
tested in the Ames Salmonella typhimurium plate incorpora-
tion assay. It was demonstrated that the mutagenic response
of these exhaust organics depends upon the method of collec-
tion and is altered by certain ambient conditions. This
study clearly demonstrates that the level of mutagenic
activity is influenced by the presence of ozone. This study
also demonstrated that the UV irradiation of a simple
mixture of propylene, NO, N02, and S02 produced muta-
genic moieties. Future experiments will continue to examine
factors within ambient air that modify mutagenic activity.
309
-------
INTRODUCTION
Most studies with diesel exhaust have not described what
effects exposure to ambient-Tike conditions would have upon
particle-associated organics, since previous studies have
used dilution-tunnel methods for the collection of exhaust
particles (1). This study exposed diesel exhaust to specific
ambient-like conditions through the use of a smog chamber.
The effect of these ambient conditions was measured by
testing the particle-associated organics in the Salmonella
typhimurium plate incorporation assay.
This study describes the generation, smog chamber exposure,
collection, and bioassay of exhaust organics from a light
duty diesel vehicle. The simulation of anticipated ambient
air conditions was achieved by varying the conditions within
the smog chamber. The Salmonella typhimurium plate incorpora-
tion assay as developed by Dr. Bruce Ames was used to evaluate
the particle-bound organics.
MATERIALS AND METHODS
THE SALMONELLA BIOASSAY
The Salmonella typhimurium plate incorporation assay described
by Ames et al. (2) was used to assay the diesel organics.
Five histTdTne-dependent strains (TA98, TA100, TA1537,
TA1538, and TA1535) were obtained from Dr. Bruce Ames,
University of California, Berkeley. The methods used followed
the Ames protocol (2) in view of the suggestions of the
Washington conference on the Ames assay (3). Solvent and
positive controls done in triplicate were conducted with
each test. When possible, the sample was assayed at five
doses applied in triplicate. Samples which are reported
together were run simultaneously within the cited strain.
The mutagenic extracts were tested in the presence and
absence of S-9 from Aroclor 1254-induced Charles River CD1
rats. The S-9 fraction was prepared according to the method
of Ames (2).
THE SAMPLES
A Mercedes Benz 240D four cylinder diesel automobile run on
an engine dynamometer using the Highway Fuel Economy Test
driving cycle (HWFET) was used to generate the exhaust
exposed to smog chamber conditions. The samples were exposed
310
-------
in the 590 m3 Calspan Smog Chamber facilities, Ashford, New
York. Irradiation was provided by 24 lighting modules
arranged in eight vertical chambers. Each array contained
two 40-watt sunlamps, eight 85-watt black lamps, and two
215-watt black lamps. The parameters which were varied
within the experimental procedure included: (1) presence or
absence of UV irradiation, (2) sampling location, (3) length
of chamber exposure, and (4) presence of ozone. A summary
of the samples is contained in Table 1. Sample 18 was a
baseline study in which propylene, S02, NO, and N02 were
injected into the smog chamber without diesel exhaust and
irradiated. Samples 20 and 21 were generated within the
same experiment. Sample 20 was an immediate sampling of the
exhaust particles while sample 21 had a 4 hour dark exposure.
Samples 29 and 30 were generated in the same manner as
samples 20 and 21; however, sample 30 had a 6 hour UV
exposure. Sample 22 was a non-irradiated exposure in which
ozone was artifically added to the chamber. The tailpipe
sample (24) was collected directly from the automobile
tailpipe at near exhaust temperatures. The exhaust gases
and particles that passed through the tailpipe filter entered
the non-irradiated smog chamber and were collected as sample
23.
After the particles were collected on Pellflex T60A20 glass
fiber filters, the filters were folded, sealed in wax paper,
placed in a manilla envelope, and stored at refrigerator
temperatures. The mutagenic activity of diesel particles
stored under these conditions has been shown to be stable
(1). Extraction of the particles was with dichloromethane
in a soxhlet apparatus. The solvent extract was blown to
dryness and solvent exchanged into dimethylsulfoxide.
311
-------
Exp
No.
1
2
3
4
5
6
7
Sample
Source:Variable
Prop/SO
Diesel
Diesel
Diesel.
Diesel.
Diesel
Diesel-
Diesel.
Diesel:
Diesel
2/NOx:Baseline
Basel ine
Baseline
Baseline
Baseline
Injected 0,
Filtered
Tailpipe
Tunnel (D-l)
Tunnel (D-2)
TABLE 1
Sample
Number
0018
0020
0021
0029
0030
0022
C023
0024
0504
0505
EXPERIMENTAL CONDITIONS
Exp
Proc a
-
U
U
U
U
C4
F
T
X
X
Irrad
I
No
No
I
I
No
No
No
No
No
Gases (ppm) Prop Exposure
Spike Time
0,max (ppmC) (hrs)
* 30 60
< 001 - 05
< 001 - 60
* - 0 5
* - 6 0
0.65 - 6 0
* 0 5
-
-
-
Experimental Procedures U - Emissions injected directly into chamber without filtering
or diluting, C = CVS dilution used, F = Emissions filtered at tailpipe and collected from
chamber, T ~ Emissions filtered and collected at tailpipe, X = Laboratory dilution tunnel
studies
Initial concentration only
Initial propane spike
*
Equipment failure
312
-------
RESULTS
In a baseline study, an artificial mixture of 3.0 ppm
propylene, 0.52 ppm S02, 0.20 ppm NO, and 0.22 ppm N0£
were added to the smog chamber without diesel exhaust. Upon
irradiation, this mixture generated background aerosols that
demonstrated mutagenicity (Figure 1). In order to assess
the effect of dark exposure, two samples were collected in
the second experiment. The first sample (sample 20) was
collected immediately after the diesel exhaust was put into
the chamber, and the remaining particles were collected
(sample 21) after a 4 hour dark exposure. The third experi-
ment also provided two samples (samples 29 and 30) in the
same manner, however, the 4 hour dark exposure was replaced
with a 6 hour UV irradiation. Similar mutagenic responses
(Figure 2) were obtained from these dark and irradiated
exhaust samples collected both immediately and after an
extended period.
A comparison of the various non-irradiated exposures demon-
strates that samples collected at the automobile's tailpipe
are significantly different from other non-irradiated
samples (Figure 3). within the linear portion of the dose
response curve, the revertants per plate for the tailpipe
sample are approximately double that of other non-irradiated
samples. Artificially added ozone produced a marked decrease
in mutagenic activity of the exhaust organics (Figure 4).
Ozone also decreased the bactericidal activity of the
exhaust organics. Figure 5 shows a comparison of bioassay
results between the smog chamber test vehicle and two
dilution samples from a Mercedes 300D automobile. There is
a highly significant difference between these two samples.
Table 2 provides the mean plate counts at each dose for the
reported samples.
313
-------
Figure 1. Mutagenic response (revertants per plate vs.
micrograms of organic extract) of aerosol organics produced
upon irradiation of propylene, NO, N02, and S02.
BACKGROUND AEROSOLS AS DETECTED IN 3 STRAINS
500
WITH S9
500
400
300
200
100
WITHOUTS9
100 200
100 200
Footnote: (A, TA100; , TA98; , TA1537)
314
-------
Figure 2. Mutagenicity of irradiated and non-irradiated
diesel exhaust organics collected immediately after gener-
ation.
IMMEDIATE, DARK, AND IRRADIATED EXPOSURES
TA98
WITH S9
WITHOUT S9
0 200 400 600
UGS ORGANIC
DOSE PER PLATE
200 400 600
UGS ORGANIC
(A, Sample 21; B, Sample 29; C, Sample 20; D, Sample 30)
315
-------
Figure 3. Responses of tail pipe (T-P), immediate (I), and
four hour smog chamber (4-HR) exposures of diesel exhaust
organic in Salmonella typhimurium TA98.
TAIL-PIPE, IMMEDIATE AND FOUR HOUR EXPOSURES
WITH S-9
TA98
WITHOUT S-9
UJ
0.
CC
UJ
o.
i
<
QC
UJ
0 200 400 600
UGS. ORGANIC
0 200 400 600
UGS. ORGANIC
DOSE PER PLATE
316
-------
Figure 4. Mutagenicity of diesel exhaust organics exposed
in a smog chamber to varying levels of ozone.
EFFECT OF OZONE
WITH S-9
WITHOUT S-9
Q-
t£
UJ
Q.
CO
UJ
CC
0 200 400 600
UGS. PER PLATE
0 200 400 600
UGS. OF ORGANIC
DOSE PER PLATE
Peak levels of ozone: 21, < .001 ppm; 22, 0.65 ppm (injected);
25, 0.012 ppm; 26, 0.24 ppm; 27, 0.54 ppm.
317
-------
Figure 5. A typical diesel exhaust organic collected from a
dilution tunnel compared to a typical smog chamber sample.
COMPARISON OF DILUTION TUNNEL AND
SMOG CHAMBER SAMPLES
TA98
WITHS-9 WITHOUT S-9
Q-
OC
LU
O.
GO
z
LU
cc
200 400 600
200 400 600
UGS. ORGANIC DOSE PER PLATE
S, Smog chamber sample
D-l and D-2, Dilution tunnel samples
318
-------
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DISCUSSION
Previous studies had shown that extracts from diesel exhaust
particles are mutagenic when tested in the Salmonella typhi-
murium plate incorporation assay. This study simulated some
of the modifying conditions found in an ambient atmosphere
containing diesel exhaust. The associated chemistry and
engineering procedures are reported more fully elsewhere
(4). This study clearly indicates that some mutagenic
products are formed by the interaction of a simple hydrocar-
bon propylene, S02, NO, N02 gases, and UV irradiation.
Earlier studies showed that the spontaneous interaction of
simple mixture produces a large variety of organics and five
aerosols (5,6). Up to 35% of the total activity of any of
the irradiated smog chamber samples could be attributed to
this interaction. As shown in Figure 2, the recovery of
total mutagenic activity is the same under dark and UV light
conditions unless some other mitigating factor, such as
ozone, is present. Compared to the remaining non-irradiated
smog chamber samples, the tailpipe sample had a marked
increase in mutagenicity. This study demonstrates that the
response of any exhaust sample, and therefore the substances
providing that response, varies markedly with the method of
sampling. Various parameters, including collection temper-
ature, concentration of exhaust gases, and rates of chemical
interaction, could account for the variation with sampling
methods. One factor that was clearly shown to modify the
mutagenic activity of particle-bound organics was ozone. As
shown in Figure 4, ozone can reduce the mutagenic activity
as detected by the Ames test; however, since differing
levels of ozone produce differing levels of oxidation and
chemical interaction, this study does not demonstrate that
all levels of ozone will reduce mutagenic activity. Unfor-
tunately the vehicle used in the smog chamber studies has
not, to date, been used in dilution tunnel studies so that
direct comparisons can be made between smog chamber and
dilution tunnel samples. The closest comparison available
is between the smog chamber vehicle and a Mercedes 300D
vehicle run with dilution tunnel techniques. The smog
chamber sample used for comparison was from a non-irradi-
ated, imediate (0.5 hour) collection. The response between
these two samples was clearly different, with the smog
chamber sample more mutagenic than the dilution tunnel
sample.
322
-------
In summary, the mutagenic response associated with exhaust
particulate organics depends upon the ambient conditions
as well as upon the methods of generation and collection.
The complex aerosol mixture produced by the interaction of
propylene, NO, N02, SO?, and irradiation was shown to
be mutagenic. Although UV irradiation without other
mitigating factors did not alter the mutagenic activity of
the collected organics, ozone was shown to alter the type
and level of mutagenic compounds detected by the Ames
test. Future experiments will continue exploring the
effect of ambient conditions on mobile source emissions.
REFERENCES
1. Huisiflgh, J. _et^ aj_. Application of Bioassay to the
Characterization of Diesel Particle Emissions. In:
Application of Short-term Bioassays in the Fractionation
and Analysis of Complex Environmental Mixtures, M.
Waters £t ^1_., eds. Plenum Press, New York, 1979.
2. Ames, B. N., J. McCann, and E. Yamasaki. Methods for
Detecting Carcinogens and Mutagens with the Salmonella/
Mammalian-Microsome Mutagenicity Test. Mutation Res.,
31:347-364, 1975.
3. de Serres, F. J., and M. D. Shelby. The Salmonella
Mutagenicity Assay: Recommendations. Science,
203:563-565, 1979.
4. Anderson, R. J. A Smog Chamber Study of Aerosol Forma-
tion and Growth Involving S02 and Diesel Exhaust.
Monthly Progress Report No. 10, U.S. Environmental
Protection Agency, Research Triangle Park, North
Carolina. 13 pp.
5. McNelis, D. N. Aerosol Formation from the Gas Phase
Reaction of Ozone and Olefin in the Presence of Sulfur
Dioxide. EPA-650/4-74-034, U.S. Environmental Protec-
tion Agency, Research Triangle Park, North Carolina,
1974.
6. Akimota, H. j^t a±. Formulation of Propylene Glycol-1,2-
Dinitrate in the Photooxidation of Propylene-Nitrogen
Oxides-Air Systems. J. Environ. Sci. and Engr.,
A13:677-687, 1978.
323
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General Discussion
J. DAISEY: Did you say how much ozone you put in the
chamber?
L. CLAXTON: No, I didn't, but I can tell you; 0.64
parts per million.
R. KLIMISCH: Did you ever get a case where ozone in-
creased the mutagenicity?
L. CLAXTON: No, we have not at this point.
R. KLIMISCH: Why do you say altered; why don't you say
reduced?
L. CLAXTON: We haven't tested a variety of levels of
ozone. We have only tested one level. I suspect that
other things can happen. You may have seen on the slide
what appeared as a variety of levels, but those were
levels of generated ozone with other mitigating factors,
such as different levels of $62 and different levels of
added reactive hydrocarbon.
P. SCHULTZ: How do you run a blank sample through an
Ames Test?
L. CLAXTON: What do you mean by blank sample?
P. SCHULTZ: A sample without the test material.
L. CLAXTON: We always run a series of controls and our
controls are always tested with the solvent but without the
compound. We have run some compounds that are inert in
Ames Tests, as well as in this system.
P. SCHULTZ: Did you have an absolute zero on those?
L. CLAXTON: You always have a level of spontaneous
mutation that is recordable.
P. SCHULTZ: Is that substracted from these results?
L. CLAXTON: No, it is not substracted from these re-
sults. You can look on the Y axis and see the point of
intersection on the Y axis and the level spontaneous.
J. HUISINGH: In the original study that we reported
several years ago, the*samples that were collected at South-
west Research Institute, blank filters were also run through
the total extraction system and were found to essentially
contribute nothing to the mutagenic activity. It takes a
lot of effort to always run blank samples, and we don't
always do that. We have done a number of studies where
blank filters were completely extracted and also fuel was
extracted and fractionated and we found no activity from
those studies.
L. CLAXTON: Probably the best example is actually the
fuel sample, which was put on the filters and run through a
portion of the fractionation scheme and then through the
Ames Test in the same way as the exhaust organics.
There was no activity there. It was all at spontaneous
levels.
324
-------
W. THILLY: I thought it was an interesting technical
feat to count thousands of colonies on a plate. Are these
plates different from ordinary bacterial plates?
L. CLAXTON: The highest count is about 2,000, and we
used an automatic colony counter.
W. THILLY: Does the colony counter saturate at around
2,000 counts per plate?
L. CLAXTON: No, but we cannot really go above 2,300
under the conditions in which we are testing colonies per
plate. After 2,000 we record too numerous to count.
W. THILLY: Under your conditions of assay, what as-
surance can you give us that your various samples did not
change those numbers of colonies observed as a result of
killing the bacteria as opposed to mutating them?
L. CLAXTON: We go back into each of our tests after-
wards and take out a number of bacteria that is at least
equal and sometimes double to the number of spontaneous
bacteria that would occur and we plate each of those col-
onies out separately to make sure that they are revertents
and true mutants.
W. THILLY: That is nonresponsive. Could you answer the
question with regard to the number, not their quality of
being true mutants.
L. CLAXTON: Are you asking how do you correct for tox-
icity?
W. THILLY: Quite.
L. CLAXTON: This is a problem in the Ames Test per se
in this type of protocol. I realize as you do that you can
have killing of almost 95 percent or even more and still
have a recoverable number of mutants that is even this
high. We have developed, and are working with statis-
ticians to model the results of an Ames Testing and a paper
will be published on the subject.
R. YASBIN: We have done the toxicity testing, and there
is essentially no more than 90 percent killing with the
various extracts that we have gotten from you. Toxicity
does not appear to be a problem.
L. CLAXTON: Yes, sometimes when you are testing very
toxic compounds, if you get into a high enough level, the
number of revertents will drop off in the higher portion of
the curve. We generally see very little of that with these
diesel samples.
T. BAINES: What was the magnitude of the difference
between the tailpipe filter that you took and the results
that you normally get from vehicles of that type when you
sample them in a dilution tunnel? Would you care to specu-
late on what you might find when you are able to test that
vehicle on a dilution tunnel.
L. CLAXTON: With the filter immediately at the tailpipe
sample, it is two to threefold higher for the same type of
325
-------
automobile as compared to most of the dilution tunnels that
we have seen. I suspect that if we were able to test the
same automobile on a dilution tunnel apparatus, it would be
in line with most others.
T. BAINES: What do you think is the difference; what
would account for that two to threefold difference?
L. CLAXTON: I don't know. You would need some chemists
to help on that.
T. GIBSON: Did you compare diesel particulate exposed
to NOX with diesel particulate exposed to just the other
things like S02?
L. CLAXTON: To NOX alone, as compared to the others?
T. GIBSON: Well no, exposed to the mixture, including
NOX, and the mixture for any conditions not including NOX.
L. CLAXTON: No, generally there is always NOX from the
automobile itself within the mixture.
T. GIBSON: There is no way to do that?
326
-------
SALMONELLA/MICROSQME MUTAGENICITY ASSAYS OF EXHAUST
FROM DIESEL AND GASOLINE POWERED MOTOR VEHICLES
Goran Lbfroth
Radiobiology Department, University of Stockholm,
Wallenberg Laboratory, S-106 91 Stockholm, Sweden
ABSTRACT
Motor vehicle exhaust from prechamber injection diesel and
gasoline powered passenger cars, sampled during US FTP 1973
test cycles and comprising both particulate matter and com-
pounds condensable at ambient temperature, has been assayed
for mutagenicity in the Salmonella/microsome test.
Mutagem'c components were to a large extent active in the
absence of the mammalian microsomal preparation. The muta-
genicity of both particulate matter and condensate from
diesel exhaust and condensate from gasoline exhaust was de-
creased in the presence of the microsomal preparation
whereas the mutagenicity of particulate matter from gaso-
line exhaust was enhanced by microsomal activation.
A comparison between the investigated diesel and gasoline
exhaust samples shows that the mutagenic effect in the Sal-
monella test of the diesel exhaust is more than ten times
higher than that of the gasoline exhaust.
Fractionation with respect to polarity indicates that the
mutagenic components mainly are distributed in neutral ali-
phatic, aromatic and oxygenated fractions.
Tests for mutagenic monofunctional nitroarenes by an anaero-
bic assay indicate that such compounds at most are margin-
ally present in the exhaust samples as compared with their
presence in airborne particulate matter collected in an
urban environment.
327
-------
INTRODUCTION
Combustion of carbon containing fuels for energy production
invariably gives rise to the formation and emission of com-
pounds with potential health effects. In a situation when
technological advances as well as administrative and polit-
ical decisions in some countries have resulted in gross
reduction of possible acute and other short-term effects
from air pollution, public, political and scientific inter-
ests have to some extent focused on long-term consequencies
including genotoxic effects, jL£. mutagenic, carcinogenic
and allied effects.
An advanced society with foresight may neither wish to al-
low practices which cause cancer and other somatic genotox-
ic effects nor want to leave a legacy of an increased muta-
tion rate to future generations. Although combustion emis-
sions are variable and complex, the evaluation of their
potential genotoxic effects are not impossible (1).
One of several tools available for the examination of geno-
toxic effects of combustion emissions is the Salmonella/-
microsome test for mutagenicity (2). This screening method
is particularly valuable in studies of complex mixtures
containing a multitude of compounds not yet available for
identification and quantification by chemical analyses.
Motor vehicle exhausts which have been assayed for mutagen-
icity by the Salmonella test in the present investigation
are examples of such complex mixtures.
MATERIALS AND METHODS
SAMPLING
Sampling of exhaust gases and conventional exhaust gas ana-
lyses were performed by the staff at the Vehicle Exhaust
Gas Laboratory of the National Swedish Environment Protec-
tion Board. The sampling technique, primarily created for
the determination of polycyclic aromatic hydrocarbon (PAH)
emission factors, has been described elsewhere (3, 4). The
technique is a system in which part of the exhaust gas pro-
portional to the total gas flow is removed and introduced
into a sampling train. The sampled exhaust, about 9 % for
gasoline and about 5 % for diesel fuel, passes through a
cooled glass condenser and the formed aqueous condensate is
collected in a glass flask. The cooled gases, at 25-50 °C,
then passes through a glass fiber filter (Gelman A/E) for
the collection of particulate matter. At the end of the
328
-------
sampling period the glass condenser and the flask are
washed with acetone and the acetone solution is added to
the condensate. All samples were stored dark at refrigera-
tor temperature during the first few days after collection
and then at -20 °C until they were processed.
Sampling was performed during complete US FTP 1973 test
cycles with warm engines.
Used crankcase oil was obtained either by removing a small
amount through the oil dipstick hole immediately after that
the engine was turned off or by sampling when the motor oil
was changed.
MOTOR VEHICLES
Exploratory studies were performed with exhaust samples
from different cars which became available as excess sam-
ples in studies (3, 4) of PAH emission factors.
The present investigation concerns two different motor ve-
hicles equipped with standard engines. The gasoline powered
car was a Volvo 245L of 1976 which was fueled with refer-
ence fuel ERF 61. The diesel powered car was a Peugot 504
of 1978 with prechamber injection which was fueled with
commercial diesel oil. Both cars conform with the Swedish
exhaust ordinance about CO, hydrocarbons and NOX for 1976
and later models which is akin to the US federal regula-
tions for 1973 models. In Table 1 are given results of the
conventional exhaust analyses expressed as averages of
three test cycles.
Table 1. Motor vehicles and exhaust analyses.
Fuel
Motor vehicle
US FTP 1973 test, no.
Fuel consumption,
g / test cycle
CO, g/ test cycle
HC, g/ test cycle
NOX , g N02/ test cycle
Diesel
Peugot 504
9017- 19
820
8.1
4.8
7.3
Gasoline
Volvo 245L
8905 - 07
950
117
17
17
329
-------
SAMPLE PREPARATION
All samples were stored frozen, mainly at -20 C, after
they were prepared and between mutagenicity assays.
Particulate matter on filter. Filters were Soxhlet extract-
ed for 16 h with 250 ml acetone. The acetone was first
evaporated under vaccuum to about 10 ml and then under a
stream of nitrogen on a heating block at < 40 °C to about
0.3-0.5 ml. This residue was diluted with dimethyl sulf-
oxide (DMSO) to a known volume, usually about 4 ml per
filter.
Condensate. Apart from that an aliquot of the original so-
lution was assayed for mutagenicity without any further
preparations the aqueous condensates were treated in vari-
ous ways.
A part of the condensate was reduced in volume under vacuum
to about 5 % of its original volume and then diluted with
an equal volume of DMSO.
A part of the condensate was evacuated mildly until most of
the acetone had evaporated. The solution was then passed
through a XAD-2 column, the column drained and then eluted
with acetone. The extract was reduced in volume and diluted
with DMSO as described for filter extracts.
The major part or the whole condensate was extracted three
times with half the volume of ji-pentane. The extracts were
combined and reduced in volume and dissolved in DMSO as de-
scribed for filter extracts with the difference that small
amounts of acetone were added before all pentane evaporated.
- The aqueous phase from the pentane extraction was also
reduced in volume under vacuum and then diluted with an
equal volume of DMSO.
Crankcase oil. Fresh and used motor oils were extracted
three times with an equal volume of DMSO after which the
three extracts were combined.
FRACTIONATION
Fractionation with respect to polarity was performed with a
modification of the method described by Wynder and Hoffmann
(5). The sample was diluted with diethyl ether and shaken
with a equal volume of aqueous 1 M sulfuric acid. The aque-
ous phase was then removed, neutralized with sodium hy-
droxide and re-extracted with ether, the ether solution
constituting the basic fraction. The first ether solution
was further shaken with an equal volume of 2 M sodium hy-
droxide. The aqueous phase was then removed, neutralized
330
-------
with sulfuric acid and re-extracted with ether, the ether
solution constituting the acidic fraction.
The ether solution from which basic and acidic components
had been extracted was then reduced in volume and the resi-
due transferred to cyclohexane while residual ether was re-
moved. The cyclohexane sample was applied to a silica gel
column which was successively eluted with cyclohexane, ben-
zene and diethyl ether and in some cases also with methanol.
The fractions are nominally designated to contain aliphatic
(cyclohexane), aromatic (benzene) and oxygenated compounds
(ether and methanol).
All fractions were reduced in volume, diluted and dissolved
in DMSO as described above.
MUTAGENICITY ASSAY
Mutagenicity was determined with the Salmonella/microsome
test with the strains TA 98 and TA 100 using the plate in-
corporation assay (6). In this test specific Salmonella
strains, having histidine auxotrophy, are exposed to the
compound(s) on agar plates deficient in histidine. A dose
related increase in the reversion frequency above the spon-
taneous rate to histidine prototrophy is scored as mutagen-
icity. The exposure is made both in the absence and in the
presence of a homogenate from mammalian tissue to simulate
mammalian metabolism. The present investigation employed
the commonly used microsome containing rat liver superna-
tant (S-9) from Aroclor 1254 induced male SPD rats supple-
mented with necessary co-factors as described in ref. 6.
All assays included tests with positive control compounds.
The bacterial strains were routinely checked for the pres-
ence of known characteristics, spontaneous reversion fre-
quency, sensitivity to ultraviolet light and crystal violet
and resistance to ampicillin. Benzo(a)pyrene was used as
positive control for the S-9 and 5 yg of this compound has
given in the range between 300 and 600 revertants per plate
at 20 and 50 yl S-9 per plate in TA 98 in the course of the
investigation.
Mutagenicity assays under anaerobic conditions were per-
formed by incubating the agar plates in a BBL GasPak anaer-
obic system during the first 16 h followed by additional
normal incubation for 32 h.
Each sample has been assayed with one plate per dose level
in at least three independent test at at least two dose
levels. The mutagenic potency, expressed as revertants per
liter exhaust, has been calculated from the linear part of
the dose response curve by linear regression using each
331
-------
plate as a data point. The standard deviation of the slope
of the regression line has also been calculated.
CHEMICALS
The nitroarenes were obtained from the following vendors:
1-nitronaphthalene from Fluka AG, FRG; 2-nitronaphthalene
from EGA-Chemie, FRG; 9-nitroanthracene from Aldrich Chemi-
cal Co., USA and 3-nitropyrene from Koch-Light, England.
HPLC analyses of the 9-nitroanthracene showed that this
sample contains three additional uv-absorbing contaminants.
No detectable levels of uv-absorbing contaminants were de-
tected in the other nitroarenes.
RESULTS
Mutagencity assays of extracts of particulate matter and
pentane extracts of the condensate showed that mutagenicity
was easily detected; see Fig. 1-3 and upper part of Table
2. Except for particulate matter from gasoline exhaust all
samples gave the highest mutagenic effect in the absence of
the S-9 microsomal preparation. The enhanced mutagencity of
gasoline particulate matter in the presence of S-9 was,
however, only observed with the strain TA 98; this enhance-
ment was maximal at an addition of 20 ul S-9 per plate when
the effect of different amounts, 10, 20 and 50 ul, was in-
vestigated.
Additional studies with portions of condensates from each
fuel showed that extraction with pentane or XAD-2 as well
as a simple concentrating result in samples with similar
mutagenicity; see lower part of Table 2. The results with
the diesel exhaust condensate indicate, however, that none
of the preparations recover the mutagenicity completely
from the original solution.
FRACTIONATION
Fractionation with respect to polarity of samples of each
type of exhaust sample showed clear differences between the
two fuels as well as with an acetone extract of urban par-
ticulate matter collected by high volume sampling at the
roof tops in central Stockholm in March 1979; see Table 3.
The recovery of the mutagencity is, however, rather low in
the fractionation of some samples.
332
-------
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334
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1/1
+J
S_
cu
0)
S-
0
liter exhaust / per plate
Figure 1. Mutagenicity of the extract of particulate matter
from the exhaust of a diesel powered car. Revertants per
plate, corrected for the spontaneous reversion frequency,
are plotted against liter of exhaust corresponding to the
amount of extract per plate. Vertical bars show the stand-
ard deviation of some representative points.
TA 100 - S-9; O TA 98 - S-9; 4- TA 98 +S-9.
400^ -
c
ro
i.
0)
o>
S-
0
DIESEL 9017- 9
/
s^
0
liter exhaust / per plate 15
Figure 2. Mutagenicity of the n-pentane extract of the
aqueous condensate from the exhaust of a diesel powered
car. Cf. figure 1.
TA 100 -S-9; X TA 100 + S-9;
O TA 98 -S-9; + TA 98 +S-9.
335
-------
O)
+->
re!
r
Q.
OJ
OJ
GASOLINE 8905-7
liter exhaust / per plate
30
Figure 3. Mutagenicity in TA 98 of extracts of exhaust sam-
ples from a gasoline powered car. Cf. figure 1.
O particulate matter, - S-9
«f» particulate matter, + S-9
0 n-pentane extract of condensate, - S-9
o
en
4->
C
O)
50
CRANKCASE OIL
,00-0
-S-9
10000
odometer, km
20000
Figure 4. Mutagenicity in TA 98 +/- S-9 of motor oil from
a gasoline powered car. Samples were collected at various
times between services over a period of 140 days. Fresh,
non-mutagenic,<2 revertants/mg, oil was used from *.
336
-------
NITROARENES AND ANAEROBIC MUTAGENICITY ASSAYS
Some nitro compounds exhibit an enhanced mutagenicity when
incubated with some Salmonella tester strains under anaero-
bic conditions. Typical examples are some mono-functional
nitroarenes whose mutagenic responses are given in Table 4.
Table 4. Mutagenicity in the absence of the microsomal sys-
tem of some nitroarenes in TA 100 and TA 98 under
aerobic (normal) and anaerobic conditions. The mu-
tagenicity is given as revertants / vg of the com-
mercial sample assayed without purification.
TA 100 TA 98
aerobic aerobic anaerobic
1-nitronaphthalene
2-nitronaphthalene
9-nitroanthracene
3-nitropyrene
15
24
1
400
1.0
2.5
<1
3700
2.2
5.8
63 a)
8200
a) this increase is caused by the major component of the
sample having a retention as expected for nitroanthracene.
Anaerobic incubation of motor vehicle exhaust samples did
not cause any considerably increased mutagenicity whereas
a significant enhancement has been detected with extracts
of urban particulate matter as is shown in Table 5.
Table 5. Relative mutation frequency of exhaust samples and
urban particulate matter in TA 100 and in TA 98
under aerobic and anaerobic conditions. The muta-
genicity is expressed as per cent of the response
in TA 98 -S-9 under normal conditions. The sam-
ples are the same as those reported in Table 3.
GASOLINE
particulate matter
condensate
TA100
aerobic
200
140
TA
aerobic
100
100
98
anaerobic
130
120
DIESEL
particulate matter 180 100 130
condensate 150 100 90
URBAN PARTICULATE MATTER 95 100 220
337
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CRANKCASE OIL
The presently employed extraction of mutagenic components
from gasoline engine crankcase oil by DMSO has so far given
the highest recovery among several tested procedures. Many
samples of fresh and used oils have been assayed resulting
in no detectable mutagenicity in fresh oils, j_.e_. < 2 rever-
tants per mg oil, and easily detecable mutagenic effects
from used oils, J_.e_. 5-150 revertants per mg oil.
Used crankcase oils contain both compounds which are muta-
genic in the absence of microsomal activation as well as
compounds which cause an enhanced mutagenicity in the pres-
ence of the microsomal system. The mutagenic potency of the
motor oil seems to increase with mileage; see Figure 4.
Attempts to assay motor oils from diesel engines have been
less successful. Extracts of such oils cause a small in-
crease in the reversion frequency but the effect is not
dose dependent which prevents an assessment of their muta-
genic potency-
DISCUSSION
Few data exist on the mutagencity of automobile exhausts.
Wang _e_t aj_. (7) give quantitative results with respect to
the weight of particulate matter from some gasoline engines
and Barth and Blacker (8) indicate that unspecified high
mutagenicity of particulate matter from diesel engines,
probably direct injection engines, is a reason for concern.
Tokiwa et_ _a]_. (9) have given some data for gasoline ex-
haust particulate matter and condensate which are very
similar to those found in the present study; the same au-
thors also mention that the mutagenicity is less from a
catalyst equipped car.
A comparison between the diesel and gasoline exhaust inves-
tigated in the present study can be made assigning an aver-
age mutagenicity of 100 revertants per liter exhaust for
diesel and 15 revertants per liter exhaust for gasoline and
using the known fuel consumption and total exhaust volume
parameters:
DIESEL GASOLINE
Revertants / liter exhaust 100 15
Revertants / g fuel consumed 2 700 190
Revertants/test cycle (12.1 km) 2200000 180000
338
-------
Subsequent to the present study, Rannug (10) investigated
the mutagenicity of diesel and gasoline exhaust samples
from passenger cars from the Volvo Co. assaying acetone ex-
tracts of particulate matter and diethyl ether extracts of
condensates. The diesel exhaust is reported to cause about
3100000 revertants per test cycle and the gasoline exhaust
between 340000-410000 revertants per test cycle in TA 100.
A catalyst equipped gasoline powered car is further report-
ed to emit much less.
Mutagenicity assays by the Salmonella test have previously
revealed the presence in emissions and in urban particulate
matter of components which are mutagenic in the test system
without requiring the mammalian metabolic activation (2).
Similarly behaving compounds are thus also present in the
exhaust from diesel and gasoline engines. The exhausts un-
doubtedly also contain mutagenic/carcinogenic polycyclic
aromatic hydrocarbons as benzo(a)pyrene which need mamma-
lian metabolism for the formation of mutagenic metabolites.
Their detection in a complex mixture by the Salmonella as-
say depends on their concentration relative the concentra-
tion and behavior of components not requiring mammalian ac-
tivation. In the present investigation only particulate
matter from gasoline exhaust had a composition which favors
their detection. Gasoline exhaust has, however, also a con-
siderable S-9 dependent mutagenicity residing in the basic
fraction (Table 3).
Techniques used in the sampling of motor vehicle exhaust,
stack gases and urban particulate matter can always be dis-
cussed with reference to possibilities of artifact reac-
tions during and after sampling. The problems can, however,
only be solved with parallel investigations involving dif-
ferent sampling techniques with relevant chemical and biol-
ogical analyses. - The presence in crankcase oil of muta-
genic components not requiring mammalian activation shows
that such compounds are formed in the engine and it is rea-
sonable to suggest that they also become a part of the ex-
haust.
The results of the present investigation underscore the im-
portance of collecting condensable gaseous components as a
large part of the mutagenicity is present in the conden-
sates. It seems likely that such components, given suffi-
cient time, may become adsorbed to particulate matter in
ambient air.
The fractionation with respect to polarity (Table 3) shows
that the distribution of components are dissimilar for the
different samples. The wide distribution of mutagenicity
among several fractions implies that no single compound or
single type of compounds alone are responsible for a major
339
-------
part of the mutagenic effect of any of the samples. - The
distribution of the mutagenicity of the urban particulate
matter among different fractions (Table 3) is similar to
that reported by Teranishi (11).
It has been shown that nitrogen dioxide and nitric acid re-
act with polycyclic aromatic hydrocarbons forming nitroare-
nes which are mutagenic in the Salmonella system (12). It
is, however, not known if such reactions occur in ambient
air or on the filter at the time of collection of particu-
late matter or in both cases.
Some nitro compounds have in the present investigation been
shown to have the property of becoming more mutagenic when
incubated with the Salmonella strain TA 98 under anaerobic
conditions (Table 4). This property has been used to inves-
tigate the presence of similarly behaving nitro compounds
in environmental samples. Urban particulate matter collect-
ed in the Stockholm area shows an enhanced mutagenicity un-
der anaerobic assay conditions as shown in Table 5, but
periods with no or little enhancement have also been en-
countered, probably reflecting differences in the composi-
tion of air pollutants. Anaerobic incubation of the diesel
and gasoline exhaust samples gave little or no increase in
the mutagenicity (Table 4) indicating that mutagenic nitro
compounds causing an enhanced mutagenic effect is much less
abundant in exhaust samples than in most samples of inves-
tigated urban particulate matter.
There undoubtedly exist mutagenic nitro compounds which do
not cause an enhanced mutagenicity under anaerobic assay
conditions. Several nitro compounds are presently being in-
vestigated.
The presence in used crankcase oil of mutagenic compounds
which do not require mammalian metabolism has been report-
ed earlier (7) as has the presence of mutagens requiring
mammalian metabolic activation (13). The extraction proce-
dure used in the present study may not extract all muta-
genic components but it favorably separates mutagenic com-
pounds from components which are toxic to or interfering
with the assay. The failure to extract readily detectable
amounts of mutagenic components from used motor oils from
diesel engines may either be due to that their content of
mutagens is low or to that the partition coefficients are
unfavorable. Mixing used motor oil from a diesel engine
with an equal amount of used motor oil from a gasoline en-
gine results in the extraction of the same mutagenicity as
is extracted from the gasoline engine oil alone; this shows
that toxic or interfering components from the diesel engine
motor oil probably are not causing a false low mutagenicity
of the diesel engine motor oil extracts.
340
-------
ACKNOWLEDGMENT
I am indepted to the staff of the Vehicle Exhaust Gas Labo-
ratory of the National Swedish Environment Protection Board
for their kind contribution in supplying well defined sam-
ples for this investigation. FK Edward Hefner has contrib-
uted with excellently performed Salmonella mutagenicity as-
says. The investigation has been supported by grants from
the National Swedish Environment Protection Board and the
Swedish Natural Science Research Council.
REFERENCES
1. Ehrenberg, L. and Lofroth, G. On the assessment of ge-
netical and carcinogenic effects. Particularly with re-
spect to chemicals associated with combustion emissions
in the oxidation fuel cycles. In: Goodman, G. T. and W.
D. Rowe (eds.). Energy Risk Management. Proceedings of
a Seminar. Academic Press, New York, in press.
2. Lofroth, G. 1978. Mutagenicity assay of combustion emis-
sions. Chemosphere, 7:791-798.
3. Stenberg, L). 1979. Emission av polycykliska aromatiska
kolvaten fran bensinmotorfordon. Report to the National
Swedish Environment Protection Board. 54 pp.
4. Stenberg, U. 1979. Jamfb'rande matning av PAH-emissionen
fran bensinmotorfordon med anvandning av bensin respek-
tive bensin/metanol som bransle. Report to the Swedish
Methanol Development Co. 21 pp.
5. Wynder, E. L. and D. Hoffmann. 1965. Some laboratory and
epidemiological aspects of air pollution carcinogenesis.
J. Air Pollut. Control Assoc., 15:155-159.
6. Ames, B. N., J. McCann and E. Yamasaki. 1975. Methods
for detecting carcinogens and mutagens with the Salmon-
el la/mammal ian-microsome mutagenicity test. Hut. Res.,
31:347-364.
7. Wang, Y. Y., S. M. Rappaport, R. F. Sawyer, R. E. Tal-
cott and E. T. Wei. 1978. Direct-acting mutagens in au-
tomobile exhaust. Cancer Letters, 5:39-47.
8. Barth, D. S. and S. M. Blacker. 1978. The EPA program
to assess the public health significance of diesel emis-
sions. J. Air Pollut. Control Assoc., 28:769-771.
341
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9. Tokiwa, H., H. Takeyoshi, K. Takhashi, K. Kachi and Y.
Ohnishi. 1978. Detection of mutagenic activity in auto-
mobile exhaust emissions. Mut. Res., 54:259-260.
10. Rannug, U. 1979. Den mutagena effekten av emissionen
fran diesel- och bensindrivna personbilar. Report to
the Volvo Co. and National Swedish Environment Protec-
tion Board. 26 pp.
11. Teranishi, K., K. Hamada and H. Watanabe. 1978. Muta-
genicity in Salmonella typhimurium mutants of the ben-
zene-soluble organic matter derived from air-borne par-
ticulate matter and its five fractions. Mut. Res.,
56:273-280.
12. Pitts Jr., J. N., K. A. Van Cauwenberghe, D. Grosjean,
J. P. Schmid, D. R. Fitz, W. L. Belser Jr., G. B. Knud-
son and P. M. Hynds. 1978. Atmospheric reactions of
polycyclic aromatic hydrocarbons: Facile formation of
mutagenic nitro derivatives. Science, 202:515-519.
13. Payne, J. F., I. Martins and A. Rahimtula. 1978. Crank-
case oils: Are they a major mutagenic burden in the
aquatic environment? Science, 200: 329-330.
General Discussion
SPEAKER (unidentified): If I understand correctly the
main result was that diesel exhaust is ten times as active
as automobile exhaust - am I right?
G. LOFROTH: Yes, on the average from this study.
SPEAKER (unidentified): In relation to carcinogenicity,
we made a long-term study in Germany with two experts and
found just the opposite. Automobile exhaust would be about
40 times as active as diesel exhaust.
G. LOFROTH: I could comment that different biological
tests give different results, but regarding the report
which you mentioned, I am a little worried about the ex-
traction procedure and preparation. I know what I have
been doing with the samples before they went into muta-
genicity assays and I was very careful not to destroy
compounds.
J. HUISINGH: The condensate that you compared to the
filter - is it a condensate that is made after filtration
of particles, and how does that compare to the other gen-
tleman's?
G. LOFROTH: No, in this case the condensate was taken
before filtration. Of course, one would wish that the
first particulate matter filtered would be a condensate.
However, from previous studies which have been reported in
342
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Sweden, both with respect to PAH in back gases and also in
mutagenicity from the same type of sample, it has been
shown that considerable mutagenic activity is present in
condensate collected after they have been filtered.
SPEAKER (unidentified): Along those same lines in view
of Dr. Bradow's results showing different mutagenicities
from different gasoline vehicles, could you describe the
particular system of that 1976 gasoline car? I would like
to know particularly whether that car had a catalyst, was
it run on leaded fuel, and any other exhaust treatment
system that may apply to a Swedish car.
6. LOFROTH: This was an ordinary noncatalyst car.
There has also been a Japanese report with a few data on a
catalyst and noncatalyst equipped car. There was con-
siderable mutagenicity in the noncatalyst car exhaust.
Subsequent to the studies we did in Sweden, there had been
a larger program in which three Volvo cars, one with cat-
alyst, one without catalyst, and then a diesel car were
tested. The same figures came out, the catalyst car had a
very low mutagenicity, particularly when the catalyst was
warm. In fact the mutagenicity was nondetectable and the
ratio between the ordinary gasoline car and the diesel was
about the same as in the study reported here, a factor of
10 or a little more.
D. DZIEDSIC: I think a comparison is good. Mouse skin
does not give any response to organ-specific carcinogens.
6. EDWARDS: We have built into Salmonella a nitro-
reductive deficiency, the enzyme that does not require
oxygen and have seen no mutagenic reactivity with any of
the polycyclic nitrated compounds we have looked at. This
would agree with the fact that you get enhanced sensitivity
in anaerobic systems implying that the bacteria is making a
hydroxylamine or some such intermediate to for a mutagenic
metabolite. Secondly, I was interested by something near
the end of your talk where you showed that used motor oil
showed increased mutagenicity with time of use. Could you
give us a couple of details about what kind of motor oil
that was; did this require activation; and did you try
other bacterial strains, etc?
G. LOFROTH: Mostly the motor oil was from my previous
car. I know what type of commerical oil I am buying and
putting into my own motor so I could give a few details.
The extraction of mutagenicity or mutagenic compounds from
used motor oil can be done rather easily by mixing with an
equal amount of DMSO and then shaking and separating the
phases. Of course one cannot be sure that this is 100
percent extraction, but at least if the concentration in-
creases in the motor oil and there are similar compounds
produced in the beginning as well as in th-e end, one hopes
that the extraction is as good in the beginning as in the
end. There are compounds which do not need metabolic acti-
vation, and then there are additional compounds presumably
343
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conventional PAH. It has been shown by chemical analysis
that these are present and increasing in used motor oil.
SPEAKER (unidentified): Some years ago there was a
report by, I believe, George Gross of Exon Corporation, to
the effect that there was observed in a variety of auto-
mobiles generation of polynuclear aromatic hydrocarbons in
the motor oil. The general hypothesis at that time was the
principal hydrocarbons, which are present in motor oil,
included cyclo paraffins which were important to the per-
formance of motor oil but could also be converted. I think
that would explain the induction with the large effect of
S9 in the material you have seen. The reports date, as I
recall, to the mid 1960's, so there is evidence in the
literature to support the hypothesis that there is some
induction of polynuclear aromatics.
344
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BIOLOGICAL AVAILABILITY OF MUTAGENIC CHEMICALS
ASSOCIATED WITH DIESEL EXHAUST PARTICLES
A. L. Brooks, R. K. Wolff, R. E. Royer, C. R. Clark,
A. Sanchez and R. 0. McClellan
Inhalation Toxicology Research Institute, Lovelace Biomedical
and Environmental Research Institute, P. 0. Box 5890,
Albuquerque, MM 87115
ABSTRACT
To estimate the human health risk of diesel particles it is
necessary to know their deposition and retention in the
respiratory tract and the rate of dissasociation of mutagenic
(and potentially carcinogenic) compounds associated with the
particles. The deposition of a chain aggregate aerosol of
67Ga203 with size and shape characteristics similar to
diesel exhaust particles has been evaluated using the Beagle
dog. Approximately one-third of the inhaled activity is
deposited in the respiratory tract with most of the particles
deposited in lung. The mutagenic activity present in dichlor-
methane, dog serum, lung lavage fluid, saline, dipalmitoyl
lecithin and albumin following incubation of the fluids with
diesel exhaust particles was determined in the Ames Salmonella
system. As observed by other investigators, large quantities
of mutagenic activity were removed by the organic solvent,
dichloromethane. A very small amount of mutagenic activity
was removed by the serum and lavage fluid over a 3 day
incubation period. No activity was detected with the other
solvents. The minimal mutagenic activity demonstrated in
the biological media following incubation with diesel ex-
haust particles may be due to a lack of removal of mutagens
from the particles or an inactivation of removed mutagens by
binding or some other process. These preliminary observa-
tions will be followed up as an aid in determining the
health risks of diesel exhaust particles.
345
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INTRODUCTION
When diesel exhaust particles are extracted with organic
solvents, such as CH2C12, mutagenic activity can be detected
in the extract using bacterial test systems (1-2). Since
there is a strong correlation between substances which are
bacterial mutagens and carcinogens in mammals (3-4) this
material must be considered a potential human carcinogen.
To understand the magnitude of the risk from exposure to
diesel exhaust particles the behavior and fate of both the
inhaled particles and their associated organic compounds
must be studied. This report describes research directed
toward determining (a) the deposition and early retention of
ultra-fine particles similar in size and shape to diesel
exhaust particles and (b) the biological availability of
mutagenic compounds associated with the diesel exhaust
particles. The research has demonstrated that a large
fraction of the ultra-fine aerosol particles are deposited
in the deep lung and a portion are retained for a number of
days. Studies with serum and lung lavage fluid suggest that
most of the mutagenic activity on diesel exhaust particles
is not biologically available either because it is not
removed from the particles or because removed mutagens are
inactivated by the biological solvent. These approaches
provide data to evaluate biological availability of the
chemicals which, combined with the data derived from the
retention of the particles can be used to estimate the
potential chemical dose to lung cells.
METHODS
The method of generating the ultrafine chain aggregate
aerosols has been previously described (5). With these same
methods ^63203 aerosols were generated and Beagle dogs
exposed by inhalation in a configuration which produced a
nose-only exposure. Gallium-67 was selected for use because
it has moderate energy gamma-emissions and decays with a
half-life of 78 hours thereby providing a useful tag for
whole-body counting and gamma camera scanning. This pro-
vides a measure of the total fraction of the inhaled material
that is deposited, where it is deposited and how long it is
retained. Details of the exposure and counting system have
been previously published (6).
Diesel particulate materials from two cars v/ere utilized to
evaluate the effectectiveness of biological solvents in
extracting mutagenic compounds. The samples were selected
because, using dichloromethane as a solvent, one had the
highest and the other the lowest mutagenic activity of the
samples tested to date. The particulate material with the
346
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highest activity (Sample 1) was collected on a glass fiber
filter, from a 5.7 liter displacement diesel engine, while
operating at idle. Sample 2 with the lowest activity was
collected on a Pall flex T60A20 filter, from a 2.1 liter
displacement engine operating on the Highway FET (Fuel
Economy Test] cycle.
Sample 1 was collected at this Institute and Sample 2 was
collected at Transportation Systems Center of the Department
of Transportation. The percent of total CH2C12 extractable
hydrocarbons was determined for both engines using 1 hour
ultrasonic treatment of the particles with CH2C12- For
mutagenesis testing, the CH2C12 extracted material was
evaporated to dryness under nitrogen, diluted in dimethyl-
sulfoxide (DMSO) and tested in Ames Salmonella strains TA-98
and TA-100 (7).
Particles were incubated at 37°C for 6, 24, 48, 72 and 120
hours in lavage fluid, serum, saline, albumin, dipalmitoyl
lecithin or dichloromethane. The concentrations of the par-
ticles in the solutions were 0, 1, 4 and 8 mg/ml for all
solvents. Additional tests were conducted using saline,
lavage fluid and serum at concentrations of 0, 0.5, 1, 5 and
10 mg/ml. Particles were filtered from the solvents and
0.1 ml added per plate to determine the mutagenic activity
in these solvents. This results in concentrations up to
1000 yg of diesel particulate equivalent per test plate.
Negative control plates were run for each solvent at each
incubation time. Positive controls were run to evaluate the
response of the bacteria using sodium azide, 2-nitro-fluorene
and 2-amino anthacene. Duplicate plates were used at each
concentration. The samples were tested both with and with-
out Aroclor induced liver microsomal enzymes (S-9) for each
exposure schedule. Cytotoxicity was determined with a 10"
dilution of the bacteria plated on histadine supplemented
agar. The colonies were counted on an Arteck automatic
colony counter after incubation for 48 hours.
The lavage fluid was freshly obtained from a Beagle dog
using a previously described technique (8). The cells were
removed by centrifugation in an ultracentrifuge at 1000 RPM
and the surfactant in the lavage fluid concentrated by
centrifugation at 20 000 RPM and resuspended in saline to a
final phosphorous concentration of 200 tig/ml. The surface
active material in the lavage fluid contains both phospho-
lipids and proteins. DPL was incubated with the particles
at 2.8 mg/ml as a representative phospholipid and albumin at
4.6 mg/ml as a representative protein. The serum was also
obtained fresh from a Beagle dog and used without dilution.
All solutions were filtered through 0.45 pm millipore filters
to remove bacteria prior to testing.
347
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RESULTS
Electron photomicrographs and measurements of aerosol proper-
ties including count median diameter (CMD), volume median
diameter (VMD) and the diffusion equivalent diameter indicate
that the diesel aerosol and surrogate aerosol are similar in
size, shape and aerodynamic properties (6,9). They are both
small chain and cluster aggregates. The inhalation studies
(6) indicated that 33% of the inhaled material was deposited
in the respiratory tract of the dog. Of the particles
deposited, 82% were in the lung and of that in the lung 63%
was in the deep aveolar portion of the lung. Twenty-three
percent of these particles were cleared rapidly with an
effective half-life of less than 1 day. Additional studies
are needed to determine the long-term retention pattern,
perhaps using an aerosol labeled with a longer lived
radionuclide.
CH2C12 was the solvent which was the most effective in
extracting mutagenic activity from diesel particles. The
mutagenic response of the extracts of the two particulate
samples following 1 hour ultrasonic extraction with CH2C12
is illustrated in Figure 1. Both TA-98 and TA-100 were used
and response was recorded both with and without the liver S-
9 microsomal fraction. This characterizes the response of
the bacteria to the different exhaust particles extracted in
CH2C12 and serves as a point of reference against which the
more biologically relevant solvents can be compared.
The particles were incubated over a number of time intervals
in the biological solvents using a range of particle concen-
trations. Figure 2 shows the mutagenic activity plotted as
a function of incubation time for strain TA-100 without S-9.
Since the addition of S-9 decreased the magnitude of the
response these data are not included in the illustrations on
the effectiveness of biological solvents in extracting
mutagenic activity from diesel particles. In this figure
the bacteria were exposed to extracts of 1000 yg of diesel
exhaust particle equivalent per plate. There was little
change in level of mutagenic activity in the solvent as a
function of incubation time.
The mutagenic activity in the solvents did not change marked-
ly as a function of incubation time, therefore the responses
were plotted as a function of concentration using the average
of all extraction times. Figures 3 and 4 show the response
for both samples tested with strain TA-100 without the
addition of S-9. The only biological solvent to show a
positive response was serum and it was very slight.
348
-------
2000
1500-
UJ
i-
CO
i-
2
DC
LU
>
UJ
cr
1000 -
500 -
0 5OO IOOO 1500
p.g DIESEL PARTICLE EQUIVALENT/ PLATE
Figure 1. The mutagenic response in TA-98 and TA-100 to
CH3C12 extracts of diesel particle from two different
light duty diesel engines.
349
-------
1
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N
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500 1000 1500
DIESEL PARTICLE EQUIVALENT / PLATE
Figure 4. Dose-response relationship for mutations induced
in TA-100 by extracts of diesel particle Sample 1 with a
high specific mutagenic activity.
When the tests were conducted using bacteria TA-98 on Sample
1 with the high mutagenic activity (Figure 5), there was an
apparent dose related increase in mutagenic activity extracted
by serum with an indication that lavage fluid could also
remove a small fraction of the activity. The slopes of the
dose response curves for TA-98 were 1.5, 0.13 and 0.11
revertants/yg diesel particle extracted for the CH^Cl?,
serum and lavage extraction. Saline extraction failed to
show mutagenic activity above that observed in the control
cultures.
352
-------
1000
500 1000
DIESEL PARTICLE EQUIVALENT/PLATE
Figure 5. Dose-response relationship for
mutations induced in TA-98 by extracts of
diesel particle Sample 1 with a high
specific mutagenic activity.
DISCUSSION
Small particle aerosols in this study resulted in a higher
lung deposition and potential lung dose in Beagle dogs than
has previously been reported for aerosols with larger (1.8
urn) particle sizes (10). The deposition in the lung of the
dog for the surrogate diesel aerosol was similar but lower
than the 88% predicted for man by the model from the Task
Group on Lung Dynamics (11). This may be a species difference
or may relate to the shape of the surrogate aerosol.
These preliminary results suggest that the deposition and
retention pattern for diesel exposures are fairly well pre-
dicted by Task Group on Lung Dynamics model (11). The pre-
ponderance of the aerosol is deposited in the pulmonary
region where clearance is slow. This may have some important
toxicological implications since lung retention time of the
particles needs to be considered when determining biological
availability of the organic compounds associated with the
particles.
353
-------
The estimated chemical dose to lung cells is much more dif-
ficult to determine and will require additional research.
This question has been addressed by a number of different
approaches. One approach was to measure the disappearance of
carcinogens from soot in human lungs (12). Another approach
involves extraction of exhaust condensates with organic
solvents and determining their mutagenic (1-2) and carcino-
genic potential (13). In this research an attempt was made
to address the question of chemical dose using extraction of
mutagenic activity from diesel particles with a range of
solvents and determining their activity with the Ames test.
The reversion response of the bacteria from CH2C12 extracts
of the two sources of diesel particles differed by a factor
of about 3 for TA-100 and about 10 for TA-98, without S-9.
Addition of S-9 did not change the magnitude of the response
of TA-98 to extracts from the less active particles (Sample
2). For Sample 1 the addition of S-9 decreased the magnitude
of the response for both TA-98 and TA-100. Research on the
response of TA-1538 to diesel extracts (2) indicates an
enhanced reversion frequency following the addition of liver
microsomal S-9 fraction.
When diesel particles were incubated with different media,
there was no increase in activity as a function of incubation
time over the intervals used in this study. This may indi-
cate that either the mutagenic activity that is soluble is
rapidly removed or that an equilibrium is reached between the
rate of dissociation of the mutagens from the particles and
their rate of inactivation. Thus, the minimal mutagenic
response produced by the biological media following incuba-
tion with diesel exhaust particles may be due to a lack of
removal of mutagens from the particles or the biological
materials bind or inactivate the mutagenic compounds after
they are removed. The bacteria may not be able to incorpo-
rate these bound mutagens and produce a positive response.
However, mutagens extracted from fly ash particles by horse
serum are capable of producing revertants in the Ames test
(14-15). Additional research on this is required to define
the movement or binding of chemicals by proteins and surface
active substances.
The slopes of the dose response curves can be compared for
the serum, lavage fluid and CH2C12 and indicate that the bio-
logical solvents extract from 0 to about 3% of the activity
extracted with CH2C12 as measured by TA-100 without S-9. For
TA-98 the mutagenic response for serum and lavage fluid are
similar with 10% of the response for Q^C^ being the
maximum. Several potential problems still exist when these
results are extrapolated to whole animals. First, they were
conducted over a short time and may not reflect the movement
354
-------
of the activity over the long times involved in lung clear-
ance. Second, the solvents themselves, i.e., lavage or
serum, may bind or detoxify the mutagenic compounds and make
them unavailable for interaction with the bacteria even
though they may be removed from the particles and interact
with mammalian cells. Finally, the environment in the lungs
consists of cells as well as a cellular fluids. The cells
may extract, detoxify or activate the mutagenic compounds in
vivo and make in vitro solvent systems less applicable in
understanding the changes induced in the whole animal.
These results provide a first estimate of the importance of
biological availability of mutagens from diesel particles
which can be used in determining the dose to lung cells.
Additional research combining in vitro inhalation and in
vitro and in vivo testing of mutagenic activity are needed to
further define the chemically important dose to lung cells.
ACKNOWLEDGMENT
Research performed under U. S. Department of Energy Contract
Number EY-76-C-04-1013 and conducted in facilities fully
accredited by the American Association for the Accreditation
of Laboratory Animal Care.
REFERENCES
1. Huisingh, J. L., S. Mesnow, L. Claxton, R. D. Curren,
L. M. Schechtman, R. E. Kouri, B. C. Casto, G. G. Hatch,
S. L. Huang, R. H. Jaskot, C. R. Christenson, T. J.
Slaga, L. L. Triplet, A. D. Mitchell, V. F. Simon,
K. E. Mortelmans, E. S. Riccio, M. M. Jotz, and E. L.
Evans. Mutagenic and carcinogenic potency of extracts
and related environmental emissions. In: International
Symposium on Health Effects of Diesel Engine Emissions
U. S. Environmental Protection Agency, Research Triangle
Park (in press).
2. Huisingh, J., R. Bradow, R. Jungers, L. Claxton,
R. Zweidinger, S. Tejada, J. Bumgarner, F. Duffield,
M. Waters, V. F. Simmon, C. Hare, C. Rodriguez, and
L. Snow. 1978. Application of bioassay to characteri-
zation of diesel particle emissions. In: Symposium on
Application of Short-term Bioassays in the Fractionation
and Analysis of Complex Environmental Mixtures, (M. D.
Waters, S. Nesnow, J. L. Huisingh, S. S. Sandhu, and
355
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L. Claxton, eds.), pp. 381-418, U. S. Environmental
Protection Agency, Research Triangle Park, f!C, EPA-
600/9-78-027.
3. McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. 1975.
Detection of carcinogens as mutagens in the Salmonella/
microsome test: Assay of 300 chemicals, Part I. Proc.
flat!. Acad. Sci. USA, 72:5135-5139.
4. Kotin, P., H. L. Falk, and M. Thomas. 1955. Aeromatic
hydrocarbons; III, presence in participate phase of
diesel-engine exhaust and carcinogenicity of exhaust
extracts. AMA Arch. Ind. Health 11:113-120.
5. Tu, K. W. and G. M. Kanapilly. 1979. A high-capacity
condensation aerosol generation system. Environ. Sci.
Techno!. 13:698-701.
6. Wolff, R. K., G. M. Kanapilly, P. B. DeMee and R. 0.
McClellan. 1979. Deposition of surrogate diesel aerosols
in Beagle dogs. Inhalation Toxicology Research Institute,
Lovelace Biomedical and Environmental Research Institute,
LF-Report 67 (in press).
7. Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods
for detecting carcinogens and mutagens with the
Salmonella/mammalian-microsome mutagenicity test. Mutat.
Res. 31:347-364.
8. Muggenburg, B. A. and J. L. Mauderly. 1975. Lung lavage
using a single-lumen endotracheal tube. J. Appl. Physiol.
38:922-926.
9. Frey, J. W. and M. Corn. 1967. Physical and chemical
characteristics of particulates in a diesel exhaust.
Am. Ind. Hyg. Assoc. J. 28:468-478.
10. Cuddihy, R. G., D. G. Brownstein, 0. G. Raabe and
G. M. Kanapilly. 1973. Respiratory tract deposition
of inhaled polydisperse aerosols in Beagle dogs. J_.
Aerosol Sci. 4:35-45.
11. Task Group on Lung Dynamics. 1966. Deposition and re-
tention models for internal dosimetry of the human res-
piratory tract. Health Phys. 12:173-207.
12. Falk, H. L., P. Kotin, and I. flarkul. 1958. The dis-
appearance of carcinogens from soot in human lungs.
Cancer 11:482-489.
13. Brune, H. 1974. Experimental carcinogenicity and bio-
assays of automobile exhaust condensate and its poly-
356
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cyclic aromatic hydrocarbons. In: Experimental Lung
Cancer, (E. Karbe and J. F. Park, Eds.), pp. 146-156,
Springer Verlag, New York.
14. Chrisp, C. E., G. L. Fisher and J. E. Lammert. 1978.
Mutagenicity of filtrates from respirable coal fly ash.
Science 199:73-75.
15. Fisher, G. L., C. E. Chrisp, and 0. G. Raabe. 1979.
Physical factors affecting the mutagenicity of fly ash
from a coal-fired power plant. Science 204:879-881.
General Discussion
K. CHEN: I have a couple of questions on your depo-
sition experiments. What exactly is the mass medium dia-
meter of the surrogate aerosols, and, specifically, what is
the fraction of particles of one micron or below in that
surrogate aerosol?
A. BROOKS: I am not an aerosol physicist, but as you
heard Dr. Kittleson say there are many ways to measure the
size of these particles, and mass medium diameter evidently
is not the only or the best way. As nearly as we can tell,
the characteristics are very similar to diesel particles.
The diffusion diameter that was shown was 0.06 microns, and
most of the particles were less than a tenth of a micron.
They are chain aggregates, and you can see by looking at
them that they are similar. They behave aerodynamically in
a similar fashion.
K. CHEN: How did you calculate your deposition ef-
ficiency for these particles in adults? Were you able to
get the exact activity or did you assume certain respi-
ratory frequency?
A. BROOKS: They know the respiratory volume, the total
activity per respiration, and they know the total depo-
sition. From those they can calculate the total fraction
deposited as I understand it. They say that 32 percent of
the material is deposited.
K. CHEN: You did find about 18 percent in the layrnx
and in the trachea?
A. BROOKS: Yes. This is much lower than what you would
find with larger particle sizes.
E. CANTRELL: In your studies of the extraction of the
biological fluids, I notice that all you had was lipid
soluble fluids. It would give the impression that the
biological material is not effective in extracting the
hydrocarbons, or whatever they are, from the smoke parti-
cles. There have been a few recent reports using arti-
ficial membranes and human cells to show that lipid ma-
terial either in the cell membrane or in artificial par-
ticles is quite effective in leeching hydrocarbons that are
loaded on a particle, asbestos in particular.
357
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A. BROOKS: The mutagenic activity wasn't available for
the bacteria. I don't know whether it was taken off or not
and tied with the proteins, or whether it was inactivated.
We did try to re-extract with that polascitate to try to
pull it off the proteins if it was there. We weren't suc-
cessful. I think that Dr. Huisingh referred to several
attempts that she had made to find out where the mutagenic
activity has gone. At least we can't get it back from the
bacteria. It may be that it is just bound with the protein
and that mammalian cells can indeed use it.
J. HUISINGH: Do you shake or sonicate or what do you do
during the period that the particles are with the serum?
A. BROOKS: It is on a roller. The particles that are
in the serum are rolling in a 37 degree incubator so it is
a very mild treatment.
D. KITTLESON: Was the 0.06 microns you gave a number
weighted mean diameter?
A. BROOKS: No, that was a diffusion diameter I believe.
D. KITTLESON: Based on number, right?
A. BROOKS: I really don't know the answer to that ques-
tion. I think there are a lot of people at our place who
would really like to talk to you, Dr. Kittleson, about the
way that measurement was made and I am sure they could do
better than me.
D. KITTLESON: I was just going to say that I was gues-
sing it was a number of mean diameter, because if you look
at something like a Volkswagen Rabbit at normal load, the
number of mean diameter is around 0.06. The volume or mass
weight of mean diameter is then around 0.15, but you have
to remember there are different weightings.
That may have confused some of the people when they were
asking about what the effects of size were.
358
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DIESEL PARTICULATE MATTER
CHEMICAL AND BIOLOGICAL ASSAYS^
T. H. Risby
Department of Environmental Health Sciences
The Johns Hopkins University
School of Hygiene and Public Health
Baltimore, Maryland 21205
R. E. Yasbin
Department of Microbiology and Cell Biology
And S. S. Lestz
Department of Mechanical Engineering
The Pennsylvania State University
University Park, Pennsylvania 16802
ABSTRACT
This study involves the chemical characterization and bio-
logical assay of Diesel particulate matter generated from
a single-cylinder, direct-injected Diesel engine. This
engine was powered with a pure hydrocarbon fuel. The
total extract obtained with this fuel had a positive bio-
logical activity by various biological assays, although the
raw particulate matter was negative. The methodology of
sampling and the assays are discussed together with the
implications of the results to date.
INTRODUCTION
Currently there is considerable scientific evidence which
suggests that the particulate emissions from a Diesel or a
compression-ignition engine have mutagenic or carcinogenic
properties. This evidence is based on the results obtained
when the organic compounds extracted from the particulate
matter are subjected to various bacterial assays. In addi-
tion to the biological activity, the physical properties
of the particulate matter have also been studied and the
average diameters of 95% of the particles were found to
be less than 1 yM. Also, these particles have active
surface areas which would be capable of adsorbing other
combustion products. The combination of this information
represents a potential public health hazard since the
biologically-active particles are respirable.
359
-------
A large number of investigators are currently attempting
to assess this potential health hazard using various
approaches. The approach which has been adopted in this
research program is unique since it is attempting to
identify the compounds or groups of compounds which have
the biological activity and to relate their formation to
the fuel and engine operating conditions. The rationale
behind this approach is that it is necessary to first
identify the origins of the biologically-active compounds
before their emissions can be reduced to "safe" levels.
A multidisciplinary program, which consists of mechanical
engineers, chemists and toxicologists, has been estab-
lished at The Pennsylvania State University and at The
Johns Hopkins University. This group has the necessary
expertise to generate and collect Diesel particulate
matter and then to characterize it by chemical and bio-
logical assays.
This program is attempting to obtain the basic information
on the nature of the emissions from Diesel engines by
controlling the many interrelated variables. A pure
hydrocarbon fuel (2,2,4 trimethylpentane and tetradecane,
50% V/V, cetane number of 53) and a low-ash synthetic
lubricating oil were used in place of full-boiling-range
Diesel fuels and the traditional lubricating oils. The
chemical composition of. a full-boiling-range Diesel fuel
suffers from inherent variability as a result of the re-
fining processes, the origin of the.crude and the aging
of the fuel. Also, the chemical composition of the
Diesel fuel is unknown since the specifications of Diesel
fuels are based on their physical properties. Since the
composition of the fuel is unknown and the cylinder and
post-cylinder combustion chemistry is unknown, it is dif-
ficult to deduce the origin of the biologically-active
species. The use of a synthetic low-ash lubricating oil
is an attempt to minimize contributions to the emissions
from any partially combusted lubricating oil. In addition
to the fuel and lubricating oil the Diesel engine was run
at a constant set of operating conditions. The rationale
behind this careful control of variables was that it should
be possible to generate and collect reproducible parti-
culate matter which may be related to the fuel and engine
condition. The numbers of compounds in Diesel fuel and
in Diesel exhaust emissions are currently thought to be
greater than 500 and 10,000 respectively. These figures
show the magnitude of the problems facing studies which
are attempting to identify and quantify the biologically-
active compounds contained in particulate matter from
full-boiling-range Diesel fuels.
This report discusses some of the preliminary results from
our program in which a light-duty Diesel engine was
360
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operated as a chemical reactor under constant conditions.
Partial chemical and biological assays have been performed
using either the raw Diesel particulate matter or the
organic soluble extract. These results are compared to
others obtained in a companion study in which an Olds-
mobile 350 Diesel engine was run using a refereed full-
boiling-range Diesel fuel.
The chemical assays were performed using gas chromatography
and/or mass spectrometry and the Ames Salmonella/microsome
mutagenicity testl and the ]3, subtilis Comp test^ were used
for the biological assays. While the Ames test and the
Comp test can never substitute for animal and human epi-
demiology studies on potential health hazards, they do
provide rapid and economical ways of obtaining information
about mutagenic and/or carcinogenic activity of a large
number of uncharacterized compounds.
EXPERIMENTAL
Engine and Sampling Train
The exhaust particulate matter and gas phase emissions
were generated using a small, single-cylinder, four-stroke
cycle, air-cooled, direct-injected, Diesel engine (Lycoming
Bernard W-51). The raw undiluted exhaust was drawn from
the exhaust stream isokinetically. The particulate matter
can be collected on either 47 mM or 142 mM membrane
filters (PalIflex type T60A20 filter) contained in temper-
ature controlled filter holders. In addition to the
collection of the particulate matter, carbon monoxide,
carbon dioxide, oxygen, the oxides of nitrogen and the
total unburned hydrocarbons were monitored. More de-
tailed descriptions of the engine and sample train have
been published previously. ' The lubricating oil (UCON
LB-525) was maintained below 70°C with a heat exchanger
to reduce thermal decomposition. Before the particulate
matter was collected, the engine was allowed to stabilize
and the gas phase was monitored before, during, and after
the particulate matter was sampled. The gas phase analyses
were used to check stability and reproducibility. The
typical engine and sampling parameters are shown in Table
Table 1. Engine and Sampling Parameters
Air In-take
Temp °C
42°C
Load
3/4 Rack
CO CO^
% %
0.6 9.0
Air In-take
Press. Atm.
1.00
RPM
2400
02 NO
% ppm
8.5 760
Air /Fuel Lube Oil
Ratio Temp °C
21 62
BHP BSFC
3.80 0.600
Total Hydrocarbons
ppm
450
361
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Table 1. continued
Mass of Particulate Sampling Temp
(142mM Filter) (47mM Filter)
g
0.127
%
0.018
°C
44
°C
500
Chemical Assay
The membrane filters were weighed and then Soxhlet ex-
tracted with dichloromethane (approximately 80-100 cycles).
The extracts from a series of filters run under identical
conditions were combined (5 extracts for the 47mM filters
and 2 extracts for the 142mM filters) and the solvent was
removed by rotary evaporation. The resulting extract
^ 4.8% for 47mM filters and ^ 2.8% for 142mM filters) was
weighed and stored in a freezer until used. During ex-
traction, evaporation, storage, and assays exposures to
ultra-violet light and plastics were avoided as much as
possible.
Before any further steps were taken a gas chromatographic
fingerprint was taken using a Micropak column using the con-
ditions shown in Table 2.
Table 2. Gas Chromatographic Conditions for Fingerprint
1.5M, 6mM od, O.SmM id glass column
3% Dexsil 300 on 120-140 mesh Chromosorb W-HP
Flow Rate lOmL/min.
150°C for 1 min. 150-330°C l6°C/min. Hold at limit 2 min.
If the fingerprint of the extract agreed with previous ex-
tracts run under the identical engine conditions within a
reasonable error then the extract was submitted for chemical
and biological assays. The extract was then analyzed by gas
chromatography-electron iiapact mass spectrometry (Finnigan
4000 GC-MS with INCOS data system). The gas chromatographic
conditions are shown in Table 3.
Table 3. Gas Chromatographic Conditions for GC-MS
1.8M, 6mM od, 4mM id, glass column
3% OV-1 on 100-120 mesh Chromosorb W-HP
Flow Rate 30mL/min.
100°C for 3 min. 1QO-300°C at 10°C/min. Hold at limit 2 min.
362
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Additionally, the extract was analyzed by chemical ioni-
zation mass spectrometry using a solids probe inlet (BIO-
SPECT, Scientific Research Instruments Corporation with a
MODCOMP data acquisition system5) and by capillary column
gas chromatography using the conditions shown in Table 4.
Table 4. Capillary Column Gas Chromatography Conditions
6M., Quadrex glass capillary column coated with Carbowax 20M
Flow Rate lOOcm/sec.
50°C for I min. 50-150°C at 30°C/min. 150°-210°C at 5°C/min.
Biological Assay
Ames Test
This test system requires the use of genetically construc-
ted mutants of Salmonella typhimurium. These strains (TA
1535, TA 1537, TA 1538, TA 100 and TA 98) have been selec-
ted for sensitivity and specificity in being reverted from
a histidine requirement (auzotrophy), back to non-requiring
prototrophy. This mutagenic capacity has been correlated
to potential carcinogenic activity.
The following procedure was used to Ames test the total
extract. A known mass of extract was dissolved in di-
methyl sulfoxide and sterile distilled water was added to
give a starting concentration in 50% dimethyl sulfoxide.
An aliquot (0.1 mL) of the sample at various concentrations
was added to top agar (2.0 mL) containing a 12-16 culture
(0.1 mL) of the tester strain. This top agar, cell, and
sample to be tested were overlayered onto minimal glucose
agar plates and incubated at 37QC for 48 hours. After
this time the number of colonies which appeared on the
test plates and control plates were counted and recorded.
In addition to the extract the raw Diesel particulate
matter was also studied. The raw particulate matter sus-
pended in dimethyl sulfoxide was heated gently and then
diluted with sterile distilled water (50% soln.) to yield
the test solution. When testing with the S-9 microsomal
fraction, an aliquot of S-9 (0.5 mL) was added to the
top agar containing the bacterial cells and sample, just
prior to pouring the overlay.
B. subtilis Comp Test
This test system measures the potential of a chemical to
induce or activate DMA repair mechanism. The repair-spe-
cific Comp Test detects carcinogens based on the activa-
tion of the "SOS" system in clutures of B, subtilis, The
363
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"SOS" hypothesis was proposed in order to explain the fol-
lowing phenomena: error-prone repair, induction of pro-
phage, "W" reactivation and "W" mutagenesis, induction
of rec A+ gene product (protein X) and inducible post-
replication repair. Transformation experiments necessary
for obtaining the results of the Comp test are carried
out as described by Yasbin and colleagues.2 Essentially,
cells are grown in GMi broth, and for 90 minutes (T90)
after cessation of logarithmic growth (To). The cells
are then diluted 10-fold into GM£ broth, and incubated
at 37°C on a gyratory shaker for 60 minutes. At this
time, DNA extracted from strain RUB818 (1 to 5 yg/mL) is
added; the culture is incubated for 20 minutes before
the addition of DNAase (10 yg/mL). After an additional
five minutes of incubation, the carcinogenic activity
of the Diesel particulate is assayed. The transformed
cells are incubated with various doses of the Diesel
particulate-DMSO solution for 30 minutes at 37°C. After
this time the cells are centrifuged, the supernatant
discarded, and the cells resuspended in IX minimal salts.
The cells are then plated onto appropriate media for deter-
mining the percent transformation. Proper controls for
cells not exposed to the test samples are handled in the
same manner. The samples for assay were prepared in a
similar manner to that used in the Ames test.
RESULTS AND DISCUSSION
Sampling and Chemical Analysis
Considerable effort was expended in order to design and
construct the engine and sample train in order to control
all the engine and sampling parameters. One hundred and
forty data runs were made in order to establish a statis-
tically significant sample. The criteria used to judge
the sample generation and collection was the reproduci-
bility of the engine parameters, gas analyses and the gas
chromatographic fingerprint of the total extracts. A
typical fingerprint of the total extract is shown in Figure
1, The fingerprint is characterized by its shape and the
relative peak heights of the various solute peaks. The gas
chromatographic column and conditions were optimized for
the separation although the efficiency and resolution of
the separation are not sufficient for qualitative or quan-
titative analysis. For comparison purposes the gas chro-
matographic fingerprint of the total extract from a full-
boiling-range Diesel fuel is shown in Figure 2, This
particulate sample was obtained from an automobile Diesel
engine (350 Oldsmobile) cradled on a dynamometer. The
differences between these two fingerprints show graphi-
cally the advantage of using the pure hydrocarbon fuel as
opposed to the full-boiling-range fuel. The broad un-
resolved peak in Figure 2 is probably due to the presence
364
-------
of paraffin!c hydrocarbons. In all the gas chromatographic
studies it was apparent that only 5-10% of the mass of the
sample was reaching the detector and was irreversibly
adsorbed on the column packing material. This loss in
sample had a serious effect when a mass spectrometer was
used as a selective detector as it limits the identifica-
tion of solute peaks.
An interesting observation was made when it was decided to
switch from 47 mM filters to 142 mM filters in an attempt
to collect larger samples more quickly. The larger filters
have a lower pressure drop as a result of filter clogging.
When the mass of the total extract from the particulate
samples collected on the different sized filters were com-
pared,, the particulate matter from the 47 mM filters had a
much greater mass extracted than the particulate matter
from the 142 mM filters (4.8% versus 2.8%). The only dif-
ference between these extractions was the size of the Sox-
hlet apparatus and sufficient data has been collected to
ensure that this result is statistically valid. This
drastic difference in extraction cannot be easily explained.
A possible explanation could be that since the particulate
matter is collected in a more dense manner in the 47 mM it
acts as a very efficient adsorption surface for any organic
molecules in the gas phase. Conversely, the particulate
matter on the 142 mM filter is less dense and the gaseous
organic molecules have a lower probability of adsorption.
Currently we do not have sufficient data to prove or dis-
prove this hypothesis. However, if this theory were cor-
rect, it would present a major impact on the study of
Diesel exhaust particulate matter because it would mean that
it is impossible to collect, by the use of filters, parti-
culate matter which is chemically representative of the
particulate matter emitted frcm the Diesel engine. Addi-
tionally, if these volatile organic molecules were the
molecules which produced the biological activity, then
the potential health impact of Diesel exhaust would have
to be totally reassessed. This would mean that the gas
phase would have to be collected and studied with chemical
and biological assays.
In order to remedy the sample loss on packed columns a pre-
liminary study was performed using capillary column gas
chromatography using a splitless injection technique. A
gas chromatogram of the total extract from the pure hydro-
carbon fuel is shown in Figure 3. Although the coating
of the capillary column used for this separation was not
the optimum, it was possible to separate this extract into
72 peaks. It is reasonable to expect that with a less
polar column and different operating conditions then many
of the peaks will be resolved into multi-component peaks,
However, it must be stressed that this extract was obtained
365
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with the pure hydrocarbon fuel and constant engine opera-
ting conditions which shows the wealth of Diesel combustion
chemistry. The sample loss through the column was small
compared to packed-columns.
Some of the total extracts have been analyzed with gas chro-
matography-electron impact mass spectrometry. Although the
column used for this study had lower efficiency than the
Micropak and capillary columns and had significant column
bleed, it was possible to make some tentative identifications
of compounds contained in the total extract. The gas chro-
matographic-mass spectrometric data was analyzed using the
Biller-Biemann peak searching routine6 and then the mass
spectrum at the peak maximum was compared to the library of
mass spectra (INCOS library of NBS mass spectra). Approx-
imately 35 peaks were obtained of which 16 were major peaks
but only 7 of these peaks could be identified with signifi-
cant confidence. The compounds which have been tentatively
assigned are shown in Table 5. The plasticizers present in
Table 5. Computer Fit of Mass Spectral Data
Peak #
1
2
3
4
Fit
970
954
922
902
876
932
909
980
980
Compound
9H-Fluoren-9-one
Benzo/C/cinnoline
Phenanthrene
1,1' - (1 , 2-ethynedyl) bisbenzene
Anthracene
Benzo/C/cinnoline
9H-Fluoren-9-one
Butyl-2-methylpropylphthalate
Dibutylphthalate
5 995 lH,3H-naphtho/l,8-CD/pyran-l,3-dione
6 974 Pyrene
972 Fluoranthene
7 894 Benzo/GHl/fluoranthene
the sample (peak #4) are probably there as contamination
which occurs as a result of the contact of dichloromethane
with any plastic material. Three total extracts were ana-
lyzed and of the 7 peaks which had a tentative assignment
6 appeared in each sample and the relative quantitations of
these peaks are shown in Table 6. There is reasonable
agreement in these relative quantitations between samples
369
-------
Table 6. Summary of Relative Quantitation
Sample #
BS6
BS7
BS8
BS6
BS7
BS8
BS6
BS7
BS8
BS6
BS7
BS8
BS6
BS7
BS8
Compound
Phenanthrene
l,l'-(l,2-ethynediyl)bisbenzene
Anthracene
Benzo/C/cinnoline
9H-Fluoren-9-one
lH,3K-naphtfto/l, 8-CD/pyran-l ,3-dione
Pyrene
Fluoranthene
Benzo/GHT/fluoranthene
% Total
18.7
14.3
18.8
4.3
2.3
2.9
21.0
7.3
14.4
26.4
23.5
21.6
2.9
1.6
1,9
particularly since the packed column could only partially
separate the total extract. The fits of the mass spectra
of other solute peaks to the library mass spectra were not
significant since it is difficult to deconvolute mass spec-
tra of mixtures of solutes at varying concentrations.
The use of a solids probe with chemical ionization mass
spectrometry was disappointing in that no new compounds
were identified. This was caused by sample loss during
the insertion of the probe and by the plasticizer impuri-
ties which overwhelmed the mass spectral data, Currently
we are attempting to avoid these problems by the use of
a temperature programmed solids probe and by coupling a
capillary gas chromatograph to a chemical ionization mass
spectrometer. The impurities due to plasticizers have been
reduced to a minimum by avoiding any contact with plastic
materials.
Bioassay
A statistically significant series of the total extracts
from Diesel particulate matter were subjected to both the
Ames test and the j?, subtilis Comp test and the data from
these studies are shown in Tables 7, 8 and 9.
The results of these tests, whichareused to screen chemical
compounds as potential carcinogens, indicate that the total
370
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extract presents a potential public health hazard. Of the
samples which gave negative results in the Comp Test (BS 1,
BS 3, BS 10, BS 11) samples BS 1 and BS 3 were later found
to have contained large quantities of plasticizers which
could cause the extract to precipitate. Samples BS 10 and
BS 11 were Comp Test negative although they were Ames Test
positive, we do not have a satisfactory explanation for the
divergence.
Some preliminary studies have been initiated in which the
raw particulate matter has been subjected to the Ames Test
and the Comp Test. In addition, the Ames Test was run
with the addition of a microsomal S-9 fraction in order to
check for the presence of mutagens which require activation.
The rationale behind this decision was that it is the raw
Diesel particulate matter which is present in the air and
not the total extract. Also, we wished to check to see if
the biological activity was changed as a result of Soxhlet
extraction. Samples of particulate matter which were gener-
ated using the pure hydrocarbon fuel and with full-boiling-
range Diesel fuels were subjected to these bacterial assays
and the data are shown in Tables 10, 11 and 12. The parti-
culate samples from the full-boiling-range Diesel fuels were
collected under different load conditions.
Table 10. Results of Ames Test Raw Diesel Particulate from, a
Pure Hydrocarbon Fuel.
Raw Particulate
RPM 2400, Full Rack
AF^20 Concentration (yg/plate)
TA 98
0 197 485 970
CSpontaneous
revertants)
61
64
39
21
41
32
36
34
50
47
32
30
49
53
35
36
With S-9 Activation
83
89
41
47
51
40
48
42
62
74
44
37
56
61
51
66
Raw Particulate
RPM 2400, Full Rack
AF *> 20
TA 100
Concentration (JJg/plate)
199
231
121
116
183
187
120
133
167
178
118
111
194
211
127
131
377
-------
Table 10. continued
Concentration 0Jg/plate)
TA 100
0 197 485 970
With S-9 Activation
212 211 184 196
214 200 199 162
140 143 134 161
137 150 143 135
The raw diesel particulate was soluble in 50 % DMSO.
Table 11. Results of Ames Test with Raw Diesel Particulate
from a Full-Boiling-Range Diesel Fuel.
Raw Particulate
1/2 Rack, 2000 RPM
AFV36 Concentration (ug/plate)
TA 98
0 56 112 224 548 1096
(Spontaneous
revertants)
50
41
41
48
59
74
64
58
58
51
71
63
64
52
With S-9 Activation
60 61 51 63 75 61
53 49 59 68 82 51
58
51
TA 100
187 184 181 181 211 196
180 167 297 183 223 201
172
169
With S-9 Activation
197 201 187 200 231 218
190 179 199 181 211 213
178
210
378
-------
Table 11. continued
Raw Particulate
3/4 Rack, 2000 RPM
TA98
0 167
(Spontaneous
revertants)
With S-9 Activation
58
60
59
49
TA 100
61
83
Concentration (yg/plate)
333
47
32
58
56
666
54
42
53
58
1332
42
51
47
41
40
41
41
49
38
60
46
41
49
43
68
52
63
67
171
191
165
181
With S-9 Activation
193
198
163
191
200
169
189
197
198
204
189
222
162
195
201
224
218
189
183
197
211
193
167
Raw Particulate
Full Rack, 2000 RPM
AFX-20
TA 98
Concentration (M g/plate)
125
250
500
(Spontaneous
revertants)
44
57
63
41
51
48
37
31
70
64
56
60
74
61
83
68
379
-------
Table 11. continued
TA 98
Concentration (jig/plate)
125
250
500
With S-9
61
58
53
51
Activation
62
49
53
59
73
83
75
89
81
59
47
84
Raw Particulate
Full Rack, 2000 RPM
AF 20
TA 100
Concentration (ug/plate)
171
192
201
200
187
183
169
191
With S-9 Activation
201 193
205 210
223 216
196 211
211
201
203
208
221
215
200
195
210
190
187
222
208
193
186
219
Raw Diesel particulate samples from the Oldsmobile Diesel
engine were not soluble in DMSO. The mass of sample to be
tested was placed in DMSO, heated gently, and sterile dis-
tilled H20 was added to yield a 50% DMSO solution.
380
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All these samples proved to be Ames Test negative with or
without microsomal fraction. Only one sample (Full^boiling-
range Diesel fuel 1/2 rack) appeared to give positive re-
sults in the Comp Test, however we do not have sufficient
results to know whether this result is significant.
CONCLUSIONS
This model approach to Diesel engine exhaust emissions has
shown that simple hydrocarbon fuels produce exhaust parti-
culate matter which has biological activity. This suggests
that it is the reaction mechanisms found to occur under
engine combustion conditions which account for this biologi-
cal activity. However, although the biological activities
were observed for the total extract the raw particulate mat-
ter did not exhibit similar properties. This anomaly may
be very significant since it could mean that the compounds
adsorbed or chemisorbed onto the particles are not readily
solubilized under physiological conditions and therefore
do not represent a serious health hazard. However, since
the particles are smaller than 1 pM in diameter they can
be deposited in the alveoli and the clearance time could
be sufficiently long to allow the compound to become bio-
available via the macrophage. An alternative explanation
is that it is purely a concentration effect since the
total extract only represents 4-8% of the mass of the raw
particulate matter. Currently studies are in progress to
measure the heats of adsorption of various compounds onto
particulate matter in an attempt to estimate the magnitude
of the adsorption energies. Also the size, shape, surface
area and porosity are under investigation. No attempt has
been made to confirm the preliminary identification of the
compounds found in the total extract nor have their biolo-
gical activities been measured, although this work is in
progress. The effect of filter size on the percent extrac-
tion is very interesting and could be very significant since
if the use of filters to collect particulate matter does
produce particulate matter enriched with organic compounds,
then the biological activity of Diesel particulate matter
may be overestimated.
Another aspect of the use of filters to collect particulate
matter is that the particulate matter may be subject to
chemical reactions as a result of volatile reactive species.
An excellent recent publication by Pitts? has shown that
henzoCa)pyrene reacts with nitrogen dioxide to produce
mononitrobenzo(a)pyrene which is mutagenic without activa-
tion. It is reasonable to expect that other compounds could
undergo similar reactions with the oxides of nitrogen which
are present in Diesel exhaust, This hypothesis may also be
significant when the overall public health assessment is
made. It is interesting to note that the bacterial
382
-------
activities of the extracts from particulate matter of the
pure hydrocarbon fuel and the full-boiling-range fuel do
not differ significantly. These results suggest that the
bacterial activities which have been observed for extracts
from particulate matter from Diesel engines are the result
of combustion and are not artifacts from the fuel. It is
expected that the methodology discussed herein will provide
chemical identification of the biologically active compounds
contained in Diesel exhaust emissions.
Tn future studies we will expand our studies with raw parti-
culate matter to include animal and macrophage tester sys-
tems. Also, we plan to vary the engine operating condi-
tions and blend additional fuels with our pure hydrocarbon
fuel.
ACKNOWLEDGEMENTS
Financial support was provided by a grant from the U.S.
Environmental Protection Agency (#806558010) administered
through the School of Hygiene and Public Health of The
Johns Hopkins University. We wish to thank the following
people who contributed to the success of this project:
Mich Dukovich, Keith Houser, James A. Yergey and William
Suits.
REFERENCES
1. Bruce N. Ames, ''A Bacterial System for Detecting Muta-
gens and Carcinogens", H. E. Sutton and M, I. Harris
(_Eds.), in Mutagenic Effects of Environmental Contami-
nants, Academic Press, New York, 57-66, 1972,
2. Ronald E. Yasbin and Rosemarie Miehl, "DNA Repair in
Bacillus subtilis: The Development of Competent Cells
into a TESTER for Carcinogens'1. In review for publica-
tion, Applied § Environmental Microbiology.
3. R, L. Bechtold and S, S. Lestz, "Combustion Character-
istics of Diesel Fuel Blends Containing Used Lubricat-
ing Oil", SAE Paper No. 760132 February, (1976).
4. S. R. Prescott, T. H. Risby, R. E. Yasbin and S. S,
Lestz, "Sampling, Chemical Characterization and Bio-
logical Assay of Exhaust Particulate Matter from a
Light-Duty Diesel Engine". In review for publication,
Environmental Science § Technology.
5. J. E. Campana, P. C. Jurs and T, H. Risby, "Principles
and Applications of a Research-Oriented Gas Chromato-
graphy-Mass Spectrometry-^Data System" Anal. Chim. Acta
Comp. Tech. Optimiz. In Press.
383
-------
6. J. E. Biller and K. Biemann, "Reconstructed Mass Spec-
tra - A Novel Approach for the Utilization of Gas
Chromatography-Mass Spectrometer Data". Anal, Lett.,
7, 515, (1979).
7. J. N. Pitts, Jr. "Photochemical and Biological Impli-
cations of Atmospheric Reactions of Amines and Benzo
(a)pyrene" Phil. Trans R. Soc. Lond. A 290, 551, (1979),
General Discussion
0. DAISEY: Could you tell us how you determined your
loss of material on your GC column, the dexal column versus
the capillary column?
T. RISBY: I made an estimate in terms of math and in
terms of response of the flamminization detector versus the
amount injected. When I was making the injections, it
seemed that the response - that is, the amounts of signals
that I should get versus the amount that I was getting, was
very much lower.
J. DAISEY: Were the amounts injected the same for both
these types of columns?
T. RISBY: For the count, yes. We were injecting one
microliter of the same concentrations and we were using
split injections.
J. HUISINGH: I don't think it is proper to say you are
testing the particle alone when you suspend it in dimethyl
sulfoxide, because it has been clearly shown by a number of
different people that the DMSO is also extracting some
materials.
384
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DIESEL PARTICIPATE EXTRACTS IN CULTURED MAMMALIAN CELLS
Colette J. Rudd
Biomedical Science Department
General Motors Research Laboratories
Warren, Michigan 48090
ABSTRACT
It has been reported that participate material from diesel
engine exhaust contains bacterial mutagens. To determine
whether mammalian cells are sensitive to these or other
mutagenic substances, extracts of the diesel particulates
(DP) have been tested in the Chinese hamster V79 cell line
by our laboratory and in the mouse lymphoma cell line
L5178Y by Litton Bionetics. Two dichloromethane extracts of
DP were tested for cytotoxicity and mutagenic activity at
concentrations up to 350 yg of extracted material per mL
culture medium.
Diesel particulates were collected from the exhaust of a
General Motors diesel engine at 100°C using Pallflex filters
or an electrostatic precipitator. Extracts were produced by
Soxhlet extraction with dichloromethane for 4 hours.
Treatment of the V79 cells with up to 100 yg diesel extract
per ml of culture medium for 5 to 16 hours decreased cell
survival substantially compared to control cells, but
resulted in no significant changes in their mutation fre-
quency. Addition of a rat liver enzyme preparation reduced
the relative toxicity of the diesel extract with no increase
in the mutation frequency. Concentrations of 200-350 yg/mL
were active in the mouse lymphoma cell assay, although the
extract was also very toxic at these levels.
385
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INTRODUCTION
Concern about the health effects of diesel exhaust particu-
lates has centered on their potential action as carcinogenic
agents. The bacterial assay system (Ames test) has detected
mutagens associated with the diesel participate. Although
valuable as a preliminary screening test, this assay may not
reliably indicate the relative potency of chemicals for
mammalian cells because of their more complex cell structure
and/or different metabolic pathways. The mutagenic activity
in an extract of diesel particulates may also vary, depending
on the type of engine and collection and extraction methods
[1,2].
We have tested extracts of diesel particulate from a GM 5.7 L
engine produced by Oldsmobile for mutagenic activity in
Chinese hamster V79 cells. In addition, one sample was
tested under contract by Litton Bionetics using the mouse
lymphoma L5178Y cell assay system described by Clive and
Spector [3]. These extracts were all mutagenic to the
bacterial strain Salmonella typhinaa-ium TA98 [4].
The V79 cells were one of the first cell lines used for the
demonstration of the chemical induction of mutations in
mammalian cells [5]. The permanent alterations in the
cellular DNA are translated into specific RNA and protein
molecules and can be detected with selective conditions which
allow the mutated cell to grow where normal cells die. The
V79 cells have been used here with the selective agents
ouabain, which is thought to select for cells with an altered
MA+,K+ATPase, and 6-thioguanine and 8-azaguanine, which
select for cells deficient in the enzyme hypoxathine-guanosine
phosphoribosyl transferase (HPRT). These selection procedures
are the ones used most frequently with the V79 cell line.
Because only one functional HPRT gene appears to be present
in the cells, a larger number of mutants can be recovered
than if two genes must be inactivated before the cells can be
selected. Although the Na , K ,ATPase gene may be present in
more than one copy, the selection process is feasible because
ouabain selects for a "dominant" mutation; only one gene must
produce an enzyme not inhibited by ouabain for the cells to
survive. In the mouse lymphoma cell assay, the thymidine
analogues 5-bromo-2' deoxyuridine or 5-trifluorothymidine
select for cells which have lost their thymidine kinase
activity. The L5178Y cells in this assay have been previously
selected so that they contain only one active thymidine
kinase gene.
386
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METHODS
The Chinese Hamster V79 cell line was initially obtained from
Dr. E. H. Y. Chu of the University of Michigan. The cells
grew as a monolayer in plastic dishes containing Dulbecco's
Modified Eagle's medium (DME) supplemented with 5% heat-
inactivated fetal calf serum. They were incubated at 37°C in
10% C02 and routinely subcultured twice a week after treatment
with 0.05% trypsin to dissociate them from the dish. The
cell number in the trypsinized cell suspensions was determined
using an electronic particle counter.
Diesel particulates were generated by a General Motors 5.7 L
diesel engine produced by Oldsmobile. Two different samples
of particulates were extracted for testing with V79 cells.
The first sample, DP(1), was collected on a Pal Iflex filter
at 100°C exhaust temperature from an engine burning Marathon
#2 fuel. The second sample, DP(2), was collected at 100°C on
an electrostatic precipitator from an engine burning Amoco 2D
federal compliance fuel. Both particulate samples were
extracted with dichloromethane on a Soxhlet apparatus for 4
hours. The solvent was then evaporated and the residue
weighed to calculate the percent extractable material. DP(1)
and DP(2) yielded 4.7 and 8.9% extractable material, respec-
tively, labeled DPE(l) and DPE(2). The particulate sample
collected for testing in the mouse lymphoma cell assay was
similar to DP(2), except it contained 11.6% extractable
material. The collection and extraction conditions are
described by Chan et al [4].
The diesel extracts were dispersed in dimethyl sulfoxide
(DMSO) to make stock solutions of up to 35 mg/ml. This was
well mixed and diluted in culture medium immediately prior to
cell exposure. The final concentration to which the cells
are exposed is referred to as the amount of diesel extract
per ml of culture medium. A typical concentration, 100
yg/mL, is 100 yg of extractable material from diesel particles
per ml of culture medium. The total amount of extract in the
exposure period is the concentration (ug/mL) times the volume
of medium on the cells (5 mL/25 cm2 flask, 10 mL/75 cm2
flask). The amount of diesel particles which would give an
equivalent exposure level is dependent on their extract-
ablility; for example, cells in 10 ml of culture medium with
100 yg/ml of DPE(2) are exposed to substances from a total of
11 mg of whole particles.
For activation in the V79 cell system, a microsomal enzyme
preparation (S9) was prepared as described in [6] from rats
induced with 3-methylcholanthrene. S9 (8-20 yL/mL culture
medium) was added in some experiments to investigate the
effect of enzyme catalyzed alterations of the diesel
387
-------
extract. Cofactors glucose-6-phosphate (0.15 mg/mL) and
NADP (0.3 mg/mL) were added with the S9 as specified. The
S9 used by Litton Bionetics was prepared from Aroclor-
induced rats; 50 yl/ml was used in the cell suspension to
study activation.
Cytotoxicity
Survival of the V79 cells in each experiment was investigated
by measuring their relative cloning efficiency. During the
first subculture of cells after the exposure period, 400
cells are plated separately in small culture dishes (60 mm
dia., 100 cells/dish). Each viable cell will attach and
divide many times (one cell division per 12-15 hours) to
form a colony about 1-2 mm in diameter within a week. These
colonies are stained and counted. One hundred untreated
cells will normally form 70-80 colonies (based on ^15 experi-
ments), giving a cloning efficiency of 70-£
In a specific experiment to measure the cytotoxicity of
DPE(2), cells were plated in 12 small dishes (4 x 105
cells/ 35 mm dia. dish) and exposed for 16 hours to concen-
trations of DPE(2) from 0 to 100 yg/mL. After exposure,
the cells were trypsinized and 400 cells from each dish were
plated and cultured as described above.
In the mouse lymphoma assay system, cytotoxicity is measured
as the reduction in growth of the cells in suspension com-
pared to the growth of untreated cells. Their plating
efficiency is measured only at the end of the expression
period for calculation of the number of viable cells treated
with the selection agent.
Mutagenicity
The mutagenicity assay conditions are listed in Table 1.
V79 cells were plated in 25 or 75 cm2 culture flasks (1-
4xl06 cells/flask, 1 flask/test condition) and incubated at
least 5 hours before exposure to test chemicals. The
substances to bef tested were first dissolved or diluted in
DMSO to make a solution 100 times the final concentration.
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]-
pyrene (B[a]P) were used as positive controls for direct-
acting and indirect-acting mutagens, respectively. To start
the exposure period, the solutions of chemicals in DMSO were
added directly to a flask containing cells and a known
volume of culture medium. For investigation of enzymatic
activation, S9 and its cofactors were mixed with the culture
medium prior to its addition to the culture flask.
388
-------
r- CO i j- «-
> Lf> 3 "O
S- _l >,-r-
S- O X E
O) fO O >>
MO) E OJ -C
W) C O T3 -t->
e - ^ i o
to c: CL- i-
^ «3 E , I 3
O) Ol _] O i
tn O E f-
OJ - OJ O -f-
O I
E U-J
389
-------
At the end of the exposure period, the cells were washed
with two changes of normal medium. Cells in each flask were
trypsinized, counted and replated. For selection with
ouabain, 106 cells or more were plated in 100 mm diameter
dishes (l-2xl05 cells/dish). Medium containing 1 mM ouabain
was added 2 days later.
For selection with 6-thioguanine (6-TG) or 8-azaguanine (8-
AG), an expression period of at least 6 days is required for
optimum recovery of the mutant cells. During this time, the
cells were subcultured 2 or more times because of their
rapid growth rate. At the final subculture, 5x1O5 cells or
more were plated (0.5-lxlO5 cells/100 mm dish). The selec-
tion agent was added after 1-3 days. Concentrations of 30
yg/mL 8-AG and 4 yg/mL 6-TG were used. The culture medium
with 6-TG consisted of DME and 5% dialyzed fetal calf serum;
undialyzed serum is reported to contain substances which
interfere with 6-TG uptake in the cells.
Cells were incubated with the selection agents at least 7
days, then were fixed with 100% ethanol and stained with
Giemsa. Colonies of more than ^50 cells were counted.
The mouse lymphoma assay was performed by Litton Bionetics
as described by Clive and Spector [3]. Briefly, the cell
suspensions were treated for 4 hours with the test materials.
Ethylmethane sulfonate (0.5 yL/ml) was used as a direct-
acting positive control; dimethylnitrosamine (0.3 yL/ml) as
a positive control for activation of mutagens by S9. After
two days the cells were plated in agar (0.35%) with the
selection agents 5-bromo-2'-deoxyuridine (100 yg/ml) or 5-
trifluorothymidine (3 yg/ml).
RESULTS
Cytotoxicity
The cellular toxicity of the diesel extracts (DPE) was an
important factor in these studies as detection of small
increases in the mutation frequency requires that a substan-
tial number of cells (105 or more) must remain viable after
the treatment. This survival factor must be balanced against
the fact that the mutation frequency (number of mutants/
number of suriving cells) usually increases with the dose of
the chemical.
Preliminary experiments with the V79 cells showed that
diesel extracts at 100 yg/mL (yg DPE/ml culture medium) were
fairly toxic, but enough viable cells could be recovered
after the treatment to determine the mutation frequency.
This dose was therefore chosen as the most likely dose for
390
-------
detecting mutagenic activity. A lower dose, 50 yg/mL, was
usually much less toxic and was also tested in some experi-
ments. The toxicity was quantitated by the cloning effici-
ency of the cells after treatment. The values are shown for
the experiments in Tables 2, 3, and 4.
The cloning efficiency of V79 cells was also measured after
exposure to additional concentrations of the extract DPE(2).
The results of this experiment are shown in Fig. 1. There
is a dose-dependent decrease in the viability of the cells
at concentrations between 25 and 100 yg DPE/mL culture
medium; at 25 yg/mL or less there is little or no difference
from the control cells. The cytotoxic effect is visible
microscopically as cells exposed to 100 yg/mL of extracts
change from the normal flat, irregular shape to a round ball
which is more loosely attached to the culture dish (Fig. 2).
Mutagenicity
The initial mutagenicity experiments with the V79 cells
investigated the activity of DPE(l), the extract of diesel
particles collected by filtration. The results with this
sample are shown in Tables 2 and 3. The method reported by
Chu [5] was followed in the first experiments, using the
selection agent 8-azaguanine. In Table 2, experiment A,
MNNG has induced the frequency of the azaguanine-resistant
cells over the spontaneous frequency seen in cells exposed
only to DMSO. B[a]P, which requires activation, and DPE(l)
have no effect. In experiment B, the mutation frequency of
cells exposed to B[a]P and S9 appears to be increased, but
DPE(l) at 50 yg/mL culture medium has no significant effect
with or without S9.
In Table 3, results of one experiment comparing three differ-
ent selection agents are shown. The response to three con-
centrations of MNNG was investigated. Duplicate flasks were
treated with each condition to compare the variability
within a particular experiment. The exposure time was also
lengthened to 16 hours, to increase the possibility of
detecting a less active compound.
No distinct colonies were detected in the control plates
(DMSO-treated). The frequency is calculated as less than 1
mutant per total number of surviving cells (at least 5 x 105
cells for each treatment). The mutations induced by MNNG
show a dose-dependent response for all selection agents,
even though the initial toxicity was high. The values of
the duplicate plates are close in most cases. DPE(l) at 50
yg/mL culture medium showed no activity at all; at 100
yg/mL, no colonies were selected with 6-TG while a few were
apparent with 8-AG and ouabain. The resulting frequency
391
-------
TABLE 2 PRELIMINARY STUDIES:
INDUCTION OF 8-AZAGUANINE RESISTANCE IN V79 CELLS
Cloning 8-AG-resistant
Expt. Conditions* Dose (ng/ml S9 (0.8%) Efficiency colonies/105
culture medium) (%) cellst
DMSO-1%
DPE(l)
B[a]P
MNNG
100
2
2
77
13
74
34
2.2
0.8
0.2
30.4
DMSO-0.5%
DPE(l)
B[a]P
50
2
67
71
52
57
57
52
1.0
0.2
0.2
1.4
0.2
5.2
*Treat 4 x 106 cells/25 cm2 flask/condition in DME for 5 hour exposure period.
tSelection agent 8-azaguanine (30 yg/ml) added 10 days (A) or 11 days (B)
after exposure period. Cells subcultured days 1, 4, and 7 (A) or days 2, 4,
6, and 9 (B) after exposure period. A total of 5 x 105 cells/condition
were plated at the final subculture for selection of resistant colonies.
392
-------
TABLE 3 COMPARISON OF SELECTIVE AGENTS:
OUABAIN, 8-AZAGUANINE, AND 6-THIOGUANINE
#Resistant Colonies/105 Surviving Cellst
Conditions* Cloning
Efficiency Ouabain 8-Azaguanine 6-Thioguanine
DMSO - 1%
MNNG
0.5 ug/ml
MNNG
1.0 ug/ml
MNNG
2.0 ug/ml
DPE(l)
50 pg/ml
DPE(l)
100 ug/ml
86
32
10.2
13.4
3.6
3.2
63
49
13
16
< 0.2
< 0.2
7.3
18.4
15.3
44.4
51.4
< 0.2
< 0.2
3.5
5.3
< 0.2
< 0.2
8.0
26.2
23.8
40.6
43.6
< 0.3
< 0.3
0.3
1.2
< 0.2
< 0.2
4.2
19.8
12.4
22.4
33.0
< 0.3
< 0.3
< 0.3
< 0.3
*Plated 106 cells/25 cm2 flask, 2 flasks/condition. Cells were exposed for 16
hours to MNNG (0.5-2 yg/ml culture medium) or DPE (50-100 ug DPE/mL culture
medium).
tAt the end of the exposure period, the V79 cells were plated for selection
with ouabain after a 2 day expression period or with 8-azaguanine and
6-thioguanine after a 6 day expression period.
393
-------
TABLE 4 EFFECT OF DPE(2) ON MUTATIONS TO OUABAIN RESISTANCE
Control
DPE(2)
DPE(2)
MNNG -
Conditions*
(1% DMSO)t
- 75 yg/mlt
- 100 ug/mltt
2 ug/mltt
Cloning
Efficiency
(%)
92
34
35
3
Ouabain
Resistant
Colonies
1
1
1
323
Mutation
Frequency/105
Surviving Cells
0.03
0.1
0.05
153
*Plated 3 x 106 cells/75 cm2 flask/condition 7 hours before exposure period.
Treated for 16 hours, then subcultured for expression and cloning efficiency.
Ouabain was added after 2 days for selection of resistant colonies.
tPlated 3 x 106 cells/30 culture dishes for selection; 500 cells/5 dishes
for cloning efficiency.
t+Plated 6 x 106 cells/30 culture dishes for selection; 1000 cells/5 dishes
for cloning efficiency.
394
-------
o
LL.
U- o.
LJU
CD
*i
O
I
O
r~
50
100
D1ESCL CXTRRCT,
Figure 1 Cytotoxicity of diesel extract for Chinese
Hamster cells. Cells were treated for 16
hours with various concentrations of DPE(2)
expressed in pg DPE/mL culture medium, as
described in Methods. Each marker indicates
the cloning efficiency of 400 treated cells.
395
-------
Figure 2 Appearance of V79 cells after 16 hour incuba-
tion with or without diesel extract. Cells
were plated (2.7 x 105 cells/75 cm2 flask) 6
hours before addition to medium containing
(a) 1% DMSO or (b) 100 pg DPE(2)/mL culture
medium.
396
-------
with 8-AG is still lower than what might occur spontaneously
(see Table 2), while the result with ouabain is higher than
expected.
For the remaining experiments, a dichloromethane extract of
diesel particles collected by electrostatic precipitation,
DPE(2), was obtained. Its effect on mammalian cells was
first investigated using ouabain as the selective agent
(Table 4). No induction of mutations by DPE(2) was seen,
with only one colony recovered from more than Id6 surviving
cells. MNNG, however, was very active.
Fig. 3 summarizes several separate experiments comparing the
frequency of thioguanine-resistant colonies in V79 cells
exposed to the test solvent DMSO (1%), DPE(2), benzo[a]-
pyrene, or MNNG. S9, a supernatant of 3-methylcholanthrene-
induced rat liver homogenate, was added to investigate the
possibility that some substances in the diesel may be acti-
vated by enzymes not present in the cells. B[a]P was tested
as a positive control for activation. The results show that
cells treated with the diesel extract are not different from
the solvent-treated cells either with or without addition of
S9. Although not shown, concentrations of 50 ug/mL diesel
extract also produced no increase in mutations. B[a]P
increases the mutation frequency when in the presence of S9,
although it is much less effective than MNNG.
Microsomal Enzymes and Diesel Extract Cytotoxicity
Although the diesel extract with added S9 was not mutagenic
to the V79 cells, the S9 did appear to reduce the toxicity
of high concentrations of the extract, even though the S9
itself was toxic. To verify this observation, the cloning
efficiency of the V79 cells was measured after treatment
with DPE(2), 0 to 100 yg/mL, in the presence or absence of
S9. The possibility of adsorption versus enzymatic conver-
sion was investigated by comparing S9-treated cells with and
without the cofactors NADP and glucose-6-phosphate, required
by most metabolizing enzymes. The results of this experi-
ment (Fig. 4) confirmed the initial observations that with
59 plus cofactors, there is little increase in toxicity from
low to high concentrations of diesel extract and that S9
addition is responsible for the largest amount of cell
death. Adsorption of toxic components in the extract to the
lipid and protein components in the S9 may provide some of
the protection, as cell survival was increased with S9
alone. Addition of the cofactors further reduced the toxi-
city of the extract, implying that the metabolizing enzymes
are also important.
397
-------
400
200
Colonies
per
105 Cells 6
3
0
O-S9
R+S9
I*
A
JQ^ *s^f
CK ^^
f^*
Treatment
Figure 3 Effect of diesel extract DPE(2) on the fre-
quency of 6-thioguanine resistant colonies.
The mutation frequency was compared for cell
populations treated with DMSO (1%), diesel
extract, benzo[a]pyrene, or MNNG, with or
without the addition of S9 (20 uL/mL) as
indicated. Cells were initially plated (3 x
106 cells/75 cm2 flask) 6 hours before treat-
ment period of 16 hours. The expression
period was 8 days. Cells were treated with
6-TG after being plated two days previously
at 5 x 104 cells/100 mm dish, using total of
^106 cells/condition/experiment.
398
-------
IQO-i
10- 1 1 r
0 20 40 60
DIESEL EXTRFiCT
80
100
Figure 4 Comparison of the cytotoxicity of diesel
extract DPE(2) with and without S9 and enzym-
atic cofactors. Cells were incubated for 16
hours in their normal culture medium (A) or
in medium containing 20 |jl/ml S9 with (x) or
without (a) the cofactors NADP and glucose-
6-phosphate. After the treatment period, the
cells were analyzed for their cloning effici-
ency as described in Figure 2.
399
-------
Mouse Lymphoma Cell Assay
The results with the V79 cells were supported by studies
performed on a similar sample by Litton Bionetics Laboratory
in the mouse lymphoma cells (Fig. 5). The growth of the
cells in normal medium after treatment with the diesel
extract was initially inhibited when compared with the
untreated cells. This is an indication of the toxic effect
of the treatment, a combination of cell death and reduction
in the growth rate. The presence of S9 in the culture
medium reduces the toxicity of the extract to the extent
that about twice as much extract is required to produce the
same effect. This enables higher concentrations to be
tested in the presence of S9 as a sufficient number of
surviving cells can be obtained for mutagenicity testing.
As reported by the testing laboratory, the results of the
mutagenicity tests show no increase in the frequency of
mutant colonies in the non-activated system (-S9) at doses
up to 140 yg/mL, but a relatively small increase at about
200 ug/mL. Similar values are'Obtained with identical con-
centrations in the presence of S9 indicating little or no
activation of the extract. Higher concentrations of the
extract, which were tested only in the presence of S9,
caused an increase in the mutation frequency. However, this
may not be the result of normal enzymatic activation of the
polycyclic aromatic hydrocarbons in the extract. In the
presence of 400 yg/mL of diesel extract, the benzo[a]pyrene
hydroxylase activity of rat liver microsomes is reduced to
95% of the initial level [7].
DISCUSSION
The diesel extracts have so far proven negative as mutagens
of V79 cells. However, because the extracts are a complex
mixture of substances, the toxicity may be inhibiting the
demonstration of mutagens. In the mouse lymphoma mutagen-
icity assay, activity of similar diesel extracts is negative
at concentrations up to 140 ug/mL. Concentrations greater
than 200 ug/mL could be tested only in the presence of S9
which reduced the toxicity without blocking the mutagenic
activity. Both studies indicate that the mutagenic effects
of the diesel extracts are relatively weak in mammalian
cells. Therefore, the effects may not occur -in v-ivo until
the dose of deposited diesel particulates in the organism is
manifested by the concurring cellular toxic effects.
400
-------
w
o
2 400-
o
300-
2 20°"
s
8
i
i
H
100
WO 200 300
DIESEL EXTRACT (pG/mL)
400
0 100 200 300 400
DIESEL EXTRACT (pG/mL)
Figure 5 Cytotoxicity and mutagenicity of diesel
extract in mouse lymphoma L5178Y cells.
Various concentrations of a dichloromethane
extract of diesel particles in culture
medium were incubated for 4 hours with cells
in suspension culture in the presence (x) or
absence (A) of S9. After removal of the test
medium, toxicity was evaluated by the relative
amount of cell growth in the next 24 hours
(a). Mutagenicity was evaluated by determin-
ation of the frequency of thymidine kinase-
less (TK~/-) cells after a two day expression
period (b).
401
-------
ACKNOWLEDGEMENTS
I wish to thank J. Dickman for providing excellent technical
assistance and T. L. Chan, J. D'Arcy, and J. T. Johnson for
collection of the diesel samples and preparation of the
extracts.
REFERENCES
1. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R.
Zweidinger, S. Tejada, J. Bumgarner, F. Duffield, M.
Water, V. F. Simmon, C. Hare, C. Rodriguez, and L.
Snow. 1978. Application of bioassay to the character-
ization of diesel particle emissions. In: Application
of short-term bioassays in the fractionation and analysis
of complex environmental mixtures, U.S. Environmental
Protection Agency, 600/9-78-027, September 1978.
2. Schreck, R. M., J. J. McGrath, S. J. Swarin, W. E.
Hering, P. J. Groblicki, and J. S. McDonald. 1978.
Characterization of diesel exhaust particulates under
different engine load conditions. 71st APCA Meeting,
7833.5, Houston Texas, June 25, 1978.
3. Clive, D., and J. F. S. Spector. 1975. Laboratory
procedure for assessing specific locus mutations at the
TK locus in cultured L5178Y mouse lymphoma cells.
Mutation Res., 31:17-29.
4. Chan, T. L., P. S. Lee, and J. Siak. 1980. Diesel
particulate collection for biological testing: compari-
son of electrostatic precipitation and filtration. In:
Proc. of the International Symposium on Health Effects
of Diesel Engine Emissions, U.S. Environmental Protec-
tion Agency, Cincinnati Ohio, December 3-5, 1979.
5. Chu, E. H. Y., and H. V. Mailing. 1968. Mammalian
cell genetics, II. Chemical induction of specific
locus mutations in Chinese hamster cells Ln vi£>io.
Proc. Natl. Acad. Sci. U.S.A., 61:1306-1312.
6. Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods
for detecting carcinogens and mutagens with the Sa&non-
e££a/mammalian-microsome mutagenicity test. Mutation
Res., 31:347-356.
7. Pederson, T. C. 1980. DNA-binding studies with diesel
particulate extract. In: Proc. of the International
Symposium on Health Effects of Diesel Engine Emissions,
U.S. Environmental Protection Agency, Cincinnati Ohio,
December 3-5, 1979.
402
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General Discussion
F. DANIEL: When you were trying to titrate out the
cytotoxicity by using S9 did you ever try just boiling the
S9 instead of leaving out cofactors?
C. RUDD: I thought about using heat inactivated S9 as a
control for absence of activation, but it precipitated
badly. I did do it, but it was a mess because of the pre-
cipitate and I didn't consider it a real control.
403
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DIESEL SOOT: MUTATION MEASUREMENTS
IN BACTERIAL AND HUMAN CELLS
H. L. Liber, B. M. Andon, R. A. Hites* and W. G. Thilly
*School of Public Health and Environmental Affairs
Indiana University
Bloomington, Indiana
and
The Toxicology Group
Massachusetts Institute of Technology
Cambridge, Massachusetts
ABSTRACT
Both hot pipe and dilution chamber samples of the exhaust
from a diesel (Oldsmobile 350) engine have been collected,
extracted with methylene chloride and those extracts have
been tested for mutagenicity in forward mutation assays in
human lymphoblasts and S. typhimurium. In the absence of a
metabolic activation system, the extract was significantly
mutagenic to the bacteria in the range of 0 to 30 yg/ml, but
induced no mutations in human cells at concentrations up to
200 yg/ml under the same conditions of assay medium.
However, when assayed in the presence of a postmitochondrial
supernatant derived from rat liver, the soot extracts were
significantly mutagenic to both bacteria and human cells
in the range of 50-100 ug/ml. Fractionation of the soot
extract on the basis of polarity by sequential elution from
a silicic acid column permitted concentration of the muta-
genic activity in the alkane/toluene eluate, as determined
by bacterial assays. Preliminary characterization of this
fraction and preliminary studies of pure compounds leads
404
-------
us to suspect the alkyl substituted anthracenes and phenan-
threnes as representing at least a significant fraction of
the mutagem'c activity of this alkane/toluene eluate.
INTRODUCTION
Based on our studies of kerosene soot mutagenicity in
bacteria (Kaden et al., 1979) and human cells (Skopek
et al., 1979), we have approached the study of diesel engine
emissions with a hypothesis that its mutagenicity (if any)
may be accounted by the summation of the products of
concentrations and specific mutagenic activities of the
soot's individual chemical components. Thus, our approach
requires a close collaboration between an analytical chemis-
try laboratory at Indiana University and a genetic toxico-
logy laboratory at M.I.T.
We have only begun this chemo- and bioanalytical approach to
diesel soot in the last six months, and wish to report our
findings to date for unfractionated methylene chloride
extracts and some broad fractionations based on polarity.
METHODS
Sample sources:
Dr. Charles Hobbs of the Inhalation Toxicology Research
Institute, Albuquerque, New Mexico supplied our first diesel
exhaust sample which was taken on a glass fiber filter by a
hot pipe sampling approach. No attempt to calibrate engine
load or performance was made in this preliminary sampling of
an Oldsmobile 350 engine burning a commercial diesel fuel.
Dr. Morton Beltzer of Exxon Research and Engineering Company,
Linden, New Jersey, substantially helped our project by
supplying us with a 2 gm single sample of diesel soot extract.
An Oldsmobile 350 engine burning blended typical refinery
diesel fuel products similar to commercial No. 2 fuels was
operated with repetitive hot start Federal Test Procedures.
The exhaust was passed through a dilution chamber and
collected on a Pall flex type T60A20 filter, 36 inches
square. The filter was extracted overnight with methylene
chloride in a soxhlet extractor. The solvent was evaporated
on a steam bath with nitrogen purge. The sample was shipped
on dry ice and has been stored by us at -80° C.
405
-------
Mutation assays:
Bacterial mutation to 8-azaguanine resistance was performed
as described in Kaden et al., 1979. Human cell mutation to
both 6-thioguanine resistance and trifluorothymidine resis-
tance was measured as described in detail in Thilly et a!.,
1980.
Source of post mitochondria! supernatant:
Liver post mitochondria! supernatant from aroclor pretreated
rats was purchased from Litton Bionetics, Rockville, Maryland.
Materials was received frozen on dry ice and stored by us at
-80°C. This preparation was consistently contaminated
with several bacteria or fungi per 5 milliliter aliquot
necessitating sterilization by a 1 megarad gamma ray expo-
sure (in dry ice). This exposure does not affect the
ability of the PMS to metabolize benzo(a)pyrene to a
mutagen in bacteria or human cells.
RESULTS
Figure 1 reports our measurement of the mutagenicity of the
two diesel extracts to bacteria, and of the dilution chamber
sample to human lymphoblasts when they were coincubated with
the extracts for two hours in the absence of any added
metabolizing system. Note that both samples were potent
mutagens for the bacteria, but no mutagenic activity toward
human cells was detected. In a subsequent experiment,
bacteria were coincubated with the extracts in the presence
of standard cell culture medium; the amount of bacterial
mutation was only slightly reduced by this change in expo-
sure medium.
Figure 2 reports our observations when the experiments were
repeated in the presence of a rat liver post mitochondrial
supernatant metabolizing system with cofactors and pH
conditions appropriate for observing maximum mutagenicity
from 40 yM benzo(a)pyrene. Exposures were for two hours;
the post mitochondrial fraction was 10% v/v in the bac-
terial studies and 5% v/v in the human cell studies. Under
these conditions, the amount of mutation induced in human
cells and bacteria is quite similar with the 99% upper
confidence limit on historical controls being exceeded at
about 50 yg/ml for the human cells and at about 100 ug/ml
for the bacterial cells. This similarity of sensitivity has
been observed by us for other combustion residues such as
kerosene soot and pure polycyclic aromatic hydrocarbons
(Kaden et al., 1979 and Shopek etal., 1979).
406
-------
5
1-0
0.6
0.3
300
100
0
100
200
300
DIESEL EXHAUST EXTRACT, ug/ml X 2 hr
WITHOUT METABOLIC ACTIVATION
Figure 1. Toxicity and Mutagenicity of Diesel Exhaust Ex-
tracts to Salmonella typhimurium and Human Lyrnpho-
blasts in the Absence of Metabolic Activation.
Bacteria or human cells were treated for 2 hr with diesel
exhaust extracts. Relative survival was determined by cloning
efficiency immediately after treatment. Mutant fractions were
determined for 8-azaguanine resistance (50 Mg/ml) in bacteria,
and 6-thioguanine resistance (5 vg/ml) or trifluorothymidine
resistance (1 yg/ml) in human cells. Error bars are 99% con-
fidence intervals. The dotted line is the 99% upper confi-
dence limit for the historical background mutant fraction in
bacteria; the dashed line is the 99% upper confidence limit
for the background mutant fraction in human cells.
O - Bacteria treated in minimal media - Hot pipe sample
A - Bacteria treated in complex media - Hot pipe sample
Q- Bacteria treated in minimal media - Dilution chamber
sample
0- Human cells, trifl uorothymidine resistance - Dilution
chamber sample
|- Human cells, 6-thioguanine resistance - Dilution
chamber sample
407
-------
0
25
50
75
100
DIESEL EXHAUST EXTRACT, ug/ml x 2 hr
WITH POST-MITOCHONDRIAL SUPERNATANT
Figure 2. Toxicity and Mutagenicity of Diesel Exhaust
Extracts to Salmonella typhimurium and Human
Lymphoblasts in the Presence of Metabolic Acti-
vating System.
Bacteria or human cells were treated for 2 hr with diesel
exhaust extracts in the presence of 10% (bacteria) or 5%
(human cells) aroclor-induced rat liver post-mitochondria!
supernatant.
O - Bacteria treated in minimal media - Hot pipe sample
/^ - Bacteria treated in complex media - Hot pipe sample
Q - Bacteria treated in minimal media - Dilution chamber
sample
0 - Human cells, trifluorothymidine resistance - Dilution
chamber sample
| - Human cells, 6-thioguanine resistance - Dilution
chamber sample
408
-------
Fractionation of the crude extracts was followed by bac-
terial mutation assay of each fraction. No human cell
assays have been performed on the fractions at this time.
As summarized in Table 1A, the most active fraction of the
ITRI hot pipe sample, both with and without metabolic
activation, was the one eluted from the silicic acid column
by a 1:1 pentane:toluene solvent after the column had been
rinsed with pentane. Similarly, for the Exxon dilution
chamber sample, the hexane:toluene fraction was the most
mutagenic fraction in the presence of a metabolic activating
system. In the absence of metabolism, the toluene fraction
appeared to be more mutagenic; however, because of the
extreme toxicity of this fraction, the data for this frac-
tion is considered preliminary. One may note that the
amount of mutaenic activity recovered from this column
separation was approximately equal to the amount originally
loaded on the column.
DISCUSSION
Insofar as gene locus mutation assays may be predictive of
human health risk, our observations lead us to consider
diesel engine exhausts with grave concern. It is our
present intention to continue our chemical analysis of the
sample obtained Fran the dilution chamber and to test the
compounds identified in it until we have accounted for the
biological activity in terms of individual mutagens. We
hope that our studies will help combustion scientists to
focus on those compounds and compound classes which must be
reduced in diesel exhausts if their mutagenic potencies are
to be reduced.
We note that our studies with human cells indicate a possibly
lesser priority for immediate studies of so-called 'direct
acting' mutagens since these were observed in bacterial but
not in human cell mutation assays. We intend to follow this
preliminary conclusion in setting our research priorities
for the coming year.
Finally, we must state our concern that without knowledge of
the physiologic distribution of inhaled soot particles,
there seems to be no logical means to use the kind of
observations made in our studies to estimate the impact on
human health. Until such knowledge is obtained by competent
scientists, predictions based on analytical logic do not
seem possible.
409
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Table 1A
MUTAGENICITY OF FRACTIONS OF THE HOT PIPE SAMPLE OF
DIESEL EXHAUST TO S.TYPHIMURIUM
% Weight of
Fraction Unfractionated Mixture
Soot (minE) 100
Soot (RPMI) 100
Pentane 18
Pentane: 13
Toluene
Toluene
7
Me thy lene
Chloride 8
Methanol 54
tested (yg/ml)
13
27
53
13
27
53
4.8
9.5
19
3.3
6.5
13
1.8
3.5
7.0
2
4
8
14
28
56
No Activation
95
165
280
90
141
190
5
4
5
42
107
235
28
40
78
23
30
56
16
23
35
± 12
i 25
± 54
t 12
+ 22
1 31
+ 2
+ 1
+ 1
+ 5
+ 13
+ 43
± 4
£ 5
+ 9
+ 3
+ 4
+ 7
± 2
+_ 3
± 4
10% ARO
34 +
40 +
67 +
34 +
47 +
59 ^
7 t
8 +
11 +
32 +
48 +
98 +
13 *-
31 +
8 +
21 +
7 +
6 +
9 +
PMS
4
4
6
5
5
6
2
2
2
5
8
14
2
4
2
3
2
2
2
Legend: * 8-azaguanine-resistant mutant fraction, with the 99% confidence interval.
Assays were performed without activation or in the presence of 10% (volume/volume)
aroclor induced rat liver post-mitochondrial supernatant (10% ARO PMS).
410
-------
MUTAGENICITY OF FRACTIONS OF THE DILUTION CHAMBER
SAMPLE OF DIESEL EXHAUST TO S.TYPHIMURIUM
% Weight of Concentration 8 AGR MF x Id5 - 99% CI
Fraction Unfractionated Mixture
Soot 100
(Unfractionated)
Hexane 27
Hexane : 7
Toluene
Toluene 7
Methylene 14
Chloride
Methylene
Chloride: 38
Methanol
(2:1)
Methylene
Chloride: 4
Methanol
(1:2)
tested (pg/ml)
30
100
300
150
300
150
300
150
300
150
300
150
300
75
150
No Activation 10% ARO PMS
67+7 12 + I
167 + 22 19+2
295 + 45 39+3
55+7 67+7
71+8 91+9
516 + 143 203 + 23
1000 105 + 11
172 + 20
337 + 60 50 + 5
134 + 100 73 + 8
92 + 12
149 +21 35+4
9 + 2 7+1
14 + 2 8 + 1
75
150
5 +_ 1
7 + 1
9 +_ 2
7 + 1
Control
10
5 + 1
Legend: * 8-azaguanine-reslstant mutant fraction, with the 99% confidence interval.
Assays were performed without activation or in the presence of 10% (volume/volume)
aroclor induced rat liver post-mitochondrial supernatant (10% ARO PMS).
411
-------
REFERENCES
Kaden, D. A., R. A. Hites and W. G. Thilly (1979). Muta-
genicity of soot and associated polycyclic aromatic
hydrocarbons to Salmonella, typhimurium. Cancer Res.
39:4152-4159.
Skopek, T. R., H. L. Liber, D. A. Kaden, R. A. Hites and W.
G. Thilly (1979). Mutation of human cells by kerosene
soot. J. Natl. Cancer Inst. 6.3:309-312.
fhilly, W. G., J. G. DeLuca, E. E. Furth, H. Hoppe IV, D. A.
Kaden, J. J. Krolewski, H. L. Liber, T. R. Skopek, S.
A. Slapikoff, R. J. Tizard, and B. W. Penman (1980).
Gene-Locus Mutation Assays in Diploid Human Lymphobl ast
Lines. Chem. Mutagens, Vol. 6, in press.
Discussion
A. BROOKS: Do I understand that all of the direct-
acting mutagenic activity we see in bacteria is of no con-
sequence in humans?
W. THILLY: Up to a constant of 200 micrograms per mil-
liter in the culture; the answer to that question is yes.
412
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STUDIES ON THE EFFECTS OF DIESEL PARTICULATE
ON NORMAL AND XERODERMA PIGMENTOSUM CELLS
J. Justin McCormick, Roselyn M. Zator,
Beverly B. DaGue and Veronica M. Maher
Carcinogenesis Laboratory
Michigan State University
East Lansing, MI 48823
ABSTRACT
Diesel engine emission participates (DP) have been shown
to contain direct acting bacterial mutagens. Our investi-
gations have shown that normal human fibroblasts (NF) and
excision repair deficient xeroderma pigmentosum figroblasts
(XP) (neither of which possesses significant amounts of the
cytochrome P448-P450 metabolizing system) are sensitive to
the cytotoxic effects of the direct-acting agent(s) of DP.
XP fibroblasts show greater sensitivity than NF to DP
suspensions in DMSO or organic solvent extracts of DP. When
these cell strains are exposed to equal amounts of DP
extracts, the slope of the XP survival curve is at least
twice as steep as that of NF. More significantly, there is
a pronounced shoulder on the survival curve. We also
observe this difference in survival when these strains are
treated with polycyclic aromatic hydrocarbons and several
other chemicals and have demonstrated that it is indicative
of tghe formation of lethal DNA aducts for which XP cells
have little or no repair capacity. We have found that short
extractions of DP with cold acetone, cold CH2CL£, warm
toluene, or a 4-hour Soxhlet CH2CL2 extraction are
equally effective in solubilizing the direct-acting cyto-
toxic component(s) of DP. Biological fluid extracts of DP
employing Ham's F10 Nutrient Medium or fetal calf serum also
contain cytotoxic chemicals. Howver, these extracts were
413
-------
only l/6th to 1/27 as cytotoxic as DMSO suspensions of the
DP. HPLC fractionation on a Bio-Rad silica (20 44 u) column
of Soxhlet-CH2CL.2 extracts of DP has provided a single
cytotoxic fraction. However, since humans are exposed to
whole diesel particles rather than organic solvent extracts
of them, it seemed desirable to examine the effect of diesel
particulate itself on human fibroblasts. We found that when
NF and XP cells were exposed directly to suspensions of DP
in tissue culture medium containing serum, the cytotoxic
effect was virtually identical to that found to organic
extracts of the same DP sample. This result suggests that
the differentially cytotoxic (DNA damaging) material on the
particles was somehow able to transfer from the DP and come
into contact with the DNA. Electron microscopy of cells
exposed directly to DP demonstrated that the cells contained
large amounts of DP. Our previous studies have shown that
agents which cause higher-cytotoxicity in XP than in NF
cells also induce mutations. Therefore, diesel exhaust
particulate represents a potential health hazard for man.
Whether it represents an actual hazard can only be deter-
mined by further study.
General Discussion
R. CHRISTIAN: I was very interested in your studies of
bioavailability and putting particles on cells. We have
not done this with diesel exhaust but we have with coal
particles ground very finely. When you put the particles
on cells, they do coat the cells and are taken in. This
can be seen with electron microscopy and you see phaco-
cytosis in normal tissue cultured cells. The serum as well
as the tissue culture medium is quite effective at leaching
toxic substances. One of the real advantages here is that
you can treat your medium with the particles that you wish
to use and grow your cells in it. I think that this is
quite an advantage.
J. MCCORMICK: Well, we think that this work is actually
modelled on your studies, and the results I showed indicate
that the tissue culture medium, or serum, are very poor
leachers of the material that is differentially cytotoxic
in these cells. So that doesn't seem to be a viable way to
go here.
R. CHRISTIAN: You do observe the particles associated
with the cells and in cases they are taken inside. Were
you suggesting that perhaps there was some phacocytosis
there?
J. MCCORMICK: Yes, that is the most likely explanation.
We haven't done the electron microscopy yet to demonstrate
it, but I suspect that that is exactly what happened.
414
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R. CHRISTIAN: I would like to" ask you whether you have
considered the possibility of these extracts being pro-
moters as well as initiators? It seems that with the pros-
pect of the exhaust being in our environment that promotion
may also be a problem seen in the future.
J. MCCORMICK: Promotion is certainly worth looking at,
but we have not looked at it in our studies.
F. DANIEL: I noticed on your HPLC profiles that you had
identified several fractions having activity. Have you
gotten anywhere on identifying the chemical components on
those fractions?
J. MCCORMICK: No, we haven't really done that yet.
A. BROOKS: As I understand it, you really don't have
any direct evidence of interaction with the DNA in these
cells. Because of that, I wonder if you have tried to get
the differential toxicity using some of the fractions that
are very cytotoxic. If after making fractionation, you
find that some of the toxicity is associated with fractions
that are nonmutagenic in the Ames Test, I wonder if you
tested any of those non-mutagenic, but cytotoxic fractions?
J. MCCORMICK: No, we have not done that.
415
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BENZO(A)PYRENE ALTERS LUNG COLLAGEN SYNTHESIS
IN ORGAN CULTURE
Rajendra S. Bhatnagar and M. Zamirul Hussain
Laboratory of Connective Tissue Biochemistry
School of Dentistry
University of California at San Francisco
and
Si Duk Lee
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
This Study Supported By
U.S. Environmental Protection Agency
Contract No. 68-03-2626
ABSTRACT
Benzo(a)pyrene is known to be a significant component of
diesel emissions. In our studies, benzo(a)pyrene markedly
altered parameters of collagen synthesis in organ cultures
of rat lungs.
Cultures exposed to benzo(a)pyrene synthesized greater
amounts of type III in relation to type I collagen. Expo-
sure to benzo(a)pyrene also markedly elevated prolyl
hydroxylase activity in lung organ cultures. No significant
changes occured in cultures treated with pyrene, a non-
carcinogenic hydrocarbon.
Collagen plays an important role in lung function, and in
regulating cell differentiation and proliferation. Altera-
tion of collagen synthesis appears to be an important aspect
of the toxicity of benzo(a)pyrene.
416
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INTRODUCTION
Collagen is an integral part of lung structure, contribu-
ting up to 20% of the dry weight, and it plays a major role
in pulmonary function (1). Qualitative and quantitative
changes in collagen, aberrations in collagen synthesis, and
altered patterns of tissue distribution of collagen are
major aspects of induced lung disease (2-4). Malignant
changes in lungs are often accompanied by morphologically
observed changes in collagen (5,6). Carcinogenesis alters
the structural and functional characteristics of tissues,
and changes in collagen synthesis may be expected to be a
part of the early events in carcinogenesis.
Collagen is a regulatory protein, controlling vital aspects
of cell function and differentiation by mediating cell-cell
interaction and cell adhesion, and it regulates cell proli-
feration (7). Since benzo(a)pyrene, a carcinogenic poly-
aromatic hydrocarbon, is a component of diesel emissions, it
is of interest to determine if it affects lung collagen
synthesis.
In another report from our laboratory presented at this
symposium (8), we reported the alteration of collagen
synthesis in lungs of rodents exposed to diesel emissions.
In the present study, we investigated the effect of benzo(a)
pyrene and pyrene on collagen synthesis in organ cultures of
rat lung. While benzo(a)pyrene markedly altered parameters
of collagen synthesis, no such changes were observed in
cultures exposed to the non-carcinogenic hydrocarbon pyrene.
MATERIALS AND METHODS
Radiochemicals
L-[3,4-3H]-proline, 25 Ci/mmol, and L-[14C(U)]-proline, 285
mCi/mmol were purchased from New England Nuclear.
Organ Culture Procedures
Lung organ cultures were prepared by a method developed in
our laboratory, and described in detail elsewhere (9). The
method consists of placing 1 mm thick slices of neo-natal
rat lung (Long-Evans) on Millipore filters (0.3 m pore
size, 2.5 cm diameter), supported on 1.0 ml of Dulbecco-Vogt
minimum essential medium containing 10% fetal calf serum, in
an organ culture dish (Falcon Plastics). Several dishes
417
-------
hydroxylase (16). The increase in aryl hydrocarbon hydrox-
ylase in this system was consistent with previous in vivo
and in vitro studies on the toxicity of this compound. An
additional aspect of chemical injury elicited by benzo(a)
pyrene in lung organ cultures was expressed as a large
increase in prolyl hydroxylase activity, paralleling the
increase in aryl hydrocarbon hydroxylase (16). Increased
tissue prolyl hydroxylase activity reflects tissue injury
and connective tissue proliferation (5,6,25).
When cultures were maintained in the presence of 10 ym or 25
pm benzo(a)pyrene for 12 hours, the activity of prolyl
hydroxylase was increased by nearly 80% (Table I). These
data indicated that benzo(a)pyrene may induce increased
collagen synthesis in lung organ cultures. There was no
increase in pyrolyl hydroxylase activity in cultures exposed
to 25 um pyrene, a structurally related hydrocarbon with low
carcinogenic potential.
The synthesis of collagen was examined in these cultures by
pulse-labelling with ^C-proline. The cultures were
maintained in the presence or absence of benzo(a)pyrene for
24 hours, and l^C-proline added for a 3-hour pulse period.
As seen in Table II, benzo(a)pyrene caused a significant
increase in the specific activity of ^C-hydroxyproline
synthesized, confirming an increased collagen synthesis.
There was no significant difference between control cultures
and cultures exposed to pyrene.
Collagen is a famil of genetically distinct proteins (26).
The proportion of different types of collagen vary from
tissue to tissue, and change during growth and development,
and in pathological conditions (27). Type I collagen is
ubiquitous and is the major collagen component of adult
lungs. Type III collagen chains are predominant in fetal
tissues, and are associated with rapidly proliferating
tissues, and the proportion of type III collagen synthesis
is markedly increased in regenerating tissues and scars.
As seen in Table III, the synthesis of type I collagen
accounted for 70% of the total collagen syntehsized in the
control cultures. Type III collagen accounted for the
remaining incorporation. In marked contrast, type III
collagen accounted for nearly half of the total collagen
synthesized in cultures exposed to benzo(a)pyrene. These
data suggest that benzo(a)pyrene induces chemical injury
resulting in increased expression of a fetal gene product,
namely type III collagen.
The present studies do not provide information concerning
the mechanism of chemical injury induced by benzo(a)pyrene
418
-------
were placed in a humidified, gas-tight chamber in which a
gas mixture, 95% air + 5% CO?, was circulated. Benzo(a)
pyrene and pyrene were dissolved in acetone before, for
addition to the cultures. Control cultures received the
same volume of acetone (2 1). Previous experiments demon-
strated that the addition of this quantity of acetone had no
biochemically or morphologically observable effects on the
organ cultures.
Analytical Procedures
The DNA content of the cultures was determined by the
diphenylamine procedure (10). Total protein content was
assayed by measuring the ninhydrin reactive material in the
hydrolyzates of the tissue, and expressed in terms of
"leucine .equivalents" (11). Collagen synthesis was deter-
mined by following the incorporation of radioactivity from
proline into radioactive hydroxyproline, assayed by a
published method (12). Prolyl hydroxylase activity was
determined in the 15,000 x g supernatants of the cultures by
previously published methods (13), and expressed in terms of
3H-proline-labeled, unhydroxylated collagen substrate per mg
of tissue DNA.
Collagen Chair Analysis
The chain composition of newly synthesized collagen was
determined as described elsewhere, in order to determine the
relative incorporation of radioactivity into type I and type
III collagens (14).
RESULTS AND DISCUSSION
Lung organ cultures have been shown to be useful for studies
on lung collagen metabolism under a variety of conditions,
including hyperoxic atmospheres, and in the presence of
several agents known to be injurious to lungs in vivo
(14-20). Lung organ cultures have also been useful in
investigations on the effects of chemical carcinogens
(21-24).
Exposure of lung organ cultures to hyperoxic environments
(14), paraquat, or heavy metals (16-20) elicited marked
increases in the rates of collagen synthesis. The bio-
chemical and morphological response to chemical injury in
lung organ cultures mimicked the response of lungs in vivt).
Exposure of lung organ cultures to benzo(a)pyrene also
caused a marked increase in the levels of aryl hydrocarbon
419
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Table I
EFFECT OF BENZO(A)PYRENE AND PYRENE
ON PROLYL HYDROXYLASE ACTIVITY IN LUNG ORGAN CULTURES
Prolyl Hydroxylase Activity
Culture Conditions lQ-4xdpm 3HHO Released/mg DNA % Change
Control 6.44
Benzo(a)pyrene,
10 ym 10.81 + 67
Benzo(a)pyrene,
25 ym 11.45 + 77
Pyrene, 25 ym 5.57 + 3
Each number is the average of five determinations. Enzyme
specific activity was calculated on the basis of the DNA
content in order to relate it to the cellularity of the
tissue.
Table II
EFFECT OF BENZO(A)PYRENE ON COLLAGEN SYNTHESIS
IN LUNG ORGAN CULTURES
Culture Conditions 10"5xdpm 14C-HyP/mMo1e LE % Change
Control 2.80
Benzo(a)pyrene,
10 ym 3.41 + 22
Benzo(a)pyrene,
25 ym 3.76 + 34
Culture conditions are described in the Methods Section.
Tissues were maintained under the described conditions for
21 hours, and then pulse-labelled with l^C-proline for 3
hours.
LE = Leucine Equivalents.
420
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which contributes to increase connective tissue prolifera-
tion. However, it is known that the metabolism of carcino-
genic hydrocarbons such as benzo(a)pyrene, results in the
generation of many free radical species, including the
oxygen-free radical anion superoxide (28). In contrast,
the metabolism of noncarcinogenic hydrocarbons, including
pyrene, does not result in a comparable flux of free radi-
cals. Superoxide free radicals has been shown to be a major
component of tissue toxicity of oxidants and other injurious
agents (29). Other studies in our laboratory have shown
that superoxide induces enhanced collagen synthesis in lung
fibroblast cultures (30), in cultures of hepatocytes (31),
and is the collagen-enhancing agent in the toxicity of
paraquat in lungs (15).
In our studies reported here, the syntehsis of collagen was
altered after short exposures to benzo(a)pyrene. In this
short period, cell transformation cannot be considered to be
a major factor for the altered biochemical benhavior of the
tissue, although this possibility cannot be entirely ruled
out. Type III collagen is always associated with rapidly
proliferating cells and tissues, and there is some evidence
suggesting that type III collagen regulates cell prolifera-
tion and differentiation (27). Benzo(a)pyrene interferes
with cell differentiation as a part of its carcinogenic
mechanism (32). It may be speculated that the initial spurt
in type III collagen synthesis may promote the various
effects attributed to benzo(a)pyrene by altering the pat-
terns of cell proliferation and differentiation. Collagen
has been implicated in carcinogenesis and significant
changes in the rates of collagen synthesis and in its nature
(i.e., scar tissue formation), have been associated with
carcinogenesis (5,6). Our limited studies support these
ideas.
ACKNOWLEDGEMENTS
We wish to thank Ms. M. Tolentino and Mr. K. R. Sorensen for
expert technical assistance.
REFERENCES
Hance, A. J., and Crystal, R. G. The Connective Tissue
of the Lung. Am. Rev. Resp. Dis. 112 657-711, 1975.
Hance, A. J., and Crystal, R. G. Collagen. In:
The Biochemical Basis of Pulmonary Function. R. G.
Crystal, Ed., Marcel-Dekker, New York, 1976, pp.
215-272.
421
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3. Chvapil, M., Peng, Y. M. Oxygen and Lung Fibrosis.
Arch. Environ. Health. _30_ 528-532, 1975.
4. Hussain, M. Z., Cross, C. E., Mustafa, M. G., and
Bhatnagar, R. S. Hydroxyproline Contents and Prolyl
Hydroxylase Activities in Lungs of Rats Exposed to Low
Levels of Ozone. Life Sci. _18 897-904, 1976.
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424
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R. BHATNAGAR: First of all I should apologize for the
briefness of my abstract, I did due submit the full ab-
stract, and planned on our discussion with personnel here
and perhaps on the basis of a letter that I talked about -
the kind of things that would be interesting. The abstract
does not entirely reflect what I said here. I should also
have named the coworkers that were involved in this. Our
morphologist is a Dr. John Belton, a Professor of Biology
at California State University at LaJolla Hayward, and
presently is at Johns Hopkins University. Other coworkers
are Dr. Muselam, Ms. Terento, and Dr. Si Duk Lee of the EPA
have been involved in some of the agent studies. Now, as I
discussed with Dr. Daniel, there has been quite a bit of
interest in the role of collagen itself in carcinogenesis.
Collagen is a mitogen. Collagen has been shown to be pre-
sent at the site of all sorts of injuries, and it has been
said many times that cancer commonly appears at the sites
of old injuries. What I was tyring to imply here was that
the type of collagen that appears in benzopyrene, treated
cultures is a type that is associated with differentiating
systems. A collagen that is associated with laboratory
preparation of cells perhaps may have some significance.
425
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Table III
RELATIVE SYNTHESIS OF TYPE I AND TYPE III COLLAGENS
IN LUNG ORGAN CULTURES EXPOSED TO BENZO(A)PYRENE
Percent Radioactivity Incorporated Into
Collagen Chains
Culture Conditions Type I Type III
Control
Benzo(a)pyrene
70
52
30
48
Cultures were pulse-labelled between 21 and 24 hours as
described in Table II and analyzed for collagen chain
composition.
General Discussion
F. DANIEL: Have you tried any other classes of car-
cinogens at this time?
R. BHATNA6ER: No, these are preliminary studies and we
have concentrated on benzopyrene and pyrene and we are
planning to look at other chemicals. We have looked at
cadmium incidentally and with cadmium we see the same kind
of thing. I should point out, though, that these changes
may have similarities to the process of carcinogenesis.
We have heard many papers on the metabolism of polyaromatic
hydrocarbons in which the metabolites eventually bind to
DNA and transform it. If you have a system with such a
process going on and there is a great increase in the poly
factor within itself, then it is conceivable that the cells
that have been transformed may somehow get amplified and we
may see more of them and that many contribute to the pro-
cess of carcinogenics.
J. VOSTAL: I would like to ask two questions. First of
all you have been talking about the addition of the benzo-
pyrene. Would you please tell us what the benzopyrene was
dissolved in, and how did you mix it with the medium in
which the tissue culture occurred? The second question is
even much more important. If I am correct, in your ab-
stract, you concluded that the changes you have seen may be
related to carcinogenic alterations and may be used as
early markers. Frankly speaking, we would like to know who
is the morphologist who gave you the impression that you
have seen any indication of the carcinogenic alterations?
426
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APPLICATION OF A BATTERY OF SHORT TERM MUTAGENESIS
AND CARCINQGENESIS BIOASSAYS TO THE EVALUATION
OF SOLUBLE ORGANICS FROM DIESEL PARTICULATES
Joellen Huisingh, Stephen Nesnow, Ronald Bradow
and Michael Waters
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC
ABSTRACT
The extractable organics from Diesel participate emissions
have been shown to be mutagenic in a bacterial screening
assay (Ames, S. typhimurium). This report summarizes the
results from a battery of confirmatory bioassays for muta-
genic and carcinogenic activity. The test systems included
in this battery are primarily mammalian cell systems,
however, one assay was conducted in insects (Drosophila) and
one in yeast (Saccharomyces). The bioassays detected the
following biological effects: gene mutations, DNA damage,
and oncogenic (neoplastic) transformation.
The Diesel particles,-extracts, and fractions were generated
from a Catepillar 3208, 4 stroke cycle, V8 engine used in
urban service vehicles. The samples included a total
dichloromethane extract (DCM) of the Diesel particles
collected after dilution on a filter; a polar, strongly
fluorescent fraction of the neutral organics (TRN) and the
most polar fraction of the neutral organics (OXY).
The gene mutational assays in mammalian cells were positive
with all samples tested including DCM, TRN, and OXY in the
BALB/3T3 mouse cells and OXY in the L5178Y mouse lymphoma
427
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cells. The X-linked recessive lethal assay In Drosophila
melanogaster was negative for the OXY fraction administered
by feeding 1 nig/ml for 3 days.
The bacterial DNA damage assays requiring either diffusion
or killing were non-conclusive with these relatively non-
toxic complex organics. The nitotic recombination assay in
Saccharomyces cerevisiae D3 was positive with DCM and OXY
and negative with the TRN fraction which caused no toxicity
at the highest doses testable. Unscheduled DNA synthesis in
WI-38 cells was negative with the OXY fraction.
Oncogenic transformation assays performed in BALB/3T3 Cl A31
mouse cells showed all three samples, DCM, OXY, and TRN to
produced morphologically transformed colonies.
For further information, see Huisingh, J., et al., "Applica-
tion of Bioassay to the Characterization of Diesel Particle
Emissions" in Application of Short-Term Bioassays in the
Fractionation and Analysis of Complex Environmental Mixtures
(Environmental Science Research, Vol. 15), M. D. Waters et
aj_., Eds., Plenum Press, New York, 1979, pp. 381-418.
428
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General Discussion
J. VOSTAL: I am really very seriously concerned. I
think you have mentioned that you have found some evidence
of mutagenicity and potential carcinogenicity. You have
been comparing the data on heavy duty motor vapors and
light duty motor vapors and I still have the same problem
that was raised yesterday, namely, why have only two re-
vertents/microgram been shown in some of our tests while
another one has been showing 200 of them. I think that we
are living in the dirt of confusion, since we are always
seeing on our slides the expression of a concentration
which is given as a microgram per milliliter, but nobody
states how it's possible to compare a microgram of the
residium obtained after fractionation with a total extract
and probably what is most important with the total mass of
the diesel particles. I can hardly accept that anything
could be really deduced from the data we have seen in this
presentation the sample, which has been tested being ob-
tained from two heavy-duty motor vapors, had about 50 or
25 percent of the extractable mass, and we are trying to
apply it to a type of engine of the future producing a
light duty vapor which barely has about five, or maximum
eight percent of extractable material. We have already
discussed it with EPA. I think it will be a great benefit
if we abstain from using the data only showing micrograms
per milliliter. We should really do the recalculation as
an activity for microgram of the particulate. After that
we should probably reassess our conclusions relating to
dose-response. Practically all the tests which are being
shown here as a documentation of the mutagenic effect are
typically dose dependent, and it was very clearly demon-
strated that you must accumulate a certain amount of mi-
crograms before you show any effect. Obviously those mi-
crograms have a completely different meaning. It could be
a product of one heavy-duty engine as compared to the 50
light-duty motor vaports.
J. HUISINGH: All right, I think you have made some
very good points. It is, of course, possible for us to
calculate our data back that way and it does probably give
a clearer idea related to the particle emissions. However,
I think that at this point, particularly in this meeting,
we would like to concentrate on testing for biological
effects and in the process determines the best ways to
test the materials, not to calculate revertents per mile
429
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and tumors per kilogram of fuel consumed, or that sort
of thing. In the evening session we will present data
from three light-duty vehicles; the Oldsmobile, the Nissan,
and a Volkswagen Rabbit, as well as a heavy-duty vehicle
compared to a gasoline vehicle - data that I think will
help address that question. It does not appear that these
vehicles which are light-duty vehicles are necessarily less
active. In some cases they are considerably more active.
There is a difference between the vehicles and that cer-
tainly is something that will make some interesting dis-
cussion. We have not and will not be presenting that data
expressed per mile, but all of the data is there if you
want to make those multiplications. I think we are just a
little hesitant at this time, until we understand more
about the systems, to express the data that way because I
think it may encourage people to jump to conclusions. We
are not looking at man exposed over a lifetime. These
cells in some cases were exposed two hours. These mam-
malian cell assays were all corrected for toxicity, but
after the two-hour exposure the cells were washed and were
then plated out or cultured in the absence of the test
substance. In the Salmonella data we have seen some dif-
ferent reported numbers, even when you correct the toxicity
by the estimated method that Dr. Thilly described, that is
an estimation of toxicity. The toxicity is not determined
and handled exactly in the same way that is possible for
the self culture assay. I think the range of Salmonella
data you referred to, two to 200 revertents per microgram,
is being confused with some fractionation work and total
extracts, because if you look at the total extracts over
the whole range of vehicles we have tested and other other
people have tested, and was done in three separate labo-
ratories, we never see that range of activity. We see very
consistent numbers between vehicles run on different days
when the samples are handled the same way.
430
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A REVIEW OF IN-VITRO TESTING SYSTEMS
APPLICABLE TO DIESEL HEALTH EFFECTS RESEARCH
Gary K. Whittnyre
Energy and Environmental Analysis, Inc.
1111 North 19th Street
Arlington, Virginia 22209
ABSTRACT
In-vitro mutagenicity and carcinogenicity testing techniques
are currently being used to assess the potential risk to man
of exposure to diesel exhaust emissions. This paper examines
general considerations of such systems, the types of in vitro
tests currently available, the advantages and disadvantages
of each cell line and type of test, the limitations of
in vitro techniques, the alternative human cell lines that
could be utilized for diesel health effects studies, and
recommendations for future research employing in vitro
methods.
1. INTRODUCTION
Diesel exhaust emissions contain numerous organic compounds,
including polycyclic aromatic hydrocarbons (PAH) which may
have a potential impact on human health (Santodonato, et
al., 1978). Various PAH compounds, which are adsorbed as
condensed material on particulate matter, are thought to be
the major contributors to the mutagenic activity of organic
extracts of diesel particulates (Briggs, et al., 1978),
Adsorption of these compounds to particulates may signifi-
cantly increase their carcinogenic effects (Katz & Pierce,
1976). Some of these PAH compounds, particularly the
lipid-soluble neutral fraction and the basic fraction, have
great affinity for lung tissues (Philpot, et.al., 1977).
431
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In vitro tests have been developed over the last decade
which can provide a rapid appraisal of the mutagenic/
carcinogenic potential of various compounds or mixtures of
compounds. Several agency and interagency groups have
reviewed current in vitro test procedures (Science News,
1979) . There are three general types of in vitro tests -
biochemical studies, bacterial mutagenesis tests, and tests
employing mammalian cell cultures:
Biochemical testing examines adduct formation of
the activated forms of the carcinogens with nucleo-
philic centers on biological macromolecules, e.g.,
protein, ribonucleic acid (RNA), deoxyribonucleic
acid (DNA). The most useful information comes
from looking at adduct formation with DNA since
this is the center of interest in terms of genetic
damage and carcinogenesis. Adduct formation with
blood cell proteins has been suggested for diesel
health effects work, although this is a very
indirect measure and may not be a good index for
the amount of reactive PAH metabolite that would
bind to DNA in low concentration ranges (U.S. EPA,
1979). DNA repair studies also fall into this
category.
In vitro microbial tests are tests for mutagenicity,
i.e., the capability of a compound or mixture of
compounds to cause a mutation. The most widely
used mutagenicity assay, and the one accepted by
EPA, is the Ames test (Salmonella). Various
assays employing cultures of other microbes sup-
plemented with mammalian microsome preparations
can also be used.
In vitro mammalian cell culture studies include
tests for mutagenicity and carcinogenicity per-
formed in primary or continuous cell lines.
Researchers ultimately would like to extend the results of
in vitro tests to man in terms of relating a given exposure
to the risk of cancer. An individual's risk of developing
lung cancer is dependent on three groups of "determinants".
These are 1) exposure to carcinogens, 2) exposure to
modifying factors in the environment (cocarcinogens, syn-
carcinogens, anticarcinogens), and 3) predisposing host
factors. Most in vitro studies address the first issue,
although the first and the second issues can also be
addressed together in properly designed in vitro studies.
Individual variation in susceptability, which is part of the
third factor may be approachable in mammalian cell systems
by using heterogeneous cell populations instead of cloned
cell lines (Harris, et. al., 1978).
432
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This paper provides a critical review of in vitro test
systems that are being applied to or could be applied to
studies of the health effects of diesel exhaust emissions.
Section 2 presents general biochemical considerations to be
kept in mind when comparing various cell systems used in
in vitro tests. Section 3 provides an overview of in vitro
nmtagenesis, cell transformation, and biochemical tests
currently available. Section 4 examines the limitations of
these tests. Conclusions and recommendations comprise
Section 5.
2. IN VITRO SYSTEMS - GENERAL CONSIDERATIONS
The final impact of a given PAH exposure to a particular
cell ultimately depends on the rate at which metabolites are
produced in a given tissue. These kinetics reflect both the
number of molecules of activating enzyme present in the cell
as well as the specific activity of the enzyme, expressed as
nanomoles of PAH activated per mg of enzyme per minute. The
higher the rate of conversion of PAH the higher is the
effective dose of PAH metabolite to the target DNA. This
rate may determine if mutagenesis or cell transformation
occurs in an in vitro system and whether the lag time
necessary to see the effect will be within the time allowed
for scoring the test results. A compound that is detected
as a mutagen or potential carcinogen by one in-vitro system
may be missed by another in vitro system due to inherent
differences in activating enzyme activities.
The major enzyme responsible for activation of certain PAH
to mutagenic and/or carcinogenic forms is aryl hydrocarbon
hydroxylase, which is part of the complex of oxidative
enzymes referred to as P-450. It is thought that two dif-
ferent pools of AHH exist in any tissue at any given time.
One represents constituent enzyme which is always present at
some minimum level. The second represents an inducible
enzyme pool whose level can be highly variable. Certain PAH
can induce enzyme levels up to almost 10-fold depending on
the species, strain, or tissue from which the cell line is
derived (Philpot, et.al., 1977). Pulmonary AHH is highly
inducible in cell lines derived from rat, hamster, and some
strains of mice. The strain dependence of this character-
istic is very strong in some animals such as mice, where the
level of AHH induction is also dependent on the inducer
compound used (Philpot, et.al., 1977). Some mouse strains,
such as B6 and B6D2 are highly inducible. Others such as D2
and the first generation obtained from crossing C3H and D2
demonstrate little or no AHH induction (Wang, et al., 1976).
Data for smokers versus nonsmokers suggest that pulmonary
AHH activity can be induced more than 10-fold in man (Philpot,
et. al., 1977).
433
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Existing evidence confirms that the concentration and spe-
cific activity of aryl hydrocarbon hydroxylase (AHH) in a
given tissue vary by an order of magnitude or more from
species to species (Philpot, et.al., 1977). Furthermore,
the metabolite pattern obtained in terms of relative
proportions of specific metabolites also varies from species
to species (Pelkonen, 1976). There are also differences in
AHH activities between sexes and between tissues, the dif-
ferences in the latter case being greater (Philpot, et. al.,
1977).
There are also significant developmental differences in AHH
activity. Cell lines derived from fetal tissue show the
same tissue variability in PAH metabolism as cell lines
derived from adult tissues. Fetal liver cells have much
greater (about 2-fold) induced AHH activity than fibroblasts
derived from fetal lungs or skin (Pelkonen, 1976), although
uninduced activities for conversion of benzo(a)pyrene (B[a]P),
dimethylbenzanthracene (DMBA) and N-2-fluorenylacetamide
(FM) are about the same (Juchau, et. al., 1978).
In humans and presumably in animals there is also significant
individual variation in AHH activity (Paigan, et.al., 1978a).
Many highly variable factors including developmental, genetic,
dietary, hormonal/ physiological, disease, and behavioral
(e.g., smoking) factors may be correlated with levels of AHH
(Boulos, 1978). Human liver AHH levels vary up to 16-fold
and may be strongly influenced by smoking and general health
(Pelkonen, 1976). There is some evidence supporting the
theory that those persons with high AHH inducibility and
activity are at higher risk of lung cancer compared to
individuals with lower activities or inducibilities (Paigan,
et.al., 1978); Busbee, et.al., 1979). AHH levels in cultured
human lymphocytes and pulmonary alveolar macrophages from
cancer patients support this correlation (Busbee, et.al.,
1979).
Generally, metabolic activation is obligatory for the expres-
sion of the mutagenic/carcinogenic potential of PAH (Nagao
and Sugimura, 1978). Many in vitro mutagenesis and cell
transformation assays are supplemented with microsomes
preparations from a mammalian species (usually rat liver) or
with another cell type to aid in conversion of PAH to their
active forms. In the case of microsomes, the animal from
which they are obtained is usually treated with a compound
that induces P-450/AHH to higher than consituent levels.
Routinely, Arochlor has been used, although this may not be
a wise choice due to its toxic and carcinogenic nature which
may cause undesirable interactions with the test material.
Some PAH such as 3-methylcholanthrene (3-MCA) and B(a)P
which are also used for this purpose in some systems have
the same inherent problems. If this problem is to be
circumvented, these compounds are easily replaced with a
434
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mixture of phenobarbitol and 5,6-benzoflavone, which is as
equally as potent an inducer as 3-MCA or B(a)P (Nagao and
Sugimura, 1978). In some instances, however, such inter-
actions as co-mutagenesis, co-carcinogenesis, and syncar-
cinogenesis are desirable in an in-vitro system as they
increase significantly the sensitivity of the system for
detecting mutagens and carcinogens.
Ideally, activation kinetics of the in vitro systems applied
to diesel particulate extracts should reflect that seen in
lung tissues, preferably human lung. This suggest adaptions
for certain currently-used protocols. For example, in
microbial (bacteria or yeast) mutagenic systems the conven-
tionally used rat liver microsomes could be replaced with
human lung microsomes, when available, to better represent
the lung metabolic processes, both qualitatively and quanti-
tatively, that occur in man.
3. TYPES OF IN VITRO TEST SYSTEMS
3.1 MUTAGENESIS
In vitro mutagenesis assays include microbial (bacteria,
yeast) and mammalian cell systems. In the forefront of the
microbial categoy is the Ames test which uses various
histidine-requiring mutants of the bacteria Salmonella
typhimuium. Mutation is detected in the Ames test as con-
version of individual cells to a histidine-independent
state. These mutants include TA 1537 and TA 1538 which are
frame-shift mutants, TA 1535 which are base-pair substitution
mutants, and TA 100/TA98 which are derived from TA 1535 and
TA 1538. The relative sensitivity of these various strains
in detecting mutagens/carcinogens varies as a function of
the compound tested (Juchau, et. al., 1978; Nagao and Sugimura,
1978).
Another type of bacterial test system involves DNA repair as
a parameter of damage in DNA polymerase-deficient mutants
(pol A.) of E coli. This test is based on inhibition of
growth of pol A, mutants by compounds that alter DNA.
Microsomal activation is required for PAH studies using this
sytem (Nagao and Sugimura, 1978).
The third type of bacterial system involves prophage (bac-
terial virus) induction in E. coli. Specifically one can
use an E. coli mutant (envA, uvrB) which produces a defec-
tive membrane (allowing facile penetration of PAH) and which
is deficient in DNA repair. Both mutations result in high
sensitivity to PAH. However, not all PAH have phage inducing
capability. Again microsomal activation of PAH is required
(Nagao and Sugimura, 1978).
Mammalian cell systems are also used for detecting mutagens
and comparing mutagenicity of various substances. The cell
435
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types employed for mutagenicity studies can be divided into
two categories: permanent cell lines which have been in
culture for several generations of cells, and cells derived
directly from fresh tissue (primary cell cultures). The
cell strains and genetic markers employed are listed in
Table 1. The fact that the PAH's need to be metabolized to
"proximate" or "ultimate" forms capable of binding to DNA
was not recognized during early efforts of mutagenesis
testing with the pure compounds. Thus, many of the pre-1970
PAH mutagenicity studies in both bacteria and mammalian
TABLE 1. TARGET CELL TYPES AND GENETIC MARKERS
EMPLOYED FOR INVESTIGATING MUTAGENESIS IN
MAMMALIAN CELLS BY PAH'S AND DERIVATIVES*
Investigators using estab-
lished cell lines (aneuploid,
high plating efficiency,
infinite lifespan)
Investigators using diploid
cell strains (diploid,
relatively lower plating
efficiency, finite lifespan
1. Chinese hamster cells 1.
a. V79 hamster lung cells
1) Brookes and colleagues;
AGr
2) Huberman and colleagues;
AGr, OUAr
3) Krahn and Heidelberger;
AGr
4) Malaveille et al.; 2.
AGr OUAr
5) Marquardt et al.;
AGr
6) Wood, Wislocki and
colleagues; AG
Syrian (golden) hamster
embryo cells
a. Barret, Ts'o and
colleagues; OUA
b. Huberman et al., OUA
Human skin fibroblasts:
normal and repair
deficient
Maher, McCormick
et al.; AGr
b. CHO hamster ovary cells
1) Hsie et al.; TGr
2) Huberman and Sachs;
ts reversion
2. Mouse hymphoma cells L5178Y
Clive; BUdRr, TGr
SOURCE: Maher and McCormick, 1978.
* Abbreviations: AG = 8-azaguanine resistance; OUA =
ouabain resistance; TGr = 6-thioguanine resistance;
ts = temperature sensitivity; BUdR = bromodeoxyuridine
resistance.
436
-------
cells did not provide metabolic activation systems and
yielded negative results. Methods used to provide metabolic
activation for mammalian mutagenesic detection systems are
outlined in Table 2. For cell lines that do not have suf-
ficient P-450 activity, activation can be provided by a
microsome preparation or a "feeder" layer of a different
cell type which does have PAH metabolic capability. An
example of the latter is the use of Syrian hamster embryo
(SHE) primary cell cultures as a feeder layer in a Chinese
hamster ovary (CHO) cell system. This particular system has
some advantages in that it can yield higher activities
(higher mutation frequencies) than microsomal preparations
and does not have the problem of nonlinear activation
responses at moderate to high S-9 concentrations (above 0.5
mg/ml). This type of system also has less variability than
a microsomally-activated assay (Carver and Felton, 1979).
TABLE 2. METHODS USED TO PROVIDE REACTIVE METABOLITES OF
PAH'S FOR ASSESSING MUTAGENICITY IN MAMMALIAN CELLS
1. Chemical or biosynthesis: reactive metabolite or
derivative prepared chemically or isolated following
enzymatic synthesis
2. Microsomal-mediated: production of reactive metabolites
from parent or intermediate compound by cell homogenate
fraction not of target cell origin
3. Cell-mediated: production of reactive metabolites by
feeder layer present with target cells
4. Host-mediated: metabolites formed in body of host
animal in presence of target cells
5. Metabolism within target cell itself: target cell has
sufficient PAH-metabolizing capability
SOURCE: Maher and McCormick, 1978.
The potency of PAH varies in such mammalian mutagenesis
systems over two orders of magnitude or more. Tables 3 and
4 show the relative mutagenic potential of various PAH
compounds in a CHO system that utilizes lethally-irradiated
rodent cells for metabolic activation (Huberman, 1978).
Some mammalian cells are more sensitive indicators of muta-
genesis than bacteria (Kuroki, 1978). Furthermore, there
are significant differences in mutagenicity between the
bacterial and mammalian test systems in terms of magnitude
of response to B(a)P metabolites. The ability to induce
mutations in V79 cells appears to be more closely related to
437
-------
carcinogenicity than induction of revertants in Salmonella
(Kuroki, 1978).
TABLE 3. INDUCTION OF OUABAIN-RESISTANT MUTANTS IN THE
CELL-MEDIATED ASSAY BY DIFFERENT CARCINOGENIC HYDROCARBONS
Number of
Concentration of Ouabain-resistant
Hydrocarbon Mutants per 10
Hydrocarbon
Control
Benzo(e)pyrene
Phenanthrene
Pyrene
Benz (a) anthracene
Chrysene
Dibenz(a , c)anthracene
Dibenz (a, h) anthracene
7 -Me thy Ibenz ( a ) anthra cene
3-Methylcholanthrene
Benzo(a)pyrene
((Jg/ml)
0
1
1
1
1
1
1
1
1
1
1
survivors
1
1
1
1
2
2
3
4
24
108
121
SOURCE: Huberman, 1978.
TABLE 4. MUTABILITY OF DIFFERENT GENETIC LOCI IN THE
CELL-MEDIATED ASSAY BY CARCINOGENIC HYDROCARBONS*
Hydrocarbon
Control
Pyrene
Phenanthrene
3-Methylcholanthrene
Benzo (a)pyrene
Number of
Temperature-
Resistant
0.6
0.9
0.7
125
170
mutants per
Ouabain-
Resistant
1
1
1
108
121
106 cells
8-Azaguanine
Resistant
60
50
80
3660
4250
* Cells were treated with 1 ug/ml of the polycyclic hydrocarbons .
SOURCE: Huberman, 1978.
438
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Induction of sister chromatid exchange (SCE) in mammalian
cell cultures represents another in vitro mutagenisis system.
V79 cultured cells have been used for this purpose. The SCE
assay is a rapid and sensitive method for evaluating the
interactions of chemicals with DNA. This assay has been
used for various PAH and environmental samples including
cigarette smoke condensate (Lockard, et.al., 1979).
3.2 TRANSFORMATION
Numerous cell lines and protocols exist for in vitro study
of chemical induction of neoplastic transformation. These
systems involve primarily two cell types - fibroblastic or
epithelial cells.
Fibroblasts are the best-characterized and perhaps the
easiest to grow, and have been employed for much if not most
of the basic research into the mechanism of cell trans-
formation. One major advantage in using fibroblasts is
that, unlike epithelial cells, chemical carcinogens induce
drastic morphological changes in fibroblasts which are
easily detected and quantitated. Such quantitative indices
of transformation of epithelial cells are lacking, and thus
the study of these cells is more difficult. Secondly,
fibroblasts have a much shorter response time than epithelial
cells. Neoplastic transformation in fibroblasts can be
detected in 1-4 weeks, whereas in epithelial cells this lag
time is measured in months.
Currently, the cells widely-used for PAH transformation
studies include the SHE cells and the various mouse lines.
SHE cells exhibit morphological changes in about 1 week, the
extent of which is dose dependent. The major advantages of
SHE are that its chromosomes are similar in number and
morphology to human cells, that methodologies involving this
cell line are well developed, and that these cells have a
low spontaneous transformation rate. The disadvantages of
SHE cells are that growth is slow in early passage numbers
and that a mixed population of cells is used (although the
cells becomes more homogeneous as they divide and grow).
Another type of system uses aneuploid mouse cell lines
including C3H mouse embryo cells, their cloned lines such as
C3H/10T^, Balb C mouse embryo cells and 3T3, which are
derived from Balb C. These cell lines are contact inhibited,
grow in a uniform monolayer, and form foci of transformed
colonies which "pile up" upon treatment with a carcinogen.
Time for obtaining results from these cell lines is usually
four to six weeks.
The major advantage of these cells is that they are cloned
populations derived from a single cell and, therefore, are
genetically homogeneous, like SHE cells, they display
variations in transformation responses depending on culture
439
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conditions, particularly the concentration and character-
istics of the serum used.
There are several different indicators of cell transforma-
tion, and, therefore, clarification is needed to delineate
which characteristic or property is heing transformed (e.g.,
neoplastic transformation, morphological transformation).
Some of these indices are listed in Table 5. Not all of the
transformations in properties can be associated with produc-
tion of an abnormal tumorigenic cell. Some changes repre-
sent just one step in the sequence of events leading to
truely neoplastic cells. For example, enhanced fibrinolytic
activity and morphological transformation of the cells are
noted soon after treatment with a carcinogen. However,
growth in soft agar, which is truly characteristic of a
TABLE 5. PHENOTYPIC TRANSFORMATIONS OF NEOPLASTIC CELLS
Phenotypes
1. Tumorigenictity
2. Loss of anchorage dependency for growth
a. Soft agar/agarose
b. Methyl cellulose
c. Teflon
3. Morphological changes
4. Enhanced fibrinolytic activity
5. Chromosomal changes
6. Indefinite life span
7. Lack of density-dependent inhibition of replication
8. Growth at low serum concentrations
9. Changes in membrane properties
a. Altered glycoproteins and glycolipids
b. Increased rate of transport
c. Loss of high molecular weight, surface glycoprotein
d. Increased agglutinability by plant lectins
e. Increased mobility of membrane proteins
f. Altered surface structure (SEM)
10. Changes in microtubules and actin cables
11. Decreased cAMP levels.
SOURCE: Barnett and Ts'o, 1978.
440
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malignant cell, is not noticed until after some 32-75 cell
divisions. Some of these indicators need weeks to develop
and may be unobserved due to use of premature analysis time
in the test protocol (Barrett and Ts'o, 1978).
3.3 ADDUCT FORMATION AND DNA REPAIR
One can initially detect DNA damage by looking at DNA repair
activities within the affected cell, or at DNA adduct forma-
tion with the active metabolites of the mutagens/carcinogens.
Unscheduled DNA synthesis (DNA repair) involves excision of
some of the damage caused to DNA, and synthesis of new
segments of DNA. A large amount of DNA damage induces high
levels of DNA repair activity, but not all the damage may be
repaired. DNA-repair deficient cell strains are especially
sensitive to mutagenesis and transformation (McCormick and
Maher, 1978). Thus, cell transformation can be related to
unscheduled DNA synthesis.
To determine the extent of DNA adduct formation, one can
examine either RNA or DNA binding of radio-labelled PAH or
metabolites, e.g., H-B(a)P. DNA adduct determinations must
be made soon after treatment of the cells with the test
compound, since the proportional composition of the adducts
changes with length of time after treatment. For B(a)P, and
presumably other PAH, adduct formation is quite a sensitive
method for some cell lines, e.g., hamster embryo cells
(Baird and Dumaswala, 1979).
3.4 ALTERNATIVE HUMAN SYSTEMS
For studies of PAH or complex mixtures of PAH, as in the
case of diesel exhaust particulate extracts, use of in vitro
systems employing human cell lines would be especially
relevant, two human cell systems suggest themselves - skin
and lung. Human foreskin cells are readily available and do
well under culture conditions. This system has already been
applied to the study of cytoplasmic PAH binding protein
activity and its role in transporting PAH to the nucleus of
the affected cell (Tejwani and Milo, 1979). Suitable lung
cells can come from lung tissue itself or from the mobile
cells within the lung, e.g., the pulmonary alveolar macro-
phages (PAM's) and lymphocytes. Some studies have success-
fully utilized mixed populations of both PAMS and lymphocytes
in studying the activation and detoxification of PAH compounds
(Marshall, et.al., 1979).
4. LIMITATIONS OF TESTS
There are several limitations to in vitro tests which place
constraints on interpretation of results obtained with them.
Some of these limitations are due to problems inherent in
the test systems themselves. Additionally, results with
in vitro systems for detecting mutagenesis/carcinogenesis
represent only rough indications of what may happen in vivo
441
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(in the whole animal) due to several irreconcilable dif-
ferences between isolated cells and the intact organism,
such as:
Differences in hormonal levels, which affect
general overall metabolic activity and rates.
Differences in thresholds, if any exist, for
mutagenic/ carcinogenic effects.
Differences in relevant phannokinetic factors such
as rate of absorption, effective dose delivered or
experienced by the tissue, rates of activation and
detoxification.
The most widely used mutagenicity assay, and the one
accepted by EPA, is the Ames test (Salmonella). There is
some controversy as to whether the Ames assay can be used as
a predictor of carcinogenesis. There are opinions and
evidence pro (Ames and Hooper, 1978) and con (Ashby and
Styles, 1978; DeFlora, 1978; Maugh, 1978a). The fact that
the Salmonella assay can yield both false positive and false
negative results (the latter being of greater consequence)
is the major restriction of this technique.
Furthermore, due to biochemical differences between animals
of various species, activation of test compounds by micro-
somes isolated from one species may yield a positive Ames
test result, while carcinogenicity testing in a different
species may yield negative results (Ashby & Styles, 1978a).
In addition, 14 variables have been identified which
influence the numerical results obtained with the Ames test
(Ashby and Styles, 1978b). In particular there is great
variability between liver microsome preparations and thus,
they should be standardized for carcinogen-activating and
carcinogen-deactivating enzymes. For example, differences
in how liver microsomes are prepared can result in a varia-
tion by more than a factor of 100 in the observed mutagenic
potency of benzo(a)pyrene (Maugh, 1978a). Critics say that
no correlation should be drawn between Ames test results and
carcinogenic potential, especially for PAH, nitrosamines,
polycholorinated cyclic compounds, aza-napthols, symmetrical
hydrazines and steroids, which yield a high proportion of
false negatives (Maugh, 1978b).
Mammalian mutagenesis systems also have their limitations.
Mammalian systems lack reproducibility for quantitative
determinations due to such things as sensitivity to varia-
tions in initial plating density and insufficient incubation
time for expression of all mutations. The latter problem is
due to the fact that PAH and PAH metabolites in mammalian
systems exhibit dose-dependent delay of the response. That
442
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is, the smaller the dose, the longer is the lag period
before mutation is expressed. This is presumably partly due
to low metabolic rates for PAH in some cell lines. This
problem can be alleviated somewhat by choosing a cell line
that has inherently high rates of activation of PAH (Maker
and McCormick, 1978).
Low metabolic rates also may be responsible for the fact
that in some mammalian mutagenesis assays, the frequency of
mutation is not proportional to dose at high doses. That
is, high doses of PAH fail to give the number (frequency) of
mutants expected on the basis of responses at lower doses.
This may be due to saturation of the AHH activation systems
in some cell lines (Maher and McCormick, 1978).
There are several things which affect cell transformation
results. These include cell density, fixation of DNA damage,
and factors in the growth media. The relationship to cell
density is that cell cultures in confluency (fully-grown
monolayer within the physical constraints of the culture
plate) have been found to be relatively resistant to trans-
formation by polycyclic hydrocarbons compared to that seen
in growing cultures. This is true for mouse cell lines and
some hamster cell lines such as SHE. This is most likely
connected with the need for cells to "fix" DNA damage via
several cell divisions before transformation can be expressed.
Experiments with mouse 3T3 cloned cells indicate that about
4 cell divisions (generations) are required to fix the
damage and result in expression. The transformation frequency
(the number of transformed foci per number of cells) decreases
with increases in initial cell density, with increases in
cell density at time of treatment, and with decreases in
number of cell generations required to attain saturation
density (confluency). Additionally, serum factors consumed
by the cells are related to inducibility of AHH. If cells
are depleted of serum factors, e.g., as in the confluent
state, then there is low AHH activity and subsequently low
sensitivity to PAH. Serum factor depletion may tie in with
the cell division requirement mentioned above in that serum
factors are required for normal cell growth. This is sig-
nificant for testing both in terms of qualitative and quan-
titative results (Kakunaga, 1978).
5- CONCLUSIONS AND RECOMMENDATIONS
There are two perspectives from which an in vitro system may
be judged appropriate for diesel health effects research.
One is on the basis of sensitivity of the system to muta-
genesis or carcinogenesis by PAH or PAH metabolites. The
other is on the basis of biochemical and physiological
similarily to the human tissues that would be exposed to and
affected by diesel exhaust emissions.
443
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Two things that can be done to enhance the sensitivity of a
mutagenicity/carcinogenicity assay is to account for inter-
ferences that reduce mutagen/carcinogen detection potential
(e.g., anticarcinogens in the growth media) and to screen
candidate cell lines for AHH activity and inducibility.
Rapid and simplified methods exist for doing the latter
(Miyazawa, et al., 1977).
For more meaningful comparisons of data between laboratories
and within laboratories, in vitro mutagenicity and carcino-
genicity test protocols should be standarized. This should
include:
the confirmation of dose-response patterns
the use of a standard latent period between the
time of treatment with the test sample and the
scoring of results for a given cell line
the use of a standardized media for a given cell
line
the confirmation of cytotoxicity or cell viability
under test conditions, provision of both activated
and nonactivated test systems where appropriate,
and provision of both positive and negative controls
the use of a battery of in vitro tests to fully
and accurately screen fractions from diesel par-
ticulate extracts.
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447
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THE DMA DAMAGE ACTIVITY (PDA) ASSAY AND ITS
APPLICATION TO RIVER UATERS AND DIESEL EXHAUSTS
Charles 0. Doudney, Mary A. Franke,
and Charles N. Rinaldi
Division of Laboratories and Research,
New York State Department of Health,
Albany, N.Y. 12201
ABSTRACT
An extremely sensitive assay has been developed
for DNA-damaging chemicals using a DNA repair-
deficient strain of Escherichia coli. Consid-
erable DNA-damaging activity has been demonstrated
by this assay in natural surface-water samples and
in diesel-exhaust particulate extracts.
INTRODUCTION
All common biological assays (such as the Ames
technique) for traces of potentially mutagenic
and carcinogenic chemicals require concentration
ranges of mutagenic chemicals far above those
encountered in the environment. It is therefore
necessary to collect relatively large amounts of
material and to extract and concentrate the muta-
gens by elaborate procedures. This severely
limits the use of these systems for examining the
envi ronment.
We therefore set out to develop an effective bio-
assay for screening surface waters and other en-
vironmental materials. Such an assay would have
448
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to be sensitive enough to detect potentially muta-
genic chemicals at environmental concentration
ranges. It must also be simple and rapid, so that
large numbers of samples could be examined with
minimum effort.
As one possibility we turned to the assay for DNA-
damaging chemicals developed fay Nishioka and Na-
bori (1,2). This assay measures the effect of such
chemicals on the increase in turbidity of cultures
of repair-deficient bacteria during a 4-h period.
Uith our modifications this technique can detect
DNA-damaging chemicals with much greater sensi-
tivity. We then developed a standard assay which
allows detection of extremely small amounts of
potentially mutagenic chemicals. Because of the
extreme sensitivity of the assay, environmental
samples, such as surface water, can be examined
directly.
In this report we describe the assay and its ap-
plication to the examination of diesel-exhaust
particulate extracts. These extracts were pre-
pared in the Division of Air of the New York
State Department of Environmental Conservation
(DEC) under a project sponsored by the U.S. En-
vironmental Protection Agency (EPA; Grant
R805934010; Dr. Richard E. Gibbs, Project
Di rector).
SPECIAL ASSAY METHODS
Strains. Escheri chi a coli strains WP2 trp, WP2S
trp uvrA, WP10 trp recA and WP100 trp uvrA recA
were obtained from Dr. Evelyn Witkin, Rutgers
University, New Brunswick, NJ.
Glassware and other materials. Because of the
sensitivity of the assay it is very important
that all materials used be pure or clean and thus
free of DNA-damaging chemicals. While well-
cleaned Pyrex glassware may be used for prelim-
inary growth of the culture, special glassware
free of chemicals which damage DNA must be used
for any direct contact with the assay medium.
Otherwise the growth of WP100 will be inhibited,
ruining the assay. In particular, any materials
sterilized with ethylene oxide gas or other
chemical sterilizing agents must be avoided.
449
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We have had little success reusing any type of
glassware, especially Pyrex. For that reason
and for convenience we used disposable glass-
ware. Plain flint, 3-oz. prescription bottles
(Brockway) were "tissue-culture" washed (i.e.,
without detergent), rinsed for 30 s with dis-
tilled deionized water, and sterilized in an
electric oven at 350°C for 2 h. For pipetting
nonsteri1ized I-ml Biotips (Schwartz/Mann)
were used. Commercially available 0.1- or
0.2-ml Biotips cannot be used, since they have
been chemically sterilized. Special 0.1-0.2-ml
Biotips, tissue-culture washed but not chemi-
cally sterilized, were prepared for us by
Schwartz/Mann Division of Becton Dickinson Co.,
Orangeburg, N.Y. We had little success using
Pyrex glass pipettes, although Thermex glass
tuberculin syringes (no tips) could be used
once but not reused.
Culture growth. Cultures were grown overnight
and kept frozen at -73°C in 0.3-ml of minimal
broth plus 10% dimethyl sulfoxide (DMSO) in
1-dram vials. The day before the assay the con-
tents of one vial was added to 20 ml of minimal
medium plus L-tryptophan and incubated for 6-7 h
at 37°C on a reciprocating shaker. The minimal
medium used routinely was Davis minimal broth
(Difco) supplemented with 20 yg of L-tryptophan/ml
and 1% glucose (MBD). Distilled deionized
water was used to prepare all media.
After this incubation 0.1 ml of the culture was
added to 20 ml of MBD (tryptophan, 40 jjg/ml) and
grown overnight (4 p.m.-7 a.m.). The culture
was then adjusted with MBD at 37°C to an absor-
bance of 0.5 at 660 nm, using a Zeiss PMQII
spectrophotometer, and was incubated with shaking
for about 90 min or until the absorbance reached
about 0.8. (If this absorbance was passed, the
culture was adjusted back to 0.8 with MBD at
37°C).
This culture must be used immediately as the
inoculum for the assay, which was usually set up
while this culture was growing.
Assay procedure. With all weighable materials
including reference materials (mutagens) 1 mg was
weighed out and dissolved in 20 ml of DMSO or
other solvent (50 yg/ml). Appropriate serial
450
-------
dilutions (usually 1 ml into 9 ml of MBD) were
then made using prescription bottles. This
was done immediately before the experiment, since
many mutagens in solution are unstable at low
concentrations. All subsequent operations were
carried out in a 37°C walk-in incubator, where
prescription bottles and all growth media had been
stored the night before to ensure temperature
equi1i bration.
Aliquots (0.1 ml) of serial dilutions of the sus-
pected DMA-damaging chemicals were added to the
bottom of these prescription bottles in an upright
position. Appropriate controls were prepared
using serial dilutions of the solvent (usually
DMSO). A suspension of the inoculation culture
was made (1 ml per each 49 ml of MBD at 37°C) and
mixed well. A prewarmed Cornwall pipetter set
at 4.9 ml was used to rapidly dispense this culture
into the prescription bottles with and without
chemicals and into empty bottles. (The highest
final concentration of the weighed material was
1 yg/ml of growth medium.) This addition must
be very rapid (bottle caps were removed before-
hand) to ensure an accurate zero time point.
In general 100 bottles could be inoculated within
2 min. At this point a timer was started, and
the bottles were shaken gently and put on their
sides for incubation. We did not use aeration,
since many mutagens are apparently sensitive to
oxidation, which reduces the sensitivity of the
assay. All samples were duplicated.
Control bottles containing only the culture
suspension were checked periodically for tur-
bidity increase to follow growth, until an A^Q
of 0.18 was reached. All bottles were then
rapidly placed in an ice bath to halt growth.
Bottles were then selected at random and warmed
rapidly to room temperature. Their contents were
mixed with a vortex mixer and their A56Q deter-
mined, using matched 3-ml-square glass cuvettes
(1-cm light path) in a Zeiss PMQ II spectro-
photometer. MBD at room temperature was the
blank. Figs. 1 and 2 show further details.
Interpretation of results. The assay relies
primarily on E_. c o 1 i strain B/r WP100 recA uvrA.
Because of genetic mutations at two loci this
strain lacks the enzymatic capacity to carry out
the two major pathways for repair of DNA after
451
-------
environmental damage. These pathways are the
excision-repair system (u_y_r/\+) and the postre-
plication recombination repair system (re_cA+).
Thus even very slight DNA damage, such as that
exerted by very low levels of mutagenic chem-
icals, is presumably nonrepairable. The failure
in DNA replication and consequently in cell di-
vision and growth are measured as an inhibition
of the expected increase in turbidity. Since
almost all repairable DNA damage has a mutagenic
effect, a DNA damage assay based on this or-
ganism is a valid measurement of potential
mutagenici ty.
Any difference in the effects on WP100 and on
the repair-proficient strain (WP2) confirms the
genetic toxicity of a given compound or sample.
Both strains are routinely tested simultaneously;
the effect on WP2 is usually very low. In ad-
dition, the recA (WP10) and uvrA (WP2s) strains
can be used to differentiate toxic effects due
to failure of excision repair as opposed to
postreplication repair, when this is of interest.
RESULTS AND EXPLANATION
The effects of three direct-acting DNA-damaging
chemicals are demonstrated in Fig. 1. Aroclor
1254, a polychlorinated biphenyl mixture, at
three concentrations (10~2, 10~6 and 10~8 yg/ml )
inhibited growth for some 70 min; recovery then
occurred rapidly. MNNG at the same concentra-
tions slowed growth but did not completely pre-
vent it. After about 120 min rapid growth oc-
curred. These data suggest a pause in growth
after turbidity has doubled in the presence of
MNNG. The effect of mitomycin-C was similar
to that of MNNG, although the increase was
slowed somewhat more effectively at first.
We believe that the increased growth during sub-
sequent incubation reflects the action of a
residual, inducible repair mechanism in WP100,
which can repair low-level chemical damage to
DNA. Peters and Jagger (3) have demonstrated
repair of near-UV lethal damage in an organism
of this genotype by an inducible recA+ gene-
independent system. Nishioka and Nabori's 4-h
assay would allow time for such recovery to
occur and would therefore be less sensitive to
DNA-damaging chemicals.
452
-------
In developing our DNA damage activity (DBA) assay,
we prevented such recovery by reducing the incu-
bation time. It is evident from Fig. 1 that when
the chemical-free control culture has reached an
A55g of 0.18, the assay has achieved maximum
sensitivity in measuring the inhibition of tur-
bidometric increase. For that reason, in our
standard assay we stop the incubation at that
point.
A typical assay for MNNG is shown in Fig. 2. It
is evident that the recA+ gene product can also
repair the DNA damage involved, since the WT and
uvrA strains are much less sensitive to MNNG.
The combination of the recA and uvrA genes in
WP100 increases the sensitivity considerably
over recA.
The recA gene renders E_. col i more sensitive to
most DNA-damaging chemicals; and the DDA assay
is sensitive to a variety of such chemicals
(Table 1). Benzo(a)pyrene, which requires meta-
bolic activation in the Ames assay for muta-
genicity, is apparently detectable without acti-
vation in the DDA assay, although its low level
of activity could be due to a contaminant. The
DDA assay also detects polychlorinated biphenyls
(PCBs), which are not mutagenic in the Ames
system. Since PCBs are a common contaminant in
surface water, this sensitivity increases the
assay's potential value in studies of environ-
mental water contamination, although perhaps
limiting its value as a specific indicator of
mutagens.
APPLICATIONS
River water. To evaluate the utility of the DDA
assay in examining the environment, we measured
DNA damage activity in river samples from two
river systems in the northern United States
(Fig. 3). The results indicated measurable con-
tamination in the Mohawk-Hudson river system in
the Schenectady-Troy-Albany urban area and down-
stream. Concentrations of DNA-damaging chemicals
in the Buffalo-Niagara river systems in the
Buffalo metropolitan area are several orders of
magnitude higher. Assessment of the ecologic
and public health significance of such contami-
nation requires further study, especially to
453
-------
INCUBATION (min)
Figure 1. Effect of three concentrations (yg/ml)
of Aroclor 1254 (Monsanto Company),
N-methyl-N'-nitro-N-nitroso-guanidine
(MNNG) (Aldrich Chemical Company) and
mi totnycin-C (Cal biochem-Behring Corp.)
on turbidity increase in E_. coli WP100
trp recA uvrA.
454
-------
o
s
0.18
0.16
0.14
0.12
0.10
wr
uvrA
1.0
Figure 2.
IQ-2
ID'
IO-6
MNNG
IO'8
Standard DDA assay of MNNG. The di-
luted cultures of the four strains are
grown with a range of concentrations
of the suspected DNA-damaging chemical
until the control culture (containing
no mutagen) reaches an Afifin of about
0.18. In carrying out
one bottle is used for
of turbidity. We take
readings every 10 m or
tures approach an Afigo
then read the turbiaity
this operation,
each reading
duplicate
so as the cul-
of 0.18 and
in 6 or more
bottles to verify the final reading
at 0.18. The effects of the various
concentrations of the chemical on tur-
bidity increase are then compared. The
results may be quantitated by pro-
jecting the point of 50% change between
the base level and the final level of
turbidity to the axis.
455
-------
TABLE 1. ACTIVITY OF SELECTED MUTAGENS
IN THE DDA ASSAY WITH E.. coli
recA uvrA
Minimum detected
Chemical concentration (yg/ml)*
MNNG
4-ni troqui no! i ne
Mi tomyci n-C
Ethyl methansulfonate
2-Ni trof 1 uori ne
Benzo (a) pyrene
Sodium bisulfite
Aroclor 1254 (PCB mix. )
10-10
10'8
io-10
io-7
10-10
10-5
10-9
10-10
* Lowest concentration giving maximum inhibi-
tion in the DDA assay, performed as in Fig. 2,
Results with the other three E_. col i strains
were comparable to those reported for MNNG
in Fig. 2.
determine whether the particular DNA-damaging
material is known or suspected to be hazardous
to humans.
This assay system should provide a rapid, low-
cost, and extremely sensitive method of sur-
veillance of contamination of surface water
by DNA-damaging chemicals. (Many sources of
public water supply are surface water.) By
working upstream from contaminated areas, sources
of contamination could be pinpointed. By fre-
quent sampling at selected points, sudden re-
leases or spills of suspect materials could be
detected. Correlation of results over an ex-
tended period with ecologic and public health
data could indicate biologic effects of chronic
contamination of streams with DNA-damaging
materi al.
456
-------
018-
Mohawk River
below Schenectady
0.10
Figure 3.
10
10-6 IO'8
DILUTION
lO-io
IO-12
10-"
DNA-damaging activity of river water
from two river systems, the Mohawk-
Hudson near Albany and the Niagara-
Buffalo near Buffalo. Only results
with the recA uvrA strain (WP100) are
reported, since the other three
strains gave much lower responses.
Tenfold serial dilutions into MBD were
made from a mixture of 9 ml of un-
diluted filter-sterilized water (kept
on ice after collection until use) and
1 ml of 10X concentrated MBD. The
pure water control was distilled-
deionized water commonly used in the
laboratory. The river water was col-
lected by dipping at the surface, and
was transferred into prescription bot-
tles or 100 ml glass-stoppered bottles,
Control pure water held in these
bottles during storage of the samples
showed no activity.
457
-------
Diesel exhaustparticulates. In collaboration
with the DEC Division "of Air we are studying the
mutagenicity of diesel exhaust participate^.
Extracts we,e prepared and fractionated as pre-
viously described (4-6). With the DDA assay
we can detect the DNA-damaging activity of as
little as 10"9 to 10'5 yg of diesel extract/ml,
in contrast to the 50 to 200 yg needed per plate
for routine Ames examination. Thus much smaller
quantities of samples need be collected and
handled in chemical isolation procedures.
Typical results are shown in Fig. 4 for extracts
from an Oldsmobile 350 and a Caterpillar 3304
each furnished dissolved in DMSO by the EPA. Re-
sults with the EPA Oldsmobile sample in DMSO were
very low in both assays, compared with a solid
0.18
0.16-
o
-------
sample subsequently received from the EPA and
weighed out and dissolved in DMSO by us. Ex-
tracts from exhaust particulates of Nissan CN6-33,
Volkswagen Rabbit and Oldsmobile 350 diesel
motors showed considerably greater activity. Re-
sults with these extracts in the Ames test
(Table 2) corresponded roughly with the DDA assay
data, suggesting a reasonable agreement between
mutagenicity and the DNA damage measured by this
assay.
TABLE 2. MUTAGENICITY IN THE AMES TEST OF 100 yg
OF THE DIESEL PARTICIPATE EXTRACTS
SHOWN IN FIG. 4*
Strain
Source TA98 TA100
Caterpiller 3304 (EPA) 0 32
Nissan CN6-33 (DEC) 390 1321
Oldsmobile 350 (EPA) 94 180
Oldsmobile 350 (DEC) 827 1488
Volkswagen Rabbit (DEC) 150 490
Basic fraction 16 0
Acidic fraction 627 228
Neutral fraction 119 166
*Colonies per plate. S9 not used. See Choudhury
and Doudney (4) for details of the fractionation
and the Ames assay.
Choudhury and Doudney (4) fractionated the Rabbit
diesel extracts. In the DDA assay the acidic
and neutral fractions were several orders of
magnitude more inhibitory than the basic frac-
tion (Fig. 5). The Ames test showed little
mutagenic activity with the basic fraction but
considerable mutagenicity with the acidic and
neutral fraction (Table 2), again supporting a
459
-------
correspondence between mutagenicity and DNA
damage.
DISCUSSION
By measuring the effect of DNA-damaging chemi-
cals on the increase in turbidity of £. col i
recA uvrA before inducible DNA repair functions
have time to develop, the DDA assay is ex-
tremely sensitive in detecting those chemicals.
0.18-
O.lOo
i.o
Figure 5.
icr2 io-4 io-6
EXTRACT FRACTION (^g/ml)
io-'2
io-14
DDA activity of acidic basic and
neutral fractions of the Volkswagen
Rabbit diesel particulate extract.
See Table 2 for Ames test results.
End-point measurements, such as survival, would
not achieve a similarly high sensitivity. Such
damage presumably is not lethal, as a recovery
mechanism exists (3).
The results presented here are preliminary and
were obtained primarily to demonstrate the
utility of the assay. The biological signifi-
cance of any given degree of contamination re-
mains unknown, and the specific responses of
460
-------
the DDA assay to representative chemicals like-
ly to contaminate the environment must be
studied in detai1.
Detection of DNA-damaging chemicals by this
system should not be considered to indicate
with certainty the presence of mutagenic and
carcinogenic agents. Excessively contaminated
material should be studied further by concen-
tration, fractionation, and isolation of groups
of chemical components, using this test to
follow activity; and the isolated DNA-damaging
materials should be tested by more traditional
methods, such as the Ames test, to confirm
mutagenicity.
Nevertheless, the extreme sensitivity of this
assay allows a number of approaches toward
examination of the environment for contami-
nation not possible heretofore. For example:
Air Particulate Sampling. The DDA assay should
greatly reduce the quantity of material needed,
so that more frequent monitoring of air for
DNA-damaging material is possible.
Mutagenic Vapors. This assay should be sensi-
tive enough for examination of vapors in air.
The low levels of potential mutagens found
even in severely contaminated air make col-
lection and testing with less sensitive systems
impractical.
Surface Contamination. The sensitivity of the
DDA assay should make detection of contamination
of surfaces by the wipe method practical.
Surface Soil Sampling. The sensitivity of the
assay should make testing of extracts of small
soil samples for DNA-damaging material practical,
Contaminants in soil surface layers are a good
indicator of air contamination.
461
-------
REFERENCES
1. Nishioka, H., and K. Nabori. 1975. Some
modifying factors on mutagenicity of a
nitrofuran compound (AF2) in E_. col j .
Mutat. Res. 31, 267.
2. Tatsumi, K. and H. Nishioka. 1977. Ef-
fects of DNA repair systems on antibac-
terial and mutagenic activity of an anti-
tumor protein, neocarzinostaten. Mutat.
Res. 48, 195-204.
3. Peters, J., and J. Jagger. 1979. Repair
of near-UV lethal damage in E^. col i by an
inducible j*e_c_-A-gene-independent system.
Abstracts, 7th Annual Meeting, American
Society for Photobiology, 154.
4. Choudhury, D. R., and C. 0. Doudney.
1979. Isolation of mutagenic fractions
of diesel exhaust particulates as an ap-
proach to identification of the major con-
stituents. This symposium.
5. Gibbs, R., G. Wolzak, S. Byer and S. Hyde,
1979. Emissions from diesel vehicles in
consumer use. This symposium.
6. Choudhury, D. R., and B. Bush. 1979.
Contribution of particulate emissions to
composition of polynuclear aromatic hydro-
carbons in air. This symposium.
462
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Session III
BIOCHEMICAL AND METABOLIC EFFECTS
OF DIESEL EMISSIONS AND DIESEL EMISSION COMPONENTS
Chairman:
Robert M. Danner
Lung Biochemistry of Rats Chronically Exposed to Diesel
Participates.
Misiorowski, R. L., K. A. Strom, J. J. Vostal , and
M. Chvapil.
DMA-Binding Studies with Diesel Exhaust Particle Extract.
Pederson, Thomas C.
The Effect of In Vivo Exposure of Rats to Diluted Diesel
Exhaust on Microsomal Oxidation of Benzo(a)pyrene.
Charboneau, J. and R. McCauley.
Benzo(a)pyrene Metabolism in Mice Exposed to Diesel Exhaust:
I. Uptake and Distribution.
Tyrer, H. W., E. T. Cantrell, R. Horres, I. P. Lee, W.
B. Peirano, and R. M. Danner.
Benzo(a)pyrene MetaboTisrn in Mice Exposed to Diesel Exhaust:
II. Metabolism and Excretion.
Cantrell, E. T., H. W. Tryer, W. B. Peirano, and R. M.
Danner.
Effect of Exposure to Diesel Exhaust on Pulmonary Prosta-
glandin Dehydrogenase (PGDH) Activity.
Chaudhari, A., R. G. Farrer and S. Dutta.
463
-------
Session III
(Continued)
Effect of Diesel Participate Exposure on fldenylate and
Guanylate Cyclase of Rat and Guinea Pig Liver and Lung.
Schneider, David R. and Barbara T. Felt.
Biochemical Alternations in Lung Connective Tissue in
Rats and Mice Exposed to Diesel Emissions.
Bhatnagar, R.S., M. Z. Hussain, K.
Sorensen, F. M. Von Dohlen, R. M.
Danner, L. McMillan, and S. D. Lee
464
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LUNG BIOCHEMISTRY OF RATS
CHRONICALLY EXPOSED TO DIESEL PARTICULATES
R. L. Misiorowski, K. A. Strom*
J. J. Vostal* and M. Chvapil
Division of Surgical Biology
Arizona Health Sciences Center
Tucson, AZ
*Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
ABSTRACT
Male rats were exposed under comparable experimental condi-
tions to diesel emissions at concentrations of 0, 250 and
1500 yg/m3 diesel exhaust particulate, for twenty hours a
day and 5-1/2 days per week. After 12, 24 and 36 weeks of
exposure, the rats were sacrificed and the lungs analyzed
by morphological and biological methods.
Body weight was not changed by the exposure to diesel
emissions. Lung wet weight, normalized to body weight, was
significantly higher (p < 0.01) after 12 weeks of exposure
to 1500 ug/m3 diesel exhaust particulates. Cell content in
the lung tissue (DNA) was significantly increased at 1500
ug/m3 after six months. The rate of collagen synthesis was
significantly increased while collagen deposition was not
affected. Total lung collagen content increased proportion-
ately with the change in lung weight. Prolyl hydroxylase
was increased only after 12 weeks exposure and its activity
decreased with the age of the rats. A significant increase
in lipids (phospholipids and cholesterol) was found in rats
exposed for 36 weeks at 1500 yg/m3. The profile of fatty
acids was not significantly changed.
The results suggest that due to the exposure of rats to
diesel emissions, lipids accumulate within the lung tissue
465
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and the reactivity of fibrogenic cells is enhanced. The
increased rate of collagen synthesis is compensated by
increased collagen degradation, resulting in no net collagen
accumulation in the lung during the investigated time period.
INTRODUCTION
Current views on the reactivity of lung tissue to inhaled
particles and vapors changed substantially with the intro-
duction of new scientific techniques to ascertain tissue
injury. It has been documented that exposure of lungs to
respirable particles of classical inert dusts such as Ti02
induce cellular reactions comparable with transient inflamma-
tory changes [1]. Intratracheal instillation of saline induced
significant changes in lung lipids, non-coilagenous proteins,
and DMA, which returned to normal within 48 hours [2,3].
Finally, it has been recognized that lung (similar to liver
tissue), plays an important role in metabolism of various
xenobiotics [4]. Accordingly, we can expect that bio-
chemical changes will be observed in the lungs of rats
chronically exposed to high concentrations of diesel particu-
lates. The nature, magnitude, and the dynamics of occurrence
of these changes was the objective of this study.
MATERIALS AND METHODS
The experimental protocol dealing with lung biochemistry is
outlined in the following Tables 1 and 2. In brief, young
adult male rats, eight in each group, were exposed for 12,
24 and 36 weeks, 20 hours daily, 5-1/2 days per week, to 250
or 1500 yg/m3 of diesel exhaust particulates. A control
group was exposed in an identical environment to ambient air.
At time of sacrifice, body weight and lung wet weight were
recorded. The content of DNA was determined [5] as a
measure of cell content and hydroxyproline was determined [6]
as a measure of collagen in the homogenized tissue. We also
studied noncollagenous proteins and some lipids [8], namely,
phospholipids [9], cholesterol [10], and the fatty acid
profile. Among dynamic parameters of tissue injury, we
measured the rate of collagen biosynthesis [11], and the
activity of prolyl hydroxylase [12,13]. In the experience
of our laboratory, the latter reflects the functional state
of fibrogenic cells. We also analyzed the activity of lysyl
oxidase, an enzyme which stabilizes collagen molecules by
covalently cross-linking them [14]. The increased activity
of this enzyme quite often coincides with the inflammatory
process. The changes in all these parameters of lung chem-
istry were related either to total lung weight (absolute
changes) or expressed per gram of lung tissue (density
changes). Finally, in order to obtain a meaningful biological
insight into the changing lung chemistry, we plotted the
results against various reference bases, such as lung weight,
466
-------
DNA content, or ratios of the above parameters. Anywhere a
reference is made to statistical significance in the results,
these are related to appropriate control group and refer to
values of p < 0.01 unless otherwise indicated.
Table 1
Experimental Design
Exposure ^
Do se__J^-- T i me
Control
250 yg/m3
1500 yg/m3
12 24 36
Total 72 rats
Exposure 20 hours/day, 5-1/2 days/week.
Rats - Rattus novvegious from Charles River
Young adult males
Table 2
Experimental Design
Lung Parameters Studied
Reference
Body weight
Lung wet weight
DNA
Collagen (Hydroxyproline)
Noncollagenous Protein
Lipids - Phospholipids
Cholesterol
Fatty Acid Profile
Fluorescent Product
Rate of Collagen Synthesis
Prolyl Hydroxylase Activity
Lysyl Oxidase Activity
Burton (1956) [5]
Stegemann (1958) [6]
Lowry et al (1951) [7]
Folch (1951) [8]
Raheja et al (1973) [9]
Zak (1965) [10]
GLC method
Tappel (1973) [15]
Juva and Prockop (1966) [11]
Mutton et al (1966) [12]
Bentley and Weiser (1976) [13]
Pinell and Martin (1968) [14]
467
-------
RESULTS
Body weights of rats in all groups increased significantly
during 36 weeks of the experiment; however, no adverse
effects of diesel particulate exposure was observed within
any one time period (Table 3).
Lung wet weights, absolute or relative (related to 100 g body
weight), were significantly higher in rats exposed to 1500
vig/m3 diesel particulates at all these sampling periods. No
significant changes in lung weight were observed in 250
yg/m3 exposed group (Table 3).
We analyzed the possible reasons for a significant increase
of wet weights of lungs of rats exposed to 1500 yg/m3 D.P.
The most logical explanation for such an increase would be the
accumulation of tissue water in the interstitium due to an
increased vessel wall permeability. Direct determination of
tissue water in these groups of rats as compared to control
lung showed, however, no edema formation, the water content
forming 77.9% in the exposed lungs and 78.4% in the controls.
Table 3
Effect of Chronic Exposure to Two Concentrations of Diesel
Exhaust Particulates on Body and Lung Weight of Rats
BODY WEIGHTS (g)
Weeks
12
24
36
12
24
36
Concentration diesel particulate (yg/m3)
0 250 1500
346.5 ± 15.8
363.1 ± 25.1
397.6 ± 17.7
336.4 ± 22.5
374.8 ± 15.0
414.6 ± 33.4
343.2 ± 17.9
376.4 ± 19.2
403.8 ± 19.9
LUNG WEIGHTS - RIGHT LOBE (g)
0.7118 ± 0.0354
0.7408 ± 0.0461
0.7571 ± 0.0416
0.6980 ± 0.0408
0.7477 ± 0.0238
0.8039 ± 0.0516
0.7508 ± 0.0485
0.8444 ± 0.0453*
1.012 ± 0.0709*
LUNG WEIGHT - RIGHT LOBE/BODY WEIGHT (g/IOOg)
12
24
36
0.2055 ± 0.0091
0.2041 ± 0.0044
0.1904 ± 0.0082
0.2078 ± 0.0085
0.1998 ± 0.0078
0.1965 ± 0.0072
0.2188 ± 0.0076*
0.2244 ± 0.0065*
0.2506 ± 0.0098*
Variability given by x ± S.D.
*p > .01.
468
-------
Cellular content (DNA) of the lung was unchanged at 250
yg/m3 exposure compared to controls for the entire observa-
tion period of 36 weeks (Fig. 1A). Surprisingly, exposure to
250 yg/m3 for three months indicated slightly reduced cell
content of the lung; the difference was of a borderline sig-
nificance. In contrast, the DNA content of the lung was
significantly increased in high exposures to diesel particu-
lates after 24 and 36 weeks and indicated an increased
number of cells in the lung. High level of deposited par-
ticulates obviously leads to a hyperplastic cellular stimula-
tion of the respiratory system. Concurrent cytological
studies reported in another paper in this symposium indicate
a several-fold increase in the number of cells freely migrat-
ing in the alveolar lumen, mainly pulmonary alveolar macro-
phages, and, later on, also neutrophilic phagocytes at the
same exposure level. No difference in cell (DNA) density was
found in any group at any time interval studied (Fig. IB).
Figure 1A Total DNA contained in the left lung lobe. Total
DNA in the left lobe was determined using the method of
Burton (1956) [5].
469
-------
-C
00
c
3 4000
E
a
Z.
Q
2
12
24
weeks
D 0 Mg
0 ZSO
1500 M«/m9
Figure IB Density of DMA in the left lung lobe. The density
of DNA in the left lobe is expressed as yg/g wet weight.
Determinations were done on rat lungs exposed to 0, 250, and
1500 yg/m3 diesel particulates at 12, 24 and 36 weeks.
Rate of collagen synthesis was significantly increased only
at the highest exposure (1500 yg/m3) to diesel particles
after 12, 24, and 36 weeks. The magnitude of overall
synthetic rate decreases slightly with the duration of the
exposure, that is, with the age of the exposed animals (Fig.
2). The increased production of collagen at 1500 yg/m3 did
not result, however, in net collagen deposition in the lung,
as both absolute amount (Fig. 3A) and density (Fig. 3B) of
this fibrous protein were significantly lower in almost all
experimental groups when compared to appropriate controls.
On the other hand, no significant changes in the rate of
collagen synthesis were detected in the low level (250 yg/m3)
exposure group (Fig. 2), in spite of the accumulation of
milligram amounts of diesel particulates and highly pro-
nounced discoloration of the lung.
The specific activity of prolyl hydroxylase was significantly
elevated in both experimental groups only at 12 weeks (Fig. 4).
470
-------
Figure 2 Rate of Collagen Biosynthesis. The specific
activity, dpm/ymol, of ^C-hydroxyproline was determined
using the method of Juva-Prockop (1966) [11]. Samples were
obtained from rat lungs exposed to 0, 250 and 1500 yg/m3
diesel particulates for 12, 24 and 36 weeks.
e
a.
x
an
Figure 3A Total Collagen in the right lung lobe. The total
collagen contained in the right lobe of rats exposed to 0,
250 and 1500 u9/m3 diesel particulate for 12, 24 and 36 weeks
was determined as hydroxyproline using the method of Stegemann
(1958) [6].
471
-------
1
5
T
12
24
weeks
36
D 0 Mg/m>
0250 Mg/m'
B 1600 »i|/n>'
Figure 3B Density of collagen in the right lung lobe.
density of collagen in the right lobe is expressed as
hydroxyproline, ug/g wet weight.
The
d aoo
GO
E aoo
ex
a
I!
12
24
weeks
II
36
D 0
250 /ig/m'
1SOO /ig/m3
Figure 4 The specific activity of prolyT hydroxylase. The
specific activity of prolyl hydroxylase, dpm/VgDNA, was
determined using the method of Mutton et al (1966) [12], as
modified by Bentley and Weiser (1976) [13].
472
-------
Phospholipids and cholesterol were both significantly in-
creased after 36 weeks of exposure to 1500 yg/m3 diesel
participates when related to whole lung. In addition,
phospholipids at 24 weeks were also significant at p > 0.05
at this particulate concentration. The density of these
lipid species followed exactly the same pattern (Table 4).
In contrast, no significant differences were observed in the
250 yg/m3 group in either analysis.
The profile of fatty acids showed significant reduction of
saturated as well as polyunsaturated fatty acids in 12 week
exposures to 250 as well as to 1500 yg/m3 particulates. No
changes were observed after 24 or 36 weeks of the exposure.
Table 4
Lipid Content in the Lung of
Control and Diesel Particulate Exposed Rats
PHOSPHOLIPIDS mg/g wet weight left lobe
Diesel particulate (yg/m3)
Time (weeks) ° 25° 150°
12 22.426 ± 3.710 19.792 ± 2.114 22.106 ± 3.924
24 22.441 + 2.299 21.686 ± 2.622 27.207 ± 3.945*
36 21.718 ± 1.802 21.918 ± 2.588 29.580 ± 2.599**
CHOLESTEROL mg/g wet weight left lobe
12 5.469 ± 0.406 5.000 ± 0.582 5.024 ± 0.537
24 7.090 ± 0.563 6.943 ± 0.330 7.566 ± 0.894
36 5.265 ± 0.488 5.582 ± 0.604 6.732 ± 0.631**
FLUORESCENT PRODUCT
relative fluorescence/g wet weight left lobe
12 13.63 ±1.98 14.92 ±1.92 16.30 ±2.25*
* p > .05
** p > .01
473
-------
DISCUSSION
An analysis of the described changes in lung chemistry,
although very complex, indicates that the lung can tolerate
the exposures to concentrations of diesel particulates as
high as 250 yg/m3 for more than 36 weeks, without any
chemical tissue reaction. This happens in spite of the
accumulation of large quantities of particulate matter in the
respiratory system. Particulate burden of the lungs was
estimated at approximately 3-4 mg of particulates for this
concentration at 36 week exposure.
Only with excessive exposures (lung particulate burden in
excess of 7-8 mg) do we observe a biochemical reaction. It
is manifested by an increased cellularity of the lung
(produced in part by the mobilization of cellular macrophages,
in particular alveolar macrophages and neutrophils in the
alveolar lumen) and increased rate of collagen synthesis.
There are also significant increases in noncollagenous pro-
teins (data not shown), phospholipids and cholesterol.
Interestingly, these reactions are accompanied by changes in
the specific activity of prolyl hydroxylase which becomes
significant after three months of exposure at the dose of 250
yg/m3 and probably reflects transient stimulation of fibro-
blasts, probably due to a specific fibroblast stimulating
factor released by pulmonary macrophages.
The rate of collagen synthesis was increased only after ex-
posures to high concentrations of diesel particulates and was
not, at all, elevated at the low level exposures. No speci-
fic inflammatory changes other than the increase in non-
collagenous proteins and lipids, which are normally observed
as a transient impact after inhalation of inert dusts or
intratracheal instillation of saline were found at any expo-
sure level or exposure time. The reasons why we have diffi-
culty in characterizing the changes in lung biochemistry as
inflammatory changes are based on the following findings:
a. no increased water content in the lung
b. no cell infiltration with increase of cell density
(DNA/weight)
c. only early transient increase in the activity of prolyl
hydroxylase
d. no increased activity of lysyl oxidase.
Several chemical parameters of tissue injury showed signifi-
cant deviation from controls only at the early time exposure
and returned to normal values after prolonged exposure.
These were prolyl hydroxylase activity, profile of fatty acids
and accumulation of fluorescent products [15]. Although it
may be difficult to interpret the meaning of such a transient
change, we speculate that the lung reacts to the early
474
-------
encounter with diesel exhaust particulates by compensatory
cell activation and proliferation of the whole lung tissue.
The cytodynamics of this type of lung injury is reminiscent
of the reparative changes in the lung after oxygen exposure
as reported by Bowden and Adamson [16]. They also observed
depression of cellular activity with longer exposure times.
The morphology of the lungs at 1500 yg/m3 exposure will be
reported later in this session and showed local inflammatory
lesions in the peribronchial tissue. This reaction may
explain the increased content of granulocytes as well as
alveolar macrophages in the lavaged fluid from this group of
rats. The finding of inflammatory foci in the lung inter-
stitial tissue appeared to be restricted and did not result
in significant changes in lung chemistry, indicating a dif-
fuse inflammatory reaction. Furthermore, several presenta-
tions at this Symposium documented that none of the pulmonary
function tests of animals exposed to diesel exhaust particu-
lates were changed, as would be expected in tissue modified
by the inflammatory process. Thus, the biochemical changes
reported in this study are further corroborated by morpho-
logical picture and evaluation of the lung functions. The
complex analysis seems to indicate that the proliferation of
the lung cells is only a compensatory reaction to the pres-
ence of diesel exhaust particulates in the alveoli.
It may, therefore, be concluded that only under conditions of
massive deposits of diesel particulates in the respiratory
system can we observe a slow dose-dependent proliferative
reaction. Lungs exposed to the low dose of diesel particu-
lates showed the first trend for change only after a prolonged
period of 36 weeks of the exposure.
The increases in lung wet weight, non-collagenous proteins
and lipids, and the accumulation of fluorescent product may
reflect a pattern usually found in non-specific inflammatory
processes. A fibroproliferative process, on the other hand,
is documented by increased specific activity of prolyl
hydroxylase and the increased rate of collagen synthesis.
Our results show that in spite of increased collagen synthe-
sis, there is no abnormal collagen deposition in the lung. The
observed change in the synthetic rate indicates, therefore,
increased collagen turnover. Presently, we are testing the
hypothesis that there is an overall increase of collagen
turnover by specific animal experiments and by experiments
with fibroblasts grown in tissue culture incubated with
diesel particles.
In order to illustrate the magnitude and dynamics of the
reported changes in lungs exposed to diesel particles, we
would like to show briefly other results presenting similar
biochemical changes in the lungs after various doses of
475
-------
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476
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quartz (Table 5). Note that within 6 days after a single
intratracheal administration of the smallest dose (10 mg)
of the standardized fibrogenic silica dust DQ-12, much more
pronounced changes occurred than after exposure to 1500
pg/m3 diesel particles for 36 weeks. Since we cannot
directly compare the effects of a single intratracheal
injection with chronic exposure, this comparison is not
meant to minimize the potential risk of the inhalation of
diesel particulates on the lung, but to show the observed
findings in perspective to the well-known fibrogenic effect
of silica particles.
It seems that our findings produced more new questions than
answers to existing problems. We certainly would like to
know the possible contribution of benzo[a]pyrene metabolism
by the lung mixed function oxidases to the susceptibility
of the tissue exposed to particulates. Another important
question is the role of the possible involvement of lung
macrophages in the development of the fibrotic lesion [17-
20]. Evidence was presented at this symposium that lung
macrophages are activated by diesel particulates. Other
evidence suggests that activated macrophages produce sub-
stances promoting the activity of fibrogenic cells.
Obviously, we can expect that many of these mechanisms
respond to the deposited mass of particulates. More research
is needed before the observed subtle changes can be described
in detail, evaluated, and assessed in the light of the
expected exposure levels to diesel exhaust on our roads.
REFERENCES
1. Moores, S.R., Sykes, S.E., Morgan, A., Evans, N., Evans,
J.C., Holmes, A. The short-term cellular and biochem-
ical response of the lung to toxic dusts: an in vivo
cytotoxicity test. Given at a Symposium on "The in
vitro effects of mineral dust." Sept. 1979.
2. Eskelson, C.D. , Stiffel, V., Owen, J.A., Chvapil, M.,
(1979). The importance of the liver in normal and
silicotic lung-lipid hemeostasis. 2. Cholesterol.
Environ. Res., 19:432-441.
3. Chvapil, M., Eskelson, C.D., Stiffel, V., Owen, J.A.,
Early changes in the chemical composition of the rat
lung after silica administration. Arch. Environ. Health,
(In press).
4. Hook, G.E.R., Bend, J.R. (1976). Pulmonary Metabolism
of Xenobiotics. Life Science, 18, 279-290.
5. Burton, K., (1956). Study of conditions and mechanisms
of diphenylamine reaction for colorimetric estimation of
deoxyribonucleic acid. Biochem. J., 62:315-323.
477
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6. Stegemann, H. (1958). Mikrobest immung von Hydroxy-
prolin tnit Chloramin-T und p-Dimethylaminobenzaldehyd.
Hoppe Seylers Z. Physiol. Chem., 311:41-95.
7. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall,
R.J. (1951). Protein Measurement with the Folin Phenol
Reagent. J. Biol. Chem., 193:265-275.
8. Folch, J., Ascoli, I., Lees, M., Meath, J.A., LeBaron,
F.N. (1951). Preparation of lipide extracts from
brain tissue. J. Biol. Chem., 191:833.
9. Raheja, R.K., Charanjit, K., Singh, A.H., Bhatia, I.S.,
(1973). New colorimetric method for quantitative
estimation of phospholipids. J. Lipid Res., 14:695-697.
10. Zak, B. (1965). Total and Free Cholesterol. In "Stan-
dard Methods of Clinical Chemistry," (S. Meites, Ed.),
5:79-89, Academic Press, New York.
11. Juva, K., Prockop, D.J. (1966). Modified procedure for
the assay of 3H or llfC-labelled Hydroxyproline.
Anal. Biochem., 15:77-83.
12. Hutton, J.J., Tappel, A.L., Undenfriend, S. (1966). A
Rapid Assay for Collagen Proline Hydroxyproline.
Anal. Biochem., 15:77-83.
13. Bentley, J.P., Weiser, K.A. (1976). A Simple Microassay
of Prolyl Hydroxylase Applicable to Skin Punch Biopsy
Specimens. Conn. Tiss. Res., 4:255.
14. Pinnell, S.R., Martin, G.R. (1968). The Cross-Linking
of Collagen and Elastin--Enzymatic Conversion of Lysine
in Peptide Linkage to a-amino-6 Semialdehyde (Allysine)
by an Extract from Bone. Proc. Nat!. Acad. Sci.,
61:708.
15. Tappel, A.L. (1973). Lipid Peroxidation Damage to Cell
Components. Fed. Proc., 32:1870-1875.
16. Bowden, D.H., Adamson, I.Y.R. (1971). Reparative
Changes Following Pulmonary Cell Injury. Arch. Path.,
92:279-283.
17. Chvapil, M., Old-New Problems of Silicotic Fibrosis.
In: Biochemistry of Silicon and Related Problems.
(Bendz, G., Lindqvist, I., Eds.) Plenum Press, New
York and London (1978).
478
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18. Heppleston, A.G., Styles, J.A. (1967). Activity of a
macrophage factor in collagen formation by silica.
Pathology, 214:521.
19. Richards, R.J., Wusteman, F.S. (1974). The effects of
silica dust and alveolar macrophages on lung fibro-
blasts in vitro. Life Sci., 14:355-364.
20. Aalto, M., Potila, M., Kulonen, E. (1976). The effect
of silica-treated macrophages on the synthesis of
collagen and other proteins in vitro. Exp. Cell Res.,
97:193-202.
General Discussion
6. SIGNER: Many of the reactions that you have talked
about are classic reactions which occur when there is in-
jury to the peripheral lung. However, at least one that
you didn't mention was changes in type II cells. Do you see
a type II cell hyperplasia such as you commonly see when
you injure the peripheral lung?
M. CHVAPIL: I believe that there is an inflammation
located mostly in the bronchial tree. There is an increase
in polymorphonuclear leucocytes not only in the lavage
system, but also in the bronchial tissue. Therefore, there
is a typical reaction only after the intratracheal ad-
ministraton of 15,000 micrograms of silicon particle. Some
changes in the type II cells have been related, but I was
not involved in this type of research.
6. SIGNER: Would an increase in some of the phospho-
lipid profiles be suggestive of that change?
M. CHVAPIL: I believe the phosphate deposited in the
lungs comes primarily from the liver. We recently have
shown that the liver recognizes injury to the lung within
a few hours and the enormous lipid centers in the liver are
active in the producton and trannsport of these lipids. We
just did a study, and the liver seems to be the major pro-
ducer of lipids not relating to the surface active lipids.
6. SIGNER: Do you see any changes in the bronchiolar
epithelium and hyperplasia?
M. CHVAPIL: Again the same answer. There will be the
morphological study of injuries to explain this. For ex-
ample, rats exposed to low oxygen tension will definitely
have an increase in the fibrinogenic subpopulation in the
lung. That has been shown. There is a cellular hyper-
plasia. If you have an increased population of fibrino-
genic cells you would also expect that they will produce
more collagen. In fact, local tissue hypoxia has been
recognized as a fibrinogenic stimulus. Therefore, I be-
lieve hypoxia should promote and potentiate the fibrino-
genic reaction to any stimulus.
479
-------
BURNS: It looks like your changes could be due just to
a simple hypoxia. It is unclear where it is corning from,
but could it happen that way?
M. CHVAPIL: I believe your suppositions of hypoxia
wouldn't apply in this case because we couldn't see any-
thing that would indicate a hypoxia reaction. You don't
want to overload your lungs with diesel particles; 75 mil-
ligrams of silicate dumped into the lung is an absolutely
huge amount which cannot be compared with anything that you
are doing with inhalation studies. What I am trying to say
is that the changes I am finding with silicate in six days
are enormous as compared with those we are finding in nine
months. In regard to your first comment, after the highest
dose there is a bronchial inflammation. Otherwise, I haven't
seen it myself.
480
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DNA-BINDING STUDIES
WITH DIESEL EXHAUST PARTICLE EXTRACT
Thomas C. Pederson
General Motors Research Laboratories
Biomedical Science Department
Warren, Michigan 48090
ABSTRACT
This study examines whether chemical components from diesel
exhaust particulates react with DNA to form covalently bound
adducts. Experiments in this report describe the in vitro
reaction of purified DNA with a dichloromethane extract of
diesel exhaust particulates in the absence or presence of
enzyme activation by rat liver microsomes. The reactivity of
the particle extract was compared to that of benzo[a]pyrene
metabolites using low temperature fluorescence techniques
which detect small quantities of polycyclic aromatic compounds
bound to DNA. Incubation of DNA with the particle extract in
the presence of microsomal enzymes produced no detectable
fluorescent adducts in contrast to model experiments using
benzo[a]pyrene. However, addition of the particle extract to
incubation mixtures containing benzo[a]pyrene markedly de-
creased formation of benzo[a]pyrene-DNA adducts because the
particle extract inhibits microsomal enzymes which activate
benzo[a]pyrene and other polycyclic aromatic hydrocarbons.
In the absence of microsomal enzymes, fluorescent material
was detected in DNA exposed to high concentrations of the
particle extract, but probably not as a result of covalent
binding because the mutagenic activity of the particle extract
remained unchanged during prolonged incubation with DNA.
This stability is in contrast to the rapid decrease in muta-
genic activity of benzo[a]pyrene-4,5-oxide during incubation
with DNA. Thus, direct mutation of bacteria by the particle
extract may require activation by bacterial enzymes as is
known to occur with nitroaromatic compounds.
481
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INTRODUCTION
Most chemical mutagens and carcinogens induce genetic damage
by forming covalent adducts with DNA [1-3]. As a result of
such modifications to the structure of DNA, errors can be
introduced in a cell's genetic information during DNA repli-
cation and cell division. In bacteria or cultured animal
cells, these errors may be recognized as genetic mutations.
Similar genetic errors are believed to be an initiating event
in a series of changes which -in vivo transform normal cells
into tumors [4].
The chemical components extracted from diesel particulates by
organic solvents are mutagenic in bacteria [5-7]. These
extracts contain benzo[a]pyrene (B[a]P) and other polycyclic
aromatic hydrocarbons (PAH) but simple unsubstituted PAH are
not the major mutagenic components because the particle
extract is mutagenic to bacteria even in the absence of
mammalian microsomal enzymes. However, certain substituted
PAH such as epoxides and nitro-derivatives are directly
mutagenic in bacteria [8-12]. Since the identity of extracted
mutagenic components and their biological mechanism of action
are unknown, it is difficult to assess what genetic effects
would be induced in human or other mammalian tissues.
The DNA binding studies described in this report were all
done in vitro, by incubating purified DNA with diesel particle
extract or reactive metabolites of B[a]P. Since the particle
extract contains both unsubstituted PAH and components
directly mutagenic to bacteria, the reaction between the
extract and DNA was studied in both the presence and absence
of the microsomal enzyme system from rat liver. The methods
used to detect covalent binding of mutagens to DNA include
the low temperature fluorescence techniques first described
by Ivanovic and coworkers [13]. Low temperatures prevent
fluorescence quenching and allows the fluorescence to be
detected in conventional spectrofluorometers. Although re-
stricted to fluorescent adducts, primarily those having
condensed aromatic ring structures, the characteristics of
excitation and emission spectra provide valuable structural
information.
METHODS
Collection and Extraction of Diesel Particulates Detailed
procedures for collecting and extracting the diesel particu-
lates have been reported by Chan and Lee [14]. Diesel
exhaust particulates were collected by electrostatic precipi-
tation from the undiluted exhaust (at 100°C) of a 1978 5.7 L
GM diesel engine built by Oldsmobile (constant speed = 1350
r/min, load = 96 N-m). The extract was prepared by Soxhlet
482
-------
extraction with dichloromethane. Solvent was removed under a
stream of nitrogen gas and the extracted material redissolved
in dimethylsulfoxide (10 or 100 mg/mL) and stored at -80°C.
Animal Treatment and Microsome Isolation Liver microsomal
membranes were prepared from rats (male, Sprague-Dawley, 200-
250 g) intraperitoneally injected 48 hours prior to sacrifice
with 3-methylcholanthrene (20 nig/kg) dissolved in corn oil.
The microsomal fractions were isolated from liver homogenates
by differential centrifugation, suspended in a solution
containing 50% glycerol and 25 mM Tris-HCl (pH 7.4) and
stored at -80°C.
Microsomal Enzyme Activity Assays Metabolism of B[a]P to
alkali-extractable metabolites was assayed by the fluorescence
method of Nebert and Gelboin [15] in reaction mixtures con-
taining 50 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 0.7 mM NADP, 10
mM glucose-6-phosphate, G-6-P-dehydrogenase (1 unit/ml) and
0.025 to 0.050 mg/mL of microsomal protein. Product formation
is expressed as fluorescence equivalents of 3-hydroxy-B[a]P
used as an internal standard. Reaction rates were determined
from the linear increase in product concentration measured by
sequential removal of aliquots from reactions incubated for
15 minutes at 37°C under air in a Dubnoff metabolic shaking
incubator.
DNA-Binding Reactions Reaction mixtures used in these DNA-
binding studies contained deproteinized salmon sperm DNA (1.0
mg/mL), 100 mM phosphate buffer (pH 7.5), 2.5 mM MgCl2, and
0.1 mM EDTA in a total reaction volume of 4 or 5 ml. Incu-
bations including enzymatic activation also contained rat
liver microsomes (1.0 mg/mL), 0.7 mM NADP, 10 mM glucose-6-
phosphate, and G-6-P-dehydrogenase (1 unit/mL). The mixtures
were incubated for 1 hour at 37°C under air in a covered
Dubnoff metabolic shaker. Reactions were stopped by placing
the reaction vessels on ice and microsomal membranes were
removed by centrifugation at 105 000 g for 60 minutes.
Isolation and Purification of DNA All operations in the
isolation of DNA were done under subdued light at 0-4°C. DNA
was recovered from the incubation mixtures by ethanol precip-
itation, redissolved in 1/10 SSC (15 mM NaCl, 1.5 mM Na-iso-
citrate) and extracted three times with ethylacetate. After
addition of sodium-4-aminosalicylate (4%, w/v) and NaCl (1%,
w/v), the solution was extracted twice with equal volumes of
phenol-cresol reagent [16] (100 g phenol, 11 mL water, 14 mL
m-cresol and 0.1 g 9-hydroxyguinoline). Remaining traces of
the phenol reagent were removed by extraction with diethyl-
ether. The DNA was precipitated by ethanol, wound on a glass
rod and washed successively in ethanol, hexane and again
ethanol. Further purification was achieved by isopycnic
centrifugation in cesium chloride gradients containing 17 mM
483
-------
TrisHCl (pH 7.4) and 9 mM EDTA. Peak fractions were combined
and chromatographed on a short column (1.2 by 10 cm) of
sephadex G-75 and preequilibrated with 60% EGW (60 ml ethylene
glycol, 40 ml 1/10 SSC). The DNA therefore eluted from the
column in the 60% EGW solvent. The concentration of DNA,
determined by its absorbance at 260 nm, was usually between
0.3 and 0.5 mg/mL.
Low-Temperature Fluorescence Measurements The equipment used
to measure the fluorescence of DNA samples in 60% EGW at
subzero temperatures is illustrated in Figure 1. The fluor-
escence instrument is a SLM Model 8000D photon counting spec-
trofluorometer equipped with double grating emission and
single grating excitation monochromators. The sample holder
was covered with a foam insulating tape and cooled by a
chilled stream of nitrogen gas regulated at temperatures from
ambient to -190°C. The nitrogen gas exhausts around a cylin-
drical quartz sample cell (4 mm i.d., 7 mm o.d.) preventing
frost formation.
Xenon Lamp
Excitation
Monochromator
Emission
Monochromator
Quartz Tube
with DNA Sample
Insulated Sample
Holder
Flow
Controller
Detector
Pressure
Regulator
Figure 1 Cryogenic equipment for low-temperature fluorescence
measurements.
Spectral Corrections All fluorescence spectra were normalized
to exciting light intensity with the response of the reference
photomultiplier corrected by use of a quantum counter (rhoda-
mine B). Excitation spectra using broad-band emission were
recorded with excitation wavelengths transmitted by a Corning
7-54 filter and emitted light transmitted by a long-pass
filter with 50% transmission at 370 nm. Digitized spectra,
484
-------
recorded by a HP9815 calculator and transferred to the GM
Research Laboratories computer center, were further corrected
for background luminescence from the cell, solvent and the
Inherent fluorescence of DNA. Fluorescence intensities are
described in either arbitrary units or quantitative units
(PFU) obtained by using pyrene as an internal standard. The
PFU is defined as the fluorescence intensity of 1 nM pyrene
in 60% EGW measured under the same conditions as the DNA
samples with excitation at 338 nm and emission at 372 nm or
over a broad-band spectrum as already described.
Chemicals 3-Methylcholanthrene, sodium-4-aminosalicylate, m-
cresol, 8-hydroxyquinoline, bovine serum albumin, NADP,
glucose-6-phosphate, G-6-P-dehydrogenase (Type XI), cesium
chloride (optical grade) and salmon sperm DNA (Type III) were
obtained from Sigma Chemical Company. Benzo[a]pyrene (Gold
Label) and 2-nitrofluorene were purchased from Aldrich
Chemical Company. Ethylene glycol (Baker Analyzed) and
phenol were obtained from J. T. Baker Chemical Company. A
sample of 3-hydroxybenzo[a]pyrene was obtained from Dr. C. A.
King of the Michigan Cancer Foundation and benzo[a]pyrene-
4,5-oxide was obtained from Dr. J. J. McCormick at Michigan
State University. Phenol and m-cresol were redistilled and
benzo[a]pyrene was recrystallized from toluene-methanol
(1:1). DNA was deproteinized by extraction with phenol-
cresol reagent. All other chemicals were reagent grade.
RESULTS AND DISCUSSION
Fluorescence Enhancement at Low Temperatures The low temp-
erature fluorescence spectra described by Ivanovic et al [13]
were measured at the temperature of liquid nitrogen, -196°C.
In this study, fluorescence enhancement was investigated at
several temperatures between +20 and -190°C. Figure 2 shows
the emission spectra of two samples - one containing pyrene
and the other DNA exposed to B[a]P and liver microsomal
enzymes. At room temperature, pyrene exhibits a well defined
emission spectra but no distinct spectral peaks are discern-
able in the fluorescence from the DNA-B[a]P sample. As the
samples are cooled to -100°C, the 60% EGW solvent is trans-
formed from a liquid to a rigid transparent glass. At this
temperature, a distinct fluorescence spectra is observed in
the DNA-B[a]P sample with peak emissions at 379 and 399 nm.
Below -100°C the fluorescence intensity of the DNA-B[a]P
sample increases only slightly in contrast to the fluorescence
enhancement with the sample containing pyrene. Since the
measurement of fluorescence spectra at -100°C presents less
operational problems than lower temperatures, the spectra of
DNA samples described in the remainder of this report were
measured at -100°C.
485
-------
A
0.6-1
EAflSSION SPECTRA OF PYRENE
IN A 60% ETHYLENE CLYCOL SOLUTION
TEMPERATURE
jgpjc
-100*0
440
460
u
u
w
a:
o
t,
B EMISSION SPECTRA OF B(a)P-DNA ADDUCT
IN A 60% ETHYLENE CLYCOL SOLUTION
0.15 -,
0.10-
0.05-
0.00
TEMPERATURE
j-jpj:
-100*0
360 380 400 480 440
WAVELENGTH, nm
460
Figure 2 Fluorescence emission spectra at ambient and sub-
zero temperatures. A - spectra of a 10 nM pyrene sample
excited at 338 nm. B - spectra of DMA incubated with 10 yM
benzo[a]pyrene and microsomal enzymes as described under
Methods. The excitation wavelength used for the B[a]P-DNA
sample was 348 nm.
Fluorescence Spectra Using Broadband Emission The fluores-
cence emission spectra shown in Figure 2 were readily measured
using excitation maxima previously described by other investi-
gators [13]. To detect other fluorescent adducts in DNA
exposed to either B[a]P or diesel particle extract, excitation
spectra were recorded while measuring fluorescence emission
over a broad band of the visible wavelength range. The
fluorescence excitation spectra of the DNA-B[a]P reaction
product measured by broad-band emission is shown in Figure 3.
Part A shows the uncorrected spectra of three samples; the
reaction product; a control sample of DNA; and the solvent
and sample cell. The corrected spectra in Figure 3B retains
only the fluorescence of the bound B[a]P metabolite(s) ex-
pressed in PFU per 10 absorbance units of DNA (~0.5 mg/mL) as
described under Methods. The excitation and emission maxima
486
-------
of the DNA-adduct isolated from this reaction mixture are the
same as those described for the products formed from the 7,8-
dihydrodiol-9,10-epoxide of B[a]P [13,17].
E A
i
I
0.8 n
0.6-
0.4 -
0.2-
0.0-
EXCITATION SPECTRA IN 60% EGW
AT -100'C USING BROAD-BAND EMISSION
SAMPLE
BWP-DNA
60J5..EGW
P.NA.
360 380
300
330
340 360
o
EL
B
15-i
10-
260
CORRECTED EXCITATION SPECTRA
OF B(a)P-DNA ADDUCT
860 300 380 340
WAVELENGTH, nm
Figure 3 Fluorescence excitation spectra of B[a]P-DNA.
A - uncorrected spectra in aribtrary fluorescence units.
B - spectrum corrected for luminescence of the solvent cell
and DNA expressed in pyrene fluorescence units.
Reaction of DNA with Diesel Particle Extract in Presence of
Liver Microsomes"DNA was exposed to diesel particle extract
at concentrations of 0.1, 1 and 5 mg/mL in the presence of
liver microsomes under the same conditions used to prepare
the DNA adducts of benzo[a]pyrene. The fluorescence excita-
tion spectra of DNA recovered from these incubation mixtures
are shown in Figure 4. There are no discernable fluorescent
components in these spectra which are shown in quantitative
comparison to the fluorescence spectrum of the DNA adduct of
B[a]P. The amount of B[a]P in the particle extract used in
these studies has not been measured, but Williams and Chock
[18] have reported exhaust particulates from a similar GM 5.7
L engine contains about 10 yg/g of extractable B[a]P. Thus
487
-------
CORRECTED EXCITATION SPECTRA OF DNA
FROM REACTIONS CONTAINING LIVER ENZYMES
12-1
10-
e-
Reaction
Conditions
0.0035 mg/mt BaW
0)
O
260 280 300 330
Wavelength, nm
340
360
Figure 4 Comparison of fluorescence detected in DNA incubated
with diesel particle extract (DPE) or B[a]P in presence of rat
liver microsomal enzymes. Other reaction conditions are de-
scribed under Methods and fluorescence spectra were recorded
as in Figure 3B.
16-,
2
<
o
t.
a.
u
2
10 -I
5-
0.1
1 10
BENZO(a)PYRENE,
100
Figure 5 The quantity of fluorescent B[a]P-DNA adduct formed
as a function of the concentration of benzo[a]pyrene. Fluor-
escence emission spectra were recorded as described in Figure
2B at a temperature of -100°C. The values plotted are from
the intensities at the 379 nm emission maximum.
488
-------
DNA binding reactions containing 5 mg/mL of the particle
extract should contain nearly 0.5 yg/mL of B[a]P plus other
PAH compounds. As shown in Figure 5, the fluorescence of
covalently bound B[a]P was detected in DNA exposed to as
little as 0.25 yg/mL. Thus the particle extract either
contains less B[a]P than estimated, or interferes with the
DNA-binding activity of B[a]P.
Inhibition of DNA-Binding Activity by Diesel Particle
Extract In Figure 5, the maximal amount of B[a]P-DNA binding
activity occurs at concentrations of 2.5 to 10 yg/mL and at
higher concentration there is a decrease in binding. This
decrease is probably due to the competition between reactions
1 and 2 as shown below. Both reactions are
B[a]P -Primary products (1)
including B[a]P-7,8-diol
B[a]P-7,8-diol -B[a]P-diol-epoxide (2)
catalyzed by the P450-monoxygenase in liver microsomes. Thus
high concentrations of the initial substrate, B[a]P, should
inhibit the formation of the diol-epoxide product which sub-
sequently binds to DNA. Other PAH compounds may also inhibit
DNA binding activity. Similar inhibition by multicomponent
mixtures has been observed in mutagenicity studies with shale
oil fractions [19] and model studies have shown one PAH
compound may either inhibit or augment the mutagenic action
of another [20,21]. Therefore, the effect of the diesel
particle extract on formation of DNA adducts of B[a]P was
examined.
Figure 6 shows the amount of fluorescent B[a]P adduct detected
in DNA from incubation mixtures which include particle extract
in addition to B[a]P and liver microsomes. At a concentra-
tion of 0.1 mg/mL DPE, there is little change in this DNA-
binding activity, but at 1.0 mg/mL, the amount of fluorescent
adduct formed is decreased by more than 90%. This decrease
in DNA binding activity can be attributed to the inhibition
of B[a]P metabolism. Table 1 describes the B[a]P hydroxylase
assays which show the inhibition by the particle extract.
Since the DNA-binding reactions contained 10 uM B[a]P, the
effect of the extract on B[a]P metabolism was measured with
this substrate concentration. These assay mixtures also
contained 1 mg/mL bovine serum albumin, BSA, as a substitute
for the 1 mg/mL of microsomal protein in the DNA-binding
reactions. Under these conditions, the inhibition of B[a]P
hydroxylase activity is similar to the inhibition of DNA-
binding activity. Therefore, the presence in diesel particu-
lates of B[a]P, or other PAH compounds requiring metabolic
activation, would probably not be detected in this DNA-
binding assay.
489
-------
INHIBITION OF B(a)P BINDING TO DNA
BY DIESEL PARTICLE EXTRACT
REACTION
CONTROL
360
400 420 440
WAVELENGTH, nm
4CO
Figure 6 Inhibition of B[a]P-DNA adduct formation in reaction
mixtures containing the diesel particulate extract. Each
reaction mixture contained 10 uM benzo[a]pyrene and microsomal
enzymes. Fluorescence emission spectra were recorded as
described in Figure 2B.
Table 1
Inhibition of B[a]P Hydroxylase by the Particle Extract
Reaction
Conditions
Benzo[a]pyrene
Hydroxylase
nmol/min/mg
80 yM B[a]P
10 yM B[a]P*
+ 0.1 mg/mL DPET
+0.4 mg/mL DPE
+1.0 mg/mL DPE
*Reaction mixtures al
Diesel particle extr
3.48
2.20
0.88
0.13
0.05
so contain 1.0 mg/mL BSA.
^act
Direct Reactions of Diesel Particle Extract with DNA in
Absence of Liver Microsomes Purified DNA was exposed to the
particle extract as previously described except the rat liver
490
-------
microsomal enzymes were omitted from the incubation mixtures.
The DNA was isolated from these reaction mixtures and exam-
ined for fluorescent adducts. Figure 7 shows the corrected
excitation spectra of DNA exposed to three concentrations of
the particle extracts. A fluorescent component was observed
in DNA from the reaction containing the highest concentration
of extract. As shown in Figure 4, this fluorescent component
was not observed when DNA had been similarly exposed in the
presence of microsomal membranes, and may be a non-covalent
association of material in the extract which was not separated
by the isolation methods employed.
EXCITATION SPECTRA OF DNA EXPOSED TO DPE
IN THE ABSENCE OF LIVER MICROSOMAL ENZYMES
< "»-
Q
B '
z
< «-
2
£ «-
ESCENCE, P
> PO
FLUOR
,
REACTION
CONDITIONS
0.1 mg/mL DPE
1 ma/mL DPE
«'*..
»' >. 5 ma/ml DPE
* % * ^W* »»»
/ ,,,4
,,..^-,
360 380 300 320 340
WAVELENGTH, nm
360
Figure 7 Fluorescence excitation spectra of DNA incubated
with diesel particulate extract (DPE) in the absence of an
enzyme activation system. Spectra were recorded as described
in Figure 3B.
A more definitive characterization of the direct reaction
between DNA and the particle extract was obtained by measuring
the stability of mutagenic activity during incubation in the
presence of DNA. The experimental procedures are illustrated
in Figure 8. The mutagenic agent is incubated in either the
absence or presence of DNA and at timed intervals, aliquots
are removed from the incubation mixtures and assayed for
mutagenic activity in the Ames assay as described by Siak et
al [22]. If the mutagen reacts covalently with DNA, it will
no longer be available to mutate bacteria. These experiments
were done using two model compounds, both being direct acting
mutagens, but distinctly different in their mechanism of
491
-------
action. One is benzo[a]pyrene-4,5-oxide which reacts directly
with DNA to form a covalently bound adduct [17]. The other
model compound is 2-nitrofluorene which is not directly
reactive towards DNA but is a direct acting mutagen because
it is activated by a bacterial nitro-reductase enzyme system
[12].
STABILITY OF DIRECT-ACTING MUTAGENS
Incubation Transfer of Plating the Counting
of Mutagens Reaction Aliquot Overlay Mixtures Mutant Colonies
Mutagen Mutagen
+
DNA
Bacteria and
Molten Top Agar
Model Compounds
Benzolalpyrene-4,5-oxide
-reacts with DNA-
2-Nitrofluorene
-not reactive-
-activated by bacteria-
Figure 8 Experimental procedure for examining in vitro DNA-
binding activity of direct-acting bacterial mutagens.
The results of the experiments with these two model compounds
and the diesel particle extract are shown in Figure 9. As
expected, benzo[a]pyrene-4,5-oxide rapidly lost mutagenic
activity when incubated with DNA in contrast to the slow loss
of activity in the control incubation mixture by hydration of
the epoxide. The mutagenic activity of 2-nitrofluorene was
not decreased at all during the 5 hour incubation with DNA.
In a similar experiment with diesel particle extract, the
mutagenic activity is not decreased during incubation with
DNA. Identical incubation mixtures had been prepared 5 days
beforehand which still retained full mutagenic activity.
492
-------
Therefore, the direct acting bacterial mutagens in the diesel
particle extract do not bind covalently to purified DNA.
INCUBATION MIXTURES
a -DNA
REVERTANTS,
0 §
1 1 1 ..,,.. n
|2-NITROFLUORENE|
CO
<
H
W
1(11
100 1
\
B days '
1 =
DIESEL PARTICLE EXTRACT!
80 160 240
TIME, minutes
320
Figure 9 Stability of direct-acting bacterial mutagens incu-
bated in the absence or presence of purified DNA. Mutagenic
activity was measured as described in Figure 8 with 0.05 ml
aliquots from reaction mixtures containing model compounds
(B[a]P-4,5-oxide, 2 yg/mL or 2-nitrofluorene, 20 yg/mL) or
diesel particle extract (1 mg/mL) in 1/10 SSC incubated with
or without DNA (2 mg/mL) at 37°C.
493
-------
CONCLUSIONS
This study of reactions between DNA and chemical components
extracted from diesel exhaust particulates emphasizes the
necessity of further characterizing the specific chemical
components and biological mechanisms of action responsible
for the activity of diesel particulates in various assays of
genetic toxicity. The inhibition by diesel particle extract
of microsomal enzymes and binding of B[a]P to DNA illustrates
the problems encountered in studying complex mixtures. Such
interference by one component of a mixture with the action of
another may or may not be of consequence In vivo. The direct
acting bacterial mutagens in the extract have been shown to
not react with purified DNA which implies bacterial enzymes
may activate these components as occurs in the mutagenic
action of nitroaromatic compounds. Therefore, a detailed
understanding of the molecular mechanisms involved in both
bacterial and mammalian systems is required to assess any
health effects which might be attributed to bacterial muta-
gens in diesel exhaust particulates.
ACKNOWLEDGEMENT
I wish to thank C. Anderson and M. Baxter for their valuable
assistance in the laboratory and acknowledge the essential
efforts of T. Chan and T. Johnson in collection of diesel
particulates and preparation of the particle extract.
REFERENCES
1. Grover, P.L., 1976. Reactions of polycyclic hydrocarbon
metabolites with DNA. In In vitro metabolic activation
in mutagenesis testing, (F. J. de Serres, J. R. Fouts,
J. R. Bend and R. M. Philpot, Eds.), North-Holland, New
York, pp. 295-312.
2. Brookes, P., 1977. Role of covalent binding in carcin-
ogenicity. In Biological Reactive Intermediates, (D.
J. Jallow, J. J. Kocsis, R. Snyder and H. Vainio, Eds.)
Plenum Press, New York, pp. 470-480.
3. Lutz, W. K., 1979. In vivo covalent binding of organic
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process of chemical carcinogenesis. Mutation Res.,
65:289-356.
4. Weinstein, I. B., Yamasaki, H., Wigler, M., Lee, L. S.,
Fisher, P. B., Jeffrey, A. and Grunberger, D., 1979.
Molecular and cellular events associated with the action
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Carcinogens: Identification and Mechanisms of Action,
(A. C. Griffin and C. R. Shaw, Eds.), Raven Press, New
494
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York, pp. 399-418.
5. Huisingh, J., Bradow, R., Jungers, R., Claxton, J.,
Zweidinger, R., Tejada, S., Bumgarner, I., Duffield, F.,
Waters, M., Simon, V., Hare, C., Rodriguez, C. and Snow,
L. Application of Bioassay to the characterization of
diesel participate emission. EPA-600/9-78-027.
6. McGrath, J. J., Schreck, R. M., Siak, J. S., 1978.
Mutagenic screening of diesel participate matter.
Presented at 71st Annual Meeting of the Air Pollution
Control Assoc., Houston, Texas, June 25-30, 1978.
7. Yergey, J., 1979. The sampling, chemical characteriza-
tion and biology assay of diesel exhaust particulate
matter from a light duty diesel engine. Presented at
Spring Meeting of Central States Section,-The Combustion
Institutes, Columbus, Indiana, Technical paper #CSS/CI-
79-06, April 9-10.
8. Cookson, M. J., Sims, P. J. and Grover, P. L., 1971.
Mutagenicity of epoxides of polycyclic hydrocarbons
correlates with carcinogenicity of parent hydrocarbons.
Nature, 234:186-187.
9. Lehr, R. E., Yagi, H., Thakker, D. R., Levin, W., Wood,
A. W., Conney, A. H. and Jerina, D. M., 1978. The bay
region theory of polycyclic aromatic hydrocarbon-induced
carcinogenicity. In Carcinogenesis, Vol. 3: Polynuclear
Aromatic Hydrocarbons (P. W. Jones and R. I. Freudenthal,
Eds.) Raven Press, New York, pp. 231-241.
10. Pitts Jr., J. N., Van Cauwenberghe, K. A., Grosjean, D.,
Schmid, J. P., Fitz, D. R., Belser Jr., W. L., Knudson,
G. B. and Hynds, P. M., 1978. Atmospheric reactions of
polycyclic aromatic hydrocarbons: Facile formation of
mutagenic nitro derivatives. Science, 202:515-519.
11. McMahon, R. E., Cline, J. C. and Thompson, C. Z., 1979.
Assay of 855 test chemicals in ten tester strains using
a new modification of the Ames test for bacterial
mutagens. Cancer Research, 39:682-693.
12. Rosenkranz, H. S. and Poirier, L. A., 1979. Evaluation
of the mutagenicity and DNA-modifying activity of car-
cinogens and noncarcinogens in microbial systems.
J. Natl. Cancer Inst., 62:873-892.
13. Ivanovic, V., Geacintou, N.E. and Weinstein, I.B., 1976.
Cellular binding of benzo[a]pyrene to DNA characterized
by low temperature fluorescence. Biochem. Biophys. Res.
Commun., 70:1172-1179.
495
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14. Chan, T. L. and Lee, P. S., 1980. Diesel participate
collection for biological testing: Comparison of elec-
trostatic precipitation and filtration. In: Proc. Int.
Symp. on Health Effects of Diesel Engine Emissions,
U. S. Environmental Protection Agency, Cincinnati, OH,
December 3-5, 1979.
15. Nebert, D. W. and Gelboin, H. V., 1968. Substrate-
inducible microsomal aryl hydroxylase in mammalian cell
culture. J. Biol. Chem., 243:6242-6249.
16. Kirby, K. S. and Cook, E. A., 1967. Isolation of DNA
from mammalian tissues. Biochem. J., 104:254-257.
17. Daudel, P., Duquesne, M., Vigny, P., Grover, P. L. and
Sims, P., 1975. Fluorescence spectral evidence that
benzo[a]pyrene-DNA products in mouse skin arise from
diol-epoxides. FEBS Letters, 57:250-253.
18. Williams, R. C. and Chock, D. P., 1980. Characteriza-
tion of Diesel Particulate Exposure. In: Proc. Int.
Symp. on Health Effects of Diesel Engine Emissions,
U. S. Environmental Protection Agency, Cincinnati, OH,
December 3-5, 1979.
19. Pelroy, R. A. and Peterson, M. R., 1979. Use of Ames
test in evaluation of shale oil fractions. Environ.
Health Persp., 30:191-203.
20. Kubitschek, H. E. and Haugen, D. A., 1979. Biological
activity of effluents from fluidized bed combustion of
high-sulfur coal. Presented at International Society for
Occupational and Environmental Health, Park City Environ-
mental Health Conf., Park City, Utah, April 4-7, 1979.
21. Hermann, M., Durand, J. P., Charpentier, J. M., Chaude,
0., Hofnung, M., Petroff, N. and Weill, N., 1980.
Correlations of mutagenic activity with polynuclear
aromatic hydrocarbons content of various mineral oils.
In: Proc. Fourth Int. Sympo. Polycyclic Aromatic
Hydrocarbons, Battelle Columbus Laboratories, Columbus,
OH. October 2-4, 1979.
22. Siak, J., Chan, T. L. and Lee, P., 1980. Diesel
Particulate extracts in bacterial test systems. In:
Proc. Int. Symp. on Health Effects of Diesel Engine
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Cincinnati, OH., December 3-5, 1979.
496
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General Discussion
D. HOFFMAN: Isn't it possible that your dihydroxide
epoxide has reacted with some other "tar constituents" from
diesel exhaust? You have not made the control experiment
in which you have added the dihydroxide epoxide and that
would have shown that there is no binding.
T. PEDERSON: That is true. That is another experiment
that could be done, however, it did inhibit AHH activity.
497
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THE EFFECT OF IN VIVO EXPOSURE OF RATS
TO DILUTED DIESEL EXHAUST ON
MICROSOMAL OXIDATION OF BENZO[a]PYRENE
J. Charboneau and R. McCauley
Wayne State University
School of Medicine
Department of Pharmacology
540 East Canfield
Detroit, Michigan 48201
ABSTRACT
The effect of exposure of Fischer 344 rats to diesel
exhaust at two concentrations, 250 pg/M3 and 1500 yg/M3
of diesel particulates on several parameters including
the metabolism of benzo[a]pyrene to polar metabolites and
to metabolites capable of alkylating denatured DNA has
been studied. A years exposure had no effect on the size
of the animals or their livers; however after the higher
exposure lung weight was increased. After one year, the
ability of lung microsomes to oxidize benzo[alpyrene was
impaired at either exposure level. At present it is not
certain whether this effect is an artifact due to contam-
ination of the lung microsomes by diesel particles or a
genuine biochemical pathology. Liver microsomes seemed
to be less able to oxidize the carcinogen at six and
twelve weeks after exposure but were no different than
control after a years exposure.
INTRODUCTION
Polycyclic aromatic hydrocarbons are environmental pollu-
tants which are formed as products of the incomplete
combustion of organic materials. Many of the chemicals
are substrates for the mixed function oxidase system
located in the enodplasmic reticulum of mammalian cells.
498
-------
In rats, for example, these chemicals are oxidized to
epoxides, phenols and diols by a microsomal electron
transport system with spectral characteristics, immunologic
properties and catalytic specificities that are different
from the usual system responsible for metabolism of
xenobiotics (1). Among the products of these oxidations
catalyzed by mammalian microsomes are certain epoxide
metabolites which have been shown to be mutagenic in in
vitro assays (2). Benzo[a]pyrene is a carcinogen which
has been shown to be a prototypical substrate for this
polycyclic aromatic hydrocarbon metabolizing system, and
like other substrates for the mixed function oxidases,
has the interesting property of being able to induce the
enzymes responsible for its own metabolism (3). In fact,
there is good evidence that human populations that are
exposed to unusually high levels of polycyclic hydrocarbons
have an increased level of the microsomal oxidases respon-
sible for benzo[a]pyrene metabolism (4). In our study, we
have assayed the activity of the microsomal enzymes involved
in benzo[a]pyrene metabolism in both the lungs and livers
of Fischer 344 rats which were exposed to two concentrations
of diesel exhaust as part of a larger study conducted by
the General Motors Corporation. Our objective was to
decide whether under the conditions of this study sufficient
amounts of the appropriate polycyclic hydrocarbons were
introduced via inhalation to produce either a local induc-
tion of the microsomal oxidases in lung or a more wide-
spread induction involving the liver as well.
METHODS
Male Fischer 344 rats were exposed at the General Motors
Technical Center to clean air or to diesel exhaust diluted
with clean air to 250 yg or 1500 JJg of diesel parti-
culates per cubic meter. The details of the conditions of
the exposure have been described by others (5). After
various intervals of time of up to one year, groups of
six rats were fasted overnight and then removed from the
exposure chamber to be transported about 30 min by auto
to our laboratory. The animals were then weighed,
decapitated and the liver and lungs were removed, weighed
and homogenized in four volumes of 0.25 M sucrose.
Mitochondria, nuclei and cell debris were removed by
centrifugation at 17400 x gmax for 10 min, and microsomes
were isolated from the resulting supernatant by centri-
fugation at 144,000 x gmax for 60 min. These microsomal
pellets were resuspended in 0.25 M sucrose.
Oxidation of benzo[a]pyrene to polar metabolites was
measured by our modification of the method described by
Van Cantfort e_t a_l. (6). Briefly, about .1 mg hepatic or
.4 mg pulmonary microsomal protein was incubated for
30 min at 35Oc in 0.4 ml of a mixture which contained
499
-------
1 mM NADP, 6.5 mM sodium isocitrate, 0.05 mM MnCl2, 0.44
international units of pig heart isocitrate dehydrogenase
in 50 mM Tris-HCl. Incubations with hepatic microsomes
were buffered at pH 7.5 while those with pulmonary micro-
somes were at pH 8.0. The reaction was initiated by the
addition of 14c-benzo[a]pyrene in 10 \il of an acetone
solution; the final benzo[ot]pyrene concentration was 80
Umolar. Nonenzymatic oxidation of the substrate was
estimated for each tissue from each animal by performing
a similar incubation except that NADP, isocitrate and
isocitrate dehydrogenase were omitted. The reactions were
stopped by adding one ml of a mixture of 0.15 N KOH in
85% (v/v) dimethyl sulfoxide. This mixture was twice
extracted with three ml of hexane and an aliquot of the
aqueous-dimethyl sulfoxide phase was removed and neutralized
with acetic acid so that radioactivity could be estimated
by liquid scintillation using a commercially available
xylene based scintillant. The difference between the
amount of radioactivity in the complete incubations and in
the incubations without the NADPH generating system was
used to calculate the rate of enzymatic oxidation of
benzo[a]pyrene. Data are expressed as nmoles of benzo[a]-
pyrene oxidized per 30 min incubation per mg microsomal
protein and are based on estimates of protein by the Lowry
method (7).
A second incubation procedure was used to estimate the
ability of the metaholites generated in vitro to alkylate
DNA. About 0.4 mg hepatic or 1.0 mg pulmonary microsomal
protein was incubated in 2 ml of a mixture of the same
composition as was used to assay the oxidation of benzo[a]-
pyrene except that 0.5 mg per ml denatured and sheared
calf thymus DNA was also present. Incubations were per-
formed in the presence and absence of the NADPH generating
system and the reaction was started by adding 50 yl of
3.2 mM 14C-benzo [ot]pyrene in acetone. Incubations were
allowed to proceed for 60 min at 35°C and then the reaction
was stopped by adding sodium dodecysulfate to a final
concentration of 1%, and then the mixtures were further
incubated for 30 min with 8 mM EDTA and 5 Ug per ml
ribonuclease A from bovine pancreas. The ribonuclease
treated mixture was then extracted with an equal volume
(2.5 ml) of water saturated phenol for 30 min at room
temperature. The aqueous phase was then mixed with two
volumes of 95% ethanol and DNA was precipitated overnight
at -18OC. The precipitated DNA was collected by centri-
fugation at 1000 x g for 10 min and washed twice with 5 ml
aliquots of cold 95% ethanol. Finally, the precipitate
was dissolved in 1 ml of water; a small protion was
removed to estimate the absorbance at 260 nmeters and the
remainder was used to estimate the amount of covalently
found radioactivity by liquid scintillation. Data are
expressed as nmoles of benzo[a]pyrene bound to a mg of DNA
500
-------
per hr per mg of microsomal protein. Protein was determined
by the Lowry procedure (7), and calculations of the recovery
of DMA were based on the assumption that one mg of DNA
absorbs 20 A250 over a cm light path.
[7, 10-14C]Benzo[a]pyrene (5-15 Ci/mole) was purchased from
Amersham Corporation as a benzene solution. The radio-
chemical was subsequently dried under a stream of N2 and
redissolved in 3 ml of hexane. The hexane was extracted
with 3 ml 0.5 N NaOH in 80% ethanol followed by an extrac-
tion with 3 ml 1 N NaOH. Finally, the hexane layer was
collected, evaporated under N2 and the residue was dissolved
in an acetone solution of 3.2 mM unlabeled benzo[alpyrene.
The benzo[a]pyrene was stored in the dark at -18°C. Other
chemicals were purchased from either the Sigma Chemical
Company or the BioRad Corporation.
RESULTS AMD DISCUSSION
Fischer 344 rats were exposed to either clean air or
dilutions of diesel exhaust containing 250 pg per M3 or
1500 pg per M3 of diesel particulates. The conditions of
this exposure have been detailed elsewhere (5). At in-
tervals of 6, 12, 24 and 53 weeks animals were removed
and body, liver and lung weights were estimated; microsomal
fractions were prepared from liver and lungs and were used
to estimate the ability of the animals to oxidize benzo[a]-
pyrene to more polar metabolites and to metabolites capable
of covalently reacting with DNA.
In Table 1 the body weights for the three groups are shown,
and it can be seen that while the average weight gain over
the exposure period was somewhat less in the group exposed
to the highest concentration of exhaust, there were not
substantial differences between the average body weights
at any of the intervals tested. The data in Table 2
TABLE 1
THE EFFECT OF EXPOSURE TO DIESEL EXHAUST
ON RAT BODY WEIGHT (GRAMS)*
Concentration of Diesel Particles
Duration
6 weeks
12 weeks
24 weeks
53 weeks
Control
251 +_ 3
284 +_ 6
335 + 10
391 + 14
250pg/m3
249 + 6
297 +_ 6
364 +_ 9
392 + 10
1500yg/m3
261 + 4
299 +_ 3
335 + 6
366 + 17
*Each value is the mean +_ S.E.M. of from 4 to 6 individual
values.
501
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TABLE 2
THE EFFECT OF EXPOSURE TO DIESEL EXHAUST
ON RAT LIVER WEIGHT (GRAMS)*
Concentration of Diesel Particles
Duration
6 weeks
12 weeks
24 weeks
53 weeks
Control
7.0 + 0.2
7.4 +_ 0.2
7.7 +_ 0.2
9.3 + 0.6
250yg/m3
7.3 + 0.3
7.5 + 0.2
9.1 + 0.4
9.5 + 0.3
1500Ug/m3
6.9 +_ 0.2
7.1 +_ 0.1
8.3 +_ 0.2
8.7 + 0.5
*Each value is the mean +_ S.E.M. of from 4 to 6 individual
values.
indicate that neither exposure affected liver size. How-
ever, the effect of the exposure on the activity of hepatic
microsomal oxidases responsible for the conversion of
benzo[a]pyrene to more polar metabolites (Table 3) was
more complicated. Six weeks of exposure to either
TABLE 3
THE EFFECT OF EXPOSURE TO DIESEL EXHAUST
ON THE ABILITY OF RAT LIVER MICROSOMES
TO OXIDIZE BENZO[a]PYRENE TO POLAR
METABOLITES (nmoles per 30 min per mg protein)*
Concentration of Diesel Particles
Duration Control 250yig/m3 1500i_lg/m3
6 weeks 21.4^0.6 12.5^2.1 10.7^1.0
12 weeks 14.4 +_ 1.4 15.7 +_ 0.8 8.5 +_ 1.3
24 weeks 11.5 +_ 0.5 12.3 +_ 1.3 12.0 _+ 1.1
53 weeks 16.9 +_ 2.4 13.6 +_ 1.8 12.9 +_ 2.7
Each value is the mean +_ S.E.M. of from 4 to 6 individual
values.
concentration of diesel particulates appeared to reduce
the ability of the hepatic microsomes to oxidize benzo[a]-
pyrene. At twelve weeks only exposure to the highest
concentration interfered with the microsomal oxidation
and by six months neither group was significantly different
from control. The ability of microsomes to generate
metabolites of benzo[a]pyrene which were capable of
502
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alkylating DMA was also assayed (Table 4). Again at the
briefest exposure interval, the microsomal oxidation seemed
to be surpressed, but was restored to normal after longer
periods of exposure.
TABLE 4
Control
0.13 +_ 0.02
0.04 + 0.003
0.05 + 0.01
0.07 + 0.05
250yg/m3
0.04 +_ 0.01
0.04 +_ 0.003
0.05 + 0.01
0.08 + 0.04
1500yg/m3
0.05 +_ 0.01
0.04 +_ 0.004
0.04 + 0.01
0.06 + 0.01
THE EFFECT OF EXPOSURE TO DIESEL EXHAUST
ON THE ABILITY OF RAT LIVER MICROSOMES
TO OXIDIZE BENZO[a]PYRENE TO ALKYLATING
METABOLITES (nmoles bound to mg DNA per
hr per mg protein)*
Concentration of Diesel Particles
Duration
6 weeks
12 weeks
24 weeks
53 weeks
*Each value is the mean +_ S.E.M. of from 4 to 6 individual
values.
As might be expected, the exposure to diesel exhaust had
more obvious effects on the lungs. As can be seen from
Table 5, after a year's exposure to the highest exhaust
concentration, the size of a lung pair was about 50%
greater than control lungs or lungs from animals exposed
to the lowest dose. The most striking effect of the
TABLE 5
THE EFFECT OF EXPOSURE TO DIESEL EXHAUST
ON THE WEIGHT OF THE RAT LUNG PAIR (GRAMS)*
Concentration of Diesel Particles
Duration
6 weeks
12 weeks
24 weeks
53 weeks
Control
1.5 +_ 0.1
1.3 +_ 0.1
1.3 +_ 0.1
1.4 + 0.2
250pg/m3
1.5 +_ 0.2
1.2 + 0.1
1.1 +_ 0.1
1.5 + 0.1
1500pg/m3
1.0 +_ 0.1
1.5 +_ 0.2
1.6 + 0.2
2.1 + 0.1
*Each value is the mean +_ S.E.M. of from 4 to 6 individual
values.
503
-------
exposure was on the appearance of the lungs and tissue
fractions prepared from them. The lungs of exposed animals
ranged from a gray color after the shortest exposures to a
dark gray shot through with dark black deposits after longer
exposures, and microsomal membranes prepared from these
lungs were contaminated with the dark material so that
they were gray to black suspensions instead of the usual
pinkish suspension of control microsomes. Neither group
exposed to diesel exhaust was able to metabolize benzo[a]-
pyrene as well as the controls at any time interval although
this effect is most evident after 53 weeks of exposure (Table
6).
TABLE 6
THE EFFECT OF EXPOSURE TO DIESEL EXHAUST
ON THE ABILITY OF RAT LUNG MICROSOMES
TO OXIDIZE BENZO[a]PYRENE TO POLAR
METABOLITES (nmoles per 30 min
per mg protein)*
Concentration of Diesel Particulates
Duration
6 weeks
12 weeks
24 weeks
53 weeks _ _ _
*Each value is the mean +_ S.E.M. of from 4 to 6 individual
values.
By this time the highest exposure had markedly compromized
the animals ability to convert benzo[a]pyrene to polar
compounds and even exposure to 250 g/M3 had reduced the
enzymatic activity by more than 50%. The way in which
diesel exhaust produces this inhibition is not clear. It
is possible that the particulates that contaminate the
lung microsomes may interfere with the assay of benzoia]-
pyrene metabolism (e.g., reduce the availability of sub-
strate or directly inhibit the microsomal oxidations).
Alternatively, the lowered enzymatic activity may reflect
a pathological change in the lung endoplasmic reticulum
related to the exposure. This remains an open question
at present; however we have tried to evaluate the potential
of the diesel particulates to inhibit the in vitro metabolism
by mixing experiments. The inhibitory effects of boiled
control microsomes, and boiled low exposure and high ex-
posure microsomes on metabolism of benzo [otjpyrene by
untreated control microsomes are compared in the experiment
described in Table 7. The results suggest that particulates
504
Control
0.23 +_ 0.08
0.29 + 0.02
0.13 +_ 0.05
0.32 + 0.05
250ug/m3
0.12 +_ 0.05
0.16 +_ 0.02
0.10 +_ 0.06
0.15 + 0.05
ISOOyig/m3
0.16 + 0.02
0.23 ^ 0.03
0.07 +_ 0.02
0.02 + 0.01
-------
present in the microsomes prepared from the highest
exposure group may inhibit benzo[a]pyrene metabolism;
however, these results should be considered preliminary.
The production of alkylating metabolites by lung microsomes
was too low to be detected by our methods in either controls
or exposed animals.
TABLE 7
THE EFFECT OF LUNG TRAPPED DIESEL PARTICULATES
ON IN VITRO BENZO[a]PYRENE OXIDATION BY LUNG MICROSOMES*
Additions Percent of Control
None 100
Boiled control microsomes 83 +_ 23
Boiled low exposure microsomes 140 +_ 16
Boiled high exposure microsomes 65 _+ 12
*Microsomes from control lungs were incubated as usual
except the indicated additions were made. Boiled micro-
somes were from a 12 week exposure to clean air, 250 pg
per M3 and 1500 yg per M3 of diesel particulates. These
microsomes were heated at boiling for 5 min in a capped
tube and allowed to cool before mg microsomal protein
were added to a control incubation. Each value is the
mean of five determinations +_ S.E.M.
It seems possible to conclude that after slightly over a
year's exposure, neither concentration of diesel emissions
has had an effect on the growth rate or liver weight of
Fischer 344 rats. A reduced capacity for hepatic oxidation
of benzo [ct]pyrene observed after earlier exposure periods
was not evident at later periods, and may either have been
repaired or may have been due to an artifact in the exper-
imental procedure. There were, however, more evident
changes in both the size of the lungs and in the ability
of the pulmonary microsomes to oxidize benzo[a]pyrene.
After a year at the highest exposure level lung weight was
increased and benzo [otlpyrene metabolism was severely
impaired at both exposure levels. The reduced ability to
metabolize benzo[a]pyrene was unexpected, and it should be
noticed that at no time during these studies was induction
of this activity observed.
This research was supported by the General Motors Corp.
Detroit, Michigan.
505
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References
1. Thomas, P., Lu, A., Ryan, D., West, S., Kawalek, J.
and Levin, W. (1976) Mol. Pharmacol. 12: 746.
2. Jernia, P.M., page 91 in Carcinogenesis: A Comprehensive
Survey, Vol. 1, eds. R. Frendenthal and J. Jones, Raven
Press, N.Y., 1976.
3. Conney, A.H. (1967) Pharmacol. Rev. 19: 317.
4. Pekonen, 0., in Carcinogenesis: A Comprehensive Survey,
Vol. 1, eds. R. Frendenthal and J. Jones, Raven Press,
N.Y., 1976.
5. Schreck, R.M., Hering, W.E., D'Aray, J.B., Soderholm, S.C.
and Chan, T.L. (this issue)
6. Van Cantfort, J., De Graeve, J. and Gielen, J. (1977)
Biochem. Biophys. Res. Comm. 79: 505.
7. Lowry, O., Rosebrough, N., Farr, A. and Randall, R.
(1951) J. Biol. Chem. 193: 265.
General Discussion
B. DANIEL: I noticed that the AHH levels in your con-
trol animals dropped after six weeks, is that normal for
that species?
R. MCCAULEY: That is right, however, it does seem to be
a smooth drop. To answer your question, I would be very
tempted to correlate the drop with age, of course, because
that is the only thing I would like to believe has changed
in the course of these things, but I would be very reluctant
to do so.
6. SIGNER: There is mounting evidence that the metabol-
ism of benzopyrene by microsomal fractions is quite dif-
ferent from that of whole cells. That is, whole cells tend
to produce more of the diol compounds as opposed to micro-
somal fractions. It might be interesting in your studies
to compare metabolism of benzopyrene in whole cells and
microsomal fractions because there are marked differences.
R. MCCAULEY: That is an interesting notion, but really
what we were using this assay for was an indication of the
extent of the exposure to potential inducers that might be
in the diesel exhaust, fume, particulate, and so the types
of metabolites that we are generating really were not
the primary interest. In fact, we tried to avoid that
whole issue by taking an assay that would extract every-
thing that they made.
506
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E. CANTRELL: We have had some experiences with smoke
inhalation. My question is, were your controls just an-
imals from the stock or were they exposed to air?
R. MCCAULEY: The controls were kept in an environmental
chamber with the same identical conditions, except no ex-
haust.
507
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BENZO(A)PYRENE METABOLISM IN MICE EXPOSED TO
DIESEL EXHAUST: I. UPTAKE AND DISTRIBUTION
H. W. Tyrer, Cancer Research Center, Columbia, MO 65205
E. T. Cantrell, Texas College of Osteopathic Medicine,
Fort Worth, TX 76107
R. Horres, Becton-Dickinson Research Center,
Research Triangle Park, NC 27709
I. P. Lee, NIEHS, Research Triangle Park, NC 27709
W. B. Peirano and R. M. Danner, EPA,
Cincinnati, OH 45268
ABSTRACT
In this study we examined the effect of diesel exhaust (DE)
exposure on the disposition of a typical polycyclic aromatic
hydrocarbon. DE-exposed and non-exposed A/Jax mice were
divided into 3 groups and each mouse instilled intratracbe-
ally with benzo(a)pyrene (BP). One group (A) received C-BP,
and at intervals of 2, 24, and 168 hours, three mice from the
group were killed and quick frozen for whole body autoradio-
graphy. Saggital sections were cut at 0.5 mm intervals and
autoradiograms prepared. Adjacent sections were studied so
that radioactive areas werg matched to specific organs. The
second group (B) received H-BP and at 2, 24, and 168 hours
mice were killed. Livers, lungs, and testes were weighed and
frozen. From these tissues metabolites were analyzed; these
data are reported in the next paper.
Histofluorescent examination of tissues from mice instilled
with nonradioactive BP (group C) confirmed that BP was present
in lung. The autoradiography data is the basis for elucidat-
ing the BP distribution in the mouse. Within 2 hours after
instillation radioactivity was detected in the entire animals,
with most in lungs, liver, and GI tract. By 24 hours after
instillation considerable radioactivity had redistributed to
the GI tract. At 168 hours after instillation only a trace
of label was found in the GI mucosa.
508
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INTRODUCTION
The carcinogenic potential of diesel engine exhaust (DE) can
in part be evaluated by considering the fate and distribution
of the polynuclear aromatic hydrocarbons (PAHs) in DE on
animals exposed to these emissions.
It is believed that the mechanism for carcinogenesis due to
PAHs follows the steps shown in Figure 1; similar schemes
have been proposed by others (1). Regardless of the exposure
modality, the PAH is absorbed and metabolized by some if not
all tissues. In the course of enzymatic oxydation of the
PAHs, reactive intermediaries are produced which, for the
most part, conjugate to more polar (more water soluble)
metabolites which are then excreted. However, some reactive
intermediaries bind to cellular macromolecules which may
produce either cell death or exhibit a potential for tumor
transformation. PAH binding to cellular macromolecules has
been demonstrated in various proteins, RNA, and DNA. The
potential for tumor transformation in cells with DNA-bound
PAH derivatives is mitigated by the ability of repair enzymes
to delete the damaged portion of DNA. The remaining pathway
leads to transformation and tumor formation. However, "immune
surveillance" is believed to be active so that tumor cells
are recognized as foreign to the body and are killed. At
some point in time, either immune surveillance fails or the
tumor kinetics overwhelm the immune system. This results in
a tumor.
An understanding of fate and distribution of PAH metabolites
can lead to at least a partial understanding of the carcino-
genic potential of specific PAHs within the context of ex-
posure conditions. We evaluate here the distribution of
benzo(a)pyrene (BP) given intratracheally to mice which were
either exposed or not exposed to Diesel exhaust. This paper
presents the data on the distribution of BP by examining
autoradiograms made of whole body sections obtained from
mice. The following paper analyzes more quantitatively the
fate of BP by analyzing the time course of clearance and
metabolite formation in three critical tissues, liver, lung
and testes. Analogous fluorometric data will be reported
later.
MATERIALS AND METHODS
Diesel Exhaust Exposure
Emission source. The diesel exhaust was generated using a
Nissan CN6-33 diesel 6 cylinder engine and a Chrysler torque-
flite automatic transmission Model A-727 coupled to an Eaton-
Dynamometer Model 758-DG. The engine was cycled using a
repetitive series of nine driving modes known as the Federal
509
-------
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Short Cycle, used in the present fuel emission studies.
Continuous cyclic monitoring of the chamber atmospheres were
carried out for carbon dioxide, carbon monoxide, total hydro-
carbons, sulfur oxides, nitric oxides, and nitrogen oxides,
and particulates were collected daily. Further details are
available (2).
Animal exposure. The animals used in this study were wean-
ling male Strain A/J mice that were housed and exposed at the
USEPA, Center Hill Facility in Cincinnati. The diesel-
challenged mice were exposed to raw diesel exhaust which was
diluted with filtered conditioned air at a 16:1 to 18:1
dilution ratio, to achieve a 6 mg/m of particulates atmos-
phere. Exposure time was eight hours per day, seven days per
week, for nine months. All mice were maintained in stainless
steel exposure chambers. The control mice were housed in
identical chambers under identical conditions, except fil-
tered, conditioned air was substituted for the diluted diesel
exhaust. Mice were fed Purina Rat Chow and tap water ad
libitum.
Animal Preparation
Instillation. Prior to instillation the animals were anes-
thesized. To reduce mucous secretions 0.1 ml (1 mg/kg)
atropine was injected i.p. Approximately 1 minute later the
animals were injected i.p. with 0.25 ml Pentobarbital
(40 mg/kg).
After anesthetization, the animals were instilled with BP
using a restraining device, a modified surgical otoscope and
a beaded 22-gauge lumbar needle. The restraining device was
used to support the animal, hold its mouth open, and to hold
the trachea in a straight line. The otoscope speculum was
modified so that its outer diameter was approximately 2 mm.
A 22-gauge needle was modified by first removing the bevel
tip and then placing a bead of epoxy plastic near the end.
The dispenser was a Hamilton repeating dispenser with a 1 ml
reservoir and dispensing 20 microliters per increment. After
placing the animal in the restraining device, the otoscope
was used to visualize the vocal cords, and the needle was
placed through the speculum, between the cords and into the
trachea. A volume of 20 microliters gelatin solution contain-
ing BP in suspension was deposited. The solution consisted
of 0.2% gelatin and 0.05 micrograms/microliter of BP. Each
dose contained 1.0 ug of BP which was either nonradioactive
or labeled with C (O.OSyCi). Each animal was observed for
respiratory distress until recovery from anesthesia.
511
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Autoradiography
Mice were sacrificed by cervical dislocation at approximately
24hours, 24 hours, and 168 hours after instillation with
C-BP. Immediately thereafter each mouse was placed into a
50 ml plastic centrifuge tube and immersed for approximately
five minutes in liquid nitrogen. Upon removal, each mouse
was placed in a cooled polyethylene zip-lock bag and placed
in dry ice and later transferred to a freezer maintained at
-20°C. Autoradiographic procedures were based on the method
of Ullberg (3) and are detailed below.
Specimen mounting. Animals instilled with C-Benzo(a)pyrene
were received in the frozen state and were maintained at
-15°C until processed. The animals were quickly scrubbed
with a 1.3% solution of sodium carboxymethylcellulose (CMC)
(Apoteket Marden, Stockholm) in distilled water, taking care
to wet the fur. Using a removable mounting frame, a 2 cm
layer of 1.3% CMC was frozen on a 1-1/2" x 4-5/8" brass
mounting base by immersion in liquid nitrogen. The mouse was
placed on its side on the frozen layer and covered to a depth
of approximately 1 cm with the 1.3% CMC solution and quickly
immersed in liquid nitrogen, being careful to prevent the top
central portion from solidifying until the .entire block was
almost solid. The mounting frame was removed, and the block
stored for a minimum of 24 hours at -12°C before cutting.
Specimen sectioning. The blocked animals were positioned in
the jaws of a Leitz Model 1300 sleigh microtome (E. Leitz,
Wetzlar, Germany) which was modified to be hydraulically
driven at 7.5 cm/sec and installed in a custom-built -12°C
cryostat. The entire animal was cut into 50 micron saggital
sections, labeling and transferring every alternate section
on clear tape (Scotch Brand Prescription Label Tape #800).
Every 20th section was placed on an open frame for freeze
drying; all other sections were stored on waxed paper for
future reference. An average of 906 sections were made per
mouse with sections for autoradiography each half millimeter.
Microtome blades were exchanged for every mouse and were
sharpened by the Week Corporation, Research Triangle Park,
NC. The frame-mounted sections were held in a freezer at
-15°C for a minimum of 24 hours to allow for freeze drying.
Section exposure. When the outer CMC portions of the section
could be handled without melting, the 1-1/2" x 4-5/8" sections
were transferred in total darkness to 5 x 7" Kodak No-Screen
(#NS-2T) Ready Pack film (Eastman Kodak, Rochester, NY), two
sections per film. The films with sections attached were
returned to their folders and stacked in groups of 12 with
standard weight blotter paper separating each film to a
thickness of approximately 1" and pressed between two 1/8"
aluminum frames held with four #100 binder clips (IDL Mfg.
512
-------
Co., Carlstadt, NJ). The package was double-wrapped in
aluminum foil and stored for 28 days at -15°C for exposure.
Exposure controls indicated that 28 days were adequate, with
no further resolution being obtained at up to 56 days.
Exposed films were processed in total darkness at 20°C for
five minutes in Kodak Liquid X-Ray Developer & Replenisher
(#146 5335, Eastman Kodak, Rochester, NY), and 10 minutes in
Kodak Rapid Fixer (#146 4114, Eastman Kodak, Rochester, NY).
Developer and Fixer were replenished after each group of 12
films and were discarded after processing 48 films. Films
were rinsed in running tap water for 20 minutes and allowed
to air dry. The entire process was conducted with appropriate
radiation safety measures.
Histofluorescence
Mice of group C were anesthetized and each was injected with
1.0 ml of filter-sterilized bacterial protease (2 mg) via the
tail vein. After one minute the mouse was sacrificed by
cervical dislocation, and the liver, lung, and testes removed
and weighed. Half of each organ was immediately frozen for
later histologic studies, the other half was prepared for
other analyses.
Microtomy. The frozen organ aliquots were stored at -90°C
until the day of microtomy. Frozen sections were prepared
under reduced light and the sections placed on glass slides
while remaining frozen. Multiple slides were prepared for
each organ. Just prior to examination by fluorescence micro-
scopy, a slide was thawed and air dried. For visualization
of BP and the collective group of intracellular metabolites,
the sections were examined by fluorescence microscopy with
excitation provided by a Xenon lamp filtered through a UG 1
and a BG 12 Corning filter, on a epi-illumination mode.
Fluorescence above 400 nm was visualized. For selected
tissues, photographs were prepared.
RESULTS AND DISCUSSION
The characteristic blue fluorescence of BP was observed in
the lung bronchi, extending into the lobes. This confirmed
that the BP was deposited in the bronchi. The distribution
of BP throughout the tissues could not be observed because of
the low level and diffuse character of the fluorescence.
Figure 2 shows a 100X magnification of the lung section from
a nonexposed mouse which was sacrificed two hours after
instillation. The bright areas are the BP taken up by the
tissues with the brightest foci corresponding to unabsorbed
BP crystals. The three dark areas are the bronchiole which
were slightly opened by the happenstance of sectioning the
tissue at a fortunate location.
513
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Figure 2. Histofluorescence of lung section. Photomicrography
of Benzo(a)pyrene fluorescence from the bronchiolar lining.
514
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Autoradiography was performed on DE-exposed or non-exposed
mice which wage killed at 2, 24, and 168 hours after instil-
lation with C-BP. Each group was done in triplicate. Ten
sections are shown from each of two DE-exposed mice sacrificed
at 2- and 24-hr time points (Fig. 3 and Fig. 4, respectively).
Autoradiograms of 168-hour exposed mice (not presented)
revealed no radiographic exposure except for a consistent
image of the stomach.
Figure 3 is a sequence of autoradiograms made from sections
taken at the indicated distance in millimeters from the skin
on the left side of the mouse. The head is to the left.
From the original autoradiograms, compared with adjacent
tissue sections as well as anatomical analysis of intact
mice, the following is apparent. The mice received an intra-
tracheally instilled bolus of radioactive material. In some
cases the animal would pass part of the bolus to the oral-
nasal cavity and return a portion of the bolus through the
esophagus to the stomach. Animals that had been sacrificed
within 2 hours of instillation showed substantial radiographic
exposure of the entire tissue section indicating the C-BP
had been passed to the blood stream and circulated throughout
the whole animal. Specks of exposure can be seen in the
regions corresponding to the lung, further confirming the
placement of the C-BP in this organ. The images of the
kidneys and bladder are quite prominent which indicates a
rapid accumulation of the C-BP in the urine. More than 10%
of the total radioactivity given to the animal had been
excreted in the urine within 16 hours of instillation.
Images of the liver and GI tract are also quite prominent at
this time, indicating a rapid uptake of the C-BP from the
stomach and portal blood. At no time did we see the reproduc-
tive organs outlined.
Figure 4 is a similar montage of autoradiograms of tissue
sections. However, sectioning was from right to left, the
head is on the right side and the mouse had been sacrificed
24 hours after instillation. By this time visualization of
the entire animal was no longer possible. Imaged organs were
associated with 1) GI tract, liver and stomach on down; 2)
urinary tract, kidneys and bladder; and 3) A faint image of
the lung. Highly prominent are the images of the fecal
material in the intestines and the speckled character of the
bladder image. Not presented are the autoradiographic images
of the mice sacrificed 168 hours after instillation. These
showed images corresponding to the rugae of the stomach.
Apparently, the stomach lining retains large amounts of the
radioactive hydrocarbons after other tissues have been cleared
sufficiently to prevent imaging.
515
-------
5.O
12.0
7.0
'i *
14.0
9.0
15.0
10.O
1.:.-
4
9* K ' ° "ij'» - *
17.0
'eiSi *
19,0
Figure 3. Photographs of autoradiograms made from 10 tissue
sections of a mouse. Benzo(a)pyrene instillation was 2 hours
prior to sacrifice. Sections were cut from the left side and
the numbers are the distances from the first cut to the
section, in millimeters. The head is to the left.
516
-------
5.0
;,
K-
12.0
7.0
13.0
9.0
15.0
10.0
** *
17.0
11.0
Figure 4. Photographs of autoradiograms made from 10 tissue
sections of a mouse. Benzo(a)pyrene instillation was 24
hours prior to sacrifice. Sections were cut from the right
side and the numbers are the distance from the first cut to
the section, in millimeters. The head is to the right.
517
-------
CONCLUSIONS
Qualitatively there are no obvious differences between the
autoradiograms of the DE exposed mice and those that had not
been so exposed. Far greater differences occurred between
mice with the same treatment, presumably reflecting their
ability to expectorate a portion of the radioactive bolus.
14
Figure 5 summarizes the distribution of C-BB in both DE-
exposed and non-exposed mice. Briefly, the C-BP is placed
in the lungs. Within two hours it is circulating in the
blood stream and may have entered the esophagus and stomach
by what amounts to coughing and swallowing. From the blood
C-BP is accumulated in the kidneys and bladder then ex-
creted. From the blood stream and digestive tract C-BP
enters the liver. By 24 hours, accumulation occurs in the
lower GI tract, the liver and stomach, the kidneys and
bladder, as well as a slight amount in the lungs. By one
week some BP is found in the stomach.
Quantitative considerations are more sensitive in differenti-
ating DE-exposed from unexposed mice. Such an analysis is
reported in the next paper. It is apparent from these studies
that the metabolism of BP is more affected by DE exposure
than its distribution.
INPUT
ESOPHAGUS
STOMACH
J.
i. , . ' "*
^^*
G
BLOOD
r
^^ ^^
J
1
OTHER ORGANS
KIDNEY
SMALL
INTESTINE
LARGE
INTESTINE
I
LIVER
I BLADDER \
URINE
FECES
Figure 5. A block diagram of the distribution of
in mice.
518
-------
ACKNOWLEDGMENTS
This study supported in part by EPA contract #68-03-2790, NIH
grant #CA-HL-15784, and grant #1120 from the Council for
Tobacco Research-U.S.A., Inc. The authors are also grateful
for the technical assistance of C. Hernandez, H. Jones,
L. Wilson, P. Eaton, and L. McMillan.
REFERENCES
1. Boobis, A. R., R. E. Kouri, and D. W. Nebert. 1979.
Genetic differences in the binding of benzo(a)pyrene
metabolites to DNA in the mouse. Cancer Pet, and Prev.
2. Hinners, et al . 1980. Animal exposure facility for
diesel exhaust studies. In: Biological Studies of
Environmental Pollutants: Aerosol Generation and Ex-
posure Facilities, edited by K. Willeke, Ann Arbor Sci.
Pub., In press.
3. Ullberg, S. 1958. Autoradiographic studies on the
distribution of labelled drugs in the body. Proc. Intl
Conf. Peaceful Uses of Atomic Energy. 24: 248-254.
519
-------
BENZO(A)PYRENE METABOLISM IN MICE EXPOSED TO
DIESEL EXHAUST: II. METABOLISM AND EXCRETION
T. Cantrell, Texas College of Osteopathic Medicine,
Fort Worth, TX 76107
W. Tyrer, Cancer Research Center, Columbia, MO 65205
W. B. Peirano and R. M. Danner, HERL, U.S. EPA,
Cincinnati, OH 45268
ABSTRACT
In this study we examined the effect of diesel exhaust (DE)
exposure on IN VIVO metabolism of benzo(a)pyrene (BP).
DE-exposed and un-expqsed A/Jax mice of group B were instilled
intratracheally with H-BP. At each time point of 2, 24, and
168 hours after instillation 5 mice were killed and the
liver, lungs, and testes were removed and frozen. Aliquots
of the organs were homogenized in 2 ml water and each received
3 volumes of cold ethanol. Radioactivity in supernatant and
precipitate was measured. The supernatant extracts were
subjected to HPLC analysis on ALOX-T and on Zorbax ODS. The
ALOX-T method was a modification of Autrup's procedure for
conjugate assay (Biochem. Pharmacol. 28:1727, 1979). Frac-
tions were: a) free BP; b) nonconjugated primary metabolites;
c) sulfate conjugates; d) glucuronides, glutathiones and
other conjugates. By 2 hours after instillation primary
metabolites were found in liver and lung, but very little was
conjugated. The unconjugated BP was mainly in the form of
free BP and phenolic metabolite(s). The lungs of DE-exposed
mice had less capacity to dispose of "bound" BP 1 week after
instillation.
INTRODUCTION
Particulate organic matter in diesel exhaust is known to
contain approximately 1% polynuclear aromatic hydrocarbons
(PAHs) by weight. Diesel exhaust particulate matter may be
520
-------
separated into acid, base, and neutral fractions: the neutral
fraction which contains PAHs shows the highest mutagenic
effects of the three fractions and each fraction is directly
mutagenic, exhibiting frameshift activity (1). Because of
the economically attractive future for diesel power and its
carcinogenic potential, it is necessary to test diesel exhaust
for its effect on the monooxigenase enzyme system. This
enzyme system has been found to convert several polynuclear
aromatic hydrocarbons into more reactive intermediaries which
bind to cell macromolecules including DNA, RNA, and proteins
(2). Such binding may lead to cancer (3,4).
In this study, HPLC analysis was used to determine the BP
conjugates and to quantitate the disposition of the BP.
Additionally, HPLC was used in a reverse phase mode to ident-
ify the organic soluble metabolites of BP. The H-BP which
was not extractable with a polar solvent gave an estimate of
tightly bound BP in the tissues.
MATERIALS AND METHODS
Animals, exposures and instillation procedures have been
detailed in the preceding paper.
After instillation with 5 yCi H-benzo(a)pyrene (BP), animals
were sacrificed by cervical dislocation at 2 hours, 24 hours,
and 168 hours. The liver, lungs, and testes were removed at
once, weighed and frozen on dry ice. The organs were kept
frozen until homogenized. Organs were removed from the
freezer and cut into halves. One half was returned to the
freezer and the other half weighed and thawed. Each organ
aliquot was placed into 2.0 ml water and homogenized in a
glass-glass tissue grinder. Three volumes of ice-cold ethanol
was added to each homogenate and allowed to stand at least 2
hours in the refrigerator. The precipitates were sedimented
by centrifugation for 15 minutes at 50,000 x g. The aqueous
ethanol supernatant of each homogenate received 5 yl of
Vitamin E to serve as antioxidant and was then dried under a
nitrogen stream. The residue was redissolved in 1.2 ml 80%
ethanol and half was injected onto an HPLC column. Ten yl
aliquots of the remainder was taken for scintillation count-
ing. The pellet fractions mentioned above, were suspended in
2.0 ml HpO and 50 yl taken for scintillation counting. The
HPLC column was 4.6 x 100 mm, packed with neutral alumina.
The bed was previously perfused with 0.5 M phosphate buffer,
pH 7.0, followed by water, ethanol, then hexane. The 200 yl
injection was made during solvent flow of 2.0 ml/min. BP was
eluted with 10 ml hexane; hydroxy BP was eluted with 15 ml
ethanol; BP-sulfate was eluted with 15 ml water; unknown
material eluted with 15 ml 0.5 M phosphate, pH 3.0; BP-gluc-
uronide and BP-glutathione eluted with 20 ml 25% formic acid.
521
-------
The column was restored by washing with neutral phosphate
buffer, water, ethanol, and hexane. The identification of
the metabolites in each fraction was confirmed by elution of
authentic standards. The glutathione conjugates were presumed
to be eluted by formic acid (5). Radioactivity in each
fraction was determined in a Beckman LS7000 liquid scintilla-
tion counter.
For selected samples, the hexane plus ethanol fractions were
dried and the residue redissolved in 0.25 ml ethanol. A part
(0.10 ml) was injected into a Dupont Zorbax ODS column and
eluted with a linear 50-100% methanol in water gradient. The
flow rate was 1.6 ml per minute and 0.8 ml fractions were
collected in mini-vials. To each vial was added 5 ml Scinti-
verse and each vial was counted 10 minutes. The profile of
nonconjugated metabolites was plotted.
The urine of each group of mice was collected over the first
16 hours after instillation and the radioactivity determined.
A sample of caecum feces was taken from one mouse sacrificed
24 hours after instillation and the conjugate profile pre-
pared.
RESULTS AND DISCUSSION
The time course of disappearance from lung, liver, and testes
is illustrated in Figure 1. There was no difference in
clearance of soluble metabolites by the DE and control groups.
Both groups were capable of clearing the bound BP, but there
was a significantly higher amount of residual BP in lungs of
DE mice one week after instillation.
Metabolism of BP can occur in all three of the tissues we
examined (6). This notion is supported by the presence of
both primary and secondary metabolites in the tissues at the
times after instillation. Figure 2 presents a summary of the
recovery of BP and its metabolites within two hours after
instillation. The total radioactivity in the alcoholic
extract represents unity on the ordinate. The bars represent
the proportional amount of each fraction. In lung, the
majority of radioactivity was as nonmetabolized BP. In
contrast, liver and testes contained more than half of the
radioactivity as metabolites with a substantial degree of
secondary metabolism to conjugates. The DE animals appeared
to have less free BP in tissues and this may reflect on
induction of the enzymes for primary metabolism of polycyclic
aromatic hydrocarbons. Uithin 24 hours after instillation
the BP in these tissues was principally in the form of metabo-
lites, with a substantial degree of conjugation (Fig. 3).
Likewise, the BP was present mainly as metabolite by 168
hours after instillation (Fig. 4). Given the low total
radioactivity as illustrated in Figure 1, the metabolite
522
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526
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profile may not be meaningful for liver and testes. The high
total amount and proportion of unmetabolized BP in DE lung at
168 hours suggests that a small amount of BP is adsorbed to
smoke particles. These have a prolonged sojourn in the body
(7) and may provide a means whereby carcinogenic hydrocarbons
may be very slowly cleared at low levels.
The predominate primary metabolite of BP in liver was 3-
hydroxy-BP. This was evidenced by the reverse phase HPLC
profile illustrated in Figure 5. This does not indicate that
other metabolites formed, but that the 3-hydroxy-BP is more
readily retained in the cells (8,9).
In the preceding paper we had seen that there was a substan-
tial amount of radioactivity in the large bowel and in the GI
tract. We were interested in determining whether the radio-
activity was benzopyrene that had been swallowed and never
absorbed, or whether we were dealing with enterohepatic
recycling of conjugates. The contents of the caecum in a 24
hour animal contained some free benzopyrene. but we found a
large amount of primary metabolites and very little conjugates
(Fig. 6). This is consistent with the concept of hydrolysis
of conjugates in the large bowel by the bacterial flora. An
alternative explanation is that these metabolites are a
result of the mucosal cells in the small intestine metaboliz-
ing the benzopyrene and then excreting the primary metabolites
right back into the lumen.
The finding of rapid metabolism of the administered BP and
accumulation in the GI tract suggests that clearance of a
total body load is quite efficient in both groups of mice.
Indeed, even the kidneys contributed substantially to the
excretion capacity. During the first 16 hours after instil-
lation 18% of the BP was excreted in urine by non-exposed
mice and 14% of the BP was excreted in urine by DE mice.
The only meaningful difference we found in DE mice was the
suppressed ability to clear the small amount of BP. We
suggest that this BP is adsorbed to smoke particles. The
implications of this finding are more profound when one
considers the probability of human subjects combining cigar-
ette smoking with fuel smoke exposure. The-extension of time
carcinogens spend in the lung will markedly increase the
carcinogenic risk.
ACKNOWLEDGMENTS
This study supported in part by EPA contract #68-03-2790, NIH
grant #CA-HL-15784, and grant #1120 from the Council for
Tobacco Research-U.S.A., Inc. The authors are also grateful
for the technical assistance of A. Loudin, L. Oborn,
L. McMillan, L. Wilson, and P. Eaton.
527
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Figure 6. Relative amount of metabolites and free BP in
feces from a mouse taken 24 hours after instillation. See
legend of Figure 2 for details.
529
-------
REFERENCES
1. Pitts, J. N. Jr., K. van Cauwenberghe, A. M. Winer, and
W. L. Belser. 1979. Chemical analysis and bioassay of
diesel emission participates. EPA Report of Contract
No. R 806042.
2. Sims, P., P. L. Grover, A. Swaisland, K. Pall, and A.
Hewer. 1974. Metabolic activation of benzo(a)pyrene
proceeds by a diol-epoxide. Nature, 252:326-335.
3. Heidelberger, C. 1975. Chemical carcinogenesis.
Ann. Rev, of Biochem., 44:79-121.
4. Weisburger, E. 1978. Mechanisms of chemical carcino-
genesis. Ann. Rev. Pharmacol. Toxicol.. 18:395-415.
5. Autrup, H. 1979. Separation of water-soluble metabo-
lites of benzo(a)pyrene formed by cultured human colon.
Biochem. Pharmacol., 28:727-1730.
6. Lee, S. D., K. I. Campbell, D. Laurie, R. G. Hinners, M.
Malanchuk, W. Moore, R. J. Bhatnagar, and I. Lee.
lexicological assessment of diesel emissions. Presented
at the 71st Annual Meeting of the Air Pollution Control
Association, Houston, Texas, June 25-30, 1978.
7. Moore, W., J. Orthoefer, J. Burkart, and M. Malanchuk.
Preliminary findings on the deposition and retention of
automotive diesel particulate in rat lungs. Presented
at the 71st Annual Meeting of the Air Pollution Control
Association, Houston, Texas, June 25-30, 1978.
530
-------
8. Cantrell, E., M. Abreu, and D. Busbee. 1976. A simple
assay of aryl hydrocarbon hydroxylase in cultured human
lymphocytes. Biochem. Biophys. Res. Comm., 70:474-479.
9. Tyrer, H. W., E. T. Cantrell, and A. G. Swan. 1977.
Automated single cell analysis of aryl hydrocarbon
hydroxylase in human lymphocytes. Life Sciences, 20:
1723-1728.
531
-------
EFFECT OF EXPOSURE TO DIESEL EXHAUST ON
PULMONARY PROSTAGLANDIN DEHYDROGENASE (PGDH) ACTIVITY
A. Chaudhari, R.G. Farrer and S. Dutta
Department of Pharmacology
Wayne State University
School of Medicine
Detroit, Michigan 48201
ABSTRACT
There has been evidence that an acute exposure of laboratory
animals to nitrogen dioxide (N02) for a short period of time
can cause marked inhibition of pulmonary PGDH activity.
Since diesel exhaust contains N02, we have undertaken this
investigation to determine the effect of long-term exposure
of guinea pigs and rats to diesel exhaust. The present
study involves measurement of PGDH activity in the lung
tissue as obtained from these animals following exposure to
250 and 1500 yg particulates/m3 for various time periods in
relation to the appropriate time-matched controls. In
guinea pigs, the exposure of 1.5 months of low dose of diesel
exhaust seems to stimulate the PGDH activity about two-fold
while the exposure to higher concentrations of diesel ex-
haust for 3 months, as well as 6 months, seem to show con-
centration dependent lowering of PGDH activity as compared
to the time-matched controls. This study also documents
the well-known species difference in PGDH activity in that
the rat shows much lower activity of this enzyme and there-
fore this species is not suitable for determination of the
effect of diesel exposure on the PGDH activity.
INTRODUCTION
15-Hydroxy prostaglandin dehydrogenase (PGDH) oxidizes the
15-OH group of naturally occurring prostaglandins (PCs) to
their corresponding 15-keto form (1). Like cyclic nucleo-
tide phosphodiesterase, acetylcholinesterase and monamine
oxidase, PGDH is among the group of regulatory enzymes in-
activating compounds of high biological potency. Therefore,
it has been considered as one of the crucial factors go-
verning the physiological and pharmacological concentrations
and actions of PCs.
532
-------
Furthermore, it has been recently shown that pulmonary PGDH
activity can be altered by an acute exposure of guinea pigs
to nitrogen dioxide (NC>2) for a short period of time (2) .
Since diesel exhaust contains N02, we investigated the ef-
fect of long-term exposure of guinea pigs and rats to diesel
exhaust on pulmonary PGDH activity.
EXPERIMENTAL PROCEDURES
MATERIALS USED
(9-%) Prostaglandin ^20. (9-2 Ci/mmol) was purchased from
New England Nuclear Corporation, Boston, MA. Prostaglandin
F2a THAM was a generous gift of Dr. J.E. Pike, the Upjohn
Company, Kalamazoo, MI. Nicotinamide adenine nucleotide was
obtained from Sigma Chemical Company, St. Louis, MO. TLC
plates (plastic), coated with silica gel and liquid scin-
tillation cocktail, Redi-solv EP were obtained from Eastman
Kodak Company, Rochester, N.Y. and Beckman Instruments, Inc.,
Fullerton, CA., respectively. Guinea pigs (Hartley) and
rats (Fischer 344) were supplied by Charles River Breeding
Labs., Inc., Wilmington, MA.
Method of Exposure of Animals to Diesel Exhaust
Guinea pigs and rats were exposed to diesel exhaust for
various lengths of time at the General Motors Technical
Center, Warren, Mich (3). Two doses, namely 250 and 1500
yg particulates/m3 were used and the control animals were
maintained under similar conditions, breathing clean air.
Method of Assay of Prostaglandin Dehydrogenase Activity
The method of assay used was essentially that of Chaudhari
et^ al. (2). The reaction mixture consisted of 0.25 ml of
"6T25~M sodium phosphate buffer, pH 8.0; 0.05 ml of PGF2a
THAM (1 mg/ml), 0.2 ml of cytosol (100,000 x g supernate)
fraction of lung homogenate containing 1 mg protein, and
water to make up the volume to 1 ml. The mixture was in-
cubated for 15 min at 37° C and the reaction was stopped by
acidifying the solution to pH 3.5 with 0.1 N HC1. Eight ml
of ethyl acetate was used to extract the substrate and
metabolite. The organic phase was evaporated to dryness
and the residue was dissolved in 100 yl of absolute methanol.
An aliquot of 40 yl was spotted on the TLC plate and the
chromatogram was developed in a mixture of acetonetmethylene
chloride:acetic acid (40:60:1.5 v/v). After drying the
chromatogram with a hair dryer, 0.5 cm segments of the
chromatogram were cut and counted in a Beckman liquid scin-
tillation spectrometer using Redi-solv EP cocktail.
533
-------
RESULTS AND DISCUSSION
Table 1 shows the results from the study on the effect of
6 months of exposure of guinea pigs to two doses of diesel
exhaust on pulmonary PGDH activity. The control group of
animals showed an age-dependent increase in the PGDH ac-
tivity. The guinea pigs exposed to 250 yg particulate/m
(low dose) showed, in comparison to their time-matched con-
trol, an initial stimulation of the enzyme activity by about
two-fold after 1.5 months, while, paradoxically, the animals
exposed to 1500 yg particulate/m3 (high dose) did not show
any change in the PDGH activity. Three months exposure of
the guinea pigs to low dose of diesel exhaust did not have
any effect on the enzyme activity, but exposure to high
doses for the same length of time produced an inhibition of
about 26% of PGDH activity as compared to control. Further-
more, it appears that exposure of guinea pigs to low dose
for 6 months has lowered the enzyme activity but this dif-
ference is statistically not significant. Exposure of
guinea pigs to high dose of diesel exhaust for 6 months
markedly affected the PGDH activity in lung, producing a
significant inhibition of about 43% in comparison to control.
TABLE 1. EFFECT OF EXPOSURE OF GUINEA PIGS TO DIESEL
EXHAUST ON PULMONARY PROSTAGLANDIN DEHYDROGENASE ACTIVITY
PGDH Activity,
nmoles 15-keto PGF2a formed/min/mg protein
Time Following
the start of
exposure
(months)
1.5
3
6
Time-matched
Control3
0.458 ± 0.059
0.631 ± 0.053
0.757 + 0.030
Exposed to
250 yg
particulate/
m3
0.881 ± 0.116b
0.514 ± 0.032C
0.574 ± 0.117C
Exposed to
1500 yg
particulate/
m3
0.469 ± 0.054
0.464 ± 0.044b
0.428 ± 0.052b
Control animals (n=6) were exposed to clean-air under simi-
lar conditions.
Significantly different from control, p < 0.05 by Student's
t-test, n = 6.
Not different from control, p > 0.1 by Student's t-test,
n = 6.
534
-------
It is speculated that this time-dependent inhibition of PGDH
activity as exhibited by the exposure of guinea pig to the
high dose of diesel exhaust may be related to various oxidant
gases present in the diesel exhaust. Recently, Chaudhari et
al. (2) have observed a time-dependent inhibition of PGDH
activity in guinea pig due to exposure to 46 ppm N02, which
reached its maximum inhibitory level after 8 hours of ex-
posure.
Although the high dose of diesel exhaust used in the present
study contains a very low level of N02, approximately 6 ppm
(3), it is possible that an exposure for 3-6 months to the
low level of NC>2 can produce similar inhibitory effect on
PGDH activity as that of 46 ppm N02 for a short exposure of
8 hours. In the context of this probable cause and effect
relationship between NC>2 and inhibition of PGDH activity, it
is of particular interest to note that the stimulation of
PGDH activity observed during exposure to a lower dose of
diesel for a shorter period of time may have some parallelism
to initial stimulation of the enzyme observed by Chaudhari
et_ al. (2) after exposure to 46 ppm for 1 hour. Presently,
additional studies are being done to determine whether the
mathematical product of the concentration of N02 and the
duration of exposure is responsible for the biphasic effect
on this enzyme.
At this time, it is not known, either from the present study
or from the previous study of Chaudhari et al. (2), the
mechanism by which NC>2 affects this vital enzyme or whether
the inhibition of this enzyme leads to elevated prostaglandin
concentrations in the lung. However, it has been reported
by Chaudhari et al. (2) that exposure of guinea pig to 100%
02 for 48 hours also inhibits pulmonary PGDH activity.
Kinetic analysis of the enzyme inhibition by C>2 showed a
non-competitive effect with respect to both prostaglandin
F2a and NAD+ which indicates a lack of availability of
catalytic sites. Being an oxidant gas, it is possible that
N02, like 02, affects PGDH activity by some oxidative me-
chanism.
The results obtained from exposure of rats to diesel exhaust
on pulmonary prostaglandin dehydrogenase activity are sum-
marized in Table 2, where the same enzyme assay system was
used as that of guinea pigs. As it is evident from the
table, most of the samples did not show detectable amounts
of PGDH activity in Fischer Rats 344 from both control and
exposed groups. Because of this problem, a proper evaluation
of the effects of diesel particulate exposure could not be
made in this species.
535
-------
TABLE 2. EFFECT OF EXPOSURE OF RATS TO DIESEL EXHAUST
ON PULMONARY PROSTAGLANDIN DEHYDROGENASE ACTIVITY
Time Following
the start of
exposure
(months)
1.5
3
6
PGDH Activity,
nmoles 15-keto PGF2a f armed /min/mg protein
Time-matched
Control3
(n)
0.055
(2)
0.022 ± 0.002
(5)
0.019
(1)
Exposed to
250 yg
particulate/m3
(n)
0.045
(2)
0.019
(1)
0.014
(1)
Exposed to
1500 yg
particulate/m3
(n)
0.076
(1)
0.019 ± 0.002
(3)
0.030 ± 0.008
(3)
Control animals were exposed to clean-air under similar con-
ditions.
(n) Represents the number of animals having detectable amount
of PGDH activity from each group of six animals.
CONCLUSIONS
1. With low dose of diesel exhaust, a remarkable increase in
PGDH activity was noted in guinea pigs only during the
short exposure of 1.5 months.
2. With high dose, on the other hand, a clear time-dependent
inhibition of PGDH activity was observed in guinea pig.
3. Further studies, varying the dose inversely in relation to
the period of exposure, may reveal the threshold dose
necessary for stimulation or inhibition of pulmonary
PGDH activity of guinea pig.
4. The rat is not an ideal species for determination of
effect of diesel exhaust on the PGDH activity since rat
shows species difference in that it has about 10% of
the enzyme activity compared to guinea pig.
536
-------
REFERENCES
1. Marrazzi, M.A. and Anderson, N.H. 1974. Prostaglandin
Dehydrogenase. In: The Prostaglandins, Peter Ramwell,
ed. Plenum Press, New York, Vol. 2, pp. 99-145.
2. Chaudhari, A., Kandiah, S., Warnock, R., Eling, I.E.
and Anderson, M.W. 1979. Inhibition of Pumonary Pros-
taglandin Metabolism by Exposure of Animals to Oxygen
or Nitrogen Dioxide. Biochem. ^J. 184: 50-57.
3. Shreck, R.M., Hering, W.E., D'Arcy, J.B., Soderholm, S.C.
and Chan, T.L. In: Proceedings of International Sym-
posium on Health Effects of Diesel Engine Emissions,
Environmental Protection Agency, Cincinnati, Ohio,
December 3-5, 1979.
537
-------
EFFECT OF DIESEL PARTICULATE EXPOSURE
ON ADENYLATE AND GUANYLATE CYCLASE
OF RAT AND GUINEA PIG LIVER AND LUNG
David R. Schneider
and
Barbara T. Felt
Wayne State University
School of Medicine
Department of Pharmacology
540 East Canfield
Detroit, Michigan 48201
ABSTRACT
3
Rats and guinea pigs were exposed to 250 or 1500 ug/m
diesel particulate for 24 weeks at the General Motors
Research Laboratories. To determine the possibility of
a tumor response from this exposure, we measured
adenylate and guanylate cyclase activity of the liver
and lung in these and control tissues. Although we
found substantial qualitative differences throughout
the 24 week period examined, we detected no changes in
the basal activity of either enzyme system. Adenylate
cyclase activity remains stimulatible from the liver
and lung tissues of each species. Guanylate cyclase
activity of the liver also remained stimulatible. On
these findings, a tissue tumor response to these
exposures was not supported. Age related changes were
observed in the lung tissues of each species, and
guanylate cyclase activity was decreased in this tissue
after diesel exposure.
INTRODUCTION
The cyclic nucleotides are ubiquitous intracellular
compounds found in most mammalian tissues as well as
extracellular fluids (1, 4, 8, 9-10, 12). These
538
-------
compounds are the products of adenylate cyclase or
guanylate cyclase and are susceptible to alteration by
numerous hormones, neurotransmitter substances and
drugs. In this study, we have examined the changes in
these two intracellular enzymes in the liver and lung
tissues of rats or guinea pigs subjected to a chronic
exposure of diesel exhaust and particulate. This study
was suggested by previous findings of others which
demonstrated that these special enzymes and their
cyclic nucleotide products are not only measures of the
biologic viability of a tissue, but may also serve as
an index in determining if a tissue contains a tumorous
mass (5, 6, 11).
As it is now understood, under conditions of abnormal
tissue growth these enzymes have been shown to be
consistently changed when compared to normal tissue
controls (5, 6, 11). While the cyclic nucleotides have
been thought to act in concert, the changes which have
been described from several studies consist of an
increased basal activity, a substantial change of the
guanylate cyclase enzyme from a soluble to a
particulate pool, and the insensitivity of guanylate
cyclase to chemical stimulation. In recent studies,
the urinary excretion of cyclic nucleotides, and
especially cGMP, was also found to be altered in benign
or neoplastic tumors. Under these same conditions,
cyclic AMP and adenylate cyclase activity was
unchanged. Studies with in vivo tumors have suggested
that the changes which are observed in cGMP metabolism
may be associated with an altered rate of cellular
growth and differentiation (5). These latter studies
together with other studies involving in vivo
transplantable tumors, suggest that the excretion of
cyclic nucleotides in urine are consistent with changes
in the cyclic nucleotide cyclase activities which are
found in tumorous tissue. Such changes may not be a
completely reliable index of tumors however- Changes
in the responsiveness and in the basal activity of
tissue cyclic nucleotide cyclases, can serve as
sensitive indicators of the primary effects of a
variety of noxious stimuli including smoke or the
exhaust fumes from diesel engines.
Our studies have determined the activity of adenylate
and guanylate cyclase in guinea pig or rat liver and
lung tissues. Control animals as well as animals
exposed to a low (250 ug/m ), or high (1500 ug/m )
concentrations of diesel particulate were examined.
This paper reports the results of these diesel exposure
after six weeks, twelve weeks or 24 weeks under
539
-------
conditions described by Schreck, et. al. , and carried
out at the General Motors Research Laboratories.
METHODS
Animal sacrifice and tissue preparation.
Animals (rats or guinea pigs) were first weighed, then
stunned and decapitated. The liver and both lungs were
removed and washed in a small beaker with approximately
50 ml cold saline. All tissues were blotted before
weighing.
Liver microsomes were prepared by first passing the
liver tissue through a Harvard press, followed by
homogenization in four volumes of ice cold 0.25 M
sucrose. Each homogenate volume was then measured and
recorded. Ten ml of the homogenate was centrifuged for
ten minutes at 12,000 rpm SS-34 rotor of a Sorvall
centrifuge and the supernate carefully decanted. The
supernate was then recentrifuged for 60 min at 40,000
rpm in a No. 40 Beckman rotor. Following this
centrifugation, the resulting microsomal pellet was
resuspended by hand using a teflon glass homogenizer in
16 ml of 0.25 M sucrose.
To prepare lung microsomes, each lung was filled with
about 10 ml of cold saline and then cut into pieces
with scissors. The fragments were homogenized in a
Potter-Elvejham homogenizer using four volumes of 0.25
M sucrose. Homogenates were centrifuged for ten
minutes at 12,000 rpm in an SS-34 rotor of a Sorvall
centrifuge and the supernate decanted. The 12,000 rpm
supernate was removed and recentrifuged at 40,000 rpm
in a No. 40 Beckman rotor for an additional 60 minutes.
The resulting microsomal pellet was resuspended by hand
homogenization in one ml of 0.25 M sucrose.
Adenylate and guanylate cyclase protocol.
The reagent for adenylate cyclase was prepared
according to the method of Steiner et al. (3). The
reagent included, in final concentrations: 10 mM Tris-
hydrochloride, pH 7.4, 3 mM ATP (adenosine
triphosphate), 4 mM magnesium sulfate, 0.1 mM EGTA
[ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetra-
acetic acid], 0.01 mM GTP (guanosine triphosphate) 2 mM
DTT (dithiothreitol) and 3 mM IBMX (iso-butyl methyl
xanthine). Maximal stimulation of adenylate cyclase
was achieved by incubating purified liver or lung
membranes in the adenylate cyclase reagent together
540
-------
with 1 mM (final concentration) sodium fluoride.
Guanylate cyclase reagent consisted of the following
ingredients in final concentration: 50 mM Tris-
hydrochloride, pH 7.6, 10 mM theophylline, 0.1 mM EDTA
(ethylene diamine tetracetic acid), 0.1 mM DTT, 3.0 mM
GTP, and 4.0 mM manganese chloride. Sodium azide (1
mM, final cone.) was used to maximally stimulate
guanylate cyclase.
Adenylate cyclase activity was determined over a ten
min period at 37 C. for both liver and lung using the
100,000 x g microsomal membrane pellet. Guanylate
cyclase activity was determined over 10 min at 37
C. in liver and lung using both the 100,000 x g
supernate and the microsomal pellet fractions. Assays
for each time point for each of the tissues were
determined in duplicate. To each assay tube,
containing 100 ul of the appropriate reagent, 20 ul of
tissue sample was added. All reagents and samples were
held at 4 C. until incubations were started. To
initiate the adenylate cyclase or guanylate cyclase
activity, racks of tubes were placed into 37 C
waterbaths. At 3, 10, or 15 min intervals, individual
sample tubes were removed and quickly placed into a
boiling waterbath (>95 C) for three min. Following
this heat inactivation, 500 ul of 50 mM sodium acetate,
pH 6.1, was added to all samples. Tubes were then
capped and stored at -20 C. until assayed.
Radioimmunoassay of cyclic AMP and cyclic GMP.
Assay of cyclic nucleotides was performed using the
double antibody radioimmunoassay (RIA) method described
by Steiner, et. al. (7). Normalization of the cyclic
nucleotide data was accomplished by protein
determinations from individual samples to be assayed
(15). Data reduction of the results of the assay were
performed using a RIANAL package described by
Duddleson, et. al. (13), kindly provided to this
laboratory by Dr. A. Rees Midgley, University of
Michigan, Division of Reproductive Endocrinology.
Data Reduction.
Correlation of the data obtained from the various
experiments performed was determined using the Student
T-test, the Studentized Neuman-Keuls test, and
multivariate analysis of variance tests from packaged
computer programs (14).
541
-------
The specific activity of the control or treatment
tissues was calculated from the averaged assayed
activity of individual tissues. These averaged values
were separately calculated by subtracting the 10 minute
measurement from the zero minute incubation. Basal
rates, as well as maximally stimulated enzyme
activities were compared.
RESULTS
Six-Week Diesel Particulate Exposure.
Adenylate Cyclase:
Rats. The composite data of adenylate cyclase
activities following 6-week exposure to diesel exhaust
is shown in Table 1. A significant decrease is seen in
the basal activity of the liver in those animals
exposed to 250 ug/m particulate. Stimulated activity
of either the low or high exposure of diesel
particulate was unchanged from the control values at
this time. For the rat lung, a substantial decrease is
noted in the basal activity of both the 250 and the
1500 ug/m diesel particulate exposure. In contrast to
the liver however, in the lung we observed an increase
in the NaF-stimulatible adenylate cyclase, Table 1.
The stimulatible adenylate cyclase activity observed
following exposure to 250 ug/m diesel particulate
although increased, is unchanged from the control;
after 6 weeks exposure to 1500 ug/m however, a
significant change is observed from control animals (p
< 0.01). In addition, the increase observed following
this highest exposure is significantly greater than
that observed following 250 ug/m exposure (p < 0.03).
Guinea Pigs. After 6 weeks exposure to diesel
particulate, the basal levels of adenylate cyclase are
nearly doubled (250 ug/m , p < 0.05, or more than
tripled (1500 ug/m ), p < 0.01, in the liver, Table 2.
After either exposure to diesel particulate, NaF-
stimulated adenylate cyclase is significantly greater,
p < 0.01, than in the control tissues. In the guinea
pig lung, a significant increase, p < 0.01, of more
than six-fold is observed in the.basal activity of
adenylate cyclase after 250 ug/m exposure. The
activity of the enzyme remains significantly elevated,
p,< 0.01), and unchanged in animals receiving 1500 ug/
m exposure. In contrast to the liver, in the guinea
pig lung, we find no differences in the NaF-stimulated
activity compared to the control group of animals.
542
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Guanylate Cyclase:
Rat. The basal activity of the rat liver 100,000 x g
supernate fraction was increased more than five-fold, p
< 0.01, following 6-weeks exposure to 250 ug/m diesel
particulates. This same fraction was more than doubled
in those animals which received 1500 ug/m exposure,
Table 3. When liver soluble guanylate cyclase was
maximally stimulated with sodium azide, a significant
increase-was only observed in those animals exposed to
250 ug/m of diesel particuate. While no differences
could be detected in the basal activity of the
corresponding particulate fractions, significant
differences, p < 0.01, were determined for stimulated
guanylate cyclase activity after either 250 or 1500 ug/
m diesel exposures. In the rat lung, basal levels of
soluble guanylate cyclase are unchanged from enzyme
activity which was maximally stimulated by sodium
azide. The approximately 3-fold increase in basal and
stimulated levels of soluble guanylate cyclase activity
from animals exposed to 250 ug/m diesel particulate,
matches the general increase of guanylate cyclase
activity seen in liver of these same animals. When the
particulate guanylate cyclase of the rat lung is
examined, a progressive decrease in enzyme activity is
noted, with increasing diesel exposure concentrations.
The particulate guanylate cyclase activity show
stimulated enzyme activities which are essentially
unchanged from the basal rates.
Guinea Pig. While no change is observed in the basal
activity of the guinea pig liver after 6 weeks of
diesel exposure at either concentration, a dose related
increase in the sodium azide stimulatible guanylate
cyclase activity is observed after diesel exposure,
Table 4. Unstimulated particulate guanylate cyclase
activity of guinea pig liver is unchanged after any
exposure to diesel particulate; however the maximal
stimulated particulate activity is significantly
increased, p < 0.01, by_approximately 4 fold after
either 250 or 1500 ug/m diesel exposure, Table 4.
Basal activity of soluble guanylate cyclase activity of
guinea pig lung is increased, approximately 2-fold, p <
0.02, after either concentration of diesel exposure.
The maximal stimulated soluble guanylate cyclase
activity is nearly doubled at this time. A similar
two-fold increase is also observed in the basal and
stimulated activity of the particulate guanylate
cyclase in the lung, Table 5.
Twelve-Week Diesel Particulate Exposure.
545
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Adenylate Cyclase:
Rat. In the rat liver, adenylate cyclase.is unchanged
following a 12-week exposure at 250 ug/m , but is
increased more than two-fold in those animals exposed
to 1500 ug/m , Table 3. In the same tissue the maximum
sodium fluoride stimulatible.activity is doubled (250
ug/m ) or tripled (1500 ug/m ) after diesel exposure.
Basal adenylate cyclase activity in the rat lung shows
a similar pattern of response, and is increased more
than two-fold in those animals exposed to 1500 ug/m
diesel particulate. In contrast to liver, however, the
lung NaF-stimulatible adenylate cyclase activity
remains unchanged following diesel particulate
exposure.
Guinea Pigs. Adenylate cyclase activity in the liver
of the guinea pig is progressively decreased by
increasing exposure to diesel particulate, Table 4.
The sodium fluoride stimulatible adenylate cyclase
activity is significantly increased above controls in
the liver membranes of animals which have been exposed
to 250 ug/m of diesel particulate. Animals exposed to
1500 ug/m diesel particulate showed a dramatic
decrease in sodium fluoride stimulatible adenylate
cyclase activity from both the controls or animals
exposed to 250 ug/m of diesel particulate. In the
lung of the guinea pig, exposure to either
concentration of diesel particulate reduced the basal x
level of the adenylate cyclase activity by one-half.
Maximal adenylate cyclase activity following sodium
fluoride stimulation was unchanged from the control
groups after either diesel exposure.
Guanylate Cyclase:
Rat. The basal activity of soluble guanylate cyclase
for the rat liver is increased three-fold (250 ug/m )
or more than ten-fold (1500 ug/m ), Table 3. At the
same time, the maximal stimulation of soluble guanylate
cyclase activity remains unchanged from control levels
after exposure to diesel particulate. Guanylate
cyclase activity associated with the particulate
fraction from the rat liver is approximately doubled
following exposure to either diesel exposure. The
maximum sodium azide stimulated guanylate cyclase
activity was increased by more.than five-fold (250 ug/
m ) and by two-fold (1500 ug/m ) at this time. In the
rat lung, the basal activity of soluble guanylate
cyclase activity was increased more than four-fold in
those animals exposed to 1500 ug/m3 diesel particulate.
549
-------
Only stimulatlble guanylate cyclase activity was
observed in those animals exposed to 250 ug/m diesel
particulate. Guanylate cyclase activity associated
with the lung membrane fraction was found to be .
increased six-fold in the animals exposed to 1500 ug/m
diesel particulate.
Guinea Pig. The basal activity of soluble guanylate
cyclase activity in the guinea pig liver, although
variable after exposure to low concentrations of diesel
particulate, remained unchanged from control, Table 4.
Maximum soluble guanylate cyclase activity was
increased significantly after either concentration of
diesel particulate exposure. Basal activity of
particulate guanylate cyclase from the guinea pig liver
demonstrated approximately four-fold increase in
activity jjfter exposure to either concentration of
diesel paiticulate. The maximal stimulated soluble or
particulate guanylate cyclase activities were identical
to response patterns in the rat: after 250ug/m , a
more than three-fold increase of stimulated activity is
observed, while exposure at 1500 ug/m remains
essentially unchanged from control values. In the
guinea pig lung, basal activity of the soluble
guanylate cyclase activity is unchanged from control
after any exposure to diesel particulate. In the same
tissues, there is no increase in guanylate cyclase
activity on exposure to sodium azide which should
maximally stimulate the enzyme in the soluble fraction.
The basal particulate guanylate cyclase activity is
decreased after exposure to either concentration of
diesel particulate. This particulate enzyme follows
the same pattern of non-stimulation when identical
tissue extracts are treated with sodium azide.
Twenty-four Week Diesel Particulate Exposure.
Adenylate Cyclase:
Rats. After twenty-four weeks of diesel particulate
exposure, the basal adenylate cyclase activity from rat
liver membranes, although slightly decreased, was
unchanged from the control values, Table 5. At the
same time, while no differences could be detected in
maximal sodium fluoride stimulatible adenylate cyclase
activity after a low exposure to diesel particulate, a
significant decrease was observed, p < 0.05, in those
animals which received 1500 ug/m diesel exposure. In
the rat lung, the basal activity of adenylate cyclase
was significantly decreased at both 250 (66% of
control), p < 0.05, and at 1500 (33% of control), p <
550
-------
0.01, diesel exposure concentrations. In this tissue,
like the liver, while maximal sodium fluoride
stimulated guanylate cyclase activity remained-equal to
the control in the animals exposed to 250 ug/m diesel
particulate, after 1500 ug/m it was significantly
reduced.
Guinea Pig. Studies of adenylate cyclase activity
from guinea pig liver, show differences only in those
animals exposed to 1500 ug/m of diesel particulate,
Table 6. In this treatment, both the basal activity, p
< 0.05, as well as the stimulated activity, p < 0.02,
is significantly increased from control values. In the
guinea pig lung, basal adenylate cyclase activity is
significantly decreased after any exposure to diesel
particulate, p < 0.05. Maximally stimulated adenylate
cyclase activity of the lung is somewhat decreased
after 250 ug/m exposure, and is significantly
decreased after 1500 ug/m exposure.
Guanylate Cyclase:
Ra t. Basal activity of the soluble guanylate cyclase
from the liver,is significantly decreased, p < 0.05,
after 250 ug/m diesel exposure, Table 5. The
comparable maximally stimulated activity of this
soluble fraction is unchanged /250 ug/m ) or increased
more than four-fold (1500 ug/m ) at this time.
Comparison of basal particulate guanylate cyclase
activity is unremarkable in the liver. Particulate
guanylate cyclase activity in the rat liver which is
stimulated by sodium_azide is increased 33-fold by
exposure to 250 ug/m diesel exposure, and by more than
fourteen-fold after 1500 ug/m diesel exposure, Table
6. In the lung, soluble guanylate cyclase activity is
unchanged from control after 250 ug/m exposure.
However, while the basal soluble guanylate cyclase
activity is unchanged.from the control levels after
exposure to 1500 ug/m of diesel particulate, a sodium
azide stimulated activity of nearly five-fold increase
is now observed. A similar pattern is seen for the
particulate guanylate cyclase activity in this tissue.
Guinea Pig. In the liver, while no changes from
control is observed in basal guanylate cyclase activity
after 250 ug/m diesel exposure, a substantial decrease
is observed after 1500 ug/m exposure, Table 6.
Maximally stimulated soluble guanylate cyclase
activity, shows a tendency to decrease, but remains
essentially unchanged from control after either diesel
exposure. When particulate guanylate cyclase activity
551
-------
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is examined, a doubling (250 ug/m ) or an increase of
nearly ten-fold (1500 ug/m ) is observed. At this
same time, the marked activity of the particulate
guanylate cyclase in the guinea pig liver is reduced
nearly ten-fold (250 ug/m ) or one-fifth (1500 ug/m ).
In the guinea pig lung, an extremely active basal
soluble guanylate cyclase activity is reduced 38% by
250 ug/m diesel exposure, and by 95% after 1500 ug/m
diesel exposures. The maximally stimulated activity in
this tissue after 1500 ug/m diesel exposure is reduced
to one-third of the control activity. When particulate
guanylate cyclase activity of the guinea pig lung is
compared, we find no changes in basal activity compared
to control animals. However, maximally stimulated
particulate guanylate cyclase activity of the guinea
pig lung is significantly reduced, p < 0.02 or greater,
by exposure to either concentration of diesel
particulate.
DISCUSSION
From the studies performed to date, there are
indications of changes which have occurred not only in
the basal activity of adenylate and guanylate cyclase,
but also in the responsiveness of these enzymes to
maximal stimulation after diesel particulate exposure.
The most obvious changes are those in which the basal
activity of the tissues is increased with exposure to
diesel exhaust when compared to control animals and
tissues. This sequence was associated primarily with
the studies of guanylate cyclase activity, and occurred
in both the rat and the guinea pig species. Although
present after 6 weeks of diesel exposure, the trend was
more pronounced in the data observed after 3 months of
exposure. Another trend is the decrease which we find
in basal (unstimulated) adenylate cyclase activity in
each of the species following increasing diesel
particulate exposure. Because of large deviations in
these data groups however, this information is not as
apparent as the data related to guanylate cyclase.
The above patterns are evident throughout the first 24
weeks of diesel exposure to the rat and guinea pigs.
These patterns are also distinctive for both the animal
species and the tissue examined.
When compared to control animals, rat liver adenylate
cyclase activity is unchanged after either 6 weeks or
24 weeks of exposure. At 12 weeks, there is a marked
exposure related increase in NaF-stimulatible adenylate
cyclase. In the same tissue, there is a marked
553
-------
reduction in stimulatible adenylate cyclase after 24
weeks in all animals tested. In the lung, an initial
dose-related increase in NaF-stimulatible adenylate
cyclase activity is abated so that there are no changes
observed after 12 weeks of exposure. After 24 weeks of
diesel exposure, a significant reduction in
stimulatible adenylate cyclase activity is observed in
lung tissue.
Liver adenylate cyclase activity in the guinea pig,
which shows a marked increase in NaF-stimulatible
activity after 6 weeks of diesel exposure, is further
increased at 12 weeks but is substantially reduced
after 24 weeks of exposure. In the lung, there is a
definite trend to increase the basal rate of adenylate
cyclase activity with age. More importantly, a clear
trend is evident which demonstrates a reduction in NaF-
stimulatible adenylate cyclase activity with diesel
exposure. In the soluble guanylate cyclase activity of
the rat lung, beginning after 12 weeks of exposure and
increasing following 24 weeks of exposure, we observed
the appearance of guanylate cyclase which is readily
stimulated by sodium azide. The particulate guanylate
cyclase activity of rat liver shows a stimulatible pool
of enzyme present after both 6 and 12 weeks of diesel
exposure; after 24 weeks, a dramatic change in
stimulatible guanylate cyclase activity is observed.
The particulate guanylate cyclase activity of the lung
provides a clear trend throughout all treatments of a
decrease in basal activity. In addition, a significant
trend of increased stimulatible guanylate cyclase is
observed with increased diesel exposure and time.
Soluble guanylate cyclase activity in the guinea pig
liver shows an initial trend of increased activity
following diesel exposure, through week 12 of the
exposure study. After 24 weeks however, this initial
stimulation is reversed in an apparently dose related
pattern. Soluble guanylate cyclase activities in the
guinea pig lung suggest a progressive loss in basal
activity, and the appearance of a stimulatible fraction
with age. This stimulatible fraction in unaffected by
diesel exposure. The findings which we observe in
guinea pig guanylate cyclase liver particulate
guanylate cyclase activity demonstrates a marked trend
toward increased basal activity seen most clearly after
24 weeks exposure. Basal ^articulate guanylate cyclase
activity in the guinea pig lung remains unchanged
throughout 24 weeks of exposure. Interestingly, after
24 weeks of exposure, a stimulatible guanylate cyclase
activity appears in guinea pigs. This age-related
554
-------
stimulatible activity is significantly reduced by
exposure to diesel particulate.
Conclusions: In the findings reported to date, we
find no evidence of the trend of increased basal
activity of adenylate cyclase or guanylate cyclase
following exposure to diesel exhaust particulate.
Throughout the 24 weeks of exposure reported, although
substantial qualitative differences are apparent,
adenylate cyclase activity remains stimulatible from
the rat or guinea pig liver and lung tissues.
Guanylate cyclase activity also retains stimulation
effects in both the rat and guinea pig liver. On the
basis of these studies therefore, a conclusion of
tumorous response to diesel exposure cannot be
supported. In other observations, in the lung of each
species, we find an age-related appearance of
stimulatible activity. This stimulatible activity is
depressed in the presence of diesel exposure. And
finally, the data of this study supports the conclusion
that some loss of stimulatible guanylate cyclase
activity in the lung, while showing a stimulation of
this enzyme in the liver of the rat and guinea pig.
BIBLIOGRAPHY
1. Katsuki, S., Arnold, W.P., Murad, F. 1977. Effects
of sodium nitroprusside nitorglycerin, and sodium
azide on levels of cyclic nucleotides on and
mechanical activity of various tissues. J. Cyclic
Nucleotide Res., 3:239-247.
2. Nijjar, M.S. 1979. Role of cyclic AMP and related
enzymes in rat lung growth and development.
Biochem. Biophys. Acta, 586 464-472.
3. Steiner, A.L., Pagliara, A.S., Chase, L.R., and
Kipnis, D.M. 1972. Radioimmunoassay for Cyclic
Nucleotides. II. Adenosine 3',5'-monophosphate and
Guanosine 3',5'-monophosphate in mammalian tissues
and body fluids. J. Biol. Chem., 247:1114-1120.
4. Nijjar, M.S. 1979. Regulation of rat lung
adenylate cyclase by cytoplasmic factors during
development. Biochem. Biophys. Acta, 584:43-50.
5. Criss, W.E., Murad, F., Kimura, H., and Morris,
H.P. 1976. Properties of guanylate cyclase in
adult rat liver and several Morris hepatomas.
Biochem. Biophys. Acta, 445:500-508.
555
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6. Criss, W.E. and Murad, F. 1976. Urinary
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in rats bearing transplantable liver and kidney
tumors. Cancer Res., 36:1714-1716.
7. Steiner, A.L., Parker, C.W., and Kipnis, D.M.
1972. Radioimmunoassay for cyclic nucleotides.
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8. Goldberg, N.D. and Haddox, M.K. 1977. Cyclic GMP
metabolism and involvement in biological
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1969. Effects of some hormonal and other factors
on the excretion of guanosine 3',5'-monophosphate
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10. Hardman, J.G. and Sutherland, E.W. 1969. Guanyl
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11. Arnold, W.P., Aldred, R., and Murad,
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monophosphate in tissues. Science, 198:934-936.
12. Goldberg, N.D., Dietz, S.B. and O'Toole, A.G.
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Neiswender, G.D.. 1972. Computer program
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Biomed. Res., 5:205-217.
14. Afifi, A.A. and Azen, S.P.. 1979. Statistical
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193:165-175.
556
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BIOCHEMICAL ALTERATIONS IN LUNG CONNECTIVE TISSUE
IN RATS AND MICE EXPOSED TO DIESEL EMISSIONS
Rajendra S. Bhatnagar, M. Zamirul Hussain,
Keith R. Sorensen, and Frederick M. Von Dohlen
Laboratory of Connective Tissue Biochemistry
School of Dentistry
University of California
San Francisco, CA 94143
and
Robert M. Danner, L. McMillan, and S. D. Lee
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
This Study Was Supported By
U.S. Environmental Protection Agency
Contract No. 68-03-2626
ABSTRACT
With increasing use of diesel-powered vehicles, it is
necessary to assess the potential hazards of increased
diesel emissions in the environment.
The lung is the primary portal of entry of atmospheric
contaminants in the body. Lung structure and function are
dependent on the structural protein collagen, and chemical
injury to the lung elicits a connective tissue response
involving alterations in collagen content and synthesis.
We have examined the effect of inhaling diesel emissions on
lung collagen synthesis in rats and mice, our studies
showed that exposure to diesel emissions increased the rate
557
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of collagen synthesis and the levels of prolyl hydroxylase,
an enzyme intimately associated with collagen synthesis.
These observations suggest that diesel emissions may induce
connective tissue alterations in lungs.
INTRODUCTION
The increasing popularity and use of diesel-powered vehicles
makes it essential to assess the possible hazards of diesel
emissions. The lungs are likely to be a primary target for
any toxic effects of such emissions since they are not only
the primary portal of entry of airborne contamnants in the
body, but they also present the largest exposed surface.
The structure and function of lungs are dependent on connec-
tive tissue which comprises a large part of lung mass.
Collagen, the principal structural protein of connective
tissue, is responsible for maintaining the architectural
integrity of the lungs, and it actively participates not
only in the mechanical aspects of lung function, but is also
a part of the blood-gas interphase (1). Aberration in
collagen content, distribution, and synthesis accompany
pulmonary dysfunction induced by a variety of atmospheric
toxins (1,2).
We have examined the synthesis of collagen and overall
protein synthesis in lungs of mice and rats exposed to
diesel emissions. In our studies, inhalation of diesel
emissions significantly altered these parameters of lung
macromolecular synthesis on experimental animals, suggesting
that diesel emissions may induce structural changes in
lungs, and contribute to pulmonary dysfunction.
MATERIALS AND METHODS
Experimental Animals
Male Sprague-Dawley rats (CD Strain), 10 weeks of age, were
obtained from Charles River Breeding Laboratories. Strain
A/HEJ mice were obtained from the same source. The animals
were maintained on a rodent diet (Purina) and with free
access to water.
Radiochemicals
L-[3,4-3H]-proline, 25 Ci/mmol, L-[14C(U)]-proline, 285
mCi/mmol and L-[4,5-3H]-leucine, 100 Ci/mmol were products
of New England Nuclear.
558
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Exposure to Diesel Emissions
Exposure to diesel emissions was carried out as described in
detail elsewhere (3,4).
Biochemical Procedures
In vivo labelling of proteins with radioactive leucine or
proline was carried out in rats by injecting the radioactive
precursor in the tail vein, at a concentration of 1 mCi/Kg
weight of the animal.
In vitro labelling of tissues was carried out by incubating
the minced tissues in short-term organ culture in Dulbecco-
Vogt medium in a shaker-water bath maintained at 37°. The
tissue-mince was suspended in the medium in a ratio of 5 ml
medium/g tissue. The medium contained 10 Ci of radioactive
proline. Incubation was carried out for 3 hours and termi-
nated by rapidly chilling and homogenizing the tissues in a
Polytron.
Homogenates of tissues labelled in vivo or in vitro were
extensively dialyzed to remove free radioactivity and
hydrolyzed in 6N HC1 for assay for collagen and total
protein. Collagen synthesis was assayed by analyzing the
hydrolyzate for labelled hydroxyproline (5), and total
protein content was assayed by determining the total nin-
hydrin reactive material, using leucine standards (6).
Radioactive incorporation data were expressed as specific
activity terms based on dpm incorporated/ Mol leucine
equivalent.
Prolyl Hydroxylase Assay
Lungs from animals maintained in clean air or diesel-con-
taminated environments .were homogenized in a Polytron in
0.1 M TRIS-HC1, pH 7.4, containing 0.1 M KC1 and 0.1%
Triton-X-100. Aliquots of the 15,000 G supernatents of the
homogenates were assayed for prolyl hydroxylase as described
elsewhere (7). The protein content of the supernatent was
assayed by the Folin-Lowry procedure. The assay for prolyl
hydroxylase is based on the release of ^H from ^H-pro-
line-labelled unhydroxylated collagen. Enzyme activity was,
therefore, expressed in terms of dpm^H released/mg protein
in the supernatent.
559
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RESULTS AND DISCUSSION
Physical Appearance and Characteristics of Exposed Lungs
When the animals were dissected, it became apparent that the
lungs of animals maintained in diesel-contaminated environ-
ments were markedly different both in appearance and in
physical characteristics such as elasticity and consistency.
In general, the exposed lungs were considerably enlarged, no
longer pink, and'appeared grety to black as the length of
exposure was increased. Many lungs appeared mottled and
some had distinct nodules visible to the naked eye. The
homogenates of exposed lungs were sooty and oily in appear-
ance. These observations are summarized in Table I for
exposed and unexposed mice. Similar differences were
observed in case of rat lungs.
Protein Content and Protein Synthesis
The exposed lungs were enlarged both in terms of volume,
measured crudely by displacement of water in a graduated
cyliner, and also in terms of wet weight. The protein
content of the exposed lungs was markedly elevated. As seen
in Table II, the total protein content of rat lungs exposed
to diesel emissions for 42 days was increased nearly 40%.
Increased protein content in lung injury can result from
increased accumulation of circulating proteins and migratory
cells in the alveolar interstitium and in other compart-
ments. Protein content may also increase as a result of
connective tissue proliferation, and the increased deposi-
tion of connective tissue matrix proteins.
In order to determine if there was a change in the rate of
overall protein synthesis in the lungs, the tissues were
pulse-labelled in vivo, by intravenous injection of ^H-
leucine. Previous experiments had demonstrated that in
the lung the highest specific activity of unincorporated
radioactive precursors administered through the tail vein,
was achieved between 15 and 30 minutes. In these experi-
ments, the pulsed incorporation was terminated at 20 minutes
after the radioactive precursor was administered, by sacri-
ficing the animals and rapidly freezing the tissues.
In this experiment, tissues from anmals in each group were
pooled before analysis. As seen in Table III, the specific
activity of leucine incorporation was lower in the exposed
animals than in controls. Even if it is assumed that there
is some dilution of label by accumulated protein in the
lung, this observation suggests a decrease in the overall
synthesis of proteins. Decreased protein synthesis has been
observed in other forms of chemical injury to the lungs (2).
560
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The synthesis of collagen was examined under the same
conditions by injecting -^H-proline and following the
incorporation of total ^H-radioactivity and the synthesis
of ^H-hydroxyproline in the lungs. Data from this experi-
ment are presented in Table IV. Unlike leucine, which is
considerably less abundant in collagen than in other pro-
teins, the incorporation of ^H-proine and the synthesis of
^H-hydroxyproline did not appear to be significantly
altered on exposure to diesel emissions. Proline is several
times more abundant in collagen than in noncollagen pro-
teins, and significant amounts of hydroxyproline occur only
in collagen. These data can thus be interpreted as suggest-
ing a relative increase in collagen synthesis in comparison
to the synthesis of noncollagen proteins in lungs exposed to
diesel emissions.
Increased collagen synthetic activity in the exposed lungs
was also confirmed in experiments in whcih lung tissues were
labelled in short-term organ culture. In this experiment,
lungs were dissected out and minced in a small volume of
organ culture medium, and rinsed several times to eliminate
the accumulated circulating protein material, before being
labelled with l^C-proline. /\s seen -jn Table V, although
the overall incorporation of the radioactive precursor was
suppressed, the ratio of radioactivity in hydroxyproline to
the total radioactive incorporation was nearly doubled.
These data are consistent with the in vivo incorporation
studies and suggest that, although there is decreased
protein synthesis in diesel-exposed rat lungs, the propor-
tion of collagen synthesis is greater.
Prolyl hydroxylase is a crucial enzyme in the pathway of
collagen synthesis, and its levels usually reflect a tissues
potential for collagen synthesis. Prolyl hydroxylase levels
in lungs are elevated when the tissue is injured and such
increases reflect increased turnover and proliferation of
connective tissue (8-10). Increased prolyl hydroxylase
activity in exposed lungs would be consistent with injury.
We examined prolyl hydroxylase activity in young adult male
and female rats exposed to diesel emissions for 42 days, and
in rat pups, exposed to diesel emissions either in utero or
neo-nataly. These groups exhibited marked differences in
their response to diesel emissions as measured by altera-
tions in prolyl hydroxylase activities (Table VI). After
42-day exposure, adult female rats showed larger increases
in lung prolyl hydroxylase than did the adult male rats;
however, there was a greater increase in protein accumula-
tion in the lungs of male rats, and this makes the data on
male rat lungs difficult to interpret. 20 day old rats
exposed to diesel emissions in utero showed significant
increases in prolyl hydroxylase activity. These data
561
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suggest that toxic components of diesel emissions may pass
the placenta! barrier. Interestingly, when neo-natal rats
were exposed to diesel emissions for 20 days, there appeared
to be a decrease in lung prolyl hydroxylase. The reason for
this decrease is not clear at present. One possible expla-
nation is that large accumulation of infiltrating proteins
may have lowered the specific activity of the enzyme.
We also examined the effect of length of exposure to diesel
emissions on lung collagen synthesis. These experiments
were carried out using male mice of A/HEJ Strain. The
animals were exposed to diesel exhaust for varying periods.
The lungs exhibited marked changes in appearance and other
characteristics as summarized in Table I. They were exam-
ined for collagen and protein synthesis in short-term organ
cultures (Table VII). As in the case of rat lungs, there
appeared to be large increases- in protein content in the
exposed tissues, and while the specific activity terms do
not accurately reflect lung protein synthesis in these
experiments, the rate of protein synthesis appeared to be
lowered. The sysntehsis of collagen, however, appeared to
account for a greater proportion of total protein synthe-
sized, and increased with the length of exposure so that,
after a 9-month exposure, the rate of collagen synthesis
was 1.5 times that in the controls. These data support
the observations made in the experiments with rats, and,
furthermore, they suggest that continual exposure to diesel
emissions may exacerbate lung injury. These studies also
suggest a species difference in the susceptibility to the
deleterioius effects of diesel emissions in that mice may
be more resistant to such injury than rats.
Our in vivo studies on the effect of diesel emissions on
lung collagen synthesis are corroborated by other studies in
our laboratory using rat lung organ cultures (11-13). Some
of these studies are summarized in another contribution from
our laboratory at the symposium (13). Polyaromatic hydro-
carbons are the major toxic components of diesel emissions
(14). When lung organ cultures were exposed to benzo(a)-
pyrene for periods of 24 hours, they exhibited markedly
increased activities of prolyl hydroxylase (11), and synthe-
sized collagen at elevated rates (12,13). Furthermore, the
relative proportion of type I and type III collagen synthe-
sis were altered (12,13).
562
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Table I
PHYSICAL CHANGES IN LUNGS OF MICE
EXPOSED TO DIESEL EMISSIONS
Color
Insoluble White
Approx. Sinewy Material
Size Nodes After Homogenization
3.5 Month
Control
Exposed
6.0 Month
Control
Exposed
9.0 Month
Control
Exposed
Pink
Gray/Black
Pink Edges
Pink
Black with
Some Pink
Edges
Gray/Pink
Black with
White Edges
100%
120% +++ +
100%
150% ++++ ++
120% +
180% +++ +++
Size = Wet Weights and Volume.
563
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Table II
TOTAL PROTEIN CONTENT OF LUNGS
Control
42-Day Diesel Exposure
42-Day Diesel Exposure
(12 Animals/Group)
Protein
mg/g Tissue
(Wet Wt.)
54
71
78
% Change
-
31
44
The total protein content of the lungs was assayed on the
basis of total ninhydrin reactive material in tissue hdroly-
zates. In order to express the protein content in these
units (mg/g tissue), the assay was based on bovine serum
albumin used in standards adn hydrolyzed and processed
identically to the tissues.
Table III
PULSE-LABELLED INCORPORATION OF 3H-LEUCINE INTO LUNG PROTEIN
10-5xdpm 3H/g Tissue (Wet Wt.)
Control 1.17
42-Day Diesel Exposure 0.72
42-Day Diesel Exposure 0.56
(12 Animals/Group)
The incorporation of ^H-leucine into lung proteins was
assayed in the hydrolyzates and related to the protein
content as described in the legend to Table I.
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Table VI
PROLYL HYDROXYLASE LEVELS IN LUNGS OF RATS
EXPOSED TO DIESEL EXHAUST
Prolyl Hydroxylase Activity
(PPM 3H/mg Protein)
Female Control (6 Rats) 3730 ^ 1086
Female Exposed (6 Rats) 5965 jt- 1332 (p 0.05)
Male Control (6 Rats) 561 _+ 1522
Male Exposed (6 Rats) 4780 ^ 1744 (p 0.05)
Gestational Control (20 Rats) 5474 +_ 1435
Gestational Exposed (20 Rats) 7342 +_ 1534 (p 0.0005)
Neo-Natal Control (19 Animals) 6644^1321
Neo-Natal Exposed (18 Animals) 3051 _+ 376 (p 0.0001)
Lungs were homogenized in a Polytron homogenizer and pH
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ing the release of -^H from ^H-Pro-labelled unhydroxylated
collagen. Each number is the average of six determinations.
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1. Hance, A. J., and Crystal, R. G. Collagen. In: The
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570 *US GOVERNMENT PRINTING OFFICE 1980757-064/0190
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