United States
Environmental Protection
Agency
Research and Development
v-/EPA 1981
DIESEL EMISSIONS
SYMPOSIUM
Registration and
Abstract Book
Octobe/5-7,1981
The Roval Villa Hotel
Raleigh, North Carolina
Sponsored by the
U.S. Environmental
Protection Agency
Office of Research and
Development
Research Triangle Park,
North Carolina
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Foreword
The high fuel efficiency of diesel encnnes is expected to result, in a
significant increase in the production of diesel-powered passenner cars. Ma.ior
research programs were initiated in the late 1970s by governments, industry,
and the academic community in order to understand the physical and chemical
characteristics of emissions from the diesel enm'ne, and the potential
biological effects of these emissions.
In December 1979 the D.S. Environmental Protection Agency Health Effects
Laboratory at Cincinnati, Ohio, sponsored the first symposium on the Health
Effects of Diesel Engine Emissions. The purpose of this 1981 symposium,
sponsored by the Office of Research and Development, is to foster exchanoe of
the more recent scientific and technical information derived from the various
research programs.
This registration volume contains a compilation of extended abstracts of
each presentation to he made at the 19S1 Diesel Emissions Symposium on
October 5-7 at Raleigh, North Carolina. The symposium is oroanized into the
foil owing sessions:
Diesel Emissions Characterization and Control Techno! ooy
Chemical and Bioassay Characterization
Pulmonary Function
o Pulmonary Toxicology and Biochemistry
Mutagenesis and Carcinogenesis
Exposure and Risk Assessment
The 96 short papers included here represent the oral and poster
presentations. The invited overview presentations and selected papers will he
published after the meeting to provide an in-depth review of each of these
topics.
F. Gordon Hueter
Di rector
Health Effects Research Laboratory
U.S. Environmental Protection Aoency
Research Trianale Park, MC
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Acknowledgements
The assistance of the many individuals who contributed to the planning of
this symposium is gratefully acknowledged. Special appreciation is due to
Ms. 01 ga Wierbicki and Ms. Barbara Elkins of Northrop Services who serve as
symposium coordinators.
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Contents
GENERAL INFORMATION!
General Chairman
Organizing Chairman
Organizing Committee
Symposium Coordinators
Location
Registration
Luncheons and Coffee
Symposium Publications
Checks
Meeting Room
Poster Sessions
Tours
Messages
PROGRAM
October 5:
October 6:
October 7;
Session 1: Diesel Emissions Characterization
and Control Technology
Session ?: Chemical and Rioassay
Characterization
Poster Session 1
Session 3: Pulmonary Function
Session 4: Pulmonary Toxicology and
Biochemistry
Poster Session ?
Session 5: Mutaoenesis and Carcinooenesis
Session 6: Exposure and Risk Assessment
ABSTRACTS
The abstracts of the oral and poster presentations appear
in the order that they are listed in the program.
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General Information
GENERAL
CHAIRMAN
ORGANIZING
CHAIRMAN
ORGANIZING
COMMITTEE
SYMPOSIUM
COORDINATORS
LOCATION
REGISTRATION
LUNCHEONS
COFFEE/TEA
James Smith, Acting Director, RASSO, U.S. Environmental
Protection Agency, Research Trianole Park, NC 27711,
(919) 541-2909.
Joel!en Lewtas, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle
Park, NC 27711, (919) 541-3849.
Stephen Mesnow, Larry Claxton, and Ronald Rradow,
U.S. Environmental Protection Agencv, Research Trianole
Park, NC 27711.
01 ga Wierbicki and Barbara El kins, Northrop Services,
Inc., P.O. Pox 12313, Research Triangle Park, NC 27709,
(919) 549-0411. The Symposium Coordinators and their
staff will be happy to help you in case you have any
Questions or problems.
The symposium is being hosted at the Royal Villa Hotel
and Convention Center, 5339 Glenwood Avenue, Raleioh,
NC 27662, (919) 782-4433.
All participants are asked to reaister either
Sunday, October 4, 7-9 pm, in the Main Hall or
Monday, October 5, startina at 7:30 am, also in the
Main Hall. Participants arriving after commencement
of the symposium should register on arrival.
A luncheon has been catered for all three days. Participants
are encouraged to sian up for these luncheons due to the
limits of time and available restaurants. The meal fee is
$37.00. This fee also includes your coffee/tea. Those
participating for one day only may pay $13.00 for lunch
and coffee. The meals will he in Royal King's Hall III.
Coffee, tea, and sanka will be provided for the participants.
Soda will also be available for the afternoon break. Anyone
who has not paid a meal fee but wants coffee, etc., is asked
to pay a fee of $10.00 for the three days. Those here for
only one day are asked to contribute $5.00 for coffee.
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SYMPOSIUM
PUBLICATIONS
CHECKS
An abstract hook will he distributed to each attendee at the
time of the symposium. A proceedinos will he published in
book form consistina of the overview presentations and
selected papers. This proceedings is available at a discount
for $35.00 if it is ordered at the time of the symposium.
Participants may order a copy at the registration table.
Please make checks for the meal or coffee fees and/or for
the proceedinos out to Diesel Emissions Symposium.
MEETING ROOM Sessions 1-6 will he held in Royal Kino's Hall I and II.
POSTER The poster sessions are an inteoral part of the program.
SESSIONS They will be held October 5 and 6 from 5:30-7:30 pm in
Royal Kino's Hall III. Poster presenters are asked to
pin up their poster during the afternoon break of the day.
SPECIAL Cocktails will he available on a cash basis during the
EVENTS poster sessions.
TOURS A tour of selected research facilities in the Research
Triangle Park is planned for October 8. Since space in the bus
is limited, please sion up early at the reoistration table.
More detailed information will he announced during the
symposium.
MESSAGES Callers should dial (919) 782-4433, the Royal Villa Hotel
and Convention Center in Raleigh, MC. All incoming messages
will he posted near the registration table.
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Program
OCTOBER 5
SESSION 1
DIESEL EMISSIONS CHARACTERIZATION AMD CONTROL TECHNOLOGY
Chairman: Ronald Rradow, Environmental Sciences Research
Laboratory, U.S. Environmental Protection Aaency, Research
Trianale Park
8:30 am Opening Remarks
8:45 am Diesel Emissions, a Worldwide Concern
K. Springer, Southwest Research Institute
9:15 am Diesel Particle and Orpanic Emissions; Engine Simulation,
Sampling, and Artifacts
R. Rradow, U.S. Environmental Protection Aaency, Research
Triangle Park
9:45 am Diesel Rarticul ate Emissions: Composition, Concentration, and
Control
R. Williams, General Motor Research Laboratories
10:15 am Morning coffee break
10:45 am Particulate Emissions from Spark-Ignition Enoines
T. Naman, U.S. Department of Energy
11:00 am Particulate Emission Characterization Studies of In-llse Diesel
Automobiles
P. Gibbs, MY State Dept. of Environmental Conservation
11:15 am Diesel Exhaust Treatment Devices: Effects on Gaseous and
Particulate Emission and on Mutaaenic Activity
R. Gorse, Jr., Ford Motor Company
11:30 am Characterization and Oxidation of Diesel Particulate
D. Trayser, Rattelle-Columhus Laboratories
11:45 am Heavy-Duty Diesel Engine Emissions: Some Effects of Control
Technol ogy
J. Perez, Caterpillar Tractor Company
12:00 pm
Lunch
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OCTOBER 5
SESSION 2 CHEMICAL AND BIOASSAY CHARACTERIZATION
Chairman: J cell en Lewtas, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park
1:30 pm Methodology of Fractionation and Partition of Diesel Exhaust
Participate Samples
B. Petersen, Battelle-Columhus Laboratories
2:00 pm The Utility of Bacterial Mutagenesis Testino in the
Characterization of Mobile Source Emissions: A Review
L. Claxton, U.S. Environmental Protection Aaency, Research
Triangle Park
2:30 pm Emission Factors from Diesel- and Rasol ine-Powered Vehicles;
Correlation with the Ames Test
R. 7weidinger, U.S. Environmental Protection Agency,
Research Triangle Park
3:00 pm Afternoon coffee break
3:30 pm Analyses of Volatile Polycyclic Aromatic Hydrocarbons in
Heavy-Duty Diesel Exhaust Emission
W. Eisenberg, I IT Research Institute
3:45 pm The Chemical Characterization of Diesel Particulate Matter
J. Yergey, Johns Hopkins University
4:00 pm The Analysis of Nitrated Polynuclear Aromatic Hydrocarbons in
Diesel Exhaust Particulates by MS/MS Techniques
T. Riley, Ford Motor Company
4:15 pm Contribution of 1-Nitropyrene to Oirect-Actino Ames Assay
Mutagenicities of Diesel Particulate Extracts
I. Salmeen, Ford Motor Company
4:30 pm Dinitropyrenes: Their Probable Presence in Diesel Particle
Extracts and Conseouent Effect on Mutagen Activations by
NADPH-Dependent S9 Enzymes
T. Pederson, General Motors Research Laboratories
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OCTOBER 5
5:30-7:30 pm POSTER SESSION 1
Convener: Larry Claxton, Health Effects Research Laboratory,
U.S. Environmental Protection Aoency, Research Triangle Park
Mutagenicity of Particle-Bound Oraanic Chemical Fractions from
Diesel and Comparative Emissions
A. Austin, U.S. Environmental Protection Agency, Research
Triangle Park
Emission of Diesel Particles and Particulate Mutagens at Low
Ambient Temperature
J. Braddock, U.S. Environmental Protection Aoency, Research
Triangle Park
Chemical Characterization of Mutagenic Fractions o^ Diesel
Particulate Extracts
D. Choudhury, MY State Dept. of Health
Influence of Driving Cycle and Car Type on the Mutagenicity of
Diesel Exhaust Particle Extracts
C. Clark, Lovelace Inhalation Toxi col ogy Research Institute
The Rapid Analysis of Diesel Emissions Using the Taga 6000
Triple Ouadrupole Mass Spectrometer
J. Fulford, Sciex, Canada
Compounds in City Air Compete with ^H-2, 3,7,P-Tetrachl oro-
dibenzo-p-Dioxin for Binding to the Receptor Protein
J-A Gustafsson, Karolinska Institute, Sweden
GC/MS and MS/MS Studies of Direct-Acting Mutagens in Diesel
Emissions
T. Henderson, Lovelace Inhalation Toxicology Research
Institute
Evaluation of the Release of Mutagens and 1-Nitropyrene
from Diesel Particles in the Presence of Lung Macrophage Cells
in Culture
L. King, U.S. Environmental Protection Agency, Research
Triangle Park
Bacterial Mutagenicity of a Diesel Exhaust Extract and Two
Associated Mitroarene Compounds After Metabolism and Protein
Binding
M. Kohan, U.S. Environmental Protection Agency, Research
Triangle Park
%*''
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OCTOBER 5
5:30-7:30 pm POSTER SESSION 1 (continued)
Characterization of Participate Emissions from In-Use
Gasol i ne-Fuel ed Motor Vehicl es
J. Lang, Northrop Services, Inc., Research Triangle Park
Surface Reactivity of Diesel Particle Aerosols
M. Lenner, University of Gothenburg, Sweden
Effects of Ozone and Nitroaen Dioxide Present During Ssmpling
of Genuine Particulate Matter as Detected by Two Biological
Test Systems and Analysis of Polycyclic Aromatic Hydrocarbons
G. Lo'froth, University of Stockholm, Sweden
Alumina-Coated Wool as a Particulate Filter for Diesel-Powered
Vehicles
M. McMahon, Texaco, Inc.
Isolation and Identification of Mutagenie Mitroarenes in Diesel
Exhaust Particulates
J. Nachtman, University of California at Berkeley
Comparison of Nitro-PMA Content and Mutanenicity of Diesel
Emissions
M. Mishioka, Battelle-Columbus Laboratories
Capillary Column GC/MS Characterization of Diesel Exhaust
Particulate Extracts
T. Prater, Ford Motor Company
Physico-Chemical Properties of Diesel Particulate Matter
M. Ross, Johns Hopkins University
Some Factors Affecting the Ouantitation of Ames Assays
I. Salmeen, Ford Motor Company
Chemical and Mutagenic Characteristics of Diesel Exhaust
Particles from Different Diesel Fuels
D. Sklarew, Rattelle Northwest Laboratories
Fractionation and Characterization of the Oraanics from Diesel
and Comparative Emissions
C. Sparacino, Research Triangle Institute, Research
Trianole Park
Trapping Gaseous Hydrocarbons
F. Stump, U.S. Environmental Protection Aoency, Research
Trianole Park
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OCTOBER 5
5:30-7:30 pm POSTER SESSION 1 (continued)
Analytical Methods for Mitroaromatic Compounds
S. Tejada, U.S. Environmental Protection Aoency, Research
Trianql e Park
Total Luminescence Spectroscopy of Diesel Exhaust Particirtate
P. Whitby, NYS Department of Environmental Conservation
Evaluation of the Metabolic Requirements of Diesel and
Comparative Source Samples in the Salmonella Typhimurium Plate
Incorporation Assay
K. Williams, U.S. Environmental Protection Aaency, Research
Triangle Park
MS/MS Characterization of Diesel Particulates
K. Wood, Purdue University
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OCTOBER 6
SESSION 3
PULMONARY FUNCTION
Chairman: Donald Gardner, Health Effects Research Laboratory,
U.S. Environmental Protection Aoency, Pesearch Trianole Park
8:30 am Inhalation Toxicology of Diesel Exhaust Particles
R. McClellan, Lovelace Inhalation Toxicoloay Pesearch
Institute
9:00 am EPA's Inhalation Toxicolooy Study
W. Pepelko, U.S. Environmental Protection Aoency,
Cincinnati
9:30 am Pulmonary Function Testina of Rats Chronically Exposed to
Diluted Diesel Exhaust for 612 nays
K. Gross, General Motors Pesearch Laboratories
9:45 am Pulmonary Functional Response in Cats Followina Two Years of
Diesel Exhaust Exposure
W. Moorman, National Institute for Occupational Health
and Safety
10:00 am Deposition and Retention of Surroqate and Actual Diesel
Particles
R. Wolff, Lovelace Inhalation Toxicolooy Research Institute
10:15 am Lung Clearance of Radioactively Labelled Inhaled Diesel Exhaust
Particles
P. Lee, General Motors Pesearch Laboratories
10:30 am Morning coffee break
11:00 am Compartmental Analysis of Diesel Particle Kinetics in the
Respiratory System of Exposed Animals
S. Soderholm, General Motors Research Laboratories
11:15 am A Suhchronic Study of the Effects of Exposure of Three Species
of Rodents to Diesel Exhaust
H. Kaplan, Southwest. Pesearch Institute
11:30 am Response of Pulmonary Cellular Defenses to the Inhalation of
High Concentration of Diesel Exhaust
K. Strom, General Motors Pesearch Laboratories
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OCTOBER 6
SESSION 3 PULMONARY FUNCTION (continued)
11:45 am The Effect of Diesel Exhaust on Cells of the Immune System
D. Dziedzic, General Motors Research Laboratories
12:00 pm The Participation of the Pulmonary Type II Cell Response to
Inhalation of Diesel Exhaust Emission: Late Sequelae
H. White, General Motors Research Laboratories
12:15 pm Lunch
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OCTOBER 6
SESSION 4
PULMONARY TOXICOLOGY AMD BIOCHEMISTRY
Chairman: Judy Graham, Health Effects Research Laboratory, U.S.
Environmental Protection Agency, Research Triangle Park
1:45 pm Pulmonary Deposition, Retention, Inactivation, and Clearance of
Inhalerl Diesel Particles: The Role of the Pulmonary Defense
System
J. Vostal, General Motors Research Laboratories
2:15 pm Investigations of Toxic and Carcinooenic Effects of Diesel
Exhaust in Lena-Term Inhalation Exposures of Rodents
W. Stober, Fraunhofer Institute fuer Toxikoloaie unrl
Aerosol forschuno, West Germany
2:45 pm Biochemical Alterations in ^ronchopulmonary Lavane n uids After
Intratracheal Administration of Diesel Particulates to Rats
C. Eskelson, University of Arizona Health Science Center
3:00 pm Lipid Changes in Lungs of Rats After Intratracheal
Administration of Diesel Particulates
C. Eskelson, University of Arizona Health Science Center
3:15 pm Afternoon coffee-break
3:45 pm Bioavailabil ity of Diesel Particle Round [G--H]-Renzo(a)pyrene
After Intratracheal Instillation
S. Dutta, Wayne State University
4:00 pm The Potential for Aromatic Hydroxylase Induction in the Lung by
Inhaled Diesel Particles
K. Chen, General Motors Research Laboratories
4:15 pm Xenobiotic Metabolizing Enzyme Levels in Mice Exposed to Diesel
Exhaust or Diesel Exhaust Extract
W. Peirano, U.S. Environmental Protection Agency,
Cincinnati
4:30 pm Morphometric 111 trastructural Analysis of Alveolar Lungs of
Guinea Pigs Chronically Exposed by Inhalation to Diesel Exhaust
M. Rarnhart, Wayne State University School of Medicine
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OCTOBER 6
5:30-7:30 pm POSTER SESSION 2
Convener: Joel 1 en Lewtas, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park
Scanning Electron Microscopy of Terminal Airv/ays of Guinea Pigs
Chronically Inhaling Diesel Exhaust
M. Barnhart, Wayne State University School of Medicine
The Design of the Long-Term Inhalation Program within the CCMC's
Health Effects Research Program
J. Brightwell, Committee of Common Market Automobile
Constructors, Rattelle Geneva Research Center, Switzerland
Chronic Inhalation Oncogenicity Study of Diesel Exhaust in
Sencar Mice
K. Campbell, U.S. Environmental Protection Agency
Cincinnati
Species Differences in Deposition and Clearance of Inhaled
Diesel Exhaust Particles
T. Chan, General Motors Research Laboratories
Species Comparisons of Rronchoal veolar Lavages from Guinea Pigs
and Rats Exposed In Vivo to Diesel Exhaust
S-t. Chen, Wayne State University School of Medicine
Preliminary Report of Systemic Carcinogenic Studies on Diesel
and Gasoline Particulate Emission Extracts Applied to Mouse
Skin
M. Clapp, Oak Ridge National Laboratory
CCMC's Health Effects Research Program
Committee of Common Market Automobile Constructors
Emissions Research Committee, Belgium
Effects of Chronic Diesel Exposure on Pulmonary Protein
Synthesis in Rats
S. Dutta, Wayne State University School of Medicine
Fractionation and Identification of Organic Components in Diesel
Exhaust Particulate
M. Erickson, Research Triangle Institute
Preparation of Diesel Exhaust Particles and Extracts as
Suspensions for Bioassays
J. Graf, IIT Research Institute
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OCTOBER 6
5:30-7:30 pm POSTER SESSION 2 (continued)
Research Plans for Diesel Health Effects Study
H. Kachi, Japan Automobile Research Institute, Japan
Neurodepressant Effects of Uncomhusted Diesel Fuel
R. Kainz, Tulane University
The Effect of Exposure to Diesel Exhaust on Pulmonary Protein
Synthesis
R. McCauley, Wayne State University School of Medicine
Respiratory Health Effects of Exposure to Diesel Exhaust
Emissions
P. Peger, National Institute of Occupational Safety and
Health
SWRI-SFPE Diesel Health Effects Exposure Facility
K. Springer, Southwest Research Institute
Post-Exposure Diesel Particle Residence in the Lungs of Rats
Following Inhalation of Dilute Diesel Exhaust for Six Months
K. Strom, General Motors Research Laboratories
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OCTOBER 7
SESSION 5
MUTAGENESIS AMD CAPCINOGEMESIS
Chairman: Stephen Nesnow, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Trianqle Park
8:00 am Mutaqenic Activity of Diesel Emissions
J. Lewtas, U.S. Environmental Protection Aqency
8:30 am Mutaqenicity of Diesel and Spark lonit.ion Enoine Exhaust
Particulate Extract Components to Salmonella Typhimurium and
Human Lymphoblasts
T. Rarfknecht, Massachusetts Institute of Techno! ooy
8:45 am Cytotoxicity, Mutaqenicity, and Co-Mutaqenicity of Diesel
Exhaust Particle Extracts on Chinese Hamster Ovary Cells
In Vitro
A. Li, Lovelace Inhalation Toxicoloay Research Institute
9:00 am Induction of In Vivo Sister Chromatirl Exchanqe hy Diesel
Particulate and Diesel Extract
M. Pereira, U.S. Environmental Protection Aoency,
Cincinnati
9:15 am Mutaqenic Activity of Diesel Particles in Alveolar Macrophaqes
from Rats Exposed to Diesel Enqine Exhaust
J-S. Siak, General Motors Research Laboratories
9:30 am Morninq coffee break
10:00 am Skin Carcinoqenesis Studies of Emission Extracts
S. Nesnow, U.S. Environmental Protection Aqency, Research
Trianql e Park
10:30 am Dermal Carcinoqenesis sioassays of Diesel Participates and
Dichl oromethane Extract of DP
L. DePass, Rushy Run Research Center
11:00 am Respiratory Carcinoqenicity of Diesel Fuel Emissions:
Interim Results
A. Shefner, IIT Research Institute
11:30 am Carcinoqenicity of Extracts of Diesel and Related Environmental
Emissions Upon Lunq Tumor Induction in Strain 'A1 Mice
R. Laurie, U.S. Environmental Protection Aqency, Cincinnati
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OCTOBER 7
SESSION 5 MUTAGENESIS AMD CARCINOGENESIS (continued)
11:45 am The Influence of Inhaler! Diesel Enaine Emissions Upon Lunq Tumor
Induction in Strain 'A' Mice
W. Pepelko, U.S. Environmental Protection Aqency,
Cincinnati
12:00 pm Objectives and Experimental Conditions of VW/Audi Diesel Exhaust
Inhalation Study
W. Stoher, Fraunhofer Institute fuer Toxikolooie und
Aerosol forschuna, West fie many
12:15 pm Lunch
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OCTOBER 7
SESSION 6
EXPOSURE AND RISK ASSESSMENT
Chairman: Robert. Jungers, Environmental Monitoring Systems
Laboratory, U.S. Environmental Protection Agency, Research
Triangle Park
1:30 pm Projected Human Health Risks from Increased Use of Diesel
Light-Duty Vehicles in the United States
R. Cuddihy, Love!ace Biomedical and Environmental Research
Institute
2:00 pm Health Effects of Exposure to Diesel Fumes and Dust in Two
Trona Mines
M. Attfield, Mational Institute of Occupational Safety
and Heal th
2:15 pm Mutagenicity and Chemical Characterization of Carbonaceous
Particulate Matter from Vehicles on the Road
W. Pierson, Ford Motor Company
2:45 pm Afternoon coffee break
3:15 pm Emissions of Gases and Particulat.es from Diesel Trucks on
the Road
P. Kiyoura, Research Institute of Environmental Science,
Japan
3:30 pm Diesel Bus Terminal Study: Effects of Diesel Emission on Air
Pollutant Levels
R. Burton, U.S. Environmental Protection Agency, Research
Triangle Park
3:40 pm Diesel Bus Terminal Study: Characterization of Volatile and
Particle-Bound Oraanics
R. Jungers, U.S. Environmental Protection Agency, Research
Triangle Park
3:50 pm Diesel Bus Terminal Study: Mutaaenicity of the Particle-Bound
Organics and Organic Fractions
J. Lewtas, U.S. Environmental Protection Agency, Research
Triangle Park
4:00 pm Nitro Derivatives of Polynuclear Aromatic Hydrocarbons in
Airborne and Source Particulate
T. Gibson, General Motors Research Laboratories
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OCTOBER 7
SESSION 6 EXPOSURE AMD RISK ASSESSMENT (continued)
4:15 pm Risk Assessment of Diesel Emissions
R. Albert, U.S. Environmental Protection Aqency,
Washington, DC
4:30 pm Perspectives on Diesel Emission Health Research
N. Nelson, New York University Medical Center
5:00 pm Close
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Abstracts
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DIESEL EMISSIONS, A WORLDWIDE CONCERN
by
Karl J. Springer
Department of Emissions Research
Southwest Research Institute
6220 Culebra Road
San Antonio, Texas
Recent visits to Japan and Europe plus scores of visitors from other
countries have convinced me that there is a worldwide concern over the
possible health effects of diesel exhaust. Not all of these visitors come
to San Antonio to visit the Alamo or stroll by the river.
Laboratory tests with bacteria, animal cells and tissues have shown some
components of diesel exhaust to be toxic, mutagenic or carcinogenic. In
addition to gas phase compounds that have both direct and secondary effects
in the atmosphere, diesel exhaust contains particulates of both solid (soot)
and liquid (aerosol) type. The soot particles are less than one millionth
of a meter in size and provide a surface for the aerosols to condense or
absorb. For example, benzo(a)pyrene, a well known carcinogen, is but one of
the materials that are in diesel particulate.
Studies in 1977 by Southwest Research Institute's Emissions Research
Department proved that diesel passenger cars produce particulate 50 to 80
times their gasoline-fueled counterparts. A 1981 report gave 0.31 g/km
(0.5 g/mile) as an emission rate from cars with 15 percent being soluble
organics (i.e., extractable with dichloromethane solvent). It is the soluble
fraction of the particulate that has sounded the alarm since this contains
the materials that have been found to be direct acting mutagens by the Ames
bioassay test.
It is this fraction, first evaluated in the Ames test in 1977, that
resulted in the precautionary notice published by EPA that same year. It
is this fraction which has caused this symposium to be held and the previous
CRC Dearborn meeting in March 1981, the EPA Cincinnati Symposium in Decem-
ber 1979, and the EPA Ann Arbor Symposium in May 1978.. It is this fraction
which has caused the legislators, Federal officials in the DOE, DOT, EPA,
Bureau of Mines and others so much frustration and confusion. It is this
fraction which has given executives of diesel engine manufacturers and car
makers chronic nightmares. It is this fraction which has given the voices
for environmental protection another item for argument. It is also this
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fraction which has given many of us a scientific challenge.
I loosely gather the governmental policymakers, the corporate executives,
and all those for whom you work as "THEY Who Must Be Obeyed." THEY have
supported us with money, facilities and the opportunity to investigate diesel
particulate. Until recently, THEY have been, on the surface at least,
patient, realizing that such research is tedious and long term in nature.
Underneath that thin veneer of patience I sense the growing imperative to
decide, to rule, and to move on. It is that basic impatience of the U.S. or
us.
Can we decide at this conference if there is or is not a problem with
that organic fraction? If there is, do we have enough data to prove it?
If we don't, what is needed in time and money? Can we convince ourselves
and others that the studies should continue? In the meantime, what about
dieselization of the passenger cars and trucks in the U.S.? Is it business
as usual? Do we suggest diesels be limited in urban and congested areas?
Do we give diesels the green light?
THEY, who must be obeyed, are faced with these and related questions.
What to do? Do not think that because your research is incomplete or that
you need another five years to complete a health survey of a specific
population that THEY will necessarily wait. Diesels in cars and trucks are
a quick way to reduce fuel consumption, energy costs and foreign dependence.
It is clear from recent changes in environmental thinking that a new policy
is emerging. We have to prove to THEY, who must be obeyed, that there is a
clear and present danger from diesel particulate. I wonder if we are able
to do this in the next few days.
THEY, who must be obeyed, have a standard approach which goes essentially
like this. A broad program has been in progress for, say, three to four years
with no proof that diesel exhaust is hazardous beyond the positive Ames test
(which we knew of in 1977). What are the chances of proving a hazard exists,
given another three to four years?
It is the same question we ask of ourselves on many topics every day.
I submit that the delegates to this conference have a job of convincing
THEY, who must be obeyed, of the need to continue. Otherwise, THEY, who must
be obeyed, quite likely will become impatient and make those decisions for
us using whatever facts are available at the time. You may not agree, but
the decision point is very near.
The remainder of this keynote address deals with the macroscopic or big
picture particulate contributions of light- and heavy-duty vehicles. For
example, given no control and a 25 percent sales penetration (by 1985 and
thereafter) of cars powered by diesel engines, it will be 1998 before diesel
cars equal diesel truck and bus particulates. With moderate particulate
control of both classes of vehicles, particulate parity is not reached until
after the year 2000. Similar projections are given based on lower and higher
penetration of diesel cars.
To add further perspective to the diesel issue, specifically the
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substitution of many diesel cars for gasoline ones, some recent results from
microscale modeling are summarized. Self-contamination can occur in parking
garages, street canyons, tunnels, expressways, or almost anywhere crowded
conditions occur with limited dilution. The current maximum 24 hour limit
for total suspended particulate (TSP) is 260 yg/m^. For example, in a
typical above ground multilevel parking garage, the TSP can be exceeded by
having as few as 12 percent of the cars diesel-powered. These localized
concentrations may be important, if not from health, from public welfare,
such as odor and eye irritation and reduced visibility, increased soiling,
etc.
The challenge to this meeting is quite simply to assimilate and rumi-
nate and then communicate. We must absorb, digest, compare and then think
about it and get the meaning into simple terms so that THEY, who must be
obeyed, will understand. Only then, will we have done our job well.
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Diesel Particle and Organic Emissions:
Engine Simulation, Sampling and Artifacts
by
Ronald L. Bradow
The major reason for this paper is to summarize for the health scientists
among us some of the technical factors and reasoning underlying the samples so
many of us in automotive research have been generating. In virtually all
cases where material has been collected for biological testing, procedures
used in generating samples have been well-accepted engineering practices
simulating the road performance of vehicles,the emission of exhaust into
ambient air, and recovery of particles or gaseous organics on collecting media
as inert as possible to chemical changes.
In large part, the conditions were selected before any artifact problems were
known because of the time constraints in the early part of the EPA work on
diesel particles. As more becomes known about the chemical makeup of diesel
exhaust particles and organics, the potential artifacts in collection become
simpler to hypothesize. Still experiments conducted so far suggest that these
are not a major problem for samples collected in the past.
There are a number of vehicle categories which have significantly different
uses in transporting people and things. Because passenger cars, trucks and
buses are operated under different conditions of speed and load and pollutant
emission characteristics for each type of vehicle vary often dramatically,
with operating conditions, a variety of testing procedures have been deve-
loped. These attempt to simulate, while a vehicle is stationary, those forces
which would have been experienced had the vehicle been moving down a roadway.
With passenger cars, the standard way of achieving this simulation uses a twin
roll chassis dynamometer. Mechanisms are available to simulate the aerodyna-
mic drag, tire rolling resistance and inertial forces on a vehicle, all as a
function of vehicle speed. The nature of these simulations with the commonly
used dynamometer types are shown.
A variety of driving schedules are currently available for use in passenger
car testing. Since the dynamometer set up takes care of the resistive force
simulation, the driving schedules are merely speed-time listing or charts
which can be readily driven repeatably. The most commonly used of these
schedules is the Federal Test Procedure urban driving schedule. This route
-------
simulates a home-to-work commuter route in Los Angeles and in fact, the route
actually exists in that city. Other simulations for very slow central
business district driving, crowded urban expressways, and two-lane country
highways are also available for use. Literature values for particle and
organic emission rates are available for a variety of gasoline and passenger
car types and these are presented in the usual form, mass per unit distance
driven. This form is commonly used because the mass emission rate of the air
pollutant is compared in this ratio to an index of social value, the distance
driven.
There are alternative means of expressing emission results, though, and of
these, fuel-specific emission rates are the most revealing because vehicle
size tends to be normalized in this approach. Some emission rates of diesel
trucks, buses and cars are fairly close when fuel consumption is normalized.
Literature values of diesel and gasoline truck emissions of particles and of
organic matter are compared on this basis.
The compliance testing of trucks and buses is somewhat controversial right now
and, apparently, there is not wide-spread agreement on the propriety of
testing measures. Usually, just the engines and not whole vehicles are tested
and the simulation with respect to road performance is somewhat more difficult
to achieve. Further, the index of social value used to compare emissions is
different from that us'ed for passenger cars. In this case, emissions are
normalized by the useful work done and emissions are expressed in mass/unit
work (grams/kw-hr. or grams/hp-hr). Emissions expressed on this basis are
somewhat difficult to compare with conventional passenger car emission rates
and usually one resorts to fuel-specific comparisions.
However, it is possible, for research purposes, to test trucks on a chassis
dynamometer in road simulations for central business district, arterial
roadway, and expressway driving routes, just as passenger cars are tested.
Here, all the same problems of load simulation and representational character
of the driving schedule are encountered. Since this procedure is fairly new,
there are still some uncertainties associated with its use. One experimental
program has reported some results with such a procedure, however, and these
results are summarized in comparison with engine dynamometer and passenger
car data.
The emissions experienced on these schedules are conventionally measured using
constant volume sampling procedures. These sampling methods seek to accommo-
date two important test requirements. One is to provide an integrated por-
tional sample of auto exhaust and the other is to achieve some simulation of
the air-dilution process which occurs when the exhaust enters ambient air.
This process is important because the organic substances are partitioned by
this process; some enter the condensed or particle phase on cooling while
others remain gaseous.
The physical chemistry of this process has recently been an active topic of
research and current results of static and dynamic experiments are presented.
The absorption of organic substances by fine carbon particles is an important
key to understanding these processes and results from the work of Kittleson,
-------
Ross, Black and Pierson are discussed along with a model presented by McDon-
ald. It appears that both temperature and dilution ratio can have some effect
on the amount of organic material in the condensed phase. However, no serious
objection to the commonly used dilution tube procedure seems to be in evi-
dence. The contributions of diesel vehicles both to airborne carbon and
particulate organics appear to be important.
It is possible to separate the gas and particle phase material by several
methods and some comparison data for several methods is reported. To insure
that only a physical separation is made and not some recovery of gaseous
material has been no small task. While many of the currently used procedures
involve some art and guess-work, they do appear generally adequate for the
task of sampling. Equipment currently in use is described.
Recovery of heavier gaseous organics for biological sampling has been a more
serious problem. Condensation procedures used by Grimmer and by Lofroth in
Europe and by Gross in the United States are quite different in philosophy
from the air dilution procedures used in vehicle sampling. Results from a
variety of these procedures are compared and equipment for recovery of large
amounts of these organics is described.
These collection methods have some potential for artifactual generation of
biologically active material, particularly with diesel exhaust. Some preli-
minary results from experiments with N02 addition to diluted exhaust and
results from correlations of bioactivity with N02 level from the work of
Leddy and Johnson are compared. It appears that N02 levels below about 5ppm
to not constitute a serious threat to diesel particle organic integrity but
higher levels, particularly those above about 20 ppm, cause serious artifacts.
For this reason, a reasonably high dilution ratio is needed to avoid artifac-
tual generation of nitroaromatics. It appears that none of the diesel samples
generated thus far has been seriously jeopardized by this effect, but caution
might be warranted in interpreting condensation experiment data from diesel
vehicles.
Gasoline-fueled cars seem to have very low levels of N02 and no major
problems within nitroaromatic formation are known.
Thus, a reasonably useful set of engineering practices and equipment exists to
capture samples of both particle and gaseous organic materials. While there
could still be some improvements, it appears that estimates of biological
activity and chemical composition using existing samples are reasonably
correct.
-------
DIESEL PARTICULATE EMISSIONS: COMPOSITION,
CONCENTRATION, AND CONTROL
by
Ronald L. Williams
Environmental Science Department
General Motors Research Laboratories
Warren, Michigan
The application of new techniques and approaches to study diesel
emissions has increased our understanding of the formation, atmospheric
impact, and health significance of diesel particulate emissions. This
paper will review recent work on the composition of diesel particulate
and compare it with particulate from other combustion sources. Then,
estimates of the current and projected concentration of diesel particulate
in urban areas will be discussed. Finally, the limited information
available on the effects of experimental particulate-control systems
will be presented.
COMPOSITION
The size distribution of diesel particles has been studied by a
variety of instruments, including, electron microscopes, electrical
aerosol analyzers, and inertial impactors. While the mass median
diameter of diesel particulate is a few tenths of a micrometer, the
number median diameter is considerably smaller. Therefore, on a frequency
basis, the particles deposited in animal lungs will be dominated by the
smaller particles.
Despite the application of the best analytical methods, no feature of
diesel particulate has been identified which clearly distinguishes it
from particulate emitted by gasoline-burning engines or from particulate
emitted by other combustion sources. Figure 1 shows the relative amounts
of organic and elemental carbon in combustion particulate from several
different sources (1,2). The ratio for each source type appears to
depend on the air-to-fuel ratio during combustion. Measurements of the
polynuclear aromatic compounds in the organic material likewise fail to
show source-specific components of the particulate.
CONCENTRATION
Since diesel vehicles emit larger amounts of particulate carbon on
a per mile basis than gasoline vehicles, it is important to project the
increase in the ambient concentration of particulate carbon from expanded
use of diesel-powered cars and trucks. Estimates of the current input
of diesel particulate to the nation's atmosphere range from 80 to 120 x
109 grams per year. Ambient measurements show that diesel particulate
currently accounts for about 25% of the elemental carbon in urban air (3),
or 1 to 2 micrograms per cubic meter in most major United States cities.
-------
Estimates of diesel particulate concentrations for the future
depend on the total amount of diesel particulate emitted and on the
distribution of diesel vehicles geographically. Particulate emissions
from light-duty diesels result in proportionately larger increases in
urban particulate concentrations than do particulate emissions from
heavy-duty diesels which accumulate a smaller percentage of their mileage
in urban areas. We estimate that 6 to 9 million light-duty diesels with
an average emission rate of 0.6 g/mile would contribute an additional 1
to 2 micrograms per cubic meter of diesel particulate to the air of most
major United States cities.
CONTROL
The prospect for increased use of diesel engines has stimulated
efforts to develop new technologies for reducing diesel particulate
emissions. Two different control approaches have been examined to
determine the effects on the composition of the particulate as well as
on the total quantity. Because the health effects studies being conducted
predate the availability of particulate-control systems, it is important
to make a preliminary assessment of the composition changes, even though
these systems are still experimental.
One of the particulate-control systems lowered the FTP total particu-
late to 160 milligrams per mile (0.16 g/mile). With this system organic
carbon was 3 to 8 milligrams per mile and benzo(a)pyrene emissions were
more than 90% lower than normally emitted by diesels. However, enhanced
sulfate emissions occurred in higher-speed driving cycles, apparently
due to the oxidation of sulfur dioxide by the control system. During
regeneration of the particle trap of this system, a visible white cloud
of sulfate was emitted.
A second particulate-control system also reduced the FTP total
particulate substantially. More than 99% of the elemental carbon was
removed from the exhaust. Organic carbon emissions were 10 to 30 milli-
grams per mile and benzo(a)pyrene emissions were more than 90% lower
than normally emitted by diesels. Higher-speed driving cycles and
regeneration of the particulate trap in this case gave similarly reduced
emission rates of organic carbon and benzo(a)pyrene.
Diesel particulate is currently the subject of intense chemical and
biological study. While typical diesel particulate is not distinguishable
from particulate from other combustion sources, the limited evidence
available indicates that particulate-control systems may markedly
change the composition of diesel particulate in the future.
-------
REFERENCES
1. Muhlbaier, J. L. and R. L. Williams. 1981. Fireplaces, furnaces, and
vehicles as emission sources of particulate carbon. In: Particulate
Carbon: Atmospheric Life Cycle. G. T. Wolff and R. L. Klimisch, eds.
Plenum Press; New York.
2. Cadle, S. H. and P. J. Groblicki. 1981. An evaluation of methods for
the determination of organic and elemental carbon in particulate samples.
Ibid.
3. Wolff, G. T., P. J. Groblicki, S. H. Cadle, and R. J. Countess. 1981.
Particulate carbon at various locations in the United States. Ibid.
Furnace
Normal
Rich
Fireplace
Hardwood
Softwood
Synthetic
Automobiles
Pre-Catalytt Detroit
Pre-Catalytt Denver
Catalyst Detroit
Catalytt Denver
Diesel Detroit
Dieael Denver
0.2
0.4 0.6
Ce/Cr
0.8
Figure 1. The ratio of elemental carbon to total carbon from selected sources.
-------
PARTICULATE EMISSIONS FROM SPARK-IGNITION ENGINES
by
Ted M. Naman
D. E. Seizinger
U.S. Department of Energy
Bartlesville Energy Technology Center
Bartlesville, Oklahoma
Charles R. Clark
Inhalation and Toxicology Research Institute
Albuquerque, New Mexico
ABSTRACT
Experiments were conducted at the U.S. Department of Energy's Bartlesville
(Okla.) Energy Technology Center to quantify particulate and gaseous emis-
sions from current-production vehicles equipped with spark-ignition engines,
to determine the influence of fuel and ambient temperature on particulate
emissions, and to characterize particulates in terms of their carbon
content, soluble organic fractions, and biological activity.
Four 1980-81 model-year vehicles equipped with oxidation and three-way
catalysts and spark-ignition engines ranging from 1.6 liter, 4-cylinder to
4.3 liter, V8 (see table 1) were tested on a climate-controlled chassis
dynamometer using the driving cycles of the 1975 Federal Test Procedure.
The vehicles were operated at 20°, 50°, 75°, and 100° F (-7°, 10°, 24°, and
38° C, respectively) ambients on gasoline and at 75° F ambient on four fuel
blends: 90 percent gasoline/10 percent ethanol, 90 percent gasoline/10 per-
cent methanol, 93 percent gasoline/7 percent methyl tertiary butyl ether,
and a commercial gasohol.
Particulate matter was collected on 40- by 40-inch filters using the total
volume of the exhaust, and on conventional 47 mm filters using a sampling
probe and a portion of the exhaust. Bioassays of dichloromethane extracts
of the samples were carried out using the Salmonella mutagenicity (Ames)
test at the Lovelace Inhalation Toxicology Research Institute.
The results from the Federal Test Procedure (figure 1) showed a significant
reduction in particulate emissions with the alcohol/gasoline fuel blends
when compared to gasoline alone. The methyl tertiary butyl ether/gasoline
fuel blend showed a slight reduction in particulate emissions. The carbon
monoxide emissions were slightly reduced with the alcohol/gasoline fuel
blends. Hydrocarbon emissions remained relatively unchanged, oxides of
nitrogen emissions were slightly increased, and fuel economy was 2 to 3
percent lower (data not shown). Ambient temperature seemed to have a slight
effect on particulate emissions. Overall, particulates emitted from the
vehicles with spark-ignition engines were 90 to 100 times lower than partic-
ulates emitted from current-production diesel vehicles.
-------
Samples of participate extracts from vehicles fueled with gasoline and
gasohol were separated using a silica column. The results obtained from the
separation showed a higher percentage of polar compounds in the gasohol than
in the gasoline samples (figure 2).
Benzo(a)pyrene and nitropyrene levels in the gasohol particulate extracts
were at least 50 percent lower than the measured levels in particulate
extracts from gasoline-fueled vehicles.
Dichloromethane extracts of the particulate exhaust from vehicles operating
on gasoline were evaluated in Salmonella strain TA-100, with and without
the addition of a liver enzyme preparation (S-9) as a source of metabolic
enzymes. Extracts of particulate exhaust from the four vehicles operated
on gasoline and/or on alcohol/gasoline fuel blends produced direct, dose-
related increases in mutagenicity (see table 2). The addition of S-9
either decreased or did not alter the observed direct mutagenicity (data not
shown).
The addition of 10 percent ethanol to gasoline either decreased or did not
significantly change the mutagenicity of the resultant exhaust particulate
extracts (table 2). Operation of the Mercury Monarch on the methanol blend
increased the mutagenicity of the exhaust particulate extracts but decreased
mutagenicity in the Chevrolet Citation. Commercially available gasohol
resulted in particulate extracts that were less mutagenic in the Ford Escort
but not significantly different in the other cars.
The mutagenic potencies of the extracts do not reflect differences among
cars in the mass of mutagenic material associated with the particulate emis-
sion rates. Therefore, the mutagenicity data were normalized so that com-
parisons of the amount of mutagenicity could be made among cars. This was
done by dividing the mass of dichloromethane extractable material from each
filter by the number of miles of vehicle operation (26), to yield mg/mile of
particulate associated organic material. This number was multiplied by
revertants per microgram (ug) to estimate the amount of mutagenicity emitted
from each car (revertants per mile).
When compared to gasoline, the addition of 10 percent ethanol or methanol to
gasoline, or operating the vehicles on the commercial gasohol, reduced the
mass emission rate (mg/mi) of organic materials associated with the particu-
lates. This resulted in significant reductions in revertants per mile for
all of the alcohol fuel blends tested (table 2).
-------
Table 1. Test vehicles
Ford
Escort
Oldsmobile
Cutlass
Chevrolet
Citation
Mercury
Monarch
Engine displacement,
CID (liters)
Carburetion
Compression ratio
Transmission
Emission Control System:
EGR
Ai r pump
Air injection
Oxidation catalyst
Three-way catalyst
Charcoal canister
Axle ratio
Inertia weight, Ib
Actual dyno load, hp
98 (1.6) 263 (4.3) 151 (2.5) 250 (4.1)
2 bbl
8.8
Manual
4-spd
Yes
Yes
No
No
Yes
Yes
3.59
2375
6.4
2 bbl
7.5
Auto
Yes
Yes
No
No
Yes
Yes
2.29
3750
11.5
2 bbl
8.2
Auto
Yes
No
Yes
Yes
No
Yes
2.84
2875
6.6
1 bbl
8.6
Auto
Yes
Yes
No
Yes
No
Yes
2.79
3625
11.1
-------
Table 2. Influence of alcohol fuel blends on mutagenicity of
spark-ignition engine exhaust particulate extracts
Vehicle Revertants/ug
and Extract
Fuel TA-100
Ford Escort
Gasoline
Ethanol blend
Commercial gasohol
Oldsmobile Cutlass
Gasoline
Ethanol blend
Commercial gasohol
Chevrolet Citation
Gasoline
Ethanol blend
Methanol blend
Commercial gasohol
Mercury Monarch
Gasoline
Ethanol blend
Methanol blend
Commercial gasohol
10
9
4
10
5
13
17
14
11
10
16
12
26
20
Emission of
Particulate Associated
Organic Material Revertants
(mg/mi) per Mile
1.5
1.1
1.2
1.7
0.6
0.6
1.9
0.9
0.8
1.0
7.1
2.8
3.3
2.2
15,000
9,900
4,800
17,000
3,000
7,800
32,300
12,600
8,800
10,000
114,000
34,000
86,000
44,000
Slope of linear portion of dose-response curve, without S-9.
Research performed in part under U.S. Department of Energy Contract Number
DE-AC04-76EV01013.
-------
40
'e
1 30
cn~
z
o
in
i 20
UJ
Z>
o
Gasoline Gasoline Commercial Gasoline Gasoline
+ 10% gasohol +10% t7%
EtOH MeOH MTBE
Figure 1. Influence of fuel extenders
on particulate emissions.
70
60 -
50 -
40 -
30
o
K 20
10
W-K
EH3 Gasoline
Gasohol
MECL2 MEOH
NR
TEMPERATURE, 75°F
Figure 2. Particulate extracts from vehicles
operating on gasoline and gasohol.
-------
PARTICULATE EMISSION CHARACTERIZATION STUDIES
OF
IN-USE DIESEL AUTOMOBILES
by
Richard Gibbs, James Hyde, and Robert Whitby
Division of Air
New York State Department of Environmental Conservation
Albany, New York
A sample of 20 in-use diesel automobiles has been repeatedly tested over
a two-year period to accumulate emissions characterization data with major
emphasis on particulate. Each vehicle test included replicate sample col-
lection for "as received" and "control fuel, control oil" vehicle conditions.
Driving cycles tested at each vehicle condition included: FTP, CFDS (CUE),
HFET, 50 mph cruise, NYCC, and I'dle. Measurements in each test cycle included
gaseous emissions, fuel economy, and particulate emissions; also, individual
particulate samples were collected for each individual test cycle in suffi-
cient quantity for subsequent chemical and bioassay analyses. These particu-
late samples were quantified for soluble organic fraction (SOF) and Ames test
direct-acting mutagenic response by TA98 (-). These parameters are examined
for 60 vehicle tests to indicate particulate character effects for vehicle
types and test cycles. Results will be presented for three vehicles in the
sample group which have been tested over sufficient mileage accumulation
intervals to provide limited insight into vehicle aging effects.
Table I gives average values of the mutagenic activity of the SOF
directly. In general, FTP samples exhibit a higher specific activity than
samples from the same vehicle for other driving cycles. Expressed as a per-
cent of total particulate, however, the SOF (7o) is generally less for the
FTP than other cycles as seen in Table II.
When SOF mutagenic response and SOF (%) data are combined to express
mutagenic response on a particulate mass basis, these differences are mostly
indiscernable as shown in Table III.
From Table III, with some exceptions, it can be seen that the bio-activi-
ty on a total particulate basis is quite uniform within a vehicle type. This
is in spite of rather large variations in activity among the various vehicles
tested to generate the averages. Thus, we conclude that, as a first
-------
approximation, for a given vehicle test a "gram of particulate is a gram of
particulate" regardless of driving cycle. When variations in vehicle partic-
ulate emission rate per distance travelled are incorporated to the data of
Table III, the per-mile emission of bio-active material can be calculated as
given in Table IV.
Since all test cycles except the FTP begin with a warm vehicle, the
foregoing conclusions may not include parameters related to vehicle start-up.
A battery of tests were performed on a VW diesel in the Winter of 1981 to
preliminarily investigate cold-start effects on particulate, SOF, and muta-
genic activity. Overnight vehicle soak at laboratory and outdoor ambient
conditions were followed by: FTP, Bag I, 10 min. pause, FTP, Bag III to
give separate particulate samples for each bag at each condition. The cold-
ambient tests were repeated, and average values from two runs are reported.
Continuous temperature recordings of ambient temperature, crankcase lubri-
cating oil, and fuel temperature (between pump and injectors) were obtained.
When results for the normal FTP Bag III were used as a basis for comparison,
the other three test conditions yield ratios as given in Table V.
Comparison of the 0°C Bag I results to the 20°C base condition showed a
74% increase in particulate emission, 11% increase in SOF emission corres-
ponding to a 36% decrease in SOF expressed as a percentage of particulate.
SOF mutagenic response was increased by a factor of 3.6 and 4.0 when expres-
sed as revertants/mile. These results are in general agreement with those
presented above in comparison of FTP and other driving cycles for the in-use
sample group.
GM
VW
MB
Table I. Mutagenic Activity Per Microgram SOF
(Revertants/^g SOF)
FTP
HFET 50mph Cruise CFDS NYCC IDLE Sample n
3.8
11.3
4.8
2.4
10.9
4.4
2.1
8.1
3.5
2.7
11.0
5.9
1.4
16.8
1.8
2.4
3.4
2.7
57
36
18
FTP
HFET
Table II. SOF
50mph Cruise CFDS NYCC
IDLE Sample n
GM 25.1 34.1 39.3 31.2 31.7 24.6 57
VW 20.0 21.5 20.7 22.7 33.4 55.0 36
MB 14.0 13.6 15.2 14.6 14.8 14.6 18
-------
Table III. Mutagenic Activity Per Microgram Particulate
(Revertants/ug Particulate)
FTP HFET 50mph Cruise CFDS NYCC IDLE Sample n
GM 0.77 0.75 0.72
VW 1.95 2.06 1.53
MB 0.53 0.45 0.40
0.74 0.36 0.43 57
2.26 5.82 1.94 36
0.68 0.27 0.39 18
Table IV. Mutagenic Activity Per Vehicle Mile
(105 Revertants/Mile)
*
FTP HFET 50mph Cruise CFDS NYCC IDLE Sample n
GM
VW
MB
6.5
6.9
2.8
2.7
6.1
1.7
2.2
4.4
1.3
3.8
7.4
3.0
8.6
27.0
6.2
0.74
0.47
0.17
57
36
18
105 Revertants/Minute
Table V. VW Cold Start Particulate Comparisons
Vehicle Test Condition
Base Condition =
(FTP Bag III)
Mean Temoeratures °C
Overnight
Soak
20
Injector
Fuel Line
25
Note: Results below shaded areas are ratios
to base condition
Bag III after ambient
Cold Soak Bag I
Normal FTP
Bag I
Cold Ambient Soak
FTP Bag I
0
20
0
18
21
5
Crankcase
Lube
90
98
48
38
Particulate
-------
CHARACTERIZATION AND OXIDATION
OF DIESEL PARTICULATE
by
David A. Trayser and Louis J. Hillenbrand
Battelle-Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
INTRODUCTION
A study was recently completed by Battelle-Columbus Laboratories
for the U.S. Environmental Protection Agency to evaluate emissions control
on light-duty diesel vehicles by postcylinder oxidation. The primary objec-
tive of this program was to determine the feasibility of thermal or
catalytic oxidation as a means of diesel particulate emissions control.
Two major efforts in this program involved characterization of the
particulate for chemical and physical parameters relating to ignition and
oxidation, and development of a catalytic ignition concept for reducing the
ignition temperature of the particulate in the exhaust system.
CHARACTERIZATION OF THE PARTICULATE
Particulate characteristics were measured using samples collected
directly from the surface of the exhaust pipe and samples collected by filter
from a dilution system.
Physical Properties
The particulate physical properties included surface area, size
distribution, and mass concentration. These were measured using dilution
system samples, hence, representing the particulate after entering the atmos-
phere. The surface area was approximately 100 m^/g, the mass median particle
diameter was in the range of 0.1 to 0.3 ym and was observed to increase in
size as engine speed and load increased, and the mass concentration in the
engine tail pipe was observed also to vary with engine speed and load. Mass
concentrations of particulate in the exhaust gas varied from 20 mg/m3 at the
lowest speed and lightest load to 500 mg/m3 at maximum speed and load. These
values translate to 0.1 to 3.8 grams per mile, respectively, a fairly "dirty"
exhaust for an automotive diesel (and not representative of current diesel
engines).
-------
Chemical Properties
The chemical properties were measured using the exhaust-pipe
particulate samples and included soluble organics (using toluene), carbon,
hydrogen, oxygen, ash, and trace minerals. The soluble organics ranged from
2 to 10 percent, with no evident correlation with engine speed/load condi-
tions. By comparison, the soluble organic content measured in particulate
collected in the dilution system, for the same range of engine speed and load
conditions, varied from 3 to 25 percent, increasing as speed and load decreased.
Carbon content ranged between 73 and 93 percent, hydrogen content
varied from 0.5 to 1.8 percent, and ash content ranged from 0.1 to 2.2 per-
cent. Again, no correlation with engine conditions was noted for any of
these parameters. About 7 percent oxygen was found in two particulate
samples, and 23 percent in a third sample. Part of the oxygen is present in
the particulate as $04 and part may represent partial oxidation (to C02,
perhaps) but with the products still bound in the particulate.
The trace mineral analyses revealed considerable calcium, iron,
phosphorus, and magnesium in the particulate. Similar analyses of samples
of the fuel and lube oil indicated that the lube oil was most likely the
source of these elements as well as of chromium, copper, manganese, and lead,
found in lesser quantities in the particulate. These results imply that a
substantial portion of the particulate may derive from the lube oil.
Ignition Properties
A Differential Thermal Analyzer technique was used to measure
ignition temperatures and maximum oxidation temperatures for the particulate
samples obtained in the engine exhaust pipe. The mean ignition temperature
for the samples evaluated was 594 C, with a variation range of 583 to 604 C.
The only significant correlations with engine operating conditions and with
physical and chemical properties appeared to be with the presence of hydro-
carbons in the exhaust and with aluminum and lead in the particulate. Lower
ignition temperatures occurred with higher exhaust gas hydrocarbons and
higher particulate aluminum and lead.
More significant changes in ignition temperature were observed when
the bulk density and the amount of the particulate sample in the DTA were
altered. Increasing the sample size and the bulk density by factors of 10
resulted in a decrease in ignition temperature of about 150 C. This result
suggests that if a method could be devised for compacting the particulate
in a trap, it would be more easily ignited under normal exhaust-gas conditions.
CATALYTIC IGNITION
In this study the catalytic ignition of diesel particulate was
initially developed in bench-scale experiments. Final experiments were
conducted in the exhaust system of an engine to verify the applicability of
the concept to the actual engine environment.
-------
Bench-Scale Experiments
The bench-scale experiments were carried out using a hot-tube
reactor in which small samples of the diesel particulate were subjected to
a gradually increasing temperature until ignition and oxidation occurred.
Experiments were conducted to identify potentially catalytic materials, to
explore methods of catalyst application, and to determine the magnitude of
the catalytic effect in relation to the amount of catalytic material used.
Metal salts such as copper chloride, manganese chloride, and cobalt
chloride were found to be capable of reducing the ignition temperature of the
particulate by as much as 200 C. The addition of sodium or ammonium salts
to the metal salt reduced the ignition temperature another 50 C, for a total
reduction of 250 C (resulting in an ignition temperature of about 350 C for
the catalyzed particulate). For most of this work, copper chloride equiva-
lent to 7.5 mg Cu/g soot was used to achieve the catalyzed ignition.
Exhaust System Experiments
Final experiments were conducted in the exhaust pipe of a production
diesel engine, where the results of the bench-scale experiments were confirmed.
Various particulate trap materials and configurations were tried, including
quartz wool, stainless steel wool, foamed alumina, and porous ceramic honey-
comb. The porous ceramic honeycomb traps proved to be the most suitable,
providing high surface area in a small volume, good collection efficiency at
low pressure loss, and tolerance to the high temperatures reached during
oxidation of the particulate.
Successful regeneration of the trap in the engine exhaust was
achieved by intermittent injection of a water solution of the copper chloride/
sodium chloride catalyst mixture into the exhaust stream immediately upstream
of the trap, after a period of particulate collection on the trap. The
actual ignition temperature in these tests ranged from 350 to 400 C. The
amount of soot burned in each of these final trials is unknown but the copper
concentration is believed to have been somewhat greater than used in most
of the bench-scale tests.
CONCLUSIONS
The concept of using a catalyst material introduced into the exhaust
of a diesel engine in a manner which allows the catalyst to associate with
particulate collected on a trap has been successfully demonstrated. The
catalyst acts to reduce the ignition temperature of the trapped particulate
by about 250 C, which results in particulate burnoff and trap regeneration
at significantly lower exhaust gas temperatures than would be required
otherwise. This concept could provide more highly controlled burnoff of the
trapped particulate, leading to lower peak oxidation temperatures in the
trap (promoting longer trap life and improved trap reliability) and to
minimization of the danger of unscheduled trap burnoff as a fire hazard.
Practical application of this concept requires development of suit-
able hardware and identification of the optimum operation cycle for the trap-
oxidizer/catalyst injection system. The enclosed figure illustrates, in
simplified form, a particulate control system that might be developed based
on the metal-salt-catalyst concept briefly described in this paper.
-------
EXHAUST
MANIFOLD-\
ENGINE
PARTICULATE
TRAP
CATALYST-7 TRANSFER * CATALYST
INJECTOR LINE RESERVOIR
PARTICULATE
TRAP
EXHAUST
PIPE
NOZZLE
TRANSFER
rLINE
VALVE/
^SOLENOID
DIESEL ENGINE PARTICULATE CONTROL SYSTEM
BASED ON BATTELLE METAL-SALT-CATALYST
CONCEPT
-------
HEAVY-DUTY DIESEL ENGINE EMISSIONS SOME EFFECTS OF CONTROL TECHNOLOGY
by
J.M. Perez and R.V. Rower
Research Department
Caterpillar Tractor Co.
Peoria,. Illinois
Use of various control technology methods to reduce specific emissions
such as NOX or particulates usually result in changes to other constituents in
the exhaust of diesel engines. The effects of control technology on EPA
Advisory Circular No. 76 emissions are reported. Unregulated emission
trade-offs as a result of timing, EGR, catalysts, and engine modifications are
discussed. Fuel consumption increased with most changes.
Although the emission levels are changed as a result of the control
technology, the emissions pose no obvious health risk based on estimated
exposure levels and available health effects data.
Table 1. Engine Change Tradeoffs
f = Increase J = Decrease - = No Change
Change
PC^OI
EGR
Timing
Advance
Retard
Afterc ooling
Injector SAC
Volume Increase
Catalyst*
Fuel
(BaP Increase)
Partic
Total
t
t
ti
tl
tl
t
tl
ulates
SEF
tl
1
_t
t
tl
t
tl
1
HC
tl
1
~
tl
u
1
NO*
t
1
t
1
11
t
1
ALD
tl
|
tl
Jt
1
t
1
BAP
t
1
t
t
1
t
1
1
T
Fuel
Cons
1
t
*-*
t
tl
-*
*J
Power
~
n
I
t
~^
S0
NH3 f
-------
METHODOLOGY OF FRACTIONATION AND PARTITION OF
DIESEL EXHAUST PARTICULATE SAMPLES
by
Bruce A. Petersen
Battel1e-Columbus Laboratori es
Columbus, Ohio
Diesel exhaust is a complex mixture of carbonaceous matter and gaseous
compounds. As the exhaust is cooled, the gases condense or adsorb onto
the solid particles. Because of the complex nature of diesel exhaust parti-
culate, fractionation of the diesel exhaust extract into different compound
classes is required before identification of individual components.
A fractionation method was developed by Battelle in 1978 to separate the
chemical classes in diesel particulate extract samples. Several refinements
in the original procedure have been made, and considerable experience in its
use has been obtained on a wide variety of particulate sample types. There
are two primary advantages associated with this procedure which are pertinent
to the analysis of the organic material extracted from particulate samples:
It is sensitive enough to fractionate small quantities of
extracted mass. Particulate extracts containing 1 to 4 mg
of total extracted mass have been sufficient for compound
class separation and chemical analysis
It can be conveniently scaled up for work with much larger
samples. Particulate extracts containing several grams of
extracted mass have been successfully fractionated without
significant loss of material.
The procedure has already been used to separate about 800 diesel particulate
filter extracts into six specific compound classes, without significant loss
of material. The range of total organic mass was 1 to 143 mg. Recovery.of
extracted mass through this procedure ranged from 75 to 104 percent.
This procedure has also been scaled up in order to separate gram quantities
of extracts from carbon black, urban air particulate, and wood stove emissions,
Typical recovery of total mass using the scaled-up procedure has ranged from
90 to 101 percent.
-------
THE FRACTIONATION PROCEDURE
Using the Battelle fractionation method, the particulate extract is separated
into acidic, basic, and neutral components. Acids and bases are first
separated by liquid-liquid partitioning. The neutrals are further parti-
tioned by means of silica gel column chromatography. A schematic representa-
tion of the entire fractionation procedure is shown in Figure 1, and the
generated fractions are listed in Table 1. For purposes of discussion, the
fractions are referred to numerically as #1, #2, etc. In this discussion,
the method describes the procedure to fractionate approximately 50 mg of
extract.
The solvent extract of the particulate samples is first concentrated to 50 ml
and an aliquot of 10-100 yl is removed to measure the organic mass in the
extract before fractionation into the compound classes. In the fractionating
scheme, the bases are first separated from the extract by liquid- liquid
partition with a 5 percent sulfuric acid solution. Then, the acid and
phenolic compounds are extracted from the organic solution with a 5 percent
sodium hydroxide solution. Both the basic and acidic fractions are back
extracted with methylene chloride. The remaining organic solution containing
the neutral compounds is further partitioned into four fractions by open
column chromatography on 5 percent ^-deactivated silica gel.
The silica gel columns are packed with 20 g of the silica gel in a hexane
slurry. An additional column is also prepared to check the accuracy of the
silica gel deactivation before partitioning the neutral organic solution.
This is done by measuring the volume of hexane required to elute 500 ng of
anthracene. The migration of the anthracene is monitored by a 366-nm UV
lamp, and the volume of hexane is measured during the migration. When the
silica gel is deactivated 5 percent, anthracene starts to elute from the
column after the addition of 140 +_ 10 ml of hexane. For 3 percent and 7
percent deactivation, the volume of hexane required is 270 + 12 ml and 115 +
8 ml , respectively.
Upon assurance that the silica gel is 5 percent HzO deactivated, the neutral
organic compounds are further fractionated. Four elution solvents are used.
They are applied to the column and the eluent is collected in the following
sequence: 60 ml hexane, 100 ml hexane/benzene (1:1), 100 ml methylene
chloride, and finally, 200 ml methanol. The collected fractions correspond
to fraction numbers 3, 4, 5 and 6.
Alkanes and alkenes are present in the aliphatic fraction (#3). The PAH
compounds, nitrogen heterocycles and mono-nitrated PAH are presented in the
aromatic fraction (#4). Polynitrated PAH, sulfur heterocycles, and oxygenated
2 and 3 rings PAH are found in the moderately polar fraction (#5). The
highly polar fraction (#6) typically contains PAH with more than one function-
al group, oxygenated with more than 3 rings.
In this presentation, methods for fractionation of diesel exhaust particulate
samples are reviewed and Battelle-developed methods are discussed. Experi-
mental results using Battelle methods from two light-duty diesel engines and
-------
one heavy duty diesel engine are presented. Data will be given to illustrate
the quantity of mass in each fraction as well as the material balance for
each particulate sample extract. Identification of selected compounds such
as the nitro-PAH within the various fractions will also be presented.
Table 1. Compound Classes Generated by Fractionating Scheme
Fraction Fraction #
Bases 1
Acids and Phenols 2
Aliphatic Hydrocarbons 3
Aromatic Hydrocarbons 4
Moderately Polar Neutrals 5
Highly Polar Neutrals 6
-------
SoitiM EKUKI In 700 m)
solvent for 16 hours,
Concwitnt* Mtrftct to
6O ml by roury voporation
Evoportto down to 1 n
Add OMSO la 6 ml
EoapoitM lo 1 ml
Add OMSO to ml
Amaa Mutag*na*is Bioaaaay
Dry with nhydreut NO4O*
««ponl« down to 1 ml
ddhutiwtolOrm
MBftorat* down lo 1 ml
ddh«i»Mto 10ml
vapoiat* down to 1 ml
lllc* O*l Column Chronwiog
Eluvnt. (Volume)
Sampte Coda
. 1:1 (lOOrnQ
M«thv**M Chlottd* (tOO mq
Ew*pof«t* MCA F>«ction to 6 ml
110O iA tot rMidu* wM«ht
Maaaura Organic Mau in Fractions 9-6
Evcportl* Mch Fraction to 1 ml
Add OMSO loft mt
Evaporai* oach Fraction to 1 ml
Add OMSO to B M
Amas Mutagansit Bioassay on Fractions 3-6
Ev«po>M«ia1 ml
Add OMSO 10 B ml
EwBpoiat* u I ml
Add OMSO to ml
Arnaa Mut*g«o*aii Bioassay on Fraction 2
FIGURE 1. EXTRACTION AND FRACTIONATION PROCEDURE
-------
THE UTILITY OF BACTERIAL MUTAGENESIS TESTING IN THE
CHARACTERIZATION OF MOBILE SOURCE EMISSIONS: A REVIEW
by
Larry D. Claxton
Genetic Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
Since humans are and may be increasingly exposed to whole diesel
exhaust, it is important to identify, characterize, and evaluate the
biological activity of exhaust materials associated with mobile source
emissions. It is also important to understand the effects of factors such
as fuel, driving cycles, engine modifications, etc. that modify the emission
products. The purpose of this paper is to review the use of bacterial
mutagenicity testing in gathering information about the organics associated
with mobile source emissions. The mutagenic activity of organic extracts
from both diesel and gasoline exhaust particles was first established with
the Salmonella typhimurium plate incorporation assay. Efforts to date
demonstrate that mutagenic compounds condense upon and adhere to the central
carbonaceous core of mobile source (diesel and gasoline engine) particles.
In addition, bacterial mutagenesis work demonstrated that a S9 activating
system containing the mixed function oxidase enzyme system was not needed
in order to provide a mutagenic response. The response of the various
tester strains provide evidence that the mutagens are of the frameshift
type. By integrating microbial tests with chemical fractionation procedures,
the more biologically active chemical fractions (e.g., polar neutrals) have
been identified. This, in turn, has led to the identification of specific
mutagens within diesel exhaust organics such as 1-nitropyrene. Bacterial
mutagenicity studies have aided in demonstrating that the mutagens are
removed from the carbonaceous particles and become protein-bound when
incubated with physiological fluids. By exposing diesel exhaust to various
ambient-like conditions within a smog chamber and comparing the resulting
mutagenic activity, one study demonstrated an alteration of mutagenic
activity by ambient levels of ozone. Although bacterial mutagenesis studies
provide exceptional insight into the nature of the genotoxic activity of
mobile source organics, caution must also be observed in interpreting the
data since differences between bacterial and mammalian cells exist with
respect to metabolic activation, cell permeability, and particulate
processing.
-------
Emission Factors from Diesel and Gasoline Powered Vehicles;
Correlation with the Ames Test
by
Roy B. Zweidinger
Mobile Source Emissions Research Branch
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
In 1978, initial findings on the mutagenic nature of diesel extracts
were reported (1). Since that time, the Ames salmonella typhimurium
bioassay has been used extensively in the investigation of mobile
source emissions. Both government and industry have carried out numerous
studies on the many variables effecting the mutagenicity of mobile
source samples. These studies for the most part fall into six general
categories: 1) Sampling-exhaust dilution ratios, gas phase vs particulate
phase collection and examination of filter types; 2) Bioassay sample
preparation-extraction solvents, extraction procedures, solvent exchange
studies and sample storage; 3) Vehicle types-light duty diesels, heavy
duty trucks and gasoline powered cars; 4) Operational characteristics-
driving cycles, fuel types, temperature, mileage and engine malfunction
conditions; 5) Artifacts; 6) Characterization studies-qualitative and
quantitative studies on the nature of mobile source mutagens and their
precursors.
The findings and results of some of these studies are given below.
While problems exist in the quantitative comparison of Ames test results
from different laboratories or even the same laboratory over a given
period of time (2), camparisons should be valid at least in the qualita-
tive sense.
Table 1 lists some average emission factors and Ames data for
several vehicle classes. The heavy duty trucks were operated over the
proposed 1983 transient driving cycle while the light duty vehicles were
operated over the Federal Test Procedure driving cycle. Data for the
light duty gasoline cars (3) and light duty diesels (4) are from in-use
vehicle studies. A number of the gasoline cars had some form of emission
system malfunction as evidenced by the high regulated emissions observed
in some cases (eg. 1977 Dodge Aspen, NOX emissions were 6.1 g/mile).
The heavy duty trucks (5) were all 1979 models with the exception of the
-------
2 cycle which was a 1977 Detroit Diesel city bus. The heavy duty truck
data is limited and one must realize that "average" values may be
strongly affected by outliers (eg. the TA 98, +S9 data for the heavy
duty gasoline trucks is an average of two vehicles having values of 604
and 253 revertants/mile (xlO~3). One does not know which is the more
representative value).
Correlations carried out on the gasoline cars indicated that benzo(a)-
pyrene (BAP) emissions of the catalyst (unleaded) vehicles correlated
with TA 98, +S9 (r* = 0.81). This is also in accord with the fact that,
in general, gasoline cars showed higher activity with activation. A
positive correlation was also found for the soluble organic fraction
(SOF) vs TA 98, +S9 (r2- = 0.84). The THC, CO, and NOX emissions showed
no correlation with activity.
The levels of 1-nitropyrene in the light duty gasoline vehichles
were only about 3% of that observed for diesels and show no correlation
with TA 98, without activation. Emission levels of 1-nitropyrene have
not been determined for a large number of diesels. It has been found,
however, to correlate fairly well with TA 98, -S9 in cycle studies
conducted on the same vehicle (r2 =0.96) and in some artifact experiments
(r = 0.97).
Recently, some low temperature experiments were conducted on light
duty diesels (6). Results indicated indicated increased particulate and
SOF emissions wih decreasing FTP test temperatures. The major portion
of the increased SOF emissions appeared to be unburned fuel and no
correlation of temperature and TA 98 activity was observed.
While the majority of work has been conducted on diesel and gasoline
particulate extracts, attempts have also been made to examin the gas
phase organics for mutagenic activity (7). Using the porous polymer
resin XAD2 for trapping followed by elution of the organics wiht dichloro-
methane, activity in TA 98 was found to be very low, at least an order
of magnitude less than the SOF. In addition, control experiments indicated
that much of the observed activity may have been due to artifacts.
-------
References
1. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. Tejada,
J. Bumgarner, R. Duffield, M. Waters, V. Simmon, C. Hare, C. Rodriguez,
and L. Snow, "Application of Bioassay to the Characterization of Diesel
Participate Emissions: Parts I, II." in Application of Short-term
Bioassays in the Fractionation and Analysis of Complex Environmental
Mixtures, EPA 600/9-78-027, November 1978.
2. Salmeen, I. and A. M. Durisin, "Some Effects of Bacteria Population on
Quantitation of the Ames Salmonella Histidine Reversion Mutagenisis
Assays." Mutation Research 85, 109-118, 1981.
3. Lang, J. L. Snow, R. Carlson, F. Black, R. Zweidinger and S. Tejada,
"Characterization of Particulate Emissions from In-use Gasoline Fueled
Motor Vehicles", Paper 81186 to be presented at SAE Fuels and Lub.
Meeting, Tulsa, October 1981.
4. Gibbs, R.E., J.D. Hyde and S. M. Byer, "Characterization of Particulate
Emissions from In-Use Vehicles", Paper 801372 presented at SAE Fuels
and Lub. Meeting, Baltimore, October 1980.
5. Dietzman, H.E. and M.A. Parness, "Study of Emissions From Trucks Over
Transient Driving Cycles", Final report to EPA under contract No.
68-02-2993, September 1981.
6. Braddock, "Emissions of Diesel Particles and Particulate Mutagens at
Low Ambient Temperature", EPA Diesel Emissions Symposium, Raleigh,
N.C. October 5-7, 1981.
7. Stump, F., "Trapping Gaseous Hydrocarbons", Ibid.
-------
Table 1. Emission and Participate Characteristics by Vehicle Class
Emission Factors
THC, g/mi.
CO, g/mi.
NOX, g/mi.
Total Particulate, g/mi.
SOF, mg/mi .
BAP, ug/mi.
1-Nitropyrene, ug/mi.
TA 98, -S9, rev./mi.(10"3)
TA 98, +S9, rev./mi.(10"3)
Heavy Duty
Gasoline (2)c
13.5
116.3
13.4
0.74
Trucks3
4-cycle
Diesel (3)
1.88
7.53
25.5
1.49
27.6 335
39.5 2
8
111 392
428 356
.61
>»26e
2-cycle
Diesel
2.13
75.1
35.5
3.33
537
1.33
13
48.3
Light
Diesel
0.38
1.27
1.27
0.61
124
4.5d
7.4d
509
Duty Vehicles
. Leaded
(6) Gas(4)
2.74
28.5
3.52
0.10
21.1
14.5
0.20
152
258
b
Unleaded
Gas(15)
1.05
12.2
2.31
0.03
14.4
3.3
0.24
42.1
79.3
a. 1983 Transient Cycle
b. FTP cycle
c. ( ) denotes number of vehicles
d. Values derived from other vehicle data
e. One vehicle only, 1979 Cummins Formula 290
-------
ANALYSIS OF VOLATILE POLYCYCLIC AROMATIC HYDROCARRONS
IN HEAVY-DUTY DIESEL EXHAUST EMISSIONS
by
Walter C. Eisenberg and Sydney M. Gordon
Analytical Research Section
IIT Research Institute
Chicago, Illinois 60616
Joseph M. Perez
Research Department
Caterpillar Tractor Company
Peoria, Illinois 61629
The breakthrough and/or loss of polycyclic aromatic hydrocarbons (PAH)
from particulate filters was investigated during the collection of heavy-duty
diesel exhaust emissions. The concentration of PAH associated with heavy-duty
diesel particulate has been monitored for several years. During these studies
it was observed that appreciable quantities of organics including PAH pass
through the particulate filter during the sample collection (1). The emissions
from a heavy-duty diesel engine were sampled using 70-mm Pallflex TX40HI20WW
filters. A portion of the gas passing through the particulate filter was
sampled using 4.0-mm x 4.0-cm sorbent.cartridges. Chromosorb 102 and Tenax GC
were used to collect gas phase organics. The particulate filter was extracted
in a micro-Soxhlet apparatus for 6 hours with methylene chloride. The sorbent
cartridges were extracted by passing hexane/benzene (90/10, v/v) through the
cartridge at a flow rate of 0.5 ml/min. Following concentration the PAH
fraction was isolated using open column silica gel chromatography. The PAH in
particulate and gas phase samples were analyzed using gas chromatography/mass
spectrometry (GC/MS) and high performance liquid chromatoaraphy (HPLC).
In the GC/MS analysis the samples were eluted on a 15.0-m x 0.31-mm i.d.
SE-54 fused silica capillary column directly into the ion source of a Varian
MAT 311A mass spectrometer operating in the repetitive scanning mode. The data
were enhanced using a computer program by Dromey et al. (2) to locate peaks in
the raw data and provide a set of clean mass spectra of the sample components
free of contributions from background and overlapping peaks. Compounds were
identified with a library matching search algorithm, and in the case of 'complex
spectra the data was manually interpreted.
-------
Twelve parent PAH were measured in the gas phase and particulate samples
using reverse phase HPLC. The analysis was performed using two coupled
4.6-mm x 25.0-cm Zorbax ODS columns and an acetonitrile water gradient. The
eluant were monitored using an ultraviolet absorhance detector at X = 254 and
280 nm and a fluorescence detector, Xex = 280 nm and \gm = 389 nm.
Over 40 compounds were tentatively identified in the gas phase after a
particulate filter during the collection of heavy-duty diesel exhaust
emissions. Thirty-five of these compounds were volatile PAH and included
parent- and aldyl-substituted compounds ranging in size from two to five fused
rings. A distribution quotient was defined as the ratio of the concentration
of the PAH in the gas phase to their concentration in the particulate phase.
It ranged from ~ 56 for fluorene to ~ 1 for benz[a]anthracene. Experiments to
date show that at some operating conditions the concentrations of volatile PAH
were significantly lower when measured on the particulate only. Since the role
of these compounds in the formation of artifacts and mutagens is unresolved,
the methods for measuring PAH in exhaust streams need to be modified to
account for gas phase compounds.
REFERENCES
1. W.E. Pepelko, R.M. Danner, and N.A. Clarke, eds. 1980. Health Effects of
Diesel Engine Emissions. EPA-600/9-8-057a. Health Effects Research
Laboratory, U.S. Environmental Protection Agency: Cincinnati, OH
45268. pp. 138-174.
2. R.G. Dromey, M.J. Stefik, T.C. Rindfleisch, and A.M. Duffield. 1976.
Anal. Chem. 48:1368.
-------
THE CHEMICAL CHARACTERIZATION OF DIESEL PARTICULATE MATTER
by
James Alan Yergey and Terence H. Risby
School of Hygiene and Public Health
Johns Hopkins University
Baltimore, Maryland
Samuel S. Lestz
Department of Mechanical Engineering
Pennsylvania State University
University Park, Pennsylvania
INTRODUCTION
A great deal of research has been directed toward elucidating the
potential health hazards of Diesel particulate matter. The potential health
risks of the emmitted particles are due to a number of important factors.
The mass median diameter of the agglomerated particles found in Diesel
exhaust is less than 1 urn (1-4), and their surface areas greater than 50
m^/g (3,4). The large surface areas facilitate the adsorption of gas
phase combustion products, while the small diameters allow appreciable
residence times in the atmosphere. In addition, it is generally accepted
that particles less than 1 urn in diameter can be respired by humans, with a
significant portion depositing in the pulmonary regions of the lung (5-7).
An assessment of the potential health effects of Diesel particles must also
consider the fact that many of the surface adsorbed species which have been
isolated from the soluble organic fraction (SOF) of Diesel particulate
matter are potentially carcinogenic (8-10).
A number of references have appeared in the literature in recent years
regarding the identification of the individual constituents of the SOF
(11-15). The commercial Diesel fuels and lubricating oils which were used
in each of these studies contain such a wide variety of compounds that a
study of the combustion mechanisms which lead to the particle-bound products
becomes extremely difficult. An understanding of the mechanisms governing
the formation of the adsorbed species is a requisite for complete
comprehension of the potential health effects of Diesel particulate matter.
The primary objective of this research was to simplify the combustion
chemistry in order to better understand the overall mechanisms governing the
formation of the particle-adsorbed species.
-------
EXPERIMENTAL
Diesel particulate matter was generated from a single-cylinder engine,
operated on a 1:1 by volume blend of n-tetradecane and 2,2,4-trimethylpentane.
This prototype fuel was used in conjuntion with a synthetic lubricating oil,
in order to simplify the exhaust chemistry. Air and argon/oxygen oxidant
systems were employed. The Ar/02 was used in order to investigate the
products of nitrogen-free combustion. Particle samples were collected on
142 mm Pallflex filters in an isokinetically drawn sample line, and gas
phase emissions were monitored. Filters were Soxhlet extracted with
dichlorometnane, and the resulting extract blown to dryness under nitrogen
and weighed. Samples were analyzed by the Ames Salmonella and Comptest (16)
bacterial assays in order to assess their mutagenic and potential
carcinogenic capaity.
The simplifications introduced in this study allow the separation and
analysis of the entire SOF in a single pass. Particle extracts were
analyzed by capillary gas chromatograpny, using flame ionization and
thermionic specific detectors, and high performance liquid chromatography,
using UV detection. Both positive and negative chemical ionization mass
spectrometry were employed for direct analysis of the gas chromatographic
effluents, and for analysis of collected HPLC fractions, using a
Pt-filament, direct-insertion probe. Capillary gas chromatography/electron
impact mass spectrometry was utilized for substantiating identifications.
In addition', -Diesel particles were heated under vacuum, and the evolved
gases analyzed by chemical ionization mass spectrometry.
RESULTS
Figure 1 portrays a typical GC/PCIMS total ion profile for an air
oxidant sample. Table 1 illustrates the compounds which have been
identified in the air oxidant samples. Identifications were based upon gas
chromatographic retention indices (17), molecular weights derived from CIMS,
and EIMS library searchs. In general, the Ar/02 samples exhibited the
same major components, with lower concentrations. GC/TSD results indicated
a lower number of nitrogen containing compounds in the Ar/02 samples,
however, specific identifications of the nitrogenous species were not
possible. HPLC separations coupled with the mass spectra generated using
the Pt-filament probe were especially useful for identifying higher
molecular weight PAH, while GC/NCIMS data were particularly sensitive to the
oxygenated species found in the SOF
Results indicate that unsubstituted, non-linear polynuclear aromatic
compounds are the primary particle-bound combustion products from aliphatic
hydrocarbon fuel components. The identified compounds are demonstrated to
be fuel-independent products of the diffusion controlled combustion process
which exists in the Diesel engine. This fact should be considered in any
future health effects studies of Diesel particulate matter. The potent
carcinogen, nitropyrene, was tentatively identified in the soluble organic
fraction of the particles, and could possibly account for a large portion of
the observed mutagenic properties of the extractable organics.
-------
TIC? FOR GC/PCIKS Or SAH?L[ i
TOTAL ION CURRENT: 3583972
I 1
nt-
N -
T |
r
Uvl l\rtllUI_W-/
i
i
i1 ll
i\ - ;;
S i'
1 1
'i
!«
ill
[I
i I I ; : i i ; i
1 i
288
|-
i
i
I
1
\ III
i
i
i
ll
M Hill In ll
i i 1 i i i i i i i i i i i i i ; ; , i . i i i i i i i i i i i i i i i i i i i i I
111,1! l 1 1 1 1
488 688 8BC 1808 1208 1408
FIGURE 1. Total Ion Current Profile for GC/PCIMS of Air Oxidant Sample
Naphthalene
Benzofuran,7-methy 1-
Inden-1-one,2,3-dihydro-
Methylnaphthalenes
Phthalate-anhydride
Biphenyl
n-Tetradecane
l-Benzopyran-2-one
Biphenylene, or Acenaphthylene
Acenaphthene
Dibenzofuran
Fluorene
9-Fluorene
Anthracene
TABLE 1. Compounds
Phenanthrene
Me thy 1-9-Fluorenones
BenzofcIcinnoline
Fluorene Quinone
Phenanthrene Quinone
Cyclopenta-phenanthrene-5-one
Naphtho(1,8-cdlpyran-1,3-dione-
Fluoranthrene
Pyrene
MethyIpyrenes
Benzofghi]fluoranthene
Cyclopenta(cd Jpyrene
Chrysene or Triphenylene
Benzofluoranthrathenes or Benzopyrenes
Identified in Air Oxidant SOF
-------
REFERENCES
1. W. H. Lipkea, J. H. Johnson and C. T. Vuk. 1978. The Physical and
Chemical Character of Diesel Particulate Emissions- Measurement
Tecnniques and Fundamental Considerations. S.A.E. Paper No. SP-430.
2. J. A. Verrant and D. A. Kittelson. 1977. Sampling and Physical
Characterization of Diesel Exhaust Aerosols. S.A.E. Paper No. 770720.
3. J. W. Frey and M. Corn. 1967. Nature. 216:615-617.
4. M. M. Ross. 1981. Physicochemical Characterization of Diesel
Particulate Matter. Ph. D. Thesis. The Pennsylvania State University.
5. T. F. Hatch and P. Gross. 1964. Pulmonary Deposition and Retention of
Inhaled Aerosols. Academic Press: New York.
6. Task Group on Lung Dynamics. 1966. Health Physics. 12:173-208.
7. P. Kotin and H. L. Falk. 1959. Cancer. 12:147-163.
8. D. J. Earth and S. M. Blacker. 1978. Journal of the Air Pollution Control
Association. 28:769-771.
9. J. McCann, B. N. Ames, E. Choi and E. Yanasaki. 1975. Proceedings of the
National Academy of Sciences U.S.A. 72:5135-5139.
10. M. Dukovich, R. E. Yasbin, S. S. Lestz, T. H. Risby and R. E. Zweidinger.
1981. Environmental Mutagenisis. In press.
11. E. F. Funkenbusch, D. G. Leddy and J. H. Johnson. 1979. The
Characterization of the Soluble Organic Fraction of Diesel Particulate
Matter. S.A.E. Paper No. 79418.
12. D. Schuetzle, F. S. Lee, T. J. Prater and S. B. Tejeda. 1981.
International Journal of Environmental Analytical Chemistry. 9:93-144.
13. F. W. Karasek, R. J. Smythe and R. J. Laub. 1974. Journal of
Chromatography. 101:125-136.
14. M. D. Erikson, D. L. Newton, E. D. Pellizzari, K. B. Tomer and D.
Dropkin. 1979. Journal of Chromatographic Science. 17:449-454.
15. F. Black and L. High, Methodology for Determining Particulate and
Gaseous Diesel Hydrocarbon Emissions. 1979. S.A.E. Paper No. 79042.
16. M. Dukovich. The Mutagenic and "SOS" Inducing Activity of Diesel
Particulate. M. S. Thesis. (1981). The Pennsyvania State University.
17. M. L. Lee, D. L. Vassilaros, C. M. White and M. Novotny. 1979.
Analytical Chemistry. 51:768-773.
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THE ANALYSIS OF NITRATED POLYNUCLEAR AROMATIC HYDROCARBONS
IN DIESEL EXHAUST PARTICIPATES BY
MASS SPECTROMETRY/MASS SPECTROMETRY TECHNIQUES1
by
T. Riley, T. Prater and D. Schuetzle,
Ford Motor Co, Scientific Research Lab., Dearborn, MI 48121;
T. M. Harvey and D. Hunt
Dept. of Chem., Univ. of Virginia, Charlottesville, VA 22901
INTRODUCTION
Recent investigations have indicated that organic solvent extracts of light-duty
diesel exhaust particulates exhibit direct-acting mutagenicity when tested using the
Ames assay. Preliminary estimates indicate that a significant portion of this direct-
acting mutagenicity may be due to the presence of nitrated polynuclear aromatic
hydrocarbons (nitro-PAH). Mass analyzed ion kinetic energy spectrometry (MIKES)
and triple stage quadrupole (TSQ) analytical techniques used to characterize these
compounds in diesel exhaust are described.
EXPERIMENTAL
Light duty diesel exhaust particulate samples were collected on T60A20 Pallaflex
filters using a dilution tube and a chassis dynamometer test facility. Filter samples
were extracted with dichloromethane.
MIKES analyses were performed on a Vacuum Generators ZAB-2F mass spectrometer
using both electron impact (El) and negative ion methane chemical ionization (NICI)
procedures. All experiments were conducted using a magnetic sector resolution of
approximately 2000 and helium as a collision gas ( 1x10" torr).
A Finnigan TSQ mass spectrometer was used to perform collisionally activated
dissociation (TSQ-CAD) and constant neutral loss studies. All experiments were
conducted using positive ion methane chemical ionization (PICl) procedures. In the
TSQ-CAD studies the first quadrupole was set to transmit a parent ion of interest
into the second quadrupole which functioned as a collision cell (N~, 5 x 10~ torr).
The third quadrupole was scanned repetitively to collect daughter ion spectra. In the
constant neutral loss studies, the first and third quadrupoles were scanned in parallel
with a 17 amu mass deficit. Under these conditions, only ions which experience the
loss of a 17 amu neutral fragment when collisionally dissociated in the second
quadruple are detected. This ion reaction was found to be characteristic of nitro-
PAH compounds.
-------
RESULTS AND DISCUSSION
The TSQ constant neutral loss analysis was found to be a very useful screening
procedure for nitro-PAH compounds. Table I lists 20 different nitro-PAH derivatives
which were tentatively identified in diesel particulate extract using this procedure.
It must be emphasized that this technique only monitors a reaction characteristic of
nitro-PAH compounds and does not confirm their presence. The specificity of the
constant neutral loss analysis was assessed by examining the response of several
compounds representative of the classes of PAH derivatives observed in diesel
exhaust. The results of this study, as shown in Table II, indicate that the technique
exhibits good selectivity for the nitro-PAH derivatives.
Table III illustrates the concentration of 1-NP in the exhaust particulate extract from
four different diesel engines as determined by MIKES and TSQ-CAD analysis. These
quantitation studies indicated that both MS/MS techniques lacked sufficient
resolution on the first mass filter to eliminate positive interferences in the daughter
ion spectra completely. The M-16 daughter ion fragment (M+l - OH) was found to be
specific for 1-NP and was used for quantitation by TSQ-CAD, but the electrostatic
sector of the MIKES instrument did not resolve this daughter ion adequately. It was
necessary to prefractionate the OP-1 and PG-1 samples by preparative scale high
performance liquid chromatography and to use NICI techniques to accomplish an
interference-free MIKES analysis of 1-NP.
Neither MS/MS technique distinguished between nitro-PAH isomers. This information
was obtained by capillary GC-MS.
Riley, T., T. Prater, D. Schuetzle, T. Harvey and D. Hunt. 1981. The analysis of
nitrated polynuclear aromatic hydrocarbons in diesel exhaust particulates by mass
spectrometry/mass spectrometry techniques. Presented at the 29th Annual
Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN.
2
Schuetzle, D., T. Prater, T. Riley, A. Durisin and I. Salmeen. 1980. Analysis of
nitrated derivatives of PAH and determination of their contribution to Ames
assay mutagenicity for diesel particulate extracts. Presented at the Fifth
International Symposium on Polynuclear Aromatic Hydrocarbons, Columbus, OH.
-------
Table I. Nitro-PAH Derivatives Tentatively Identified in Diesel Particulate
Extracts by TSQ Constant Neutral Loss Analysis
Nitroacenaphthylenes
Nitro(acenaphthlenes, biphenyls)
Nitronaphthaquinones
Nitrodihydroxynaphthalenes
Nitrofluorenes
Nitro(methyiacenaphthalenes, methylbiphenyls)
Nitro(trimethylnaphthalenes)
Nitro(naphthalic acid)
Nitro(anthracenes and phenanthrenes)
Nitro(fluorenones and methylf luorenes)
Nitro(methylanthracenes and methylphenanthrenes)
NitroCanthrones and phenanthrones)
Nitro(pyrenes and fluoranthenes)
Nitro(dimethylanthracenes and dimethylphenanthrenes)
Nitro(methylpyrenes and methylfluoranthenes)
Nitro(pyrones and fluoranthones)
Nitro(pyrene and fluoranthene)quinones
Nitro(dimethylphenanthrene and dimethylanthracene) carboxaldehydes
Nitro(methylbenzo(a)anthracenes, methylchrysenes and methyltriphenylenes)
Nitro(benzo(a)pyrenes, benzo(e)pyrenes and perylenes)
Table II. Selectivity of the TSQ Constant Neutral Loss Analysis for Nitro-PAH
Compound Class Selectivity Ratio3
(Nitro-PAH/Compound Class)
Amines 50/1
Aldehydes
Quinones
200/1
Carboxylic Acids
Acids
interference level <5%
-------
Table III. Quantitation of 1-NP in Diesel Exhaust Particulate Extract
using MS/MS Techniques
Engine Sample
Instrument
lonization
Concentration
(ppm)
Nl-1
OL-1
OP-1
PG-1
TSQ
MIKES
TSQ
TSQ
MIKES
MIKES
MIKES
PICI
El
PICI
PICI
El
NIC I
NICI
2285+230
2080+220
204+30
77+15
£105
55+11
150+30
-------
CONTRIBUTION OF 1-NITROPYRENE TO DIRECT ACTING AMES ASSAY
MUTAGENICITIES OF DIESEL PARTICULATE EXTRACTS
by
Irving Salmeen, Anna Marie Durisin,
Thomas J. Prater, Timothy Riley, and Dennis Schuetzle
Engineering and Research Staff
Research
Ford Motor Company
Dearborn, Michigan
We have determined the percentage contribution, P, of 1-nitropyrene
(1-NP) to the direct acting Ames assay mutagenicities of dichloromethane
extracts of exhaust particles collected from three different diesel powered
passenger vehicles. For strains TA98, 100, and 1538, respectively, P was
(16, 1, and 7%) for vehicle 1; (24, 9, and 7%) for vehicle 2; and (13, 4, and
13%) for vehicle 3. We assumed:
P = C x (M1/M2) x 100%
Ml and M2 are the slopes of the linear portion of the Ames assay dose-
response functions for 1-NP and for diesel particulate extract respectively.
C, the mass of 1-NP in the particulate extracts/mass of extract, was deter-
mined by collisionally activated dissociation mass analyzed ion kinetic
energy spectrometry (CAD-MIKES). (CAD-MIKES is a double mass spectrometer
technique with which compounds often can be analyzed in complex mixtures
directly without chromatographic separation.) C for vehicles 1, 2, and 3,
respectively, was 55 ^ 11, 2030 +_ 220, and 150 + 30 ppm. We used fused-silica
capillary column GC-MS to show that the mass 247 (1-NP) CAD-MIKES spectrum
was due to 1-NP and not to some other mass 247 isomer. Several isomers of
nitrofluoranthene and nitropyrene were synthesized to assure adequate GC-MS
separation of these isomers from 1-NP.
The slopes, Ml and M2, are approximately proportional to yN, where y is
the mutation rate per concentration of mutagen and N is the total number of
histidine auxotrophs in the background lawn (1). N is proportional to the
initial inoculum and the average number of auxotrophs per individual back-
ground colony. N cannot be measured in the Ames assay and it is not neces-
sarily the same for Ml and M2. Consequently, whenever we carried out an
Ames assay of diesel particulate extract we concurrently carried out an assay
of IrNP using equal amounts of the same broth culture, thereby ensuring that
the initial inocula were equal. We then obtained photomicrographs of the
-------
background lawn (100X) on each plate at the time of counting revertants and
from these photomicrographs counted the background colonies and estimated
their average size. If the background colony counts and their average sizes
were the same for both 1-NP and diesel particulate extracts, we took this as
evidence that N was the same and then used the corresponding slopes to
calculate P.
Finally, we did experiments in which various known amounts of 1-NP were
added to a constant amount of extract and obtained the Ames assay dose-response
function of the 1-NP in the presence of the diesel extract. The added amounts
of 1-NP were chosen to be within the same order of magnitude as that of the
1-NP already in the particulate extracts as measured by mass spectrometry.
We found that the slope of the 1-NP dose-response function in the presence of
particulate extract was the same as that for 1-NP alone in solution. This
result supports an assumption, implicit in the above equation, that the
mutagenicity of 1-NP is not altered by the other components in the mixtures.
These data show that 1-NP is an important contributor to the mutagenicity
of these diesel particulate extracts. Even a 1% contribution is important,
considering the thousands of compounds present in the particulate extracts.
This observation is very encouraging to experimenters seeking to identify
mutagens in complex mixtures because it suggests the possibility that a small
number of compounds may account for the majority of the mutagenicity of these
complex mixtures.
REFERENCES
1. Salmeen, I. and Durisin, A., Mutat. Res., 85, 101-118, 1981.
-------
DINITROPYRENES: THEIR PROBABLE PRESENCE IN DIESEL PARTICLE
EXTRACTS AND CONSEQUENT EFFECT ON MUTAGENIC ACTIVATIONS
BY NADPH-DEPENDENT S9 ENZYMES
T. C. Pederson and J-S. Siak
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
The direct-acting mutagenic activity of diesel exhaust particle extracts in the
Salmonella mutation assay may be mostly due to nitroaromatic compounds activated by
bacterial nitroreductase enzymes. Chromatographic separations have demonstrated
that much of the extract's mutagenic activity is associated with components that are
more polar than 1-nitropyrene or other monosubstituted nitro-PAH (Pederson and Siak,
1981, J. Appl. Tox., 1(2):54-60). The present studies, employing TLC and HPLC
separation techniques and the recently developed dinitropyrene-resistant Salmonella
strains (Rosenkranz et al, 1981, Mutation Res., 91:103-105), investigate the probable
presence of dinitro-, trinitro-, or tetranitropyrenes in the polar mutagenic fractions of
diesel particle extracts.
The direct-acting mutagenic activity of a diesel particle extract, reduced by 40% in the
niridazole-resistant strain TA98NR, was decreased by 70% in the dinitropyrene-
resistant strain TA98/l,8-DNPg. After fractionation of the extract by silica gel TLC,
all mutagenic fractions exhibited reduced activity in TA98/1,8-DNP_, as shown in the
figure. The most marked reduction occurred with the fractions which co-chromato-
graphed with reference samples of the multisubstituted nitropyrenes. The mutagenicity
of these fractions and multinitropyrenes was markedly reduced in TA98/l,8-DNPfi, but
not in TA98NR. HPLC separations on a cyano phase-bonded silica column indicate that
1,8-dinitro-and 1,6-dinitropyrene are the predominant mutagenic components in the
material recovered from TLC fractions 11 through 15. Some mutagenic activity was also
attributed to 1,3-dinitro- and 1,3,6-trinitropyrene. The dinitropyrenes may account for
15-20% of the mutagenic activity in the particle extract, but they are very potent
bacterial mutagens and would be present at concentrations of less than 1 ppm in the
exhaust particulate.
The nitroreductase-deficient bacteria have also been used in the SalmoneZZa/SS muta-
tion assay to examine the effect of mammalian enzyme activities on the mutagenicity
of diesel particle extracts and the nitropyrenes. Under appropriate conditions, the
activation of mutagens in diesel particle extract by rat liver S9 enzymes was evident as
a difference between assays with and without NADPH (Pederson and Siak, 1981, J. Appl.
Tox., l(2):61-66). 1-Nitropyrene was similarly activated by S9 enzyme activity. The
activation of 1-nitropyrene is located in the microsomal fraction of the S9 preparation,
but activation of diesel particle extract was more evident with the cytosol fraction.
-------
The mutagenicity of each dinitropyrene was greatly reduced by the NADPH-dependent
activity of S9 enzymes. As shown in the table below, the NADPH-dependent
inactivation is catalyzed by microsomal enzymes, but with the cytosol fraction, the
mutagenic activity of the dinitropyrenes is increased. The NADPH-dependent increase
in mutagenicity of the dinitropyrenes includes both a cytosol-independent reaction,
which is probably a direct reduction of the compounds by reduced pyridine nucleotide,
and an enzyme-catalyzed reaction. NADH is just as effective as NADPH in both the
cytosol-dependent and independent activation reactions.
The NADPH-dependent increase in mutagenricity of diesel particle extract in the
Salmonella/SB assay involves both multiple extract components and multiple S9
enzymes. The dinitropyrenes presumably contribute to the cytosol-catalyzed activa-
tion. The much smaller effect of microsomal enzymes on the mutagenicity of the
particle extract must reflect competing activation and inactivation reactions as
evidenced by the difference between 1-nitropyrene and the dinitropyrenes.
Net TA98NR Revertants/Plate
1,6-Dinitropyrene -NADPH
10 tig/plate +NADPH
Change
1,8-Dinitropyrene -NADPH
2 ng/plate +NADPH
Change
Diesel Particle -NADPH
30 ug/plate +NADPH
Change
plus
S9 microsomes
282 +17
19 ± 5
-95%
196 ±13
7 ± 6
-95%
326 ± 6
415 ± 8
+25%
plus
S9 cytosol
347 ± 16
878 ± 98
+150%
392 ± 21
1129 ± 51
+190%
344 ± 17
605 ± 20
+75%
ASSAY
CONDITIONS
CH TA98
E3 TA98NR
I TA98/1.8-DNP6
15 20
TLC Fraction
25
30
-------
MUTAGENICITY OF PARTICLE-ROUND ORGANIC CHEMICAL FRACTIONS
FROM DIESEL AND COMPARATIVE EMISSIONS
by
Ann Austin, Larry Claxton, and Joellen Lewtas
Genetic Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
A variety of mobile and stationary sources emit particle-bound organics
that have demonstrated mutagenicity. The objective of this study was to
measure the mutagenicity of the chemical class fractions derived from the total
extracted organics for diesel and several comparative emission sources.
The four sources of combustion organics were diesel engine exhaust
particles, a cigarette smoke condensate, a coke oven main sample, and roofing
tar emissions. The diesel exhaust particles were collected from a 1978
Oldsmobile 350 diesel vehicle operated on the highway fuel economy test cycle
(HWFET) with No. 2 diesel (Union 76) fuel. The particles were collected on
Pallflex T60A20 filters and the organics were removed by Soxhlet extraction
with methylene chloride as previously described (1). The 2RI Kentucky
reference cigarette smoke condensate was generated according to the method of
Patel (1977) at Oak Ridge National Laboratory (1). The coke oven main sample
was collected from a separator located between the gas collector main and the
primary coolers within a coke oven battery at Republic Steel in Gadsden, AL,
about 60 miles northeast of Birmingham. The roofing tar sample was generated
and collected using a conventional tar pot containing pitch-based tar, enclosed
within a chamber and heated to 360° to 380°F, a normal temperature for
commercial use. The evaporative emissions were collected using a small bag
house fitted with Teflon filter bags (1). The solvents used to extract or
condense the organics from each of these samples were removed by evaporation
under nitrogen. The total extracted organics were class-fractionated into
organic acids, organic bases, cyclohexane insolubles, polar neutrals, non-polar
neutrals, and polynuclear aromatics (PNA). The PNA fraction was further
fractionated chromatographically using gradient elution on high pressure liquid
chromatography (HPLC) such that a purified fraction containing PNA hydrocarbons
was obtained (PNA1) by elution with 2% dichloromethane (DCM) in hexane.
Elution with more polar solvents resulted in three additional fractions
(PNA2-4) that contained compounds of intermediate to higher polarity. Each
class of organics was chemically characterized using GC/MS (2).
-------
The chemical fractions for each emission source were bioassayed using the
Salmonella typhimuriurn/microsomal plate incorporation test as developed by Ames
et al. (3). Due to the large number of samples to be bioassayed at one time in
this study and the limited amounts of some of the samples, only the mutant
strain (TA98) of Salmonella typhimuriurn was used. The protocol described by
Ames et al. (3) was followed with minor modifications (4). The data was
analyzed using a non-linear model (5) to determine the slope of the
dose-response curve. Weighted mutagenicities were determined for each fraction
based on the mutagenicity model slope (rev/yg) and the percent of the total
mass recovered from each fraction represented. The weighted mutagenicities
were then used to determine the percent of mutagenicity attributed to each
chemical fraction. Based on this data as summarized in Table 1 the following
summary can be made:
1. Olds Diesel. Although the non-polar neutral fraction (NPN)
represented the greatest percent of the total mass recovered upon
fractionation, it accounted for very little (< 2%) of the mutagenic
activity. From 45 to 50% of the mutagenic activity was found in the
polar neutral fraction (PN). Polar neutral compounds having limited
solubility in cyclohexane would appear in the cyclohexane insoluble
fraction (CI), which contained from 15 to 31% of the mutagenic
activity. Both of these fractions (PN and CI) contained
direct-acting mutagens.
2. Cigarette Smoke. The cyclohexane insoluble fraction (CI) represented
the greatest percent of the total mass recovered upon fractionation.
The purified polynuclear aromatic fraction (PNA1) was the most active
fraction; however, when the model slopes were weighted according to
the percentage each fraction represented of the total organic sample,
the basic fraction (BASE) accounted for the majority of the mutagenic
activity in the presence of metabolic activation (57%), and a polar
neutral PNA contaminant (PNA4) accounted for the majority of the
mutagenic activity in the absence of metabolic activation (87%).
3. Coke Oven Mains. The greatest percent of the total mass recovered
after fractionation was represented by the cyclohexane insoluble
fraction (CI). The basic fraction (BASE) and the cyclohexane
insoluble fraction (CI) contained the largest percentage of the
mutagenic activity in the presence of metabolic activation (41% and
34%, respectively). A polar neutral PNA contaminant (PNA4) accounted
for the majority of the mutagenic activity in the absence of
metabolic activation (76%).
4. Roofing Tar. The chemical fractions representing the greatest
percent mass were the non-polar neutrals (NPN) and a purified PNA
fraction (PNA1). Although mutagenic activity was associated with
several of the fractions, the cyclohexane insoluble fraction (CI)
accounted for the majority (> 50%) of the mutagenic activity both in
the presence and absence of metabolic activation.
-------
This study demonstrated significant biological differences among the four
emission sources. Within each source, the relative mutagenicity of each
fraction was significantly different in the presence and absence of metabolic
activation. The two sources which showed some similarities were the cigarette
smoke and the coke oven mains. These sources had similar profiles in the
percent of mutagenic activity attributed to each fraction both with and without
metabolic activation; however, chemical characterization showed significant
differences in the compounds identified in these two sources (2). Further
chemical characterization of the constituents of each fraction is required to
determine which specific chemicals are biologically active within a single
source.
REFERENCES
1. Huisingh, J.L., R.L. Bradow, R.H. Jungers, B.D. Harris, R.B. Zweidinger,
K.M. Gushing, B.E. Gill, and R.E. Albert. 1980. Mutagenic and
Carcinogenic Potency of Extracts of Diesel and Related Environmental
Emissions: Study Design, Sample Generation, Collection, and
Preparation. EPA Report EPA-600/9-80-057b. U.S. Environmental
Protection Agency, Research Triangle Park, NC. pp. 788-800.
2. Sparacino, C.M., R. Williams, and K. Brady. 1981. Fractionation and
characterization of the organics from diesel and comparative emissions.
Presented as a poster abstract at the U.S. Environmental Protection
Agency Diesel Emissions Symposium, Raleigh, NC.
3. Ames, B.N., J. McCann, and E. Yamasaki. 1975. Methods for detecting
carcinogens and mutagens with the Salmonel 1 a/mammalian-microsome
mutagenicity test. Mutat. Res. 31:347-364.
4. Claxton, L.D. 1980. Mutagenic and Carcinogenic Potency of Diesel and
Related Environmental Emissions: Salmonella Bioassay. EPA Report
EPA-600/9-80-057b. U.S. Environmental Protection Agency, Research
Triangle Park, NC. pp. 801-809.
5. Stead, A.G., V. Hasselblad, J.P. Creason, and L. Claxton. 1981. Modeling
the Ames test. Mutat. Res. 85:13-27.
-------
Table 1. Percent of Mutagenic Activity Attributed to Each Chemical Fraction
from Comparative Sources (Reported as Percent of Weighted Slope)3
TA98 Without
Fractions
Acid
Base
PN
NPN
PNA1
PNA2
PNA3
PNA4
CI
Olds
Diesel
9.56
4.63
44.90
0.00
0.00
9.44
0.74
0.21
30.53
Cigarette
Smoke
2.22
0.00
0.00
0.00
0.00
0.00
10.84
86.94
0.00
Activation
Coke
Oven
0.00
0.00
0.00
0.00
0.00
0.00
24.14
75.86
0.00
Roofing
Tar
0.00
0.00
4.67
0.00
0.00
6.85
0.03
0.07
88.38
TA98 With Activation
Olds
Diesel
3.88
3.66
49.94
1.26
0.19
15.87
9.67
0.30
15.23
Cigarette
Smoke
0.46
57.39
0.00
1.99
0.20
0.00
0.18
8.37
31.41
Coke
Oven
0.15
40.89
8.99
7.04
4.31
0.00
2.90
1.84
33.87
Roofina
Tar
1.09
5.91
17.22
4.21
4.79
7.84
0.14
0.96
57.83
aPercent of mutagenic activity (% M) determined by: weighted mutagenicity of
each fraction (model slope [rev/yg] x % mass of fraction) x 100 * total
weighted mutagenicities of all the fractions.
-------
EMISSION OF DIESEL PARTICLES AND PARTICIPATE MUTAGENS
AT LOW AMBIENT TEMPERATURE
by
James N. Braddock
Environmental Sciences Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
Gaseous and participate exhaust emissions from two 1980 model diesel-
powered passenger cars, an Oldsmobile 98 and a Volkswagen Rabbit, were
measured over the urban dynamometer driving schedule of the Federal Test
Procedure (FTP) as a function of ambient temperature (23°F-84°F). Gaseous
emissions analysis included total hydrocarbons (HC), carbon monoxide (CO),
nitrogen oxides (NO ), and fuel economy (MPG). All were measured as a
function of the 3 individual test phases (i.e., cold transient-phase 1, stabi-
lized-phase 2, and hot transient-phase 3) of the FTP. Particulate emissions
analysis included total particulate matter, particulate organic emissions,
and molecular weight distribution measurements (also measured as a function
of FTP individual test phase), polynuclear aromatic hydrocarbon measurements
including benzo-a-pyrene (BaP), nitropyrene, and pyrene, and Ames Salmonella
bioassay of the particulate organics in strain TA-98. See figure detailing
diesel emissions analysis scheme below:
DIESEL EMISSIONS ANALYSIS SCHEME
*HC (g/mi)
r,n -i
*NOX (g/mi)
*MPG
PARTICULATE EMISSIONS
*TOTAL PARTICULATE MATTER (mg/mi)
EXTRACT WITH CH2CI2
(DISCARD INERT CARBONACEOUS MATERIAL)
PARTICULATE ORGANIC EMISSIONS (mg/mi)
'MOLECULAR WEIGHT DISTRIBUTION
(C-|2 C38, mg/mi)
AMES SALMONELLA BIOASSAY
(rev//jg, rev/mi)
POLYNUCLEAR AROMATIC HYDROCARBON ANALYSIS
BaP NITROPYRENE PYRENE
(ng/mg, ^jg/mi) (ng/mg, pg/mi) (ng/mg, pg/mt)
'MEASURED AS A FUNCTION OF FTP PHASE 1, 2, 3 AND COMPOSITE FTP.
-------
The data trends may be conveniently divided into two general categories,
gaseous emissions and participate emissions. Gaseous emission trends: HC,
CO, and NOX emissions increased slightly (0.10 < r2 < 0.75) with decreas-
ing test temperature. Oldsmobile HC emissions ranged from 0.20 to 0.29 g/mi,
CO emissions from 1.1 to 1.4 g/mi, and NOX emissions from 1.2 to 1.4 g/mi;
Volkswagen HC emissions ranged from 0.26 to 0.32 g/mi, CO emissions from 1.0
to 1.2 g/mi, and NOX emissions from 1.0 to 1.2 g/mi. Fuel economy was more
temperature dependent (r2 > 0.80) with the Oldsmobile's decreasing from 21.6
to 18.0 mpg and the Volkswagen's decreasing from 45.7 to 41.0 mpg.
Particulate emission trends: total particulate matter and particulate
organic emissions (i.e., the CH2C12 extractable particulate matter)
increased with decreasing test temperature. Comparison of overall FTP data at
82°F (median high temperature) versus 45°F (median low temperature)
indicates that Oldsmobile total particulate emissions increased from -567 to
739 mg/mi (+30%) and particulate organics increased from -94 to -153 mg/mi
(+63%). For the Volkswagen, total particulate emissions increased from -361
to ~423 mg/mi (+17%) and particulate organics increased from ~72 to ~101
mg/mi (+40%). Molecular weight distributions of the particulate organ-
ics in the C]2-^38 carbon number range, determined by gas chromatography,
indicated that much of the organic matter associated with the particulate
appears to be uncombusted diesel fuel. This is evident when comparing a
lower temperature FTP to a higher temperature FTP. Using the Oldsmobile for
example, at 32°F, 61% of the overall FTP particulate organic emission rate
of 127 mg/mi is attributable to C]3-C22 while at 82°F, only 40% of the
particulate emission rate of 83 mg/mi is attributable to C]3~C22- This
uncombusted diesel fuel effect is even more pronounced in the FTP test phase
1 molecular weight distributions: at 32°F, 68% of the Oldsmobile organic
emission rate of 137 mg/mi is attributable to ^3-^22 while at 82°F, only
35% of the particulate emission rate of 105 mg/mi is attributable to C-|3-
C22-
Polynuclear aromatic hydrocarbon (PAH) analysis of selected FTPs indicat-
ed decreasing (r2 ~0.60) BaP, nitropyrene, and pyrene emissions (in ng/mg
extract) with decreasing FTP test temperature. Overall Oldsmobile BaP emis-
sions ranged from 0.5 to 1.1 yg/mi (average 0.7 ±0.2); nitropyrene emissions
ranged from 6.6 to 13.6 yg/mi (average 9.8± 2.2); pyrene emissions ranged
from 82.1 to 133.7 yg/mi (average 106.6± 7.4). Overall Volkswagen BaP
emissions ranged from 15.5 to 20.2 yg/mi (average 18.2± 1.5); nitropyrene
emissions ranged from 4.2 to 12.6 yg/mi (average 8.3* 2.8); pyrene emissions
ranged from 260.4 to 354.5 yg/mi (average 293.9± 33.7).
Ames activity levels, in terms of revertants per microgram of particu-
late organic emissions, correlated moderately (r2 = 0.73) with FTP test
temperature indicating decreasing mutagenic activity with decreasing test
temperature. Activity levels also correlated moderately (r2 - 0.73) with PAH
emissions indicating decreasing mutagenicity (rev/yg) with decreasing PAH
emissions (ng/mg extract). Mutagenic activity was greater without metabolic
activation (-S9). For the Oldsmobile, activity with metabolic activation
ranged from 1.1 to 1.6 rev/yg (average of 1.3± 0.1) and without metabolic
activation from 2.4 to 3.8 rev/yg (average of 3.4 ± 0.9). Corresponding
-------
Oldsmobile rev/mi x 10^ ranges and rates were 105 to 185 (average of 152 ±
29) with S9 and 297 to 475 (average of 383 ± 66) without S9. The Volkswagen
displayed slightly greater mutagenic activity than the Oldsmobile. Activity
with metabolic activation ranged from 1.8 to 2.9 rev/vg (average of 2.2 ±
0.3)and without metabolic activation from 3.2 to 5.7 rev/yg (average of 4.5 ±
0.80. Corresponding Volkswagen rev/mi x 10^ ranges and rates were 163 to
226 (average of 192 ± 23) with S9 and 341 to 426 (average of 385 ± 28)
without S9.
CONCLUSIONS
1. The regulated gaseous emissions (HC, CO, and NOX) of diesel-powered
passenger cars were slightly temperature dependent with decreasing FTP test
temperature slightly increasing HC, CO, and NOX emissions. Fuel economy was
moderately temperature dependent with decreasing FTP test temperature de-
creasing fuel economy.
2. Total particulate matter and particulate organic emissions were moder-
ately temperature dependent. Decreasing FTP test temperature increased
total particulate and particulate organic emissions.
3. Increases in particulate organic emission rates at low FTP test tempera-
tures may be primarily attributed to uncombusted diesel fuel.
4. There appeared to be moderate correlation between polynuclear aromatic
hydrocarbon emissions and FTP test temperature with PAH emissions decreasing
with decreasing test temperature.
5. There appeared to be moderate correlation between Ames test mutagenic
activity (without metabolic activation) and FTP test temperature with muta-
genic activity decreasing with decreasing test temperature. There also
appeared to be moderate correlation between mutagenic activity and PAH emis-
sions with mutagenicity decreasing with decreasing PAH emissions.
-------
CHEMICAL CHARACTERIZATION OF MUTAGENIC FRACTIONS OF DIESEL PARTICIPATE EXTRACTS
by
Dilip R. Choudhury
Toxicology Institute
Division of Laboratories and Research
New York State Department of Health
Albany, New York
Projected increased use of diesel-powered automobiles has stimulated
considerable interest in research on health effects of the particulates and
identification of deleterious compounds adsorbed to the particulates. Diesel
particulates are highly respirable and may present significant inhalation
health hazard. It is now well recognized that organic extracts of diesel
emission particulates exhibit significant mutagenicity as detected by Ames
Salmonella bioassay and several other short-term mutagenicity assays. A great
deal of effort has been directed to identification of known as well as hitherto
unrecognized mutagens in the particulate extracts.
We have applied Ames Salmonella reversion assay to determine mutagenic
potencies of diesel particulate extracts and to aid in isolation of mutagenic
fractions for in-depth chemical characterization. In this presentation I will
discuss the chemical characterization of mutagenic fractions employing a
combination of complementary analytical techniques. Extracts of particulates
collected from three vehicles run on a chassis dynamometer-dilution tube have
been examined. On-line high performance liquid chromatography-mass
spectrometry (HPLC-MS), HPLC-ultraviolet spectroscopy, and gas chromatography-
MS provided definitive characterization of a number of compounds in mutagenic
fractions. Several polar derivatives of polycyclic aromatic hydrocarbons
(PAHs) including some nitrated PAHs have been identified. Some NOj>-PAHs are
presently known to be bacterial mutagens. However, it is likely that several
polycyclic carbonyl compounds detected in the extracts may also be mutagenic.
-------
INFLUENCE OF DRIVING CYCLE AND CAR TYPE ON THE MUTAGENICITY
OF DIESEL EXHAUST PARTICLE EXTRACTS
C. R. Clark, A. L. Brooks, R. 0. McClellan
Lovelace Inhalation Toxicology Research Institute
P. 0. Box 5890, Albuquerque, NM 87185
and
T. M. Naman and D. E. Seizinger
U. S. Department of Energy, Bartlesville Energy Technology Center
P. 0. Box 1398, Bartlesville, OK 74003
Solvent extracts of particles collected from the exhaust of diesel trucks
and automobiles are known to be'mutagenic in bacterial test systems. To
reasonably predict the potential health hazard of diesel exhaust emissions,
differences in toxicity likely to be produced by different cars, or under
various driving conditions, were studied. Extracts of exhaust particles
collected from Oldsmobile, Peugeot, Fiat, Mercedes, Audi and Volkswagen die-
sel automobiles were evaluated for mutagenicity in standard and dinitro-
pyrene-resistant Salmonella tester strains.
EXPERIMENTAL
Diesel exhaust particles were collected from the exhaust of cars operated on
a climate-controlled chassis dynomometer at the Bartlesville (Oklahoma) Ener-
gy Technology Center. Cars were acquired new from the dealer or on loan from
the manufacturer and allowed a 4000 mile break-in period before testing. All
test vehicles were operated according to the EPA Federal Test Procedure (FTP).
To study the influence of driving cycle, exhaust samples were also collected
while driving the Oldsmobile on the Highway Fuel Economy Test (HFET) and the
New York City Cycle (NYCC). The exhaust was diluted in a tunnel sized to
cool the air/exhaust mixture to below 125°F, and the particulate portion col-
lected on 40 x 40 inch Pall flex T60A20 filters. All tests were conducted
with the same standard #2 diesel fuel.
The organic material associated with the exhaust particles was extracted by
ultrasonication in dichloromethane and the extract evaporated to dryness
under nitrogen. A portion of the extract was fractionated by high pressure
liquid chromatography (HPLC) on a Biosil A column and eluted in a gradient
from 94% isooctane to 100% dichloromethane. Only the gamma-1 fraction (1)
-------
was evaluated in these studies. The extracts were evaluated for mutagenicity
in Salmonella strains TA 100, TA 98, or TA 98-1,8-DNPR, without the addition
of a liver enzyme homogenate. Five concentrations of each sample were tested
in triplicate, and the results reported as the slope of the dose-response
curve (revertants/yg) calculated by linear regression analysis. Since re-
sults of mutagenicity testing reflect only the genetic toxicity of the mater-
ial extended from the filters, the results were normalized for differences in
the amount of extractable material associated with the particles (extractable
fraction) and particle emission rates. This provided an estimate of the
amount of mutagenic activity emitted from the exhaust per mile of vehicle
operation (revertants per mile).
RESULTS
Influence of Car Type - Extracts of exhaust particles collected from all six
cars demonstrated direct, dose-related increases in mutagenicity in TA 100
(Table 1). The amount of dichloromethane extractable material associated
with the exhaust particles (extractable fraction) produced by the cars varied
markedly, and was inversely related to mutagenic potency of the extracts.
The particle emission rates varied by about 3-fold in the six cars. Normal-
izing the mutagenic potency for extractable fraction and particle emission
rates yielded "revertants per mile" values of a different ranking than that
shown by the revertants per yg value.
Influence of Driving Cycle - Operating the Oldsmobile on highway, urban and
congested urban driving cycles did not markedly influence the mutagenicity of
exhaust particle extracts but dramatically changed the particle emission rate
and extractable fraction (Table 2). Increasing the extent of stop and start
driving increased the particle emission rate but decreased the amount of ex-
tractable material associated with the particles. Thus estimates of mutagen-
icity emitted from the exhaust (revertants per mile) were similar for the
three driving cycles.
Mutagenicity in a Nitroreductase-Deficient Strain of Salmonella - Because of
the occurrence of nitro-substituted polycyclic aromatic hydrocarbons (PAH) in
diesel exhaust extracts (1) and the extreme potency of nitro-PAH in Salmonel-
la, presumably due to their high nitroreductase activity (2), the extracts
were evaluated in a strain shown to be resistant to the mutagenicity of some
nitro-PAH. Mutagenicity of the gamma-1 HPLC fractions of the extracts, pre-
viously shown to contain nitro-PAH (1), were markedly lower (20-60%) in TA
98-1,8 DNPR than the standard TA 98 tester strain.
CONCLUSIONS
The similar mutagenic potencies of extracts of particles collected from six
different diesel cars operated on the same fuel suggest that engine design
has very little influence on mutagenicity of the particle associated organic
materials. Driving cycle also did not significantly alter mutagenicity of
the particle extracts. The large variability in extractable fraction and
particle emission rates between different cars, and in the same car operated
on different driving cycles, emphasizes the need to include these variables
-------
when estimating the quantities of potentially hazardous materials emitted
from the exhaust. The lower response of nitro-PAH containing fractions of
the extracts in a nitroreductase deficient Salmonella strain is difficult to
interpret since it is not known if the enzymes which activate nitro-PAH to
mutagenic metabolites in Salmonella are unique to bacteria. (Research per-
formed in part under DOE Contract Number DE-AC04-76EV01013.)
REFERENCES
1. Scheutzle, D., F. S. C. Lee, T. J. Prater, and S. B. Tejada. 1981. The
identification of polynuclear aromatic hydrocarbon (PAH) derivatives in
mutagenic fractions of diesel particulate extracts. Int. J. Environ.
Anal. Chem. 9:93-145.
2. Mermelstein, R., K. K. Demosthenes, M. Butler, E. C. McCoy and H. S.
Rosenkranz. 1981. The extraordinary mutagenicity of nitropyrenes in
bacteria. Mutat. Res. 89:187-196.
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Table 1. Influence of Car Type on Mutagenicity of Diesel Exhaust
Particle Extracts
Test Vehicle
Fiat 131
Peugeot 504
Audi 5000
Oldsmobile D-88
VW Rabbit
Mercedes 300
Rev/pg
Extract
(TA-100)
6
13
13
17
16
15
Extractable
Fraction
(*)
71
29
43
20
26
13
Particle
Emissions
(g/mi)
.34
.21
.51
.38
.17
.26
Revertants
per mile
(x 103)
1500
800
2900
1300
700
500
Federal Test Procedure, hot start used for all tests
Table 2. Influence of Driving Cycle on Mutagenicity of Diesel
Exhaust Particle Extracts
Driving
Cycle9
HFET
FTP
NYCC
Average
Speed
(mph)
50
20
7
Rev/yg
Extract
(TA-100)
15
16
13
Extractable
Fraction
(%)
35
21
12
Particle
Emissions
(g/mi)
.22
.33
1.23
Revertants
per mile
(x 103)
1100
1100
1900
a01dsmobile Detla-88 used in all tests
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THE RAPID ANALYSIS OF DIESEL EMISSIONS USING
THE TAGA 6000 TRIPLE OUADRUPOLE MASS SPECTROMETER
by
J.E. Fulford, T. Sakuma, and D.A. Lane
SCIEX, Inc.
Thornhill, Ontario, Canada
Because of the increasing consumption of diesel fuels by cars, trucks,
heavy equipment, and industry, the environmental scientist is concerned with
the atmospheric loading of toxic combustion products and their detrimental
biological effects.
The conventional analysis of diesel fuel combustion products is very
time-consuming and difficult, since the quantity of toxic pollutants such as
polycyclic aromatic hydrocarbons (PAH) and their nitro derivatives is low, and
since they are often associated with other contaminants (for example, unburned
diesel fuel). The analysis entails sample trapping, extraction, substantial
clean-up, and determination by capillary column gas chromatography combined
with mass spectrometry.
Using MS/MS, the particulate extract can be directly analyzed for
m'tropolycylic aromatic hydrocarbons without sample clean-up. The sample is
deposited unto a temperature program direct insertion probe operated at
atmospheric pressure, and is desorbed over a period of twenty minutes. In the
negative mode, parent ions which yield a daughter ion of m/z 46 [N02~] can be
scanned, or target compounds can be quantitated by integrating the desorption
curve or the m/z 46 daughter ion in the multiple ion monitoring mode.
Calibration plots for nitropyrene in spiked samples of diesel particulate
extract are linear (r = 0.99), and the extrapolated detection limit for
nitropyrene in the diesel extract is in the ppb range.
Since the vapor phase emission of diesel engines are also of interest, the
exhaust has been sampled directly by the TAGA 6000 MS/MS. In this study,
emissions of a diesel-powered vehicle were transported through a heated (150°C)
Teflon pipe at 2 L/sec, and a small portion of this flow was admitted directly
into the atmospheric pressure ion source. The mass spectrometric analysis was
based on:
(i) the identification of particular compounds in the exhaust gas;
(ii) the rapid screening of the exhaust gas for nitro compounds;
(iii) the analysis of the exhaust gas for specific nitro-PAH.
-------
COMPOUNDS IN CITY AIR COMPETE WITH 3H-2,3,7,8-TETRACHLORO-
DIBENZO-p-DIOXIN FOR BINDING TO THE RECEPTOR
J.-A. Gustafsson, R. Toftgard, J. Carlstedt-Duke, G. Lofroth
Dept. of Medical Nutrition and Pharmacology,
Karolinska institute, S-104 01 Stockholm, and Dept. of
Radiobiology, University of Stockholm, S-106 91 Stockholm
It.is well known that filter collected urban particulate matter contain
compounds which are mutagenic in the Ames'' Salmonella assay in the absence of
rat liver metabolic activation, showing that these compounds are different
from conventional polycyclic aromatic hydrocarbons (PAH). In the present
study, we have shown that such particulate matter also contains compounds with
an affinity for the rat liver receptor protein which specifically binds
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The content of conventional PAH
cannot account for the degree of binding.
Urban particulate matter used in the investigation was collected on glass
fiber filters by high volume sampling at roof top levels in central Stockholm
and a suburban site. Collection, Soxhlet extraction with acetone, extract
preparation in dimethyl sulfoxide (DMSO) and mutagenicity testing of the ex-
tracts in the Salmonella assay have been described elsewhere. The affinity of
the particulate fraction of the air samples to the rat liver receptor protein
was measured by competition for the binding of H-TCDD, as described below.
Liver cytosol was prepared and diluted to 3.5 A9Rf, ,.. n/ml 2-2.5 mg
protein/ml). Each experiment consisted of a series of ten incubations of one
ml of cytosol with 1.5 nM H-TCDD. Two experiments were carried out in the
presence of 20 yl of DMSO (total binding). Two incubations contained 150 nM
radioinert 2,3,7,8-tetrachlorodibenzofuran (TCDBF) (non-specific binding).
The remaining six incubations contained varying amounts of the extract of the
particulate fraction of an air sample. 2.5-20 pi of the extracts, dissolved
in DMSO, was added to the incubation mixture and the volume of DMSO was then
adjusted to 20 yl/incubation. After incubation at 0 C for 60 min, the incuba-
tions were treated with dextran-coated charcoal and the amount of H-TCDD
bound, to the receptor in each of the ten incubations was measured by iso-
electric focusing carried out in golyacrylamide gel as described previously.
The non-specific binding of H-TCDD was obtained from the two incubations
in the presence of 150 .nM radioinert TCDBF and was subtracted from the total
binding in each of the other incubations (= specific binding). The specific
binding of H-TCDD in the presence of extracts of the air samples was express-
ed as a percentage of the specific binding in the control incubation ( H-TCDD .
+ DMSO). The relative binding affinity for the individual air samples was cal-
culated from log-logit plots of the competition for TCDD-binding, where logit
b = ln(6/l-6). The log-logit plots were calculated using linear regression
analysis. Each extract was analysed 3-4 times at a suitable dilution.
An unused air filter (blank filter) was extracted in the same manner as
-------
the other samples and tested for competition for receptor binding. The results
were expressed as an equivalent volume of air. Benzo(a)pyrene and TCDBF were
dissolved in dioxane and the competition for binding to the receptor measured.
The results are shown in Table 1, in which are given the mutagenic effects
in Salmonella TA 98 and TA 100 in the absence of mammalian metabolic activation
and the relative binding affinities expressed as the concentration of air sample
extract that competes for 50% of the TCDD-binding to the receptor (ED,-0; m
air/ml cytosol). Log-logit plots for the different air samples indicated
that they competed for the same binding site. There seemed to be a gross
correlation between the binding affinity and the pollution level as measured
by mutagenic effects. Samples collected in the summer had higher ED,, "s than
samples collected in the winter and spring.
Several compounds are known to bind to the receptor protein including many
PAH present in urban particulate matter. Some of the particulate samples were
analysed for PAH. Assuming that all PAH present at concentrations above 0.1
ng/m have the same binding affinity for the receptor protein as benzo(a)pyrene
(B(a)Pl, it can be calculated that the ED _ value should be 1.7, 15.3 and
0.07 m of air for sample 148, 173 and S-258, respectively. The observed
values were 0.035, 0.14 and 0.015 m , i.e. known PAH may account for about
2.4, 0.8 and 22.4% of the binding, indicating that other types of compounds
are of major importance.
TCDD and TCDBF have the highest binding affinities for the receptor among
investigated compounds. Two samples have been analysed for TCDD and TCDBF, and
the concentrations were below the detection levels of 2 pg/m . Consequently,
these compounds cannot account for the binding.
The affinity of a compound for the TCDD receptor is well correlated to the
magnitude of aryl hydrocarbon hydroxylase (AHH) induction caused by that
compound. Strains of mice with high AHH inducibility are more susceptible to
pulmonary cancers caused by 3-methylcholanthrene than strains with low induci-
bility, indicating a link between AHH activity and appearance of pulmonary
tumors. The rat lung has a high content of receptor protein, and the capability
of the human lung to metabolise B(a)P indicates the presence of the receptor
protein also in this tissue. Although TCDD has been shown to be a potent
carcinogen in chronic feeding studies, it is apparent that TCDD shows no or
very little mutagenic activity in in vitro bacterial test systems such as
the Ames' test and a very low covalent binding to DNA in vivo. In a recent
study, however, it was shown that TCDD is a potent promoting agent for
hepatocarcinogenesis initiated by diethylnitrosamine. TCDD and possibly also
other compounds with an affinity for the same receptor may thus better be
described as cocarcinogens and tumor promoters rather than carcinogens. The
presence of compounds with affinity for the receptor in urban particulate
matter may be of importance with regard to the health implications of urban
air pollution. This type of compounds may or may not be similar to the
components that are mutagenic in the absence of mammalian metabolic activation
in the Ames' Salmonella assay.
Conclusions
Acetone extracts of filter-collected urban atmospheric particulate
matter contain compounds which can displace H-2,3,7,8-tetrachlorodibenzo-
p-dioxin from the rat liver receptor protein. The concentration of conven-
tional polycyclic aromatic hydrocarbons or chlorinated dioxins and dibenzo-
furans cannot account for more than 0.8-22% of the displacement.
-------
Table 1. Competition for TCDD receptor-binding and mutagenicity in Salmonella
TA 98 and TA 100 in the absence of mammalian metabolic activation by extracts
of filter-collected urban particulate matter. Samples were collected at roof
top levels for 24 h starting about 6 a.m. Samples 148 and 174 were collected
at a suburban site 22 km NNW Stockholm and the others in central Stockholm.
The ED,.Q is the concentration of competitor that competes for 50% of the spe-
cific binding of H-TCDD. The ED "s (in nM) for 2,3,7,8-tetrachlorodibenzo-
furan (TCDBF), benzo(a)pyrene (Bla)P), 3-naphthoflavone (BNF) , 3-methylchol-
anthrene (3-MC) and benz(a)anthracene (BA) are given for comparison. The muta-
genic response is highest in the absence of mammalian metabolic activation and
decreases by addition of S9 from rat liver; the decrease is dependent on the
amount of S9 added. Samples 258, 264 and 268 were tested for mutagenicity both
prior to (Feb. 1980) and after (Sept. 1980) the completion of the TCDD recep-
tor analyses; there were no detectable changes in the mutagenic response.
Sample
149
148
173
174
S-258
T-262
S-264
T-268
Blank
filter
TCDBF
B(a)P
BNF
3-MC
BA
Q
Receptor affinity
EV
(m air/ml cytosol)
0.049 - 0.014
0.035 - 0.017
0.137 - 0.073
0.302 - 0.147
0.015 - 0.007
n.d.
0.039 - 0.010
0.049 - 0.026
2.18 - 0.640
2.69 - 1.91 nM
18.21 - 9.88 nM
7.0 nM
2.9 nM
3.8 nM
Mutagenic response Sampling date
/ / 3, and site
(revertants/m )
TA 98 TA 100
19 11 79 04 10 inner city
14 8 79 04 10 suburban
3 n.d. 79 07 05 inner city
1 n.d. 79 07 05 suburban
59 79 80 02 04 inner city
92 102 80 02 05 inner city
30 20 80 02 06 inner city
9 7 80 02 07 inner city
<0.2 <0.4
values represent the means - standard deviation (three to four determina-
tions) .
Blank filter extracted with the same volume of acetone. The competition is
expressed as an equivalent volume of air.
n.d. Not determined.
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GC/MS AND MS/MS STUDIES OF DIRECT-ACTING MUTAGENS IN DIESEL EMISSIONS
by
T. R. Henderson, J. D. Sun, R. E. Royer and C. R. Clark
Lovelace Inhalation Toxicology Research Institute
Albuquerque, New Mexico
T. M. Harvey and D. F. Hunt
Department of Chemistry
University of Virginia
Charlottesville, Virginia
J. E. Fulford, A. M. Lovett, and W. R. Davidson
Sciex, Inc.
Toronto, Canada
While the direct-acting mutagens in diesel emissions have not been un-
equivocally identified, evidence has accumulated showing that nitro-PAHs
(polycyclic aromatic hydrocarbons) may be a major source of mutagenic activ-
ity (1,2). We have found that diesel fuel-PAHs are devoid of mutagenic
activity in Salmonella test strains. After reaction with N02, the nitro-
fuel-PAH mixtures are direct-acting, frameshift mutagens (200-1000 revert-
ants/ug) in TA98, but were not active in TA1535, a base-pair substitution
indicator strain (2). The cytotoxicity to CHO cells also increases after
reaction with N02(2). These biological responses are very similar to
those observed with diesel soot extract fractions although the activity of
diesel extracts is much lower.
The fuel-nitro-PAH mixtures have been found useful as a positive control
and as a reference mixture for MS/MS (triple stage quadrapole mass spectrom-
etry) analyses. Although the fuel-nitro-PAHs are not resolvable by capil-
lary GC/MS, the fuel-PAHs before reaction with N02 are readily separated
and identified by GC/MS. Since the fuel-PAHs extracted with DMSO (M62SO)
are readily identified by GC/MS, correlations can be made with the nitro-PAH
masses observed in MS/MS spectra of nitro-PAH mixtures. These are used for
MS/MS interpretation since MS/MS does not differentiate between isomers of
the same molecular weight in most cases. When possible, these interpreta-
tions should be confirmed by independent methods (1).
MS/MS was carried out to compare the relative intensities of ions in
samples of widely different mutagenic potencies. In this way, it may be
possible to estimate which of the nitro-PAHs detected in diesel soot ex-
tracts (1) may make major contributions to the total mutagenic activity.
-------
The MS/MS analyses involved: R-NO^ R-NOg-H"1" (ionization under
isobutane chemical ionization conditions) + R-N+0 (collisionally induced
dissociation by collision with N£ in quadrupole #2). The MS/MS instrument
was a modified Finnigan 3200, which has been described previously(S). The
first quadrupole was scanned from 80 to 350 amu with a scan time of
1.3 sec. The second quadrupole was operated (with RF voltage only) as a
collision chamber. The third quadrupole was scanned the same as #1, but
17 amu behind it. In this way, only ions which lost 17 amu in passing
through the collision chamber were detected. The instrument was tuned with
1-nitropyrene (parent-H+ ion m/z 248) and the N2 pressure adjusted for
maximum m/z 231 daughter ion (m - 17). Extract samples (40 ug) were vola-
tilized into the source using a thermal desorber (temperature programmed
from 50 to 350° C in 10 minutes).
MS/MS analyses were done on selected samples with an APCI/MS/MS (triple
quadrupole MS/MS with an atmospheric pressure chemical ionization source) at
the laboratories of Sciex, Inc., Toronto, Canada. The reaction monitored
was:
-
R-N02 + 02 - R-M02 -j R + NO 2
The first quadrupole was scanned over a 100 to 300 amu range or single ion
monitoring was done at m/z 247". The third quadrupole was set for con-
stant daughter scans at m/z 46". The corona discharge current was
6 uamp constant current at 4 to 6 kV and an ion energy of 65 eV. Thus in
this reaction, only negative ions of 100 to 300 molecular weight were de-
tected which yielded N02 on collision with argon gas.
Diesel soot extracts were fractionated with DMSO to yield aliphatic,
aromatic and polar fractions for MS/MS analyses. This fractionation method
has been described previously (4-6). The aromatic fraction from a single-
cylinder Swan diesel engine was used for comparison with an Oldsmobile ex-
haust aromatic fraction because of differences in mutagenic activity. The
aromatic fraction recovered by DMSO fractionation of diesel soot extracts
typically contains 50-80% of the direct mutagenic activity of different ex-
tracts and is concentrated 5- to 10-fold in specific activity. The aromatic
fraction of diesel exhaust is very similar to the y fraction isolated by
Schuetzle .et _a]_. (1) in containing mononitro-PAHs and dinitro PAHs of 2 ring
PAHs.
The mutagenic activities of the extracts in TA98 (no S-9) under standard
Ames bioassay conditions (7) were: nitro-fuel -PAHs 435 rev/ug; Swan
diesel soot aromatic fraction--57 rev/up; Olds diesel soot aromatic frac-
tion--21 revertants/yg. These samples, differing by about 20-fold in
mutagenic specific activity, were compared by MS/MS. The unfractionated
extracts were low in the intensity of certain (M - 27) ions, the polar frac-
tions contained only a minor part of the total mutagenicity (< 20%) and
the aliphatic fractions contained very low intensities of nitro-PAH ions.
Figure 1 shows that the even-mass ions (dinitro-PAHs) appeared to cor-
relate with increased mutagenic activity of the three extracts, while odd-
mass ions (mononitro-PAHs) negatively correlated with mutagenic activity
-------
with the exception of m/z 171. The even mass ions of particular signifi-
cance and their tentative identification were: m/z 252, dinitrophenan-
threnes; m/z 256, dinitromethylbiphenyls; m/z 230, 244 and 258, dinitro-
naphthalenes containing 2, 3 and 4-methyl groups. These compounds might be
particularly important in the total mutagenicity of diesel soot extracts.
Mitropyrenes (m/z 231) did not significantly correlate with differences in
the mutagenicity of these samples, although these compounds may account for
5-10% of'the total mutagenicity(4).
Figure 1 also shows that the same nitro-PAH (M - 17) masses were present
in all three types of samples. The fuel-nitro-PAHs, having been treated
with excess N02, appeared to be lower in mono-nitro-PAH type masses and
higher in the even masses (dinitro-PAHs) discussed above. Since most of the
nitro-PAH ions present in diesel exhaust are also present in fuel aromatic
fractions treated with M02 and these masses are relatable to the PAHs
present in the fuel burned by the two engine types, this suggests that the
fuel PAHs contribute to the exhaust nitro-PAHS. One possible mechanism is
reaction of unburned fuel PAHs with N02-
Studies involving the addition of pyrene to the fuel for the single
cylinder diesel engine was performed to further test the hypothesis that
fuel PAHs may contribute to the formation of nitro-PAHs in exhaust soot ex-
tracts. It was found that addition of 0.01 to 1.0% w/v pyrene to diesel
fuel (less than 0.01% pyrene by GC/MS) resulted in increased pyrene/phenan-
threne ratios in soot extracts. With no addition of pyrene, this ratio was
less than 2, but increased to-12 with 1% pyrene addition to the fuel. The
mutagenicity in TA100 increased 2 to 3 fold by 0.01 to 0.1% pyrene addition,
but decreased somewhat with 1% pyrene addition. MS/MS analyses of these
extracts by APCI/MS/MS showed increased ion intensities of mono- and
dinitropyrenes in the soot extract for pyrene additions up to 0.1% in the
fuel, but the intensities of dinitropyrenes decreased at 1% ovrene addition
to the fuel. (Supported in part by U.S. Department of Energy under DOE
Contract No. DE-AC04-76EV01013.)
-------
REFERENCES
Schuetzle, D., T. Riley, T. J. Prater, T. M. Harvey and D. F. Hunt.
1981. The identification of nitrated derivatives of PAH in diesel
participates. Anal. Chen., in press.
Henderson, T. R., A. P. Li, R. E. Royer and C. R. Clark. 1981.
Increased cytotoxicity and mutagenicity of diesel fuel after reaction
with N02« Environ. Mutag. 3: 211-220.
Hunt, D. F., J. Shabanowitz and A. B. Giordani. 1980. Collision
Activited Decompositions of negative ions in mixture analysis with a
triple quadrupole mass spectrometer. Anal. Chem. 52: 386-390.
Henderson, T. R., R. E. Royer and C. R. Clark. 1981. MS/MS
Characterization of Diesel Emissions. Proceedings of 29th Annual
Conference of Mass Spectrometery and Allied Topics (in press-extended
abstract).
Henderson, T. R., C. R. Clark, R. L. Hanson and R. E. Royer. 1980.
Fractionation of environmental organic extracts with dimethylsulf-
oxide. Applications to diesel exhaust particulates. Proceedings of
28th Annual Conference on Mass Spectrometry and Allied Topics, p.
243-244. (extended abstract).
Henderson, T. R., C. R. Clark, T. C. Marshall, R. L. Hanson and C. H.
Hobbs. 1981. Heat degradation studies of Solar Heat Transfer
Fluids. Solar Energy (in press).
Ames, B. N. 1979. Identifying environmental chemicals causing
mutations and cancer. Science 204: 587-593.
-------
io
CO 10
z
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10
i
o
D
O
I
171
230
O 244
209
I
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o
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a o
* 6
k
258
256
252t
OT
231 8J
n DA
\fl
?
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1
O
a
ISO I70 I90 2IO 230
MASS (M/Z)
250
270
290
FIGURE 1
Figure 1. MS/MS Comparisons of Nitro-PAHs from Diesel Fuel and Exhaust
Filter Extracts. --A-- Nitro-fuel-PAHs; -a Aromatic
Fraction from Swan Engine Exhaust Soot Extracts; --0-- Aromatic
Fraction, Olds Exhaust Soot Extracts. The masses represent the
(M - 17) ions, the parent nitro-PAH being 17 amu more. The ion
intensity is the total ion counts summed over the entire run,
usually 300 scans.
-------
EVALUATION OF THE RELEASE OF MUTAGENS AND 1-NITROPYRENE FROM DIESEL PARTICLES
IN THE PRESENCE OF LUNG MACROPHAGE CELLS IN CULTURE
by
Leon C. King, Silvestre B. Tejada, and Joellen Lewtas
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
Diesel particles have been shown to contain organic components which are
mutagenic in short-term bioassays (1). Nitroaromatics including 1-nitropyrene
now appear to account for a portion of the mutagenic activity observed in these
organics (2). It is important to determine whether these potentially
carcinogenic components are released from particles under physiological
conditions. We have shown in a previous study (3) using the Ames Salmonella
typhimurium plate incorporation assay that serum and lung cytosol were very
effective in removing mutagenic activity from diesel particles (3). This was
determined by measuring the activity extractable from the particles before and
after treatment of the particles.with serum or cytosol. Direct addition of
serum or lung cytosol to these organics reduced the mutagenic activity
detectable by 80 to 90%. The objective of this study was to evaluate the
removal and release of mutagenic activity and 1-nitropyrene from diesel
particles in the presence of lung cells in culture. The lung cells used in
these studies were alveolar macrophages obtained from rabbits by lung lavage.
The diesel particles used in this study were obtained from a Datsun Nissan
220C, 4-cylinder passenger car previously described (3). The particles were
sonicated in tissue culture medium (M199 with Hank's Salts) at 37°C for 30 min
to deagglomerate and disperse the particles. Particle size analysis showed the
majority of the particles to be 2.0 to 2.5 un after this treatment.
Rabbit alveolar macrophage (RAM) cells were lavaged and cultured with the
diesel particles according to previously published procedures (4) except that
the final serum concentration was reduced to 10% and the culture time was
increased to 40 h. In brief, RAM cells were added to individual wells of
cluster dishes containing suspensions of the diesel particles from 15 to
1,500 yg/ml. Under these conditions, at concentrations of diesel particles
above 75 yg/ml, over 95% of the particles were phagocytized after 20 h of
culture. After 40 h of culture, cells were harvested by trypsinization. The
final exposure conditions for the mutagenesis and 1-nitropyrene analyses were
selected to maximize cellular exposure to the diesel particles while minimizing
cellular toxicity. The final concentration of diesel particles selected was
375 yg of particles/ml. At this concentration, RAM cells engulf over 95% of
the particles and after 40 h of exposure, less than 7% cell lysis was observed
and cell viability was 63% of the control cultures.
-------
In order to evaluate the effect of the lung macrophage cells on the
removal of mutagens and 1-nitropyrene from diesel particles, particles were
exposed to the culture medium at 375 pg/iiil with and without lung macrophages
and cultured for 40 h. After incubation the medium control treatment dishes
were combined and the medium was separated by centrifugation from the particles
which were washed once with water. The harvested macrophages were separated
from the culture medium and sonicated to release engulfed particles.
Dichloromethane and methanol (DCM:MeOH, 1:1) were used to extract the medium,
free particles, engulfed particles, cell sonicate, and water washes. Each of
these fractions was analyzed for 1-nitropyrene by LC/fluorescence (5) and
compared to untreated particles extracted with DCM:MeOH.
Each of the fractions was also assayed for mutagenicity in the Ames
Salmonella typhimurium plate incorporation assay in TA98 as previously
described (3).The media, cells, and washes were assayed without extraction
and due to a high background of activity in both the medium and cells, no
detectable activity was observed as a function of the treatment group.
Mutagenicity was detected in the DCM:MeOH extract of the macrophage-engulfed
particles; however, it was only 2% of the mutagenicity originally present on
the particles. Comparison of this activity with that of the media control
particles, in which 6% of the mutagenicity was recovered, showed that the
presence of macrophages decreased the mutagenicity 62%.
Nitropyrene analysis of the medium control group showed 96% recovery of
the 1-nitropyrene with 31% of the nitropyrene found in the medium and washes
and 66% remaining on the particles. Significantly less 1-nitropyrene was
recovered in the macrophage treatment group (76%). Since over 95% of the
particles were phagocytized and only 27% of the original 1-nitropyrene was
recovered from the engulfed particles, it appears that the macrophages may have
metabolized the 1-nitropyrene to a nondetectable form.
Comparison of the recoveries shows a greater loss of mutagenicity than
1-nitropyrene suggesting that other compounds detected in these particles may
contribute more to the mutagenicity than the 1-nitropyrene alone.
REFERENCES
1. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. Tejada,
J. Bumgarner, F. Duffield, M. Waters, U. Simmon, C. Hare, C. Rodriguez,
and L. Snow. 1978. Application of short-term bioassay to the
characterization of diesel particle emissions. In: 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 L. Claxton, eds. Plenum Press: New York.
pp. 383-418.
-------
2. Nishioka, M., B. Peterson, and J. Lewtas. 19P1. Comparison of nitro-PNA
content and mutagem'city of diesel emissions. Presented at the
U.S. Environmental Protection Agency Diesel Emissions Symposium,
Raleigh, North Carolina.
3. King, L.C., M.J. Kohan, A.C. Austin, L.D. Claxton, and J.L. Huisingh.
1981. Evaluation of the release of mutagens from diesel particles in
the presence of physiological fluids. Environ. Mutagen. 3:109-121.
4. Garrett, N.E., .J.A. Campbell, H.F. Stack, M.D. Waters, and Joellen Lewtas.
1981. The utilization of the rabbit alveolar macrophage and Chinese
hamster ovary cell for evaluation of the toxicity of particulate
material. Environ. Res. 24:345-365.
5. Tejada, S.B., R.B. Zweidinger, J.E. Sigsby, Jr., and R.L. Bradow. 1981.
Identification and measurement of nitro derivatives of PAH in diesel
exhaust particulate extract. Presented at the Chemical Characterization
of Diesel Exhaust Emissions Workshop, Dearborn, MI.
-------
BACTERIAL MUTAGENICITY OF A DIESEL EXHAUST EXTRACT AND TWO ASSOCIATED
NITROARENE COMPOUNDS AFTER METABOLISM AND PROTEIN RINDING
by
Mike Kohan and Larry Claxton
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
Previous work has demonstrated that nitroarenes are associated with
organic extracts from diesel exhaust (1,2,3). This study was designed to
characterize two nitroarenes and to determine whether these compounds
demonstrate the same type of mutagenic response as diesel exhaust organics.
An extract of the particulate from exhaust of a VW diesel automobile,
2,7-dinitrofluorenone, and 1-nitropyrene were tested in the Salmonella
mutagenicity assay using strain TA98 and two nitroreductase-deficient strains
(TA98FR1 and TA98/l,8DNPs) to establish a proper dose for a multivariant
experiment. Each of the three samples was tested with and without a 9000g liver
homogenate (S9) prepared from Arbclor 12F>4-induced rats. In the multivariant
experiment, the following treatments (both with and without the
NADPH-generating system) were used:
(a) no activating system (-S9),
(b) microsomes derived from the original S9 by centrifuging for
90 min at lOO.OOOg,
(c) the cytosol fraction of the original S9, and
(d) boiled S9.
In addition, the samples were tested in the presence of both boiled and
unboiled bovine serum albumin (BSA). Salmonella tester strains TA98 and
TA98FR1 were used in the multivariant study. These treatments were used to
determine the similarities of the three samples in the presence and absence of
treatments with differing enzymatic and protein binding capabilities. This
work is an extension of similar efforts by Wang et al. (4) and Pederson et
al. (5).
The results using the diesel exhaust sample are seen in Table 1. When
compared to the untreated (no activating system) situation, all of the
treatments with one exception gave reduced mutagenic activity. Only the
cytosol fraction gave an increase in activity. In comparison to the
non-activated situation the cytosol activation gave a relative increase to 111%
-------
for strain TA98 and to 164% for strain TA98FR1. In addition, it was noted that
TA98 was the strain most responsive to the diesel exhaust extract, followed by
TA98FR1, and then TA98/1,8DNP6. 1-Nitropyrene activity was decreased by half
in TA98/1,8DNP6 but almost abolished in TA98FR1.
As in the diesel exhaust particulate sample, the mutagenic activity of
2,7-dinitrofluorenone was reduced in the majority of the different treatments.
An increase to 170% of the untreated situation was seen when using the cytosol
fraction with strain TA98FR1.
A reduction in the mutagenic activity of 1-nitropyrene was observed in all
of the treatments without the generating system. However, in contrast to
2,7-dinitrofluorenone and the diesel exhaust particulate sample, a twenty-fold
increase in the mutagenic activity of 1-nitropyrene was seen with the microsome
treatment when using the generating system and strain TA98FR1. Smaller
increases in activity were also observed with the S9 and cytosol fractions when
using this same strain and generating system. The mutagenic activity of
1-nitropyrene also was increased by the microsome fraction with the generating
system in strain TA98. This microsomal activation of 1-nitropyrene, in
addition to the response pattern obtained with the nitroreductase-deficient
tester strains, indicates that 1-nitropyrene may not be the major mutagenic
component in this sample of extractable organics from VW Rabbit Diesel particle
emissions.
REFERENCES
1. Pederson, T.C., and J.S. Siak. 1981. The role of nitroaromatic compounds
in the direct-acting mutagenicity of diesel particle extracts. J. Appl.
Toxicol. 1:54.
2. Claxton, L.D., and J.L. Huisingh. 1980. Comparative mutagenic activity
of organics from combustion sources. In: Pulmonary Toxicology of
Respirable Particles. Proceedings of the Nineteenth Annual Hanford Life
Sciences Symposium at Richland, WA. P.L. Sanders, F.T. Cross,
G.E. Dable, and J.A. Mahaffey, eds. pp. 453-465.
3. Lofroth, G. 1980. Salmonella/microsome-mutagenicity assays of extract
from diesel and gasoline-powered motor vehicles. In: Health Effects of
Diesel Engine Emissions: Proceedings of an International Symposium.
EPA-600/9-80-057a, Vol. 1. U.S. Environmental Protection Agency:
Cincinnati, OH. pp. 327-342.
4. Wang, Y.Y., R.E. Talcott, D.A. Seid, and E.T. Wei. 1980. Antimutagenic
properties of liver homogenates, proteins, and glutathione on diesel
exhaust particulates. Cancer Lett. 11:265-275.
5. Pederson, T.C., and J.-S. Siak. 1981. The activation of mutagens in
diesel particle extract with rat liver S9 enzymes. >1. Appl. Toxicol.
1:61-66.
-------
Table 1. Mutagenic Activity of Diesel Exhaust Particle Extracts
Using Different Treatment Conditions
Without Generating System With Generating System3
Treatment TA9S TA98FR1 TA98 TA98FR1
Untreated
S9
Microsomes
Cytosol
Boiled S9
BSA
Boiled BSA
1039
467
781
687
595
354
343
(ino)b
(45)
(75)
(66)
(57)
(33)
(33)
788
242
488
332
369
264
232
(ino)c
(31)
(62)
(42)
(47)
(34)
(29)
inni
696
826
inn
577
(inn)
(70)
(82)
(111)
(58)
680
649
557
1118
305
(inn)
(95)
(*2)
(164)
(45)
^NADPH-generating system: NADPH, G-6-P, MgCL?, and KCL.
"Average net revertants per plate at 100 ug organic extract.
cRelative response to untreated sample expressed as percent.
-------
CHARACTERIZATION OF PARTICIPATE EMISSIONS
FROM IN-USE GASOLINE FUELED MOTOR VEHICLES
by
John M. Lang, Roy A. Carlson, Linda Snow
Northrop Services, Inc.
Research Triangle Park, North Carolina
Frank M. Black, Roy Zweidinger, Silvester Tejada
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
One of the primary tasks of those concerned with the study of environmental
quality is estimating population exposure to air pollutants and determining
the risk associated with exposure. Mobile sources generally contribute
significantly to the population's exposure to hydrocarbons (HC), carbon
monoxide (CO), oxides of nitrogen (NO ), and fine particulate matter.
Much of the mobile source data available in the literature has been
obtained from well-maintained engineering test vehicles. However, emissions
from consumer-operated vehicles can vary considerably from well-maintained
vehicles.
Because of growing interest in diesel power for light-duty motor vehicles,
it has become necessary to study particulate emissions comprehensively
to determine the potential impact of dieselization on the public health.
Assessment of exposure and risk requires knowledge of the emission rates
and composition of diesel particulate matter and of the gasoline particu-
late matter being replaced by diesels.
Our study examined particulate emissions from twenty consumer-operated,
light-duty gasoline fueled cars and trucks. The emissions characteristics
observed were compared with those previously reported by Gibbs, et al.,
for nineteen consumer-operated, light-duty diesels (1).
A test fleet of twenty light-duty gasoline passenger cars and trucks was
obtained from local residents and rental agencies. Four of the vehicles
were fueled by leaded gasoline; the remaining sixteen fueled by unleaded
-------
gasoline. Vehicles from model years 1970 to 1981 were represented and
ranged in mass from 907 kg (2,000 Ibs) to 2,268 kg (5,000 Ibs) and
accumulated mileage from 565 km (353 mi.) to 130,490 km (81,559 mi.). A
variety of emission control configurations were represented, including
oxidation catalysts, oxidation catalysts with air pumps, and three-way
catalysts. All vehicles were tested as received.
Three gasoline fuels were purchased locally for the program; two leaded
and one unleaded. Road load simulation was achieved with a Clayton
CTE-50-0 direct-drive, water brake dynamometer. The vehicles were tested
with a daily routine involving a cold-start Federal Test Procedure (FTP)
cycle followed by repetitive Highway Fuel Economy Test cycles (HWFET).
The FTP simulates city driving after the car has not been started for at
least twelve (12) hours. The HWFET simulates highway driving after the
car has been warmed up.
A positive-displacement pump Constant Volume Sampling (CVS) system
collected the exhaust gases and allowed dilution air to flow through the
sample train in order to maintain the dilution exhaust temperature at or
below 52° C (125° F). Before entering the CVS system, the dilution
exhaust flow passed through three 20 x 20 inch filters for collection
of particulate matter (2). Pallflex T60A20 PTFE glass fiber filters were
used for collecting the particulate.
The 20 x 20 filters were soxhlet extracted for eight hours with dichloro-
methane (DCM) to remove the soluble organic fraction (SOF). The extracted
organics were examined for nitro-pyrene (NCL-P), benzo(a)pyrene (BaP),
and pyrene (Py) content by high pressure liquid chromotography (HPLC),and
bioassayed with Ames Salmonella strain TA-98 for mutagenic potency. The
activity (revertants/ microgram SOF) was determined as the slope in the
linear portion of the dose-response curve. Five organic doses, 0 (solvent
blank) to 100 ug, were used to define the dose-response curve.
A review of the regulated emission rates indicate that the test fleet
included properly functioning vehicles and vehicles with a variety of
emission control malfunction conditions. HC emission rates ranged from
0.07 to 24.5 g/mi., CO had a range of 0.08 to 60.95 g/mi., and NO was
from 0.37 to 6.43 g/mi.. Comparison with the diesels indicate tha£ the
gasoline fleet average emission rates exceed the diesel rates and a
broader range was seen in the gasoline data.
The total particulate emission rates from leaded gasoline vehicles were
2.7 times greater during the HWFET than the FTP. On the other hand,
HWFET total particulate emission rates from unleaded gasoline vehicles
were 81% of the FTP value. Overall, the leaded vehicles emitted more
particulate than the unleaded vehicles during both cycles, 3.2 times
more during the FTP and 10.8 times more during the HWFET.
The light-duty diesel total particulate emission rates reported by
Gibbs, et al., compared to the gasoline vehicle rates as follows:
(FTP) diesel ^5.9 x leaded rate; 19.1 x unleaded rate.
(HWFET) diesel ~ 1.3 x leaded rate; 13.5 x unleaded rate.
-------
Fleet average DCM soluble particulate organic emission rates during the
FTP and HWFET compared with the light-duty diesels as follows:
(FTP) diesel^ 5.9 x leaded rate; 8.6 x unleaded rate.
(HWFET) diesel "~ 3.4 x leaded rate; 6.9 x unleaded rate.
Nitro-pyrene emission rates were similar for leaded and unleaded gas-
oline vehicles. Diesels emit about 20 to 30 times as much NCL-P as
gasoline vehicles.
Gasoline vehicle BaP emissions were greater during cold-start FTP driving
than during either hot-start FTP or HWFET driving. Functioning catalyst
systems appeared to effectively reduce polynuclear aromatic hydrocarbon
(PAH) emissions. During the cold-start FTP, BaP emission rates from
leaded vehicles exceeded BaP rates from diesels; BaP emission rates from
unleaded vehicles were similar to the diesel results. During the HWFET,
BaP emission rates from both categories of gasoline vehicles were less
than the rates from diesels.
Generally, the Ames TA-98 mutagenic activity of the gasoline particulate
organics was higher with metabolic activation than without metabolic
activation. Ames TA-98 revertant per mile levels were substantially
higher for the leaded gasoline vehicles than for the unleaded gasoline
vehicles under both FTP and HWFET conditions.
The higher total particulate and particulate organic emission rates of
diesels are compensated somewhat by lower Ames TA-98 mutagenic activities
when compared to gasoline vehicles. Relative activities (without metabolic
activation) were as follows:
(FTP) diesel^- 0.6 x leaded activity; 0.5 x unleaded activity.
(HWFET) diesel^ 0.4 x leaded activity; 0.4 x unleaded activity.
Fleet average Ames TA-98 revertant per mile levels (without metabolic
activation) for FTP and HWFET conditions compared with light-duty
diesels as follows:
(FTP) diesel^ 3.4 x leaded rate; 12.1 x unleaded rate.
(HWFET) diesel^ 1.5 x leaded rate; 9.1 x leaded rate.
In conclusion, replacing the gasoline passenger cars represented by the
test fleet of this program with diesel passenger cars would decrease HC,
CO, and NO population exposures, and increase total particulate and
mutagenic particulate organic exposures (as indicated by Ames Salmonella
strain TA-98).
-------
Table 1. Characterization of Particulate Emissions,
Diesel versus Gasoline
Diesel *
FTP HWFET
Total participate, mg/mi.
Dichloromethane soluble organics, mg/mi.
Benzo-a-pyrene, ug/mi.
Nitro-pyrene, ug/mi.
TA-98,-S9, rev/mg
TA-98,+S9, rev/ug
TA-98,-S9, rev/mi. (x 10"3)
TA-98,+59, rev/mi. (x 10"3)
607(1)
124(1)
4.5(3-7)
7.4(8)
4.1(1)
**
509(1)
**
345(1)
79.7(1)
2.7(3-7)
6.8(8)
3.0
**
239
**
Leaded
FTP HWFET
103
21.1
14.5
0.20
7.31
12.5
152
258
276
23.5
0.89
0.39
8.55
10.6
163
232
Unleaded
FTP HWFET
31.7
14.4
3.3
0.24
7.57
13.4
42.1
79.3
25.6
11.5
0.61
0.16
7.39
7.43
26.4
25.2
* Number in parenthesis indicates reference.
** Information unavailable at this time.
-------
REFERENCES
1. Gibbs, R.E., J.D. Hyde, and S.M. Byer. Characterization of particulate
emissions from in-use diesel vehicles. SAE paper number 810081,
February, 1981.
2. Killough, P., and J. Watson. Filter-type, high volume particulate
samples for automotive diesel emission studies. ES-TN-79-13. Northrop
Services, Inc., December 1979.
3. Huisingh, J.L., and R.L. Bradow, R.H. Jungers, B.D. Harris, R.B.
Zweidinger, K.M. Gushing, B.E.Gill, and R.E. Albert. Mutagenic and
carcinogenic potency of extracts of diesel and related environmental
emissions: study design, sample generation, collection and preparation.
Health Effects of Diesel Engine Emissions: Proceedings of an Inter-
national Symposium, EPA-600/9-80-056b. November 1980.
4. Kraft, J., and K.H. Lies. Polycyclic aromatic hydrocarbons in the
exhaust of gasoline and diesel vehicles. SAE paper number 810082.
February 1981.
5. Williams, R.L.,and D.P. Chock. Characterization of diesel particulate
exposure. Health Effects of Diesel Engine Emissions: Proceedings of
an International Symposium. EPA-600/9-80-057a. November 1980.
6. Hare, C.T., and T.M. Baines. Characterization of particulate and
gaseous emissions from two diesel automobiles as a function of fuel
and driving cycle. SAE paper number 790424. February 1979.
7. Williams, R.L., and S.J. Swarin. Benzo(a)pyrene emissions from
gasoline and diesel automobiles. SAE paper number 790419.
February 1979.
8. Tejada, S.. Particulate NO -pyrene emissions from a 1980 Oldsmobile
and a 1980 VW diesel, FTP and HWFET cycles. Unpublished data. June
1981.
-------
SURFACE REACTIVITY OF DIESEL PARTICLE AEROSOLS
by
Magnus Lenner, Oliver Lindqvist
and Evert Ljungstrom
Department of Inorganic Chemistry
University of Gothenburg and
Chalmers University of Technology
Inger Lundgren and Ake Rosen
Volvo Car Corporation
Gothenburg, Sweden
INTRODUCTION
In 1979 a research project, concerning exhaust emissions from passenger
cars, was initiated by Volvo Car Corporation in collaboration with the
University of Gothenburg and Chalmers University of Technology. The first
report (1), which appeared in September 1979, comprised of investigations
of exhaust particulates both from gasoline powered and from diesel powered
passenger cars, as well as chemical analyses by several methods, of gaseous
and particulate matter in exhaust samples from Volvo cars.
The present work deals with the influence of diesel particles on the
oxidation of nitric oxide to nitrogen dioxide at different conditions of load,
temperature and dilution. Secondly, spectroscopic investigations of how the
chemical composition of diesel particle surfaces may be modified in the
atmosphere have been performed. A full report (2) will be published in October
1981.
EXPERIMENTAL
Determination of Conversion Rates
To determine the effect of diesel particles on the conversion NO -» NO-,
two series, each comprising of eight rate constant determinations were made in
bag samples of exhausts from a Volvo passenger diesel. The samples were
-------
analysed for nitrogen oxides concentrations at intervals during ^24 h after
sample collection. A Monitor Labs. 8440 Nitrogen Oxides Analyser was used.
For the first series of samples the engine of the car was run at
approximately 1500 rpm idle, while for the second series the car was driven
at 40 km/h with a road load of 12.5 horse powers in a chassis dynamometer.
The parameters temperature (0° or 23° C), dilution rate (~1/60 or~1/120)
and presence/absence of diesel particles were varied. The samples were
collected in Tedlar bags contained in an aluminum barrel, which could be
evacuated to make the bag extract the appropriate volume of exhaust gases.
Dry air from a gas cylinder was used for dilution. Diesel particles were
removed by a filter for the particle-free samples.
Spectroscopic Methods
For infrared spectroscopy, samples were collected with an Electrical
Cororna Sampler (3) on gold covered metal plates and analysed by a
reflection-IR method, with a Nicolet MX-1 FTIR instrument.
ESCA samples were collected on Millipore filters and the electron
spectra were recorded with a Hewlett-Packard 5960 A electron spectrometer.
RESULTS AND DISCUSSION
Conversion Rates
v.2
The formation of N02 from NO obeys the relationship:- = k [N0|
Tbe rate constant k is commonly given in either of the dimensions
1/ JMJ per second or 1/ppm per minute.
2 1
The values for k ([M] x sec. ), calculated for the respective 16
experiments, are summarized in Table 1. The rate constant has a negative
temperature dependence, especially at higher dilution rates. The reaction is
enhanced by the presence of diesel particles. The latter effect, though, is
not as strong as the catalytic effect of street and wall surfaces, reported
by Lindqvist et al. (4).
Spectroscopic Results
ESCA measurements were made on three kinds of samples of diesel
particles, namely unexposed samples, samples which had been exposed to 2 ppm
N0~ for 48 h and finally samples which had been exposed simultaneously to
N02 and UV-light for 6 h. Nitrogen (1s) responses were obtained only from
the latter kind of sample. Signals at 400 eV and at 402 eV were assigned
to emanate respectively from N and NH4 by comparison with the results of
Chang & Novakov (5).
The infrared spectra obtained by reflection of IR-light through diesel
particles precipitated on a gold film showed absorption peaks at 1290/cm
and at 860/on, corresponding respectively to C - N stretch in primary
-------
aromatic amines and to N - 0 stretch in aromatic nitro compounds. Unexposed
samples gave the same kind of spectra as samples which had been exposed to
N02 and UV-light.
REFERENCES
1. Lundgren, I., Rosen, A. and Lindqvist, 0. 1979. Unregulated pollutants.
Measurements and analysis of exhaust gas and particulates from Volvo
light-duty vehicles. Volvo Car Corporation: Gothenburg. 80 pp.
2. Lenner, M., Ljungstron, E., Lindqvist, 0., Lundgren, I., and Rosen, A.
1981. Reactivity and catalytic activity of diesel particles. Studies
of NO and particle emissions from a Volvo passenger diesel. Volvo
Car Corporation: Gothenburg. 63 pp. In press.
3. Van de Vate, J. F., Plomp, A., de Jong, C. and Vrins, E. L. M. 1977.
A battery-operated portable unit for electrostatic and impaction
sampling of ambient aerosols for electron microscopy. Presented at the
5th Conference of the Gesellschaft fur Aerosolforschung, Karlsruhe,
W. Germany.
4. Lindqvist, 0., Ljungstrcm, E. and Svensson, R. 1981. Low temperature
thermal oxidation of nitric oxide in polluted air. Atm. Environment.
In press.
5. Chang, S. G. and Ifovakov, T. 1975. Formation of pollution particulate
nitrogen compounds by NO-soot and NH.,-soot gas-particle surface
reactions. Atm. Environment 9:495-509.
-------
Table 1. Calculated Fate Constants, k is the slope of the function
1 / [NO] t - 1 / [NO] o = kt, calculated from measurements of
|i6] at intervals after the start of an experiment at
t = 0. The values have been multiplied by 10~4.
No.
1
2
3
4
No.
9
10
11
12
Temp.
0
0
23
23
Temp.
0
0
23
23
Particles
(°C) Oil. rate
1/60
1/120
1/60
1/120
40_k
Particles
CO Oil. rate
1/60
1/120
1/60
1/120
__1500_r
k
2.11
2.39
1.81
1.82
m/h^Poaa
k
2.18
2.43
1.97
1.92
ES-idle
No.
5
6
7
8
-load:.
No.
13
14
15
16
No
Temp.
0
0
23
23
No
Temp.
0
0
23
23
particles
(°C) Oil. rate
1/60
1/120
1/60
1/120
particles
(°C) Oil. rate
1/60
1/120
1/60
1/120
k
1.74
2.04
1.56
1.57
k
1.75
1.99
1.59
1.53
-------
EFFECTS OF OZONE AND NITROGEN DIOXIDE PRESENT DURING SAMPLING OF
GENUINE PARTICULATE MATTER AS DETECTED BY TWO BIOLOGICAL TEST SYSTEMS
AND ANALYSIS OF POLYCYCLIC AROMATIC HYDROCARBONS
by
G. Lbfroth
Department of Radiobiology
University of Stockholm
S-106 91. Stockholm
R. Toftgard, J. Carlstedt-Duke and J-A. Gustafsson
Departments of Medical Nutrition and Pharmacology
Karolinska Institute
S-104 01 Stockholm
E. Brorstrbm, Po Grennfelt and A. Lindskog
Swedish Water and Air Pollution Research Laboratory
S-402 24 Gothenburg
Urban particulate matter was collected in wintertime at -5-0 °C during
24 h periods on glass fiber filter with two simultaneously operating high
volume samplers. One of the samplers was equipped with an ozone or nitrogen
dioxide dosage system enhancing the concentration with about 200 ppb ozone or
960 ppb nitrogen dioxide. Filters were Soxhlet-extracted with acetone and the
extracts .analyzed with respect to eight polycyclic aromatic hydrocarbons
(PAH), mutagenicity in the Salmonella/microsome assay and ability to displace
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the rat liver receptor
protein.
Polycyclic Aromatic Hydrocarbons
A part of or the whole acetone extract of each filter was subjected to a
clean up procedure with respect to PAH and eight components were quantified
by gas chromatography on a Carlo Erba equipment with a SE-54 glass capillary
column. The range of concentrations detected is given in Table 1.
Exposure to ozone had very little effect on the concentrations of the
PAH. Significant degradation occurred only in one of the experiments. At this
occasion the concentrations of nitric oxide and nitrogen dioxide in ambient
air were rather high and it seems likely that the degradation may have been
caused by nitrogen oxides formed by ozone oxidation of nitric oxide.
Statistical analysis between the nitrogen dioxide exposed samples and
the simultaneously collected non-exposed samples showed a significant degra-
dation for pyrene, benz(a)anthracene and benzo(a)pyrene being on the average
about 20, 40 and 60 %, respectively.
-------
Salmonella Mutagenicity
Portions of the acetone extracts were reduced in volume, but not to dry-
ness, at <40 QC and then diluted with dimethyl sulfoxide (DMSO). Each of the
DMSO samples was assayed at at least three different occasions for mutageni-
city by the Salmonella plate incorporation method with bacterial cultures
fully grown overnight. Tests were performed with the strains TA 98 and TA 100
in the absence and presence of the microsome containing liver supernatant
from Aroclor 1254-induced male rats (S9) and with the nitroreductase defi-
cient strains TA98NR, TA98/1,8DNPg and TA100NR in the absence of S9. The
S9 was used at a level of 20 yl per plate and was added together with neces-
sary co-factors.
The response expressed as revertants per cu.m was calculated from the
linear or approximately linear dose response curves. The results for assays
in the absence of S9 are given in Table 2. The addition of S9 either de-
creased or did not change the mutagenic response.
Exposure to ozone did not generally alter the mutagenic response except
for an increase in the experiment which also resulted in degradation of PAH.
Exposure to nitrogen dioxide increased the mutagenic response in nitro-
reductase proficient as well as nitroreductase deficient strains. The average
enhancement found in the three investigated experiments was a 3-4-fold
increase.
Affinity to the Rat Liver TCDD-Receptor Protein
Assays for the ability to displace TCDD from the rat liver receptor
protein were performed by adding different amounts of the DMSO samples to
one ml of rat liver cytosol containing tritium-labeled TCDD and determining
the amount of TCDD which remained bound to the receptor. After correction
for non-specific binding, the relative specific binding was calculated from
log-logit plots and was expressed as the amount that competes for 50 % of
the specific TCDD-binding, EC5Q, cu.m per ml cytosol.
Two experiments from the nitrogen dioxide and one from the ozone expo-
sure were analyzed and the results are given in Table 2. Neither nitrogen
dioxide nor ozone altered the affinity.
Conclusions
High volume sampling of ambient particulate matter on glass fiber filter
in the presence of a high level of ozone does not cause a significant degra-
dation of PAH, alter the mutagenic response which is detected by the Salmon-
ella assay or change the affinity to the TCDD-receptor protein.
Sampling in the presence of a high level of nitrogen dioxide causes a
significant degradation of reactive PAH and increases the mutagenic response
which is detected by the Salmonella assay, but does not change the affinity
to the TCDD-receptor protein.
Simultaneous sampling of genuine particulate matter without and with en-
hanced concentrations of reactive gases may be the best method for studying
artifact reactions during sampling. Further studies are in progress with
nitrogen dioxide, nitrous acid and nitric acid.
-------
Table 1. Concentration ranges of analyzed PAH components; ng/rrr*.
PAH component
Phenanthrene
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene / Triphenylene
Benzo(b&k)fluoranthenes
Benzo(e)pyrene
Benzo(a)pyrene
8 samples
non-exposed
Oo42-2o2
0.64-22
1.1 -8.0
1.1 -5.2
0.8 -4.0
1.6 -7.8
0.39-3.6
0.17-2.4
3 samples
03-exposure
200 ppb
0.66-2.0
1.7 -3.6
1.5 -3.6
- 0.93-2.0
1.1 -2.1
2,0 -3.5
0.48-1.0
0.16-0*66
5 samples
N0?-exposure
960 ppb
0.39-1.9
0.81-4.5
0.81 -5.8
Oo82-3.8
1.1 -4.0
2.2 -7.6
0.36-2.2
0.07- 1.0
Table 2. Mutagenic response in the absence of mammalian metabolic activation
and affinity to the TCDD-receptor protein of extracts of particu-
late matter collected without and with enhanced concentrations of
nitrogen dioxide or ozone; n.d. not determined,,
Revertants per
Sample
800225 AMB
N02
800226 AMB
N02
800228 AMB
N02
800326 AMB
°3
800327 AMB
°3
800331 AMB
3
TA 98
62
87
14
37
11
76
4.3
4.6
28
31
10
16
TA 98 NR
37
48
8
24
6
35
n.d.
n<>do
11
12
n.d.
n.d.
^,8m.
o
28
32
3.2
6o5
5
20
n.d.
n.d.
4.5
4.4
n.d.
n.d.
m
TA 100
76
142
14
41
20
110
2.9
3,3
21
25
8.4
14
TA 100 NR
36
62
5
20
5
41
n.d.
n.d.
5
7
n.d.
n.d.
Receptor
affinity
EC50±s.d.
(n = 4)
3
nr/ml
n.d.
n.d.
0.17 + 0.04
0.09 ±0.04
0.07 ±0.02
0.10±0.03
n.d.
n.d.
0.08 ±0.02
0.06 ±0.04
n.d.
n.d.
-------
ALUMINA COATED METAL WOOL AS A
PARTICULATE FILTER FOR DIESEL POWERED VEHICLES
by
M. A. McMahon, W. T. Tierney,
K. S. Virk, and C. H. Faist
Pending federal regulations will probably require 1985 and later
diesel powered vehicles to be equipped with aftertreatment de-
vices to control particulate emissions. Filtering devices which
employ alumina coated steel wool as the filter matrix show pro-
mise of being a practical means for removing submicron size
particles from diesel exhaust. Our diesel exhaust filters (DEF's)
consist of two alumina coated stainless-steel-wool filled car-
tridges inserted into an outer housing about the same size as a
conventional muffler (Figure I). The alumina coating, which is
applied to the steel wool by a proprietary process, characteristi-
cally retains soot particles which come in contact with it.
In our design, metal wool packing density, alumina coating thick-
ness and the physical dimensions are the dominant performance
determinants for the collection efficiency and exhaust back
pressure. The surface area of the alumina in the DEF, which is a
function of all of these, correlates well with collection effi-
ciency.
Our tests have demonstrated that particulate removal in the 70%
range in FTP testing can be achieved at acceptable backpressures.
Measurements also show that trapping efficiency is constant over
a gas flow range of 0.7 to 20 ft/sec. The trapping efficiency of
a given size filter can be increased above 80% but only at an
increased backpressure penalty. Satisfactory filters have been
made for diesel engines varying in size from 2 to 14 litres.
Generally, filters capable of trapping soot with efficiencies in
excess of 70% have a pressure drop of 3 to 4 inches of water per
inch of filter at gas velocities of 15 ft/sec. For example, a
filter mounted on a vehicle equipped with a 1980 Oldsmobile
engine, having a trapping efficiency in this range demonstrated a
pressure drop of 31 inches of water at 40 mph level road condi-
tions.
-------
In addition to removing soot from diesel exhaust, alumina coated
steel wool filters remove significant amounts of hydrocarbons and
sulfates. Importantly, a significant percentage ( 50%) of the
polynuclear aromatics, as indicated by benzo (a) pyrene measure-
ments, is removed. Noise is attenuated to levels equivalent to
those observed using conventional mufflers.
Since the volume of soot generated by current diesel engines is
quite large, soot collected on DBF's must be periodically removed
for the filter to trap effectively for extended mileage. Burning
at temperatures above 1000°F is the most feasible method for
doing this. Since diesel exhaust temperatures at moderate loads
are generally below 1000°F, consideration is being given to
mounting torches in the exhaust system to increase exhaust gas
temperature when regeneration is required. In that conventional
torches present difficult operating problems when employed in
exhaust systems, a catalytic torch is being developed in our
laboratories. With this kind of torch, hydrocarbons injected
into the exhaust are catalytically oxidized to increase the
exhaust gas to regeneration temperature. The catalytic converter
used was made by applying a noble metal catalyst to alumina
coated steel wool. Although the torch performed satisfactorily
using either propane or diesel fuel, propane was used in most of
the development work because of experimental convenience.
To date, about 3,000 miles have been accumulated on a 1980 Olds-
mobile vehicle equipped with a diesel exhaust filter. During
this test, the filter was regenerated at 150-200 mile intervals
with our catalytic torch and a trapping efficiency of greater
than 65% was maintained throughout the entire test. Since the
amount of heat released from burning soot in the limited DEF
volume can be large, the regeneration interval is determined by
this factor rather than by the potential for high backpressure
due to filter loading.
In addition to the durability experience achieved with diesel
engines, it is worth noting that alumina coated mesh filters have
undergone millions of miles of durability testing as a lead
particulate filter in the exhaust of a variety of gasoline
engines.
-------
ALUM IN A-COATED METAL WOOL
SUBSTRATE
INSULATION
INLET
GAS
OUTLET
GAS
PERFORATED
BAFFL ES AND
RETAINERS
GAS
SPREADER
FIGURE 1 - Typical Texaco Diesel Exhaust Filter Design
-------
ISOLATION AND IDENTIFICATION OF MUTAGENIC NITROARENES
IN DIESEL-EXHAUST PARTICIPATES
by
X.B. Xu*, Joseph P. Nachtman, 7.L. Jin*, E.T. Wei,
and Stephen Rappaport
Department of Biomedical and Environmental Health Sciences
School of Public Health
University of California, Berkeley, CA
and
A.L. Burlingame
Space Sciences Laboratory
University of California, Berkeley, CA, and
Department of Pharmaceutical Chemistry
School of Pharmacy
University of California, San Francisco, CA
Particulate matter emitted from diesel engines contains chemicals which
are active in the Ames Salmonella typhimurium assay. The major portion of this
mutagenic activity is liver enzyme independent and thus indicates that diesel
exhaust contains a different class of mutagens than unsubstituted polynuclear
aromatic hydrocarbons, which are liver enzyme dependent.
Diesel exhaust parti culates were collected on glass fiber filters from
heavy-duty engine test apparatus. A total of 14 filters were extracted for 24
hours with 6 L dichloromethane in a Soxhlet apparatus, the extract filtered and
concentrated by rotary evaporator. The combined extracts contained
approximately 225 g of organic matter which yielded 0.46 net TAP8 revertants/yg
(1.0 x 108 net TA98 revertants).
The CH£Cl2 extract was further fractionated on a preparative silica column
with successively increasing solvent strength: hexane, chloroform, and
methanol. The intermediate polarity fraction had the highest specific
mutagenic activity and was further separated on gel permeation, high
performance normal and reverse phase chromatography. Mutagenic activity was
detected in virtually all fractions, so that fractions containing the highest
specific activity were selected for further analysis.
*Members of the Institute of Environmental Chemistry, Chinese Academy of
Sciences, Beijing, People's Republic of China.
-------
High resolution mass spectrometry (HRMS) was performed on selected
subfractipns from the reverse phase separation. Each sample was evaporated
under N2 in the probe which was inserted into the electron impact source.
Identification of nitroarenes was based upon accurate mass determinations (± 15
ppm at a resolution of 9000) of the molecular ion and of fragment ions
corresponding to losses of the neutral fragments NO and/or N02« Some
nitroarenes tentatively identified are listed in Table 1. Those with an
asterisk (*) were found to have the same HRMS spectrum and high pressure liquid
chromatography (HPLC) retention volume as their corresponding standard.
In this study, about 50 nitroarenes have been tentatively identified by
HRMS. The variety of nitroarenes in diesel exhausts is extensive and, because
only a few of the mutagenic fractions were examined, it is likely that more
nitroarenes will be characterized. This complexity is not surprising if one
considers the numerous PAH substrates available for aromatic ring nitration: a
chemical process which will readily occur in the presence of even low
concentrations of nitrogen oxides. Positive identification of each nitroarene
is made difficult by the small quantity of nitroarenes relative to co-eluting
oxygenated and some sulfur-containing PAH and also by the absence of synthetic
standards. Comparison of mass spectra and retention volumes with available
standards, however, support the identifications which have beep suggested.
A number of nitroarenes are potent mutagens in the Ames Salmonella assay,
because nitroreductases in the tester strains facilitate reduction of
nitroarenes of electrophilic intermediates which, in turn, react with nucleic
acids. This raises the question of whether nitroarenes in diesel exhaust or
any other environmental source pose a significant human health hazard. From a
qualitative viewpoint, nitroarenes are hazardous because compounds such as
2-nitronaphtalene and 2-nitrofluorene are carcinogenic to animals. However,
from a quantitative viewpoint, issues of the human dose and the biologic
potency of nitroarenes have not yet been established. It is also not known if
nitroarene emissions are independent of or bear a reciprocal relationship to
the amount of unsubstituted PAH that is emitted. The development, of convenient
techniques to separate and quantify nitroarenes and metabolites may expedite
the acquisition of data to answer these questions.
-------
TABLE 1
Mass Possible Compound
161.048 Nitroindene
197.048 Nitroacenaphthylene
199.063 Nitrobiphenyl*
211.063 Nitrofluorene*
213.079 Nitro-methylbiphenyl
223.063 Nitroanthracene*
225.079 Nitro-methylfluorene
247.063 Nitropyrene*
261.079 Nitro-methylpyrene
287.095 Nitro-methylchrysene
225.043 Nitro-.fluorenone*
241.074 Nitro-hydroxymethylfluorene
253-038 Nitro-anthraquinone
256.048 Dinitrofluorene*
340.143 Dinitro-(C6)alkylfluorene
348.111 Dinitro-(Cii)alkylpyrene
371.112 Trinitro-(C5)alkylfluorene
-------
COMPARISON OF NITRO-PNA CONTENT AND MUTAGENICITY OF DIESEL EMISSIONS
by
Marcia G. Nishioka and Bruce A. Petersen
Battelle Columbus Laboratories
Columbus, Ohio
and
Joel 1 en Lewtas
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
INTRODUCTION
The increasing number of automobiles and light-duty trucks powered by
diesel engines has generated concern over the emissions associated with these
engines. The diesel engines have a higher particulate emission rate than
gasoline catalyst engines. The extractable organics from both diesel and
gasoline particle emissions have been found to be mutagenic and
carcinogenic (1). Recently, nitro-substituted polynuclear aromatic
hydrocarbons (nitro-PNA) have been identified in diesel particle extracts (2).
Several of these nitro-PNAs are very potent bacterial mutagens (3).
A study was carried out to identify and quantitate nitroaromatic compounds
in the extract of particulate material from three different diesel engines
(Datsun Nissan diesel 220C, Oldsmobile diesel 350, and VW Rabbit diesel) and
one gasoline engine (Ford Mustang II). The operating and sampling conditions
have been described elsewhere (4). Mutagenic assay data was also collected on
these extracts using the Salmonella typhimurium TA98 bioassay. The results of
these two studies were compared to determine whether the amount of
nitroaromatics detected can fully account for the direct-acting mutagenic
activity indicated by the bioassay data.
METHODS AND RESULTS
Chemical
Two separate methods were developed to analyze the emission extracts:
1) combined high performance liquid chromatography/mass spectrometry (HPLC/MS)
with positive chemical ionization (PCI); and 2) on-column injection high
resolution gas chromatography/mass spectrometry (HRGC/MS) with negative
-------
chemical ionization (NCI). The total dichloromethane extract was screened
first by the PCI HPLC/MS method, which consisted of a Supelco normal phase HPLC
column (Supelcosil LC-Si, 5 ym) interfaced to a Finnigan 4000 mass spectrometer
via a microbore capillary and a polyimide moving belt. Nitropyrene and
nitrophenanthrene/anthracene were detected by this method in all of the diesel
engine extracts, but not in the gasoline engine extract.
The extracts were fractionated on silica gel by open-bed liquid
chromatography into four compound class fractions: 1) hexane - aliphatic
hydrocarbons, 2) hexanerbenzene - polycyclic aromatic hydrocarbons and
mononitroaromatics, 3) methylene chloride - moderately polar neutrals,
including di- and trinitroaromatics, and 4) methanol-highly polar neutrals,
primarily oxygenated compounds. Over 94% of the total extract was recovered
for all four engines.
The three fractions expected to contain nitro-PNA (2,3,and 4) were
analyzed by the NCI HRGC/MS system, which consisted of a JaW DB-5 bonded fused
silica capillary column interfaced to a Finnigan 4000 mass spectrometer. The
quantisation of nitroaromatics was based on response factors for eight standard
nitroaromatics relative to the internal standard dy-nitronaphthalene calculated
over a concentration range of 100. More than twenty nitroaromatics were
detected in the diesel engine extracts but only 1-nitropyrene was detected in
the gasoline engine extract. In all cases the 1-nitropyrene was the
nitroaromatic detected in greatest quantity and its concentration in the
extracts is shown in Table 1. Quantitative data on the other nitroaromatics
will be presented.
At masses higher than nitropyrene, the mono nitro derivatives of the
molecular weight 228 (benz[a]anthracene, chrysene) and molecular weight 252
(B[a]P, B[e]P, perylene, benz[o]fluoranthenes) PNAs were also detected. Two
nitropyrenone isomers were tentatively identified in the Nissan and Oldsmobile
samples and three dinitropyrene isomers were identified in the VW sample.
Most of the compounds detected in the methanol fraction of the Nissan and
VW samples were quinones. The methanol fraction represented 30$ and 17% of the
total organic mass for the Nissan and VW extracts, respectively.
Biological
The total dichloromethane extractable organics from each of the emission
samples were bioassayed in the Salmonella typhimurium plate incorporation assay
with minor modifications as reported elsewhere (5).The slope of the
dose-response curve (rev/ug) for each of these samples with and without 59
activation was determined using a non-linear model (6) and is shown in Table 1.
The emissions from the gasoline catalyst (Mustang II) differ from the diesel
emissions in that these organics are significantly more mutagenic in the
presence of the S9 activation system.
-------
DISCUSSION
Both the PCI HPLC/MS and NCI HRGC/MS methods are capable of detecting
nitroaromatic compounds. However, the greater chromatographic resolution and
lower detection limit of the NCI HRGC/MS method favor the use of this method
over the PCI HPLC/MS method. The limit of detection by NCI HRGC/MS is
approximately 0.05 ng for the mononitroaromatics, but only about 100 ng for
nitropyrene by the PCI HPLC/MS method.
The concentration of 1-nitropyrene detected in each of the samples in not
highly correlated with the direct-acting mutagenic activity. The total nitro-
aromatic content does account for a substantive portion of the direct-acting
mutagenicity of the Olds diesel, VW Rabbit diesel, and Mustang II gasoline
vehicles. However, the nitroaromatics detected cannot account for the signifi-
cantly higher mutagenic activity associated with the Nissan diesel extract.
It is possible that the quinones detected in the Nissan and VW extracts
are the oxidation productions of nitroaromatics in the stored extracts, as
similar extracts were shown to increase in toxicity with time (6). A greater
concentration of quinones in the Nissan sample may indicate that a greater
concentration of nitroaromatics may have been originally present in the
extract. This is consistent with the fact that the Nissan extract originally
had higher mutagenic activity than the VW extract.
REFERENCES
1. Nesnow, S., and J.L. Huisingh. 1980. Mutagenic and carcinogenic potency
of extracts of diesel and related environmental emissions: Summary and
discussion of the results. In: Health Effects of Diesel Engine
Emissions, Vol. II. W.E. Pepelko, R.M. Danner, and N.A. Clarke, eds.
EPA-600/9-80-057b. U.S. Environmental Protection Agency: Cincinnati,
OH.
2. Petersen, B.A., C. Chuang, W. Margard, and D. Trayser. 1981.
Identification of mutagenic compounds in extracts of diesel exhaust
particulates. Proceedings of the 74 annual APCA Meetings, Philadelphia,
PA.
3. Rosenkranz, H.S., E.C. McCoy, D.R. Sanders, M. Butler, O.K. Kiriazides,
and R. Mermelstein. 1980. Nitropyrenes: Isolation, identification and
reduction of mutagenic impurities in carbon black and toners. Science
209:1039-1043.
4. Huisingh, J.L., R.L. Bradow, R.H. Jungers, B.D. Harris, R.B. Zweidinger,
K.M. Gushing, B.E. Gill, and R.E. Albert. 1980. Mutagenic and
carcinogenic potency of extracts of diesel and related environmental
emissions: Study design, sample generation, collection, and
preparation. In: Health Effects of Diesel Engine Emissions, Vol. II.
W.E. Pepelko, R.M. Danner, and N.A. Clarke, eds. EPA-600/9-80-057b.
U.S. Environmental Protection Agency: Cincinnati, OH. pp. 788-800.
-------
5. Claxton, Larry D. , 1980. Mutagenic and carcinogenic potency of diesel and
related environmental emissions: Salmonella bioassay. In: Health
Effects of Diesel Engine Emissions, Vol. II. W.E. Pepelko, R.M. Danner,
and N.A. Clarke, eds. EPA-600/9-80-ri57b. U.S. Environmental Protection
Agency: Cincinnati, OH. pp. 801-807.
6. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. Tejada,
J. Bumgarner, F. Duffield, V.F. Simmon, C. Hare, C. Rodriguez, L. Snow,
and M. Waters. 1979. Application of bioassay to the characterization
of diesel particle emnissions. Part II. Application of a mutagenicity
bioassay to monitoring light-duty 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, S. Nesnow, J.L. Huisingh, S.S. Sandhu, and L. Claxton, eds.
Plenum Press: New York. pp. 400-418.
Table 1. Quantification of 1-Nitropyrene in Engine Exhaust Extracts
and Mutagenic Activity of the Extracts
Sample
Cone. 1-Nitropyrene
wt ppm
Mutagenic Activity, rev/yg
TA98/-S9
TA98/+S9
Nissan diesel
Olds diesel
VW Rabbit diesel
Mustang II gasoline
407
107
589
2.5
20.8
2.1
5.2
2.1
15.1
1.4
6.1
8.6
-------
CAPILLARY COLUMN GC/MS CHARACTERIZATION OF DIESEL
EXHAUST PARTICULATE EXTRACTS1
by
T. J. Prater, T. Riley and D. Schuetzle
Analytical Sciences Department
Scientific Research Laboratory
Dearborn, Michigan 48121
INTRODUCTION
Previous studies have shown that most of the direct-acting Salmonella typhimurium
mutagenic activity (>50%) in diesel exhaust particulate extracts is concentrated in
chemical fractions which contain compounds of moderate polarity (2). Analytical-
scale normal phase high performance liquid chromatography(NP-HPLC) (3), packed-
column GC/MS and high resolution MS analysis were used in that work to determine
that these moderately-polar fractions consisted primarily of polynuclear aromatic
hydrocarbon (PAH) derivatives. The objective of this study was to extend the
previous work by developing preparative scale NP-HPLC fractionation followed by
fused silica capillary GC/MS analysis in order to improve component resolution.
EXPERIMENTAL
Light duty diesel exhaust particulate samples were collected on T60A20 Pallaflex
filters using a dilution tube and a chassis dynomometer test facility. Filter samples
were soxhlet extracted with dichloromethane (DCM).
HPLC analysis was performed on a Varian Model 5600 LC equipped with a 7.8 mm i.d.
x 30 cm long Microporasil 10 (4 column. The solvent program consisted of 100%
hexane for 5 min, then 1% DCM/min for 5 min, followed by a linear gradient to 100%
DCM in the next 25 min, 100% DCM for 10 min, then 10% acetonitrile/min for 10
min, and a final 10 min flush with 100% acetonitrile. The chromatographic
separation was monitored by UV at 254 nm and by fluorescence at 254/320 nm.
Further details of this technique are presented elsewhere (3).
GC/MS analyses were performed on a VGMM ZAB-2F mass spectrometer equipped
with a 30 m long x 0.25 mm i.d. SE54 fused silica capillary column interfaced directly
to the mass spectrometer source. Samples were injected directly on-column and
temperature programming was 80° to 270° at 4°/min. Electron impact ionization
techniques were used.
-------
RESULTS AND DISCUSSION
This investigation emphasized the analysis of HPLC fractions containing nonpolar and
moderately polar PAH derivatives. Nonpolar aliphatic and highly polar HPLC
fractions were not characterized by GC/MS. The nonpolar and moderately polar PAH
derivatives which were identified are listed respectively in Tables I and II. Many of
the compounds identified have a large number of isomers as indicated by the
parentheses in the tables. This is illustrated by the mass chromatograms in Fig. 1
which show the increasing complexity of the isomer series as methyl substituents are
added to anthracene and phenanthrene. There are probably more isomers for the
methylated anthracene and phenanthrenes than we were able to resolve even with the
high-re solution fused-silica capillary column. It would be difficult and of limited
utility to identify every specific isomer present in these fractions. For this reason,
synthesis of standards and identification of isomers are only being undertaken on
those groups of PAH and PAH-derivatives which yield a relatively high level of
direct- or indirect-acting Ames activity. This has been found to be the case for the
nitrated-PAH derivatives which tend to show high levels of direct-acting
mutagenicity compared to other PAH and PAH derivatives in these samples.
The combination of normal phase HPLC fractionation followed by capillary GC/MS
analysis proved to be a very useful approach to the qualitative characterization of
diesel exhaust particulate extracts. The quantitative analysis of these diesel extracts
by capillary GC/MS is complicated by the labile nature of some of the PAH
derivatives. The quantitation technique found to be most accurate and presently
being used in our laboratory employs the deuterated analog of the PAH derivative of
interest which is added to filter samples prior to extraction. The deuterated standard
exhibits the same chemical characteristics as the native compound, but it can be
distinguished mass spectrometrically. Therefore, losses which occur during sample
workup and analysis can be accounted for.
1. Prater, T. J., T. Riley, and D. Schuetzle. 1981. Capillary column GC/MS
characterization of diesel exhaust particulate extracts. Presented at the 29th
Annual Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN.
2. Schuetzle, D., F.S.-C. Lee, T. 3. Prater, and S. B. Tejada. 1981. The
identification of polynuclear aromatic hydrocarbon (PAH) derivatives in
mutagenic fractions of diesel particulate extracts. Intern. J. Environ. Anal.
Chem. 9:93-144.
3. Schuetzle, D., and J. M. Perez. 1981. A CRC cooperative comparison of
extraction and HPLC techniques for diesel particulate emissions. Presented at
the 74th Annual Meeting of the Air Pollution Control Association, Paper //81-
564, Philadelphia, PA.
4. Levine, S. P., and L. Skewes. 1981. High performance semi-preparative liquid
chromatography of diesel engine emission particulate extracts. J. Chromatogr.
In preparation.
-------
Table I. Nonpolar PAH Identified in Diesel Exhaust Participate Extract
dibenzothiophene
anthracene and phenanthrene
methyl dibenzothiophene isomers(3)
methyl (phenanthrene and anthracene) isomers(4)
dimethyl dibenzothiophene isomers(7)
dimethyl (phenanthrene and anthracene) isomers(13)
fluoranthene and pyrene
trimethyl dibenzothiophene isomers(9)
BaP, BeP, perylene,and isomersO)
trimethyl (phenanthrene and anthracene) isomers(15)
tetramethyl dibenzothiophene isomers(12)
tetramethyl (phenanthrene and anthracene) isomers(16)
benzo(g,h,i) fluoranthene
benz(a)anthracene, chrysene, benzo(c)phenanthrene,
triphenylene isomers(2)
methyl benz(a)anthracene isomers(4)
pentamethyl dibenzothiophene isomers(4)
dimethyl benz(a)anthracene isomers(2)
methyl (fluoranthene and pyrene)isomers(7)
Table II. Moderately Polar PAH Derivatives Identified in Diesel Exhaust
Particulate Extract
benz(a)anthracenedione
methyl (anthrone and phenanthrone) isomers
thiozanthone isomer
dimethyl (anthrone and phenanthrone) isomers
pyrenone
trimethyl (anthrone and phenanthrone) isomers
methyl thioxanthone
dimethyl thioxanthone isomers(2)
benz(d,e)anthrone and isomersO)
1-nitropyrene
1,1'biphenyl-ol
9-fluorenone
(pyrene or fluoranthene) carboxaldehyde
dibenzofuran carboxaldehyde
phenanthrone
anthrone isomer
9-xanthone
xanthene carboxaldehyde
(anthracene or phenanthrene)dione
dibenzothiophene carboxaldehyde
methyl (anthracene or phenanthrene)dione
phenanthrene carboxaldehyde
anthracene carboxaldehyde
methyl (anthracene and phenanthrene)
carboxaldehyde isomers(9)
dimethyl (anthracene and phenanthrene)
carboxaldehyde isomers(S)
-------
Figure 1. Mass chromatograms of phenanthrene(P) and anthracene(A) (178),
methyl-A and -P (192), dimethyl-A and -P (206), and trimethyl-A
and -P (220)
M/Z
178
M/Z
192
M/Z
206
M/Z
220
25
INTENSITY
9168
11040
16860
30 35
TIME (MIN)
-------
PHYSICO-CHEMICAL PROPERTIES OF DIESEL PARTICULATE MATTER
by
Mark M. Ross and Terence H. Risby
Division of Environmental Chemistry
Department of Environmental Health Sciences
The Johns Hopkins University School of Hygiene and Public Health
Baltimore, Maryland 21205
Samuel S, Lestz
Department of Mechanical Engineering
Pennsylvania State University
University Park, Pennsylvania 16802
Ronald E:, Yasbin
Department of Microbiology
Pennsylvania State University
University Park, Pennsylvania 16802
Numerous studies have dealt with the identification and quantifi-
cation of the compounds sorbed onto Diesel particulate matter. The
ultimate environmental significance of these sorbed species depends upon
the relative bioavailabilities which, in turn, depend upon the nature
and strength of interactions prevailing at the gas-solid interface.
This research focused on the fundamental adsorptive properties and sur-
face characteristics of Diesel particulate matter. In addition, the free
radical nature of the particles and the associated reactivity with
selected stimuli was investigated.
This study was carried out using a graphitized carbon black, Spheron
6, as a "reference" solid. Two Diesel particulate samples were used.
The first, DPM-PSU, was collected from a single-cylinder engine operated
with a prototype fuel and a lubricant free of trace inorganic compounds.
The second, DPM-EPA, was collected from an Oldsmobile 350 engine operated
with a commercial fuel and lubricant at the US E.P.A.
Electron micrographs and elemental compositions revealed the common
spherical carbon particle structure of all the samples. Yet, the Diesel
samples had lower bulk densities and higher external surface areas, as
calculated from mean particle diameters. Nitrogen B.E.T. surface areas
-------
were measured in order to determine internal plus external surface areas.
The surface area of DPM-EPA was found to be dependent on outgassing
pretreatment. The surface area accessible to nitrogen increased from
41 M2/g at 50°C degassing to 112 M2/g at 400°C degassing. DPM-PSU was
measured to have a surface area of approximately 104 M2/g and showed
little change upon increasing activation temperature. 0Nitrogen porosity
experiments revealed the existence of pores of 100-200A in diameter in
both Diesel samples.
Isosteric heats of adsorption of a variety of organic compounds on
the particulate samples were measured with gas-solid chromatography.
The heats on DPM-PSU were consistently greater than those on graphitized
carbon blacks (gcb). The variations of adsorption energies with adsor-
bate surface coverage were determined by measurement of adsorption
isotherms at different temperatures. DPM-PSU exhibited adsorption
characteristics similar to those of gcb and the few differences are
attributed to a more polar and energetically heterogeneous DPM-PSU sur-
face. DPM-EPA was determined to have markedly different properties due
to the increased quantity of presorbed material. The DPM-EPA surface
was found to be relatively non-polar and homogenous. Removal of the
presorbed species caused the surface to become more active and similar
to that of DPM-PSU. The significance of these results is that compounds
close to the carbon surface will .be more difficult to remove than those
adhering to presorbed layers. Sorbed compound bioavailability and sur-
face properties of Diesel particulate matter are dependent upon the
nature and amount of presorbed material.
A related study of the reactivity of Diesel particulate matter with
respect to atmospheric stimuli was performed using electron paramagnetic
resonance (EPR) spectrometry. The EPR signals of the three samples were
monitored after selected heat and evacuation treatments, gas (CL, NO,
NOp) exposures, and ultraviolet/visible irradiation. The sample signals
differed with respect to line widths but all signals narrowed upon sample
evacuation and heat treatment and broadened upon exposure to oxygen and
nitric oxide. The Diesel particulate sample signals were extremely
sensitive to nitrogen dioxide and irradiation. Exposure of N0? caused
an increase in the free radical concentration in the Diesel samples.
Irradiation effects were varied depending on sample conditions but the
greatest signal increase occurred with evacuated Diesel particles. With
EPR, the existence of free radicals in Diesel particulate matter and the
reactivity of these species with respect to selected treatments were
demonstrated. The results provide evidence of potential photochemical
reactivity of airborne particulate matter.
-------
SOME FACTORS AFFECTING THE QUANTITATION OF AMES ASSAYS
by
Irving Salmeen and Anna Marie Durisin
Engineering and Research Staff
Research
Ford Motor Company
Dearborn, Michigan
The simple theory of bacterial mutation experiments of Luria and
Delbrlick (1) starts with the assumption
f=yn(t) (1)
where m is the number of mutants, n is the number of wild type, and y is the
mutation rate coefficient. We have shown (2) that, in the absence of cell
killing, this equation predicts for the Ames assay
M =.aCN (2)
where M is the number of revertants per plate, a is the mutation rate per
concentration, C, of mutagen and N is the total number of histidine auxo-
trophs in the background lawn. Thus the dose-response function is linear,
but the slope is proportional to N. In the Ames assay, N ~ n0P, where n0
is the initial inoculum and P is the average number of bacteria per background
colony of histidine auxotrophs.
In a series of experiments we determined dose-response curves as a
function of n0; estimated n0 by counting background colonies in photo-
micrographs (100X) of the background lawn-; and estimated the volume of
individual background colonies which is proportional to P. We found that P
depends nonlinearily on n0; P is much larger at low r\q than it is at high n0
presumably because at lower n0 there is less competition among the colonies
for the trace histidine. We observe that N decreases by about 1/3 when n0
is decreased from 10^ to 10? bacteria/plate. N is roughly independent of n
when n0 is less than about 5x10° bacteria/plate. For merely detecting
mutagens, neglect of the dependence of slopes on N, while conceptually
incorrect, may have no serious effect. For quantitative experiments, such
as determining the contribution of a compound to the mutagenicity of a
mixture, failure to take into account the dependence of the slopes on N may
-------
cause errors by factors of 2 to 3.
When the test compound causes killing, then equation (1) becomes:
$f= yn(t) - kbm (3)
where kb is the killing rate coefficient for mutants and, now, n(t) must
include a specific killing term for the histidine auxotrophs. If we assume
that
n(t) = n0exp[(Y - kfl)t] (4)
where y is the growth rate coefficient and ka is the killing coefficient for
histidine auxotrophs, and if we also assume that killing and mutations are
independent events and that the killing coefficients of auxotrophs and
revertants are equal and proportional to the concentration of mutagen, i.e.,
ka=kb=kC, then the number of revertants per plate is of the general functional
form
M ^ aCN1 exp(-kC) (5)
where N1 is the total number of histidine auxotrophs in the background
population when kC«l . The concentration which yields the maximum in the
dose-response function is Cm=l/k.
We have obtained dose-response functions using diesel particulate
extract which have a maximum followed by a monotonic decrease to zero. We
determined the killing coefficient k in three ways: (1) from the value of
the concentration corresponding to the maximum of the dose-response function;
(2) from a classical dilution-plating killing curve; and (3) from a killing
curve developed from counting colonies in photomicrographs of the background
lawn. The killing rates determined from these 3 methods agree to within
about 15%, suggesting that this simple model is a good approximation to the
mutation-killing kinetics, at least for these samples.
This model is free of adjustable parameters in the sense that the two
parameters can be determined directly from the data. Functional forms
similar to equation (5) have sometimes been used ad hoc in statistical curve
fitting routines to describe Ames assay data; thelibove derivation provides
theoretical support for use of this function. We will show data to illustrate
that the apparent slope of the initial approximately linear portion of non-
linear dose-response functions obtained with several diesel -particulate
extracts can over estimate, by as much as a factor of 2, the actual mutation
frequency in the Ames assay.
REFERENCES
1. Luria, S. and Delbruck, M., Genetics 28, 491 1943.
2. Salmeen, I. and Durisin, A., Mutat. Res. 85, 109 1981.
-------
CHEMICAL AND MUTAGENIC CHARACTERISTICS OF DIESEL EXHAUST
PARTICLES FROM DIFFERENT DIESEL FUELS
by
D. S. Sklarew, R. A. Pelroy and S. P. Downey
Pacific Northwest Laboratory operated by Battelle
P. 0. Box 999
Richland, Washington 99352
R. H. Jungers and J. Lewtas
U. S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
A potential for increased use of a wide variety of diesel fuels has in-
creased the importance of studies to determine whether the nitrogen content
of different fuels affects the chemical and mutagenic characteristics of the
particles produced during combustion. In this study, the exhaust particles
from five diesel fuels with various nitrogen and aromatic hydrocarbon contents
were examined. The fuels included minimum quality #2 (Min. Qual.), jet fuel
(JP-7), shale diesel fuel-marine (DFM), a base fuel plus heavy aromatics and
hexylnitrate (BF+HAN+HN), and the base fuel plus isoquinoline (BF+IQ). Table 1
indicates the aromatic and nitrogen contents of these fuels. Particles used
in the study were generated by Southwest Research Institute for EPA using a
Mercedes 240D vehicle driven through five consecutive Highway Fuel Economy
Tests.
Experimental
The particle samples were soxhlet extracted with methylene chloride and
these extracts and the fuel samples were fractionated by acid-base and silica
gel column chromatography methods. Six fractions resulted from each material:
base, acid, aliphatic hydrocarbon, aromatic HC, moderately polar neutral, and
highly polar neutral. Several fractions were analyzed by selective detector
gas chromatography and by gas chromatography-mass spectrometry. A 30m fused
silica Carbowax column was used for the moderately polar and highly polar
fractions, and a 30m SE-52 column was used for the aromatic hydrocarbon frac-
tion.
All fractions were assayed for mutagenicity in the Ames histidine rever-
sion test with Salmpnell^typhin^ All samples were
-------
tested with and without metabolic activation with Aroclor induced rat liver
(S9) homogenates.
Because of the current interest in nitroaromatics in diesel particles
(1,2,3), an experiment was done to estimate the recovery of smaller ring ni-
troaromatics from filters. 2-Nitrofluorene was added to one of the filter
samples (BF+HAN+HN) and this "spiked" filter as well as a control filter of
the same fuel were extracted, fractionated, and assayed in the Ames test with
TA98 minus S9.
Results and Discussion
The amount of material extracted with DCM from the diesel particles
ranged from 9% to 16%. Recovery of material in the fractionation procedure
ranged from 82% to 105%. The weight distribution in the six chemical class
fractions did not appear to correlate with either the aromatic or nitrogen
content of the parent (uncombusted) fuel. Nitroaromatic standards distributed
between the aromatic hydrocarbon and moderately polar neutral fractions.
Gas chromatography with a nitrogen selective detector was used to compare
the nitrogen-containing components from the five particle samples. The mod-
erately polar fractions showed similar patterns in all five samples, as did
the highly polar fractions (Fig. 1). However, particles from the diesel fuel
marine and minimum quality fuels appeared to contain more nitrogen compounds
than did the other fuels. There did not appear to be a correlation between
the major nitrogen-containing peaks in the" fractions and the nitrogen content
of the fuel.
The aromatic hydrocarbon fraction of the shale diesel fuel-marine parti-
cles was analyzed by GC-MS. It contained numerous polycyclic aromatic hydro-
carbons including phenanthrene, anthracene, fluoranthene, pyrene, benz(a)an-
thracene, chrysene, benzofluoranthenes, and benzpyrenes. The moderately polar
fraction of the particles from minimum quality #2 contained a number of car-
boxy lib acids, carboxylic acid methyl esters, phenanthrenequinone, alkylated
phenols, fluorenone, alkylated fluorenones, and possible alkanones. Compounds
tentatively identified in the highly polar fraction of particles from the base
fuel plus isoquinoline include a number of phenols, carboxylic acids, benzoic
acid, quinoline, xanthenone, and benzofuranone. Another peak with a probable
molecular weight of 179 is tentatively identified as an anhydride, possibly,
a nitrogen-containing phthalic anhydride.
The fractions from the parent fuels showed no mutagenic activity in the
Ames assay. The mutagenic activity of the fractions from the diesel particles
is shown in Figure 2. After fractionation mutagens were recovered in four
fractions: acid, moderately polar neutrals, strongly polar neutrals, and
aromatic hydrocarbons. Recoveries of mutagenic activity in the diesel frac-
tions were low; however, standard reference mutagens, both direct and indirect,
did not appear to be destroyed by the fractionation procedure. In particular,
in the experiment with the 2-nitrofluorene spike, much of the mutagenic activ-
ity was recovered in the aromatic hydrocarbon and moderately polar neutral
fractions. It is of interest to note that ^50% of the recovered mutagenic
-------
activity from the samples is not in the fractions in which the 2-nitrofluorene
was concentrated but instead is in the highly polar neutral fraction.
The three most mutagenically active crude DCM extracts were derived from
the diesel fuels that contained relatively high concentrations of aromatic
compounds. No correlation was observed between increased mutagenicity and
nitrogen content of the parent diesel fuels.
References
1. Wang, Y. Y., S. M. Rappaport, R. F. Sawyer, R. E. Talcott, E. T. Wei.
1978. Direct-acting mutagens in automobile exhaust. Cancer Letters 5:
39-47.
2. Yu, M. L., and R. A. Kites. 1981. Identification of organic compounds
on diesel engine soot. Anal. Chem. 53:951-954.
3. Schuetzle, D., T. Riley, T. J. Prater, T. M. Harvey, D. F. Hunt. 1981.
The identification of nitrated derivatives of PAH in diesel particu-
lates. Anal. Chem., in press.
Table 1. Aromatic and Nitrogen Content of Diesel Fuels
Aromatics Nitrogen
(vol %) (ppm)
DFM 29.9 5
Min. Qual. 34.6 240
BF+HAN+HN 30.8 718
BF+IQ 6.6 930
JP-7 2.7 <1
-------
D
C
zzo
±ii s
"A
Z
m
C
O
z
CA
o
n
P
P
2Om
-<30
0(0
O
m
C
O
(0
Fig. 1. Gas chromatogram with nitrogen selective detector of the moderately
polar and highly polar fractions from two diesel participate samples.
-------
DIESEL FILTERS
2.0
1.5
1.0
0.5
2.5
2.0
1.5
1.0
0.5
TA98+S9
TA98-S9
U.P.
CRUDE EXTRACT
1
BASES
B2&3 DFM
EMJ MIN. QUAL.
^B BF + HAN + HN
I I BF+IQ
P'SMi JP-7
ALIPHATIC HC AROMATIC HC
MOD. POLAR
NEUTRALS
HIGHLY POLAR
NEUTRALS
Fig. 2. Ames mutagenicity data of fractions from five diesel filters.
-------
FRACTIONATION AND CHARACTERIZATION OF THE ORGANICS
FROM DIESEL AND COMPARATIVE EMISSIONS
by
C. Sparacino, R. Williams, K. Brady
Research Triangle Institute
Research Triangle Park, North Carolina
R. Jungers
Environmental Protection Agency
Research Triangle Park, North Carolina
Semi-volatile materials were analyzed from various media including
diesel soot, tar from coke oven residues, roofing tar and cigarette
smoke condensate (CSC).
The diesel exhaust particles were collected from a 1978 Oldsmobile
350 diesel vehicle operated on the Highway Fuel Economy Test Cycle
(HWFET) with No. 2 diesel (Union 76) fuel. The particles were collected
on Pallflex T60A20 filters and the organics were removed by Soxhlet
extraction with methylene chloride as previously described (1).
The 2RI Kentucky Reference cigarette smoke condensate was generated
according to the method of Patel (1977) at Oak Ridge National Laboratory
(1).
The coke oven main sample was collected from a separator located
between the gas collector main and the primary coolers within a coke
oven battery at Republic Steel in Gadsden, Alabama, about 60 miles northeast
of Birmingham.
The roofing-tar sample was generated and collected using a conventional
tar pot containing pitch-based tar, enclosed within a chamber and heated to
360°-380°F, a normal temperature for commercial use. The evaporative emissions
were collected using a small bag house fitted with Teflon filter bags
(1).
The solvents used to extract or condense the organics from each of
these samples was removed by evaporation under nitrogen.
The nature and number of organic compounds associated with samples
of the type addressed in this program render them among the most complex
of environmental samples. At the present time no direct determinant
approach, regardless of the resolving power, is available for routine
characerization. The analytical problems can be minimized by pretreatment
of the sample in order to distribute the sample compounds into fractions
of similar chemical or physical properties. This class division provides
useful information regarding the sample's overall make-up, yields conven-
ient and meaningful subfractions for biotesting purposes, and lessens
the analytical burden for ultimate characterization. This procedure is
-------
based on work by Novotriy et jil., (2) which, after significant alteration,
was used for this study. The entire scheme is depicted in Figure 1.
Each sample was subjected to this fractionation procedure to yield
six fractions of various chemical properties and polarities. The acid
fraction contains both weak (e.g., phenols) and strong (e.g., carboxylic
acids) acids. The base fraction contains organic, Bronsted bases (e.g.,
amines). The neutral fraction is subdivided into 3 main fractions based
on compound polarity. The non-polar neutral (NPN) fraction is comprised
of compounds less polar than ^ naphthalene. Paraffinic materials are
characteristic of this fraction. The PNA fraction contains compounds of
intermediate polarity, and is selective for condensed ring aromatics.
All neutral materials with polarities greater than PNA hydrocarbons are
found in the polar neutral (PN) fraction. Prior to the subfractionation
of the neutral fraction, the latter must be dissolved in cyclohexane.
All components are not soluble in this solvent. The insoluble material
is collected as a separate fraction (CI), and is comprised of intermediate
and highly polar compounds.
Spillover of various compounds into all fractions is a natural
feature of solvent partitioning processes. Polar neutral material was
removed from the PNA fraction by silica gel chromatography. The PNAs
were chromatographed using gradient elution such that a fraction containing
only PNA hydrocarbons was obtained (PNA-1). Other fractions (PNA 2-4)
were collected that contained compounds of intermediate to high polarity.
Most fractions were directly analyzed by capillary GC/MS. The
fractions enriched in polynuclear aromatic hydrocarbons (PNAs) were
further purified by column chromatography, and the collected subfractions
were analyzed by GC/MS. A large portion of each sample, after fractionation,
was submitted to the EPA for biotesting. Theremainder of each sample
was used for all analytical work.
Approximately 1 g of each sample was partitioned. Recovery of
material after application of the fractionation scheme was generally ca.
80%. Overall mass balances are shown in Table 1. The recovery for CSC
was uncharacteristically low (47.6%). Extensive emulsions were not
formed during partition; the formation of insoluble material upon dissolution
in methylene chloride prior to fractionation may account for the low
figure.
The mass distribution for each sample is shown in Table 2. These
results represent approximate quantities since any solvent partition
process is a rough separation method. The cyclohexane insoluble (CI)
fraction contains significant proportions of material in some samples.
This fraction is a measure of the amount of sample, after acid/base
removal, that is not soluble in cyclohexane, and is therefore presumably
polar neutral material. The CSC and coke oven samples both contain
major quantities of such material.
-------
The CSC sample contained a significant amount of organic bases, while
the acid fraction was more important for the diesel soot extract. The large
amount of non-polar neutral material associated with the diesel soot is not
unexpected; aliphatic hydrocarbons are known to constitute a major proportion
of such mixtures (3). The roofing tar sample is notable for the size of the
PNA fraction.
The partition scheme proved effective in providing some fractions
that were amenable to direct GC/MS analysis. Acid fractions require
derivatization before comprehensive GC/MS analysis can be carried out.
The bases, NPN, PNA and CI fractions can be successfully approached via
GC/MS, although clean-up is required for some fractions. The PN fraction
represents a very difficult analytical problem that probably requires
LC/MS, derivatization, further fractionation, etc. The actual extent to
which any fraction can be comprehensively analyzed by any GC technique
is unknown. More work is clearly required in this area.
The partition scheme fractions showed, after GC/MS analysis, more
or less the expected compositions. The acid fractions were shown to
contain phenolic materials; carboxylic acids such as fatty acids were
detected infrequently (derivatization required). The non-polar neutral
fraction was, for all sample types, highly enriched in saturated and
unsaturated aliphatic hydrocarbons. The compounds covered a molecular
weight range corresponding to ca. CIQ-COA paraffins. The CSC fractions
contained several plant natural products. The PNA fraction from CSC was
too small to permit both adequate bioassay and complete analysis. The
PNA fraction from all other samples showed the presence of PNAs and
methylated PNAs containing 2-5 rings. Other compounds found were dibenzo-
thiophene, dibenzofuran and oxygenated fluorenes.
The preparative chromatography of the PNA fraction provided separation
of PNA hydrocarbons from the more polar contaminants of that fraction.
The amount of material recovered in the chromatographic fractions (PNA
2-4) was usually quite small; only phthalate esters and unknown species
were indicated. Based on similar chromatographic schemes (4), nitro-
arenes, oxygenated PNAs and non-basic nitrogen containing PNAs (e.g.,
carbazoles) would be expected constituents of these fractions.
The polar neutral fractions showed, for all samples, significant
spillover of PNAs. Some oxygenated PNAs (e.g., benzanthrone, anthraquinones,
hydroxyaromatics) were also identified in this fraction.
References
1. Huisingh, J. L., R. L. Bradow, R. J. Jungers, B. D. Harris, R. B.
Zweidinger, K. M. Gushing, B. E. Gill, R. E. Albert. 1980
Mutagenic and Carcinogenic Potency of Extracts of Diesel and Related
Environmental Emissions: Study Design, Sample Generation, Collection,
and Preparation. EPA Reports (EPA Report EPA-600/9-80-057b, pp.
788-800.
-------
2. Novotny, M., P.L. Lee, K.D. Bartle. 1974. J. Chrom. Sci. 12:606.
3. Rodriguez, C.F., J.B. Fisher, and D.E. Johnson. 1980. Health Effect of
Diesel Engine Emissions: Proceedings of an International Symposium,
Vol. 1. EPA-600/9-80-057a. U.S. Environmental Protection Agency:
Cincinnati, OH.
4. Erickson, M.D., D.L. Newton, K.B. Tomer. 1980. Analytical
Charactierization of Diesel Exhaust Particulate Extracts. Third Annual
Report. EPA Contract No. 68-02-2767.
Table 1. Mass Balance Results from Fractionation of Comparative Samples
Amount fractionated (mg)
Fractional totals (mg)
Mass balance ( % )
Diesel
Vehicle
803.0
696.9
86.8
Cigarette
Smoke
913.8
435.0
47.6
Coke
Oven
936.8
783.1
83.6
Roofing
Tar
1071.2
895.9
83.6
Table 2. Percent of Total Mass Recovered Upon Fractionation
of Comparative Samples
Fractions
Acid
Base
PN
NPN
PNA1
PNA2
PNA3
PNA4
CI
Diesel
Vehicle
3.6
1.0
7.6
74.2
1.4
1.7
0.9
1.6
7.8
Cigarette
Smoke
1.0
12.1
11.5
0.6
0.02
0.0
0.1
0.9
73.7
Coke
Oven
0.4
4.3
7.1
15.2
4.9
0.0
4.0
0.7
63.3
Roofing
Tar
2.3
2.9
13.1
40.4
31.8
1.2
0.02
0.3
8.0
-------
Figure 1.
SOLVENT PARTITIONING FRACTIONATION SCHEME
SAMPLE
DCM (CH2CL2) EXTRACTION
ACID WASH
BASIFY (pH 10)
ORGANIC
BASE
BASE
CYCLOHEXANE
SOLUBLES
NON-POLAR
NEUTRALS
NPN
BASE WASH
ACIDIFY (pH 3)
ORGANIC
ACID
I
CYCLOHEXANE
NEUTRALS
MeOH WASH
MeN02 WASH
SOLUBLES
PNA +
POLAR NEUTRALS
MeOH
SOLUBLES
POLAR
NEUTRALS
I
PN
HPLC
CYCLOHEXANE
INSOLUBLES
CI
25 DCM
IN HEXANE
PNA-1
50/50
HEXANE OCM
PNA-2
,
DCM
PNA-3 | Pto
MeOH
IN DCM
-------
TRAPPING GASEOUS HYDROCARBONS
Fred Stump
Environmental Sciences Reserach Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
The gas phase mutagen research was initiated at the request of the Office
of Mobile Source Air Pollution Control for the development of a procedure to
trap gas phase hydrocarbons (HC) and then to use the method in a series of
studies with the objective of comparing the Ames Bioassay activity of the gas
phase with the particle-bound vehicle emissions.
Several of the hydrocarbon absorbents in use by other researchers were
procured for evaluation as to applicability for HC collection in the diesel
fuel range. A thorough examination of the known physical characteristis of
the mediums resulted in the selection of coconut charcoal and XAD-2 resin
(divinylbenzene polystyrene polymer) as the best possible candidates.
Preliminary evaluations consisted of making injections of diesel fuel
into a small dilution tunnel and trapping the hydrocarbons with subsequent
recovery determinations proving both to be excellent HC absorbers but the
XAD-2 was the better release medium. Although, several different solvents and
solvent combinations were used as extraction agents to improve recovery
efficiency from the charcoal, a recovery greater than 60% could not be achiev-
ed and work with charcoal was discontinued. Further recovery work showed the
XAD-2 to be quantitative by both chromatographic and gravimetric analysis for
diesel fuel range hydrocarbons. The recovery and qualification experiments
were performed using A.D. Little (ADL) developed odor traps, 3/8" O.D. x 2"
length, filled with the absorbents.
With completion of the qualification tests several samples were taken
from both a 1978 Oldsmobile and a 1978 Datsun 220C diesel powered passenger
car with the resin filled ADL traps. An extended sample time in the order of
5-10 hours was necessary, due to the low trap flow rate and limited resin
capacity, to obtain sufficient materials for Ames Bioassay. This long sample
time not only tied up equipment and personnel excessively but also posed some
-------
question as to the possibility of artifact formation on the resin. In order
to help clarify the artifact question, an experiment was conducted by making
injections of diesel fuel and NOX on resin filled traps (ADL) and then
submitting the extract for Ames testing using the TA 98 strain. The test
results indicated that considerable artifact, sample level in some situations,
could be generated under these long exposure conditions and further hydro-
carbon collecting with these tubes was discontinued.
To eliminate the low flow and the long sample time problems, a larger 2"
x 2" trap with about 100 times the resin capacity of the ADL tubes was fabri-
cated and carried through the same qualification experiments as the smaller
traps with comparable recovery efficiencies (greater than 95%).
These large traps were then used to sample a series of different cycles
(FTP'S, NYCC, HWFET's) from a VW Rabbit diesel passenger car. These samples
were submitted for Ames testing with the results showing the gas phase mater-
ials to be active but this activity was only 9-16% (depending on test cycle)
of the particle-bound activity. Although, artifact formation is probably
still present the trap activity data indicates that it had been substantially
reduced.
These tests completed the diesel studies. Since the gasoline system had
little N0£ present, with possible minimum artifact formation, an XAD-2
trapping system, 20" x 20" x 2" bed, having 100 times (due to low gasoline
emissions) the resin capacity of the 2" x 2" traps was fabricated to collect
the hydrocarbons. A series of three gasoline powered vehicles were tested:
(1) a 1972 Chevrolet Impala using unleaded fuel; (2) a 1981 Dodge van with a
light duty catalyst system also using unleaded fuel; and (3) a 1970 Ford van
using commercially available leaded fuel.
A sample from each of the vehicles was then submitted for Ames Bioassay
testing with the XAD-2 trapping results being quite different from that
observed in the diesel vehicle. The activity of the gaseous materials
trapped by the XAD-2 was at background level for all three vehicles. The
Dodge van particle-bound HC, without S9 activation, had about twice the act-
ivity in reverents/microgram as the Impala, and four times the activity of the
Ford, with the higher Dodge activity density probably due to the oxidative
properties of the emissions control system since the catalyst was the major
parameter difference between the Dodge and Impala. The Ford had the lowest
activity of the three vehicles and this could possibly be attributed to the
lower fuel aromatic content (44.4% unleaded and 27.8% leaded).
A comparison of the diesel and gasoline vehicles activity (reverents/
microgram) indicates that for the HWFET cycle,(the only cycle tested common to
all vehicles) without activation the diesel and 1972 Chevrolet are about the
same activity, the Dodge has twice the diesel activity, and the Ford about one
half the diesel activity. Although the gasoline emissions have activities
comparable on a revertent/microgram basis, when the emissions are observed on
a reverent/mile basis, without activation, the worst of the gasoline vehicle
emitters (Ford) has an activity that is only 11.8% and, with activation, only
4.5% of the diesel particle-bound activity.
-------
These trapping studies have clearly indicated the low level of activity
associated with the diesel gas phase hydrocarbons and the extremely low or
background levels present in gasoline gas phase emissions.
The ability of XAD-2 to effectively collect diesel fuel range HC has been
well demonstrated and studies are currently in progress to characterize the
XAD-2 collectability of gasoline fuel range hydrocarbons.
-------
ANALYTICAL METHODS FOR NITROAROMATIC COMPOUNDS
by
Silvestre B. Tejada
Mobile Source Emissions Research Branch
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
A number of methods have been used to detect and/or measure nitroaromatic
compounds in environmental samples (l-5). The analysis usually involves a
combination of fractionation schemes - solvent-solvent extraction, thin
layer chromatography (TLC), open column chromatography, high performance
liquid chromatography (HPLC) - and followed by analytical finish using TLC,
HPLC with UV and fluorescence detection, gas chromatography (GC), and a
variety of mass spectrometric (MS) techniques. Most of these methods are
labor intensive and some are plagued by poor sensitivity and interference
problems.
We have developed a reverse phase HPLC-fluorescence method using water-
methanol solvent for the detection, identification and measurement of selected
nitroaromatic compounds with sensitivity at low and sub-nanogram levels. The
detection technique is based on on-column catalytic conversion of the non-
fluorescent nitroaromatic compounds to the highly fluorescent amine analogs.
Compound selectivity is achieved by appropriate choice of wavelengths for
fluorescence measurements. Stop-flow techniques and spectral scanning of the
trapped peaks were used to establish chemical identify by comparison with
spectra of standard samples.
The heart of our analytical system is a platinum-rhodium catalyst column
(maintained at 60-80 degrees Celsius) between two reverse phase ODS columns.
Initial separation is achieved in the first ODS column, reduction to amine
analog is immediately accomplished in the catalyst column and the final
analytical separation of the aminoaromatic compounds from interfering compon-
ents is achieved in the second ODS column. By allowing only selected amino-
aromatic peaks through the second ODS column, we have managed to conveniently
eliminate the tedious sample clean-up prior to analysis. Figures 1 and 2
illustrate the use of this technique in the analysis of nitro-pyrene.
Precision of ± 3% at 1 nanogram level is routinely obtained for nitro-pyrene
analysis. Minimum detectable quantity of nitropyrene under our present
analytical configuration is about 20 picograms.
-------
The catalyst has been observed to reduce nitro compounds to the corresponding
amine reproducibly under fixed conditions of flow rate, temperature and
solvent composition. The following nitroaromatic compounds are converted with
better than 99% conversion efficiency: nitro-naphthalenes, nitro-anthrance,
nitro-fluorene, nitro-chrysene, nitro-BaP, dinitro-pyrenes and nitro-fluor-
enones. We have obtained fluorescence spectra of the amine analogs of most of
the nitroaromatic compounds available to us. The amines, especially the
diamines, were observed to be unstable under UV light. Adjustment of the
solvent pH to about 8 with NaOH helped to stabilized the diamines adeqately to
make reproducible spectral scans of the trapped peaks.
The present analytical system has been used to measure nitro pyrene in complex
matrices such as diesel exhaust particulate extracts, leaded and non-leaded
automotive exhaust particulate extracts, gas trap extracts, fly ash extracts
as well as biological extracts. Samples dissolved in DMSO intended for Ames
tests are likewise amenable to analysis without additional sample clean-up.
Other nitroaromatic compounds can be detected and measured by appropriate
choice of chromatographic elution windows coupled with the optimum wavelengths
for fluorescence measurements.
REFERENCES
1. Jager, J., "Detection and characterization of nitro derivatives of some
polycyclic aromatic hydrocarbons by fluorescence quenching after thin layer
chromatography: Application to air pollution analysis", 0. Chrom. 152,
575-578 (1978).
2. Schuetzle, D., Lee, F.S. -C., Prater, T.J., Tejada, S.B., "The Identifica-
tion of polynuclear aromatic hydrocarbon derivatives in mutagenic fractions of
diesel particulate extracts", Intern. J. Environ. Anal. Chem. 9_, 1-53, (1981).
3. Gibson, T.L., Ricci, A. I., Williams, R.L., "Measurement of Polynuclear
Aromatic Hydrocarbons, Their Derivatives and Their Reactivity in Diesel
Automobile Exhaust" in "Chemical Analysis and Biological Fate: Polynuclear
Aromatic Hydrocarbons", Cooke, M. and Dennis, A.J., eds., Battle Press, 1981,
p.707.
4. Schuetzle, D., Riley, T., Prater, T.J., Harvey, T.M. and Hunt, D., " The
Identification of Nitrated Derivatives of PAH in Diesel Particulates", in
press.
5. Rozenkranz, H.J., McCoy, E.C., Sanders, D.R., Butler, M., Kiriazides,
O.K., Mermelstein, R.."Niropyrenes: Isolation, identificatin, and reduction
of mutagenic impurities in carbon black and toners", Science 202, 515-519
(1978).
-------
I-JW
Figure 1
Neat diesel extract (25 ug) through ODS column only ( );
through CDS and catalyst columns ( ). Note peak
enhancements due to formation of aminoconpounds. Detection
wavelengths: Excitation (360 nm), Bnission (430 ran), UV (254 ron).
Figure 2
Neat diesel extract (25 ug) through ODS-Catalyst-ODS columns.
Only the nain aminopyrene peak was injected into the second
ODS column. ( ) Ov and fluorescsnce profiles of the sanple
through ODS-Catalyst columns. Note removal of interferrent
peaks after passage through second ODS column.
-------
TOTAL LUMINESCENCE SPECTROSCOPY
OF DIESEL EXHAUST PARTICULATE
by
Gregory Wotzak, Ph.D.
Cleveland State University
Cleveland, Ohio
Robert Whitbv. P.E.
New York State Department of Environmental Conservation
Division of Air
Albany, New York
Total Luminescence Spectroscopy (TLS) analysis of organic extract
material from diesel exhaust particulate matter has been previously described
by Wotzak et al(l). TLS is the determination of the luminescence intensity
as a function of all accessible excitation and emission wavelengths. TLS
data are typically obtained from between 50 and 200 emission spectra, each
taken at a specific excitation wavelength. Contour mapping of the points of
equal luminescence intensity on an excitation vs. emission wavelength grid
has been chosen as a convenient means of representation for TLS data. TLS
analysis thus encompasses the computerized data acquisition, manipulation,
display, and interpretation of such luminescence data.
The New York State Department of Environmental Conservation, Automotive
Emission Evaluation (AEE) unit has been studying the utilization of TLS in the
characterization of diesel particulate organic extract using 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 micropro-
cessor controller which are linked to a host Data General Nova 3 computer
system with 128K words of core and a 10 megabyte disk. Software was pro-
vided by Baird Corporation and modified by AEE computer personnel to include
data smoothing algorithms. Contour plots are produced on a Houston Instru-
ments Complot X-Y recorder.
More recent advancement in raw data reduction has been achieved by wave-
length correction and digital smoothing using Fast Fourier Transform methods.
Extract samples, fractionated by column chromatography, have been analyzed,
generating TLS contour maps for each fraction. These fraction contours may
be added by computer software routines, using appropriate weighting by cut
recovery factors, and compared with spectra for the original bulk sample.
-------
This reconstitution work was performed as an internal consistency test for
both the acidic, basic and neutral cuts of the raw extract, as well as the
sub-cuts of the neutral fraction.
A variety of tasks have been performed in order to obtain a general in-
dication of the utility of this relatively new analytical procedure. Se-
quential dilutions of extract sub-fractions were performed in order to
determine the extent of internal absorption of fluorescent radiation. TLS
spectra of the diesel fuel and lubricant were subtracted from appropriate
spectra in order to facilitate further analysis, and determine the partition
of unburned fuel and lubricant among extract sub-fractions. The neutral
fraction was analyzed qualitatively and quantitatively for several known
compounds .
Wotzak, G., R. Gibbs, and J. Hyde, 1980, A Particulate Characterization
Study of In-Use Diesel Vehicles. In: Health Effects of Diesel Engine
Emissions: Proceedings of an International Symposium, Volume 1.
W.E. Pepelko, R.M. Danner, and N.A. Clarke, editors. U.S. Environmental
Protection Agency, EPA-600/9-80-057a. pp. 113-137.
-------
EVALUATION OF THE METABOLIC REQUIREMENTS OF DIESEL AND COMPARATIVE SOURCE
SAMPLES IN THE SALMONELLA TYPHIMURIUM PLATE INCORPORATION ASSAY
by
Katherine Williams and Joel!en Lewtas
Genetic Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
The mutagenic activity of a mobile source sample (Nissan diesel exhaust
extract) and three comparative source samples (coke oven mains, cigarette smoke
condensate, and roofing tar extract) were examined in the Ames plate
incorporation assay using strain TA98. The mutagenic and carcinogenic activity
of these complex mixtures had previously been determined in a bioassay test
matrix (1). The comparative sources were less mutagenic in the Ames assay
compared to the mobile source sample. However, they were more active in
mammalian cell mutation and mouse tumor initiation bioassays. The objective of
this study was to determine whether modifications in the S9 activation system
would alter the mutagenic activity in the Ames bioassay. The modifications
examined included altering both the species from which the liver S9 was
prepared and the concentration of S9 on the plate.
Both Aroclor 1254-induced and -uninduced S9 were prepared from CD rats and
Syrian golden hamsters. Livers were pooled from groups of at least 6 male
animals for each preparation. The protein concentration of each S9 was
determined by the method of Lowry et al. (?.}. Four doses of S9 were run:
0.31, 0.63, 1.25, and 2.5 mg/plate, chosen to encompass the usual concentration
in the Ames test (ca 1.5 mg/plate). Aliquots were adjusted to contain the
required dose in a 0.5-ml volume as used in the Ames test.
Three doses of each source sample (a low, medium, and high range) were
tested at all four S9 concentrations for each S9 preparation. The dose varied
between samples and were selected to be below the toxic level for each, yet
high enough to have mutagenic activity. For Diesel Nissan and cigarette smoke,
the doses were 30, 100, and 300 yg/plate. For coke oven mains and roofing tar,
they were 5, 50, and 100 ug/plate.
Experiments were run in duplicate, on different days, using triplicate
plates for each point. In any experiment all 3 doses of 2 samples were tested
with all 4 concentrations of both uninduced and induced S9 from one species.
For each experiment fresh dilutions were prepared from aliquots of the S9
-------
preparations, which were held at -80°C. The time between duplicate experiments
ranged from 3 to 9 days. The data is presented as the mean ± SE for duplicate
experiments.
The optimum S9 dose for all samples, with the exception of the Diesel
Nissan which did not require metabolic activation, was either 1.25 or
2.5 mg/plate for both induced and uninduced rat and hamster S9, as shown in
Table 1 for the induced rat liver S9.
Recently, several studies have noted that induced hamster S9 is more
effective than induced rat S9 in activating such compounds as aromatic
amines (3) phenacetin (4) and diethylnitrosamine (5), while Aroclor-induced rat
S9 is more effective than hamster S9 with polycyclic aromatic hydrocarbons (3).
This study showed no difference in effectiveness between hamster S9 and rat S9
in activating the Diesel or the comparative source samples.
The mutagenic activity of the comparative source samples was higher when
Aroclor-induced S9 was the metabolic activator as compared to uninduced S9,
whether rats or hamsters were the source of the S9. The Diesel Nissan sample
was again the exception; the mutagenic activity was higher with uninduced S9
than with induced.
In conclusion, for the three comparative source samples, all of which
require metabolic activation for maximum mutagenic activity in the Ames test:
the optimum S9 dose is 1.25 to 2.5 mg/plate; Aroclor-induced S9 is more
effective as an activator than uninduced S9 regardless of species; and rat and
Syrian golden hamster S9 are equally effective in activating these complex
mixtures. These results would suggest the lower mutagenic activity of the
comparative source samples in the Ames test as compared with other mutagenicity
or carcinogenicity bioassays was not due to the exogenous metabolic activation
system.
REFERENCES
1. Nesnow, S., and J.L. Huisingh. 1980. Mutagenic and carcinogenic potency
of extracts of diesel and related emissions: Summary and discussion of
the results. In: Health Effects of Diesel Engine Emissions.
Proceedings of an International Symposium, Vol. 2. W.E. Pepelko, R.M.
Danner, and N.A. Clarke, eds. EPA-600/9-80-057b. U.S. Environmental
Protection Agency: Cincinnati, OH. pp. 898-912.
2. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein
measurement with the golin phenol reagent. J. Biol. Chem. 193:265-275.
3. Raineri, R., J.A. Poiley, R.J. Pienta, and A.W. Andrews. 1981. Metabolic
activation of carcinogens in the Salmonella mutagenicity assay by
hamster and rat liver S9 preparations. Environ. Mutagen. 3:71-84.
-------
Nagao, M., T. Sugimura, and T. Matsushima. 1978. Environmental mutagens
and carcinogens. Ann. Rev. Genet. 12:117-159.
5. Prival, M.J., V.D. King, and
diabyl nitrosamines in the
1:95-104.
A.T. Sheldon. 1Q79. The mutagenicity of
Salmonella plate assay. Environ. Mutagen.
Table 1. Effect of Metabolic Activation Dose in Mutagenicity
of Diesel and Comparative Source Samples3
Revertants/plateb
Samp!
e
Diesel Nissan
Coke oven mains
Cigarette smoke
Roofing tar
aSalmonel
la
typhimurium
0.31
816 ±
492 ±
68 ±
59 ±
TA98.
c
26
57
0
1
Samples
0
625
727
83
86
at
.63C
± 39
± 8
± 4
± 2
100
yg/plate,
1.
491
861
74
94
i
25C
± 50
± 21
± 6
± 3
2.5<
334 ±
874 ±
64 t
113 ±
31
27
3
9
"Mean ± SE of two experiments with triplicate plates.
cRat Aroclor 1254-induced S9, mg/plate.
-------
MS/MS CHARACTERIZATION OF DIESEL PARTICULATES
by
Karl V. Wood
Fuels Analysis Laboratory-Chemistry Building
Purdue University
West Lafayette, Indiana 47907
James D. Ciupek and R. Graham Cooks
Department of Chemistry
Purdue University
West Lafayette, Indiana 47907
Colin F. Ferguson
School of Mechanical Engineering
Purdue University
West Lafayette, Indiana 47907
INTRODUCTION
Analysis of the organic constituents adsorbed on diesel exhaust particu-
lates has become increasingly important with the increase in the number of
diesel engine automobiles. Extraction followed by GC/MS has been the usual
analytical method of choice for these unknown organic constituents. Mass
spectrometry/mass spectrometry (MS/MS) offers a possible means of direct
analysis with either minimal or no separation. This technique enables a
fast characterization of the organic constituents of the whole diesel par-
ticulates. Selectivity and specificity of particular compounds or classes
of compounds of interest using MS/MS can be improved by the appropriate choice
of the chemical ionization reagent gas as well as the choice of positive or
negative ion detection.
EXPERIMENTAL
The MS/MS experiments described in this study were run using a Finnigan
triple stage quadrupole mass spectrometer.1 The diesel particulate sample
is introduced into the source with the direct insertion probe which is
heated in steps to obtain temperature profiles of the organics adsorbed on
the particulates. The sample is ionized using the chemical ionization
technique. The ion of interest is mass selected by quadrupole 1, undergoes
collisionally induced dissociations in quadrupole 2 with the resulting
-------
fragment ions being mass analyzed with quadrupole 3. Typcial ion axial
energies into quadrupole 2 are 20 eV relative to the source. The collision
gas used for these studies was argon at a pressure ca. 2.2 mTorr.
The diesel engine employed in this study was an AVL model^ 520.005
naturally aspdrated single cylinder direct injection engine. The diesel
exhaust particulates are sampled using a mini dilution tunnel system.3 AS
this system was designed both the dilution ratio and the temperature of the
particulate filter can be varied.
RESULTS
The initial emphasis of this study was to identify constituents af
diesel exhaust particulates by direct analysis using MS/MS. This identifi-
cation is done by comparing the MS/MS spectrum of a particular ion in the
diesel exhaust particulate sample with the MS/MS spectrum of the correspon-
ding ion of a standard reference compound. For example, the MS/MS spectrum
of the m/z 143 ion from a diesel exhaust particulate sample has two major
fragment ions, m/z 128 (100%) m/z 115 (15%) besides the main beam ion m/z
143 (20%). These ion ratios are nearly identical to that found in the
MS/MS spectrum of 2-methylnaphthalene, suggesting its presence in the
diesel exhaust particulate sample. It is not possible to say which methyl-
naphthalene is present, if only one is, or the relative concentration of the
two in a probable mixture of both. The case of the m/z 143 ion suggesting
the presence of predominately only one constituent and its positional isomers
is not unique. However, as would be expected the MS/MS spectra of many ions
are suggestive of the presence of more than one type of structure. An
example of this is the ion at m/z 139 in the diesel exhaust particulate
sample which is strongly indicative of the presence of hydroxybenzoic acid
isomers through comparison with standard reference compounds. However, there
are other relatively intense fragment ions in'the MS/MS spectrum which can-
not be resulting from hydroxybenzoic acid. One other compound that may
account for the remaining fragment ions is decalin.
Along these lines is the investigation of the selectivity and specifi-
city of the MS/MS technique as it relates to the direct analysis of diesel
exhaust particulates. For example the comparison of positive and negative
ion isobutane chemical ionization of a diesel exhaust particulate sample can
yield information about specific classes of compounds. An example of this is
the identification of carboxylic acids in the diesel exhaust particulate
samples. In positive ion chemical ionization a carboxylic acid will be
protonated as will an aromatic hydrocarbon at the same nominal mass. How-
ever, in negative ion chemical ionization the carboxylic acid will lose a
proton to give a (M-H)~ ion whereas the aromatic hydrocarbon will be ionized
by electron transfer to give a M~ ion. This technique was used to confirm
the identification of the components in the m/z 139 ion MS/MS spectrum
discussed previously. Thus the use of negative ion chemical ionization allows
the separation of different classes of compounds for MS/MS identification in
the direct analysis of diesel exhaust particulates.
Another example of selective ionization to allow a more accurate MS/MS
identification to be made is the use of differing reagent gases. While
-------
isobutane chemical ionization is a general protonating agent, ammonia can
be used to protonate relatively basic compounds, like amines or aza compounds
While this technique has been used successfully in the analysis of coal-
derived liquids^ it has not been as useful with diesel exhaust particulates.
Besides these studies, two variables associated with sampling the
diesel exhaust particulates have also been investigated. These variables,
dilution ratio and particulate filter tmperature were utilized to gain a
better insight into the complex problem of particulate sampling.
CONCLUSION
MS/MS provides a means for the rapid direct analysis of diesel
exhaust particulates. The use of selective ionization techniques further
enhances the positive identification when different types of compounds are
present at the same nominal mass.
REFERENCES
1. Slayback, J.R.B. and M.S. Story. 1981. Chemical Analysis Problems Yield
to Quadrupole MS/MS. Industrial Research FEB; 129-134.
2. Pischinger, R. and W. Cartellieri. 1972. Combustion System Parameters
and Their Effect Upon Diesel Engine Exhaust Emissions. SAE Paper
720756.
3. MacDonald, J.S., S.L. Plee, J.B. O'Arcy, and R.M. Schreck. 1980.
Experimental Measurements of the Independent Effects of Dilution
Ratio and Filter Temperature on Diesel Exhaust Particulate Samples.
SAE Paper 730834.
4. Zakett, D., V.M. Shaddock and R.G. Cooks. 1979. Analysis of Coal
Liquids by Mass Analyzed Ion Kinetic Energy Spectrometry. Anal.
Chem. 51:1849-1852.
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INHALATION TOXICOLOGY OF DIESEL EXHAUST PARTICLES
by
R. 0. McClellan, A. L. Brooks, R. G. Cuddihy, R. K. Jones,
0. L. Mauderly and R. K. Wolff
Lovelace Inhalation Toxicology Research Institute
P. 0. Box 5890
Albuquerque, NM 87185
Studies of the inhalation toxicology of diesel exhaust particles (DEP)
have been directed toward answering several inter-related questions. Does
exposure of laboratory animals or people to high levels of DEP place them at
increased risk for developing health effects? If health effects are ob-
served, what are the mechanisms by which DEP produces health effects? If
health effects are observed, does consideration of the specific kinds of
health effects and the mechanisms by which they are produced provide a basis
for extrapolating these health effects to exposure levels of DEP likely to be
encountered in occupational or environmental settings?
With these questions in mind, an idealized experimental approach to
assessing the toxicity of inhaled DEP will be presented. Using this approach
as a reference point, the current state of our knowledge on the toxicity of
DEP will be reviewed. This will include consideration of the deposition and
retention of DEP; dissociation, detoxification and activation of organic
species associated with DEP; the effective dose of the organic species to
various tissues of the respiratory tract and other organs; and carcinogenic
and other effects produced by exposure to DEP. Each of these areas will be
reviewed to determine the adequacy of our current knowledge and to identify
information needs that have not yet been adequately addressed relative to
answering the questions that have been posed.
ACKNOWLEDGEMENTS
Research performed under U.S. Department of Energy Contract No. DE-AC04-
76EV01013 and conducted in facilities fully accredited by the American Asso-
ciation for Accreditation of Laboratory Animal Care.
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U.S. ENVIRONMENTAL PROTECTION AGENCY'S INHALATION TOXICOLOGY STUDY
by
William E. Pepelko
Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio
Due to increasing use of diesel engines in passenger cars and light
trucks, the U.S. Environmental Protection Agency (EPA) over the past several
years has undertaken an investigation of the health effects of diesel engine
emissions. This program included studies in the areas of epidemiology,
bioassays of collected diesel particulate matter, and animal inhalation
studies. The inhalation studies described are those carried out by the EPA
Health Effects Research Laboratory at Cincinnati, Ohio. The types of studies
include an assessment of the effects of inhaled diesel engine emissions upon a
variety of toxicological, carcinogenic, and heritable mutagenic endpoints.
Nonheritable mutation will be described elsewhere.
Due to limitations on chamber space resulting from the wide variety of
planned experiments, a single exposure level was selected. Because of the
Agency's interest in carcinogenic and mutagenic risk assessment and because of
its plans to use linear non-threshold models to make risk assessments, an
exposure level near the maximum chronically tolerated concentration was
selected. After a two-month preliminary study to aid in estimating tolerance
levels, an exhaust concentration was selected such that the total suspended
particulate matter equaled 6 mg/m3. This was achieved by a daily adjustment of
the dilution ratio which was usually near 18:1. After about one year of
exposure, it became apparent that this concentration was not producing overt
signs of toxicity, such as decreased food consumption and body weight gain. As
a result, a decision was made to decrease the exhaust dilution ratio to about
9:1, and to increase the particulate concentration to 12 mg/m3. The
particulate concentration was maintained at this level until completion of
exposures, approximately 14 months later.
The animals were housed in wire cages and exposed in 24 100 cubic-foot
stainless steel chambers. Exhaust was produced by a 6-cylinder 198 cubic-inch
displacement Nissan diesel engine developing 90 horsepower. City driving
conditions were simulated by operating the engine under the Federal Short Cycle
mode. The engine was operated 8 h/day, 7 days/week. Carbon dioxide, carbon
monoxide, nitrogen oxides, nitrogen dioxide, sulfur dioxide, total
hydrocarbons, total suspended particulate matter, temperature, and relative
-------
humidity were monitored regularly. Aliphatic aldehydes, ammonia, and sulfates
were measured periodically.
A wide range of experiments were carried out during the 124 weeks the
engines were operated. Carcinogenesis studies included lung tumor induction in
Strain "A" mice and Syrian hamsters, tumor induction in SENCAR mice treated
with promoters and initiators, and liver island assays. No conclusions could
be reached from the hamster study due to the loss of a large number of animals
from Tyzzer's disease during the exposure.
The other three studies have been completed and will be reported on at
this conference. The effects of exhaust exposure on heritable mutations was
investigated using mice and fruit flies. These studies were reported at the
1979 Diesel Health Effects Symposium held at Cincinnati. Other toxicological
endpoints studied included reproductive effects in mice; teratological effects
in rats and rabbits; pulmonary function changes in mice, Chinese hamsters, and
cats; lung pathology in mice, rats, hamsters, and cats; behavioral and
neurophysiological effects in rats; biochemical alterations in the lungs of
mice, rats, and cats; resistance to infection in mice; deposition and clearance
in rats; and enzyme induction in mice. The majority of these experiments have
been completed and many were reported on at the previous symposium. Two that
will be presented here include pulmonary function changes in cats exposed to
diesel exhaust for two years and the enzyme induction studies. Two other
experiments, a multigeneration reproduction study in mice and detailed
morphometric analysis of chronically exposed cats' lungs, are not yet
completed.
Following completion of these studies, no further inhalation exposures are
planned at EPA's Cincinnati facility.
-------
PULMONARY FUNCTION TESTING OF RATS CHRONICALLY EXPOSED
TO DILUTED DIESEL EXHAUST FOR 612 DAYS
K. B. Gross
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
Diesel engine emissions contain participate matter composed of a multitude of organic
compounds, and of a size that may be readily inhaled and retained by the lung. It thus
has the potential for interacting with the deep lung, and possibly altering the structure
and function of this organ. The study was designed to address the question of whether
the chronic inhalation of diluted diesel exhaust may affect pulmonary function.
Twenty-five Fischer-344 rats were exposed to diesel exhaust, diluted with clean air at a
ratio of 1:15 (particulate concentration = 1500 ug/m ) for 20 hrs/day, 5-1/2 days/week,
for 612 days. Twenty-five control animals were treated in a similar manner, but
exposed to clean filtered air. Noninvasive pulmonary function testing, which produced
no apparent harmful effects, were performed on the animals at mean times of 11, 23, 30,
38, 51, 65, and 87 weeks on the exposure regimen. Animals were anesthetized
(Halothane, 5%), transorally intubated, and placed in a plethysmograph. Measurements
of lung volumes and flows were made while the animals were spontaneously breathing
and during forced expiratory maneuvers. Functional residual capacities were computed
using the Boyles law principle. All data was normalized by each animal's own forced
vital capacity in order to compensate for animal growth and interindividual variability.
Through the first year of testing, no significant differences between the two groups
were found for any of the measured parameters. During the second year of exposure,
seven of the eighteen measured indices displayed a significant difference as indicated
by analysis of variance. The normalized functional residual capacity (FRC) and its
component volumes - expiratory reserve (ER) and residual volume (RV) - maximum
expiratory flows at 40% (MEF4Q) and 20% (MEF«Q) of the lung volume remaining, and
the forced expiratory volume in 0.1 sec (FEV.i fwere all greater in the diesel-exposed
animals. The normalized inspiratory capacity (1C) was significantly larger in the
control group, but the test point of greatest difference for this parameter does not
exceed 4%, and at all other test points is 2% or less. The statistical significance of the
1C is interpreted as being the result of the very low variability of this parameter, rather
than a result of a clinically important change in the pulmonary function. The
significantly larger values for relative normalized FRC, ER, and RV in the diesel-
exposed animals could be indicative of chronic obstructive lung disease and are similar
to the changes seen in other studies in which an emphysema-like condition was induced
in rats by intratracheal instillation of elastase, as well as in reported clinical data on
chronic obstructive lung disease. However, this interpretation of the changes in the
three lung volumes is contradicted by the MEF.g, MEF2Q, and FEV l values. If chronic
lung disease was occurring, these parameters would be expected'to decrease in the
-------
diesel-exposed animals compared to the controls. The fact that they increased in the
diesel group, suggesting an improvement in airway caliber, is not consistent with what
would be expected based on studies reported in the literature for human pulmonary
disease. In conclusion, the majority of the measured parameters did not differ
significantly between the control and diesel-exposed groups, and while one cannot
exclude the possibility that the differences that were observed in this experiment
between the diesel-exposed and clean air controls may be attributable to the chronic
inhalation of the diesel exhaust, the results are not consistent with documented clinical
findings on chronic lung disease.
la
0.3-
0.2-
t,
o
CK
t.
0.0
0.1
Legend
CONTROL
EXPER _
DlF(c-e)
0 100 200 300 400 500 600 TOO
DAYS ON EXPOSURE.REGIMEN
1b
^ 8-
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C
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if ~i
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^ - CONTROL
^\ EXPER _ _
DIF(c-e)
100 200 300 400 500 600 700
DAYS ON EXPOSURE REGIMEN
Figure la Functional residual capacity (FRC) of diesel exposed ( ) and control
( ) animals as a fraction of each animal's forced vital capacity (FVC). The bottom
curve ( ) is the difference between the means of the two groups (control -
experimental).
Figure Ib Normalized maximum expiratory flow rate at 20% of vital capacity (MEF9ft).
Legend same as Figure la. ' ^°
-------
PULMONARY FUNCTIONAL RESPONSE IN CATS
FOLLOWING TWO YEARS OF DIESEL EXHAUST EXPOSURE
William J. Moorman and John C. Clark
National Institute for Occupational Safety and Health
Division of Biomedical and Behavioral Science
4676 Columbia Parkway
Cincinnati, OH 45226
and
William E. Pepelko and Joan Mattox
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Cincinnati, OH 45268
INTRODUCTION:
Both NIOSH and the EPA have responsibilities to assess potential health
effects from exposure to diesel engine exhaust. These responsibilities are
mandated under both the OSH and Clean Air Acts, respectively. While EPA's
responsibility relates to the general population, NIOSH's responsibility is
to the worker, especially those at high exposure risk in the mining and
transportation industries.
The advantages of diesel power are manyfold; however, the prime motivation
for the use of diesels relates to the reduced cost of operation due to longer
engine life, decreased maintenance, cheaper fuel, increased specific energy
(2.4 X that of gasoline), and increased safety. The increase in safety is
related to diesel fuel's lower volatility, flash point with a concomitantly
reduced explosive hazard when compared to gasoline. As a result of our
proximal locations in Cincinnati, the NIOSH, DBBS Cardiopulmonary Laboratory
participated with EPA-HERL Laboratory to apply comprehensive pulmonary
function testing to the cats exposed to the controlled diesel atmospheres.
BACKGROUND:
Evaluation of pulmonary function responses to experimentally controlled
diesel exhaust is primarily confined to investigation of the past few years.
Previous studies are largely epidemiologic and while they are important
guides, they lack qualitative and quantitative characterization, essential
for analytical, dose-response toxicology.
At the last symposium on Health Effects of Diesel Engine Emissions, Gross
reported pulmonary function findings in Fischer 344 rats following 38 weeks
of exposure at 1500 yg/m3 for 5-1/2 days/week, 20 hrs/day(l). He found no
differences in mechanical properties, lung volumes, or dynamic ventilatory
performance. In a later report presented at the Society of Toxicology meet-
ing (1980), he found higher function residual capacity and maximum expiratory
-------
flow rates at 20% of vital capacity in the diesel exposed group. These re-
sults are not interpretable or consistent with dysfunction. His conclusion
was that no clinically important alterations were observed after 65 weeks of
exposure(2).
In another report, O'Neil et al., reported on functional and morphological
consequences of diesel exhaust exposure in mice following 3 months of expo-
sure to a 1:18 dilution (diesel and air) of diesel exhaust with particulate
levels of 6.4 mg/m^. He found no statistically significant findings in lung
volume or diffusing capacity(3).
In a third report on functional responses in rodents, Vinegar et al., de- '
scribed significant decrements in vital capacity, residual volume, and
diffusing capacity in Chinese hamsters exposed to a dilution of diesel
exhaust with a particulate level of 6.4 mg/m^(4).
In the only larger animal exposure, we reported findings from one year of
exposure in cats(5). The exposure was conducted at the Cincinnati EPA, HERL
laboratory. A 1:18 dilution diesel exhaust with 6.4 mg/m3 was used. Our
results indicated no response in mechanical properties, lung volumes,
distribution, diffusing capacity or ventilatory performance following one
year of exposure.
METHODS:
In an effort to enhance functional response characteristics, the diesel
exposure was increased by decreasing the dilution ratio from 1:18 to 1:9
after the first year. A summary of exposure is provided in Table I. During
this 'second year, 19 of the original 21 male cats were exposed to 11.7 mg/m
particulate, 4.37 ppm nitrogen dioxide, 5.03 ppm sulfur dioxide, and 33.30
ppm carbon monoxide with total hydrocarbons of 7.72 ppm. Twenty male cats
served as controls. All cats were young adult males obtained from Liberty
Laboratories and were born and maintained in a disease-free environment and
inbred for several generations. They were of uniform size (3.63 ± 0.46 kg)
and within two weeks of the same age.
Prior to pulmonary function testing, the cats were fasted for one day. The
testing followed 18-20 hours of no diesel exposure. On a random schedule,
the cats to be tested were anesthesized with ketamine and acepromizine at a
dose of 42 mg/kg. Following induction of the anesthesia, an esophageal
balloon was placed in the lower third of the esophagus and an 18-22 F
endotracheal tube (largest possible) was inserted into the trachea with the
aid of a laryngoscope. All testing, except compliance (CL) and resistance
(RL) was performed with the cats placed in the prone position in a variable
pressure plethysmograph. CL and RL tests were performed with the cats re-
cumbent to facilitate measurement of transpulmonary pressure. Figure I
shows the general pulmonary testing situation diagrammatically.
Pulmonary mechanics were obtained from simultaneous volume, flow and trans-
pulmonary pressure. Dynamic compliance (CL(jym) was measured from volume
and transpulmonary pressure at points of no flow. Average flow resistance
(RLave.flow) was measured from change in transpulmonary pressure at equal
-------
volumes, divided by the sum inspiratory and expiratory flow. All mechanics
were obtained while the cats were spontaneously breathing (15-25 breaths/min).
The pulmonary function tests requiring breathing maneuvers [lung volumes,
forced expiratory flows (FEF% Vol), diffusing capacity (DLC!RQ), nitrogen
washout (AN2), and closing volume (CV)] were performed using a variable
pressure plethysmographic chamber previously described(6). The methods of
Brashear et al.(7) and Mitchell et al.(8) were combined to obtain values for
DLC18O and total lung capacity (TLC). The calculations for DLC180 were per-
formed according to the methods described by Wagner et al., for Cl8o(9).
All gas analyses were done using a respiratory mass spectrometer (Perkin-
Elmer MGA1100). Distribution was studied using the single-breath nitrogen
washout and closing volume adapted from the human methods described by Buist
and Ross(10). All data was tested statistically by nonparametric, Kruskal-
Wallis one-way rank analysis of variance.
RES HILTS:
Following the first year of exposure, no significant differences were found
in mechanical properties, diffusing capacity, uniformity of distribution or
ventilatory performance. In contrast to the negative findings following the
first year, we now have clearly defined responses at the end of two years.
Table II presents all parameters studied for control and exposed cats con-
trasting the values for one and two years.
The reduction in inspiratory capacity, vital capacity, and total lung
capacity with normal values for ventilatory function (mechanics of breathing)
indicates that a lesion is present which restricts breathing but does not
cause airway obstruction or loss of elasticity. This restrictive disease
found in this study is compatible with a diagnosis of pulmonary fibrosis of
the interstitial or intraalveolar type. Concurrent status may include
chronic inflammation, interstitial edema, or vascular engorgement. Addi-
tional support for the diagnosis of interstitial disease is the finding of
impaired diffusing capacity. Distribution of this disease appears nonuni-
form as indicated by the significantly elevated nitrogen washout values for
the exposed group.
DISCUSSION:
Pathological description of pulmonary responses to diesel exhaust at similar
concentrations has been previously characterized(11,12). The observations
include: (1) marked accumulation of black pigment laden macrophage in the
interstitum localizing around blood vessels and respiratory bronchioles; (2)
hyperplasia of the alveolar lining cells with focal thickening of the inter-
stitium; (3) interstitial pneumonitis; (4) traces of, or no emphysema or
peribronchiolitis. While the pathological examination of the cats' lung
not complete, the above description is consistent with our physiologic find-
ing of restrictive lung disease.
-------
REFERENCES:
1. Gross, K.B. 1980. Pulmonary Function Testing of Animals Chronically
Exposed to Diluted Diesel Exhaust. Presented at the Environmental
Protection Agency International Symposium on Health Effects of Diesel
Engine Emissions. Cincinnati, Ohio.
2. Gross, K.B. 1981. Noninvasive Pulmonary Function Testing of Fischer 344
Rats Chronically Exposed to Diluted Diesel Exhaust for Fifteen Months.
The Toxicologist. Vol. 1, No. 1.
3. O'Neil, J.J. et al., 1980. Functional and Morphological Consequences
of Diesel Exhaust Inhalation in Mice. Presented at the Environmental
Protection Agency International Symposium on Health Effects of Diesel
Engine Emissions. Cincinnati, Ohio.
4. Vinegar, A., et al. 1980. Pulmonary Function Changes in Chinese Hamsters
Exposed Six Months to Diesel Exhaust. Presented at the Environmental
Protection Agency International Symposium on Health Effects of Diesel
Engine Emissions. Cincinnati, Ohio.
5. Pepelko, W.E., et al. 1980. Pulmonary Function Evaluation of Cats After
One Year of Exposure to Diesel Exhaust. Presented at the Environmental
Protection Agency International Symposium on Health Effects of Diesel
Engine Emissions. Cincinnati, Ohio,
6. Moorman, W.J., et al. 1975. Maximum Expiratory Flow Volume Studies on
Monkeys Exposed to Bituminous Coal Dust. J. Appl. Physiol. 39:444-448.
7. Brashear, R.E., et al. 1966. Pulmonary Diffusion and Capillary Blood
Volume in Dogs at Rest and With Exercise. J. Appl. Physiol. 21:520-526.
8. Mitchell, N.M., et al. 1968. Application of the Single-Breath Method of
Total Lung Capacity Measurement to the Calculation of Carbon Monoxide Dif-
fusing Capacity. Am. Rev. Resp. Dis. 97:581-584.
9. Wagner, P.O., et al. 1971. Diffusing Capacity and Anatomic Dead Space
for Carbon Monoxide [C18o]. J. Appl. Physiol. 31:817-852.
10. Buist, A.S., et al. 1973. Quantitative Analysis of Alveolar Plateau
in the Diagnosis of Early Airway Obstruction. Am. Rev. Resp. Dis.
108:1078-1087.
11. Wiester, M.J., et al. 1980. Altered Function and Histology in Guinea
Pigs After Inhalation of Diesel Exhaust. Environ. Res. 22:285-297.
12. Karagiones, M.T., et al. 1981. Effects of Inhaled Diesel Emissions and
Coal Dust in Rats. Am. Ind. Hyg. Assoc. J. 42:382-391.
-------
Table I. EXPOSURE CHAMBER COMPONENT CONCENTRATIONS, STUDY AVERAGES
Weeks #1-61
Weeks 62-124
Dilution Factor (air:diesel)
Parti culate Mass, mg/m
Nitrogen Oxides
Nitric Oxide, ppm
Nitrogen Dioxide, ppm
Sulfur Dioxide, ppm
Total Hydrocarbons, ppn
Carbon Monoxide, ppm
Carbon Dioxide , %
DF
M
NOX
NO
N02
S02
THCcorr.
CO
CO2
18.16 ±
6.34 ±
11.64 ±
2.68 ±
2.12 ±
4.15 ±
20.17 ±
0.30 ±
1.72:1
0.81
2.34
0.80
0.58
0.97
3.01
0.04
9.37 ±
11.70 ±
19.49 ±
4.37 ±
5.03 ±
7.22 ±
33.30 ±
0.52 ±
1.13:1
0.99
3.80
1.19
1.03
0.85
2.94
0.04
Table II. PULMONARY FUNCTION PARAMETERS COMPARING THE CONTROL
GROUP TO THE DIESEL EXPOSED GROUP AFTER 1 YEAR AND 2 YEARS
Mechanical
Properties
CLdyn.
^ave.flow
One
YP
1 Exposed
23
10
.5 ±
.7 ±
7.2
4.6
23
10
ar
Control
.7 ±
.3 ±
9.3
4.4
Two
1 I
I 1 Exposed
27.5 ±
5.6 ±
4.9
3.2
Years
26.
5.
Control
2 ±
7 ±
7.1
2.3
Lung Volumes
TLC
FVC
FRC
ERV
RV
RV/TLC%
1C
415 ±
348 ±
158 ±
69 ±
86 ±
20.3 ±
279 ±
56.0
43.5
35.6
24.6
36.9
6.9
44.8
449 ±
368.9 ±
165 ±
67 ±
104 ±
22.7 ±
301 ±
74.5
42.1
42.2
19.0
37.7
5.9
49.6
± 56.34-*
± 42.34-*
± 26.24-*
± 24.0
±14.3
428
369
145
79
67
15.6 ± 1.9
291 ± 44.14-*
484
410
163
83
80
16.4 ± 4.5
328 ± 58.6
± 68.3
± 57.6
± 36.9
± 34.5
± 28.2
Ventilatory
Performance
FEV.5%
PEFR
FEF50
FEF10
FEF40%TLC
Diffusion
DLCO
84.3 ±
1016 ±
728 ±
490 ±
196 ±
8.4
185
196
186.8
107.4
486 ± 252.6
1.18 ± .43
81.S ± 6.4
1042 ± 174
761 ± 160
481 ± 199.5
222 ± 156.8
557 ± 248.0
1.22 ± .40
86.9 ± 6.1
887 ± 98 4-*
802 ± 125
518 ± 154
223 ± 109
586 ± 173
86.9
952
864
574
234
625 ±
5.9
110.7
121
153
102
213
0.89 ± .27 4-* 1.01 ± .14
Distribution and
Closing Volume
%N2/25%AC
CV
0.32 ± .20
25.6 ± 13.44-*
0.29 ± .30
36.0 ± 16.1
*Statistically significant P < 0.05
0.39 ± .27
27 ± 17.6
0.21 ± .181
25 ± 19.3
-------
SLAVE
CYLINDER
NEOPRENE
DIAPHRAGM
SPIROMETER
(11. Dry rolling seal)
4-WAY
VALVE
ASSEMBLY
HYDRAULIC
PUMP,
7 !/2 HP
MOTOR
EQUILIBRATION
VALVE
LIMIT
SWITCH
STAINLESS
STEEL
CHAMBER
TRANSDUCER
PNEUMOTACH
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FLOWRATE
AUTO. MANUAL
O O O O !«0
IN OUT IN OUT
MASS
SPECTROMETER
A-D Converter
CPU
Disc Drive
Tnn« Stnr
CONTROL PANEL
Diagram of Pulmonary Function Lab.
-------
DEPOSITION AND RETENTION OF SURROGATE AND ACTUAL DIESEL PARTICLES
by
R. K. Wolff, L. C. Griffis, G. M. Kanapilly and R. 0. McClellan
Lovelace Inhalation Toxicology Research Institute
P.O. Box 5890
Albuquerque, New Mexico 87185
INTRODUCTION
Data on deposition and retention of diesel particles are an important
need in assessing their toxicological impact. Deposition and retention
experiments v/ere carried out with 67Ga aggregate aerosols (surrogate
diesel particles) in Beagle dogs to provide good estimates for human depo-
sition. Experiments were also carried out with these particles in Fischer-
344 rats to provide comparative information in a small laboratory animal
and also to estimate lung burdens in chronic exposures to diesel exhaust.
A method was developed to quantitate actual diesel soot burdens in rats
exposed to diesel exhaust. These values were found to be comparable to
these predicted from the surrogate particle deposition experiments.
METHODS
Aggregated particles of ^Ga^Os, 0.02 and 0.1 urn volume median
diameter (VMD), were produced using heat treatment of ^Ga tetramethyl-
heptanedione using methods described previously (1). Ten Beagle dogs from
the Institute's colony were exposed for 1/2 hr in a nose-only exposure unit
equipped with a plethysmograph for pulmonary function monitoring. Whole
body counting and gamma camera analysis v/ere used to measure total amounts
of activity deposited and their regional distribution. A total of 144
Fischer-344 rats were exposed 5 hrs/day for either 1 or 3 days in the same
style multi-tiered exposure chamber used in the chronic diesel exhaust ex-
posure study. Whole body counting and also sacrifice and tissue counting
methods were used to measure deposited radioactivity.
The diesel exhaust exposure system consisted of an Oldsmobile 5.71
diesel engine connected to a dynamometer with the engjne load and speed
determined by an analog control system. The engine was operated on a
7 mode urban cycle and the fuel was U.S. Department of Energy Reference
fuel 8007 (Phillips Chemical Co.). The entire engine exhaust was diluted
in a large stainless steel tunnel and then further diluted in 3 stages with
filtered air which produced average particulate concentrations of 4150, 990
and 200 ug/m3. The diluted exhaust was drawn through the 2.2 m3 ex-
posure chambers (Hazleton Systems, Inc., Aberdeen, MD) at 560 1/min. The
exposure schedule was 7 hrs/day, 5 days/week.
-------
Laboratory reared Fischer-344 rats were 12-13 weeks old at the initia-
tion of the study. They were exposed for a cumulative period of 541 hours
over 18 weeks. At the end of this period 8 rats from each exposure group
were sacrificed,, Lungs were removeds homogenized, and centrifuged to pro-
duce a cell pellet. This tissue pellet was dissolved in 1 ml H20 and
2 ml tetramethy1arnmoniurn hydroxide. The remaining "soot" particles were
suspended in 5 ml ^0. Light absorbance at 690 nm was measured and com-
pared against standards prepared from known weights of diesel particles
collected on filters from the dilution tunnel of the inhalation exposure
system. For these collections the same engine cycle was used as for the
animal exposures.
RESULTS AMD DISCUSSION
Table 1 shows the mean total and regional deposition values measured in
the Beagle dogs for the 0.02 and 0.01 ym 676a203 particles. Depo-
sition was higher in all compartments for the 0.02 urn particles. Despite
the overall high variability in deposition 9 of the 10 dogs had higher
deposition at 0.02 ym than 0.1 ym and the difference was statistically
significant (P < .05). Although most of the material was deposited in
the pulmonary region, deposition in the nasopharyngeals and tracheobron-
chial regions was becoming increasingly significant as particle size
decreased. Figure 1 shows pulmonary deposition of the 0.02 and 0.1 ym
particles is in good agreement with the trends in deposition observed pre-
viously in humans (293) and Beagle dogs (4) at larger particle sizes. The
deposition values are lower than predicted by the ICRP Task Group on Lung
Dynamics (5) but are in good agreement with predictions by Yeh and Schum
(6) and also Yu (7).
Deposition of the 0.1 ym ^63203 particles was somewhat lower
in rats than had been found in dogs. Lung deposition (bronchial and pul-
monary) was estimated to be 15 ± 3%. This estimate was based on measured
lung burdens and assuming minute volumes as measured in free standing
Fischer-344 rats at this Institute. Absolute lung deposition was found to
be 2.4 yg/hr for a particle mass concentration of 1000 yg/m^. Using
these initial deposition values and a measured lung half-life of 75 days
for °'Ga2039 lung burdens could be calculated for various exposure
periods. Table 2 shows the predictions for the exposure period and concen-
trations experienced by the animals in the chronic diesel exhaust exposures.
Measured diesel soot burdens following the 18 week exposure are also
shown in Table 2. The observed values were in good agreement at the high-
est exposure level but overestimated deposition at the two lower levels.
Either deposition was higher or clearance was slower at the high exposure
level compared to the lower levels.
The degree of agreement between predicted and observed burdens shows
that deposition and retention behavior of the surrogate particles is simi-
lar to actual diesel particles. The observed -long-term retention of "'63203
in rats was very similar to the 62-day half-time reported by Chan, et al.,
(8) following acute exposures to ^-labeled diesel particles. These
observations give confidence to extrapolations made from observations with
-------
0.1 ym Ga203 aggregate particles. These data do show that pul-
monary deposition is relatively high for diesel particles, and retention
times are relatively long.
ACKNOWLEDGEMENTS
Research performed under U.S. Department of Energy Contract Number
DE-AC04-76EV01013 and in facilities fully accredited by the American
Association for the Accreditation of Laboratory Animal Care.
REFERENCES
1. Wolff, R.K., G.M. Kanapilly, P.B. DeNee, and R.O. McClellan. 1981.
Deposition of 0.1 pm chain aggregate aerosols in Beagle dogs.
0. Aerosol Sci. 12:119-129.
2. Lippmann, M. 1977. Regional Deposition of Particles in the Human
Respiratory Tract. Handbook of Physiology, Section A: Reactions
to Environmental Agents, The American Physiological Society,
Chaper 14, pp. 213-232.
3. Chan, T.L., and M. Lippmann. 1980. Experimental measurements and
emperical modelling of the regional deposition of inhaled
particles in humans. Am. Ind. Hyg. Assoc. J. 41:399-409.
4. Cuddihy, R.G., D.G. Brownstein, O.G. Raabe, and G.M. Kanapilly. 1973.
Respiratory Tract Deposition of Inhaled Polydisperse Aerosols in
Beagle Dogs. J. of Aerosol Sci., 5, 35-43.
5. Task Group on Lung Dynamics. 1966. Deposition and Retention Models
for Internal Dosimetry of the Human Respiratory Tract. Health
Physics, 12, p. 173-207.
6. Yeh, H.C., and G.M. Schum. 1980. Models of Human Lung Airways and
Their Application to Inhaled Particle Deposition. Bulletin of
Mathematical Biology, 42, pp. 461-480.
7. Yu, C.P. 1978. A Two-Component Theory of Aerosol Deposition in Lung
Airways. Bulletin of Mathematical Biology, 40, p. 693-706.
8. Chan, T.L., P.S. Lee, and W.E. Hering. 1981. Deposition and clearance
of inhaled diesel exhaust particles in the respiratory tract of
Fischer rats. J. Appl. Toxicol. 1:77-82.
-------
PULMONARY DEPOSITION
.6r
.4
O
en
\ /Theory (ICRP)
Human and Dog
Data
Theory
(fth and Schum)
aos
0.1
0.5 1.0
10
Volume Median Diameter]« Mass Median Aerodynamic-*]
Diameter
Figure 1. Comparison of mean pulmonary deposition (± S.D.) of
0.1 urn irregularly shaped polydisperse aerosols () with
that of spherical monodisperse aerosols. The mid range of
deposition data (///) taken from human experiments (Lippmann
1977 (2); Chan and Lippmann, (3)) and dog experiments
(Cuddihy et _aJL 1973, (4)) is shown. Also shown are
theoretical predictions for depositions in humans by ICRP
Task Group on Lung Dynamics (5) and also by Yeh and Schum (6),
-------
Table 1. Comparison of Total and Regional Deposition of Ga^ Particles in
Beagle Dogs
Particle Size
Compartment
Nasopharyngeal
Tracheobronchial
Pulmonary
n.l urn
n
n
25%
TOTAL 39%
0.2 gm
9%
12%
32%
53%
Table 2. Lung Burdens of Diesel Soot in Rats One Day After 18 Weeks Exposure
to Diluted Exhaust
Lung Burden (pg)
Average Aerosol
Concentration3
Predicted Observed
200 ± 70 . 100 36 ± 8
990 ± 390 500 224 ± 39
4150 ± 1460 2100 1926 ± 335
± S.D. of average daily values.
-------
LUNG CLEARANCE OF RADIO-ACTIVELY LABELLED
INHALED DIESEL EXHAUST PARTICLES
P. S. Lee, T. L. Chan, and W. E. Hering
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
The fate of inhaled diesel exhaust particles was studied in male Fischer 344 rats using
radioactive diesel particles generated from a single cylinder diesel engine and tagged in
the insoluble particulate core with radioactive llfc. The particle size, extractability
and 1!*C distribution in the diesel exhaust were characterized at various engine load
conditions. At full load, only 1% of the radioactivity of diesel particles was extractable
by dichloromethane and the mass median aerodynamic diameter of the particles was
0.12 ym [J. Appl. Tox., l(2):77-82, 1981]. Radioactive carbon dioxide was removed from
the exhaust by a diffusion scrubber prior to exposures via a "nose-only" inhalation
chamber. Rapid elimination of the inhaled 1I*CO2 from the blood in the expired air, and
urine of test animals indicated that the correction for increased radioactivity due to
the inhaled carbon dioxide was necessary only for the initial deposition measurement.
The amount of blood and its contribution of lf*CO2 activity was accounted for in the
excised organs.
Test animals were exposed to diluted diesel exhaust at two particulate concentrations
with similar total inhaled dose (7000 yg/m3 for 45 minutes, and 2000 yg/m3 for 140
minutes) and had comparable deposition efficiencies. After the exposure, the animals
were housed in a clean air environment and the clearance of the radioactively tagged
particles was determined over an extended period of time. Up to the period of 28 days
after exposure, no significant difference in particle clearance has been observed. This
indicates that thus far, the differences in the concentration of inhaled particles did not
cause any significant alteration in the alveolar clearance process after a single, short-
term inhalation exposure, at least within the studied concentration range.
The retention of inhaled particles in animals exposed to radioactive diesel exhaust at
7000 yg/m3 particulate concentration has been investigated, thus far, to 126 days after
the exposure. The particle retention data (Figure 1), analyzed by a curve stripping
procedure, indicated three components with approximate half-times of 1 day, 8 days,
and 80 days, respectively. The biological meaning of these components can be
understood in terms of clearance mechanisms. The first mechanism deals preferentially
with particles deposited in the tracheobronchial tree and represents their rapid
transport by the mucociliary escalator. The particles are finally cleared through the
gastrointestinal tract, and their elimination is clearly documented by the presence of
C activity in the feces. The second mechanism is interpreted as the transport of
material deposited in the most proximal respiratory bronchioles, where only a short
-------
distance is required for transferring the participates to the mucociliary escalator. The
third mechanism removes the particulate matter from the alveolar region, and the
clearance mechanism may involve endocytosis, passive and active absorption, and
dissolution or metabolism. The extended data base in continuing studies is expected to
provide further information on the presence of additional clearance phase(s) of inhaled
diesel particles and to assess their clinical significance.
100
O
K
50 -
DAYS POST-EXPOSURE
Clearance of inhaled diesel exhaust particles in Fischer 344 rats. The vertical lines
represent standard deviations.
-------
COMPARTMENTAL ANALYSIS OF DIESEL PARTICLE KINETICS
IN THE RESPIRATORY SYSTEM OF EXPOSED ANIMALS
S. C. Soderholm
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
One key element in assessing the potential health effects of diesel engine emissions is
determining to what extent inhaled particles deposit in the respiratory system and how
long they remain before being cleared. The deposition and clearance of diesel
particulate is also of interest as an example of a seldom studied interaction between
the lung and submicron particles which are assumed to be insoluble. Several
investigators have collected data relevant to the deposition and clearance of diesel
particulate in animal models. The development of a model of the kinetics of insoluble
submicron particles in the respiratory system should provide a basis for organizing,
explaining, and comparing these experimental data.
A review of available information on the deposition, transport, and clearance of
insoluble submicron particles suggests a model with compartmental divisions illustrated
in Figure 1. The model consists of a set of differential equations specifying the rate of
transport of particulate mass among compartments. The kinetics are assumed to be
first order, since it is the simplest assumption which can be made and since it leads to
exponential clearance curves, the type found experimentally.
The major conceptual difference between this model of particulate kinetics in the lung
and some previous ones [1,2] is that the model describes a wide range of experimental
inhalation conditions, and provides compartments and parameters that apply even to
exposures using nearly maximum tolerated doses. Although the ICRP clearance model
includes a nasopharyngeal compartment and emphasizes particle dissolution, these
features were not considered in the present model because they are irrelevant to the
character of insoluble diesel participates. Inclusion of the "free particulate" compart-
ment does not significantly influence the fit to experimental data, but serves as a
reminder that particulate resides on the lung surface for a short time.
General solutions of the model for two types of experiments are presented. In a Type I
("post-exposure") experiment, the animals are exposed for only a short period, and their
changing particulate lung burdens are measured starting at the end of the exposure. In
a "long exposure" experiment, the animals are exposed continuously or nearly continu-
ously over a long period, and particulate lung burdens are periodically measured.
The major contribution of the particulate kinetics model to the analysis of the
experimental data is to point out that the long-term clearance phase may be
-------
interpreted in two ways: it may be clearance of participate out of the deep lung with a
half-time of 62 days as originally interpreted, or it may be a combination of deep lung
clearance and sequestering. The amount of participate in the lungs of chronically
exposed animals suggested a linear build-up over time and indicated that after
excessive exposure some portion of the deposited particulates may be retained in the
lung for longer times. Consequently, the new model emphasizes that the slope of the
buildup may be related to the clearance half-time from the "macrophage" to the
"sequestered particulate" compartment, to the overall clearance rate from the "macro-
phage" compartment, and to the deep lung particulate deposition rate.
The application of the model has so far shown that no single set of model parameters fit
all the data available from all experimental approaches. The differences may be
related to deficiencies in the model or to actual differences in the respiratory system's
response to particulate in the different exposure situations.
REFERENCES
1. Task Group on Lung Dynamics (Paul E. Morrow, Chairman), Deposition and
retention models for internal dosimetry of the human respiratory tract. Health
Physics, 12:173,1966.
2. R. G. Thomas, An interspecies model for retention of inhaled particles. Asse-
ssment of Airborne Particles, W. Stober et al, eds., Springfield, IL. Charles C.
Thomas, p. 405,1972.
-------
iiiillillllllllllilllllllimilViMiiimiiiiiiE
Figure Compartments and parameters in the model.
Compartments:
T = "tracheo-bronchial" S
F = "free particulate" on L
deep lung surfaces 6
n = "macrophages" and other LNG
scavenger cells
Parameters:
RT = deposition rate into T RF
RT = SDT * C RF
SDT = specific deposition SDF
rate into T
SDT = DET * VM SDF
DET = deposition efficiency DEF
of compartment T
C = concentration of VM
airborne particulate
HTxy = clearance half-time from compartment "x" to compartment "y"
"sequestered particulate"
"lymph nodes" draining lung
"GI tract"
"total lung"
deposition rate into F
SDF « C
specific deposition
rate into F
DEF « C
deposition efficiency
of compartment F
minute volume
-------
A SUBCHRONIC STUDY OF THE EFFECTS OF EXPOSURE
OF THREE SPECIES OF RODENTS TO DIESEL EXHAUST
by
Harold L. Kaplan, Ph.D.
Department of Fire Technology
Southwest Research Institute
San Antonio, Texas
William F. MacKenzie, D.V.M.
Department of Comparative Medicine
University of Texas Medical School
Houston, Texas
Karl J. Springer
Department of Emissions Research
Southwest Research Institute
San Antonio, Texas
Richard M. Schreck, Ph.D.
and
Jaroslav J. Vostal, M.D., Ph.D.
Biomedical Science Department
General Motors Research Laboratories
Warren, Michigan
A subchronic study of toxicologic and carcinogenic effects of inhalation
exposure of rodents to diesel exhaust was conducted in preparation for a
15-month chronic investigation of three dose levels of the exhaust. More than
five hundred rodents, consisting of male Fischer 344 rats, Syrian hamsters
and Strain A/J mice, were exposed to diluted diesel exhaust containing
1500 (jg/m3 of particulate 20 hours per day, seven days per week. A
5.7-liter Oldsmobile engine was operated continuously at 40 mph to generate
the diesel exhaust. Hydrocarbons, CO, CO2, NO and particulates were
monitored on a periodic basis and dilution was adjusted, as necessary, to
maintain the 1500 pg/m5 particulate level. Control groups of animals were
exposed to the same filtered air used to dilute the. raw exhaust. At the end
of three months of exposure, a portion of each species of animals was
randomly selected for measurement of organ weights (lungs, heart, kidneys,
liver and spleen) and for histopathological examination. The remaining
animals were held for a six-month post-exposure recovery period, with the
exception of the majority of mice which were held for four and one-half
-------
months following termination of exposure. Growth rate of all animals was
monitored by biweekly measurements of body weights during the exposure and
recovery periods. At the end of four and one-half months of recovery, most
of the Strain A/J mice were sacrificed at nine months of age for evaluation of
pulmonary adenoma response. After six months of recovery, animals of each
species were sacrificed for histopathology and organ weight measurements.
Exposure to diesel exhaust did not affect animal health, as indicated by
mortality, growth rate and organ weights, in any of the three species. In
the Strain A/J mice, neither the average number of adenomas per animal nor
the incidence of these tumors was significantly different in control and
exposed animals. In contrast, intraperitoneal injection of urethane at a dose
of 1 mg/g of body weight produced a large increase in both tumor para-
meters. The results of light microscopic examination of tissues were con-
sistent in the three species. After three months of exposure, particulate was
distributed diffusely and in foci in the alveolar spaces of the lungs, both in
free form and in macrophages, and in the regional lymph nodes. After six
months of recovery, partial removal of particulate from the lungs and redis-
tribution into focal accumulations had occurred. Most of the particulate was
found inside macrophages or within protean matrices. Clearance of the par-
ticulate via lymphatic channels was evident in both the exposed and recovered
animals. Species differences in clearance of particulate were apparent, with
hamsters exhibiting the largest clearance of particles and mice the least. In
both exposed and recovered animals of each species, alveolar fibrosis associ-
ated with large accumulations of particulate was minimal and not considered of
clinical significance. Lesions in organs other than the respiratory system
were also minimal, of similar incidence in control, exposed and recovered
animals and not considered exposure-related.
-------
RESPONSE OF PULMONARY CELLULAR DEFENSES TO
THE INHALATION OF HIGH CONCENTRATIONS OF DIESEL EXHAUST
Kenneth A. Strom
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
Bronchopulmonary lavage was used to explore the responses of the pulmonary phago-
cytic defense in rats exposed to diesel exhaust at concentrator of 250, 750, or
1500 yg/m diesel particles for 26 to 48 weeks. Figure 1 shows the quantities of
alveolar macrophages, polymorphonuclear leukocytes and lymphocytes obtained by
lavage of the respiratory airways (millions of cells in 40 mL). There is no difference in
the number or kinds of cells obtained from control and from 250 yg/m exposed animals
after either 6 or 11 months of exposure. However, after exposure to 750 or 1500 yg/m »
the number of alveolar macrophages increases by 25-33% and lflO-150%, respectively.
When the inhaled particulate concentration exceeds 250 ug/m , the quantity of the
lavaged alveolar macrophages shows dependence on the inhaled concentration of the
diesel particulate after both 6 and 11 months of exposure. In contrast, polymorpho-
nuclear leukocytes, which are not observed in lavage fluid from ^control rat lungs, are
obtained in high numbers from rats exposed to 750 or 1500 ug/m for 11 months or 1500
ug/m for 6 months. The quantities of lavaged cells are, therefore, dependent primarily
on the concentration of exposure and secondarily on the length of exposure.
Since equal amounts of diesel particulate are delivered to the lungs during exposure to
1500 yg DP/m for 6 months or to 750 yg DP/m for 12 months, comparison of the
quantities and kinds of cells obtained by lavage points out differences in the response of
the phagocytic defense to the inhaled concentrations. When identical amounts of
particulates are administered over twice the length of time, the number of alveolar
macrophages is increased by 25% above control values, compared to 150% after shorter
exposure to a higher concentration. The,counts of polymorphonuclear leukocytes are 5.1
± 1.5 x 10 compared to 19.1 t 4.4 x 10 at the shorter time of higher level exposure.
This comparison demonstrates that the pulmonary cellular systems respond primarily to
the rate of submicron particles entering the lungs, rather than to the total amount of
particulate delivered to the lung. Lymphocytes which are not obtained from control rat
lungs are lavaged from.animals exposed for 11 months to both 750 and 1500 ug DP/m ,
but not to 250 yg DP/m . The presence of the nonphagocytic lymphocytes in the lavage
fluid may represent a slow immune response to the presence of large numbers of
polymorphonuclear leukocytes and particle-filled alveolar macrophages in the lung. The
enzyme contents of the acid phosphatase and 3-glucuronidase in alveolar macrophages
lavaged from control and 250 yg/m exposed animals were identical. At higher inhaled
concentrations, the cellular enzyme content per mL of lavage fluid increased, but due
to the variability of the cell counts and types with the continuing diesel exposure, the
contribution of each cell type to the total enzyme activity was not determined.
-------
Under the exposure regime used in these experiments (20 hrs/day, 5-1/2 days/week), a
threshold rate for response of, the phagocytic defense occurs between inhaled concen-
trations of 250 and 750 ug/m diesel particles. The response consists of compensative
immigration into the lungs, at first, of alveolar macrophages and later on, also, of
polymorphonuclear leukocytes. After excessive exposures (11 months of exposure to 750
or 1500 yg/m diesel particles), mononuclear leukocytes (lymphocytes) are also lavaged
in high numbers. The probable cause of the observed effects is the continuous
phagocytosis of the diesel particles by the macrophage, which results in the cellular
accumulation of excessive amounts of "indigestible" carbonaceous particle nuclei.
When the macrophage renewal rate in the lungs is too slow to cope with the influx of
particles, the macrophages eventually become overloaded. Release of humoral factors
from active macrophages may stimulate the recruitment of more alveolar macrophages.
Polymorphonuclear and mononuclear leukocytes most probably respond to other not yet
known humoral agents from degenerating particle-laden macrophages.
40
30
20
10
o -
LAVAGED CELL POPULATIONS
Q macrophigei
B neutrophila
lymphocytes
6 Months
n
i
n n I
Y
I.
40 -
30 -
20 -
10-
o.
12 Months
1
f\ it rk
j.
i
%
IL
Figure Lavaged cell populations. The proportion
of each cell type in lavaged cells was multiplied
times the total number of cells obtained from
each rat. The numbers were averaged for each
exposure group (six rats) and charted as millions
of cells in the total lavage volume vs. the diesel
exposure concentration. The line shows the stan-
dard deviation range of the data.
0 260 760 1600
DieMl Exposure Concentration
-------
THE EFFECT OF DIESEL EXHAUST ON CELLS OF THE IMMUNE SYSTEM
D. Dziedzic
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
Inhalation of diesel engine exhaust results in the deposition of submicron carbonaceous
particles in the respiratory airways. The particles are phagocytized by the pulmonary
alveolar macrophages, and are cleared from the respiratory tract via the mucociliary
escalator or through lymphatic channels. Lung clearance via lymphatics results in an
accumulation of particles in the regional lymph nodes, and literature data suggest that.
the presence of hydrocarbons or carbonaceous particles in high doses might affect
immune functions [1,2].
Two approaches have been used in the present work to determine whether inhalation of
diesel exhaust could be immunotoxic. In one series of experiments, dichloromethane
extract of diesel particles was injected in massive doses (10-50 mg/kg) intraperitoneally
over a 7 day period into C,-7 Bl mice in two separate protocols. First, the splenic
lymphocytes were isolated and studied for ability to respond to polyclonal stimulation
of B or T cells by E. coli lipopolysaccharide or concanavalin A, respectively. Secondly,
dinitrofluorobenzene-induced contact hypersensitivity reaction was measured as a
reflection of T cell function by quantifying changes in ear thickness after an irritative
challenge. In both experiments, a small deterioration in the immuno-defensive ability
of lymphocytes from extract-treated animals was observed. In mitogen response
assays, lipopolysaccharide response (LPS) was reduced by about 20% compared to
vehicle control groups. Similarly, a 20-50% reduction was seen in concanavalin A (CON
A) stimulated cultures. In T cell mediated contact hypersensitivity reaction, all of the
treated animals showed decreased ear thickness response. In none of the experiments,
however, was a direct dose-response relationship observed. Furthermore, fluctuations
in liver weights from experimental animals indicated the possibility that hepatic
changes induced by the excessive doses of injected hydrocarbons may be involved in the
observed effects.
The approach used in this series of experiments is clearly limited, since 1) by use of high
doses of diesel particle extract, the question of bioavailability of hydrocarbon from
particles is ignored; 2) the large doses of extract may overwhelm normal defense
mechanisms; 3) the route of exposure allows for system distribution of material which
may not occur when inhalation occurs; and 4) in the case of mitogen responsiveness,
splenic rather than lymph node lymphocytes were studied. Nonetheless, the possibility
that the diesel exhaust particle extract administered in high doses may potentially
affect the immune system is at least tentatively raised, and should be further verified
in inhalation studies.
-------
The second approach was used to detect immunotoxicity of diesel particles by studying
lymph nodes, blood and spleen from guinea pigs exposed to diluted diesel exhaust at a
particulate concentration of 1500 ug/m . In this experiment, immune system organs
were studied for shifts in lymphocyte subpopulations counts. Alterations in this
parameter have been observed in several forms of human diseases, including active
forms of lupus nephritis or chronic glomerulonephritis, inflammatory bowel disease, and
other disorders. In addition, exposures to environmental toxins such as lead, poly-
brominated biphenyls, and cigarette smoke may also be associated with changes in
subpopulation proportions. The data from the present experiments, however, show that
no shift in subpopulation occurred in the mediastinal lymph node, the site of primary
diesel particle deposition. In spleen and blood, small fluctuations of no more than ± 5%
were observed, which is well within the limits of variability described in the literature,
and no other significant biological effects were identified.
In summary, small functional differences were seen in lymphocyte responsiveness of
C57B1 mice after treatment with diesel particle extracts. However, limitations of the
experimental protocol preclude direct extrapolation to possible findings during inhala-
tion exposure to diluted diesel exhaust. In a test system where the more realistic
inhalation mode of exposure was employed, no major effects were seen, and more
studies are needed to focus on lung immune system reactivity before the immunotoxic
potential of diesel particulates deposited in the mediastinal lymph nodes can be
definitely assessed.
REFERENCES
1. D. E. Bice, et al, Drug Chem. Toxic., 2,1979.
2. A. Zarkower, Arch. Environ. Health, 26,1972.
INCREASED EAR THICKNESS VS. TIME
(MEAN + S.E.)
30
CO
CO
UJ
g 25
5»
Efc 20
si15
z *
w° 10
CO -*
uj 5
oc
S o
24
48
VEHICLE CONTROL
<
10MG/KGDOSE
k
25MG/KGDOSE
72
HOURS AFTER DNFB CHALLENGE
-------
THE PARTICIPATION OF THE PULMONARY TYPE II CELL
RESPONSE TO INHALATION OF DIESEL EXHAUST EMISSION:
LATE SEQUELAE
H. J. White and B. D. Garg
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
Among the earliest cellular responses of the lung to inhalation of diluted diesel exhaust
is the focal proliferation of the alveolar Type II cell, the cell responsible for the
synthesis of surfactant. This reaction takes place relatively within the same time
frame as that for phagocytosis by the alveolar macrophage, and can be seen in rats as
early as twenty-four hours post-exposure to a diesel particle concentration of
6000 yg/m [1]. The proliferation is focally within alveoli, sometimes showing several
cells in a line. The variation in the staining of their nuclei supports the idea of a fairly
rapid proliferation, although mitoses have not been seen. There is no evidence in our
hands that this proliferative response is of a reparative nature secondary to damage of
the Type I cell. The Type II cells release considerably increased amounts of surfactant,
which accounts for a morphologic change in the phagocytic alveolar macrophage which
now takes on a more foamy appearance. TJie transition between the early macrophage
(one-day exposure to diesel at 6000 yg/m to that of 6 weeks' exposure) reveals the
gradual accumulation of surfactant material. Some of the phagosomes can be seen to
contain recognizable myelin figures of Type II provenance changing to a more
filamentous form, suggesting an unraveling of the more compact tightly-wound phos-
pholipid. The mechanism of accumulation of cholesterol is certainly not clear, although
plate-like crystalline structures can be easily identified within the phagosomes,
suggesting cholesterol ester formations. The excess cholesterol could well have its
origin from the phagocytosed surfactant material which then at some later stage is de-
esterified to form the familiar elongated acicular structures of cholesterol. This foamy
cell is apparently more sluggish, and tends to accumulate near the terminal bronchioles
where further Type II cell activity is elicited.
3 3
With prolonged exposures to 9 weeks at 6000 yg/m and 1500 yg/m for two years,
cholesterol also begins to accumulate within the phagosome. Later, the crystals grow
and tend to become extracellular. This can be demonstrated by both light and electron
microscopy. Build-up of the cholesterol deposits in these late stages has been found to
be associated with increased collagenosis of the septal wall in which mast cells are also
present. The reaction of mast cells to the release of extracellular cholesterol is also
obscure. One could speculate that the heparin of the mast cell is involved in activation
of lipoprotein lipase in an attempt to clear the excess of lipid release. In some way, the
amine component of the mast cell is also released at this time to provoke the laying
down of collagen. In addition, there is an apparent intimate association between laying-
-------
down of extracellular cholesterol, lipids, septal mast cells and collagenosis; the
collagenosis is, however, focal and the integrity of the septal architecture is preserved.
Occasionally, focal cholesterol granulomas have been observed. These are quite similar
in appearance to those seen after prolonged inhalation exposure to Sb^O, [2] and
marihuana smoke [ 3]. The phenomenon of "benign" focal alveolar collagenosis seems
to be a consequence of high participate burden that stimulates an increased production
of phospholipids, and not directly of the effect of the diesel particle per se. The
observation that diesel participates, sequestered in the thoracic lymph nodes for up to
two years, do not provoke a fibrotic reaction and supports this contention.
REFERENCES
1. White, H.J. and Garg, B.D. (1981), Early pulmonary response of the rat lung to
inhalation of high concentrations of diesel particles. J. Appl. Tox. 1:104-110.
2. Gross, P., Brown, J.H., and Hatch, T.F. (1952), Experimental endogenous
pneumonia. Am. J. Path., 28:211-221.
3. Fleischman, R.W., Baker, J.R., and Rosenkrantz, H. (1979), Pulmonary pathologic
changes in rats exposed to marihuana smoke for one year. Tox. and Appl.
Pharmacol., 47:557-566.
-------
PULMONARY DEPOSITION, RETENTION, INACTIVATION
AND CLEARANCE OF INHALED DIESEL PARTICLES:
THE ROLE OF THE PULMONARY DEFENSE SYSTEM
J. J. Vostal
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
The submicron size of diesel particles permits an easy penetration of the particles into
the deepest parts of the respiratory system, and allows the possibility of their prolonged
contact with the sensitive cells at the lowest levels of the bronchopulmonary regions.
Since a knowledge of the level of the interaction between the particle surface or of the
adsorbed hydrocarbons with the intimately adjacent cells of the respiratory epithelium
is essential for the assessment of their potential toxicity and adverse respiratory
effects, the exact determination of the inhaled dose and residence time of particles
retained in the respiratory airways is important in predicting the potential health
impact of the wider use of diesel-powered vehicles on our roads.
As a direct continuation to our previous program studying the health effects of long-
term, high-level exposures in extensive laboratory experiments, the deposition, reten-
tion, inactivation, and clearance of inhaled particles have been intensively investigated
in animal models during the past year. Specifically, investigations were concentrated
on the detailed analysis of the conditions responsible for the handling of the particles by
the pulmonary defense system.
In various animal models, investigations with freshly-generated, radioactively-labelled
particulates indicated that even under conditions of extremely high exposures, not more
than approximately 15-17% of the inhaled dose is retained in the respiratory tract of
most laboratory animals used in the studies. After a single short-term exposure,
approximately one-fifth and one-third of the retained dose is cleared from the
respiratory tract of Fischer 344 rats within the first 48 hours, and during the first two
weeks, respectively; the remaining portion of the lung burden is removed more slowly
with a clearance half-time of approximately 60-80 days [Lee et al, 1981]. The fact that
other species of laboratory animals may clear the particulates from the lung by
different mechanisms and at different clearance rates [Chan et al, 1981al makes a
direct comparison among animal species and the extrapolation of the laboratory data to
man particularly difficult. Consequently, our conclusions are restricted to a relative
assessment of specific clearance or deposition mechanisms in individual animal models
with the final intention of correlating the administered dose with the observed effects.
Unfortunately, most of the accumulated data were obtained after local administration
of high doses or inhalation of highly elevated concentrations of diesel particulates.
-------
Although the first comparisons of single short-term exposures between either extremely
high or moderately high concentrations did not reveal a significant effect of inhaled
concentration on the rate of clearance of diesel particles [Lee et al, 19811, other data
obtained in animals after prolonged exposures to high doses of particulates (particulate
lung burden > 10 mg) seems to indicate that the removal of the particles may be
significantly slowed in excessive exposures and contribute to an artifically exaggerated
accumulation of particles in the respiratory system - a phenomenon which is not
expected after breathing expected ambient levels of diesel particulates [Chan et al,
1981bl.
A similar threshold effect of the inhaled concentrations has also been observed in the
response of pulmonary cellular defense mechanisms, in which the first measurable
quantitative increase of the macrophage cell counts occurred in the bronchopulmonary
lavage only when the inhaled concentrations were higher than 250-750 ug of particu-
lates per cubic meter [Strom, 1981]. Similarly, qualitative changes in the composition
of the lavageable mobile cells (immigration of polymorphonuclear leukocytes, and later
on, of lymphocytes) in the lavage fluid were observed only after excessively high
concentrations were inhaled.
Furthermore, a high rate of particulate influx to the deep alveolar region can
potentially overload the physiological clearance mechanisms, and significantly influence
the rate of accumulation and storage of the inhaled particulates. This appears to
happen via the creation of a new sequestering compartment in the form of focally
aggregated macrophages with particulates in the subpleural and terminal bronchiolus
regions of the lung [White et al, 1981al; again a phenomenon which probably does not
exist after inhalation of low concentrations.
However, even if we assume that the macrophage sequesteration and aggregation are
not typical physiological mechanisms, it is important to note that the sequestered
particles' are well enclosed in the aggregated macrophages, and form a stationary
deposit which may persevere in the lung tissue without any negative reaction for a long
period of time [Strom et al, 1981; Soderholm, 19811. Although it can significantly
contribute to a measurable burden of retained particles, the macrophage aggregate
effectively prevents the contact of particles with the sensitive respiratory epithelium.
The tissue reaction to the presence of the aggregates remains negative for a long period
of time. Only after long and excessive exposures can we observe an intracellular build-
up of phospholipids and cholesterol deposits, which may later be accompanied by a
locally increased number of collagen fibers in the septal walls, as well as by focal mast
cell activation. Even then, the tissue reaction remains focal and without significant
clinical impact [White et al, 1981bl. Therefore, this tissue reaction must be classified
as a non-specific reaction to the high level of particulate burden, rather than a direct
effect of the presence of the diesel particle or its adsorbed hydrocarbons.
The pulmonary alveolar macrophage is obviously not only capable of scavenging and
sequestering particles which penetrate deeply into the respiratory system, but can also
contribute to the inactivation of their biological activity, as indicated by the observed
loss of mutagenic activity of diesel particulate extracts from particles which had been
incorporated into the macrophage phagosomes [Siak et al, 19811. Although we cannot
clearly determine at this time if the inactivation occurs either by dissolution,
metabolism, or simple relocation of mutagenic hydrocarbons from the particulate
surface, it is also important to note that this process prevents the immediate contact of
sensitive respiratory cells with the concentrated hydrocarbons on the particle surface.
-------
Inactivetion of the biological effects of diesel particulates deposited deeply in the lung
is further corroborated by the absence of hydrocarbon metabolizing enzyme induction in
the lung, even after long-term inhalation exposures to high concentrations of diesel
particulates [Chen et al, 19811. Since published studies of the structural and functional
responses of the respiratory system do not reveal changes considered to be clinically
significant, despite conditions of excessive exposures, the effective role of the
pulmonary defense mechanisms, which provide significant protection against the effects
of inhaled diesel particles, has been clearly demonstrated.
-------
INVESTIGATIONS OF TOXIC AND CARCINOGENIC EFFECTS
OF DIESEL EXHAUST IN LONG-TERM INHALATION EXPOSURES
OF RODENTS
by
U. Heinrich, L. Peters, W. Funcke, F. Pott, U. Mohr and W. Stb'ber
Fraunhofer-Institut flir Toxikologie und Aerosolforschung
Mlinster, Federal Republic of Germany
Extended Abstract
INTRODUCTION
The purpose of this study was to provide experimental evidence of the
theoretically expected carcinogenicity of diesel exhaust. It is known that the
emissions from internal combustion engines contain minute amounts of poly-
cyclic aromatic hydrocarbons (PAH), some of which are proven to be carcinogens
in animals. These carcinogenic PAH show rather low vapor pressures and tend to
condense on airborne particles. Since diesel engines produce much larger
amounts of finely dispersed particles during fuel combustion than gasoline
engines, the particulate matter in diesel exhaust with its surface condensate
of various organics is suspected to constitute a special carcinogenic potent-
ial which deserves a careful investigation into the chronic effects of con-
tinuous inhalation exposure.
EXPERIMENTAL
A total number of 864 female Syrian golden hamsters [Hoe: SYHK(SPF Ars),
breeding farm of Frankfurt-Hoechst, West Germany] was subdivided into three
groups of 288 animals each. One group was to inhale clean air, the second
group was exposed to the diluted original emissions of a diesel engine, the
third one breathed the same diluted emissions but void of any particulate mat-
ter. The animals were exposed to the exhaust in wire cages placed into a
chamber ventilated with the diluted exhaust atmosphere. The exposures lasted
for 7 to 8 hours per day, 5 days per week for about two years.
In addition to the golden hamsters, two groups of 20 rats were exposed
for 18 to 24 months for subsequent testing of some lung functions in com-
parison to a control group of the same size.
-------
The exhaust was generated under constant operating conditions with a
Daimler-Benz DB 200 D diesel engine on a test bench. The emissions were
cooled and, without passing the dew point, diluted with refrigerated clean
air (1 part of exhaust into 7 parts of air) before drawing them through the
inhalation exposure chambers. Two locations were selected for sampling the
exposure atmosphere : one port was inserted into the ducts about 5 meters up-
stream of the flow entrance of the inhalation chamber, the other probe drew
directly from within the chamber. Table 1 gives the results for all measured
components as average values of all data obtained over the whole study period.
Apparently, there were no significant differences between the gaseous compo-
nents of the original exhaust and the exhaust void of particles. In addition
to these results, the concentrations of 14 PAH on the particles .in the in-
halation chambers were recorded and found to be low (e.g. 7.0 yg of benzo(a)-
pyrene per gram of particles). The particulate matter in the exposure cham-
bers was highly dispersed, the mass median aerodynamic diameter being below
0.1 ym.
In designing the experiments, it was anticipated that the probability of
directly proving an existence of the theoretically expected carcinogenicity
of diluted diesel exhaust with a limited number of animals was rather low.
Thus, an attempt was made with some of the test animals to produce a base Tine
tumor induction rate by applying a known carcinogen at the beginning of the
inhalation exposure and to investigate the influence of the exhaust inhalation
on this base line rate. Each of the three exposure groups of 288 hamsters was
subdivided into 6 sub-groups (48 hamsters each) and pre-treated in the follow-
ing manner :
(1) no additional treatment (controls)
(2) 2 mg pyrene in 20 weekly intratracheal instillations of
0.1 mg each (second controls)
(3) 2 mg dibenzo(a,h)anthracene, intratracheally instilled
as under (2)
(4) 6 mg dibenzo(a,h)anthracene, intratracheally instilled
as under (2), but 0.3 mg each
(5) 1.5 mg diethyl nitrosamine per kg body weight,
subcutaneously injected
(6) 4.5 mg diethyl nitrosamine per kg body weight,
subcutaneously injected
During the course of the long-term exposure, 11 biochemical and 7 hemato-
logical parameters were determined repeatedly. In addition, the impact of the
diesel exhaust inhalation on the lung clearance of a radioactively labelled
iron oxide aerosol and on some respiratory parameters was measured for the
rats.
RESULTS
A detailed report on the results will be given in the final paper. Two
of the findings of the long-term study were particularly conspicuous :
- the tumor induction rate in the respiratory tract produced by the
subcutaneous injection of diethyl nitrosamine was enhanced for the
hamster groups exposed to the exhaust atmospheres (Fig. 1),
-------
- the lung clearance of the chronically exposed rats was de-
creased (Fig. 2).
It appears to be premature to draw firm conclusions from these prelimi-
nary results obtained with rather small animal groups. The findings certain-
ly need a confirmation by results of additional experiments.
MEASUREMENT
IN THE CHAMBERS
BEFORE THE CHAMBERS
CO tppm 1
C02 [vol%J
S02 [ppml
CNHM IPP*]
CH4 tPP")
CNHH - CH4 Ippm]
NO tppm 1
NOX IPP")
N02 IPP"1'
02 IvoHl
PARTICLES [»g/m3:
A
16,9 (+3,0)
0,63 (+0,14)
4,7 (+1,6)
8,5 (+3,4)
2,1 (+0,8)
6,3 (+3,0)
15,8 (+7,1)
16,3 (+7,2)
0,45 (+0,42)
20,0 (+0,7)
-
B
17,9 (+3,3)
0,67 (+0,14)
4,9 (+0,9)
8,9 (+2,8)
2,5 (+0,9)
6,3 (+2,6)
15,6 (+6,9)
15,9 (+6,5)
0,40 (+0,28)
19,5 (+0,6)
4,2 (+O,3)
A
16,3 (+3,2)
0,52 (+0,14)
7,9 (+3,8)
5,3 (+3,8)
1,0 (+0,33)
4,3 (+3,7)
17,6 (+8,5)
17,7 (+10,2)
0,10 (+0,09)
20,0 (+0,7)
-
B
18,0 (+3,4)
0,57 (+0,16)
7,0 (+1,4)
4,2 (+1,7)
1,0 (+0,321
3,3 (+1,4)
16,0 (+8,8)
16,4 (+10,3)
0,21 (+0,27)
19,1 (+0,5)
-
A: EXHAUST WITHOUT PARTICLES
B: TOTAL EXHAUST
Table 1 : Analytic average data on emission concentrations during exposure
V. HAMSTERS WITH RAPILLOMAb IN LAKYNX/
80
70-
60
50
40'
30
20
10-
n
T
DEN -4,5-conlrols
DEN -4,5-exh.without part.
T DEN -4,5-total exhaust
0 DEN -1,5-controls *
D DEN -1,5-«xh.withoul part
V DEN -1,5- total exhaust
» .
»
1
V O
o B
f 1 ° a
iKAUHtA urr/oj
T
V T
B
9 *
0
V V V
D o g
0 0
20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-99 100-109 110-119
WEEK
Figure 1 : Papillomas in pre-treated Syrian golden hamsters
-------
CLEARANCE OF 59Fea03 FROM RAT LUNGS
20
diesel exhaust
Tl|2"92.4:14.3days
Y = 5g.4 e -0.00751
Rs =0.8387
o control group
TIB-46.514,2 days
Y.47.7e -O
q3.0.9400
Figure 2 : Lung clearance of
59r
aerosols in rats
-------
BIOCHEMICAL ALTERATIONS IN BRONCHOPULMONARY LAVAGE FLUID AFTER
INTRATRACHEAL ADMINISTRATION OF DIESEL PARTICULATES TO RATS
by
C.D. Eskelson, M. Chvapil, E. Barker, J.A. Owen
Department of Surgery, Division of Surgical Biology
University of Arizona Health Sciences Center
Tucson, Arizona 85724
J.J. Vostal
Biomedical Science Department
General Motors Research Laboratories
Warren, Michigan 48090
Male Sprague Dawley rats weighing 180-200 g were given intratracheally
5 mg of diesel particulates (DP) in 0.75 ml saline. Control rats were given
saline. Five days after administering the DP and three hours before the rats
were sacrificed they were injected with a pulse of 20 yC of 14C-acetate.
The lungs were intubated before they were removed and layaged 3 times
with 5 ml of saline. The combined lavage fluids were lyophylized. The
lipids were extracted from the lyophylized lavage fluid with chloroform:
methanol (2:1) and its cholesterol (C) and phospholipid (PL) content
determined. The radioactivity incorporated into the lipids was determined by
separating the lipids by a TLC method and scraping the spots of interest into
counting vials.
Pulmonary lavage fluid from the rats given DP contained 4 times more PL,
C, and protein than in control rats (Table 1). Total radioactivity
incorporated into lecithin was twice that of controls and was 3.5 times
greater than the radioactivity found in the other PL studied. The lavaged
lungs from the control and experimental rats were lyophylized and homogenized
in a chloroform:methanol (2:1) solution. The lipid analysis showed no
difference in the PL and C levels between the control and experimental lungs
(Table 2).
The fatty acid (FA) profile of the lavage fluid determined by a GLC
method indicated a three-fold increase in palmitic acid and arachidonic acid.
Stearic, oleic and linoleic acids were not significantly altered (Table 3).
These studies imply that the lipid loading observed in lungs exposed to
5 mg of DP for 5 days are a result of increased deposition of pulmonary sur-
factant (extracellular lipids) and are not a result of intracellular lipids.
-------
Table 1. Analysis of Pulmonary Lavage Fluid from
Rats Intratracheally Exposed to 5 mg of Diesel Particulate
Experimental
Control
Student's t
Phospholi
mg
1.90
0.49
7.82 P
pids
SD
0.48
0.18
< .001
Choi
mg
.539
.151
6.37
esterol
SD
.040
.081
P < .001
Protein
mg
8.47
2.09
8.25
SD
0.33
1.04
P < .001
Results expressed as mg of lipids in the total lavage fluid
Table 2. Analysis of Lavaged Lungs from Rats Intratracheally
Exposed to 5 mg of Diesel Particulate
Experimental
Control
Student's t
Phospholipids
mg SD
34.9 1.34
31.30 10.6
.453 NS
Cholesterol
mg
15.9
13.1
1.18
SD
1.02
3.23
NS
Results expressed as mg of lipid per lung
-------
Table 3. Fatty Acid Profile from Lung Lavage Fluid of
Rats Exposed to 5 mg Diesel Particulate
'16
'18
'18:1
'18:2
'20:4
Experimental
SD
Control
SD
Student's t
1.77
.07
0.541
0.195
8.50
P < .001
0.115
.033
.097
.103
NS
0.152
.016
0.152
.015
NS
0.152
.030
0.225
.322
NS
0.104
.015
.030
.021
4.61
P < .01
Results expressed as mg of the fatty acid methyl ester per total lavage fluid
-------
LIPID CHANGES IN LUNG OF RATS AFTER
INTRATRACHEAL ADMINISTRATION OF DIESEL PARTICULATES
by
C.D. Eskelson, E. Barker, M. Chvapil, J.A. Owen
Department of Surgery, Division of Surgical Biology
University of Arizona Health Sciences Center
Tucson, Arizona 85724
J.J. Vostal
Biomedical Science Department
General Motors Research Laboratories
Warren, Michigan 48090
Lung, liver and serum from 180-200 g male Sprague Dawley rats were
analyzed for various lipids 5 days after the rats were given intertracheally
in a saline solution 1 mg of diesel particulates (DP). The lipogenic
activity occurring in these rats was studied by giving i.p. 20 yC of
llfC-acetate one hour before they were sacrificed.
Phospholipids and cholesterol content of the lungs were significantly
increased while that of triacylglycerols were not changes significantly (see
Table 1).
Contrariwise to pulmonary lipids, hepatic phospholipids and cholesterol
levels were decreased in rats exposed to diesel dust while hepatic triacyl-
glycerol (TG) levels were not significantly altered. Accompanying the loss
of hepatic lipids are an increased phospholipid, cholesterol and TG specific
activity indicating increased hepatic lipogenesis. To determine if the loss
of hepatic lipids were due to their being mobilized to the serum from the
liver serum lipids were determined and were found not to be significantly
altered. However, the specific activity of serum phospholipids, cholesterol
and triacylglycerols were all significantly increased in rats intratracheally
given the diesel particulates. A corresponding doubling of radioactivity in
pulmonary phospholipids and cholesterol was also detected in these animals.
The results obtained here are similar to those reported earlier for rats
intratracheally given silica dust (1-3) and suggest that a participate insult
to the lungs results in the lung producing lipotrophic factors which
stimulate the liver to increase lipogenesis and lipid export to the blood.
The lung in turn picks up the lipids from the serum and remodels them to meet
pulmonary lipid need. To further study this hypothesis, rat hepatocytes were
isolated and incubated in a pH 7.0 phosphate buffer containing 2 yC
-------
ll*C-acetate and several cofactors. To this hepatocyte suspension was added
lung slices and the system thence incubated for 2 hours at 37°C.
Phosphatidyl choline (PC) was isolated from each sample by TLC and the
PC spots from each TLC scraped into counting vials. The amount of
radioactivity incorporated into the PC spots of the 5 samples for each
experimental manipulation was averaged and is presented in Figure 1.
This study clearly demonstrated increased lipogenesis above that of the
sum of lung slice PCgenesis and hepatocyte PCgenesis. The results from the
in vitro studies strongly support the concept that a pulmonary lipotropic
factor exists which stimulates lipgenesis in the liver and that these de novo
synthesized lipids are utilized in part to maintain lipid homeostasis in the
lung.
Table 1.
Pulmonary Lipids from Rats Intratracheally Exposed
to 1 mg of Diesel Particulate
Phospholipids
Cholesterol
Triacylglycerols
Experimental
Control
Student's t
mg
51.7
30.8
6.15
SD
4.64
4.66
P < .001
mg
11.95
8.27
5.15
SD
1.11
0.63
P < .005
mg
20.00
24.52
2.32
SD
3.15
1.24
NS
Results expressed as mg of lipids per lung
Table 2.
Experimental
Control
Hepatic Lipids from Rats Intractracheally Exposed
to 1 mg of Diesel Particulate
(mg/g of liver)
Phospholipids
Cholesterol
Triacylglycerols
mg
27.9
31.3
SD
1.9
1.7
mg
3.36
4.02
SD
0.10
0.11
mg
8.57
9.07
SD
1.43
0.88
Student's t
2.52
P < .05
8.60
P < .001
.53
MS
-------
Figure 1.
Lecithin Formation in Hepatocytes
and Lung Slices from 14C-Acetate
T P<-05
20 n
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References
1. Eskelson, C.D., Stiffel, Virginia, Owen, J.A. and Chvapil, M. The
importance of the liver in normal and silicotic lung-lipid homeostasis.
2. Cholesterol. Environ Res 19:432-441(1979).
2. Eskelson, C.D., Stiffel, Virginia, Owen, J.A. and Chvapil, Milos. The
importance of liver in normal and silicotic lung-lipid homeostasis:
3. Triacylglcerols. Physiol Chem Physics TJ_:135-141(1979).
3. Eskelson, C.D., Stiffel, Virginia, Owen, J.A., and Chvapil, Milos. The
importance of liver in normal and silicotic lung-lipid homeostasis.
1. Phospholipids. Accepted for publication in J Environ Path Tox.
-------
BIOAVAILABILITY OF DIESEL PARTICLE BOUND
[G-3H-] BENZO(a)PYRENE (3H-BP)
AFTER INTRATRACHEAL INSTILLATION
by
P.K. Medda, Sukla Dutta and Saradindu Dutta
Wayne State University School of Medicine
Detroit, Michigan 48201
Recently, we have investigated whether pulmonary prostaglandin dehydro-
genase (PGDH) activity is affected by long-term exposure to low doses of
diesel exhaust (1) and whether high doses (6.0 mg/m3) for a short period (2-
8 weeks) can affect such biochemical functions as (a) blood methemoglobin
level, (b) reduced glutathione levels, (c) angiotensin coverting enzyme
activity, and (d) mixed function oxidase activity (2, 3). In general these
studies have shown that as far as the above set of biochemical parameters is
concerned, there exists no particular adverse effect of the diesel exhaust in
the exposed animal. Because of these findings and the observation made by
Siak £t a\_., (4) that simulated biological fluids elute no significant mut-
agenic activity from the diesel particles, we have contended that the many
polycyclic hydrocarbons, such as benzo(a)pyrene (BP) which are known to be
present in the diesel particles (DP), probably remain unavailable to the
pulmonary tissue. In order to provide support for this contention attempts
have been made to determine the bioavailability of 3H-BP following intratra-
cheal administration of this agent bound to diesel particles in an albumin-
saline suspension.
In order to determine the bioavailability of benzo(a)pyrene as bound to
diesel particles, 3H-BP (120 uCi/1.79yg/1.0 ml ethanol) was allowed to bind
diesel exhaust particles by adsorption. After removal of free 3H-BP by re-
peated suspension and centrifugation, nearly 90-95 yCi of 3H-BP remained
bound to 1 mg of DP. Tightness of the binding was tested by continuously
washing 125 ug of 3H-BP bound DP with 6.0 ml either Krebs-Henseleit (K-H)
solution or K-H with 3.2% albumin solution or DMSO or dichloromethane for one
hour. Results showed the least dissociation in K-H solution (2.1%) and the
highest in the presence of dicholoromethane (72%). For the bioavailability
experiments, female guinea pigs (Hartley) were lightly anesthetized with ether
in preparation for instillation of 1 mg labelled DP. When animals were appro-
priately anesthetized, diesel particles as suspended in 0.2 ml K-H solution
containing 3.2% albumin was introduced slowly into the trachea over a period
of 10-15 minutes by inserting a PE10 polyethylene tubing through the tracheal
ring and pushing deep inside the broncheal tree. After intratracheal instil-
lation of labelled particles guinea pigs were divided into two groups
Five guinea pigs of one group were immediately sacrificed within 20-25
-------
minutes, while six guinea pigs belonging to the second group were moved to
Nalgene(R) metabolic cages, held there individually for collection of urine
and feces for 48 hours and then sacrificed for excision of selected organs
such as liver, kidney, intestine and collection of blood for measurement of
radioactivity.
Results of these studies showed that intratracheal instillation of 1 mg
labelled DP did not produce any obvious symptoms in these animals. Further-
more, except for individual difference, no abnormality was noted in feeding
and excretory profiles of these guinea pigs. At autopsy, lobar localization
of DP was clearly visible as distinct patches of black marks of 1-2 cm
diameters. Percentages of radioactivity retained by guinea pig lungs immedi-
ately after intratracheal instillation of DP bound to ^H-BP, when calculated
on the basis of the administered dose, showed wide variability in that 77-95%
of the theoritical radioactivity (90-95 yCi 3H-B(a)P/mg DP) was actually mea-
sured in the lungs of these guinea pigs that were sacrificed within 20-25
minutes of instillation. As observed by Henry and Kaufmann (5), in the pre-
sent study also the discrepancy between the amount of dose actually intended
for delivery and the amount actually measured in the lungs after intratracheal
instillation could not be accounted for any loss due to regurgitation of the
suspension.
Percentages of radioactivity retained by six guinea pig lungs at 48 hours
following intratracheal instillation of DP bound ^H-BP, when calculated on the
basis of the administered dose, showed a mean disapperance of 42%±6 of radio-
activity during the 48 hour time period. However, the observation that actual
delivered dose was somewhat less than the dose intended for instillation meant
that the lungs might have lost much more radioactivity during the 48 hours
time interval. It was also noted that at 48 hours radioactivity had distri-
buted widely in that all the organs studied such as liver, kidney and intes-
tine showed about 1-2% as much ^H-BP content per gram in comparison to the
radioactivity retained per gram of lung tissue. Furthermore, during 48 hours
25% +_ 3 of the radioactivity was excreted in urine and feces in these animals.
In conclusion, these studies show that -^H-BP dissociates from the labelled
diesel particles upon instillation in the lungs and appears in urine and
feces. This rapid dissociation of ^H-BP from the diesel particles implies
that by the existing method of labelling of DP by adsorption with ^H-BP we
may not have simulated the forces by which benzo(a)pyrene binds to diesel
particles under engine condition.
-------
REFERENCES
1. Chaudhari, A., R.G. Farrer and S. Dutta. 1981. Effect of exposure
of diesel exhaust of pulmonary prostaglandin dehydrogenase (PGDH)
activity. J. Appl. Toxicol. 1: 132-134.
2. Chaudhari, A. and S. Dutta. 1982. Alteration in tissue glutathione
and angiotensin converting enzyme due to inhalation of diesel exhaust.
J. Toxicol. Envir. Heal. (In Press).
3. Navarro, C., J. Charboneau and R. McCauley. 1981. The effect of
in vivo exposure to diesel exhaust of rat hepatic and pulmonary
microsomal activities. J. Appl. Toxicol. 1: 124-127.
4. Siak, J., T.L. Chani and P. Lee. 1979.. Diesel particulate extracts
in bacterial test system. Presented at the U.S. Environmental
Protection Agency Symposium on Health Effects of Diesel Engine
Emissions. Cincinnati, Ohio.
5. Henry, M.C. and D.G. Kaufman. 1973. Clearance of benzo(a)pyrene
from hamster lungs after administration of coated particles. J.
Nat. Cane. Inst. 51: 1961-1964.
-------
THE POTENTIAL FOR AROMATIC HYDROXYLASE INDUCTION
IN THE LUNG BY INHALED DIESEL PARTICLES
K. C. Chen, and J. J. Vostal
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
Diesel exhaust particles contain trace amounts of a wide spectrum of polyaromatic
hydrocarbons (PAH) adsorbed on the surface and, when extracted by an organic solvent,
produce mutagenic effects in short-term microbial laboratory tests. Since the
mutagenic or carcinogenic effects of hydrocarbons are frequently initiated by a
metabolite rather than by the parent molecule, and since the activity of the
metabolizing enzymes can be easily increased by preceding administration of powerful
inducers, the enzyme induction could theoretically predetermine the potential for the
adverse health effects of inhaled diesel exhaust emissions.
The effects of long-term inhalation of diluted diesel exhaust on aryl hydrocarbon
activity (AHH) and cytochrome P450 content in lung and liver microsomes were
investigated in male Fischer-344 rats and compared with repeated parenteral adminis-
tration of organic solvent extracts of hydrocarbon adsorbed on the diesel particulate
surface during the combustion process. No significant effects of long-term inhalation
exposure were observed in liver-microsomal AHH activity. The animals were exposed
to concentrations of 750 yg m or 1500 yg m of diesel particulates from a 5.7 GM
diesel engine at 20 hours per day, 5-1/2 days per week for up to 9 months, or treated by
repeated IP injections of diesel particulate extract dissolved in corn oil, from the same
engine at the several dose levels for 4 days. A decrease in lung microsomal AHH
activity was found in rats following % months of exposure to diesel exhaust at the
particulate concentration of 1500 yg m .In contrast, 1.4- to 9-fold increases in AHH
activity were observed in liver and lung microsomes of rats pretreated by intraperi-
toneal doses 10-15 times larger (25-125 mg/kg BW) than the most conservative estimate
of the deposited lung burden [J. Appl. Tox., 1(2):27, 19811.
Since the intraperitoneal injection of diesel particle extract may not fully represent the
activity of PAH deposited on the inhaled diesel particles in the respiratory airways,
direct intratracheal instillation (ITI) of various doses of extract was used, and
microsomal enzyme induction was investigated in the lung as well as in the liver in
order to detect the local and systemic response to hydrocarbons deposited in the
respiratory system. Diesel particulate extract or pure benzofa] pyrene, dissolved in a
gelatin-saline solution and used as a reference compound, were administered by ITI at
several dose levels. The results show that direct intratracheal administration of the
diesel particle extract required doses as high as 6 mg/kg BW before the activity of the
induced enzyme in the lung was barely doubled (Figure 1). The induction was slow and
-------
occurred selectively in lung only (Figure 2), indicating that diesel particulate extract
probably does not absorb easily into the lung circulation, and is not distributed to other
organs. The data suggest that the absence of AHH activity induction in rat lung
exposed to diesel exhaust is due to the inavailability of hydrocarbons for distribution in
the body and insufficient quantities for enzyme induction. All data seem to indicate
that the inhaled diesel particles would not be capable of inducing aromatic hydroxylase
in the lung unless the total deposited dose in the lung reaches approximately 6-8 mg of
the particle extract per kilogram of body weight. Since the extractable portion
represents only 10-15% of the total particulate mass, the required pulmonary deposits of
diesel particles in a 70 kg man would be excessive to become a significant step in
promotion of a potential neoplastic process.
Lung
N = 4
2
468
Dose (mg/kg)
10 12
Liver
N = 4
c
'55
o
O.
O)
si
> c
o E
<\
-i- O.
1-
< I
o
n
o>
o
Q.
1000
800
600
400
200
0
/\
/ ^v
/ \j T DP-Ext = 12 mg/kg
^ T ^Ss^ i.
I B(a) = 5 mg/kg T
i l I i i i
0 24 48 72 96 120 144
Hours After Administration
-------
XENOBIOTIC METABOLIZING ENZYME LEVELS IN MICE EXPOSED TO
DIESEL EXHAUST OR DIESEL EXHAUST EXTRACT
by
William Bruce Peirano
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio
Diesel exhaust (DE) contains organics, specifically polycyclic ar-
omatic hydrocarbons (PAH), which are mutagenicU) and also potentially
carcinogenic. These PAH, for the most part, must be metabolized via the
xenobiotic metabolizing enzymes to become active mutagens and/or carcino-
gens. The primary functions of these enzymes are to detoxify and/or to
make these PAH more readily excretable. Unfortunately, a percentage of the
metabolites formed are reactive electrophiles, which can bind to proteins,
RNA and/or DNA to cause mutations and/or cancer. Moreover, the body can
respond to an environmental PAH assault by increasing the levels of the
metabolizing enzymes; thus, potentially increasing the formation of active
metabolites and the potential risk of cancer. Therefore, the effects of DE
on the xenobiotic metabolizing enzymes levels were determined as one
approach to assessing the potential carcinogenic risk from DE exposure.
The inhalation study involved the chronic exposure of Strain A/J
male mice 8 hours/day, 7 days/week for periods of 6 and 8 months to clean
air or DE diluted to 6 mg/m^ of particulates at the U.S. Environmental
Protection Agency, Center Hill facility in Cincinnati, Ohio'^). In this
study, lung and liver microsomal aryl hydrocarbon hydroxylase (AHH)
activities and liver microsomal cytochrome P448/450 levels were determined
using the slightly modified methods of Van Cantfort (1977) et al., and
Omura and Sato (1964), respectively. The results (Table 1) indicated that
there were no statistical differences in the liver microsomal cytochrome
P448/450 levels and liver microsomal AHH activites between clean air and DE
exposed mice both at 6 and 8 months. Small differences were noted in the
lung microsomal AHH activities, but these are believed to be artifactual
differences, due to increases in non-microsomal lung protein present in
the microsomal preparations. The only significant differences found were
in the weight of the animals' lungs. The DE exposed mice were found to have
significantly increased wet lung weights, which could be attributed, in
-------
part, to the deposition of diesel participates in the lungs and possibly
to lung physiological and biochemical changes caused by the DE insult on
the lungs.
A follow-up study was conducted to assess the ability of extracted
diesel particulate organics to cause changes in the levels of liver
cytochrome P448/450- This was done to see ^ changes in these enzymes
levels could be produced from intraperitoneal (i.p.) injections of diesel
exhaust extract (DEE) given at a maximum tolerated dose to mice. The
extract of the diesel particulates was used because it should be fully
bioavailable to the body's systems, whereas there are still doubts as to
the degree of bioavailability of the DE organics when adsorbed onto diesel
carbon particulates and then deposited in the body. The experimental
approach involved the i.p. injection of male and female Strain A/J mice
with DEE, the positive enzyme inducing compounds phenobarbital (PB, a
cytochrome P45Q inducer) or 3-methylcholanthrene (MC, a cytochrome PWQ
inducer), or the appropriate vehicle controls for two days and sacrificing
the animals the third day. Liver microsomal preparations were immediately
prepared after sacrificing and the liver microsomal cytochrome P448/450
levels were determined. The total doses given per kg body weight were DEE
- 500 mg, PB - 160 mg, MC - 40 mg, and vehicle controls (DMSO and saline)
- 300 u 1. The DEE was derived from the 24-hour soxhlet extraction, without
cellulose thimble, of DE particulates collected on teflon coated pallflex
TbOAZO type filters, using methylene chloride as an elutant. The extract
was then made up to the desired injectable concentration in DMSO via
solvent exchange using a nitrogen atmosphere for methylene chloride
removal. The results (Table 2) showed that PB and MC did result in the
induction of the respective P448 and P45Q enzymes in both sexes as
expected. The injected DEE caused significant increases in the liver
cytochrome P448/450 levels in male but not female mice. This increase,
however, was smaller than those seen in the PB and MC induced animals, and
the male DEE induced enzymes were found spectrally between where the
cytochrome P448 and P45Q enzymes were found for PB and MC, respectively.
It is therefore concluded, from the chronic inhalation and the i.p.
injection studies, that enzyme inducing chemicals are present in DE and
that the absence of enzyme changes found in the mice exposed to DE via
inhalation may be due to 1) the enzyme inducing organics associated with DE
were not bioavailable to the body system and/or 2) the inhalation dose was
not sufficient to elicit a detectable change in the enzyme levels.
REFERENCES
1. Pitts, Or., J.N., K. Van Cauweberghe, A.M. Winer, and W.L. Belser.
1979. Chemical Analysis and Bioassay of Diesel Emission Particu-
lates. U.S. Environmental Protection Agency Report of Contract No.
R806042.
-------
2. Hinners, R.6., J.K. Burkart, M. Malanchuk, and W.D. Wagner. 1980.
Animal Exposure Facility for Diesel Exhaust Studies. In: Generation
of Aerosols. K. Willeke, ed. Ann Arbor Science Publishes: Ann Arbor,
Mich., pp. 525-540.
3. Van Cantfort, J., J. DeGraeve, and J.E. Gielen. 1977. Radioactive
Assay for Aryl Hydrocarbon Hydroxylase. Improved Method and Biolo-
gical Importance. Biochem. Biophys. Res. Comm. 79: 505-512.
4. Omura, T., and R. Sato. 1964. The Carbon Monoxide - Binding Pigment
of Liver Microsomes. J. Biol. Chem. 239: 2370-2378.
-------
Table 1. Liver Cytochrome P448/450 Levels and Liver Aryl Hydrocarbon
Hydroxylase Activity in Mice Exposed to Clean Air or Diesel Exhaust
_ Cytochrome P448/450 Level
Values = X H^ SEM in (nMoles/mg microsomal
protein)
*n = sample size
Aryl_Hydrocarbon Hydroxylase Activity
Values = X + SEM (pMoles/min./mg microsomal
protein)
*n = sample size
Months
Exposed
Control
Diesel Exposed
Control
Diesel Exposed
1.52 + 0.073
n = 10
1.54 + 0.066
n ^ 11
48.41 + 2.40
n ^ 10
44.08 + 1.26
n ^ 11
1.61 + 0.067
n = 9
1.62 + 0.081
n = 8
50.84 + 2.57
n = 9
49.04 + 1.76
n = 8
*Each sample consisted of pooled microsomes from two mice.
-------
Table 2. LIVER CYTOCHROME P448/450 LEVELS
Values = X +_ SEM in (nMoles/mg microsomal protein)
n = Sample Size
Saline (control)
100 Hl/30gm BW
Phenobarbital
160 mg/Kg BW
in 100 pi saline
DMSO (control)
300 Hl/30gm BW
Diesel Exhaust
Extract
500 mg/KG BW
in 300 pi DMSO
3-Methylcholanthrene
40 mg/KG BW
in 300 pi DMSO
Males
Females
1.386 + 0
n = 4
1.302 + 0
.049
.067
2.426
n
3.013
+ 0.020
= 4
+ 0.127
1.096
n
1.106
+ 0.059
= 8
+ 0.056
1.346
n
1.186
+ 0.080
= 6
+ 0-.066
1.524
n
1.512
+ 0
= 6
+ 0
.077
.056
n = 4
n = 4
n = 7
n = 5
n = 6
-------
MORPHOMETRIC ULTRASTRUCTURAL ANALYSIS
OF ALVEOLAR LUNGS OF GUINEA PIGS
CHRONICALLY EXPOSED BY INHALATION
TO DIESEL EXHAUST (DE)
Marion I. Barnhart, Steven 0. Sal ley*,
Shan-te Chen and Henry Puro**
Departments of Physiology, Anesthesiology* & Pathology**
Wayne State University School of Medicine
Detroit, MI 48201
Sixty-four Hartley guinea pigs were sacrificed at intervals up to two
years during chronic inhalation exposure to 0, 250, 750, 1500 or 6000 yg DE/m3.
Preliminary findings are published on short term experiments with chronic in-
halation of DE (1,2). The cellular uptake of DE particulates (DEP) was strik-
ingly accomplished by alveolar and interstitial macrophages as well as by
epithelial type I cells (Epi I) and recruited eosinophils (Fig. 1). Cellular
DEP (bulls-eye profiles of 0.073 ± 0.01 ym diameter) was confined to phago-
lysosomes and there was no evidence of cytotoxicity. Arithmetic and harmonic
mean tissue thicknesses of the air-blood barrier were occasionally but signif-
icantly increased (p < 0.05) during DE exposures greater than 250 yg; the 6
mon 1500 yg set was 2 fold greater than controls. However, morphometric
diffusion capacity was relatively unaffected. Epithelial type II cell (Epi II)
prominence and increased interstitial thickness were evident as early as 2
weeks with 750 yg DE. Increased interstitial celiularity (up to 4 fold con-
trol values) suggests the presence of local chemotactic substances. However,
adaption to the DE challenge and burden may be occurring since comparison of
the duration effects reveal a reduced but still abnormal interstitial celiu-
larity; eg. 183 x 106 interstitial cells occurred by 6 mon contrasting with
only 64 x 106 cells by 18 mon 1500 yg DE. Non-cellular interstitium of 250
and 750 yg sets was not significantly different in absolute volume from con-
trols through 6 mon but 1500 \\g DE promoted more interstitial fibers. Epi-
thelial type II cells increased in both numbers (3.2 times control of 18 x 106
cells/cm3) and absolute mass (1.7 x controls). In contrast, Epi I by 3 mon.
at 750 yg DE had decreased numbers (30-60% of controls) but cell volumes were
increased. Endothelial cell numbers were generally increased up to 3 fold
the control values and volumes were decreased. Alveolar macrophages increased
2-3 times control numbers with little significant changes in volume per cell.
Particularily notable are comparative data from animals experiencing possibly
equivalent dose-duration DE exposures. One comparative set contrasts 6 mon
1500 yg DE with 12 mon 750 yg DE. There was a 2-4 fold increase in celiular-
ity in the short duration high dose partner or the longer duration equivalent
partner set was less responsive. Although certain responses to chronic DE
were dose dependent the absence of linearity suggests adaptive responses to DE
challenge. (Aided in part by General Motors Research Laboratory, Warren, MI).
-------
REFERENCES
Barnhart, M.I., Chen, S. and H. Puro. 1980. Impact of diesel engine ex-
haust (DEE) particles on the structural physiology of the lung. Health
Effects of Diesel Engine Emissions: Proc. Internat. Symp., Vol. 2, pp.
649-672. Center for Environ. Research Information EPA, Cincinnati, OH.
Barnhart, M.I., Chen, S., Sal ley, S.O. and H. Puro. 1981. infrastructure
and morphometry of the alveolar lung of guinea pigs chronically exposed
to diesel engine exhaust: Six month's experience. J. App. Tox. 1: 88-
103.
Fig. 1. Uptake of DEP by lung parenchymal cells.
1A - DE-laden alveolar macrophages can pass through pores of Kohn in
alveolar lung.
IB - DEP in epithelial type I cell of lung from 6 mon 250 pg DE ex-
posure.
-------
SCANNING ELECTRON MICROSCOPY OF TERMINAL AIRWAYS OF
GUINEA PIGS CHRONICALLY INHALING DIESEL EXHAUST (DE)
Marion I. Barnhart, Fatma Mohamed and Ahmet Kucukcelebi
Department of Physiology
Wayne State University School of Medicine
Detroit, MI 48201
The structural physiology of airways near gas exchanging alveoli was
documented to establish any changes induced by DE exposure. Preliminary
findings are published on effects of DEP inhalation on alveolar macrophages
(1,2). Here scanning electron microscopy was used to reveal cell interrela-
tions and to resolve distribution of DE particulates (DEP) along the terminal
airway. Thirty guinea pigs inhaled either 0, 250, 750 or 1500 yg DE/m3 for
110 hr/week for 2 weeks, 3 and 12 mon while fifteen rats were exposed for 10
weeks 6000 yg DE, 6 mon 750 yg DE and 12 mon 1500 yg DE. Peripheral airways
were selected for study and photography when they were of sufficient length
to provide structural information from terminal bronchiolus to alveolar out-
pockets. Airways were evaluated without knowledge of the animal's history.
The relative amount and distribution of deposited particulate, was graded on
a scale of 1 to 5+. Decoding was done later followed by final interpretations.
At least 10 terminal airways/animal were extensively photographed. DEP was
identified as free individual particles, 0.1 + 0.03 ym, (Fig. 1). DEP was
adherent to epithelium and irregular patches of particles were prominent at
airway bifurcations (Fig. 1A). Proximal airway, characterized by an epithel-
ium of secretory and ciliated cells, had even larger agglomerates of particu-
lates especially in 12 mon 1500 and 10 week 6000 yg DE sets. These agglomer-
ates consisted of various sized particles, only some of which had sizes appro-
priate for DEP. Quite likely some of the admixture was secretory granules and
congealed proteins; which tend to be larger and more irregular than particles
suspected of being DEP (Fig. IB). Terminal bronchioles often were crowded
with macrophages and granulocytes, exiting th.e lung. Surface domes were
prominent on Clara cells which may be increased in number in DE sets. More
pneumocyte II cell clusters occurred at bronchiolar-alveolar junctions in
heavily exposed than in age-matched control animals. Broad expanses of ter-
minal airway in DE exposed animals appeared relatively clean, but still con-
tained more particulate than companion controls. Alveoli opening off termin-
al bronchioles had more particulates than oth.er alveoli. The morphology and
0.1 ym size of the spherical particles and relative sparcity of such in con-
trols suggests that this is a visualization of the DEP burden but is insuffi-
cient for absolute identification. However the highest DE exposure conditions
were associated with the dustier terminal airways. (This study was aided in
part by General Motors Research Laboratory, Warren, Michigan).
-------
REFERENCES
Barnhart, M.I., Chen, S. and H. Puro. 1980. Impact of diesel engine exhaust
(DEE) particles on the structural physiology of the lung. Health Effects
of Diesel Engine Emissions: Proc. Internat. Symp., Vol. 2, pp 649-672.
Center for Environ Research Information EPA, Cincinnati, OH.
Barnhart, M.I., Chen, S., Salley, S.O. and H. Puro. 1981. Ultrastructural
and morphometry of the alveolar lung of guinea pigs chronically exposed
to diesel engine exhaust: Six month's experience. J. App. Tox. 1: 88-103.
Fig. 1A. Terminal airway of guinea
pig exposed to 1500 yg DE for 12
mon. Note patches of particles
whose individual size is 0.1 ym
and could be DE deposits.
Fig. IB. Terminal bronchiole ad-
jacent to alveolus in rat exposed
to 6000 yg DE for 2 mon. Small
dust particles are 0.1 ym in
diameter and probably DEP.
-------
THE DESIGN OF THE CCMC'S LONG-TERM INHALATION PROGRAM TO
INVESTIGATE THE POSSIBLE TOXICOLOGICAL EFFECTS OF
DIESEL AND GASOLINE ENGINE EXHAUST EMISSIONS
by
J. Brightwell, R.D. Cowling, X. Fouillet, R.K. Haroz,
H. Pfeifer, and J.C. Shorrock
Center for Toxicology and Biosciences
BATTELLE
Geneva Research Centres
Switzerland
The health effects program of the Committee of Common Market Automobile
Constructors (CCMC) on diesel and gasoline engine emissions is presented in
another poster. Part of this program, the long-term inhalation study, is being
carried out by Battelle-Geneva, and in this poster we present the design of the
equipment and the experimental protocol.
Four different types of emissions--diesel (D), filtered diesel (OF),
gasoline (G), and gasoline with converter (GC)are generated by three engines
(VW Rabbit 1.5 litre diesel and two Renault RIP 1.6 litre gasoline) running on
the FTP (US-72 hot start cycle). Two species of animals (Syrian hamsters and
Fischer-344 rats) will be exposed for.up to 24 months, 16 hours per day, 5 days
per week.
These emissions can be diluted to three dose levels: high (H), medium
(M), and low (L). From this 4x3 matrix of exhaust types and dose levels,
three have been omitted as being of little potential interest. An indicative
matrix of dose levels selected for exposure are shown in Table 1.
Table 1. Matrix of Dose Levels
Dose Levels D DF G GC
H 8.3 8.3 3.6 3.6
M 2.8 2.8 1.2 1.2
L n.Q2 -
-------
The figures given in the above table are the calculated mean exhaust gas
concentrations (% volume) when the exhaust gases from one cycle are diluted by
the same volume of air for all engines. In this example the dilution air
volume per cycle for the high dose levels is 300 Nm3 (equivalent to 40
Nm3/mile). The M and L dose levels have been set at 1/3 and 1/9 of the H
levels.
Such dose levels are considered to be directly comparable across the table
since they are based on equal mileage. This comparison takes into account, the
different internal dilution taking place in the engines and also the different
fuel efficiences of the automobiles being compared.
In Table 2, the high dose levels, HO and HG, are expected to correspond to
the following concentrations of the regulated components (running the gasoline
engine at lambda =1).
Table 2. High Dose Level and Concentration Correspondence
Components
Exhaust gas in air
Particulate matter
CO
Nox (N02 equivalent)
THC
Units
%
mg/m3
ppm
ppm
mg/m3
Diesel
(HD)
8.3
5.5
20
15
9.?
Gasoline
(HG)
3.5
-
203
49
37
The figures given in these tables are all calculated from the data
supplied by the automobile manufacturers. They are currently being evaluated
in our system and, if necessary, the flow rate of air to the dilution tunnels
will be modified with a view to keeping concentrations of the biologically
critical components in HD and HG at as high a level as is considered compatible
with a chronic study.
Although slight differences exist in the distribution systems for each
engine, the basic principle remains the same and is described below for one
engine.
The exhaust gases are injected from the tail pipe directly into a dilution
tunnel where they are mixed with a constant flow of conditioned air (filtered
and dried to a water content of 7 g/kg air). The air is dried to compensate,
at least partially, for the high water vapour content of the gasoline engine
emissions so that condensation does not take place in the dilution tunnel, and
to ensure that the relative humidity in the inhalation chambers is not too
high.
-------
A fraction of the diluted exhaust gas is drawn off from the mixing tunnel
into a buffer tank where it is held for about one minute. The need for this
tank is being investigated, and it will be removed from the system if not
required. Its role is to attenuate the high peak values of NO;? and CO that are
produced by the engines over short periods during the US-7? cycle to a level at
which they do not appreciably affect the breathing pattern of the animals.
The dilution tunnels are run at the high dose level. The medium and low
dose levels are achieved by further diluting the high dose level with air. The
dose levels are computed directly from flow rate measurements using rotameters.
The high dose level streams will be continuously monitored for CO and NOX
for safety purposes. The concentrations of the regulated components and
certain non-regulated components will be checked in the inhalation chambers at
reqular intervals.
Battelle-Northwest designed Hazleton-1000 inhalation exposure chambers
will be used, each chamber housing one treatment group of 72 male and 72 female
rats or 156 male and 156 female hamsters. The control groups (fresh air) will
contain 288 rats and 624 hamsters.
Initial and interim sacrifices will be made on 8 animals of each sex from
each group after 0, 6, 12, and.18 months of exposure. These animals will be
used for respiratory physiology, haematology, urinalysis, and blood chemistry
investigations.
Complete autopsies will be carried out on all animals in the study and the
animals in the highest dose levels and the control groups will be subjected to
a histopathological examination of the respiratory tract. Any anomalies
detected during autopsies will also be subjected to a histopathological
diagnosis. Other organs will be stored in formalin and be available for
further examination if required.
System testing of the experimental facility and some short-term exposures
at the high dose levels were carried out up to August 1981. The plan to start
up the long-term exposure using staggered intakes of animals during the months
of September to December 1981 has unfortunately been delayed by several months
due to an accident. Rebuilding and testing of part of the facility is now
necessary before the long-term exposure can be started.
-------
CHRONIC INHALATION ONCOGENICITY STUDY OF
DIESEL EXHAUST IN SENCAR MICE
by
K. I. Campbell, E. L. George. I. S. Washington. Jr.,
P. K. Roberson. and R. D. Laurie
Health Effects Research Laboratory
U. S. Environmental Protection Agency
Cincinnati, Ohio
ABSTRACT
A large number of Sencar mice were used in an investigation to assess
the long term potential inhalation oncogenicity of automotive diesel
emissions. After exposure of parental mice from before mating on through
gestation, the offspring continued in exposure 8 hours daily for 15 months
to an atmosphere of diluted automotive diesel engine exhaust bearing 6 mg
particulate/m-^. Subgroups for testing initiation (A), promotion (B), and
whole carcinogen (C) potentials of diesel exposure were administered,
respectively, weekly i.p. injections of promoter (butylated hydroxy-
toluene) for about 1 year, an initial single i.p. injection of initiator
(urethan), and neither promoter or initiator. Matching controls were
exposed to purified air. Each subgroup initially numbered 260, equally
divided by sex. Additional negative, positive, and vehicle control groups
were used.
Over all groups, survival was 13 percent less in diesel-exposed than
in control mice (75 vs 65 percent), the initiation test group males being
the most affected. Survival was least in initiation-test mice, due
apparently in large part to consequences of frequent i.p. BHT-in-oil
injections. In all subgroups except A females survival was greater in
control than in diesel-exposed mice. In general, males were more sus-
ceptible than females to reduced-survival effects of diesel exposure, and
survival effects were least severe in mice receiving diesel exhaust only
(i.e., not receiving promoter or initiator).
-------
In periodically weighed sample groups, body weight gain was depressed
in diesel-exposed mice of both sexes in all subgroups compared to
corresponding clean air controls, the effect ranging from 11 to 24 percent
of control mean weight gain. Similarly, mean terminal body weights taken
for all survivors showed lower values (ranging 7 to 17%) for both sexes in
all subgroups of diesel-exposed compared to control mice.
Brief inspection of histopathology results indicate no remarkably
consistent differences in lung or other tumor incidences associated with
diesel exhaust exposure. However, for several types of respiratory lesions
there were consistently and greatly increased incidences in diesel-exposed
compared to control mice. These lesions included: alveolar macrophages,
black alveolar pigment material, perivascular and peribronchial mono-
nuclear cells, focal fibrosis, alveolar interstitial thickening, rhinitis
(females), and black pigment in mediastinal lymph nodes. Predictably,
serositis was a common lesion in mice of the BHT-in-oil injected groups of
both control and diesel atmospheres.
-------
SPECIES ntPFERENCES IN DEPOSITION AND CLEARANCE
OF INHALED DIESEL EXHAUST PARTICLES
T. L. Chan and P. S. Lee
Biomedical Science Department
General Motors Research Laboratories
Warren, Michigan 48090
Deposition of inhaled diesel particles and their subsequent clearance from the gas
exchange regions of the respiratory tract may play important roles in the question of
potential health impact of diesel emissions. The initial particulate deposition in the
lungs depends on the physical characteristics of the particles and the airways
morphometry may also affect the regional deposition within the lungs. For example,
the narrow nasal passages can shift the particle deposition in the nasal region
proximally for particles larger than a micron in small experimental animals, and the
final deposition patterns in the lungs will be significantly different from those of larger
species or man. Although diesel particles are not large enough to deposit by impaction
in the upper respiratory tract, species differences can still exist in alveolar clearance
mechanisms, clearance pathways and kinetics. Table 1 compares the estimated initial
particulate dose to the lungs in different species exposed to 0.1 um particles at
250 ug/m for an hour. Although the absolute particulate burden by weight is highest
in man, the immediate local dose to the lung tissues is expected to be five times higher
in the dog and guinea pig. The relative dose is even higher in rats and hamsters by at
least a factor of ten.
Male Hartley guinea pigs and Fischer 344 rats were exposed in a nose-only inhalation
chamber to radioactive diesel exhaust particles. The particles, tagged in the insoluble
carbonaceous core with llfC, were generated by combustion of (1- C)-n-hexadecane in
a single cylinder diesel engine operated at full load [1]. The 1!*C activity in the lungs
and lymph nodes were determined for groups of exposed animals sacrificed immediately
after the 45-minute exposure and others at scheduled intervals for an extended period
of time. Although the initial lung deposition efficiencies and mucociliary clearance
half-times were comparable in both species, the amount of inhaled diesel particles
cleared from the upper respiratory airways in the guinea pig accounted for only 17% of
the initial lung burden, compared to 34% in the rat. Furthermore, the alveolar
clearance of diesel particles in the guinea pig was extremely slow, with more than 80%
of the initial dose retained after 105 days (Figure 1). The pulmonary clearance half-
time for inhaled diesel particles in the guinea pig is estimated to exceed 300 days which
strongly contrasts with 60-80 days in rats (determined by fitting experimental data
collected so far to two- or three-phase clearance models.) The differences observed in
this study demonstrate a greater long-term retention of inhaled diesel particles in the
guinea pig possibly caused by slower clearance processes in the deep lung of this
species. The actual biological dose to the respiratory epithelium would also be
-------
different in both species. This clearly indicates the difficulty in comparing studies on
potential health effects of inhaled diesel particles among different species and in
extrapolating experimental animal data to man.
TABLE 1. ESTIMATED INITIAL LUNG DEPOSITION DOSE IN
DIFFERENT SPECIES AFTER INHALATION OF 0.1 ym
PARTICLES AT 250 yg/m FOR 1 HOUR
Species
Body
Weight
(g)
Minute
Volume
(mL)
Deposition
Efficiency
(%)
Lung
Wt
(g)
Particulate
Burden
(yg)
Particles/g
Lung Tissue
(yg/g)
Man 70K 7000 25 1000 26
Dog 12K 3100 27 80 12
G. Pig 400 125 20 3.0 0.4
Rat 250 150 17 1.5 0.4
Hamster 92 61 20 0.4 0.2
0.025
0.15
0.15
0.25
0.50
REFERENCES
1. T. L. Chan, P. S. Lee, and W. H. Hering, Deposition and clearance of inhaled
diesel exhaust particles in the respiratory tract of Fischer rats. J. Appl.
Tox., 1:77-82, 1981.
-------
Lung Retention of Inhaled Diesel Particles
20 H
Fisch«r Rat
x HortUy Guinea Pig
F ^
10 20 30 40 50 60 70 80 90 100 110
Days Post-exposure
-------
SPECIES COMPARISONS OF BRONCHOALVEOLAR
LAVAGES FROM GUINEA PIGS AND RATS EXPOSED IN VIVO
TO DIESEL EXHAUST (DE)
Shan-te Chen, Mary Ann Weller and
Marion I, Barnhart
Department of Physiology
Wayne State University School of Medicine
Detroit, MI 48201
This ultrastructural, biochemical and functional study involved 93 Hartley
guinea pigs and 63 Fischer rats divided into control and DE exposed sets of 3
animals. Preliminary reports on certain aspects of this study have been pub-
lished (1-3). Clean air or DE exposures of 250, 750, 1500 and 6000 yg DE par-
ticulates/m3 were inhaled for a few days or chronically up to 18 mon. Broncho-
lavage with Dulbecco's phosphate buffered saline provided free cells, which in
controls, were 89-100% macrophages. Viability according to dye exclusion was
90-99% in all specimens. The absolute number of alveolar macrophages increased
1.4 to 2 times control values (7.63 ± 1.11 million cells) by 6 and 12 mon ex-
posure to 750 and 1500 yg DE in guinea pigs. In a short term study in rats
after 2 mon 6000 yg DE macrophage number was 5 times controls. Macrophage size
also increased, excepting 250 yg DE sets. Maximum diameters and surface areas
were measured on scanning electron micrographs of cytocentrifuged lavage speci-
mens. Calculated macrophage volume increased up to 4 times the controls. After
tne in vivo DE exposure there were ultrastructural signs of macrophage activa-
tion. DE particulates were phagocytized without cytotoxic effects since phago-
somal membranes remained intact and macrophage lactic dehydrogenase activity
(a signal of cell lysis) was not in lavage fluids. Cytofluorometry revealed
partial blockade of macrophage phagocytosis of ex vivo fed latex. Macrophages
from 12 mon 750 and 1500 yg DE/m exposures had reduced staining for acid phos-
phatase while the cell free lavage fluids showed 2-5 fold increases in that
enzyme, Comparison of equivalently dosed animals revealed similar magnitudes
of change in macrophage number and granulocyte recruitment. Upon DE exposure
granulocytes became a significant percent of the free cell population by 1 mon
at 6000 yg DE, 2 mon at 750 and 1500 yg DE and 12 mon at 250 yg DE. In rat
lavages neutrophilic granulocytes appeared contrasting with eosinophilic granu-
locyte mobilization in guinea pigs. Lymphocytes appeared in lavages of both
species after 2 mon. While rats appear to be less responsive to the DE chal-
lenge, both species show elevations of albumin, IgG and total protein in the
longer duration and higher exposure sets (Table 1). Both granulocyte emigration
and elevations in proteins at the high DE doses are features of a classic in-
flammatory response, but the 250 yg DE exposures even to 18 mon elicited few and
generally insignificant changes over controls. Defense capabilities of these
healthy rodent species appear adequate to cope with chronic DE challenges at
the tested concentrations.
-------
REFERENCES
1.
2.
3.
Chen, S., Weller, M.A. and M.I. Barnhart. 1980. Effects of diesel engine
exhaust on pulmonary alveolar macrophages. Scanning Electron Microsc,
3:327-338.
Weller, M.A., Chen, S. and M.I. Barnhart. 1981. Acid phosphatase in al-
veolar macrophages exposed in vivo to diesel engine exhaust. Micron 12:
89-90.
Barnhart, M.I., Chen, S. and H. Puro. 1980. Impact of diesel engine ex-
haust (DEE) particles on the structural physiology of the lung. Health
Effects of Diesel Engine Emissions: Proc. Internat. Symp., Vol. 2, pp.
649-672. Center for Environ. Research Information EPA, Cincinnati, OH.
Table 1. Comparison of Dose-Duration Effects
on Protein & Enzyme Content of Acellular
of Diesel Exhaust Exposure
Broncholavage Fluids
ANIMALS (#) &
CONDITIONS
TOTAL PROTEIN
mg/ml
ALBUMIN IgG ACID
mg/ml mg/ml PHOSPHATASE
n M/hr/mg prot
GUINEA PIGS (58)
CONTROLS (16)
2 WK-18 MON
250 Pg DE (15)
2 WK & 2,4,6 MON
12 & 14.5 MON
18 MON
750 yg DE (14)
2,6 & 8 MON
12 MON
1500 yg DE (13)
2 WK, 2 & 6 MON
12 MON
RATS (38)
CONTROLS (12)
2-18 MON
2_DE (11)
4.78 ±1,35 1.55 ±0.66 0.14+0.10 7.97+4.58
4.82 ± 0.93
4.74 ± 1.01
10.57 ± 4.19
7.89 + 2.64
11.50 ± 3.77
8.55 ± 0.80
17.88 ± 5.64
1.27 ± 0.65
1.41 + 0.66
3.95 ± 2.59
2.19 ± 0.94
5.00 ± 2.61
2.17 + 0.17
4.75 ± 0.90
0.12 ± 0.10
0.26 ± 0.17
0.72 ± 0.19
0.39 ± 0.27
0.54 ± 0.21
0.25 ± 0.07
0.92 ± 0.43
10.32 ± 5.07
12.60 ± 6.74
11.54 ± 5.85
16.41 ± 3.25
29.18 + 7.94
13.72 ± 3.07
61.76 ±23.48
1.94 ± 0.21 0.34 ± 0.19 0.05 ± 0.01 19.10 ±12.34
__
2 MON
12 & 14.5 MON
18 MON
750 yg DE (9)
2, 5 & 8 MON
12 MON
1500 ug DE (6)
2 MON
12 MON
1.99 ± 0.45
1.84 ± 0.18
2.75 ± 0.08
2.38 ± 0.15
3.55 + 0.41
2.06 ± 0.23
7.26 ± 1.09
0.28 ± 0.15
0.28 ± 0.16
0.33 ± 0.08
0.41 ± 0.18
0.89 ± 0.26
0.59 ± 0.41
4.18 ± 2.67
0.04 ± 0.02
0.06 ± 0.01
0.11 ± 0.03
0.07 ± 0.01
0.08 ± 0.0
0.04 ± 0.03
0.27 ± 0.02
29.24 ±15.58
28.30 ±10.88
23.40 ± 3.56
27.26 ± 6.95
42.14 ± 6.7
43.07 ± 2.98
70.93 ±10.82
ACKNOWLEDGEMENTS:
Laboratories.
This work was partially supported by General Motors Research
-------
PRELIMINARY REPORT OF SYSTEMIC CARCINOGENIC STUDIES ON DIESEL AND GASOLINE
PARTICULATE EMISSION EXTRACTS APPLIED TO MOUSE SKIN
by
N.K. Clapp, M.A. Henke, T.L. Shock, T. Triplett, and T.J. Slaga
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee
and
S. Nesnow
Carcinogenesis and Metabolism Branch
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
Emission participates may constitute a potential health hazard to persons
constantly exposed. We are determining if emission components given by skin
application might cause carcinogenesis in other organs. To the skin of SENCAR
mice, we applied dichloromethane extracts from particulates collected by
filtration of cooled diluted emissions from Oldsmobile (OLDS) (1 mg/mouse),
Nissan (10 mg/mouse), and Volkswagen (VW) (10 mg/mouse) diesel, and Mustang V-R
(3 mg/mouse) gasoline fueled engines.. Appropriate controls including
benzo(a)pyrene (BP) (0.054 mg/mouse), 12-0-tetradecanoylphorbol-13-acetate
(TPA), and aging untreated mice (CONTROLS) were maintained. In each treatment
group, 40 male and 40 female 6-week-old mice were treated for 52 weeks
following one of two protocols: 1) A single initiation dose of the compound
was followed twice weekly by applications of TPA (2 ug); or 2) the test
compound was applied twice weekly (OLDS only, 4 mg/mouse/week). Surviving mice
were killed 52 weeks after initiation and examined grossly; tissues from
20 different organs were routinely taken for histologic examination. The doses
chosen were those that gave maximal tumor-initiating activity (1,2).
Survival and tumorigenesis for the experimental groups are shown in
Table 1. The numbers of surviving mice were significantly reduced by TPA alone
and all experimental groups given TPA reflected this treatment with decreased .
survival as compared with controls. The only group that was further reduced in
survival by the test compound was Nissan + TPA, which had the lowest survival.
Lung tumor incidences varied with treatment groups but were not different from
controls and TPA lone. Tumors of other organs were observed randomly in the
treated groups but showed no consistent increased incidences associated with
treatments; tumors were found in the mammary gland, uterus, pituitary gland,
cervix, and liver. No evidence of leukemogenesis was seen in killed animals,
although 35% (6/17) of dead males had leukemias in BP + TPA group. In mice
-------
that died prior to the kill date significant numbers exhibited squamous cell
carcinomas of the skin with in to 25% metastases to regional lymph nodes and
lungs in Nissan mice. We have also observed high incidences (40 to 60%) of
amyioidosis, primarily in the spleen and liver, and pyelonephritis and
papillary necrosis in the kidneys of mice given TPA with or without test
compounds as initiators. The relationships and pathogenesis of these diseases
are now under investigation. The sex effect, which shows a difference in
survival as well as tumorigenesis, is not consistent between treatment groups,
and its relationship to the tumor process is unclear at this time. When OLDS
was given repeatedly over the 52-week treatment period (protocol 2), tumor
incidences were not different from untreated controls; it was not effective as
a complete carcinogen with this dose and protocol.
Further analysis of remaining treatment groups and complete of
observations on mice through 24 months of age will provide information about
temporal advancement and tumor incidence modifications by various treatments.
t
(Research jointly sponsored by the EPA under Interagency Agreement
40-728-78, and the Office of Health and Environmental Research, U.S. Department
of Energy, under contract W-7405-eng-26 with Union Carbide Corporation.)
REFERENCES
1. Slaga, T.J., L.L. Triplett, and S. Nesnow. 19RO. Mutagenic and
carcinogenic potency of extracts of diesel and related environmental
emission: Two-stage carcinoegenesis in skin tumor sensitive mice
(SENCAR). In: Health Effects of Diesel Engine Emissions. Proceedings
of an International Symposium, Vol. 2. W.E. Pepelko, R.M. Danner, and
N.S. Clarke, eds. EPA-600/9-80-057b. U.S. Environmental Protection
Agency: Cincinnati, OH. pp. 874-987.
2. Nesnow, S., L.L. Triplett, and T.J. Slaga. (in press). Tumorigenesis of
diesel exhaust and related emission extracts on SENCAR mouse skin. In:
Short-Term Bioassays in the Analysis of Complex Environmental Mixtures,
1980. Michael D. Waters, Shahbeg S. Sandhu, Joellen Lewtas Huisingh,
Larry Claxton, and Stephen Nesnow, eds. Plenum Press: New York.
-------
Table 1. Survival and Tumor Induction in SENCAR Mice Surviving 1 Year Given Diesel and
Gasoline Particulate Emission Extracts to the Skin
Tumors
Treatment
Untreated
controls
TPA
BP + TPA
OLDS + TPA
Mustang + TPA
Nissan + TPA
VW + TPA
OLDS (no TPA)
Sexa
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
No.
surviving
mice
(%)
100
97
67
71
81
56
74
95
80
71
47
46
68
75
97
97
T- posit ive
(paps)
(«)
0
0
19
4
74
100
27
42
31
12
94
89
54
50
0
0
Skin
No. paps/
No. surviving
--
0.23
0.04
3.10
4.70
0.38
0.67
0.46
0.12
4.50
3.40
1.00
1.00
__
-
T-positive Lung
carcinomas (%)
0
0
0
4
0
14
3
3
9
0
11
5
8
3
0
0
3
0
4
4
6
4
14
19
3
8
0
5
0
3
n
3
Misc.
(*)
0
0
0
4
13
0 .
3
0
0
0
5
n
4
0
3
3
Leukemias
(X)
3
0
0
0
0
0
0
6
0
0
0
0
0
0
0
0
F = female; M = male.
-------
CCMC'S HEALTH EFFECTS RESEARCH PROGRAM
by
the Members of the Emissions Research Committee of the
CCMC (Committee of Common Market Automobile Constructors)
Brussels - Belgium (*)
1. GENERAL
In the past decade much progress has been achieved in cleaning
the air by a concerted effort of the governments and the automotive
industry. With more health effect data becoming available, further
legislative measures involving more stringent car emission standards
become more and more questionable. Before stricter standards for
emissions of motor vehicles are legislated, resources for research
programs on health effects must be deployed on an increased scale.
Already in 1974 in the U.S. the National Academy of Sciences (NAS)
established that there were virtually no results available relating to
the effects of automobile emissions. Since then the situation has not
altered greatly. It is only with the concern about cancer with regard
to diesel-powered cars that larger research programs have now been
started in the U.S.
Although a limited number of research investigations have shown
no carcinogenic effects of human exposure to diesel-engined vehicles'
exhaust gases up to now, the European car manufacturers have also
initiated a large scale research project on "an investigation into
possible toxicological and carcinogenic effects of diesel and gaso-
line engine exhaust emissions". The project is sponsored by the
Committee of Common Market Automobile Constructors (CCMC).
(*) Mrs. Chevrier (Renault), A. Henriet (Peugeot S.A.),
H. Klingenberg (VW), H. Metz (BMW), 0. Montabone (Fiat),
N. Pelz (Daimler-Benz), A. Piccone (Alfa Romeo),
S. Wallman (Volvo), J.H. Weaving (BL)
-------
2. OBJECTIVES
This program is considered to be a first step in what will
probably be a continuing investigation by the European industry on
the potential hazards associated with the emissions from automobiles.
The objectives of this first CCMC program are
- to compare potential toxic and carcinogenic effects
of diesel and gasoline engine emissions,
- to check out the presumed beneficial effect of the
catalytic converter of gasoline engines,
- to check out the often assumed relative harmlessness
of the gaseous fraction of the diesel emissions,
- to investigate the mutagenic properties of diesel
and gasoline engine particulates and condensates.
3. PROGRAMS
The project is divided into two parts:
(1) a "long term" inhalation exposure of rats and hamsters
to gasoline and diesel engine exhausts;
(2) a "short term" program of in vivo and in vitro tests on
the effects of particulate extracts and condensates from
gasoline and diesel engine exhausts.
3.1 Long Term Program
The "long term" inhalation program has been contracted
out by the CCMC to the Geneva Division of the Battelle
Memorial Institute.
With about 6000 hamsters and rats the combined effects of
different concentrations of total exhaust from gasoline engines
with and without catalyst, and a diesel engine with and without
particulate matter, removed by filtration, operating according
to the U.S. FTP cycle, are being investigated to determine the
dose/response relationship, and to make a carefully controlled
comparison of diesel and gasoline engines, with respect to
toxicity and carcinogen!city. The running time of the project
is 3 years and costs will amount to 4 million U.S.. dollars.
-------
The program is described in detail in another paper in
this Symposium.
3.2 Short Term Program
The "short term" program, including three main approaches,
is being conducted in separate laboratories in England, France
and Italy.
In different short term in vivo and in vitro tests with
bacteria, mammalian cells, rats and mice, the effects of partic-
ulate extracts and condensates from gasoline and diesel engine
exhaust, collected at Fiat, Italy, are being investigated in an
endeavour to identify the mutagenic components.
The running time of this project is three years and the
costs will amount to 0.73 million U.S. dollars.
Three main approaches were taken:
3.2.1 "in vitro" assays - detection and identification of
mutagens (Microtest, University of York, United Kingdom)
The four basic preparations (particulate and con-
densate, both diesel and gasoline) in total and addi-
tionally fractionated in accordance with the EPA pro-
cedure, are subjected to a modified AMES procedure
(bacterial mutation assay) using five strains of sal-
monella typhimurium (TA 1535, TA 1537, TA 1538, TA 98
and TA 100). The preparations are also tested for the
ability to elicit unscheduled DNA synthesis (UDS) in
cultured human fibro-blast, (DNA repair assay using
Hela cells in culture) and in vitro transformation in
rodent cells (mammalian cell mutation assay using mouse
lymphoma L 5178 Y cells). All these assays have been
combined with a liver monoxygenase enzyme fraction.
The tests are also carried out with extracts of
the particulate fractions.
3.2.2 skin painting tests - detection and identification of
promoters (Institute of Scientific Research on Cancer
(CNRS), Paris, France)
In the short term work sebaceous gland and hyper-
pi as ia tests are performed with the different exhaust
preparations. Investigation is being undertaken to sepa-
rate potential cancer initiators and cancer promoters.
-------
Female 45-day old Swiss mice housed individually and
randomised between the different groups receive, on well
delineated skin areas previously clipped (3 days before),
0.05 ml of acetone solution of the studied substance.
The treatment is repeated on alternate days up to a total
of 3 skin applications (more applications may be necessary)
Eight days after the first treatment, the mice are killed
and the areas of treated skin fixed, sectioned and stained
for histological examination. The thickness of the
epidermis and the number of sebaceous glands present are
determined in 2 to 4 microscopic fields of each of 6 sec-
tions that are cut from each skin specimen. The micro-
scopic examination is carried out on code-numbered slides
which carry no details of treatment given.
For each term or treatment, 25 to 30 mice are being
used. To control the model and to evaluate the relative
activities of the test compounds, adequate positive
(benzo-a-pyrene and forbol ester-tpa) and negative
(solvent) control groups will be provided for each assay.
3.2.3 in vivo tests - possible extraction of mutagens from
particulate matter in the whole animal (bioavailability)
(University of Naples, Department of Biochemistry, Italy)
An important aspect of the CCMC short term project
is the bioavailability of any mutagen or possible car-
cinogen and although inhalation is the preferred test
method, due to the long term nature of this process,
additional short term tests are being conducted with
animals.
These tests consist of assimilating the sample
(diesel particulates) into an appropriate medium, such
as corn oil, bovine serum albumen and human blood serum
and injecting this into the peritoneum and subsequently
subjecting the urine to appropriate AMES assays to test
for mutagenicity. These tests are being carried out
using male and female Sprague-Dawley rats. Three
animals for each dose are being used.
-------
FRACTIONATION AND IDENTIFICATION OF ORGANIC COMPONENTS IN
DIESEL EXHAUST PARTICULATE
Mitchell D. Erickson, David L. Newton, Michael C. Saylor, Kenneth B. Tomer,
and E. D. Pellizzari
Research Triangle Institute
P. 0. Box 12194
Research Triangle Park, NC 27709
Roy B. Zweidinger and Sylvestre Tejada
Mobile Source Emissions Research Branch
Environmental Protection Agency
Research Triangle Park, NC
Diesel exhaust particulate, generated using production model passenger
car engines on a chassis dynamometer, was extracted from Teflon-coated glass
fiber filters with methylene chloride and fractionated using either a solvent
partition scheme partition .scheme or low pressure liquid chromatography
(LPLC). The solvent partition scheme(l) generated two acid, two base, a
cyclohexane insoluble, polar neutral, non-polar neutral, and PNA fractions.
The LPLC scheme generated fractions which elute from a silica gel (Lobar )
column with 10% CH2C12/90% hexane (Fraction Fl and part of F2), 50% CH2C12/
50% hexane (part of F2 and F2A), 100% CH2C12 (F3 and F4), 10% CH3OH/90%
CH2C12 (F5 and F6), 50% CH3OH/50% CH2C12 (F7 and F8) and 100% CH3OH (F9 and
F10). A hexane-insoluble (HI) fraction was analyzed using the same prepara-
tive LC technique. The fractions generated by both schemes were analyzed by
normal phase HPLC, glass capillary GC/MS/DS (electron impact [El], chemical
ionization, and negative ion chemical ionization [NICI]), direct probe
NICIMS, direct probe El high resolution MS, FTIR and (GC)2/FTIR.
Including isomers, 52 polycylic aromatic hydrocarbons and alkyl deriva-
tives, 35 PNA-ketone and di-ketone derivatives, 20 aromatic aldehydes and
cyclic anhydrides, 26 nitrogen-containing PNAs (including nitro PNAs) and 30
other compounds (including alkanes and some possible background contaminants)
were identified.
The compounds of particular interest are the various PNA ketones (e.g.,
fluorenone) and the nitro PNAs(e.g., nitropyrene) and their alkyl-substituted
homologs. These compounds are listed in Tables 1 and 2. The nitro PNAs are
of interest in light of recent findings that some of them are highly mutagen-
ic.(2-4) The nitro PNAs were found in the more mutagenic fractions. Thus,
it may be speculated that these compounds are contributing much or possibly
most of the mutagenic activity to these fractions and therefore to the
diesel exhaust particulate itself.
-------
REFERENCES
1. Hughes, T.J., L.W. Little, E.D. Pellizzari, C.M. Sparacino, G. McCue,
L. Claxton, and M. Waters. Mutation Res.. 76_, 51-83 (1980).
2. Schuetzle, D., J.S.-C. Lee, T.J. Prater, and S.B. Tejada, Int. J.
Environ. Anal. Chetn., £, 93-144 (1981).
3. Lofroth, G., E. Hefner, I. Alfheim, M. M011er, Science, 209, 1037-1039
(1980).
4. Rosenkranz, H.S., E.G. McCoy, D.R. Sanders, M. Butler, O.K. Kiriazides,
R. Mermelsteim, Science, 209, 1039-1043 (1980).
-------
Table 1. POLYCYCLIC KETONES AND DIONES IDENTIFIED IN NISSAN DIESEL EXHAUST PARTICULATE
Number of Analysis
Isomers rr/Fiuc rr /MTPTMC UDMC rr/criD nth Fraction(s) Contai ni ing
Identified Compound Identified w-'tll ^/nitiMb HKMS, bl/MlK utner compound Identified
naphthoquinone
9-fluorenone or C,,HQ0
isomer '* B
methyl fluorenone isomer
or C14H100 isomer
anthrone or phenanthrone isomer
C,-alkyl fluorenone isomers or
C,rH,-0 isomers
C,-alkyl-fluorenone isomers
or C1&H140 isomers (tent)
C.-alkyl fluorenone isomers
C,,H,gO isomers (tent)
xanthone (tent)
anthraquinone
4H-cyclopenta(def)phen-
anthrene-4-one (tent)
benzanthrone isomers
methyl -4H-cyclopenta(def)-
phenanthrene-4-one isomer (tent)
benzofluorenone isomers (tent)
C,gH,.0 ketone isomers (tent)
A
C^gH^O. dione isomer (tent)
6H-benzo(cd)pyrenone isomers
or cigHigO isomer (tent)
C,-alkyl-4H-cyclopenta
(def)phenanthren-4-one
isomer (tent)
Cr-alkyl fluorenone isomer
or C1QH100 isomer (tent)
lo lo
1 x
1 XX
3 x x
1 x
4 x,
4 x x
2 x x
1 x
1 x ?3
1 X
3 x
2 x
2 x
2 x x
1 x x
3 x x
2 x
1 x
F4
F4;F3;F2
F4;F3;F2
F2
F3;F2
F3;F2
HI;F2
F3
F3;F2
F3;F2
F3;F2
F2
F2
F2
F2
F2
F2
F2
, See text for fraction identifications.
3 Other possible isomers include perinaphthenone and benzoindenone isomers.
4 Tentative identification.
c Possible isomers include naphthacenone, triphenylenone, chrysenone, and methylbenzanthrone
Possible isomers include di-ketones of naphthacene, chrysene, and triphenylene.
isomers.
-------
Table 2. NITROGEN CONTAINING AROMATICS IDENTIFIED IN NISSAN DIESEL EXHAUST PARTICIPATE
Identified Compound
N-phenylnaphthylatnine
isomer
C--al ky 1 -N-pheny 1 naphthy 1-
amine isomer (tent)
benzo(c)cinnoline
methylbenzo(c)cinnoline
i somers
C13HgO isomer (tent)
nitroanthracene isomer or
Number of
Isomers
Identified
1
1
1
3
1
1
GC/EIMS
X
X
X
X
X
X
Analysis ,,
GC/NICIMS HRMS GC/FTIR Other ^^n^lSSntlHiS'
x F4;F2;F5;F6;F3;F8;F1G
F5
F5;HI
F5
F6
x x F1G;F2
nitrophenanthrene isomer
methylnitroanthracene or
methylnitrophenanthrene
isomers (tent)
C.-alkyl nitroanthracene or
C.-alkyl nitrophenanthrene
isomers (tent)
C,-alkyl nitroanthracene or
C,-alkyl nitrophenanthrene
isomers (tent)
nitropyrene isomer or
C,,HDNO, isomer
103 2
methylnitropyrene isomer or
nitrobenzofluorene isomer (tent)
C18H11N02 1'somer
F1G;F2
FIG
FIG
F2
F1G;F2
F2
2 See text for fraction identifications.
It is possible that these are polycyclic ketones of the formulas C,,HgO and C,.H,,,0. However, their mass
spectra more closely resembled those for benzo(c)cinnoline in standira spectra. These compounds were
also later eluting than 9-fluorenone and its alkyl homologs. Further elucidation of these compounds
is currently underway for fraction F5 of the refractionated HI sample by means of GC/FTIR and HRMS to
, determine whether these are indeed benzo(c)cinno1ines.
This eluant gave a mass spectrum similar to that of acridine or benzoquinoline, but only a trace
. quantity of the compound was present.
, Possible isomers include nitrochrysene, nitronaphthacene, and nitrotriphenylene isomers.
Fraction Fl was further fractionated to yield subfractions F1A through FIG.
-------
EFFECT OF CHRONIC DIESEL EXPOSURE OF PULMONARY PROTEIN SYNTHESIS
IN RATS
by
R. G. Farrer, Sukla Dutta and S. Dutta
Wayne State University School of Medicine
Department of Pharmacology
Detroit, Michigan 48201
There is evidence that when rats are subjected to acute exposure to cig-
arette smoke, hepatic protein synthesis is inhibited and the extent of inhi-
bition is positively correlated with the dosage of smoke. The present study
has been undertaken to determine the effect of diesel smoke on pulmonary pro-
tein synthesis. For this study, male Fischer rats have been exposed to
diesel exhaust (6 mg/m^) for 2, 4 and 8 weeks. At the end of the desired ex-
posure periods, the lungs have been removed respectively from the exposed and
time-matched control rats and placed in an isolated lung apparatus.
The apparatus was devised by modifying the design of Fisher £t a]_., (2)
for the perfusion of lung excised from rats. Briefly, for perfusion of each
lung, the animal was anesthetized with pentobarbital intraperitoneally and
trachea was cannulated and connected to a Harvard respirator. At this point,
by means of a pressure transducer placed in the in-flow route of the respira-
tor, we recorded in situ ventilation pressure for 3-4 minutes in an eight
channel recorder. This allowed us to compare in vivo tracheo-bronchiolar air
resistance under inspiratory pressure of 10 cm H20 with that of the resist-
ance when the lungs would be under ex vivo condition. Following this proce-
dure, thoracotomy was conducted and the lungs were excised by carefully sepa-
rating pulmonary artery from the aorta. Once the pulmonary artery was clear-
ly dissected out from the other mediastinal structures, heparin (0.5 units/g)
was injected through this. After a few minutes of circulation of heparin,
the pulmonary artery was separated from the right ventricle and the open end
was cannulated for delivery of perfusate at 15 ml/min by means of a Harvard
peristaltic pump from the reservior. While perfusate was going through the
lungs, a small incision was made in the left artrium so that the perfusate
might flow freely and wash blood out of the pulmonary vascular bed. When
the lungs were cleared of blood, they were transferred to a water-jacketed
perfusion chamber maintained at 35°C. During the transfer, we kept respiring
the isolated lung while interrupting the perfusate only for a few seconds.
The peristaltic pump was then switched on and upon perfusion of the isolated
lungs the perfusate freely drained into the perfusion chamber and from there
by means of Tygon tubing back to the reservoir. Thus, a fixed volume of
-------
perfusate continuously recirculated through the lungs for the entire duration
of the experiment. Moment to moment performance of the preparation was
monitored by recording continuously pH and p02 of the perfusate before it
entered the lungs and the pressures required for tidal ventilation and for
delivery of perfusate at 15 ml/min, through the pulmonary artery.
One exposed and one control Fischer rat was tested in one day, and two
such runs were conducted each week. It required approximately two hours to
complete one perfusion experiment which consisted of 30 minutes of equilibra-
tion followed by one hour of ^H leucine incorporation. During the equilibra-
tion period, the lungs were perfused by recirculation with 50 ml Leibovitz
solution and watched for any change in pH, p02» ventilation and perfusion
pressures. If all these parameters looked normal, which was the case for all
the 24 experiments needed to complete the series, the perfusion medium was
changed to fresh 50 ml Liebovitz solution containing approximately 1 yCi/y
mol/ml of ^H-leucine. Thus, the perfusion technique made it possible to
provide a constant supply of substrate, ^H leucine, with a desired specific
activity all throughout the incorporation period.
At the end of ^H leucine incorporation period, samples were taken for
autoradiography and, then, the rest of the lungs were subjected to homogeni-
zation in 0.02 KH P04 and TCA precipitation of protein which was washed with
polar and non-polar solvents. The washed protein residue was dissolved in
4.0 ml of 2.0 N NaOH at 50°C. The protein was determined by the method of
Sedmak and Grossberg (3). Total pulmonary DNA was determined by using the
modified diphenylamine technique of Burton (4) following precipitation of
DNA. Radioactivity obtained from incorporated 3H-leucine was measured in 1.0
ml samples of the dissolved protein using a Beckman LS-100 counter. Because
there were differences in quenching between the diesel exposed (very dark,due
to presence of diesel particles) and control samples, internal standard (^H
tolune) was used to correct the observed CPM before expressing the results in
DPM which was converted to nmole 3H-leucine by using the known specific acti-
vity. Results were normalized on the bases of mg protein as well as mg DNA
as obtained per gram of lung tissue. The results of ^H leucine incorporation
as shown in table 1 revealed that 8 weeks of exposure of male Fisher rats to
6.0 mg particulates/nr of diesel engine exhaust had no significant effect on
the lungs to incorporate -^H-leucine into the TCA insoluble protein. Similar
results were obtained after 2 and 4 weeks of exposure to diesel exhaust in
comparison to respective time matched controls. Also, electron microscopic
autoradiographic grain counts as obtained from the diesel exposed vs. control
rats showed no particular difference among various groups.
Table 1. Incorporation of H-leucine by the perfused lungs as obtained from
rats after 8 weeks of exposure and their time matched controls.
nmoles leucine nmoles leucine
Experiment per mg protein per mg DNA Protein/DNA
8 weeks of air 3.53+0.23* 41.3+3* 11.8+0.4*
exposure
8 weeks of diesel 3.30+0.48 46.3+6 13.9+1.1
* Means + S.E.
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REFERENCES
1. Garrett, R.J.B., and M.A. Jackson 1979. Effect of acute smoke
exposure on hepatic protein synthesis. J. Pharm. Expt. Therap.
209: 215-218.
2. Fischer, A.B., C. Dodia and J. Linadk 1980. Perfusate composition
and edema formation in isolated rat lungs. Expt. Lung Res. 1: 13-21
3. Sedmark, J.J. and S.E. Grossberg 1977. A rapid, sensitive and
versatile assay of protein using Coomassie Brilliant Blue G250.
Anal. Biochem. 79: 544-522.
4. Burton, K. 1955. The relation between the synthesis of deoxyribo-
nucleic acid and the synthesis of protein in the multiplication of
bacteriophage T2. Biochem. J. 61: 473-483.
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THE EFFECT OF EXPOSURE TO DIESEL EXHAUST ON PULMONARY PROTEIN SYNTHESIS
by
C. Filipowitz, C. Navarro, and R. McCaulev
Department of Pharmacology
Wayne State University School of Medicine
Detroit, Michigan
Previous work performed in collaboration with the Riomedical Research
Division of General Motors Corporation had indicated that exposure of rats to
diluted diesel exhaust for periods of up to one year did not induce the
activity of microsomal benzo[a]pyrene-oxidizing enzymes in lung tissue.
Several explanations for this observation, including the possibility that
exposed animals are unable to respond to inducing agents, have been suggested.
In this report, we will discuss the ability of animals which have been exposed
to exhaust in the concentration of 6 mg/m3 of diesel particles to synthesize
pulmonary proteins as judged by in vivo 3H-leucine incorporation and to respond
to 3-methylcholanthrene by the induction of pulmonary oxidative metabolism of
benzo[a]pyrene.
(This research was supported by a grant from General Motors Corporation,
Warren, MI.)
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PREPARATION OF DIESEL EXHAUST PARTICLES AND EXTRACTS
AS SUSPENSIONS FOR BIOASSAY
Jean L. Graf
IIT Research Institute
Fine Particles Research Section
10 West 35th Street
Chicago, Illinois 60616
A bioassay program is being conducted at IITRI to evaluate the acute
toxicities and carcinogenic potentials of diesel engine exhaust components,
cigarette smoke condensates and organic solvent extracts of roofing tar
volatiles and coke oven emissions. The test materials were administered to
hamsters by the intratracheal route. Administrations in both the acute
toxicity and carcinogenic potential bioassay experiments have been completed.
The test materials were supplied through the U.S. EPA Biomedical Research
Branch. The diesel engine exhaust components supplied were a whole particle
exhaust consisting of carbonaceous soot with adsorbed liquid and gaseous
species, and a dichloromethane extract of the whole particle exhaust. The
cigarette smoke condensate was supplied as a concentrated solution in acetone.
Both the roofing tar and coke oven emission extracts were supplied as
dichloromethane solutions.
The intratracheal administration route required preparation of stable
suspensions and emulsions of the test materials in fluids compatible with the
hamster respiratory tract fluids. Saline was the obvious suspending fluid to
be used but additional ingredients were required to enable suspension of the
particles and the solvent-free extracts. Examination of the as-received
whole particle exhaust revealed that very large (up to 150 ym) diesel parti-
cle aggregates were present. These large particles
preparation and were not suitable for intratracheal
a research program was conducted to develop methods
hindered suspension
suspension. Therefore,
for preparing saline
suspensions of the whole particle exhaust in particle size ranges amenable
to intratracheal instillation and saline emulsions (liquid-liquid suspensions)
of the various extracts.
For the short-term acute toxicity studies, a simple wet ball milling
-------
method was developed which both reduced the size of the diesel engine exhaust
particles and suspended them in saline. The hydrophilic nature of the diesel
particles required that they first be wetted with propylene glycol in the
glass milling jar before the saline suspending fluid and the glass milling
beads were added. Gelatin was also added to the saline to serve as a pro-
tective colloid.
Simple hand emulsifying techniques were used to prepare the diesel en-
gine exhaust extracts and the other extracts condensate as stable emulsions
in the gelatin-saline fluid for the short-term acute toxicity studies. Stan-
dard glass tissue grinders proved to adequately emulsify the four types of
organic extracts, once the solvent had been removed and they were wetted with
propylene glycol and a surface active agent. To maintain the emulsion sta-
bilities for more than 30 minutes, gum arabic was also added to the saline
to provide a stronger protective colloid.
The greater materials requirements for the larger scale carcinogenic
potential bioassay experiments required development of a semi-mechanized
method to prepare the emulsions of the test materials. Trial emulsion pre-
parations with a Polytron tissue homogenizer proved successful and a unit
was purchased. The Polytron is a high speed mixing device which employs
both mechanical, shear action and ultrasonics to accomplish homogenization
of liquid samples. Various types of homogenizing probes are available and
provide a wide range of shear and ultrasonic energies. One probe design
provides sufficient shear energy to reduce particle sizes of soft solid mater-
ials such as the diesel engine exhaust particles.
Thus, for the long-term carcinogenicity bioassay experiments, development
of protocols to prepare the suspensions and emulsions with the Polytron were
conducted. The whole particle diesel engine exhaust suspensions were easily
prepared as stable suspensions in gelatin-saline, once the particles had been
wetted with propylene glycol. Particle concentrations as high as 25 mg/ml
were attainable. The primary advantages of the Polytron milling over ball
milling to prepare the particle suspensions were shorter preparation times
(one hour versus 10 days elapsed time), elimination of glass milling con-
taminants, and reduction of the reagglomeration tendency after milling was
completed. The diesel engine exhaust extract was also easily prepared as an
emulsion in gelatin-saline once the extract had been wetted with procylene
glycol (after solvent removal). The use of the high energy Polytron to
emulsify the diesel extract as well as the other organic extracts in saline
eliminated the necessity of adding a surface active agent. However, gum
arabic was still required as an additional protective colloid to maintain
emulsion stability.
Assay and characterization methods for the prepared suspensions were
also developed. While the emulsions could be characterized microscopically,
no practical assay methods could be developed.
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RESEARCH PLANS FOR DIESEL HEALTH EFFECTS STUDY
by
Hironari Kachi and Tadao Suzuki
First Research Department
Japan Automobile Research Institute, Inc.
Yatabe-cho, Tsukuba-Gun, Ibaraki-ken
305 Japan
1) PAST RESEARCHES ON AUTOMOBILE EXHAUST IN JAPAN AUTOMOBILE RESEARCH
INSTITUTE, INC. (JARI)
JARI was granted research contracts from the Japan Automobile Manufacturers
Association, Inc. (JAMA) for studying the reduction of automobile exhaust
and their health effects around 1970 when automobile exhaust were a wide
public concern in Japan on the grounds that they may cause atmospheric pollu-
tions typically exemplified by the phenomenon of photochemical smogs.
JARI started research on these contracts in 1971 including field surveys
using a mobile smog chamber, tracing'of photochemical reactions using a sta-
tionary smog chamber and chemical analysis of emission components. A behav-
ioral assessments on animals were started in 1975, and basic researches on
health effects of NOX and 03 were started in 1976. Preliminary researches on
diesel emissions were started around this time. Small-scale animal inhala-
tion system with modified Rochester type inhalation chambers were designed
and tested. Studies on the health effects of diesel emissions to rats were
done preliminary for one month and then three months. Investigations on
respiratory system revealed that morphological changes in early stage of
exposure are attributed mostly depend to the gaseous components, and it seems
that particulate matters amplify the changes depending on the particulate
concentrations.
The facility for Ames test was completed in 1980. A preliminary Ames test
was conducted on extracts from diesel particulates using Salmonella Typhimu-
rium TA100 and TA98. It was found that PAHs contained as neutral fraction
components showed a relatively high mutagenicity. Examinations of sampling
methods which would allow to obtain artifact-free diesel particulates are
currently conducted comparing results of Ames tests and those of chemical
analysis.
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2) HEALTH EFFECTS RESEARCH PROGRAM (HERP)
In recent years, the health effects of diesel emissions has become an impor-
tant subject in Japan. However, the research works in this field is still in
an embryonic stage. Under these circumstance, JAMA referred this subject to
some researchers of automobile engineering, medical and chemical fields. As
a result, HERP has been drafted in 1981.
An outline of the HERP is presented below.
Period : From 1981 to 1985
Organization (Committee of HERP)
Chairman : Dr. Atsushi WATARI
President of the JARI
Prof. Emeritus of Univ. of Tokyo
Chairman of Steering Committee : Dr. Noburu ISHINISHI
Prof, of Hygiene & Public Health
Kyushu University
Subcommittees : 1. Diesel Exhaust Generation and Sampling
2. Analysis and Custoday
3. Inhalation Studies
4. Small Animal Experiment
5. Culture Cell Experiment (I)
6. Culture Cell Experiment (II)
7. Mutagenicity Test using Microorganisms
8. Miscellaneous including environmental assessment
Secretariat : JARI Officier
Research items :
Facilities for particulate generation, sampling and analysis and a full-scale
facility for the inhalation experiment will be built at JARI. Preparations
are underway to complete these facilities in 1982.
It is planned that JARI will participate principally sampling, analysis,
storage and delivery of diesel tars and the inhalation experiment. The
various in vitro and in vivo tests on diesel emission samples will be con-
ducted in some research institutes and laboratories of the national and
private universities.
Two types (large and small) of diesel engines will be used in the project
considering that properties of emission materials might depend on diesel
engines.
Chronic toxicity tests and carcinogenicity tests will be conducted by the
inhalation for over two years. Intratracheal instillations, skin painting
and other tests of the extracts from diesel particulates will be conducted
-------
on small animals. Mutagenic test of the extracts from diesel participates
will be conducted on culture cells and microorganisms (such as the Salmonella
Typhimurium).
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NEURODEPRESSANT EFFECTS OF UNCOMBUSTED DIESEL FUEL
BY
Robert J. Kainz, Sc.D.
Environmental Industrial Safety Consultants
Fredrick, Maryland
LuAnn E. White, Ph.D.
Tulane University School of Public Health and Tropical Medicine
Department of Environmental Health Sciences
New Orleans, Louisiana
INTRODUCTION
Studies were conducted to characterize the short term neurotoxic effects of
the inhalation of uncombusted diesel fuel vapors. Since diesel fuel contains
indentified harmful hydrocarbon constituents which may exert neurological
effects, the studies in this research effort were designed to screen for
neurotoxic effects of the uncombusted diesel vapors. Mice were exposed at
concentrations of 0.204 mg/1, 0.135 mg/1 and 0.065 mg/1 of uncombusted
diesel vapor for 8 hours/day, 5 consecutive days.
METHODS
Three groups of mice were maintained throughout the experiment: an exposure
chamber group, a control chamber group, and a vivarium control.group. Ten
mice each were in the exposure chamber and the control chamber groups;
five mice were in the vivarium control group. Conditions of the exposure and
control chamber groups were identical except for the presence of the
uncombusted diesel vapor.
Five tests were selected to identify changes in performance which are
related to interference of the nervous system. These tests were: the square
box activity test, used to evaluate activity of the mice by suggesting either
depression or stimulation of activity; the rota rod test which indicates
alterations of the integrity of neuromuscular junctions and coordination; the
inclined plane test, which serves to evaluate neuromuscular junction inte-
grity of neuromuscular strength or paralysis; the corneal reflex test, used
to screen for spinal cord depressant activity; and the hot plate test, a test
of analgesic response. General observations were made during testing and for
30 minutes after mice were returned to their cages. The tests were adminis-
tered to exposure and control chamber groups 24 hours prior to the first day
of exposure, after completion of each day of exposure, and 24 hours after
the last day of exposure. Results of the tests were compared between the
exposure chamber and control chamber groups and expressed as percent of
control within standard error. Exposure was conducted using an inhalation
chamber which exposed mice primarily via the respiratory system with minimal
ingestion and cutaneous exposures. Vapor generation was directly from complex
liquid state to the vapor state and varied +_ 10% for the duration of the study.
-------
s
RESULTS
Square Box Activity Test: Mice exposed to 0.204 mg/1 showed 50 to 90% less
activity than the control chamber mice. Those mice exposed to 0.135 mg/1
demonstrated activity comparable to that of the control chamber mice, while
mice exposed to 0.065 mg/1 had increased activity as high as 150 percent as
that of the control chamber mice. Comparison 24 hours after removal from
exposure to the diesel vapor resulted in similar values between exposure and
control chamber groups.
Rota Rod Test: When compared to the control chamber group, mice exposed to
0.204 mg/1 initially demonstrated a slight increase in performance which
drastically deteriorated as exposure continued. Mice exposed to 0.135 mg/1
had a slight increase in performance followed by a slight decrease in
performance when compared to control chamber mice. Those mice exposed to
0.065 mg/1 showed no relative difference in performance between exposure and
control chamber mice. Comparison after 24 hours of recovery from last
exposure indicated little change between exposure and control chamber mice.
Hot Plate Test: Results of the exposure to 0.204 mg/1 indicated an initial
increase in heat sensitivity followed by tolerance to heat. At 0.135 mg/1 a
slight increase in heat sensitivity was observed for the entire exposure.
Exposure to 0.065 mg/1 identified a substantial increase in heat sensitivity
for the exposure mice as compared to the control mice in the chambers. No
relative difference could be observed between exposure and control chamber
groups 24 hours after termination of exposure.
Corneal Reflex Test: No difference in response was noted between groups for
any concentrations.
Incline Plane Test: Both the exposure and control chamber groups had
negative results at each concentration.
All test result variations are depicted in Figure 1.
General Observations: All mice exposed to 0.204 mg/1 displayed severe
discoloration of the tail indicating vasodilation after three days of
exposure. Severe dehydration was observed in all mice. Grooming habits
deteriorated after day two of the exposure. While walking, five (50%) of
the mice displayed tremors through day three. Half of the mice with tremors
died at day three or sooner; the rest continued to have tremors but recovered..
A weight loss of 30% was observed in the exposure group. The mice in the
exposure chamber group were generally less active than the control chamber
group when returned to their cages.
At an exposure of 0.135 mg/1, five (50%) of the mice displayed tail discolora-
tion after 3-4 days of exposure; slight dehydration was also apparent.
Tremors were evident in three (30%) of the mice while in motion; however, no
deaths occurred. Grooming was poor but less so as compared to the 0.204 mg/1
exposure group.
-------
In mice exposed to the 0.065 mg/1 concentration no appreciable difference
were displayed between the exposure and control chamber groups.
CONCLUSIONS
The exposure of the mice identified general trends in the effect of uncombus-
ted diesel vapor on the nervous system. Comparison of exposure to control
chamber groups suggests a positive central nervous system involvement.
Exposure appears to be concentration and duration dependent. At the
concentration 0.204 mg/1 depression of the inhibitory neuron occurs followed
by extensive depression of the stimulatory neuron. Concentrations of 0.135
mg/1 appear to have little effect when compared to controls and suggest
depression of inhibitory neuron and slight depression of the stimulatory
neurons while the concentration of 0.065 mg/1 causes depression in the
inhibitory neuron.
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175
150
O
O
UJ
O
O
UJ
CL.
oo
125
100
75
50
25
Activity Test
Rota Rod Test
Hot Plate Test
Questioned Data
Point
0123456
Concentration 0.204 tng/1
0123
Concentration
456
0.65 mg/1
0123456
Concentration 0.135 mg/1
DAYS OF EXPOSURE
Figure 1. Results of Screening Tests as Percent Control for All Three Concentrations of Uncombusted
Deisel Vapor
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RESPIRATORY HEALTH EFFECTS OF EXPOSURE TO DIESEL EXHAUST EMISSIONS
(Bus Garage Mechanics; Salt, Potash, Metal, and Coal Miners)
by
R.B. Reger
Epidemiological Investigations Branch
National Institute for Occupational Safety and Health
Appalachian Laboratories
Morgantown, West Virginia
A comprehensive research program has been mounted relating to chronic and
acute respiratory health effects of diesel emissions exposure. Special
attention has focused on occupational groups exposed in enclosed spaces. This
study involves over 5,000 workers engaged in various types of mining
occupations as well as bus garage mechanics. These subjects were given chest
radiographs, asked questions on respiratory symptoms, smoking and occupational
histories, and given spirometric tests. These data have been coupled with
industrial hygiene information to evaluate relationships between selected
health parameters and component measures of diesel exhaust emissions. This
paper reports the results for each group of workers separately.
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SwRI-SFRE DIESEL HEALTH EFFECTS
EXPOSURE FACILITY
by
Karl J. Springer
Department of Emissions Research
Southwest Research Institute
6220 Culebra Road
San Antonio, Texas
The future of the fuel efficient automotive diesel passenger car engine
has been clouded by the possibility that its exhaust particulates have car-
cinogenic properties. To determine whether this is a possibility, auto
manufacturers and the federal government are seeking to determine the pos-
sible long term health effects of diesel exhaust particulates. What the
National Research Council called the most comprehensive effort in this area
is being conducted by Southwest Research Institute and its sister organiza-
tion, Southwest Foundation for Research and Education. The project is
sponsored by General Motors Corporation.
The facility is the largest and most advanced of its kind. The build-
ing consists of three rooms. The engine room houses the diesel engine and
mechanical equipment for conditioning the dilution air. The exposure levels
are monitored and the operation of the experiment is handled from the con-
trol room. The four large exposure chambers are located in the chamber
room and are in close proximity to the diesel engine exhaust, yet isolated
by a soundproof wall.
Each chamber is 8 ft wide by 8 ft long by 8 ft high. They are large
enough to handle about 1250 rats, mice, and hamsters for long term exposure
to diesel exhaust for a total of about 5000 animals. We wished to simulate
levels of exposure not unlike those that might be experienced on the street.
From previous experience we know that the maximum dose one might experience
behind a city bus is about 1 part of exhaust in 120 parts of air. So, one
chamber is operating at that level. Another chamber is operating at twice
that dose, or 1 part of exhaust to 60 parts of air. A third chamber is
operating at about one-third that level, 1 part of exhaust to 360 parts of
air. A fourth chamber receives no diesel exhaust at all, just purified air
and therefore is the control group for comparison to the other three.
Two Oldsmobile 5.7 liter diesel engines are mounted on individual
stationary dynamometers. One is operated and the other is a back-up when
-------
necessary. The engine operates at an equivalent 40 mph cruise condition
(1,350 rpm and 71 ft Ibs torque) for 20 hours each day. Individual samples
of exhaust are directed to the top entrance of each of the three pyrimidal
shaped chambers for subsequent dilution and mixing. The engine operates
on a type 2D emissions test fuel. The engine room also houses the air
conditioning and air filtration equipment used to condition and purify the
dilution air.
The control room contains the automatic controls to maintain engine
speed and power output, the environmental controls for maintaining the
chamber temperature at 74°F and relative humidity at 50 percent with a
slight negative pressure of 0.5 in hLO. Monitoring of the gaseous emissions
is on a semi-continuous basis. Each chamber is automatically monitored for
10 minutes each hour for hydrocarbons by heated FID, CO and C02 by non-dis-
persive infrared analyzers, and for NO/NO by chemiluminescence analyzer.
These and pertinent engine and chamber environment data are recorded each
10 minutes.
The most important measurement is that of total particulate in each
chamber. This is performed by collecting a sample of the chamber atmosphere
on a 47 mm diameter plastic coated fiberglass filter media. The weight
gain is used with the sample volume to compute the particulate concentration
within each chamber. These measurements are made at least once each day.
The amount of exhaust sample admitted into the mixing area prior to the
chamber is adjusted as required to maintain the concentration of particu-
lates as close to specification as possible.
The entire facility was designed for long term, continuous, trouble-
free operation. Redundancies are provided in terms of backup engine
dynamometer and controls, backup power for emergency air conditioning,
backup pumps and air conditioning units and backup controllers etc., that
may malfunction and result in an emergency condition. The totally inte-
grated design located equipment items to simulate exhaust exposures as
close to that in the field as possible. The system typically operates 20
hours each day (4 hours for animal and cage hygiene) and on a 7 day per
week basis.
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POST-EXPOSURE DIESEL PARTICLE RESIDENCE IN THE LUNGS OF RATS
FOLLOWING INHALATION OF DILUTE DIESEL EXHAUST FOR 6 MONTHS
K. A. Strom and B. D. Garg
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
Due to its submicron size, 15-17% of inhaled diesel participate deposits in the airways of
the lung. In the alveoli of the lung, the alveolar macrophages scavenge the diesel
particles, phagocytize them and diesel particulate-laden macrophages were found in
lung lavage fluid even 90 days following an exposure of 16 days to diesel emissions at
6 mg/m [1,2]. This indicated that the diesel particles may have a long residence time
in the macrophages within the lung, rather than being rapidly eliminated via the ciliated
airways or lymphatics. The studies describe the results of biochemical, morphological
and physical measurements on the alveolar rnacrophages, as well as the histology of the
lung after exposure of rats to 250 yg DP/m for 6 months, and serial sacrifice up to 16
months post-exposure.
Male Fischer 344 rats (COBS CDF F-344/CrlBR) were exposed to diesel exhaust
particulate concentration of 250 yg/m for 20 hrs/day, 5-1/2 days/week for 6
months. Control animals were exposed to clean air. Lungs of exposed rats were
lavaged in situ with Hank's Balanced Salt Solution (without calcium or magne-
sium). Differential cell counts and assays of the enzymatic activities of acid
phosphatase and beta-glucuronidase were performed on the lavaged cells.
Light microscopic investigations of the lung revealed that immediately after the
exposure, diesel-laden macrophages were diffusely distributed throughout the
pulmonary alveoli and also focally aggregated in some alveoli. Some macrophages
containing diesel particles were also observed in the bronchus-associated lymphoid
tissue (BALT) and in the lymphatics. Subpleural pigmentation consisted primarily
of aggregations of alveolar macrophages containing diesel particulate. Initially, ninety-
five percent of the lavaged macrophages were completely pigmented with phagocytized
diesel particles, such that only the nucleus of the cell was visible and the cytoplasm was
opaque. The percentage of these macrophages declined with time, showing an
exponential decay with a 6 week half-time. The percentage of lavaged macrophages
which were free of diesel participates rose linearly at a rate of 2.5 percent per week.
The rest of the lavaged macrophages contained some diesel particulate-filled
phagosomes within the cytoplasm. The macrophages were obtained in the same
numbers as those from control animals, and had comparable cell size and activities of
the lysosomal enzymes, beta-glucuronidase and acid phosphatase.
-------
With increasing time post-exposure, diesel-laden macrophages were no longer observed
diffusely distributed throughout the lung, but remained even after 16 months (life span)
in focal accumulations in the alveoli (some of which had thickened alveolar walls),
particularly noted in the subpleural regions, also in the BALT and in the lymphatics.
Initially, the lung tissue contained 0.663 ± 0.075 mg (n=2) diesel particulate. After 16
months of clearance, diesel particulate in the lung had declined to 0.250 ± 0.090 mg
(n=5) with only 0.014 mg (n=10) in the regional lymph nodes.
The rapid decline of the diesel-laden macrophages which was much faster than the
overall diesel particulate removal from the lung, suggests that under conditions of
prolonged exposures to high concentrations disappearance of particulate-laden macro-
phages from the lavageable pool of cells seems to be due to the formation of aggregates
of alveolar macrophages rather than transport out of the lung. The overall clearance of
diesel particulate after extensive exposures seems to be slow and proceeds by as yet
unknown mechanisms. After 16 months post-exposure, alveolar macrophages containing
small amounts of diesel particulate can still be identified in the lavage fluid. In
addition, polymorphonuclear leukocytes are present among the aggregated macrophages
suggesting that the incoming alveolar macrophages and polymorphonuclear leukocytes
may be involved in or contribute to the breakdown of the macrophages aggregates.
REFERENCES
1. S. D. Lee, K. I. Campbell, D. Laurie, R. G. Hinners, M. Malanchuk, W. Moore, R.
J. Bhatnagar and I. Lee, Toxicological assessment of diesel emissions. Abstract of
presentation to Air Pollution Control Assoc., 71st Annual Meeting, Houston, TX,
25-30 June 1978.
2. W. Moore, J. Orthoefer, J. Burkart, and M. Malanchuk, Preliminary findings on the
deposition and retention of automotive diesel particulate in rat lungs. Abstract of
presentation to Air Pollution Control Association, 71st Annual Meeting, Houston,
TX. 25-30 June 1978.
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-
CO
C\J
1
=3"
FIGURE LEGENDS
Fig. la
Fig. Ib
Fig. Ic
250 yg/m for twenty-five weeks and 8 weeks post-exposure:
appearance of the exposed lung.
speckled
250 ug/m for twenty-five weeks and sixty-nine weeks post-exposure.
Diesel-laden macrophages are still present in association with the pleural
surface region.
3
250 yg/m for twenty-six weeks and forty-five weeks post-exposure.
Scanning electron micrograph of macrophage aggregation in a pleural region
from an area shown in Figure Ib.
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MUTAGENIC ACTIVITY OF DIESEL EMISSIONS
by
Joellen Lewtas
Genetic Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
Incomplete combustion of fuel in diesel vehicles results in the emission
of very small carbonaceous particles which, after cooling and dilution, contain
varying quantities (e.g., 5 to 50%) of extractable organic constituents. These
organics generally have been found to be mutagenic in bacteria.
Bioassay-directed fractionation and chemical characterization studies
suggest that polar neutral compounds in general and nitrated polynuclear
aromatic (NOz-PNA) compounds in particular may account for a significant
portion of the bacterial mutagenicity.
Confirmatory bioassays in mammalian cells provide the capability of
detecting chromosomal and DMA damage in addition to gene mutations. Those
assays performed in cell lines, however, usually reauire the addition of
microsomal enzymes to metabolize polynuclear aromatic hydrocarbons (PAH) and
may lack the nitroreductases responsible for metabolizing the NO£-PNAs present
in Salmonella strains. Mammalian cells capable of directly engulfing whole
particles also provide a means of measuring cellular availability of the
organics. In order to evaluate the mutagenicity of these organics in mammalian
cells, extractable organics from particle emissions from a series of diesel and
gasoline emissions have been compared in a battery of microbial mammalian cells
and in vivo bioassays shown in Table 1. The mammalian cell mutagenicity
bioassays were selected to detect gene mutations, DNA damage, and chromosomal
effects. Carcinogenesis bioassays conducted included short-term assays for
oncogenic transformation and skin tumorigenesis.
Diesel and gasoline particle emissions collected on Pallflex T68-20
Teflon-coated fifters after dilution and cooling in a standard dilution tunnel
were Soxhlet-extracted with dichloromethane and prepared for bioassay as
previously described (1). The samples examined were obtained from heavy- and
light-duty diesel and gasoline-powered vehicles and engines.
The bioassays examined in Table 1 were generally conducted in a similar
manner. Assays were conducted at 5 to 7 doses, after a preliminary toxicity
range-finding test to select the proper doses. Data analysis was performed to
-------
determine the slope of the dose-response curve. In some assays increasing
responses were not obtained by increasing the exposure concentration.
The objective of this presentation is to review these results, comparing
different cell systems (e.g., CHO vs L517BY) and different biological endpoints
(e.g., gene mutation vs DNA damage). Mammalian cell data available on chemical
fractions and individual compounds (e.g., benzo[a]pyrene and 1-nitropyrene)
will be compared to the mutagenicity of the total extractable organics, the
whole particles, and gaseous emissions.
REFERENCES
1. Lewtas Huisingh, J., R.L. Rradown, R.H. Jungers, D.P. Harris,
R.B. Zweidinger, K.M. Gushing, R.E. Gill,"and R.E. Albert. 1980.
Mutagenic and carcinogenic potency of extracts of diesel and related
environmental emissions: Study design, sample general, collection, and
preparation. In: Health Effects of Diesel Engine Emissions.
EPA 600/9-80-057b. U.S. Environmental Protection Agency: Cincinnati,
OH. pp. 788-800.
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Table 1. Mutagenesis and Carcinogenesis Rioassays Used in the Evaluation of
Diesel Emissions
A. Mutagenesis Bioassays
1. Gene Mutation Assays
a. Bacterial assay
1. Salmonella typhimurium, reverse mutation at the histidine
locus
b. Mammalian cell assays
1. Mouse lymphoma L5178Y cells, forward mutation at the
thymidine kinase locus using tri-flourothymidine resistance
2. Mouse embryo fibroblasts, Balb/C 3T3 cells, forward
mutation using ouabain resistance
3. Chinese hamster ovary, CHO cells, forward mutation using
6-thioguanine resistance
2. DNA Damage Assays
a. Yeast assay
1. Saccharomyces cerevisiae D3 recombinogenic assay
b. Mammalian cell assays
1. Syrian hamster primary cells, DNA strand breaks by
sedimentation in alkaline sucrose gradients
2. Rat liver primary cells, DNA repair assay using
autoradiographic unscheduled DMA synthesis
3. Chinese hamster ovary, sister chromatid exchange assay
3. Chromosomal Aberrations
a. Mammalian cell assays
1. Chinese hamster ovary, chromosomal aberrations after jji
vitro exposure
2. Human lymphocytes, chromosomal aberrations after in vitro
exposure
B. Carcinogenesis Bioassays
1. Oncogenic Transformation Assays
a. Mouse embryo fibroblasts, Ralb/C 3T3 cells using morphological
transformation
b. Syrian hamster primary embryo cells using viral (SA7)
enhancement of morphological transformation
2. Skin Carcinogenesis Assays
-------
MUTAGENICITY OF DIESEL AMD SPARK IGNITION ENGINE EXHAUST PARTICULATE
EXTRACT COMPONENTS TO SALMONELLA TYPHIMURIUM AND HUMAN LYMPHOBLASTS
by
Thomas R. Barfknecht, Barbara M. Andon. and William G. Thilly
Toxicology Group
Department of Nutrition and Food Science
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Ronald A. Hites
Department of Chemistry
School of Pub.lic and Environmental Affairs
Indiana University
Bloomington, Indiana 47405
Ercole L. Cavalieri
Eppley Institute for Research in Cancer
University of Nebraska Medical Center
Omaha, Nebraska 68105
-------
MUTAGENICITY OF DIESEL EXHAUST COMPONENT PAH
In a previous report it was shown that a methylene chloride extract of
diesel exhaust particulate collected from a 350 CID Oldsmobile diesel
engine was mutagenic in a Salmonella typhimurium 8-azaguanine resistance
forward mutation assay in the presence or absence of Aroclor-induced rat
liver postmitochondrial supernatant (PMS) (1). In addition, the same
diesel exhaust particulate extract was mutagenic to human lymphoblasts only
in the presence of PMS.
In order to determine what components of the diesel exhaust extract
were responsible for its mutagenicity, a fractionation was carried out
based on polarity. An equal parts hexane/toluene polycyclic aromatic
hydrocarbon (PAH) containing fraction was found to be most mutagenic to
S. typhimurium in the presence of PMS (1,2). Analysis of this fraction by
gas chromatography/mass spectrometry has identified 27 major PAH and
derivatives (2).
Initial testing of PAH in human lymphoblasts has shown that fluoran-
thene, 1-methylphenanthrene and 9-methylphenanthrene induced significant
trifluorothymidine resistant mutant fractions at concentrations of 2 uM, 5
|jM and 4 uM, respectively (Table 1). These three PAH represent approximately
0.4% by weight of the total methylene chloride extractable organic matter
of our diesel exhaust sample; however, these same PAH can account for up to
40% of the total mutability of this sample to human lymphoblasts. Fluor-
anthene alone may be responsible for up to 30% of the total mutagenicity of
our extract.
Because fluoranthene plays a major role in determining the mutagenicity
of our diesel exhaust extract, we have initiated studies to determine what
metabolites of fluoranthene are responsible for its mutagencity. One of
the two possible trans-2,3-dihydrodiols of fluoranthene was found to be
significantly mutagenic to human lymphoblasts in the presence of PMS at a
concentration of 2 uM, however, this same dihydrodiol was much less active
in the bacterial mutation assay system requiring a concentration of 33 uM
to induce a significant mutant fraction (Table 1). A trans-2,3-dihydrodio1-
1,lOB-epoxide of fluoranthene proved to be an ultimate mutagen to S. typhi-
murium inducing a significant mutant fraction at a concentration of 0.5 jjM
without metabolic activation. However, this diol-epoxide derivative of
fluoranthene was not mutagenic to the human lymphoblasts up to a concentra-
tion of 1.0 pM (Table 1).
-------
MUTAGENICITY OF CYCLOPENTENO(c,d)PYRENE AND DERIVATIVES
Cyclopenteno(c,d)pyrene is a major PAH component of spark ignition
engine exhaust condensate (3,4). We have found that cyclopenteno(c,d)pyrene
(CPEP) was mutagenic to S. typhimurium and human lymphoblasts at the concen-
trations of 6-7 uM with PMS activation. CPEP was significantly mutagenic
to bacteria at a concentration of 1 uM when activation was by rat liver
microsomes indicating that microsomes are a more efficient source of meta-
bolic activation for CPEP than PMS. Cyc1opentano(c,d)pyrene (CPAP) which
lacks the 3,4 double bond of CPEP was significantly less mutagenic to both
bacteria and human cells (Table 1). Incubation with microsome did not
increase the mutagenicity of CPAP to bacteria.
A 3,4-arene-oxide of CPEP was significantly mutagenic to both S. typhi-
murium and human lymphoblast without activation at the concentrations of
0.7 uM and 0.4 p.M respectively. These data indicate that CPAP-3,4-oxide
is an ultimate mutagen of CPEP.
CPAP-3,4-trans-dio1 was weakly active in the bacterial system inducing
a significant mutant fraction at a concentration of 90 uM. However, it was
not mutagenic to human lymphoblast up to a concentration of 80 uM. In
contrast, CPAP-3,4-cis-diol. was as mutagenic as CPEP inducing a significant
mutant fraction at a concentration of 6 uM in both bacterial and human cell
mutation assay systems. When activated by microsomes, CPAP-3,4-cis-dio1
was as mutagenically potent as the direct-acting CPAP-3,4-oxide in the
S. typhimurium mutation assay inducing a significant mutant fraction at a
concentration of 0.5 uM.
CPAP-3-OH, CPAP-4-OH, CPAP-3-one and CPAP-4-one were significantly
mutagenic to S. typhimurium at the (jM concentrations of 14, 20, 14 and 20
respectively. When microsomes were utilized for activation, CPAP-3-OH and
CPAP-4-OH were mutagenically active at the concentrations 90 pM and 10 jjM
respectively. CPAP-3-one and CPAP-4-one were inactive up to a concentration
of 120 p.M. Only CPAP-4-OH was significantly mutagenic to human lymphoblasts
(Table 1).
These data suggest that there are four distinct metabolic pathways for
CPEP and its derivatives: 1) the formation of CPAP-3,4-oxide which is the
predominant pathway of activation for CPEP, 2) a pathway specific to CPAP-
3,4-ci_s-diol, 3) a pathway of activation for CPAP and CPAP-4-OH, and 4)
metabolic activation of CPAP-3-OH, CPAP-3-one and CPAP-4-one requiring the
cytosolic fraction of PMS. This final pathway apparently does not produce
metabolites that are mutagenic to human cells.
-------
REFERENCES
3.
4.
Liber, H. L.
Diesel soot:
Environmental
404 pp.
, B. M. Andon, R. A. Hites and W. G. Thilly. 1980.
Mutation measurements in bacteria and human cells. U.S.
Protection Agency. EPA-600/9-80-0576a. Cincinnati, OH
Yu, M.-L., and R. A. Hites. 1981. Identification of organic compounds
on diesel engine soot. Anal. Chem. 53:951-954.
Grimmer, G.
I.A.R.C. Sci.
1977. Analysis
Pub. 16:29-39.
of automobile exhaust condensates.
Grimmer, G., K.-W. Naujack, and D. Schneider. 1980. Changes in PAH -
profiles in different areas of a city during the year. In: Polynuclear
Aromatic Hydrocarbons. A. Bjorset and A. J. Dennis, eds. Battelle
Press: Columbus, OH pp. 107-125.
-------
TABLE 1. MUTAGENICITY OF POLYCYCLIC AROMATIC HYDROCARBONS AND DERIVATIVES
TO SALMONELLA TYPHIMURIUM AND DIPLOID HUMAN LYMPHOBLASTS3
Compound
Phenanthrene
1-methylphenanthrene
2-me thy Iphenan th rene
3-methylphenanthrene
9-methylphenanthrene
Pyrene
1-methylpyrene
Cyclopenteno (c , d) pyrene
CPAPd
CPAP-3,4-oxide (-PMS)3
CPAP-3,4-cis-diol
CPAP-3,4-trans-diol
CPAP-3-OH
CPAP-4-OH
CPAP-3-one
CPAP-4-one
Benz (a) anthracene
Chrysene
Triphenylene
Fluor anthene
Fluoranthene-2 ,3-trans-diol
Fluoranthene-2,3-trans-diol-l.
S. typhimurium
yM Concentration
- 300b
+ 80
+ 40
-1000
+ 80
+ 90
+ 180
+ 6
+ 12
+ 0.7
+ 6
+ 90
+ 14
+ 20
+ 14
+ 20
+ 65
+ 45
+ 44
+ 5
+ 33
+ 0.5
Human lymphoblasts
yM Concentration
+ 100
+ 5
- 200
NTC
+ 4
- 300
- 100
+ 7
+ 40
+ 0.4
+ 6
- 80
- 80
+ 40
- 80
- 80
+ 9
+ 6
" + 20
+ 2
+ 2
1
10B epoxide (-PMS)'
(continued)
-------
TABLE 1. (continued)
S. typhimurium Human lymphoblasts
Compound yM Concentration yM Concentration
Benzo(a)pyrene +4 +1
Experiments were performed in the presence of 5% v/v Aroclor-induced rat
liver postmitochondrial supernatant (PMS) except where indicated.
(+) the tested compound induced a significant mutant fraction at the indi-
cated yM concentration, or in the case of a (-) negative response, the high-
est concentration tested is indicated.
°NT, not tested.
CPAP, cyclopentano(c,d)pyrene.
-------
CYTOTOXICITY, MUTAGENICITY AND COMUTAGENICITY IN DIESEL EXHAUST
PARTICLE EXTRACTS ON CHINESE HAMSTER OVARY CELLS IN VITRO
by
A. P. Li, R. E. Royer, A. L. Brooks, and R. 0. McClellan
Lovelace Inhalation Toxicology Research Institute
P. 0. Box 5890, Albuquerque, NM 87185
Diesel exhaust particle extracts were found cytotoxic to Chinese hamster ovary
(CHO) cells. Extracts from cars of different manufacturers had different
cytotoxicity. The emission rates of cytotoxic chemicals were calculated for
the different cars using the cytotoxicity of the extracts, the percentage of
extractable chemicals on the exhaust particles, and particulate emission
rates. The ranking of emission rates of cytoxic chemicals for the different
cars were found to be the reverse of the ranking of the cytotoxicity of the
extracts (1). Our data indicate the need to include emission data other than
the activities of the extracts, when the emission of noxious agents from dif-
ferent vehicles are compared.
The cytotoxicity of diesel exhaust particle extracts is antagonized by serum,
lung and liver cytosols, and sulfhydryl agents in vitro (2) [Figures 1, 2].
The detoxifying effects of the cytosols is enhanced further by the addition
of cofactors (NADP and glucose-t-phosphate); therefore, suggesting enzymatic
detoxification in addition to protein binding. Our data suggest that similar
detoxification of the toxic chemicals associated diesel exhaust particles may
occur jji vivo.
All diesel exhaust particle extracts had low mutagenicity towards CHO cells.
This low activity was observed using different endpoints including sister-
chromatid-exchange and mutation at the hypoxanthine-guanine phosphoribosyl
(HGPRT) gene locus (3). The mutagenicity was slightly enhanced by the ad-
dition of exogenous aroclor 1254-induced liver S9. Although the extracts
had only low mutagenicity, they were found to have definite co-mutagenic
activities (4). Treatment of CHO cells with a combination of a mutagen
(N-methyl, N'-nitro, N-nitrosoguanidine or benzo(a)pyrene) and diesel exhaust
extract yielded a 2-3 fold higher mutant frequency than that calculated by the
mutagenicity of the mutagen and the diesel exhaust extract alone [Table 1].
This co-mutagenicity was observed for all extracts tested, using three dif-
ferent endpoints: mutation at the HGPRT gene locus, mutation at the Na+-Ka+-
ATPase gene locus, and sister-chromatid-exchange. We have shown that diesel
exhaust particles are associated with chemicals with cytotoxic, mutagenic,
and co-mutagenic properties. Engineering variables, biological detoxifying
molecules, and other environment mutagens/carcinogens, all could possibly
modify the health-effect of the diesel exhaust emission.
-------
REFERENCES
1. Li, A. P., R. E. Royer, A. L. Brooks, R. 0. McClellan, W. F. Marshall, and
T. M. Naman. Cytotoxicity of diesel exhaust particle extracta com-
parison among five diesel passenger cars of different manufacturers.
Manuscript in preparation.
2. Li, A. P. 1981. Antagonistic effects of animal sera, lung and liver
cytosols, and sulfhydryl compounds on the cytotoxicity of diesel ex-
haust particle extracts. Toxicol. Appl. Pharmacol. 57:55-62.
3. Li, A. P., and A. L. Brooks. 1981. Use of Chinese hamster ovary cells
in the evaluation of potential hazards from energy effluents--applica-
tion to diesel exhaust emission. Lewis, M. (ed.). "Proceedings, the
International Symposium of Health Impact of Different Sources of Ener-
gy", jointly sponsored by WHO/UNEP/IAEA, Nashville, TN, June 22-26,
1981. In press.
4. Li, A. P., and R. E. Royer. 1981. Diesel exhaust particle extract en-
hancement of chemical-induced mutagenesis in cultured Chinese hamster
ovary cells: Possible interaction of diesel exhaust with environmental
carcinogens. Mutat. Res. In press.
-------
Table 1. Co-mutagenicity of Diesel Exhaust Particle Extracts
in the Presence of Exogenous Activation System3
(Li and Royer, 1981)
Treatment
Mutant Frequency
-B(a)P +B(a)P (o.5 pg/ml)
Expected13
Observed
Enhancement0
Solvent (DMSO)
Control
7 (A)
56 (B)
Exhaust Extracts
(60 pg/ml)
Car A
Car B
Car C
Car D
Car E
28
6
11
9
25
77
56
60
58
74
218
170
194
184
229
2.8
3.0
3.2
3.2
3.1
An Aroclor 1254 - induced rat liver cytosol/cofactors mixture was used
for exogenous activation.
DExpected mutant frequency = mutant frequency (B(a)P alone) (B) + mutant
frequency (diesel exhaust particle extract alone) - mutant frequency
(DMSO alone) (A).
'Enhancement = observed mutant frequency * expected mutation frequency.
-------
1.0-
[LATIVE SURVIVAL
O
en
i
M4
IX
o
^^*""
1
51
1 1
OX
5%
U
1 1
1 1
1 ]
DMSO DIESEL HUMAN CALF CYSTEINE SERINE GLUTA- OXIDIZED MERCAPTO- ETHYLENE
SERUM SERUM THIONE GLUTATHIONE ETHANOL GLYCOL
1 1
+ DIESEL
Figure 1 (Li, 1981). Effects of animal sera, sulfhydryl compounds and their
non-sulfhydryl analogs, on the cytotoxicity of diesel
exhaust extract.
-------
Liver S9
Cofactors
Lung S9
+ Cofactors
Lung S9
- Cofactors
Liver S9
- Cofactors
0 100 200 " 0 100 200
CONCENTRATION frig/ml) CONCENTRATION Gug/ml)
Figure 2 (Li, 1981). Effects of lung (A) and liver (B) cytosols on the
cytotoxicity of diesel exhaust extract.
-------
INDUCTION OF IN VIVO SISTER CHROMATID EXCHANGE BY
DIESEL PARTICULATE AND DIESEL EXTRACT
by
Michael A. Pereira and Lofton McMillan
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
O.K. Gulati, C.L. Stephens and P. Kaur
Environmental Health Research and Testing Inc.
Cincinnati, Ohio 45220
The use of diesel fuel is no longer limited to heavy duty engines
because our energy consious society is gradually shifting to diesel powered
automobiles. The safety of this change has been questioned because diesel
engines generate 30 to 50 times more particulate matter in comparison to
gasoline engines (J. Air Poll. Control Assoc., 28: 760, 1980). In-
vestigations were carried out to study the effect of "cTTesel particulate and
its methylene chloride extract on the induction of sister chromatid
exchanges (SCE) in the bone marrow cells of mice. Diesel exhaust particles
were generated by a Nissan CN-6 diesel six cylinder engine and a Chrysler
torgue-flite automatic transmission Model A-727 along with an Eaton-
Dynamomter Model 758-DG, operated by Federal Short Cycle-type driving
modes. The diesel exhaust particles were collected on teflon coated
pallflex T60A20 type filters. An extract of the diesel particulate was
prepared in a soxhlet extraction apparatus using methylene chloride as the
elutant. The extract was transfered to dimethyl sulfoxide by solvent
exchange using a stream of nitrogen to remove the methylene chloride.
Three month old male mice were injected intraperitoneally with diesel
particulate matter at 300 mg/Kg body weight or diesel extract at 800 mg/Kg
body weight. Each animal received a single intrapentoneal injection. After
1, 2, 5, and 14 days of treatment, mice were sacrificed by cervical
dislocation and intact femurs removed. Six animals were sacrificed at each
time point. The bone marrow cells were flushed from the canal with 0.075 M
KC1. The cell suspension was centrifuged at 1,000 g for 10 minutes and the
cells fixed. After two additional fixations, the cell suspension was
dropped onto cold wet slides. A combination of Hoechst dye and Giemsa was
used to stain the cells. Twenty metaphases were evaluated for the number of
SCE. The mitotic index was estimated as total number of dividing cells per
1,000 cells.
-------
Diesel participate matter when administered ip, caused a substantial
enhancement in the number of SCE per metaphase and per chromosome in the
bone marrow cells.. In mice treated with diesel particulate matter and
sacrificed 1, 2, 5, and 14 days post-treatment, average frequency of SCE per
metaphase in the bone marrow cells was 5.4, 11.3, 6.7, and 5.1, re-
spectively. The average number of SCE in the bone marrow cells of solvent
treated animals was approximately 6 per metaphase. It is evident from these
observations that the genotoxic effect of diesel particulate matter appears
on the second day of treatment. By the fifth day post-treatment, the
genotoxic substances on the diesel particles were no longer capable of
inducing SCE. The methylene chloride extract of diesel particulate matter
also substantially enhanced the frequency of SCE/metaphase in the bone
marrow cells. Experimental animals sacrificed 1 and 2 days after the
treatment, revealed approximately 60% higher frequency of SCE than the
corresponding controls. When the treatment was extended to 5 or 14 days,
the number of SCE was equivalent to the spontaneous rate. These findings
suggest that diesel particulates and its methylene chloride extract contain
genotoxic substances.
-------
MUTAGENIC ACTIVITY OF DIESEL PARTICLES IN ALVEOLAR MACROPHAGES
FROM RATS EXPOSED TO DIESEL ENGINE EXHAUST
J-S. Siak and K. A. Strom
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090
Diesel engine exhaust contains submicron size carbonaceous particles. Dichloro-
methane extracts of these particles collected by filtration or electrostatic
precipitation elicited mutagenic responses in bacterial mutagenicity assay. Currently,
there are no data on the mutagenic properties of inhaled diesel particles that are
deposited directly in the lung. The purpose of this experiment was to determine the
mutagenicity of the inhaled diesel particles and the interaction between the particles
and alveolar macrophages.
3
Adult male Fischer 344 rats were exposed to 6 mg/m of diesel particles for 3 days (20
hrs/day). Alveolar macrophages were obtained by bronchopulmonary lavage imme-
diately after exposure and at 1, 4, and 7 days thereafter. Macrophages from forty
animals were pooled for each data point, sized and counted. The mass of diesel
particles phagocytized in alveolar macrophages was determined by a spectrophoto-
metric method (Rudd and Strom, J. Appl. Tox., l(2):83-87, 1981). The alveolar
macrophages were concentrated by filtration on pre-washed fiberglass filters and dried
at room temperature to constant weight. The filters were extracted with dichloro-
methane in a Soxhlet apparatus for 4 hours (20-25 solvent cycles). The resulting
extracts were oily, indicating cellular lipids and surfactant were extracted from the
macrophages. The Salmonella typhimurium strain TA98 was used for mutagenicity
assay. For thin layer chromatography, Whatman LK6 plates were used and the
developing solvent was toluene:hexane (5:1).
Table 1 shows the diesel particle mass recovered in alveolar macrophages from exposed
rats. The mass of diesel particle recovered from the lavage accounted for 45-50% of
the particles deposited in rat lungs. The extracts of diesel particles (DPE) collected
from the exposure chamber by filtration were used as reference for the thin layer
chromatographic and mutagenic analysis of the macrophage extracts. The TLC
fluorescence banding pattern of the samples from macrophages obtained immediately or
one day post-exposure were similar to that of chamber DPE. However, the extracts of
macrophages recovered on the fourth and seventh day post-exposure lost their fluores-
cence patterns. Figure 1 shows the results of the mutagenicity assay. The data indicate
that the cellular lipids extracted from macrophages mitigated the mutagenic response
of the airborne DPE, but a positive result was still detectable in the extracts of the
macrophages obtained immediately, and one day after the exposure. In contrast, the
mutagenic activity of extracts from macrophages obtained on the fourth and seventh
-------
day after the exposure was undetectable. The data indicate that: 1) inhaled diesel
particles contain extractable mutagenic compounds - whether they are the same as
those found in the particles collected by other means has yet to be resolved; 2) alveolar
macrophages have the ability to release or transform the fluorescent and mutagenic
extractable hydrocarbons from phagocytized diesel particles over a period of several
days and thus may significantly influence their biological activity in the respiratory
system.
Table 1
DIESEL PARTICLE MASS RECOVERY
IN ALVEOLAR MACROPHAGES FROM EXPOSED RATS
GROUPS
DP ug/mL
Lavage Fluid
DP ug/10
Macrophages
Total Recovery
(mg)
IMMEDIATELY
1 DAY-POST
4 DAY-POST
7 DAY-POST
5.0
5.9
6.3
6.6
Mutagenic activity of airborne diesel
particle extract and macrophage
extracts.
Airborne diesel particle
extract.
Airborne diesel particle
extract + 800 \ig control
macrophage extract.
Macrophage extract from
exposed rats immediately
after exposure.
Macrophage extract from
exposed rats 7 days after
exposure.
41.3
34.3
25.5
25.8
8.4
10.3
10.6
11.1
01 0' 06 01 10
EQUIVALENT DIESEL PAHTICULATE MASS Im9l
PER PLATE
-------
DERMAL CARCINOGENESIS BIOASSAYS OF
DIESEL PARTICULATES (DP) AND DICHLOROMETHANE (DCM)
EXTRACT OF DP.
by
Linval R. DePass, Lynn G. Peterson and Carrol S. Weil,
Bushy Run Research Center, Export, Pennsylvania
K.C. Chen
Biomedical Science Department, General Motors
Research Laboratories, Warren, Michigan
INTRODUCTION
The present studies were designed to assess the potential of DP
and DCM extract of DP as complete carcinogens and as initiators or
promoters of carcinogenesis using a mouse skin model.
MATERIALS AND METHODS
The test agents were applied as suspensions in acetone to the
dorsal skin of 40 male C3H/HeJ mice (Jackson Laboratory, Bar Harbor,
Maine) per group at the highest concentrations that were sufficiently
flowable for dosing. In some studies lower concentrations were also
used to obtain information on dose-response relationships. The
samples were shipped on dry ice from General Motors to Bushy Run
Research Center on a regular basis throughout the study. Suspensions
for dosing were stored at -12°C in amber-colored bottles except during
application to the animals. Dosing was performed 3 times weekly in
the initiation and complete carcinogenesis studies and 5 times weekly
in the promotion studies. Positive control groups received repeated
applications, 38 ug each, of benzo[a]pyrene (BaP, Eastman Kodak) for
complete carcinogenesis or a single application of BaP>230 ug,
followed by repeated applications, 1.5 ug each, of phorbol myristate
acetate (PMA, PL Biochemicals) for the promotion studies. The test
agents were applied in place of BaP in the complete carcinogenesis
and initiation studies and in place of PMA in the promotion studies.
Mortality and tumor incidence data were analyzed by methods
based on the Kaplan-Meier distribution (1). Comparisons were made
using the Mantel-Cox (1) and Breslow (1) tests with a probability
of 0.05 (2-tailed) required for rejection of the null hypothesis.
-------
RESULTS
Complete Carcinogenesis Studies
One tumor-bearing animal has been observed at the highest dosage
of DCM extract (12 mg/day) in the complete carcinogenesis study. The
tumor was in the treatment area and was diagnosed as a squamous cell
carcinoma. One mouse which received 1 mg/day DCM extract died with a
subcutaneous sarcoma of the left lateral surface. Thirty eight tumor-
bearing animals were observed in the positive control (BaP) group. No
tumors were observed in the negative controls or in any other dosage
group.
Initiation Studies
Six and five tumor-bearing mice were observed in the mice ini-
tiated with DP (2.0 mg) and DCM extract (12.0 mg) respectively. Four
tumor-bearing animals have been observed in a negative control group
initiated with acetone, and one tumor-bearing animal was seen in a
second negative control group initiated with PMA.
Promotion Studies
In the promotion studies, one and two tumor-bearing animals have
been observed in the groups which received 12.0 and 5.1 mg/day of DCM
extract, respectively. No tumors have been observed in the groups
treated with DP (2.0 mg/day), acetone, or a group which was untreated
after the initiating dose of BaP.
In a positive control group which received repeated applications
of PMA after an initiating dose of BaP, 19 tumor-bearing animals were
observed.
DISCUSSION
The results to date suggest that DP and DCM extract of DP have
little, if any, tumor-initiating or tumor-promoting activity under
the conditions of this assay. This conclusion is based on the absence
of a statistically significant increase in tumor incidence (or re-
duction in time to tumor) in any dosage group in the initiation and
promotion studies.
However, the observation of a tumor-bearing mouse in the high
dosage DCM extract group of the complete carcinogenesis study must
be considered in assessing the oncogenic potential of diesel emissions.
Although the presence of a single tumor is clearly not statistically
significant, its importance must be considered in the light of ex-
tensive historical control data. The C3H/HeJ strain has been found to
have an extremely low spontaneous skin tumor incidence in this labo-
ratory. Of 474 acetone-treated controls, only a single mouse with a
squamous cell carcinoma of the eyelid has been observed. No tumors
have been seen in the treatment area. Thus, the tumor in the treat-
-------
merit area of a DCM extract-treated mouse may have toxicological sig-
nificance.
Since some of these studies are still in progress, it would be
inappropriate to draw any final conclusions at this time. Prelim-
inary conclusions based on gross observations will be possible after
the death of all the animals.
REFERENCE
1. Gart, J.J., K.C. Chu and R.E. Tarone 1979. Statistical issues
in interpretation of chronic bioassay tests for carcinogenicity.
J. Natl. Cancer Inst. 62:957-974.
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RESPIRATORY CARCINOGENICITY OF DIESEL FUEL EMISSIONS - INTERIM RESULTS
by
Alan M. Shefner, Bobby R. Collins, Arsen Fiks,
Lawrence Dooley and Mauriine M. Preache
IIT Research Institute
Life Sciences Research Division
10 West 35th Street
Chicago, Illinois 60616
An experiment is in progress in which diesel engine emission particles
(DP), organic solvent extracts.of diesel particles (DE), extracts of roofing
tar volatiles (RT) and of coke oven emissions (CO), and cigarette smoke
condensate (CS) are being evaluated for their carcinogenic potential when
administered by intratracheal instillation to hamsters. Appropriate control
animal groups including untreated colony controls (CC), solvent (SV),
solvent plus ferric oxide (SF), benzo(a)pyrene (BP) as a positive control,
and gel-saline plus ferric oxide (GS) were included in the study. Because
of the number of hamsters being treated, the experiment was conducted in
two replicates of identical design with the exception of the inclusion of
a gel-saline control in the second replicate.
All five test materials were prepared as mixtures with equal weights
of ferric oxide and delivered as suspensions or emulsions in appropriate
solvents. Diesel emission particles were also prepared without admixed
ferric oxide and constituted the sixth test material. It was prepared as
a suspension in SV. Test materials were delivered at doses of 5, 2.5, and
1.25 mg/treatment/week with additional equal weights of ferric oxide for
five of the test materials. Intratracheal administration was carried out
once weekly for fifteen weeks for each test and control treated group.
Hamsters were twelve-to-thirteen weeks of age at initiation of treatment.
Only male hamsters were used in the study and animals were obtained from
Charles River Breeding Laboratories. Group sizes in each replicate
consisted of 60 hamsters per dose level for each test group, 60 SV controls,
150 SF controls, 60 BP animals and 120 colony controls. An additional 60
GS controls were included in the second replicate. Thus an initial group
of 1470 hamsters was included in the first replicate and 1530 hamsters in
the second. When the hamsters reached 12 months of age a sacrifice of
randomly selected animals from each group was carried out in each
replicate. This consisted of 10 hamsters per dose level for each test
group, 10 SV controls, 25 SF controls, 10 BP controls, 20 CC animals and
-------
10 GS controls. Sacrificed animals were subjected to a complete gross
pathology examination, major organs were weighed and an extensive group of
tissues were submitted for histologic processing. Histopathologic
examination of tissue slides from the scheduled sacrifice animals from the
first replicate has been carried out and similar studies on second
replicate animals are in progress.
High dose hamsters in most treatment groups gained weight more slowly
than their respective controls during the treatment period. Upon cessation
of treatment high dose animals generally gained weight at a faster rate
and body weight differentials gradually disappeared. At 61 weeks on test
in the first replicate and 44 weeks on test in the second there are no
consistent treatment-related trends in body weights in surviving hamsters.
First replicate hamsters were housed three to a cage in suspended
polycarbonate cages with filter sheets over the shelves of the racks. A
considerable amount of fighting occurred between cage mates resulting in
a 94-100 percent incidence of lumbosacral skin lesions secondary to
fighting. Early deaths in these hamsters were not treatment or dose related
and were attributed largely to wound-related causes. Cage dividers of
stainless steel were designed and fabricated and the hamsters were rehoused
two per cage separated by a solid partition. Skin lesions cleared up over
time after the hamsters were physically isolated from cage mates. The
hamsters in the second replicate were individually housed in these
partitioned cages from the fourth week of test onward. Overall survival
rates in the second replicate have been consistently higher than that
observed in the first replicate. This absence of fighting-associated
deaths in the second replicate should make possible the determination of
test material effects on survival time should such exist.
Group mean organ weights of test hamsters from the first replicate
showed no significant effects of test materials as compared with their
solvent controls. Histopathologic findings on.these animals were generally
less severe than those observed in earlier dose response studies where
hamsters were sacrificed five weeks after the end of treatment. This
observation tends to reinforce the apparent recovery from immediate
treatment effects implied by the increased body weight gains observed
following cessation of treatment.
Various lesions of the lung were noted and those which appeared to be
treatment related generally reflected the quantity of particulate material
administered as well as showing differences in reactivity to specific test
substances. In general inflammatory reactions and granuloma were more
prevalent in diesel particle, diesel extract and coke oven extract animals
than in cigarette smoke or roofing tar treated groups. Adenomatous
hyperplasia and papillary hyperplasia were either found in a low incidence
or not observed in solvent control and colony control hamsters and to a
trace to mild degree in solvent plus ferric oxide animals. The incidence
and severity of these lesions was greatest in both diesel particulate
groups, intermediate in the coke oven extract treated animals, and lowest
in the diesel extract, cigarette smoke, and roofing tar groups.
-------
Bronchiolization was greatest in high dose diesel particle groups, found to
a lesser degree in coke oven and roofing tar animals, least frequent in
diesel extract and cigarette smoke animals and absent in control hamsters.
Squamous metaplasia was observed primarily in coke oven treated animals and
the few adenomas that were found were present in diesel particle and diesel
extract treated animals. In general the frequency and severity of
hyperplastic and metaplastic changes in the lung were greatest in the two
diesel emission particle test groups, somewhat lower in the coke oven
extract and in the diesel extract groups, and least in the cigarette smoke
and roofing tar extract hamsters.
Particles were found in high density in the thoracic lymph nodes of
particle treated animals and reactions to the presence of these particles
were common in all treatment groups but appeared more commonly in diesel
particle treated hamsters.
Whether any of these lesions will progress over the lifespan of the
remaining hamsters now on test, and whether such progression will be
related to test material and dose remains to be seen.
(This study was supported by EPA Grant No. R806929-01).
-------
CARCINOGENICITY OF EXTRACTS OF DIESEL AND RELATED
ENVIRONMENTAL EMISSIONS UPON LUNG TUMOR INDUCTION IN
STRAIN 'A' MICE
R. D. Laurie, W. B. Peirano, W. Crocker,
F. Truman, J. K. Mattox and W. E. Pepelko
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio
INTRODUCTION
The predicted increase in the use of diesel engines has resulted in a
regulatory need for data assessing the relative carcinogenicity of diesel
exhaust. Since cigarette smoke, roofing tar and coke oven emissions have
been shown to be carcinogenic, a matrix of experiments was designed to
compare the relative potency of these pollutants with diesel exhaust
particulate. The present study is one of several, including skin painting
of Sencar mice, intratracheal instillation in hamsters and _TJT_ vitro
testing, designed to provide such a comparison.
METHODS
Compounds
Nissan diesel particulate matter was collected with a high volume
sampler using Pallflex T60A20 (teflon coated) filters. The samples were
collected from a large mixing chamber containing exhaust. diluted with
about 9 parts clean air to produce a particulate matter concentration of 12
mg/m^. Exhaust was produced with a 6 cylinder, 90 cubic inch displacement
Nissan diesel engine run on the Federal Short Cycle. For details see
Hinners et al (1979). The Oldsmobile sample differed from the Nissan sample
in that it was generated at a steady state (40 mph). Both samples were
Soxhlet extracted with dichloromethane. Cigarette smoke condensate (CSC)
was supplied by the Chemical Repository and Tobacco Smoke Chemistry
Division of the Tobacco and Health Research Institute, University of
-------
Kentucky. The CSC was produced from Kentucky Reference 2RI Cigarettes. For
details of collection and generation see Patel (1977). The coke oven
emissions were collected using massive volume samplers from the top of a
coke oven battery at Republic Steel in Godsden, Alabama. The roofing tar
emissions were collected using a baghouse filter fitted with special
nonreacting filter bags. Details of collection procedures for both coke
oven and roofing tar have been described in detail by Huisingh et al
(1979). Most samples were dissolved in DMSO: for the Nissan- particulate
matter 5% of the DMSO solution was EL620.
Animals Used and Experimental Design
Strain A/Jax mice approximately 8 weeks of age, were randomly
assigned to 9 groups: 1) uninjected controls, 2) vehicle controls, 3)
positive controls, 4) Nissan generated particulate matter, 5) Nissan
particulate extract, 6) Oldsmobile particulate extract, 7) cigarette smoke
condensate, 8) coke oven emissions, and 9) roofing tar. The doses for each
group are listed in Table 1. Because of limited availability of Oldsmobile
extract only males were injected. Due to high mortality rates among mice
injected with Nissan particulate the dose level was halved in the second
experiment. The experiment was carried out in 2 parts because of the
limited availability of animals and manpower. The mice were injected 3
times weekly for 8 weeks with the test substances. The injection volume was
50 microliters, delivered by Hamilton syringes fitted with 26 G needles.
The urethane positive controls received only one injection as the start of
the experiment.
Collection and Analysis of Data
The mice were sacrificed at 9 months of age with an overdose of
nembutal. The lungs were removed and placed in buffered formalin. After 2
weeks the lobes were detached from the bronchi and the number of adenomas
visible on the surface counted. Questionable, areas were examined micro-
scopically. Analysis of variance compared the number of tumors per mouse
among groups; Chi Square analysis compared the frequency of mice with
tumors.
RESULTS
From the data presented in Table 1 it is clear that a significant
increase in number of lung adenomas per mouse and percent of mice with
tumors occurred in the positive controls (urethane injected). In experi-
ment 1 a significant increase in number of tumors per mouse was noted in
males injected with Nissan diesel extract compared with controls or those
injected with Oldsmobile extract. In experiment 2, a significant increase
in lung tumor rates was detected in females injected with coke oven
emissions. After combining the data from both experiments, no statis-
tically significant differences were noted, except for the positive
controls.
-------
DISCUSSION
There were no consistent, statistically significant differences
between the exposed and control groups with respect to either number of
adenomas per mouse or number of mice with adenomas. The response to
urethane was equivalent to the numbers predicted from numerous previous
studies indicating that the animals were sensitive to tumor induction
(Shimkin and Stoner, 1975). It was concluded that either the carcinogens
present in the test substances were very weak or that an insufficient
concentration reached the lungs to produce a positive result.
REFERENCES
Hinners, R.G., O.K. Burkart, M. Malanchuk and W.D. Wagner. Animal exposure
facility for diesel exhaust studies. Health Effects of Diesel Engine
Emissions: Proceedings of an International Symposium, Vol. 2: 681-
697, 1979.
Huisingh, J.L., R.L. Bradow, R.H. Jungers, B.D. Harris, R.B. Zweidinger,
K.M. Cushing, B.E. Gill and R.E. Albert. Mutagenic and carcinogenic
potency of extracts of diesel and related environmental emissions:
Study design, sample generation, collection and preparation. Health
Effects of Diesel Engine Emissions: Proceedings of an International
Symposium. Vol. 2: 788-800, 1979.
Patel, A.R. Preparation and monitoring of cigarette smoke condensate
samples. In Report No. 3, Toward less hazardous cigarettes. The third
set of experimental cigarettes, G.B. Gori, Ed., DHEW Publication No.
(NIH) 77-1280.
Shimkin, N.B. and G.D. Stoner. Lung tumors in mice: Application to
carcinogenesis bioassay. Adv. Cancer Res. 21:1-58, 1975.
-------
Table 1 - Strain A Mouse Data for Induction of
Adenomas by Environmental Mixtures
Percent of Av. Number
Group
Uninjected
Controls
DMSO +
5% EL620
Urethane
Nissan Diesel
Particulate
Nissan Diesel
Extract
Olds. Diesel
Extract
Cigarette Smoke
Condensate
Coke Oven
Roofing
Tar
Injected
Controls
DMSO +
5% EL620
Urethane
Nissan Diesel
Particulate
Nissan Diesel
Extract
Olds. Diesel
Extract
Cigarette Smoke
Condensate
Coke Oven
Roofing
Tar
Sex
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
K
F
Mice with
Tumors
40
47
40
47
100A
100A
40
20
73
55
37
-
59
54
54
31
41
44
13
26
44
26
95A
100A
27
32
33
50
33
-
30
21
31
65
44
22
of Tumors
per Mouse
0.6 + 0.2
0.6 + 0.2
0.9 + 0.5
0.7 + 0.2
22.5 * 1.9A
21.8 + 1.5A
0.4 + 0.1
0.5 _+ 0.1
1,4 + 0.3B
1.0 + 0.3
0.4 + 0.1
-
0.8 + 0.3
1.1 + 0.2
1.2 + 0.3
0.5 + 0.2
0.7 + 0.2
0.7 _+ 0.3
Experiment 1
0.2 + 0.1
0.4 +0.2
0.5 + 0.2
0.3 + 0.1
7.3 + 0.7A
11.3 + 0.9A
0.3 + 0.1
0.3 + 0.1
0.4 + 0.1
0.7 + 0.1
0.4 + 0.1
-
0.4 + 0.1
0.2+0.1
0.4 + 0.1
0.9 _+ 0 . 2A
0.7 + 0.2
0.3 _+ 0.1
Experiment 2
Percent
Surviving
75
85
50
75
75
85
33
33
50
67
63
-
73
80
87
87
73
47
100
100
80
95
100
96
68
42
60
71
69
-
77
80
83
89
91
91
Dose
-
-
0.05 ml/injection
20 mg/mouse
4 mg/injection
1 mg/injection
1 mg/injection
0.20 mg/injection
0.02 mg/injection
0.02 mg/injection
_
-
0.05 ml/injection
10 mg/mouse
2 mg/injection
1 mg/injection
1 mg/injection
0. 20 mg/injection
0.02 mg/injection
0.02 mg/injection
Significantly different from uninjected and injected controls (p<0.05).
Significantly different from uninjected controls and Olds, diesel extract
(p < 0.05).
-------
THE INFLUENCE OF INHALED DIESEL ENGINE EMISSIONS
UPON LUNG TUMOR INDUCTION IN STRAIN'A1 MICE
by
William E. Pepelko, John G. Orthoefer, W. Bruce Peirano,
Wai den Crocker, and Freda Truman
Health Effects Research Laboratory
U. S. Environmental Protection Agency
Cincinnati, Ohio
The Strain 'A1 mouse was one of several animal models chosen by the
Environmental Protection Agency to assess the carcinogenic risk of exposure
to diesel engine emissions. The Strain 'A' mouse was selected because: it
has been one of the most extensively used models for assessment of lung
tumor induction; the test is well validated; exposure times are fairly
short for a cancer assay; the test is relatively straightforward to perform
and evaluate and finally; it is one of the most sensitive lung tumor
bioassays available (1). The results presented are from experiments in
which mice were exposed to exhaust with a particulate concentration of 12
mg/m^. These studies are a continuation of earlier work in which exposure
levels were about one half those used in the present experiments (2).
Details of the exposure conditions and experimental procedures have
been published previously (2, 3). Briefly, Strain 'A' mice obtained from
Jackson or Strong Laboratories were exposed 8 hrs/day, 7 days/week from 6
weeks to either 9 or 12 months of age. The mice were housed in wire cages
and exposed in 100 cubic feet stainless steel chambers. Exhaust was
produced by a 6 cylinder, 90 cu inch Nissan diesel engine. In order to
simulate city driving conditions, the engine load and speed were varied
cyclically using the Federal Short cycle. After completion of exposure, the
animals were sacrificed and the lungs fixed and observed for the presence of
pulmonary adenomas.
Three experiments were carried out. In the first, 360 animals, 180 of
each sex, were exposed to clean air or diesel exhaust. One half of each
group received a single intraperitoneal injection of 5 mg urethane prior to
the start of exhaust exposure. In the second study, 115 males and 143
females were exposed to diesel exhaust, whi le 108 males and 142 females were
-------
exposed to clean air. Conditions differed from that of the first study in
that the exposure occurred during the dark portion of the daily cycle when
the animals were presumably awake, active and respiring at a greater level.
In the final study in which only males were used, the mice were sacrificed
at 12 months instead of 9 months of age. The number of mice with tumors were
compared using Chi Square testing. Results are shown in Table 1. Exposure
of mice to diesel exhaust until 9 months of age resulted in a significant
decrease in the mean tumor incidence in females, and in males and females
combined (P <.05). Decreases were also noted in males, but differences were
not significant. In comparing groups treated with an initiating dose of 5
mg urethane prior to exposure, a decreased tumor incidence was again noted
in the exhaust exposed mice, (P < .10) for males, (P <.001) for females,
and (P < .001) for males and females combined. Exposure to diesel exhaust
during the dark portion of the daily cycle also resulted in a decreased
tumor incidence compared with clean air controls (P < .01) for males, (P<
.001) for females, and (P< .001) for males and females combined. Finally,
exposure of males to diesel exhaust until 12 months of age again resulted in
a decrease in lung tumor incidence (P < .01).
Overall, there was no indication that exposure of Strain 'A' mice to
diesel engine emissions resulted in an increase in lung tumor incidence. On
the contrary, the studies consistently showed that tumor rates were
decreased in exhaust exposed mice. Such a decrease following exposure to a
potentially carcinogenic pollutant is rare, but is not unknown. Nettesheim
et al., (4) reported that inhalation of a combination of ozonized gasoline
and ferric oxide particles inhibited the tumorigenic effects of injected
diethylnitrosamine on the respiratory tract. Kotin and Folk (5) also showed
that exposure of C57BL mice for a lifetime to an atmosphere of ozonized
gasoline resulted in a significantly lower incidence of malignant lymphomas
and hepatomas compared to mice breathing clean air. Finally, Pereira (6)
found fewer gamma glutamyl transpeptidase positive liver islands in rats
following exposure to diesel exhaust than in clean air controls.
An explanation of the present results must await further study.
Possibly, diesel exhaust inhalation inhibits the induction of enzymes
responsible for converting procarcinogens to their active forms. The
immunocompetence of the animals could also have been altered as a result of
the inflammatory reaction to deposited exhaust particulate. The results
cannot be explained by increased mortality of mice susceptible to tumor
induction since survival rates were not significantly altered by the
exposure positions.
-------
REFERENCES
1. Shimkin, M.B., and C.D. Stoner. 1975. Lung Tumors In mice: Ap-
plication to Carcinogenesis Assay. Adv. Cancer. Res. 21:1-58.
2. Orthoefer, J.G., W.Moore, D. Kraemer, F. Truman, W. Crocker, and Y.Y.
Yang. 1980. Carcinogenicity of Diesel Exhaust as Tested in Strain 'A'
Mice. Presented at the U.S. Environmental Protection Agency Interna-
tional Symposium on Health Effects of Diesel Engine Emission. Cin-
cinnati, Ohio.
3. Hinners, R.G., J.K. Burkart, M. Malanchuk, and W.D. Wagner. 1980.
Facilities for Diesel Exhaust Studies. Presented at the U. S.
Environmental Protection Agency International Symposium on Health
Effects of Diesel Engine Emissions. Cincinnati, Ohio.
4. Nettesheim, P., D.A. Creasia, and T.J. Mitchell. 1975. Carcinogenic
and Co-Carcinogenic Effects of Inhaled Synthetic Smog and Ferric Oxide
particles. J. NCI. 55: 159-169.
5. Kotin, P. and H.L. Falk. 1956. The Experimental Induction of
Pulmonary Tumors and Changes in the Respiratory Epithelium in C57BL
Mice Following Their Exposure to an Atmosphere of Ozonized Gasoline.
Cancer. 11: 473-481.
6. Pereira, M.A., H. Shinozuka, and B. Lombardi. 1980. Test of Diesel
Exhaust Emissions In the Rat Liver Foci Assay. Presented at the U. S.
Environmental Protection Agency International Symposium on Health
Effects of Diesel Engine Emission. Cincinnati, Ohio.
-------
Table 1. Effects of Inhaled Diesel Engine Emissions Upon Lung Tumor
Incidence in Strain 'A' Mice
Number of Mice
with Tumors
Age
Treatment
Clean Air
Diesel Exhaust
at Sacrifice
(Months)
9
9
Illumination
During Exposure
Light
Light
Sex
M
F
M+F
M
F
M+F
Number of Surviving
Mice
10/44
11/43
21/87
5/37
4/43
9/80
P Values
NS
< .05
< .05
Clean Air
+
5 mg Urethane
Diesel Exhaust
+
5 mg Urethane
9
9
Light
Light
M
F
M+F
M
F
M+F
32/38
34/37
66/75
26/39
16/36
42/75
< .10
< .001
< .001
Clean Air
Diesel Exhaust
12
12
Light
Light
M
M
22/38
11/44
< .01
Clean Air
Diesel Exhaust
9
9
Dark
Dark
M
F
M+F
M
F
M+F
28/97 '
31/140
59/237
13/111
9/139
22/250
< .01
< .001
< .001
-------
OBJECTIVES AND EXPERIMENTAL CONDITIONS OF A
VW/AUDI DIESEL EXHAUST INHALATION STUDY
by
U. Heinrich, F. Pott and VI. Stb'ber
Fraunhofer-Institut fur Toxikologie und Aerosolforschung
Hannover, Federal Republic of Germany
H. Klingenberg
Volkswagenwerk AG, Forschungsabteilung MeBtechnik
Wolfsburg, Federal Republic of Germany
Extended Abstract
OBJECTIVES
The rationale of this project is to make a most sophisticated effort in-
to contesting the contention that, even under aggravated conditions, exposure
to automobile exhaust, particularly to emissions of a VW diesel engine, does
not produce irreversible long-term health effects and does not warrant emiss-
ion regulations resulting in very expensive motor vehicle technologies. There-
fore, this investigation is designed to reveal the existence of potential
toxic effects of the exhaust of a VW diesel engine in long-term inhalation
studies with rats, Syrian golden hamsters and mice. In particular, attention
will be focussed on the impact on lung functions and the incidence of lung
tumors. The study takes advantage of the results of an earlier investigation
on the long-term diesel exhaust inhalation exposure of Syrian golden hamsters
and is related to a program on the inhalation exposure to gasoline engine ex-
haust presently in progress.
EXHAUST GENERATION AND ANALYTICS
The exhaust is taken from a VW diesel engine on a test bench. The engine
is operated by a computer simulating continuously the US-72 (hot start) Fe-
deral Test Procedure (FTP) cycle. The exposure chamber air is sampled for the
analysis of a number of gaseous emission components as well as a variety of
polycyclic aromatic hydrocarbons (PAH) and the physical parameters and the
chemical composition of the airborne diesel soot particles.
-------
EXPERIMENTAL
Nine stainless steel exposure chambers of special design, sized 3x2x2
meters each (depth x width x height), were built into a new inhalation toxic-
ology facility. The design provides a horizontal ventilation of the chambers
by the diluted exhaust emissions. The total number of animals utilized in
the project is about 850 rats, 1150 Syrian golden hamsters and 1700 mice. The
rats and mice are held under barrier conditions, while the hamsters are kept
conventionally.
The exhaust inhalation exposure lasts 16 to 18 hours daily on 5 days per
week and is to be continued for about 2 years. In a range-finding test ex-
periment of 3 months, 4 different exhaust dilutions are applied and the maxi-
mum exhaust concentration that may not reduce the natural life expectancy of
the 3 animal species will be determined by means of some clinical-chemical
measurements and histological evaluations. If the species will respond dif-
ferently, correspondingly different exhaust concentrations will be employed.
During the long-term experiments, periodical tests are performed to pro-
vide data on the clinical chemistry of blood and liquids of lung lavages.
Furthermore, lung function tests are conducted on hamsters and rats.
The focus of the experimental design is on the question of a potential
carcinogenicity of the exhaust of this diesel engine. Since the chances of
inducing tumors in a straightforward exposure of healthy animals are rather
slim, some of the test animals will be pre-treated with either a nitrosamine
or a carcinogenic PAH similar to an earlier experimental design already de-
scribed elsewhere. This pre-treatment should produce a base line tumor in-
duction rate of about 20 percent so that a possibly existing disproportionate
additional effect on the tumor induction by the exhaust emissions stands a
better chance of statistical significance.
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PROJECTED HUMAN HEALTH RISKS FROM INCREASED USE OF DIESEL LIGHT DUTY
VEHICLES IN THE UNITED STATES
by
R. G. Cuddihy, R. 0. McClellan, W. C. Griffith,
F. A. Seiler and B. R. Scott
Lovelace Inhalation Toxicology Research Institute
P. 0. Box 5890
Albuquerque, NM 87185
Diesel light duty vehicles are expected to comprise 20% of the total
light duty vehicle fleet in the United States within 15 years. Their use
should grow because of a perceived advantage over gasoline engine vehicles in
fuel costs, but it will be limited by the capacity to produce sufficient
diesel fuel economically. To estimate the potential health risks from expo-
sures of people to diesel vehicle emissions in the environment, it is neces-
sary to identify any toxic components in their exhaust, to project future
levels of these pollutants in congested urban environments and to estimate
the potential magnitude of the related human health risks.
Chemical analyses of diesel emissions have identified potentially toxic
gases including nitrogen oxides and carbon monoxide along with particles that
contain known carcinogenic compounds including polyaromatic hydrocarbons and
nitroaromatics. However, other industrial and transportation sources of
these pollutants contribute substantially more to the environment than can be
projected from the increased use of diesel light duty vehicles. Therefore,
in well-mixed urban atmospheres little of the total health risk attributable
to inhaled pollutants could be related to the future use of light duty diesel
vehicles.
In congested urban areas with limited air circulation such as street
canyons and parking garages, vehicle exhausts may reach sufficiently high
levels to produce respiratory symptoms. Both gasoline engine vehicles and
diesel vehicles contribute to these problem areas, however, diesel exhaust
contains significantly higher concentrations of nitrogen oxides and parti-
cles. Empirical relationships between measured levels of carbon monoxide in
vehicle exhaust and its concentrations in urban street canyons have been
developed based upon studies near streets in New York, St. Louis and San
Jose. These relationships indicate that if diesels were to comprise 20% of
the total light duty vehicles, they may add 10 yg/m3 to the particle concen-
trations of air and more than 50 yg/m3 to the nitrogen oxide concentrations.
Other models indicate that in parking garages, diesel particle concentrations
could reach 200 yg/m3 and nitrogen oxide concentrations 1000 yg/m3 during
-------
periods of high vehicle activity. The average concentrations of diesel
particles over large city areas are expected to be between 1 and 2 yg/irn.
Diesel emissions of nitrogen oxides are not expected to produce measurable
changes in these ambient air concentrations.
The potential health effects of exposures to nitrogen oxides have been
summarized in two recent reports by the National Academy of Sciences and the
Electric Power Research Institute. In general, prolonged exposures to nitro-
gen dioxide above 100 yg/m3 can lead to chronic bronchitis in children and
adults, and above 150 yg/m3 respiratory function changes may occur. Expo-
sures to nitrogen dioxide above 1000 yg/m3 for several hours can lead to
acute metabolic and respiratory function changes. Therefore, people who work
in congested urban environments with restricted air circulation are likely to
be exposed to irritant levels of nitrogen oxides. This is especially true on
days with little sunlight when the chemical balance favors formation of
nitrogen dioxide over formation of nitric oxide. However, it should also be
noted that the contribution of gasoline engine vehicles to nitrogen oxides in
these atmospheres is also significant.
The potential health effects of diesel particle emissions are more
difficult to evaluate due to very limited observations of their effects on
people or laboratory animals. Because diesel vehicles are expected to add
less than 2 yg/m3 to urban environments, their emissions will not add sig-
nificantly to health risks that are solely related to the general levels of
particulate pollution.
Concern has also been expressed for health risks due to carcinogenic
organic compounds associated with diesel particles. Diesel particle extracts
have been shown to be mutagenic to cells in culture, to cause cell transfor-
mations and to induce tumors in the skin of rats. Further studies have also
shown that diesel particle extracts are not markedly different from extracts
or condensates of cigarette smoke, coke oven emissions, or urban soot in
their ability to cause these biological effects per unit mass. Therefore, we
summarized information on the exposures of these human populations to air-
borne particles and on their lung cancer incidences (Table 1).
Table 1. Summary of Population Exposures to Airborne Particles and Annual
Cancer Risks
Study
Population
Rural Nonsmokers
Urban Nonsmokers
Average Air
Concentration
mg Parti cles/m3
0.03
0.1
Annual Lung
Cancer Risk Per
100,000 People
3
7
Annual
Cancer Risk Per
100,000 People
Per mg/m3
100
70
Smokers (cigarettes/day)
1 - 9 2-16 26 3
10 - 19 18-35 47 2
20 - 39 36-71 80 2
40 + 73+ 107 1
Coke Oven Workers 3 400 130
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By assuming that all of the lung cancers in these populations were caused by
their exposures to the particles, we obtained an upper estimate of lung
cancer risk that would be expected in people exposed to diesel exhaust par-
ticles. The risk estimator was taken to be 0.0015 cancers per year per mg/m3
lifetime exposure to diesel particles. Combining this risk factor with
projected air concentrations of diesel particles in urban environments in
future years, we estimated that less than 30 lung cancers per year could be
related to the use of diesel light duty vehicles in the United States.
Two additional analyses of the carcinogenic risks from exposures to
diesel emissions became available during 1981. The first analysis was con-
tained in a report to the Diesel Impacts Study Committee of the National
Research Council (1). This analysis was mainly based upon studies of London
bus garage workers who were exposed to high levels of diesel engine exhausts
between 1930 and 1974. Their lung cancer incidences were compared to other
transit workers including engineers, bus drivers, conductors and subway
motormen. Although the studies failed to show a definite increased lung
cancer risk in the garage workers, based upon statistical considerations,
Harris calculated an upper limit for their lung cancer risks. The upper 95%
confidence limit on the increased lung cancer risk was 1% per yg/m3 particu-
late exposures.
A second study was completed by DuMouchel and Harris (2) that estimated
lung cancer risks from diesel emissions based upon laboratory studies of
mutagenesis and viral cell transformations produced by diesel particle ex-
tracts. The relative potency of the diesel particle extracts in these test
cell systems was estimated and compared to roofing tar vapors and coke oven
emissions. The results of epidemiology studies were also used to estimate
the absolute lung cancer risks per unit of exposure to roofing tars and coke
oven emissions. By this technique DuMouchel and Harris (2) estimated an
upper 95% confidence limit for exposures of people to diesel particles at
1.8% increase in lifetime lung cancer risks per yg of particles/m3 of expo-
sure. As shown in Table 2, all of these estimates of lung cancer risk to
people exposed to diesel emissions are reasonably similar.
Table 2. Estimates of the Proportional Increased Risk of Lung Cancer Using
Three Approaches
Data Sets Used
Proportional Increased
Risk of Lung Cancer Per
yg/m3 Particulate
Reference
Cigarette Smokers and
Coke Oven Workers
London Garage Workers
London Garage Workers,
Viral Cell Transformations
and Salmonella Tester
Strains
0.5%*
1.0%**
1.8%**
Cuddihy et al_. (3)
Harris (2J
DuMouchel and Harris
(2)
*Largest estimate
**Upper 95% confidence limit
-------
ACKNOWLEDGEMENTS
Research performed under U.S. Department of Energy Contract No. DE-AC04-
76EV01013 and conducted in facilities fully accredited by the American Asso-
ciation for Accreditation of Laboratory Animal Care.
REFERENCES
1. Harris, J. E. 1981. Potential risk of lung cancer from diesel engine
emissions. Report to the Diesel Impacts Study Committee, Assembly of
Engineering, National Research Council, National Academy Press, Washing-
ton, DC.
2. DuMouchel, W. H. and 0. E. Harris. 1981. Bayes and empirical bayes
methods for combining cancer experiments in man and other speices.
Technical Report No. 24, Department of Mathematics, Massachusetts
Institute of Technology.
3. Cuddihy, R. G., F. A. Seiler, W. C. Griffith, B. R. Scott and R. 0.
McClellan. 1980. Potential health and environmental effects of diesel
light duty vehicles. Lovelace Inhalation Toxicology Research Institute
Report LMF-82, UC-48.
-------
HEALTH EFFECTS OF EXPOSURE TO DIESEL FUMES AND DUST IN TWO TRONA MINES
by
M.D. Attfield
Appalachian Laboratories
National Institute of Occupational Safety and Health
Morgantown, West Virginia
The industrial environment is often a useful situation in which to study
the effect of health hazards, as workers usually receive higher exposures than
does the general population. This is particularly so for miners exposed to
diesel fumes underground, since the restriction on ventilation acts to
concentrate the fumes. This study involves 700 workers engaged in trona
(Na2C03 NaHC03 2H20) mining. These miners were given chest radiographs,
asked questions on chest symptoms, smoking and work history, and given
spirometric tests. In addition, comprehensive industrial hygiene surveys were
undertaken at the two mines which were studied. The data available from these
surveys is being explored for dose-response relationships between health
indices and measures of diesel-engine-related pollutants. This paper will
report the results.
-------
MUTAGENICITY AND CHEMICAL CHARACTERISTICS OF CARBONACEOUS
PARTICULATE MATTER FROM VEHICLES ON THE ROAD
by
Hilliam R. Pierson, Robert A. Gorse, Jr., Ann Cuneo Szkarlat,
Wanda W. Brachaczek, Steven M. Japar, and Frank S.-C. Lee*
Engineering & Research Staff - Research
Ford Motor Company
P.O. Box 2053
Dearborn, Michigan 48121
Roy B. Zweidinger and Larry D. Claxton
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
Two experiments were conducted in the eastbound tunnel of the Allegheny
Mountain Tunnel of the Pennsylvania Turnpike in 1979 to evaluate the bacterial
mutagenicity of the organic solvent extracts of particulate emissions from
heavy-duty Diesels and from (predominantly light-duty) gasoline-powered vehi-
cles in highway operation. Filter samples (PTFE-Tefl on-impregnated glass
fiber and PTFE membrane) collected during periods dominated by Diesel traffic
as well as periods dominated by gasoline-powered vehicles were Soxhlet-extract-
ed with dichloromethane (CH2C12) followed by acetonitrile (CHsCN). Concur-
rently collected ambient-air samples (in the ventilation intake fan rooms and
at a tower on the mountaintop) were treated the same way in order to distin-
guish between vehicle and ambient contributions to the mutagenic activity of
the tunnel samples, and also to compare mutagenic activity of vehicle and
ambient particulate-matter extracts. Total tunnel air flow, traffic volume,
and traffic composition were monitored to permit calculation of emission rates
per unit distance driven, for Diesels and for gasoline-powered vehicles (e.g.,
Fig. 1).
Mutagenicity was determined by the Salmonella typhimurium plate incorpo-
ration assay [Ames test (1)] using several tester strains, with and without
microsomal activation by S9 rat-liver homogenate. The number of revertant
colonies per km travelled was calculated for each sampling run and plotted
against traffic composition (e.g., Fig. 2) to obtain revertants/km averages
for gasoline- and Diesel -powered vehicles separately. High performance
liquid chromatography (HPLC) profiles were obtained on the CH2C12 and
*Present address: Amoco Research Center, Standard Oil Company of Indiana,
P.O. Box 400, Naperville, Illinois 60566
-------
extracts. Gas-chromatographic (GC) molecular-weight distributions (retention-
time distributions) were obtained on the CH2C12 extracts and resolved as above
according to vehicle type.
The main findings are as follows:
(1) The Diesel-produced aerosol in the Allegheny Tunnel is similar to that
encountered in dilution tubes, with respect to all criteria, viz., per-
centage extractable into CH2C12 (24 +_ 3%), Ames mutagenicity in revertants
per km travelled or revertants per yg of CH2Cl2-extracted material, HPLC
fluorescence profile, and molecular-weight distribution.
(2) Expressed as revertants per vg of CH2Cl2-extracted material, the mutagenic
activities of the Diesel-produced aerosol in the Allegheny Tunnel are of
the same order of magnitude as the mutagenic activities of the ambient
aerosol in the vicinity at Allegheny.
(3) Expressed as revertants per km travelled, the mutagenicity of the CH2C12
extract of the particulate emissions from Diesels is several times that
from gasoline-powered vehicles.
Some of the mutagenicity results are summarized in Tables 1 and 2.
REFERENCES
Ames, B. N., J. McCann, and E. Yamasaki, 1975. Methods for determining
carcinogens as mutagens with the Salmonella/mammalian microsome muta-
genicity test. Mutation Research 31:347-364.
-------
Table 1. Mutagenicities, Thousands of TA98 Revertants per Kilometer
Travelled;CH2C12 Extracts, Allegheny Mountain Tunnel 1979.
Without S9
With S9
May/ June
Aug/Sept
May/June
Aug/Sept
Gasol ine-
powered
Vehicles
39+24b
19+10
26+14
12+4
Diesel
Trucks
(a)
211+113
80+20
181+40
51+7
Average gross weight approximately 35 tons.
Error quoted is the standard deviation.
Table 2. Mutagenicities, TA98 Revertants per Microgram of
CH2Cl2-extracted Material, Allegheny Mountain Tunnel
1979.
Without S9
With S9
May/June
Aug/Sept
May/ June
Aug/Sept
Gasoline-
powered
3+2
4+3
2+1
2.4+1.6
Vehicles
Diesel
Trucks
1.1+0.6
0.4+0.1
0.9+0.2
0.27+0.04
Ambient Air
Over-all
1 .3
0.6
1.0
0.4
Fan
Rooms
0.9
0.6
0.4
0.4
Tower
0.2
0.08
-------
200
180
160
140
120
100
80
60
40
20
y 186.2-1.845-% gasoline
r--0.988
Spork-igniliwi'4.8± 2.8mg/km
Diesel!-18616 mg/km
0 10 20 30 40 50 60 70 80 90 100
% gasoline-powered (s|00-X)
Figure 1. Plot of mg/km emission rate of CH2Cl2-extractable particu-
late matter vs. traffic composition, Allegheny Mountain
Tunnel August/September 1979. Intercept at 0% gasoline-
powered vehicles is the emission rate (186_+ mg/km) from
Diesels.
60000
50000
^ 40000
^
V
a.
~ 30000
o
> 20000
V
or
10000
y = 51133-402.95'% gasoline
r= - 0. 868
SparK-ignition =(l I.7±3.8)xl0 rev/km
Diesels =(51.1 ±7.5)x|03 rev/km
I
J_
0 10 20 30 40 50 60 70 80 90 100
% gasoline-powered (siOO-X)
Figure 2. Plot of revertants/km vs. traffic composition, CH2C12 ex-
tracts, tester strain TA98 with microsomal activation (+S9),
Allegheny Mountain Tunnel August/September 1979.
-------
EMISSIONS OF GASES AND PARTICULATES FROM DIESEL TRUCKS ON THE ROAD (2)
by
Raisaku Kiyoura
Research Institute of Environmental Science
4, 5-Chome, Kojimachi, Chiyoda-ku
Tokyo, JAPAN
(A) Experiments have been conducted to estimate the diesel trucks emission
rates at the Nihonzaka Tunnel. The Tunnel is a 4-1ane dual tunnel (2 eastbound
lanes through one tube, 2 westbound lanes through the other), ?. km long, on a
slight grade upward (2.5%, 1.2 km), then downward (-1.84ft, 0.8 km toward the
east), located 170 km west of Tokyo. The vehicle traffic in the tunnel is very
high with an average 1,107 - 1,148 cars/h; the percent of diesel trucks is
30 - 85%. Magnetic counts of eastbound traffic are by car length of 6 ± 0.5 m.
(Over 6 m is almost diesel-truck.) The 90% of the diesel trucks is 6.5 - 22 t
car weight. The 53% is 10 - 22 t. Intake fans above each of the tunnels force
ventilation air into the tunnel through overhead louvers at 303 m3/s. Air is
drawn in also through the vehicle entrance portal by the ramming action of the
traffic. All of the air leaves via the vehicle exit portal, at volumes
averaging 380 m3/s. Truck speed was 80 km/h. Sulphur content of fuel oil was
0.4%. Measurement procedures are almost similar to the Allegheny tunnel study
by William R. Pierson and Wanda W. Brachaczek (1). The preliminary study was
done in 1972; the present study was started in 1979, and will continue to 1981.
(B) Average emissions rates of diesel truck were found as in Table 1 and
Table 2. Particulates are ~0.03 \m by electronmicroscope.
(C) The overall sulphur dioxide conversion to sulphate in the Tunnel was
2% (1980), 3% (1979). The measurements of the ambient are on the way.
Sulfuric acid particulates of 2 - 10 micron spheres were observed on the thymol
blue dye coated films exposed in the ambient 40 meters distant from the Tunnel
portal, when relative humidity was +90%.
REFERENCES
1. Pierson, W.R., and W.W. Brachaczek. Particulate matter associated with
vehicles on the road. Automotive Engineering Congress and Exposition,
Detroit, MI, Feb. 23-27, 1976. Paper No. 760039.
-------
Table 1. Diesel Gaseous Emission Rates on the Road (g/km)a
Year
NO
NO?
NOX
CO
S02
T-HC
CH4
NM-HC
1980-Oct.
6.42 ± 9.7%
0.62 ± 15 %
7.03 ± 8.8%
5.04 ±11 %
1.27 ± 20 %
1.73 ± 9.7%
1.08 ± 9.2%
0.63 ± 17 %
1979-Oct.
5.02 ± 7.4%
0.77 ± 6.5%
5.79 ± 7.3%
1.14 + 27 %
--
aln 1980, numbers of measurements: n=36.
In case of S02 measurement, n=12. Pearson's correlation coefficient, p < 0.05.
In 1979, n=24. In case of S02 measurement, n=8, p < 0.05.
Table 2. Diesel Particulates Emission Rates on the Road (g/km)
Year
Total particulatesa
Sulphate particulates
Nitrate particulates
Ammoniate particulates
1980-Oct..
1.03 ± 4.9%
0.041 ± 25 %
0.003 ± 37 %
0.005 ± 44 %
1979-Oct.
0.92 ± 5.4%
0.051 ± 19 %
0.003 ± 15 %
0.004 ± 33 %
aParticulates of under -10 pm were measured by high-volume air sampler.
In 1980, numbers of measurement: n=36. Pearson's correlation coefficient,
p < 0.05.
In 1979, numbers of measurement: n=24, p < 0.05.
-------
DIESEL BUS TERMINAL STUDY
EFFECTS OF DIESEL EMISSIONS ON AIR POLLUTANT LEVELS
BY
Robert M. Burton, Robert Jungers, Jack Suggs
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
INTRODUCTION
The New York City Bus Terminal Diesel Study was designed to measure,
collect, chemically characterize, and bioassay diesel exhaust as it exists
after becoming resident in the ambient atmosphere. Concentration levels of
size fractionated particle mass, organic vapors and inorganic vapors were
determined. Gram amounts of size fractionated particle samples were col-
lected for detailed chemical analysis and bioassay (1) screening.
STUDY DESIGN
A semi-enclosed area of the NYC bus terminal in which approximately
1400 buses operate daily was used for the diesel exhaust collection site
(indoor, Site #1); a second site (outdoor, Site #2) on 9th Avenue upwind
of the terminal, was used as a particle and organic vapor background sampl-
ing and collection site. The background site was used for ambient control
and for comparative chemical and p-.ysical characterization of the terminal
diesel enriched pollutants and the unaffected outdoor pollutants. Back-
ground inorganic gaseous data were obtained from nearby ambient monitoring
stations which were also unaffected by the terminal pollutants.
The terminal site was selected so that the majority of the ambient
pollutant loading would be specifically concentrated diesel exhaust origi-
nating from diesel vehicles operating in a specified area. Street canyons
have the disadvantage of being traffic pattern and meteorology dependent
and are confounded by stationary sources and gasoline powered vehicles.
The traffic count of buses passing by the indoor site remained
consistent throughout the study with the weekday daily count averaging
1,415 (1,336 minimum; 1,440 maximum) and the weekend daily count averaging
615 (579 minimum; 643 maximum). Monitoring and sample collection began
July 16, 1979 and ran daily on a 24-hour schedule through July 30, 1979.
-------
TSP hi vol (2), Dichotomous (2), and massive air volume (3) particle
samples were collected; volatile organic compounds were collected on Tenax
cartridges; and primary pollutant inorganic vapors were measured by con-
tinuous sensors.
The Tradescantia plant system (4) developed by Brookhaven National
Laboratories for on-site detection of toxic air pollutants, was also
operated at the indoor site. The Tradescantia system allows ambient air
to be screened for the presence of mutagenic chemical vapors.
The majority of buses using the terminal were equipped with Detroit
two-stroke heavy diesel engines.
RESULTS AND DISCUSSION
Results of aerometric measurements reveal that some pollutants were
elevated considerably at the indoor site, while others there remained at
background level. Particle measurements revealed very high levels of fine
particulate matter being generated in the terminal building.
The average Total Suspended Particulate (TSP) 24-hour average level in
the terminal was 325 yg/m while the same TSP outdoors was 120 yg/m .
Dichotomous measurements gave a 240 yg/m3 24-hour average for indoor parti-
cles less than 2.5 microns in diameter compared to 60 yg/m at outdoor
Site #2. The 24-hour average coarse fraction of the dichtomous sample
(2.5-15 microns) averaged 46 yg/m indoors and 20 yg/m outdoors. Weekend
particle mass levels dropped considerably more indoors than outdoors. Re-
sults of 24-hour TSP sulfate, nitrate, and lead levels indoors were at the
same approximate concentration as at background Site #2. The outdoor levels
were influencing the indoor air for the three pollutants. Ratios of sulf-
ate, nitrate, and lead to TSP were all lower indoors than at the outdoor
background site.
The massive air volume size fractionating particle collectors were
operated at both sites throughout the study. The amount of material
collected is shown in Table 1.
Like the size distribution of the particle mass collected by the
dichotomous samplers, the massive volume samplers show the majority of
the elevated particulate mass at the indoor site to be aerosol in the
fine size range.
Comparison of Daily Maximum Hourly Averages for the Gaseous Primary Pol-
lutants
Real time continuous measurements of the gaseous primary pollutants
revealed peaks to occur at 8.a.m. and 5 p.m. at the indoor site each day
when bus traffic was at a maximum. More variation in maximum hourly
averages occured at the indoor site than outdoors for all gases measured.
A summary of gaseous pollutant measurements follows:
-------
Sulfur dioxide (SOp). Based on 15-day averages, there was no difference
(statistically) between indoor and outdoor maximum hourly averages. Both the
Mable Dean Bacon School (outdoor) site and the Central Park (outdoor) site
SOp levels are equivalent to those found inside the terminal, thus indicat-
ing very low SCL emissions from the buses inside the terminal. There is no
significant difference between weekday and weekend maximum hourly values
inside the terminal.
Nitrogen dioxide (NOp). For NOp levels, the data indicate more varia-
tion in maximum hourly averages for the indoor site than outdoors. The
maximum hourly average indoor values (mean of 1.36 ppm) are on the average
significantly higher than those for outdoors (mean of .09 ppm) based on
15-day average of maximum hourly values. Weekend maximum hourly values
indoors are significantly lower compared to weekdays. The N02 mean of 1.36
ppm is 10 times higher than the maximum 24-hour level of the Rational Ambi-
ent Air Quality Standard.
Nitric oxide (NO). NO maximum hourly values indoors throughout the
study were significantly much higher than those outdoors. This is consist-
ent with other diesel exhaust products measurements where NO has been shown
to be emitted at high concentration levels.
Ozone (Op). There were no detectable 03 levels indoors at the bus
terminal during the study. Outdoors, the maximum hourly 03 values ranged
from 0.0 to .12 ppm with an average of .04 ppm over the sample period. With
the NO levels exceeding 7 ppm and N02 levels exceeding 1.0 ppm, it is safe
to assume all of the 03 at indoor Site #1 was reacting with NO to form N02.
Carbon monoxide (CO). There was significantly more day-to-day variation
in the maximum hourly CO values for the indoor site as compared to the out-
door site. This was apparently due to lower weekend values for indoor Site
#1 maximum hourly averages. Averaged over the sampling period, the maximum
hourly average for indoors (17.79 ppm) was significantly higher than the
outdoor 10-day average of 2.6 ppm.
Total hydrocarbons (THC). No ambient THC data were available for com-
parison with indoor Site #1 levels. Peak hourly maximum averages for in-
door Site #1 was at the 10 ppm level.
Comparison of Indoor Diurnal Patterns for the Gaseous Primary Pollutants
The indoor generated gaseous pollutants were also analyzed for diurnal
variation of concentration. Weekday (Monday-Friday) hourly averages and
standard deviations for the study period were computed; weekend (Saturday-
Sunday) hourly averages with standard deviations were also computed. As
described below for each gaseous pollutant, the diurnal concentration levels
for weekdays (1400 buses) were always considerably higher than for weekends
(600 buses) for all gaseous pollutants except SOp.
Sulfur dioxide (SOp). Indoor SOp diurnal patterns are similar for
both weekends and weekdays. Both were influenced by peak hour traffic
-------
activity occurring at 8 a.m. and 5 p.m. each day. The indoor average 5
p.m. value was significantly higher during the weekday as compared to week-
end levels. The ambient background SCL levels did not experience 8 a.m.
and 5 p.m. peaks. As mentioned earlier, there were no significant differ-
ences between indoor and outdoor peak daily hourly averages for the dura-
tion of the study.
Nitrogen dioxide (NO,,). Diurnal patterns indicate significantly
higher average levels for weekdays compared to weekends for the 8 a.m. and
5 p.m. indoor hourly averages. For weekdays the hourly averages range from
.06 ppm during early morning hours (3-4 a.m.) to 1.55 ppm during 5 p.m.
rush hour. For weekends the hourly averages range from .08 ppm during
morning hours (3-5 a.m.) to 0.45 ppm during 5 p.m. averaging time. Had
more ozone been present in the terminal, much higher N02 levels may have
been expected.
Nitric oxide (NO). Trends indicate a higher indoor NO average level
during peak hours 8 a.m. and 5 p.m. for weekdays compared to weekends. The
values for weekday peaks were beyond the range of the instrument but are
estimated to be approximately 10 ppm.
Carbon monoxide (CO). Indoor weekday 8 a.m. and 5 p.m. hourly aver-
ages were significantly higher than weekend averages during the same aver-
aging times for weekends. During weekdays, hourly averages were slightly
elevated compared to weekend averages for hours before 8 a.m. to after 5
p.m. This cannot be concluded about other gaseous pollutants examined in
the study. The differences for CO were not significant on an hour-by-hour
basis during this time period, even though the hourly averages at 8 a.m.
and 6 p.m. were elevated above the rest of the hours in the day.
Total hydrocarbons (THC). Diurnal patterns indicate elevations in
indoor hourly averages during rush hour activity (8 a.m. to 5 p.m.) for
weekday measurements. The 5 p.m. measurement averaged 10.3 ppm during
weekdays and is significantly higher than the weekend average of 3.6 ppm.
Effect of Buses on Particle Levels
A statistical analysis for describing the relationship between the bus
activity and particulate levels both indoors and outdoors was performed. A
paired t-test was used to statistically examine the difference between the
indoor and outdoor sites for TSP, dichotomous (fine, coarse, and total),
sulfate, nitrate, and lead. The inside measurements were on the average
significantly higher (= = .05) than outside for TSP, (0-2.5y) and (0-15y)
particles. These were the only significant differences. There was np_
significant difference between indoor and outdoor (2.5-15p) coarse fraction
particles.
Correlation coefficients between daily bus activity and pollutant
levels were calculated. Several correlations were determined to be signifi-
cantly different from zero. These were specifically TSP indoors .84; (0-
-------
2.5) indoors, -.74; (0-15y) indoors, .87; sulfate indoors, -.65_; sulfate
outdoors, -.65.
An important observation is that the bus activity does not correlate
very highly with indoor sulfate, nitrate, and lead. Indoor levels of
these pollutants are essentially the same as outdoors. Obviously the
bus emissions contribution to sulfate, nitrate, and lead are lower than
levels of these pollutants already resident in the atmosphere.
CONCLUSIONS
Sulfate, nitrate, and lead emissions from the buses were at a low
level. Sulfur dioxide from the bus emissions were also at a low level,
since no significant difference between the indoor and outdoor S02 levels
was found. Small particles below 2.5y aerodynamic diameter, and the gaseous
pollutants of NO, N02, THC, and CO were all emitted at high levels from the
buses. The indoor site was somewhat shielded from ultraviolet radiation,
and its absence could have an effect on the organic exhaust products found
in the atmosphere (5). Ozone was below detectable limits due to its use
in the production of NO.
-------
TABLE 1. Amount of Size-Separated Ambient Particles Collected
by the Massive Air Volume Sampler
STAGE I (20-3.5y) Stage II (3.5-1.7y) Stage III (1.7-Op)
Site #1 7.67 gm 1.72 gm 61.89 gm
Indoors
Site #2 6.06 gm 1.18 gm 14.68 gm
Outdoors
-------
REFERENCES
1. Huisingh, J. et. al. "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, et. al. eds. Plenum Press, New York, 1979.
2. Rodes, C. "Inhalable Particulate Network Operations and Quality
Assurance Manual," U.S. EPA Office of Research and Development,
Environmental Monitoring Systems Laboratory, Research Triangle Park,
N.C. 27711, May 1980.
3. Mitchell, R.I., et. al. "Massive Volume Sampler for Gram Quantities
of Respirable Aerosols." APCA Proceedings, June 22-24, 1977, Toron-
to, Canada.
4. Schairer, L.A., et. al. "Measurement of Biological Activity of Ambi-
ent Air Mixtures Using a Mobile Laboratory for JJT^ Situ Exposures:
Preliminary Results from the Tradescantia Plant Test System."
pp. 419-440 in Application.
5. Claxton, L. and H.M. Barnes. "The Mutagenicity of Diesel Exhaust
Exposed to Smog Chamber Conditions as Shown by Salmonella Typhimu-
rium," submitted to Mutation Research for publication.
-------
DIESEL BUS TERMINAL STUDY:
CHARACTERIZATION OF VOLATILE AND PARTICLE BOUND ORGANICS
by
Robert H. Jungers and Joseph E. Bumgarner
U. S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
Charles M. Sparacino and Edo D. Pellizzari
Research Triangle Institute
Research Triangle Park, North Carolina 27709
The New York City Bus Terminal was selected as a unique source of heavy
duty diesel bus engines. To evaluate emissions from these bus engines in
comparison to ambient air a semi-enclosed site was selected inside the ter-
minal where approximately 1400 diesel buses operate daily and a second site
was selected outside and upwind of the terminal. Volatile organic compounds
were collected on Tenax cartridges, recovered by thermal desorption and
introduced into a high resolution gas chromatographic column for separation.
Characterization and quantification of these compounds were accomplished by
mass spectrometry measuring total ion current and mass fragmentography.
Volatile chemicals selected for quantitation were benzene; toluene; xylenes;
ethylbenzene; 1,1,1-trichloroethane; trichloroethylene; tetrachloroethylene;
benzaldehyde; n-octane; and n-hepane. These were selected to represent
chemicals associated with diesel engines, i.e., octane, heptane, benzalde-
hyde, chemicals not associated with diesels, i.e., chlorinated hydrocarbons
and general aromatic chemicals which could be found in the atmosphere.
The quantitation of the volatile organic compounds collected over a
two-week period showed that the chemicals which had consistently higher con-
centration inside were n-octane, n-heptane, 1,1,1-trichloroethane, xylenes,
and toluene. The chemical which was higher outside was tetrachloroethylene
while benzaldehyde, trichloroethylene, benzene, and ethylbenzene were about
the same concentration. Week day (1400 buses/day) concentrations were higher
for all ten chemicals than on weekends (600 buses/day).
Air particles were collected inside and outside (at the same site as the
volatile samplers). Total suspended particulate (TSP) measurement was done
by the standard Hi-Volume sampler method (1) and size fractionated particles
were collected using a massive air volume sampler (MAVS) which separates the
particles into three size ranges. Table 1 presents data on samplers, mass,
organic extractable and benzo-orpyrene analysis.
-------
The smallest size range (1.7 urn and below) of all samples collected
inside the terminal were combined into a single sample. This procedure was
also followed for the outside samples. This was done to insure sufficient
quantity of sample for chemical and biological analysis.
The air particle concentration, organic concentration and the percent
organic extractables were, generally, considerably higher inside than outside
the terminal while the benzo-orpyrene concentration was higher outside than
inside the terminal.
The air particle samples were subjected to a fractionation procedure to
yield six fractions of various chemical properties and polarities. The acid
fraction contains both weak (e.g., phenols) and strong (e.g., carboxylic
acids) acids. The base fraction contains organic, Bronsted bases (e.g.,
amines). The neutral fraction is subdivided into three main fractions based
on compound polarity. The non-polar neutral (NPN) fraction is comprised of
compounds less polar than * naphthalene. Paraffinic materials are charac-
teristic of this fraction. The PNA fraction contains compounds of intermedi-
ate polarity, and is selective for condensed ring aromatics. All neutral
materials with polarities greater than PNA hydrocarbons are found in the
polar neutral (PN) fraction. Prior to the subfractionation of the neutral
fraction, the latter must be dissolved in cyclohexane. All components are
not soluble in this solvent. The insoluble material is collected as a sepa-
rate fraction (CI), and is comprised of intermediate and highly polar com-
pounds.
Spillover of various compounds into all fractions is a natural feature
of solvent partitioning processes. Polar neutral material was removed from
the PNA fraction by silica gel chromatography. The PNAs were chromatographed
utilizing HPLC such that a fraction containing only PNA hydrocarbons was
obtained (PNA-1). Other fractions (PNA 2-4) were collected that contained
compounds of intermediate to high polarity.
Most fractions were directly analyzed by capillary GC/MS. The fractions
enriched in polynuclear aromatic hydrocarbons (PNAs) were further purified by
column chromatography, and the collected subfractions were analyzed by GC/MS.
A portion of each sample, after fractionation, was prepared for bioassay by
removal of the fractionating solvent and addition of dimethylsulfoxide
(DMSO).
Comparison of the mass distribution of each chemical fraction inside and
outside the bus terminal showed several significant differences. The organ-
ics from the outside air contained a higher percent mass of one of the PNA
subfractions (PNA-3), the polar neutral fraction as well as the acids and
bases. The non polar neutral fraction was present at a higher percent mass
inside the bus terminal. This appears to be due to higher concentrations of
alkanes from unburned fuel. Bioassay analysis of the non polar neutral
fraction (2) suggests that this fraction may contain substantial amounts of
polynuclear aromatic hydrocarbons.
-------
REFERENCES
Code of Federal Regulations. 1980. Title 40, part 50, appendix B.
Reference Method for Determination of Suspended Particulates in the
Atmosphere (High Volume Method). General Service Administration:
Washington, DC. pp. 531-535.
Huisingh, J. , R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S.
Tejada, J. Bumgarner, F. Duffield, V.F. Simmon, C. Hare, C.
Rodriguez, L. Snow, and M. Waters. 1978. Application of bioassay
to the characterization of diesel particle emissions. Part I.
Characterization of Heavy Duty Diesel Particle Emissions. Part II.
Application of a mutagenicity bioassay to monitoring light duty
diesel particle emissions. 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 L. Claxton,
eds. Plenum Press: New York. pp. 381-418.
Table 1
SAMPLER
particle
size range
Hi-Vol
(0-50 urn)
MAVS I
(3.5-20 urn)
MAVS II
(1.7-3.5 Mm)
MAVS III
(0-1.7 urn)
Particle
Concentration
ug Parti cle/M3
I
325.0
58.5
6.5
214.5
0
120.0
24.0
6.0
66.0
Organic
Concentration
ug Organics/M3
I
14.95
7.37
0.84
60.49
0
4.08
0.98
0.31
9.57
% Organic
Extractable
ug Organics/
100 ug Particle
I
4.6
12.6
13.0
28.2
0
3.4
4.1
5.2
14.5
BaP
ug BaP/g
Particle
I
15.4
5.9
11.9
0.8
0
32.0
25.2
34.3
5.9
I = Inside
0 = Outside
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DIESEL BUS TERMINAL STUDY: MUTAGENICITY OF THE PARTICLE-ROUND ORGANICS AND
ORGANIC FRACTIONS
by
Joellen Lewtas, Ann Austin, and Larry Claxton
Health Effects Research Laboratory
U.S. Environmental Protection Aaency
Research Triangle Park, North Carolina
Emission testing of both heavy-duty diesel engines and light-duty diesel
cars using tunnel dilution and filtration to collect diesel particles has shown
the organics associated with these collected particles to be mutagenic in the
Ames Salmonella typhimurium assay (1). The mutagenicity of these organics has
been shown to be dependent on fuel quality (2) as well as engine type (3). The
organics associated with particle emissions from heavy-duty diesel engines have
generally been less mutagenic than the organics from light-duty cars.
Fractionation and bioassay studies suggest this is due to a greater
concentration of nonmutagenic aliphatic compounds emitted from unburned fuel.
Over 90% of the mutagenic activity has been observed in the polar neutral
fractions not requiring metabolic activation (1). Mutagenicity studies of the
organics associated with urban ambient air particles have also reported
mutagenic activity in the organics extracted from particles (4,5).
In order to evaluate the impact of emissions from heavy-duty buses on the
mutagenicity of ambient air, this study was designed to compare the mutagenic
activity of the total extractable organics from size-fractionated air particles
and chemical class fractions both inside and outside a diesel bus terminal.
The mutagenicity of the ambient air inside and outside the New York Port
Authority Bus Terminal was compared using a microbial mutagenesis bioassay.
Approximately 1400 diesel buses operate daily in the semi-enclosed bus
terminal. Air particles were collected simultaneously using both the Massive
Air Volume Sampler (MAVS) (6) and the standard Hi-Volume air sampler (Hi-Vol).
The dichloromethane-extractable organics from these air particles were
bioassayed in the Salmonella typhimurium plate incorporation assay (7) in TA98
with and without metabolic activation with minor modifications (3). The slope
of the dose-response curve (rev/pg) was determined using a nonlinear model (8).
The air particle concentration inside the bus terminal was nearly 3 times the
outside concentration based on the Hi-Vol TSP. Comparison of the Hi-Vol and
MAVS data showed the increased concentration of particles inside was due
primarily to increased concentrations of particles less than 1.7 micron in
size. These small (less than 1.7 micron) particles inside the terminal had a
higher concentration of extractable organics than the small particles outside
the terminal. Although both the small particle and organic concentrations
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were lower outside the terminal, the mutagenicity of the organics from outside
was significantly greater (nearly 10 times) than inside the terminal. Both
direct-acting and indirect-acting mutagens were detected in these samples. The
mutagenic activity of the air in revertants per cubic meter provides a direct
comparison of the mutagenicity of the inside and outside air. Using data from
either the Hi-Vol samples or the smallest particles from the MAVS, the outside
air was approximately twice as mutagenic as the air inside the bus terminal.
Fractionation and mutagenesis bioassay of the organics from the less-than-
1.7-micron particles were conducted to compare the chemical composition inside
and outside. The mutagenicity (rev/yg) of each fraction and the mass
percentage of each fraction were used to calculate weighted mutagenicities.
The percent of the total mutagenicity attributable to each chemical fraction
was determined and compared inside and outside the terminal. The diesel
emissions inside the bus terminal increased the concentration of aliphatic
hydrocarbons found in the non-polar neutral fraction. The higher mutagenicity
in the outside ambient air appears to be due to higher concentrations of
organic acids and direct-acting moderately polar neutral compounds. The highly
polar neutral fraction showed more direct-acting mutagenic activity inside the
terminal.
REFERENCES
1. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. Tejada,
J. Bumgarner, F. Duffield, V.F. Simmon, C. Hare, C. Rodriguez, L. Snow,
and M. Waters. 1979. Application of bioassay to the characterization
of diesel particle emissions. Part I. Characterization of heavy duty
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, S. Nesnow,
J.L. Huisingh, S.S. Sandhu, and L. Claxton, eds. Plenum Press: New
York. pp. 382-400.
2. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. Tejada,
J. Bumgarner, F. Duffield, V.F. Simmon, C. Hare, C. Rodriguez, L. Snow,
and M. Waters. 1979. Application of bioassay to the characterization
of diesel particle emissions. Part II. Application of a mutagenicity
bioassay to monitoring light duty 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, S. Nesnow, J.L. Huisingh, S.S. Sandhu, and L. Claxton, eds.
Plenum Press: New York. pp. 400-418.
3. Claxton, L.O. 1980. Mutagenic and Carcinogenic Potency of Diesel and
Related Environmental Emissions: Salmonella Bioassay. EPA Report
EPA-600/9-80-057b, U.S. Environmental Protection Agency: Research
Triangle Park, NC. pp. 801-809.
-------
4. Tokiwa, H., H. Takiyoshi, K. Morita, K. Takahashi, N. Soruta, and
Y. Ohnishi. 1976. Detection of mutagenic activity in urban air
pollutants. Mutat. Res. 38:351-359.
5. Lewtas Huisingh, J. (in press). Rioassay of particulate organic matter
from ambient air. In: Short-term Rioassays in the Analysis of Complex
Environmental Mixtures. 1980. Michael D. Waters, Shahbeg S. Sandhu,
Joellen Lewtas Huisingh, Larry Claxton, and Stephen Nesnow, eds. Plenum
Press: New York.
6. Jungers, R., R. Burton, L. Claxton, and J. Lewtas Huisingh. (in press).
Evaluation of collection and extraction methods for mutanenesis studies
on ambient air particulate. In: Short-term Rioassays in the Analysis
of Complex Environmental Mixtures, 1980. Michael D. Waters,
Shahbeg S. Sandhu, Joellen Lewtas Huisingh, Larry Claxton, and Stephen
Nesnow, eds. Plenum Press: New York.
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. Stead, A.G., V. Hasselblad, J.P. Creason, and L. Claxton.
the Ames test. Mutat. Res. 85:13-27.
1981. Modeling
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NITRO DERIVATIVES OF POLYNUCLEAR AROMATIC HYDROCARBONS IN
AIRBORNE AND SOURCE PARTICIPATE
by
Thomas L. Gibson
Environmental Science Department
General Motors Research Laboratories
Warren, Michigan
INTRODUCTION
Direct mutagenic activity is observed in the Ames Salmonella bioassay
of organic extracts from ambient airborne particulate and source emissions.
Nitro derivatives of polynuclear aromatic hydrocarbons (nitro-PNA), some
of which are strong direct-acting mutagens, are considered to be probable
contributors to this activity.
SAMPLING AND ANALYSIS
Particulate samples were collected on dexiglas filters and extracted
with benzene-ethanol (80:20 v/v) with a Soxhlet apparatus. Automobile
exhaust samples were obtained using a chassis dynamometer and dilution
tube. An analytical method for nitro-PNA was recently developed which
involves reduction of these compounds to the corresponding amino-PNA and
their determination by HPLC with fluorescence detection.1 With HPLC
methods, the concentrations of 1-nitropyrene, 6-nitro-BaP, pyrene, and
BaP were measured in samples of the various particulates. In all of
these samples, part of the nitro-PNA may have resulted from the reaction
of oxides of nitrogen found in emissions and in ambient air with PNA
bound to the particles on the filter. For example, an increase in
nitro-PNA was measured when diesel exhaust particulate was exposed to
filtered diesel exhaust gases, suggesting a strong likelihood that nitro
artifacts are formed during filter sampling.1 The direct mutagenic
activity was determined by the Ames Salmonella bioassay (Litton Bionetics,
Kensington, MD) using tester strain TA-98 without metabolic activation.
Each particulate extract was dissolved in DMSO and tested at five doses
using triplicate plates with equal numbers of bacteria from the same
starting culture. The slope of the initial linear part of the dose
response curve was considered as the specific activity.
-------
AMBIENT AIRBORNE PARTICULATE
Ambient particulate collected at an urban and a suburban site
during Spring and Summer contained 0.2-0.6 ppm (i.e., ng/mg particulate)
of 1-nitropyrene corresponding to airborne concentrations of 0.016 to
0.030 ng/m3, and also contained 0.9-2.5 ppm 6-nitro-BaP corresponding to
0.04-0.28 ng/m3. As shown in Figure 1, the fluorescence spectra of the
nitro-PNA were scanned to determine their identity by comparison with
authentic standards. The direct mutagenic activity of the suburban
samples in the Ames bioassay was 0.15-0.56 revertants/microgram of
particulate corresponding to 7-20 revertants/m3 airborne mutagenicity.
Based on its reported specific activity, the 1-nitropyrene in the particu-
late could account for less than 0.3% of the total activity.
AUTOMOBILE EXHAUST PARTICULATES
Samples were collected from a few of the numerous sources of particu-
late emissions including a 1981 2.5-L 4-cylinder catalyst car, a 1980
4.3-L 8-cylinder car with no catalyst using unleaded gasoline, a 1974
5.7-L 8-cylinder car with leaded gasoline, and 5.7-L 1980 8-cylinder
diesel cars made by General Motors divisions. The catalyst car gave
particulate with a lower concentration of 1-nitropyrene (0.63 ppm) than
the noncatalyst cars (3.9-4.3 ppm) and production diesel car (8.0 ppm).
The catalyst car particulate had 0.21 ppm 6-nitro-BaP, the noncatalyst
cars 17-33 ppm, and the diesel car less than 0.4 ppm. On a per mile
basis also, the catalyst car emitted less of the PNA, nitro-PNA, and
direct mutagenic activity than the other vehicles. The level of direct
acting mutagenicity seemed to be directly related to the total nitro-PNA
concentration of the particulate and not to PNA concentrations.
An experimental (noncommercial) low emission diesel car was also
tested. Compared to the production model of the 1980 diesel, the low
emission diesel gave much lower particulate, PNA, nitro-PNA, and mutagenic
emissions.
STATIONARY SOURCES
Particulate samples from a wood-burning fireplace did not contain
levels of the nitro-PNA above the minimum detection limits (less than
0.1 ppm). The concentrations of pyrene and BaP found in these samples
were low compared to automobile particulates and depended on how the
samples were collected: averages of 3-4 ppm in particles collected from
the. raw flue gases with an EPA Method 5 sampling train (heated filter
and impingers) compared to 30-60 ppm when collected from emissions
diluted 25-fold with air. The increased levels of PNA in particulate
from cool, diluted fireplace emissions suggests that much of the organics
remain in the.vapor phase in. emissions sampled by the EPA method.
Particulate emissions measured by this method should not be compared to
vehicle emissions determined by the dilution tube method and may lead to
-------
erroneous estimates of emissions from stationary sources (sampled from
raw flue gases). Similarly, a particulate sample from a coal-fired
boiler, collected from hot, undiluted flue gases, showed low or undetect-
able levels of PNA (less than 0.5 ppm) and nitro-PNA (less than 0.02
ppm).
CONCLUSIONS
Nitro-PNA are found in ambient airborne particles and various
source emissions. Because of the very limited data from only a few of
the possible sources and the complicating effects of differences in
sampling methods, filter artifact formation, and atmospheric reactions,
source allocation for PNA derivatives in ambient particulate is not
feasible at the present time.
1. T. L. Gibson, A. I. Ricci, and R. L. Williams, "Measurement of
Polynuclear Aromatic Hydrocarbons, Their Derivatives and Their
Reactivity in Diesel Automobile Exhaust," in Polynuclear Aromatic
Hydrocarbons: Chemistry and Biological Effects. A. Bjorseth and A.
J. Dennis, Eds., Battelle Press (in press), presented at the 5th
International symposium on Polynuclear Aromatic Hydrocarbons,
Columbus, OH, Oct. 28, 1980.
-------
1-AMINOPYRENE
oo
LoJ
o
a:
o
LU
o:
EXCITATION
EMISSION
250 300 350 400 400
WAVELENGTH (nm)
450 500
10
TIME
Figure 1. HPLC chromatogram of ambient participate extract after treatment
with a reducing agent (Conditions, see reference1) Fluorescence
Detector -- excitation 365 nm, emission 430 nm. Stop-flow
scanning gave the emission and excitation spectra shown, which
match those of authentic 1-aminopyrene.
-------
RISK ASSESSMENT OF DIESEL EMISSIONS
by
R. Albert
Institute of Environmental Medicine
New York University Medical Center
New York, New York
and
T. Thorslund
U.S. Environmental Protection Agency
Washington, District of Columbia
The observations made a number of years ago, which have since been
verified, to the effect that diesel exhaust particulates are mutagenic and
contain agents that are recognized carcinogens, established the position that
diesel particulates are likely to be carcinogenic in humans. However, the
unanswered question is how potent are these particulates and what is the
magnitude of the cancer hazard to the general population. In view of the
absence of any direct animal experiments or epidemiologic data, an approach to
risk assessment several years ago which seemed reasonable was to use the
available epidemiologic data that involved exposure to combustion products
having similarities to diesel particulates and to compare the relative potency
of these materials with diesel exhaust particulates. The epidemiologic studies
that were chosen involved cigarette smoking, coke oven emissions, and roofing
tar. An extensive series of studies including mutagenesis, cell
transformation, skin painting, inhalation, and intratracheal intubation have
been undertaken to compare these materials with diesel particulates. The
present status of the carcinogen risk assessment in terms of the epidemiologic
and laboratory studies will be presented.
-------
Notes
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