Report


     WORKING CONFERENCE ON HEALTH INTELLIGENCE


            FOR FUELS AND FUEL ADDITIVES
               January 5 - 7, 1973

              Durham, North Carolina
                  Convened by:
Fuels and Fuel Additive Registration Program
National Environmental Research Center - RTF
      Environmental Protection Agency

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                                 Contents
  I.   Participants
 II.   Foreward:  John F. Finklea, M. D."
                 Di rector
                 National Environmental Research Center - RTF
III.   Introduction
 IV.   Development of Toxicologic Screen for Fuels and Fuel Additives
      A.   Chemical Indicators of Toxicity
      B.   General Toxicology
          1.  Introduction
          2.  Methods
              a.  Sources and types of emissions
              b.  In_ Vitro methods
              c.  In Vivo methods
      C.   Carcinogenesis
          1.  Introduction
          2.  Methods
              a.  In Vitro methods
              b.  j£ Vivo methods
      D.   Mutagenesis
          1.  Introduction
          2.  Methods
              a.  In Vitro methods
              b.  Th~ Vivo methods
  V.   Background Information — Engineering and Chemical Considerations
                              _***_

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                               FOREWORD
     The composition of fuels and fuel additives has the potential  of
altering emissions from combustion processes and the performance of
emissions control systems.  Such alterations may be either beneficial
or detrimental to the public health and welfare.  In cognizance of this
fact, Section 211 of the Clean Air Act of 1970 gives EPA the authority
to require the conduct of tests "to determine the potential  public
health effects" of fuels or fuel  additives and to control or prohibit
the use of fuels or fuel additives when emission products "will endanger
the public health or welfare."  Furthermore, the demands posed by new
emission control systems may dictate considerable modification in exist-
ing fuels and new fuel additives.  A systematic effort is required to
evaluate emissions to ensure that increased public health risks do not
ensue, either from the introduction of new toxic chemicals or by an
elevation of toxicants attributable to fuel blends and fuel  additives
now in use.
     Because of the complexity of the toxicological  problems imposed by
the number and diversity of fuel  additives already developed and the
many altered primary and secondary pollutants which may be potential
toxicants, a significant research program will be required.
     The Conference on Health Intelligence for Fuels and Fuel  Additives,
the report of which follows, was  convened for the purpose of developing
the basis for protocols so that preliminary work on toxicological  evalua-
tion of fuel and fuel additives could begin without delay.

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                                - 2 -
     I should like to take this opportunity to express the appreciation
of the Environmental Protection Agency to the members of the Conference
who devoted several days of their time to this fruitful  effort.
                                  <
                                ,   John F. Finklea, M. D.
                              
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            WORKING CONFERENCE ON HEALTH INTELLIGENCE

                   FOR FUELS AND FUEL ADDITIVES


                       List of Participants
Dr. John Bertram
McArtle Laboratory
University of Wisconsin
Mr. Ronald L. Bradow
National Environmental
  Research Center - RTF
Environmental Protection Agency

Dr. Paul Brubaker
National Environmental
  Research Center - RTP
Environmental Protection Agency
Mr. Kirby Campbell
National Environmental
  Research Center - Cincinnati
Environmental Protection Agency
Dr. David L. Coffin
National Environmental
  Research Center - RTP
Environmental Protection Agency
Dr. Timothy Crocker  .
Department of Preventive Medicine
University of California
Dr. Basil Dimitriades
National Environmental
  Research Center - RTP
Environmental Protection Agency
Dr. Samuel S. Epstein
Department of Environmental
  Health and Human Ecology
Case Western Reserve University
Dr. John F. Finklea
National Environmental
  Research Center - RTP
Environmental Protection Agency
Dr. Gustave Freeman
Life Sciences Division
Stanford Research Institute
Dr. Donald E. Gardner
National Environmental
  Research Center - RTP
Environmental Protection Agency
Mr. James Gentel
Dow Chemical Company
Mr. Thomas Gleason
Office of Research & Monitoring
Environmental Protection Agency
Dr. F. Gordon Hueter
National Environmental
  Research Center - RTP
Environmental Protection Agency
Dr. K. E. Klinksiek
German Society for Mineral Oil
  Science and Coal Chemistry
Hamburg

Dr. Marvin Kuschner
Department of Pathology
Health Sciences Center
State University of New York

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List of Participants (cont.)
Dr. Sidney Laskin
Department of Environmental
  Medicine
New York University Medical Center
Dr. J. Douglas MacEwen
Toxic Hazard Research Unit
University of California - Dayton
Mr. Henry Miller
National Environmental
  Research Center - RTP
Environmental Protection Agency
Dr. Richard L. Miller
Life Support Branch
Environmental Sciences Division
USAF School of Aerospace Medicine
Mr. John B. Moran
National Envi ronmental
  Research Center - RTP
Environmental Protection Agency
Dr. B. Richardson
Environmental Sciences Division
USAF School of Aerospace Medicine
Dr. Donald Rounds
Pasadena Institute of Medical
  Research
Dr. Eugene Sawicki
National Environmental
  Research Center - RTP
Environmental Protection Agency
Dr. Klaus Schmidt
Institute for Experimental
  Toxicology and Chemotherapy
German Cancer Research Center
Heidelberg
Dr. Frederick de Serres
Mutagenesis Branch
National Institute of Environmental
  Health Sciences
Dr. Phillippe Shubik
Eppley Institute for Research
  in Cancer
University of Nebraska
Mr. John Sigsby
National Environmental
  Research Center - RTP
Environmental Protection Agency
Dr. Daniel Strauss
Mutagenesis Branch
National Institute of Environmental
  Health Sciences
Dr. Theodore R.Torkelson
Chemical Biology Research
Toxicology and Industrial Hygiene
Dow Chemical Company
Dr. Benjamin L. Van Duuren
Institute of Environmental Medicine
New York University Medical Center
Dr. Michael D. Waters
National Environmental
  Research Center - RTP
Environmental Protection Agency

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    DEVELOPMENT OF TOXICOLOGIC SCREEN FOR FUELS AND FUEL ADDITIVES

     Registration and testing procedures must be directed toward minimizing
the risk of adverse effects on human health that may reasonably be attri-
buted to changes in fuels or fuel additives.  Such effects may be linked
to individual additive components, complete additives, blended fuels or
gaseous and particulate emissions from mobile and stationary sources.
Effects of concern include increased susceptibility to cancer, mutagenic
and teratogenic aberrations, respiratory infection, chronic pulmonary
diseases, subtle changes in cellular morphology and enzymatic function,
and increased pollutant burdens.
     Because it is not possible to completely define chemically the combus-
tion products and the likely chemical and biological interactions of the
various components in the exhaust effluent, it appears essential to approach
the problem of fuel additive toxicology by testing the whole effluent.
Basically, this would consist in comparing the toxicity of emissions from
a reference fuel with the same fuel including additives at the concentra-
tion prior to combustion specified by the manufacturer.  Whole exhaust
emission or condensates of whole exhaust effluent would be the best samples
for biological testing.
     Because of the large number of toxicological tests which may be required
and the need to obtain preliminary information quickly, it is recommended
that in vitro methods be incorporated in the toxicologic screen.  It would
appear that such methods can be utilized in hierarchical screening systems
to detect first effects and develop priorities for further study of toxic
chemicals in the environment.

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                                   -2-
     The purpose of this report is to present methods for toxicologic
screening that incorporate suggestions for in vitro and sub-whole animal
studies in an orderly screen together with validation by the more con-
ventional whole animal methods now commonly employed so that ultimately
some insight may ensue into the relative merits of various tests with
regard to cost, ease of applicability, sensitivity, reliability, etc., as
well as validity for correlation with heatlh effects in man.  At this stage
it is fully realized that in the employment of sub-whole animal tests to
develop toxicologic information, certain tests now regularly employed for
toxicologic evaluation must be included for purposes of validation.  However,
it is strongly felt that, in dealing with the multiplicity of toxicological
problems of the immediate future, the obvious potential advantages of
in vitro methods, other sub-whole animal tests, and tests employing specific
interactants should be given immediate attention in the screen so that
results can be compared with more conventional tests that require considerable
time.
     The following report contains the recommendations of the Conference on
Health Intelligence for Fuels and Fuel Additives.  It is a synthesis of
the various viewpoints of the members of the panel and thus is not likely
to wholly reflect the opinions of any one member.

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                                   -3-
1.  Chemical Indicators of Toxicity
    a.  Objectives:  (1) To determine if the parent fuel additive is a
known carcinogen, mutagen, teratogen, or alkylating agent.  If so, it should
be considered as a screening prospect on the grounds that the material may
act as a direct consumer hazard; also, a certain percentage of the unpyrolysed
parent compound  is likely to appear in the exhaust.  (2) To determine
whether mixing or burning additives in fuel gives rise to certain chemicals and
chemical structural types that are known or suspected to have deleterious
health effects.  These include carcinogens, mutagens, teratogens, etc.  A
limited group  of compounds and compound  types can be identified as chemical
indicators of potentially increased or decreased toxic effects.  This informa-
tion may provide guidance in the selection of appropriate in vitro and in vivo
bioassays.
     b.  Test Methods:  The following compound types and possible analytical
techniques are proposed:
         Compound Type                             Analytical Method
     (1) Aromatic and heterocyclic compounds,      G.C.  (Mass spec)
         including carcinogens and non-
         carcinogens
     (2) Aliphatic hydrocarbons                    G.C.
     (3) Olefinic hydrocarbons                     G.C.
     (4) Alkylating Agents                         Colorimetric
     (5) Phenols and Polyphenols                   Colorimetric/G.C.
     (6) Trace Metals                              Atomic Absorption
     (7) Aldehydes
     (8) Nitroso compounds                         G.C., TCC

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                                    -4-
     When marked Increases In levels of these agents are observed, refined
analysis and structure determination will  be required.
     New Compounds:  When compounds, such  as known carcinogens, mutagens,
teratogens or alkylating agents, or close  congeners of such compounds, are
isolated in the test condensate, they should be considered to be predictors
of potential human toxicity and evaluated  in the absence of further toxicologi-
cal information.

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                                    -5-

B.  General Toxicology

    1.  Introduction:  The adoption of a systematic "screen" for biological

effects of emissions from gasoline burned together with a series of unknown

"additives" in an automobile engine is of great importance.   The basis for

any such comparative study would be an identified and characterized "standard"

fuel.  The undesirable biological effects would constitute a "profile" of

toxicity, which is available in part for both irradiated and unirradiated '

whole emissions.  However, it can be anticipated that much of the profile

is unknown.  Profiles include observations in man, the whole animal, organ

systems, and cellular systems and take into account all categories of cellular

and biochemical responses.  Effects from standard fuel emissions would be

compared with those from fuels plus additives.

     The main objective is to provide EPA with evidence about whether or not

an emission with a particular additive effects a profile different from that

of the "standard" fuel emission and whether differences are biologically

more or less desirable.  Effects, according to the spectrum of observations

from among the several systems to be selected, that will constitute a profile,

are to be described in quantitative terms.

     2.  Methods:

     a.  Source and Type of Emission:   This is essentially the responsibility
     of engineers and chemists.They should determine the types of fuels,
     characterize their emissions chemically as far as reasonable, provide
     the materials to biologists, and monitor the quality (quantitatively)
     of the materials provided ~ or,  as an alternative, provide biologists
     with manageable techniques to do likewise.  Irradiated, as well as
     non-irradiated, emissions will be considered, if not studied.  Exhaust
     control devices affect the tendency of exhaust emissions to partici-
     pate in photochemical smog development.  The importance of this factor
     for future automobile emissions is at this time unclear because of
     the uncertainty of what control devices will ultimately be adopted.
     Thus, it would appear important to provide for irradiation of exhaust
     as part of the test system.  For reasons of feasibility, non-irradiated
     automotive emissions will probably be the initial source of material,

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                               -6-

both for atmospheric exposure of biologic test systems and
for collection of specific or whole crude samples (e.g., condensates).
However, piror work has shown that irradiated effluents have had more
significant biologic effects in rodents than non-irradiated effluents.
Therefore, every effort should be made to provide for irradiation
of automotive exhaust as part of the test system.  If irradiation
cannot be incorporated into the exhaust testing process at the outset,
early provisions to include this feature must be made so that irradi-
ated samples will be available for the determination of hazards,
as new engines, fuels, additives, and control devices are developed
for use in areas having a high vulnerability for photochemical pollu-
tion.

b.  First Approximation for Identification of Biological Effects;
    Approach:  The purpose of the vt± vitro segment of the screen is to
determine first effects and to establish priorities for further
testing.
    (1) Primary animal cells should be used in preference to
    cell lines for toxicity studies because such cells are
    closer to the tissue of origin before undergoing modification in
    in culture; and because pulmonary alveolar macrophages,
    tracheobronchial, alveolar epithelial and conjunctival cells
    can be included:  normal cells of these types are not available
    in continuous line culture.

    (2) Cell lines of human fibroblasts (as WI-38) may be of
    value because of differences which may exist between human
    and rodent fibroblasts.  Parallel rodent fibroblasts may be
    used, but should be in a stage that precedes spontaneous
    transformation.

    (3) Applicable studies in above systems, additional to
    macrophage studies, include (with emphasis on epithelial
    cells): growth rate; duration of components of the mitotic
    cycle; replicative fraction versus non-replicative (senescing)
    fraction; preservation of capacity to differentiate as
    respiratory epithelial cells; various functional parameters
    in pulmonary macrophages.

    (4) Organ cultures:  In selected instances requiring identifica-
    tion of cell types in biologic responses, this system can be
    employed at a secondary test level rather than as a screen-
    ing method.

    (5) Organ Preparations:  Ciliary beat, mucous secretion,
    and transport of particles can be seen in the excised fresh
    organ.  This system should be considered for secondary (and po
    possibly primary) screens.

c.  Exposure methods for In Vitro Tests:

    (1) Condensates:  Whole crude or fractionated condensates
    or filtrates of automotive exhaust can be added to culture
    media for testing of cellular responses, by methods described
    above.

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                               -7-

    (2) Airborne Effluent:  Appropriate systems for in vitro
    exposure are needed to permit screens of atmospheric
    contaminants suspended in air.  These systems are incompletely
    developed, but can be expected to become available.   In such
    systems cellular observations will be similar to those
    described above.

d.  In Vivo Tests:  Generally, animals will  be divided into three
groups, one to be exposed to filtered air, a second to "standard"
fuel emission, and the third to the emission produced by the
"standard" fuel with the additive to be tested.

Relatively sensitive and inexpensive species (two or more) should be
selected.  Preference should be given to growing animals so that
growth rate may be observed also.  (Subsequent studies could include
animals with increased susceptibilities.)

    (1) Prepared standard condensates (non-volatile portion)
    would be administered by parenteral and natural routes
    in parallel experiments.  Volatile fractions should be
    trapped and administered concomitantly.

    (2) Because "acute" and "chronic" are arbitrary terms, both
    early and continual observations will need to be made as
    indicated.

    (3) Duration of observations will depend upon the time of
    appearance of positive effects and/or upon-the urgency to
    have data at arbitrary points in time.

A special toxicological problem is associated with exposure of
animals to automobile exhaust by inhalation, namely that the toxic
effect of carbon monoxide may mask changes which might develop from
other chemicals present.  Therefore, unless a practical  way can be
found to remove the carbon monoxide in the large volumes of exhaust
required for animal exposure, there must be reliance on methods of
exposure other than inhalation of whole effluent. However, since
toxicologic strides on whole effluent have been largely unsuccess-
ful (except for carbon monoxide and oxidant) it would appear that
exposure of whole animals should be carried out with emission
condensates.  This will be important for validation of the in
vitro methods, as well as defining primary profiles for whole"
animal exposure.

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                                    -8-
C.  Carcinogenesis
     1.  Introduction:  Automobile exhaust in the absence of additive compounds
contains known carcinogens, such as polycyclic hydrocarbons and trace metals
and potentially co-carcinogenic materials, such as phenols.  Therefore, any
test system devised to examine the possible hazardous effects of additives
must be designed to identify increments in activity conferred by the addi-
tive.  Additionally, since some of the additive may emerge unchanged in the
exhaust, the toxicity of the parent compound should be considered, as well
as its possible contribution to overall toxicity after combustion.
     It would appear reasonable to devise a test system which would approach
the problem of potential carcinogenicity by evaluating the whole effluent.
For tests employing in vitro methodology, per cutaneous application, subcu-
taneous inoculation and intratracheal  instillation, it would seem most
practical to utilize cryogenic condensates containing as much exhaust effluent
as it is possible to collect by this method, as well as volatile materials
collected by this means.  If exposure by aerosol was to be performed, this
could be accomplished either by aerosolization of the whole extract, or by
use of the whole effluent atmosphere.   In view of the low degree of success
achieved by exposure to carcinogens via aerosol, it would appear that such
exposures should be reserved for specific purposes and not routinely employed
in the screen specifically for carcinogens.
     In view of the many condensates which must    eventually be examined
for evidence of carcinogenicity, it would appear to be of value to devise a
hierarchical screen of tests including in vitro, sub-whole animal and whole
animal, so that the experience derived from preliminary work might be
applied to the subject of carcinogenesis testing for later studies, hopefully
reducing the time involved in reaching conclusive data.

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                               -9-

The proposed screen is as follows:

In Vitro

Cell Culture:

  1. Hamster embryo
  2. Mouse fibroblast
  3. Paramecia

l£ Vivo

  1. Mouse skin painting
  2. Adult rat subcutaneous inoculation
  3. Neonatal mouse subcutaneous  inoculation
  4. Intratracheal injectipn/F203
  5. Inhalation of aerosolized tar
  6. Inhalation whole exhaust.


2.  Methods:

    a.   In Vitro:  The induction  of cancer in vitro has been basically
    achieved in two cell systems:  secondary cultures of hamster embryos
    and cell lines of mouse fibroblasts.   In both systems malignant
    transformation is demonstrated by an  alteration in morphology of the
    cells, with changes from a pattern of order to one of disorder
    (cross-crossing and piling-up).  Validation of these systems
    depends upon the ability of such altered cells to produce malignant
    tumors when injected into the species of origin.

    These i_n_ vitro test systems possess the attributes of speed (2-6
    weeks versus 1 - 2 years in whole animal) and consequent economy,
    but also allow more close control of  experimental parameters.  In
    addition, doses may be used that are  precluded in the whole animal
    for reasons of toxicity.  Furthermore, although relatively large
    doses are employed in whole animal systems, they produce only a
    small total mass, as compared with in vitro systems, which have
    the additional advantage of requiring much smaller amounts of
    inoculem (since they do not employ repeated doses).  This latter
    is  an obvious advantage as it is often difficult to derive large
    amounts of the test material  for preliminary studies.  These tests
    should be looked on as range-finding  and priority-setting steps
    in  the screen.

    Listed below are the requirements of  an in vitro screen for
    carcinogenic activity, and means of meeting these requirements by
    the currently available systems.

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                           -10-

    (1) The morphological transformation caused by the
    carcinogen should be produced rapidly and be readily
    scored.

    (2) The system should react in a quantitative manner
    to all known carcinogens.

    This goal may be unattainable in anything other than
    a battery of tests yet to be devised.  In the present
    systems two major problems need to be overcome:
    (a) Many carcinogens require metabolic activation and
    it cannot be expected that any one cell type will
    activate all such carcinogens (The present systems
    activate many, but not all).  The use of specifically
    chosen feeder cells and host-mediated assay require more
    extensive evaluation,  (b) The present systems appear
    to be insensitive to weak carcinogens.  However, it
    has been shown that the in vitro sensitivity to smog
    abstracts can be greatly enhanced by infection with an
    RNA tumor virus.  Likewise, concurrent administration
    of X-rays, repair inhibitors, or low doses or a strong
    carcinogen may expose the effects of high doses of a
    weak carcinogen.  More research is needed in these
    areas.               '

    (3) The system should be quick and economical to set
    up and reproducible in its response.  The moust fibre-
    blast system, being a cell line, is preferrable in
    all three respects to the hamster system.

Paramecia:  The photodynamic bioassay employing paramecia or other
protozoa has been useful in demonstrating sensitization of the
organisms by a previous exposure to polycyclic and heterocyclic
compounds.  In studies performed on many carcinogenic and non-
carcinogenic compounds there has been a strong statistical
association of positivity in this test and carcinogenicity.   This
method might provide a quick, relatively simple means of obtaining
preliminary information on a very large number of extracts regarding
the presence of polycyclic and heterocyclic compounds with presumed
carcinogenic potential, and it might be evaluated together with
the preceding in vitro methods and the more conventional whole
animal tests described below.
b.  In Vivo:  A number of methods employing the response of whole
animals are more or less regularly utilized for evaluating the
carcinogenicity potential of various substances.  Besides being
"time honored" in the field of toxicology, these methods have certain
other advantages, such as utilizing to a greater or lesser extent

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                           -li-

the whole potential of the animal  as a reactor, which obviously
cannot be duplicated in the in vitro systems employing a single
cell line in isolated culture.  Thus, whole animal  systems
should be used to validate the in vitro tests and give  more
definitive reference points.

    (1) Skin Painting of the Mouse:  This method will require
    a large amount of material -- up to 100 grams per experi-
    ment.  The test substance should be utilized as a whole
    carcinogen and as a parameter following DMBA initiation.
    If positive results ensue, experiments should be repeated
    to determine dose response.

    (2) Subcutaneous inoculation of adult rats:  This requires
    somewhat less material for testing and gives additional
    Information in that cells of more than one germinal layer
    may be affected.  The neonate mouse test has the virtue
    of utilizing much less material and while presenting
    problems of interpretation, also probably yields better
    insight as to the nature of the carcinogen contained in
    the inoculated material.

    (3) The intratracheal injection method of testing
    material combined with FepO^ is the most practical approach
    to the determination of the potential of the test sub-
    stance to elicit tumors of the lung.

    (4) Inhalation methods seemingly should provide the most
    naturalistic methods for evaluation of the potential
    of a substance to elicit tumors of the lung in experi-
    mental animals.  However, it is fair to point out that
    this method has been relatively unproductive in demonstrat-
    ing carcinogenicity of even the most potent hydrocarbon
    carcinogens administered in high concentrations.  Thus,
    it is unlikely that the comparative carcinogenicity of
    the effluents of the reference fuels and the contained
    fuel additives will be detected by such means until
    better methods are devised for such experiments.  If
    inhalation exposures were to be undertaken, large amounts
    of test substances would be required up to one kilogram
    of tar for aerosolization or long continued exposure
    to whole exhaust.  Such tests would offer the possibility
    of combining exposure with other air pollutants,
    cigarette smoke, asbestos, etc., for research, into the
    influences of interactants, but it is difficult to fore-
    see their immediate value as a bioassay system for the
    stated purpose of fuel additive evaluation.

A summary of the various test systems discussed appears in
the table which follows.

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TEST SYSTEM
MATERIAL REQUIRED
SYSTEMS NEEDED
A-  IN VITRO TEST SYSTEMS

    1. Cell  Culture
       a.  Hamster embryo
       b.  Mouse fibroblast examined
          for transformation  jn
          vitro and validated by
          tumor production on
          re-inoculation.
       c.  Paramecia - photodynamic
          effect to detect carcino-
          genic polycyclic and
          heterocyclic compounds.
B.  IN VIVO TEST SYSTEMS

    1. Mouse skin painting as
       whole carcinogen and as
       promoter following DMBA
       initiation.  Dose response
       if positive.
    2. Adult Rat Subcutaneous
    3. Neonatal Mice Subcutaneous
    4. Intratracheal  injection
       combined with F6203
    5. Inhalation
       a. Whole exhaust (diluted
       b. Aerosolized tar
Whole condensate - if cyto-
toxic, then appropriate frac-
tions -- mgs per test.
   ^
Material should be soluble in
acetone, DMSO, or water.

Microgram amounts required.

(Useful for rapid screening
of large numbers of fractions
and subfractions.)
100 Gms per experiment
50 Gms per experiment
5 Gms per experiment
1 kilo
Examination and development of human cell
lines, particularly those characterized by
genetic instability (e.g., Fasscessis, or  .
X.P. cells) as hypersensitive indicators.

Development of in vitro systems for examina-
tion of promotion.

Possibility of enhancement by viruses of
transformation as a sensitive indicator of
activity (see Rihm, et al. Nature  29:
103-107, 1972.)

Exploration and improvement in techniques of
organ culture.

Examination of cells and tissue derived from
pre-exposed animals.
Fractionation if positive to identify the
"new" carcinogen.
Combined exposures with air pollutants,
cigarette smoke, asbestos, etc.
                                                                              ro.
                                                                               i

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                                    -13-
B.  Hutagenesis                                                      .;

     1.  Introduction;
     The general objective of mutagenicity testing is to determine if
a new environmental chemical will produce genetic damage either alone or
in combination with other agents in the environment.  Since the vast
majority of genetic damage is considered to be harmful either to the indi-
vidual (by making him more susceptible, for example, to the development of
cancer) or to his progeny, every effort should be made to prevent an increase
in the various types of genetic damage already present in the human popula-
tion.  The genetic basis of many human diseases has been well established
and increases in the frequencies of these diseases in the population can
be avoided by careful screening of new man-made chemicals before they are
released into the environment.  Since we do not have reliable methods for
monitoring the human population to determine whether the genetic load is
changing, it is especially important to establish a comprehensive screen
which can detect mutagenic activity before new chemicals are released for
widespread use.
      2. Methods:  Test systems for evaluating chemicals for mutagenic
activity fall  into two main  classes:
      a.  In vitro test systems:  These include bacteria, fungi, insects,
         higher plants and mammalian cells in culture.
      b.  In vivo test systems:  These are assays performed on laboratory
         animals, and include the dominant lethal test, the host-mediated
         assay and in vivo cytogenetics.
      a.  In Vitro Test Procedures:
     In general, the in vitro tests provide an assay of the mutagenicity of
the original chemical on somatic cells.  The assay of the original chemical
and its potential metabolites is limited by the metabolism of the test

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                                   -14-

organism.  In many cases these metabolites may be quite different from those

found in mammalian test systems.  In this respect negative results with

in vitro assays must not be interpreted as indicating that a chemical  is

safe for man, but positive results are interpreted as indication of a

potential hazard.

     Various methods are under development to mimic in vitro the type  of

metabolic activation that occurs in vivo.  These include various methods

of chemical activation as well as biological  activation with microsome

preparations from mammalian liver and other organs.  These in_ vitro activation

systems should be able to increase the sensitivity and general  utility of

all in vitro test systems by providing a mechanism for rapid evaluation of

potential products of mammalian metabolism.

    (1) C h romo s ome a be rra t i ons (non-disjunction and chromosome rearramgements)

       (a) Mammalian cells in culture - Mouse lymphoma cells, human
           fibroblasts, etc., can be used to determine whether chromosome
           aberrations have been produced.  By using synchronized popu-
           lations of cells, it is possible to look for cell-stage
           specificity.  Periods of timed exposure with varying concentra-
           tions can be used to obtain dose-response curves for particular
           types of chromosome aberrations.

    (2) Gene mutations (point mutations and deletions)  Gene mutations in
        man can be due to alterations in structure (point mutations) or
        physical removal  from the chromosome (deletions).

        Bacteria - In Salmonella typhimurium a series of histidine-requiring
        mutants have been developed by Ames and his colleagues (Ames,  et al.,
        1973) which can be used to detect the production of particular
        types of genetic alterations that can produce point mutations.
        The assay consists of a spot test, in its simplest form, that
        provides semi-quantitative data.  It assays for the production of
        reverse mutations over a wide range of concentrations.   The assay
        is limited to the detection of particular types of genetic altera-
        tions; but the development of highly sensitive strains (e.g.,  lacking
        repair enzymes and a normal cell wall) which can detect specific
        types of damage makes this assay especially valuable.

-------
                                   -15-


        Fungi - In Neurospora crassa by using a two-component heterokaryon
        heterozygous for specific genetic markers, it is possible to detect
        forward-mutations at two specific loci in the ad-3 region (de Serres
        and Osterbind, 1962).

        Gene mutations at each locus result from both point mutation and
        chromosome deletion so that this test system can detect the same
        range of genetic alterations that can give rise to gene mutations
        in man.

        By giving various acute levels of exposure to cell suspensions over
        a constant period of time, dose-response curves can be obtained
        which can be used to extrapolate to lower (chronic) levels of
        exposure.

     (3) Mitotic recombination (reciprocal and non-reciprocal exchange
        [or gene conversion])
        Mitotic recombination in heterozygous diploid organisms provides a
        mechanism for making deleterious recessive genes homozygous.
        Homozygosity provides a means for expression of deleterious effects
        which would not be seen in heterozygous individuals.

        In diploid yeast strains of Saccharomyces cereviseae heterozygous
        for linked markers as well as (hetero; alleles at the same locus,
        it is possible to assay for effects on mitotic recombination.
      b.   In  Vivo Test  Procedures
     In general, the in vivo test procedures are used to evaluate effects

on germ cells of the original compoundsas well as its metabolites.  No

really practical test exists which can be used to evaluate the production

of gene mutations in germ cells and it is generally accepted that the use

of specific locus test developed by Russell (1951) should be restricted to

particular situations because of the time, facilities and special training

required to make this test.

    (l) Chromosome aberration (non-disjunctive and chromosome rearrangements)

       (a) Dominant lethal test - The dominant lethal test in mice is used
           to obtain presumptive data on the production of chromosome
           aberrations.  By mating treated males to new batches of females
           at frequent intervals, it is possible to look for effects on
           various cell stages during spermatogenesis.  The detection of
           a significant increase of dead embryos over the control level
           is usually attributed to the production of germ cells with

-------
                             '..  -16-           .

       abnormal  numbers of chromosomes,  chromosome fragments  and
       rearrangements.

       Breeding  tests on the viable progeny obtained in such  tests  can
       be used to determine the  frequency of transmissible chromosome
       rearrangements.   Since Generoso,  et al.  (1973) have shown that
       the frequency of spontaneous translocation is low,  this  assay
       may provide a more sensitive test for the  detection of chromosome
       rearrangements.

       In the dominant lethal test several  acute  levels of exposure
       can be given to obtain dose-response curves.   The sensitivity
       of the test is limited by the high frequencies of spontaneous
       dominant  lethality which  makes it difficult to detect  signifi-
       cant increases at low levels of exposure or positive effects of
       weak mutagens.

    (b)In vivo cytogenetics - Cytogenetic evaluation of cells that  are
       a normal  component of the animal  can be  made by putting  bone
       marrow cells or peripheral  lymphocytes  into short-term tissue
       culture.   Cytogenetic evaluation  of these  cells in  culture can
       be used to obtain data from metaphase preparations  to  determine
       whether chromosome aberrations have been produced.

(2) Gene mutations - No practical  test exists  for detecting gene mutations
    in either somatic cells or germ cells of laboratory animals. To
    compensate for this deficiency, a new technique was developed by
    Legator and  his associates (Gabridge and Legator, 1969) which
    introduces indicator cells into the  peritoneum of animals after
    they have been treated with  a mutagen.  The cells are  incubated
    in the peritoneum for varying periods of time and then removed
    for genetic  assay.   In this  host-mediated  assay many test systems
    have been used as indicator organisms to detect gene mutations.
    These include Salmonella, Neurospora and mammalian cells  in culture.

    The problems with the technique are  the host  reaction  against the
    indicator organism shown by  Mailing  (1972)  and the fact that metabolic
    activation may be organ-specific, mutagenically active metabolites
    may not be present in sufficiently high concentrations in the
    peritoneum (Mailing and Chu, 1973).

    Under development is the utilization of specific locus assays in
    mouse lymphoma cells in culture to overcome the problems  in both
    areas.

(3) Mitptic recombination - No in vivo assay for  mitotic recombination
    is known, and the test with  diploid  yeast can be performed  utiliz-
    ing yeast as the indicator organism in the  host-mediated  assay.  This
    approach permits a more thorough test for mutagenicity of any
    products that may be unique  to mammalian metabolism.

-------
                                   -17-                           .
Preparation of Samples
 -   In general, samples must be sterile and solubilized in water.  Dimethyl-
sulfoxide (spectrophotometric grade), dimethylformamide, tricaprylin
(redistilled), or other relatively non-toxic solvents should be used.
In each case controls will be performed to test possible toxic or mutagenic
effects of the solvents themselves.  Controls will also be performed to
test the sterility of samples.
Positive controls
    Positive controls will be performed using standard quantities of known
mutagens and mutagen precursors to monitor the responsiveness of tester
strains or cell lines and the activity of microsomal  enzyme fractions.
Where necessary, controls will be performed along with each experiment to
monitor the genetic integrity of tester strains and cell lines.
    One problem which may arise from the use of unfractionated or partially
fractionated materials in the mutagenesis tests is that a high background
level  of toxicity and/or mutagenicity or some component or components in the
reference sample may mask the detection of new mutagens appearing in the
test samples.  Another possibility is that some components of the reference
sample will  inactivate the liver microsome preparations used for activation
or mutagen precursors.  To approach these possible problems, positive
controls will be performed in which standard quantities of known mutagens
and mutagen precursors (e.g., 2-aminopurine, ICR 191, 2-aminofluorine)
will be added to the reference sample, and the resultant mixtures will  be
run through the mutagen screening tests.  In cases in which these positive
control experiments fail  to register positive results, this will be taken
as an indication that further fractionation of the reference and test samples
is necessary.

-------
                                    -18-
Testing Protocols
     To simplify the testing and to make it possible to detect potentially
hazardous mutagens in the most efficient manner, the short-term tests are
preferred.  The utilization of simpler in vitro tests singly, and in combina-
tion with various chemical or biological systems that mimic mammalian metabolism,
makes it possible to rapidly screen the original compound  and some of its
metabolites for genetic activity.  The test chemical should be tested alone
and in combination with those chemicals with which it will be associated
under the conditions of its intended use, over a wide range of concentrations.
These should be acute exposures for a fixed period of time (e.g., two hours)
under conditions that give 100% survival to 99.9% inactivation of cells.
Absence of the production of various types of genetic damage under conditions
that produce inactivation of cells shall be considered evidence for the lack
of a genetic effect.  It is important to note that it must be shown with
the appropriate positive controls that mutagenic activity could have been
detected if it were present.  In other words, the assurance must be given
with the appropriate positive controls that the presence of cytotoxic compounds
is not masking out the activity of mutagenic compounds.
Interpretation of Test Data
     In general, in each of the in vitro and in vivo tests the spontaneous
frequency of genetic damage can be measured in the controls.   A positive
result will be indicated when the treatment with the test chemical alone or
in combination with those chemicals with which it will be associated under
conditions of proposed use, give a doubling of the spontaneous frequency.
All other results will be considered to provide a negative test.

-------
                   -19-
Engineering and Chemical  Considerations
            January 5,  1973

-------
                                  -20-

                        National Significance

     Transportation, in general, is the major source of carbon monoxide,
hydrocarbons, and nitrogen oxides.   In 1970, emissions from all trans-
portation sources in the United States contributed 50% of the total  hydro-
carbons, 70% of the carbon monoxide, and 30% of the nitrogen oxides.  The
principal source of such emissions  is the gasoline-powered motor vehicle.
     Gasoline-powered motor vehicles constitute three major types:
passenger cars (93 million in 1972), light-duty trucks, and gasoline-
powered heavy duty trucks.  The total vehicular population in the U. S.
in 1972 exceeds 110 million.   The average vehicle is driven 10,000 miles
per year at an average fuel  consumption of 1 gallon per 12.5 miles.  Com-
position of typical gasolines commercially sold in the U.  S.  is shown in
Tables 1 and 2.  Figure 1  reflects, schematically, the basics of petro-
leum refining practice.  Table 3 indicates typical ranges  of key basic
components in commercial gasolines.

-------
                                        -21-
                                    Table  1
                        Commercial Gasoline Properties:
Research Octane
Motor Octane
Reed Vapor Pressure
% Olefins
% Aromatics
% Saturates
Initial Boiling Point
Final Boiling Point
Gravity (API)
Premi urn
99.9
92.4
13.1
4.4
29.7
65.9
81°F
410°F
60.5
Premi urn
99.4
91.5
13.0
2.6
28.0
69.4
82°F
398°F
59.9
Regular
93.1
87.0
12.9
7.4
21.9
70.7
84° F
420°F
60.7
Regular
91.9
85.6
12.3
10.6
20.3
69.1
88° F
390° F
63.0
Non-Lead
90.3
82.8
10.1
12.6
12.0
75.4
9f°F
368°F
67.5
          The range of several factors measured in 30 commercial fuel samples
          Including premiums, regulars, and 91 octane low and non-leaded
          fuels is shown below:

-------
                                                                -22-
                              Table 2
                           PREMIUM GASOLINE



                        Typical  - 98 - 100 RON
10-20% butane
10-20% Olefins
10 - 30% Lt. FCCU - (olefins)     20 -  35% Aromatics
40 - 50% Overall reformate
Balance paraffins
10-20% Udex extract
+ 1000 ppm TEL
10 - 40% Alkylate
                           REGULAR GASOLINE
                         typical  yt -  so KIM
10 - 20 % butane
20% + olefins
20 - 40% Heavy FCCU
15 - 30% aromatics
10 - 20% Light straight run
Balance paraffins
 0-10% Udex raffinate
+ 400 ppm TEL
20 - 40% Lt. CRU
30 - 50% Overall  reformate

-------
                                               GASOLINE MANUFACTURE
Natural gas condensate
 C4 -  C.JQ hydrocarbons  - boiling point - 70°F H

	»• butane, isobntane, Cg's to gasoline
                                                                                                               to
Crude oil

C5"C35
paraffins
 jpe
  &
Dis





11



ti nation
i I iuht °

paraffi
1 1 +1 C\fC
(1)
tralght Run Gasoline - RON-40-70 5-10%
ns & cycloparaffins Cg-C7 B.P. <250°F
on f\r\y ,- 	 -- —
1« «,o «41  nuuvy i.
Desu
/ ,
Vac.
Still
1
Residu
Asphal
Coker


y v, i v. yjuo u i i ' \^i uv,r>ci
\acid clay
catalyst
Nn ? Nn /I -furl nil .... 	

Diesel fuel
(6)
Overall

_,. . - _}, p(j(j| y-| | Kc 1 OiiiICi ' —
Pt. t
C-n 1 a 1 \/c +• 	 .. . , — k
v^uiaiyii. — ~*
nm __..,... IT c
t till"
1 	
(9)



. Olefinic-

. . k n-i e
• » u is
till

-*• butane
(4)
Gases
	 »-Lt. FCCU
B.P. <250
(3)
^ nsovy r vw
chemicals
C4-C3's to
— >• alkylation
-60% olefins
°F
— >• Premi urn
gasol i ne
U— *• Regular
60% olefins 9asol1ne
j , Mostl

' IVCVJU 1
(5)
-*• Heavy CRU 	 »• Mostl
— »• Bottom

Udex
s ?

y paraffins
ar gasoline
y aroma tics
(7)
t aroma tics
(8)
	 k. Ra-F-finate nar'ira-finc
                                                                                                                       ™
                                                        C3' C4
                                                        Olefins
                                                     Isobi
                                       Alky
    (10)
-» Al kyl ate
                                                                                   branched
                                                                      ns

-------
                                  -24-
                               Table 3

                 Range of Key Components in Gasoline:

Trace Elements:
                 Fe   0.5 to 1.0 wt %
                 Ca   0.5 to 0.9  "
                 Mg   2 to 3
                 Mn   0.2 to 0.3  "
Lead:  .26 to 3.6 gms/gal.
Chlorine:  .002 to .058 ppm
Bromine:  .001 to .049 ppm
Phosphorous:  0.1 to 22.8 ppm
Sulfur:  12 to 1100 ppm
HC HO:  1.9 to 34.0 ppm
Carbon:  84.2 to 86.4 wt %
Hydrogen:  12.7 to 14.3 wt%
     Typical additives used commercially in various fuels are shown in
Tables 4 through 12.   A general classification of fuel additives by chemi-
cal type and the uses of fuel additives are also outlined in Table 13.

-------
                        Table 4                            .26-
    1969 CONSUMPTION OF ADDITIVES BY FUNCTION IN U.S.
Additive Function
Anti- Foams
Anti -Icing Compounds
Anti -Knocks
Anti -Oxi dants
Anti -Rusts -j
Anti -Squawk Compounds
Anti -Wear Compounds
Biocides
Buffers
Cetane Improvers
Cold Flow Improvers
Corrosion Inhibitors
Deposit Modifiers
Detergents
Detergent-Di spersants
Dispersants
Extreme Pressure Additives
Lead Scavengers
Lubricity Agents
Metal Deactivators
Oxidation Inhibitors
Pour Point Deoressants
Rust Inhibitors
Tackiness Agents
Storage Stabilizers
Viscosity Index Improvers
Viscosity Modifiers
Total
MM#
0.070
66.500
620.920
72.935
0.428
12.287
42.513
2.250
1.069
4.707
0.432
128.023
11.195
229.586
6.983
238.853
89.804
179.423
9.601
24.352
10.048
12.770
86.496
0.036
0.708
129.775
1.226
1,982.990
MM$
0.099
8.650
221.550
26.877
0.173
4.749
11.325
0.910
0.126
1.612
0.159
29.118
4.071
43.529
4.240
71.662
19.415
25.506
1.482
10.716
1.970
3.216
15.220
0.003
0.384
21.972
0.625
529.359
Includes Friction Modifiers and Anti-Chatter Compounds.

-------
                                 -27-
                               Table 5
                         ADDITIVES FOR FUELS


                     1969 CONSUMPTION BY FUNCTION

  Additive Function                   MMI                 MM$

Anti-Icing                          66.500               8.650
Anti-Knock                         620.920             221.550
Anti-Oxidants                       15.385               8.774
Cetane Improvers                     4.707               1.612
Cold Flow Improvers                  0.432               0.159
Corrosion Inhibitors                21.320               9.942
Deposit Modifiers                   11.195               4.071
Detergents                           8.537               2.984
Lead Scavengers                    179.423              25.506
Metal Deactivators                   3.712               4.936
Rust Inhibitors                     10-]41               ?-°??
Storage btabi nzers                  0./u8               0.364
              Total                942.980             290.607

-------
                                   Table 6
                                             -28-
                              GASOLINE ADDITIVES
A.  Antiknocks
                       best in paraffinic fuels- 1000 ppm
                             10  Ibs/year
                      >• best in aromatic fuels
- Tetraethyl lead —

  Tetramethyl lead -

  Equilibrated mixtures/'""* in certa1n sPec1al  blend "ses
  Methyl cyclopentadienyl  manganese_
  Tricarbonyl
                                               T
                                                              »• best in aromatic
                                                               fuels-  400-100 ppm
B.  Scavengers


C.  Detergents
    Anti-icing,
    Deposit modifiers,
    what-have-you!
D.  Corrosion
    Inhibitors

E.  Antioxidants
F.  Deposit Modifiers

G.  Metal Deactivators
H.  Hydrocarbons
- Ethylene dichloride / ,nn nnm
  Ethylene dibromide_/~ IUU ppm

- Now mostly nitrogenous - 400 ppm+
  HTA (Esso)
  F-310 (Chevron)
  LZ-580 (Mobile, Amoco & others)

  Still some amine phosphates -
  DMA - 4     Shell  & others
  DMA - 5A    (going out)

- Mainly carboxylic acids
  Di-sec. butyl  p-phenylenediamine - 10-20 ppm
  Di-t-butyl cresol

  Phenyldicresyl  phosphate

  Di salicyl propanediamine

  Carrier oils -  1000	>• 5000 ppm
  Polymers - 100	•»• 300 ppm

-------
                    Table 7
-29-
ADDITIVES USED IN AUTOMOTIVE GASOLINE - PREMIUM
Additive
Anti -Knocks
Tetraethyllead
Tetramethyllead
Possible Minor Use
Methyl cycl opentadi enyl -
maganesetri carbonyl
Subtotal
Anti-Oxidants
2,4-dimethyl 6-tert
butyl phenol
2,6-di-tert butyl-4-
methyl phenol
2,6-di-tert butyl
phenol
N^-bis (1,4-dimethyl
pentyl) p-phenylene
diamine
N ,n-butyl -p-ami no
phenol
N,N -diisopropyl-p-
phenylene diamine
N.^-di-sec-butyl-p-
phenylene diamine
Subtotal
1969 CONSUMPTION
Use
Price Level
$/lb wt, vol %
.35 2.34
cc/gal
.36 1.24
cc/gal

7#/
0.59 mbbls
7#/
0.57 mbbls
6#/
.37 mbbls
6#/
.87 mbbls
6#/
.66 mbbls
7#/
.62 mbbls
5#/
.97 mbbls

MM#
285.100
60.690

345.790
1.624
0.738
2.953
0.316
0.189
0.368
1.054
7.242
MM$
101.290
22.150
•
123.440
0.058
0.420
1.092
0.274
0.124
0.228
1.022
4.118

-------
                         -29-
ADDITIVES USED IN AUTOMOTIVE GASOLINE - PREMIUM (Cont.)
Additive
Corrosion Inhibitors
Alkyl and amine
phosphates
Alkyl -di amine naphtha-
lene sulfonate
Fatty acid amines
Fatty acid esters
Subtotal
Deposit Modifiers
Cresyl diphenyl phos-
phate
Methyl diphenyl phos-
phate
Methyl phenyl phos-
phates, mixed
Trimethyl phosphate
Possible Minor Use
Tris (B-Chloroisopropyl)
thionophosphate
Subtotal
Detergents
Fatty acid amides
Surface active alkyl
ammonium di alkyl
phosphates
Subtotal
1969
Price
$/lb
0.51
0.20
0.35
0.25
0.30
0.33
0.35
0.55



0.30
.41

CONSUMPTION
Use
Level
wt, vol %
101
mbbls
50 ppm
50 ppm
50 ppm
6 Ibs
mbbls
6 Ibs
mbbls
6 Ibs
mbbls
6 Ibs
mbbls



50 ppm
2 Ibs
mbbls

MM#
8.966
0.664
0.664
0.664
10.958
2.531
1.899
0.633
1.265

6.328

3.322
1.582
4.904
MM$
4.572
0.132
0.232
0.166
5.102
0.759
0.626
0.221
0.695

2.301

0.996
0.648
1.644

-------
                                     -30-
           ADDITIVES USED IN AUTOMOTIVE GASOLINE - PREMIUM (Cont.)
Additive
Lead Scavengers
Ethyl ene di bromide
Ethylene dichloride
Possible Minor Use
1969
Price
$/lb
0.20
0.09

CONSUMPTION
Use
Level
wt, vol % MM#
0.617
cc/gal 43.315
0.653
cc/gal 53.582

MM$
8.663
4.822

Calcium sulfonate and
  dichlorotoluene
Cresyl diphenyl phosphate
      Subtotal                                       96.897       13.485
Metal Deactivators
N,N]-disalicylidene-                 1.5 Ibs
  l.?-di-aminopropane      1.40      mbbls            1.580        2.120
  Possible Minor Use
N,N2-disalicylal ethy-
  lene diamine
Salicylal-orthoamino-
  phenol
Rust Inhibitors
Alkyl amine salts of
orthophosohoric acids 0.47
Linoleic acid
derivatives 0.15
Possible Minor Use
2 Ibs/
mbbls
7 Ibs
mbbls

0.843 0.396
4.430 0.664

Ammonium dinonyl naphtha-
  lene sulfonate
Fatty acid amides

-------
                                   -31-
           ADDITIVES USED IN AUTOMOTIVE GASOLINE - PREMIUM (Cont.)
   Additive
Isononyl phenoxy tetra-
  ethoxy ethanol
Isooctyl phenoxy tetra-
  ethoxy ethanol
      Subtotal
      Total
                               1969 CONSUMPTION
Price
$/1b
  Use
 Level
wt, vol %
MM#
MM$
                           5.273
                         478.972
                              1.060
                            153.270

-------
                    Table 8
-32-
ADDITIVES USED .IN AUTOMOTIVE GASOLINE - REGULAR
Additive
Anti -Knocks
Tetraethyllead
Tetramethyllead
Subtotal
Anti-Oxidants
2,4-dimethyl 6-tert
butyl phenol
2,6-di-tert-butyl-4-
methyl phenol
2,6-di-tert-butyl
phenol
N.N'-bis (1,4-dimethyl
pentyl) p-phenylene
diamine
N,n-butyl-p-amino
phenol
N,N -diisopropyl-p-
nhenylene-diamine
N.N^di sec butyl-p-
phenylene diamine
Subtotal
Corrosion Inhibitors
Alkyl and amine
phosphate
Alkyl -diamine naphtha-
lene sulfonate
Fatty acid amines
1969
Price
$/1b
0.35
0.36
0.59
0.57
0.37
0.87
0.66
0.62
0.97
0.51
0.20
0.35
CONSUMPTION
Use
Level
wt, vol %
2.03
cc/gal
0.8
cc/gal
7#/
mbbls
7#/
mbbls
8#/
mbbls
6#/
mbbls
6#/
mbbls
7#/
mbbl<:
5#/
mbbls
10#/
mbbls
50 ppm
50 ppm
MM#
227.750
36.028
263.770
1.499
0.681
2.7e6
0.292
0.175
0.340
0.973
6.686
8.276
0.613
0.613
MM$
80.900
13.160
94.060
0.884
0.388
1.008
j-
0.254
0.115
0.210
0.943
3.802
4.220
0.123
0.215

-------
                           -33-
ADDITIVES USED IN AUTOMOTIVE GASOLINE - REGULAR (Cont.)
Additive
Fatty acid esters
Subtotal
Deposit Modifiers
Cresyl diphenyl
phosphate
Methyl diphenyl
phosphate
Methyl phenyl phosphate,
mixed
Trimethyl phosphate
Possible Minor Use
Tri (B-chloroisopropyl )
thionophosphate
Subtotal
Detergents
Surface active alkyl
ammonium u! alkyl
phosphate
Fatty acid amides
Subtotal
Lead Scavengers
Ethyl ene di bromide
Ethylene dichloride
1969
Price
$/lb
0.25
0.30
0.33
0.35
0.55


0.41
0.30
0.20
0.09
CONSUMPTION
Use
Level
wt, vol %
50 ppm
5#/
mbbls
5#/
mbbls
5#/
mbbls
5#/
mbbls


2#/
mbbls
25 ppm
0.51
cc/gal
0.538
cc/gal
MM#
0.613
10.115
1.947
1.460
0.487
0.973

4.867

1.460
1.534
2.994
33.114
40.749
MM$
0.153
4.711
0.584
0.481
0.170
0.535

1.770

0.528
0.459
1.057
6.622
3.667

-------
                                -34-
           ADDITIVES USED IN AUTOMOTIVE GASOLINE - REGULAR (Cont.)
                               1969 CONSUMPTION
                                       Use
                           Price      Level
   Additive                $/lb      wt, vol %         IW          MM$
    Possible Minor Use
Calcium sulfonate and
  dichlorotoluene
Cresyl diphenyl phosphate
      Subtotal                                       73.863       10.289
Metal Deactivator
N.^-disalicylidene-                 2#/
  1,2-diaminopropane       1.40      mbbls            1.946        2.724
    Possible Minor Use
   2
N,N -disalicylal ethylene diamine
Salicylal-orthoaminophenol
Rust Inhibitors
Alkyl amine salts of                 2#/
  orthophosphoric acids    0.47      mbbls            0.779        0.366
Linoleic acid                        7#/
  derivative               0.15      mb^s            4.089        0 613
    Possible Minor Use
Ammonium dir.onyl naph-
  thalene sulfonate
Fatty acid amides
Isononyl phenoxy tetra-
  ethoxy ethanol
Isooctyl phenoxy tetra-
  ethoxy ethanol
      Subtotal                                        4.868        0.979
      Total                                         369.109      119.392

-------
             Table 9
-35-
ADDITIVES USED IN AVIATION GASOLINE
1969 CONSUMPTION
Additive
Anti -Knock
Tetraethyllead
Anti-Oxidants
N,N -diisopropyl-p-
phenylene-diamine
N.^-di-sec butyl-p-
phenylene-diamine
2,4 dimethyl -6-tert
butyl phenol
2,6-di tert butyl 4-
methyl phenol
2,6-dl tert butyl
phenol
Mixed tertiary butyl
phenols
Subtotal
Corrosion Inhibitors
Alkylaminoalkyl phos-
phate in kerosene
Fatty acid amines
Esters of fatty acids
Alkyl diamine naph-
thalene sulfonates
Amine salt of mixed
alkyl acid phosphates
in kerosene
Subtotal
Pri ce
$/lb
0.35
0.62
0.97
0.37
0.57
0.37
0.44
0.51
0.35
0.25
0.20
0.51

Use
Level
wt, vol %
2.61
cc/gal
8#/
mbbls
8#
mbbls
6#/
mbbls
6#/
mbbls
m
mbbls
8#/
mbbls
!#/
mbbls
2#/
mbbls
2#/
mbbls
2#/
mbbls
2#/
mbbls

MM#
11.360
0.011
0.045
0.017
0.017
0.059
0.034
0.183
0.023
0.003
0.003
0.003
0.003
0.035
MM$
4.050
0.007
0.044
0.006
0.009
0.022
0.015
0.103
0.012
0.001
0.001
0.005
0.0014
0.016

-------
                                    -36-
                  ADDITIVES USED IN AVIATION GASOLINE (Cont.)
    Additive
Lead Scavengers
Ethylene dibromide
Metal Deactivates
Alkylamine alkyl phos-
  phate in kerosene
      Total
1969 CONSUMPTION
Use
Price Level
$/lb wt, vol % MM#
1.52
0.20 cc/gal 8.663
hos- !#/
e 0.51 mbbls 0.029
20.270
MM$
1.732
0.015
5.916

-------
                  Table  10
-37-
ADDITIVES USED IN INDUSTRIAL DIESEL FUEL OIL
Additive
Cetane Improvers
Amyl Nitrate
Hexyl Nitrate
Possible Minor Use
Methyl cycl opentadi eny 1 -
manganesetr i carboxy 1
Subtotal
Cold Flow Improver
Nitrated organics
(e.g. Oronite OFA 410)
Delerytinlb
Ethyl MPA-D
Lubrizol 560
Subtotal
Storage Stabilizers
Methacrylate polymers
in kerosene
Organic amine
Subtotal
Total
1969 CONSUMPTION
Use
Price Level
$/lb wt, vol %
0.36 .1-vol
0.33 .1-vol
0.37 .001-wt
0.44 .02-wt
0.45 .02-wt
0.53 .002-wt
0.73 .OC2-wt
MM#
0.202
0.431
0.633
0.006
0.075
0.040
0.115
0.072
0.007
0.079
0.833
MM$
0.072
0.142
0.214
0.002
0.033
0.018
0.051
0.038
O.C05
0.043
0.310

-------
                                  Table 11
                                                                   -38-
                       ADDITIVES USED IN JP-4 JET FUEL
Antl-Icing Compounds
Ethylene glycol mono-
  methyl ether and
  glycerine
Anti-Oxidants
2,4-dimethyl-6-tert
  butyl phenol
2,6-di tertiary butyl
  4-methyl phenol
2,6-di tertiary butyl
  phenol
N.N  diisopropyl-para
  phenylene diamine
N,N  di-secondary butyl
  para phenylenediamine
Tertiary butyl
  phenols
      Subtotal
Corrosion Inhibitors
Alkylamino alky! phos-
  phates in kerosene
Amine salts of mixed
  alkyl acid phosphates
  in kerosene
      Subtotal
Metal Deactivators
Alkylamino alkyl phos-
  phate in kerosene
      Total
1969
Price
$/lb
0.13
0.59
0.57
0.37
0.62
! 0.97
0.44
0.57
0.51
0.51
CONSUMPTION
Use
Level
wt, vol %
.15-vol
8#/
mbbls
6#/
mbbls
7#/
mbbls
mbbls
8#
mbbls
6#/
mbbls
1.250/
mbbls
2#/
mbbls
1.25#/
mbbls
MM#
66.500
0.209
0.078
0.319
0.052
0.210
0.078
0.946
0.082
0.130
0.212
0.038
67.696
MM$
8.650
0.123
0.044
0.118
0.032
0.203
0.034
0.554
0.047
0.066
0.113
0.017
9.334

-------
                                  Table 12
                -39-
                       ADDITIVES USED IN JP-5 JET FUEL
Additive
Anti-Oxidants
2, 4-dimethyl- 6- tertiary
butyl phenol
2, 6-di- tertiary butyl
4-methyl phenol
2, 6-di -tertiary butyl
phenol
N,N -di-isopropyl-para
phenylene diamine
N,N -di -secondary butyl -
para phenylenediamine
Tertiary butyl phenols,
mixed
Subtotal
Metal Deactivator
Alkylamino alkyl phos-
phate in kerosene
Possible Minor Use
1969 CONSUMPTION
Use
Price Level
$/lb wt, vol % MM#
8#/
0.59 mbbls 0.076
6#/
0.57 mbbls 0.029
6#/
0.37 mbbls 0.099
8#/
0.62 mbbls 0.019
8#/
0.97 mbbls 0.076
6#/
u.4t> moDis 0.02y
0.328
2.5#/
0.51 mbbls 0.119

MM$
0.045
0.016
0.037
0.012
0.074
U.UM
0.197
0.060

Amine salts of mixed alkyl
  acid phosphates in
  kerosene
  V
      Total
0.447
0.257

-------
                              Table 13
                                          -40-
     GENERAL CHEMICAL CLASSES OF  REGISTERED GASOLINE ADDITIVES
Chemical Class
Number of
Chemical Compounds
Present in Class
Total  of
Registered Fuel
Additives Containing
Chemical Compounds
Listed in Class
Alcohols
Alkyl Hal ides
Alkyl Phosphates
Alkyl Poly amines
Amines
Anthroqui nones
Aroma tics
Aromatic Phosphates
Azo Compounds
Azo-Naphthels
Imines
Lead Compounds
Naphthalenes
Phenels
Phenylenedi amines
Pyrimidines
Trace Substances
29
79
23
30
42
22
33
26
16
67
4
95
11
29
32
3
10
154
720
97
97
73
44
130
58
32
363
37
912
4
111
175
3
15
Total Individual  Additives Registered for Use  in  Gasoline as of
December 31, 1972 = 322.

-------
                              -41-
     Average exhaust gas emissions for U.  S.  vehicles by age group
are reflected below.  These data are for vehicles tested at the
condition specified in the cities shown:
                                Average Exhaust Emissions*
City and
Year Group
Los Angeles
pre 66
66-67
68-69
70
Chicago
pre 66
66-67
68-69
70
2500
HC (ppm)

390
220
190
130

410
330
220
120
ppm
CO («)

3.1
1.3
1.2
0.9

2.9
2.4
1.2
0.9
HC (ppm)

720
370
350
230

690
590
350
250
idle
CO (%)

4.9
3.1
3.7
2.4

4.4
4.4
3.5
2.6
      *  Voelz, et al;   Journal  of the Air Pollution Control
         Association,  December 72.

-------
                                  -42-
  Gaseous Emissions Relative to Fuel Composition and Fuel Additives
                         John E. Sigsby, Jr.
                         Dr. Richard Miller
                      Dr. Theodore R. Torkelson
Nature:
     The gaseous emissions of automobiles operated on a dynamometer are
typically shown in Table I.  The emissions from the same vehicle operated
with a catalytic system are shown in Table II.
     All tests were made on a car operated on Shell no-lead gasoline.
These tables are very brief summaries and do not include all  of the
detailed composition.  Almost all hydrocarbons can be detected and all
fuel components are directly reflected in the emissions.
     Possible oxygenated materials are reflected in Tables III, IV, and
V.  Nitrogen containing organic compounds is usually found at very low
levels.
     Operational parameters such as break-in, history, vehicle type, fuel
composition, and operating cycle are all critical  to the results and
must be standardized.
     Fuel composition is directly reflected in exhaust composition and
also in particle oxidation and cracked products.  Benzaldehyde and styrene
relfect the C+ mono alkyl benzene concentration.

-------
                                   -43-
'71  Ford
             Table  I

          Current Systems

Emissions in gm/mile operated on LA-4 cycle (Fed. Procedure)
 HC
 CO
 NOX

 Paraffin
 Olefin
 Aromatic

 Dilution  Ratio
 Exhaust Vol  CF/Mile

 Methane
 Ethane
 Acetylene

 iso  Butane
 n Butane
 iso  Pentane
 n Pentane
 Hexanes
 Heptanes
 Total

 Ethylene
 Propylene
 Butenes
 2-me-l-butene
 3-m3-l-butene
 Others
Benzene
Toluene
Tehylbenzene
o, p-xylene
m-xylene
Mesitylene
1,2,4-trimethylbenzene
Others
           Cold Start

        2.32   ±  0.065
       39.30   ±  1.910
        4.02   ±  0.41

        1.13   ±  0.049
        1.07   ±  0.058
        0.15   ±

       10.11   ±  0.15
       92.87   ±  1.65

        0.169  ±  0.005
        0.031  ±  0.001
        0.208  ±  0.004
                                                         Hot  Start
0.004
0.045 ±
0.051 ±
0.008
0.111 ±
0.120
0.342 ±
0.272 ±
0.139 ±
0.077 ±
0.012 ±
0.007 ±
0.164 ±
0.670 ±
0.188 ±
0.201 ±
0.046 ±
0.078 ±
0.070 ±
0.011 ±
0.054 ±
0.061 ±
0.724 ±

0.006
0.005

0.017

0.087
0.006
0.005
0.002
0.001
u.OOl
0.009
0.022
0.008
0.007
0.001
0.005
0.004
0.001
0.004
0.003
0.023
1.81 ±
15.60 ±
4.06 ±
0.90 ±
0.84 ±
0.10 ±
11.23 ±
82.27 ±
0.091 ±
0.021 ±
0.158 ±
0.005
0.077 ±
0.085 ±
0.019 ±
0.108 ±
0.118
0.432 ±
0.212 ±
0.117 ±
0.026 ±
0.011 ±
0.003
0.146 ±
0.572 ±
0.142 ±
0.142 ±
0.031 ±
0.050 ±
0.047 ±
0.005
0.035 ±
0.040 ±
0.504 ±
0.078
2.230
0.13
0.05
0.036
0.007
0.14
0.47
0.010
0.005
0.011

0.016
0.013
0.001
0.002

0.083
0.002
0.006

0.002

0.039
0.050
0.024
0.020
0.001
0.002
0.003

0.002
0.002
0.045

-------
                               -44-

                                 Table II

                              With Catalyst

'71  Ford    Emissions  in gm/mile  operated on LA-4 cycle (Fed. Procedure)

                       Hot  Start,  No Air Added
                HC                            0.16
                CO                            5.69
                NOV                          4.38
                 A          ..

                Paraffins                .    0.11
                Olefins                      0.035
                aromatics                    0.010
                Dilution
                Dilution Ratio              11.16
                Exhaust Vol,  CF/Mile         82.06

                Methane                      0.025
                Ethane                       0.008
                Acetylene                    0.003
                                             0.036

                Isobutane                      0
                n-butane                     0.004
                Isopentane                    0.005
                C(.                            u.OOi
                C^                            0.005
                C7                            0.001
                 1                            0.016

                Ethylene                     0.019
                C3=                          0.006
                C4=                          0.006
                C5=                          0.001
                C6=                          0.001
                Others                       °-004
                                             0.037

                Benzene                      0.022
                Toluene                      0.016
                Ethyl benzene                  0.004
                o,p-xylene                    0.006
                n-xylene                     0.006
                Mesitylene                    0.001
                1,2,4-trimethylbenzene       0.003
                Others                       0.004

-------
                            -45-
                         Table IV

     Oxygenates in Exhaust from Simple Hydrocarbon Fuels


         Oxygenate                      Concentration range, ppm

Acetaldehyde               .                   0.8-4.9
Propionaldehyde (+ acetone)                   2.3-14.0
Acrolein                                      0.2- 5.3
Crotonaldehyde (+ toluene)                    0.1- 7.0
Tiglaldehyde                                 <0.1- 0.7
Benzaldehyde                                 <0.1-13.5
Tolualdehyde                                 <0.1- 2.6
Ethyl benzaldehyde                            <0.1- 0.2
o-Hydroxybenzaldehyde (+ C,Q                 <0.1- 3.5
 aromatic)*11                .
Acetone (+ propionaldehyde)                   2.3-14.0
Methyl ethyl ketone                     ,       <0.1- 1.0
Methyl vinylketone ( + benzene)                 0.1-42.6
Methyl propyl (or isopropyl)
 ketone                                      <0.1- 0.8
3-Methyl-3-buten-2-one                       <0.1- 0.8
4-Methyl-3-penten-2-one                      <0.1- 1.5
Acetophenone                                 <0.1- 0.4
Methanol                                      0.1- 0.6
Ft.hannl                   ^                  <0.1  0.6
C5 alcohol (+ GS aromatic)'                   <0.1- 1.1
2-Buten-l-ol (+ C5H80)                       <0.1- 3.6
Benzylalcohol                                <0.1- 0.6
Phenol + cresol(s)                           <0.1- 6.7
2,2,4,4-Tetramethyltetrahydrofuran           <0.1- 6.4
Benzofuran                                   <0.1- 2.8
Methyl phenyl ether                              <0.1
Methyl formate                               <0.1- 0.7
Nitromethane                                 <0.8- 5.0
CrHfiO                                        <0.1- 0.2
CO                                       <0.1- 0.3
 Values represent concentration levels in exhaust from a'M test fuels,

 Data represent unresolved mixture of propionaldehyde + acetone.
 Chromatographic peak shape suggests acetone to be the predominant
 component.

cToluene is the predominant component.

 The C]Q aromatic hydrocarbon is the predominant component.
p
 Benzene is the predominant component.

 The aromatic hydrocarbon is the predominant component.

-------
                                Table  V
Observed  and  calculated level* (ppm) of oxygenates In exhaust from  simple hydrocarbon fuels
Fuel 	


2 -Me thy 1-
2-butene
+ Iso-
pentone
Obg.
Calc.
2 -Me thy 1-
2-butene
f Iso-
octane
Obs. |Calc.
2-M thyl-
2- utene
+ Iso-
octene
Obs.
Calc.
2 -Me thy 1-
2-butcne
+ toluene
Obs.
Calc.
2-Methyl-
2-butene
+ o-xylene
Obs. |Calc.
Isooctene
+ 180-
octane
Obs.
Calc.
Isooctane
+ toluene
Obs. [Calc.
Isooctane
+ Iso-
pentane
Obs. [Calc.
Isopencane
+ .o-xylene
Obs. [Calc.
Zaooctene
+ £-xylene
Obs. [Calc.
Isooctane
* o-xylene
Obs. |Calc.
2-Methyl-
2-butene
+ Isooctane
+ o-xylene
Obs.
Calc.
Isooctane
t- Isooctene
+ toluene
Obs. [Calc.
                                   HLDEMYDKS

Acroletn -f propylcne oxide
Prop tonaldeh yd 6
Methacroleln + methylfuran
Tlglaldfhyde 	


Ethyl benzaldehyde

Total aldehydes 	
6.3
3.0
3
3 1
1.0
.7
1 2
1
.5
.0
16.2
8.2
3.6
t,
2.2
.4
1
7
1
2
.0
15.9
7.8
2.6
,1
4.0
.9
.9
.8
2
.3
.0
17.6
6.2
2.3
.3
1.4
.3
.2
.3
.1
.1
.0
11.2
6.1
2.5
.0
.4
.6
1.4
1.1
2
.1
.3
12.7
7.2
3.4
.4
1.5
1.2
.6
.7
.7
.1
.0
15.8
4.9
3.4
0
1.5
.0
.4
8.3
1
.2
.0
18.8
6.8
2.5
2
1.5
.5
.1
4.0
1
.1
.0
15.8
8.5
3.5
1
5.3
.0
.0
2.2
10.7
.1
.1
30.6
4.9
3.2
2
1.4
.2
.1
.7
7.5
.2
.1
18.5
4.2
2.3
.0
.1
.0
.5
.5
.2
.2
.0
8.0
5.0
2.3
.3
.2
.9
.6
.5
.2
.1
.0
10.1
3.2
2.5
.1
.4
.0
.0
8.4
.1
.2
.1
15.0
4.4
1.4
. 1
.1
.0
.1
3.3
. i
.1
.0
9.6
9.9
4.7
.0
1.7
.7
.0
.3
1
.5
.0
17.9
4.8
1.6
.2
.4
.1
.1
.4
.1
.2
.0
7.9
KE TONES

Kcthv Ivlny Ike tone .........
Methyl ethylKptonc >•<* .

3-Kethyl-3-buten-2-one. . . .
4-Methyl-3-pentcn-2-one. . .
Total kctones 	
7.9
2.3
1.1
3
.5
2
2
12.5
5.2
1.8
. 7
2
.3
1
1
8.4
9.1
2.3
1.0
2
.7
.0
2
13.5
6.1
1.5
.5
1
.2
1
1
8.6
11.8
3.8
1.2
4
1.0
3
2
18.7
10.4
2.3
.7
1
f 2
.5
1
14.3
8.8
.0
.5
.0
.0
3
2
9.8
4.3
1.4
.5
1
.2
.0
1
6.6
9.5
.0
1.0
.0
.7
.1
3
11.6
3.7
1.3
.5
1
. 2
0
1
5.9
8.5
.8
.3
1
.0
.4
1
10.2
8.6
1.0
.3
. 1
.0
.6
1
10.7
2.8
.0
.0
0
.0
.0
2
3.0
3.2
.1
.1
0
.0
.0
1
3.5
3.7
.8
.3
.1
.0
.0
2
5.1
3.5
.3
.2
.1
.1
.1
.1
4.4
6.8
3.9
.4
1. 1
.0
.0
4.4
17.8
.2
.4
35.1
3.5
2.4
1
.4
.1
.0
.8
7.0
.2
.1
14.6
1.7
2.4
.0
.0
.0
.2
3.4
5.9
.2
.2
14.0
4.2
2.8
.2
.1
1.0
.6
.9
6 9
.2
.1
17.0
2.4
1.1
.1
.8
.0
1.4
8.7
3.1
.2
.0
17.8
3.1
2.1
.1
.1
.0
.1
.5
6 8
.1
.1
13.0
9.8
4.5
.2
2.0
.0
.0
2.1
6.8
.2
.2
25.8
5.5
2.6
.0
.9
.2
.1
.5
4.7
.1
.1
14.9
2.4
2.4
.2
.0
.0
.0
9.4
2
.2
.0
14.8
5.2
2.0
.2
.2
.7
.4
2.5
.1
.1
.0
11.4

2. 1
.0
.0
.0
.9
.0
.3
3.3
1. 1
.1
.1
1
.0
.1
1
1.7
6. 1
.0
.2
.9
.0
.0
.2
7.4
7. 1
.9
.3
1
.0
.6
1
9.1
2.6
.0
.0
.0
.0
.0
.2
2.6
2.8
.2
2
1
.1
.1
2
3.7
9. 7
1.3
.4
.0
.2
.1
2
11.9
4.1
1.0
.3
.1
.2
.0
1
5.8
4.6
.0
.0
.0
.0
.0
.1
4.7
6.8
.8
.2
.1
.0-
.4
.1
8.4
N0NCARBONYLS


Methyl formate 	
E thanol • • • • *
Tetramethyltetrahydrofuran
Propeneni t rile . • .

C H, -0 	
c H 6 	

C.H O 	

r H 0 	
Benzo Cuiran ••*««...« •

Phenol + cresol. .........


2.3
.0
1.9
4
.0
.8
6.8
.5
.7
.4
.0
.0
.1
.1
.0
.0
14.0
42.7
2.3
.0
4.1
5
.0
4
4.7
.0
1.1
.1
.0
.0
.0
.1
.0
.0
13.3
37.6
2.3
.0
.6
.5
1.0
,3
1.3
.3
.9
.0
.0
.0
.0
.0
.0
.0
7.2
38.3
2.2
.0
3.4
.4
.1
.3
2.8
.1
.2
.0
.1
.0
.0
.1
.0
.0
9.7
29.5
1.8
.0
.4
5
.0
4
4.1
.6
.3
2.8
.0
.0
.1
.1
.0
.0
11.1
42.5
2.3
.0
3.2
5
.0
.1
3.0
.1
.2
3.2
.1
.0
.0
.0
.0
.0
12.7
42.8
1.7
.0
.1
6
.0
5
1.8
.6
.0
.6
.0
.0
.0
.1
.0
.4
6.4
35.0
1.5
.3
2.9
3
.0
5
2.4
.1
.2
.0
.1
.0
.0
2
.0
.0
8.5
30.9
1.4
.0
.0
.0
.0
.7
1.2
.0
.5
.0
.0
.0
.0
.2
.2
.8
5.0
47.0
1.3
.0
2.6
3
.0
I
2.3
.1
.2
.0
.1
.0
.0
5
.1
3
7.9
32.3
1.6
.0
1.-'.
5
.0
8
2.6
.0
.2
3.2
.0
.0
.1
.1
.0
.1
10.6
28.8
2.5
.0
1.3
2
.1
1
1.8
.1
.0
.0
3.5
.0
.1
1
.0
1
9.9
30.7
1.4
.0
1.2
8
.0
5
1.0
.0
. .0
.0
.0
.1
.0
1
.3
1 2
6.6
24.6
1.8
.2
.8
. 1
.1
3
1.1
.0
.0
.0
.0
.0
.0
2
.0
.0
4.6
17.7
3.3
.0
2.1
1 2
.6
5
3.8
.0
.0
.2
.0
.0
.0
1
.0
.1
11.9
34.9
2.3
.0
1.5
.2
.1
3
2.6
.0
.6
.1
.0
.0
.0
.1
.0
.0
7.8
20.1
1.6
.0
.0
o
.0
4
2.3
.0
.0
.0
.0
.0
.1
.5
.5
1.6
7.0
45.4
1.4
.0
.6
1
.0
2
2.1
.0
.6
.1
.0
.0
.0
.4
.1
3
5.9
22.2
1.3
.0
.5
1 0
.0
8
2.6
.4
.0
4.2
.0
.0
.0
.4
.2
1.1
12.5
33.9
1.9
.0
.6
.2
.0
0
1.4
.1
.0
3.9
.0
.0
.0
.5
.1
.3
9.0
35.1
1.4
.0
.0
.0
.0
.5
.5
.0
.0
.0
.0
.0
.1
.4
.2
1.1
4.2
24.8
1.7
.0
.8
.1
.1
.1
1.2
.0
.0
.0
.0
.0
.0
.5
.1
.3
4.9
21.6
2.2
.0
.0
.0
.0
.9
.9
.0
.4
.0
.0
.0
.0
.3
.3
.7
5.7
43.4
1.7
.0
2.2
.3
.0
.2
2.1
.1
.1
.0
1.0
.0
.0
.3
.1
.2
8.3
29.0
1.7
.0
.7
.5
.0
.3
.6
.0
.1
.7
.1
.0
.0
.1
.1
.6
5.5
25.3
2.2
.2
1.0
.2
.0
.2 '
1.4
.1
.0 ..
2.6
.0
.0
.1
.2
.0
.6
8.2
28.0
                                                                                                                       en

-------
                             -47-
     Sampling of the exhaust stream depends upon the ultimate goal
of the study., "Constant Volume Sampling Systems" (CVS), see Federal
Register, November 1972, are adequate for the gross analysis of controlled
species, i.e., CO, N(L and total hydrocarbon.  Such systems can be used
for detailed hydrocarbon analysis.  This latter is performed for
between 100 and 200 individual compounds by programmed gas chroma-
tography.
     Oxygenates in exhaust are extremely likely.  Any surface that may
be moist will remove large amounts of them.  The relative humidity
must be maintained at less than 50% to prevent this phenomenon.
     Any sample-handling system must be thoroughly evaluated for its
adequacy in handling the specific compounds of interest.
     Oxygenates are determined by a variety of techniques including
direct gas chromatoqraphy,  gas  chromatoqraphy-mass spectoscoD.v.
and gas chromatography of chemical derivatives.  There are no satis-
factory continuous analyses.
     Large volume samples can be collected by cryogenic trapping
(see attached) or by collection on packed traps.  Unpacked traps may
not be quantitative due to the passage of particles through the trap.
Packed traps may react with components of the system through changing
the composition of the samples.  Reactions of condensed phase
materials with other exhaust gas components, such as NO^ are very
common and must be avoided.  These reactions should be determined
analytically by comparing gas phase concentrations and composition
with lhat of the condensed phase.

-------
                                  -48-
     Whatever witchcraft is necessary to obtain an accurate sample
must be performed.
     The effects of emission control  systems  may be major.   The obvious
primary effect is to lower the concentration  of the controlled components,
In addition, composition changes may  occur depending upon its  operating
condition.

-------
                          -49-

             Report of Particulates Subcommittee
     Members                            Affiliation
1.  Mr. Kirby Campbell            EPA - Cincinnati
2.  Mr. Thomas Gleason            EPA - ORM
3.  Dr. Sidney Laskin             NYU - School  of Environmental  Medicine
4.  Dr. R. L. Bradow              EPA - RTP

Sampling for lexicological Studies
     Practicality of toxicological experiments  with 200 to 1000
animals suggests an ultimate need of at least 15 grams, preferably
50 to 100 grams of material.  The sampling system used in collection
of particulate must, therefore, be compatible with  large scale collection
of particulate material.  Automobile exhaust for typical leaded  fuels
contains mostly lead particles with MMED less than  0.2 p.  Typical
emission rates of these particles are in the order of 0.5 grams/mile. •
Filtration of the whole exhaust can yield adequate  amounts of material
in about 100 miles of operation, mostly of lead particles.  With
unleaded fuels typical emissions rates are in the order of 0.04  g/mile.
Concequently, 300 - 400 miles of LA-4 operation or 40 to 50 22-minute
cycles are required.  Results of extraction experiments suggests that
the organic matter is essentially completely extractable in appropriate
solvents.  Therefore, it appears that standardized  filtration experiment
is the most appropriate route.
     The combination of methods including cryogenic trapping and
adiabotic expansion plus filtration may result  in higher collection
rates.  An alternative scheme might employ centrifugation as a means

-------
                          -50-

of collection of large volumes of participates.

Analytical Results
     Analysis of inorganic matter in particulate matter has been
the main thrust of DOW Chemical Company research for EPA.   With
leaded fuels, mixed lead halides and oxyhalides are the primary
constituents of the total particulate.  Other inorganic compounds
found in the particulate include PbSO^, Ni 0, NH4 Br, PbgC(CPOJ3,
Quartz, calcite, NaCl, NH4C1, 4Pb Pb S04, Fe304, Fe203, Fe 0 OH, ZnO,
Fe.
     Particle sizes characteristic of these compounds were obtained
in this work by atomic absorption spectroscopy of Andersen sampler.
Results for a few characteristic elements are shown below in Table 1.
The Table indicates that organic matter is present primarily in the
very small particles.  Under most conditions, lead particles are also
very fine, about 0.1 u in effective median diameter, there are conditions
in which the particle size distribution is shifted toward  larger
particles.
     Organic substances in the particulate range in molecular size
from about C,r to about C™ as judged by a boiling point  type
G. C. separation.  This molecular weight range is similar  to that
of organics in air samples.  Although filters differ in collection
efficiency, the molecular weight distribution of compounds extracted
from various filter batches is similar.  With non-leaded fuels, the

-------
                            -51-
majority of the collected parti oil ate matter is solvent extractable
although protic solvents may be necessary to complete this extraction.
With leaded fuels, the solvent soluble fraction composes 2 to 6%
of the total.  Table 2 presents some of the detailed analytical data
obtained in the DOW work showing small amounts of carboxylic acids
(presented as wt. % benzoic acid), phenolics, aldehydes and aromatic
ketones.  Carbon, hydrogen, and nitrogen analysis of the overall
organic particulate and of the extract have been made in a few cases.
The data for the material on the filter and for the extract is quite
similar; the majority of the extract weight is accounted for by
carbon and hydrogen and the C/H ratio is approximately 2.  Nitrogen
accounts for about 2% by weight of the extract.  Therefore, there is
H lllfPllhnnH that cnmo nvnanir- ni t^nnonnuc mal-ov*-! al i.iill k« nv.*%<-'M»4-
                  "•••" -'*/	- 	., ^ ....-«._ . . . «• ~ s» f  I M	 »**. ftt.v'W*!**
as a background even with no N-bearing additive present.
     Table 3 presents high resolution mass spectrometric analysis
of the overall particulate indicating appreciable quantities of both
nitrogen and oxygen bearing substituents.  Table 4 presents possible
hydrocarbon types by mass spectrometry.  It is apparent that the
great majority of the organic matter is aliphatic with appreciable
amounts of alkyl benzenes and smaller amounts of higher aromatic
compounds.  In a number of runs with 0.5 g/gal TEL bearing fuels,
the BaP content of the total particulate ranged from 30 to 1000 ppm
by weight.

-------
                                -52-


                               Table 1

               Mass Median Equivalent Diameter, Microns
                                Component

   Fuel       Run No.        Pb        Cl       Br       Organics
Gasoline 1+      7          0.1        0.1      0.1         0.1
3 g. TEL as
motor mix

Gasoline 2 +    23         <0.1       <0.1     <0.1        <0.1
3 g. TEL as
motor mix

Gasoline 1+9          2.0        1.2       —        <0.1
3 g. TEL + EDC

Amoco No Lead   29          0.1        3.3     <0.10       <0.1

-------
Fuel
Run No.
                                             Table 2

                             Organic Analysis of Collected Particulates
  Benzene and
Methanol Soluble
     wt. %
  Acidity as    Phenolics as
wt. % C00H    (fr-OH. wt. %
  Carbonyl
Asbenzophene   Aldehyde
   wt. %        wt. %
Gasoline 1 +
3 cc TEL
Amoco
no lead
Gasoline 2 +
3 cc TEL
Amoco + 0.5 g TEL

19

22

24
40

8.6

55

1.7
6.5
	 1—
0.7

0.3

0.1
0.8

0.08

0.2

0.02
0.2

<1.7

2.0

2.5
1.7

—

1

0.6
2.5
                                                                                                                I
                                                                                                                in
                                                                                                                CO

-------
                   -54-
                Table 3
  Percent of Total Ion Content by High
Resolution Mass Spectrometry of Extract
Ion Types
CH
CHO
CH02
CHN
CHN2
Sample A
90.2
4.2
4.3
1.3
0.03
Sample B
89.3
3.2
6.8
0.7
0.01

-------
                                 -55-
                              Table 4

                    HYDROCARBON TYPE ANALYSIS
                 HIGH  RESOLUTION MASS SPECTROMETRY
                  Sample A*           Sample B
 Hydrocarbon   7 mg  10% Organic   122  mg  <1.0%
Type
Orqa
nic
% Volume
C H
n 2n
CnH2n
Cn"2n
CnH2n
CnH2n
CM,.
n 2n
cnH2n
f 1 1
"n"2n
CnH2n
CnM2n
CnH2n
CLI
«"o«
n 2n
CnH2n
CnH2n
CnH2n
C H0
n 2n
+ 2


- 2
- 4
- 6
- 8

- 10
1 0
1 i-
- 14
- 16
- 18
- 20

- 22
- 24
- 26
- 28

21.0

23
!4.
3
26
10

1
A r>
U . O
0.02
0.83
0.45
0.05

--
--
--
.. «.

46.

20
14
3
16
<0.

0.
^
u .
0.
-
0.
_

0.
0.
-
_

0





1

1

-------
                          -56-
     Apparently the exhaust particulate is composed primarily
of paraffinic and olefinic hydrocarbons, alkylbenzenes with lesser
amounts of carboxylic acids, probably esters and polynuclear aromatics
Small but significant amounts of nitrogenous species are also present.

-------
                                -57-    •     '  '      ..'. .  Y  '.




            Automotive Exhaust Emissions Control Devices





     The 1970 Clean Air Act Amendment requires that exhaust emissions



from motor vehicles be reduced by at least 90% from 1970 levels.   Such



reductions are to be achieved by 1975 for carbon monoxide and hydrocar-



bons and by 1976 for oxides of nitrogen.  As a result of these required



emissions reductions, much attention has been paid to add-on catalytic



devices, engine modifications, etc.   It appears, at the present time,



that catalytic devices will provide  the only reasonable method for



achieving the emissions standards in model years 1975 and 1976.  Although



little data are available which reflect the effect of such devices on



emissions other than those regulated (HC, CO, NO ), the following dis-
                                                /\


cussion provides research data currently available on prototype devices.

-------
                           -57-
  Effect of Emission Control Devices on Participate Emissions

     Attached is a summary of data which shows the effect on
participates of some control devices which are currently being
considered for use on vehicles.
     These devices were tested on a 1972 400 CID Pontiac engine.   The
particulate was collected on fiberglass filter pads, and in an
Andersen impactor.  The sampling period in all cases was 2 hours.
The federal 23 minute LA-4 cycle does not give enough particulate
collected for a meaningful analysis.
     The devices are proprietory, and therefore are coded.  Catalysts
B and C are oxidation catalysts, while A is a reducing catalyst.
     Some preliminary conclusion are as follows:
     1.  Grams/mile particulate increased at 60 mph for all
catalysts, compared to the baseline.
     2.  At 30 mph, grams/mile increased in one instance, but
decreased in 2 cases.
     3.  Aldehydes in the conden^ate decreased in all cases.
     4.  NH3 did not appear to be significantly changed.
     5.  Carbon percentage in the particulate decreased with
use of the catalysts.
     6.  BaP was somewhat less, but not significantly so.
     7.  Trace metals which could be attributed to catalyst degradation
did not increase as a percent of the total particulate.
     8.  Grams/mile particulate decreased with time and miles on
vehicle studies.

-------
Baseline, Unleaded Fuel, 400 CID Pontiac Engine
Test Mode
30
60

30
60

30
60

30
60
mph
mph

mph
mph

mph
mph

mph
mph
Grams/Mile
Parti cul ate
.0238
.0167

.0968
.0905

.0048
.1052

nil
.0554
rrn i
HCHO
210
360

14
12

19
5

28
5
ii ounu-iiaa i.
NH3
6
10
Catalyst A,

4
Catalyst B
36
.9 7
Catalyst C

.3
c rrn
Hydrocarbon
275
145
Unleaded Fuel ,
75
20
, Unleaded Fuul
40
5
, Unleaded Fuul
37.5
1
10 n i
in Parti cul ate

<.153
400 CID Pontiac

.05
, 400 CID Pontiac

.02
, 400 CID Pontiac

.09
o
in Parti cul ate
76
59
Engine
<1

Engine
7
2
Engine

11
.9
.4

.0
.6

.9
.9


.1
(jpiii oar \J\J\
in Parti cul ate N00
11
1

<.
2 850
2 850

35 450
1

7 330

in
NO
260
575

570
1250

145
1

1

0 65

6
9
1600

1100
1500
                                                                                    00

-------
                         -59-
                PARTICULATE COMPARISONS
1 CFM FILTER - 142 mm fiberglass

VEHICLE    VEH/MILES    CONV/MILFS    MODE     G/MILES
                                                  •
BASELINE   2886            -         60 MPH   ,0049
BASELINE   8572            -         60 MPH   ,0067

61314      4816            *         60 MPH   ,0544
61314     34000            *         60 MPH   ,0066

61329      6700          3200         60 MPH   ,0519
61329     11300          7800         60 MPH   ,0291

PONTiAC    4325           455         60 MPH   ,0504
           6000          2130         60 MPH   ,0360
          10841          6971         60 MPH   ,0385
          15851         11981         60 MPH   ,0257
  CONV, MILES UNKNOWN

-------
                            -60-

             Polycyclic Aromatic Hydrocarbons (PAH)
         and Other Possible Hazards in Automotive Exhaust

     In terms of carcinogenic bioassay studies, as to some extent in
analytical studies, the following factors are of importance:  The
sample should be large enough for biological study.  Two types of
samples must be collected -- vapor phase and particulate material.
The sample to be meaningful must be representative of automotive
exhaust material in the air as breathed by human beings.  The integrity
of the sample  must be maintained over the period of time necessary
for analytical and biological  studies.
     Since another group is  covering direct sampling of automotive
exhaust, we will briefly mention sampling of ambient air mainly polluted
with automotive exhaust materials.  The vapor phase can be collected
on wood charcoal, a chromosorb or some other gas chromatographic
material, a liquid phase organic material bonded to an inorganic
phase.  The reason for collecting a vapor is because some carcinogenic
materials are vapors or partial vapors, e.g., tetracyclic aromatic
hydrocarbons, azo arenes (such as some of the methyl benzacridines),
long chain aliphatic hydrocarbon epcxides, diepoxides, slkyl sulfonates,
aultones, nitrosamines, etc.  Only the polynuclear compounds have been
identified in automotive exhaust or in air polluted by exhaust fumes.
However, EPA has a contract to sample atmospheric vapors and assay
for the above types of carcinogens.  Phenols are of some importance
here since they have been shown to have some carcinogenic effect.

-------
                            -61-
A list of some of these phenols found in auto exhaust is shown
in Table I.  Phenols are emitted in much larger amounts from the
automobile than are the PAH.  They are usually collected in an impinger
containing 0.1 N NaOH.  A small proportion of the monocyclic phenols
is found in the particulate phase.  EPA has a contract to develop a
better method of sampling for phenols.  In this case a solid device
will be used for collection.  Total phenols can be determined by
several colorimetric methods, e.g., 4-aminoantipyrine and p-nitro-
benzenediazonium salts are usually used.
     For analysis of the various individual vapors gas chromatography
has been the method of choice.  It is probably the best method.  For
nitrosamines gas chromatography - mass spectroscopy has been used to
assay and characterize nitrosamines found in the environment.
     The particulate phase of automotive exhaust-polluted air is usually
collected on glass fiber filters.  Studies are available on efficiency
and reproducibility of collection.  Some of the hydrocarbons (vapor
and particulate) that have been found in automotive exhaust are shown
in Table II.  Those compounds with carcinogenic activity are starred.
About a dozen of the compounds are carcinogenic.  Some of the methyl
derivatives,  such as the methylfluoranthenes, and methylbenzanthracenes
could have  biological activity.
     Another group of compounds that could be of importance are the
long chain aliphatic hydrocarbons.  These can be analyzed by molecular
sieve chromatography, iodination to get rid of the olefins and
chromatography followed by gas chromatography.  The total aliphatic
hydrocarbons are of possible importance as carcinogens.

-------
                             -62-



                           Table I

                    Phenols in Auto Exhaust
         Phenol
         0-Cresol
         m-Cresol
         p-Cresol
         2,4-Dimethylphenol
2,3-Dimethylphenol
3,4-Dimethylphenol
2,3,5-Trimethyl phenol
Salicylaldehyde
                           Table II

        Polycyclic Aromatic Hydrocarbons in Auto Exhaust
Naphthalene'
Acenaphthylene
Anthracene, A
Alkyl As
Phenanthrene
Trimethylphenanthrenes
Benz(a)anthracene, BaA*
Methyl BaA
Dimethyl BaA
Chrysene, C*
Methyl C
Dimethyl C
Fluoranthene, Ft
Methyl Ft
Pyrene, P
Methyl P
11 H-Benzo(b)fluorene, BbF
Methyl BF
Triphenylene
Naphthacene
Benzo(a)pyrene, BaP*
Methyl BaP
Dimethyl BaP
Benzo(e)pyrene, BeP*
Methyl BeP
Dimethyl BeP
Benzo(ghi)fluoranthene
Benzo(b)fluoranthene*
Benzo(j)f1uoranthene*
Benzo(k)fluoranthene*
Dibenz(a,h)anthracene*
Dibenzofluorenes
Pentaphene
Perylene
Anthanthrene
Benzo(ghi)perylene
Dibenzo(a,l)naphthacene
Dibenzo(a,e)pyrene*
Di benzo(a ,h)pyrene*
Dibenzo(a,ljpyrene*
Indeno(l,2,3-cd)fluoranthene
IndenoO ,2,3-cd)pyrene*


Coronene
Dibenzo(b,pqr)perylene

          8
Tribenzo(h.rst)pentaphene
*Carcinogenic

-------
                                                  -63-
            'Multi-Stage   Cryogenic   Trapping  System

                   JAMES P. CONKLE, M.S., JAMES W. REGISTER, B.S., and GORDON  L. WORTH
  A portable, easily  operated, multi-stage  cryogenic  trapping
system contained in a box 86 x 66  x 61 centimeters has been
developed.  Liquid nitrogen, gaseous nitrogen, ice, dry ice and
110 volt 60 cycle power required for  operation of the system are
available to most military installations.
  Ice formation in the -78:C trapping  cylinder entrance tube
and liquid oxygen formation in the -175:C trapping cylinder
were eliminated in the design of the system. Catalytic conversion
of trapped materials was minimized by use of stainless steel and
Teflon.  Operation of  the system was simplified by inclusion of
a liquid nitrogen level-controller.
  Partial separation of compounds was  accomplished by oper-
ating the  trapping cylinders of the system  at three  different
temperatures.   Several compounds are  listed according to  the
temperature at which  they are expected to  be  concentrated  in
significant quantities.  The system is clTicient for concentration
of micro and  macro contaminants in an atmosphere.  The con-
centration of a contaminant in a sample area may be estimated
from the total trapping time, the flow through the system during
trapping and  the concentration  of the contaminant in the trap-
ping cylinders.
       DETERMINATION of trace contaminants in a
 •*• spacecraft, an  aircraft  or  the  ambient atmosphere
     1 .  .!•
     DC  111
sample  collection, sample concentration,  separation of
the individual contaminants, identification and quanti-
tation.
  Techniques that  have been used for sample collec-
tion, sample  concentration and separation of the indi-
vidual contaminants include gas pressurization in steel
bottles,*  activated carbon bed sampling1-1-*  and cryo-
genic trapping.1  Each of these  methods has its  dis-
advantages.  Gas pressurization in the steel  cylinders
does not concentrate the sample appreciably.  Carbon
bed  sampling, while  it  effectively  concentrates  the
sample,  requires  additional  operations to separate the
contaminants from the carbon. In carbon  bed sampling
catalytic reaction may  cause degradation  of a contami-
nant or  interaction between contaminants.  One cryo-
genic system5 that has been used effectively to concen-
trate  samples is  constructed of delicate  material  and
requires  that the  sample be maintained at the temper-
ature at which  it was taken,  or  that  the  sample  be
transferred to another container for shipment or storage.
  One approach which does not have the disadvantages
of other techniques  is multi-stage  cryogenic  trapping.
This technique permits greater concentration than the
prcssurization approach,  separates the  contaminants
according to their vapor pressures and degradation is
minimized  due to the relatively  low temperatures  in-
volved.  This paper will describe the design and evalu-
  From the Chemical Support Section, Environmental  Systems
Branch, Bioastronautics Department, USAF School of Aerospace
Medicine, Brooks Air Force Base, Tcx.is.
 ation of such a system and discuss the potential uses
 of this device.
                     METHOD

   The air stream from which the contaminants are to
 be removed and concentrated is passed through sample
 cylinders maintained at  three different  temperatures.
 The trapping system uses an ice bath at 0CC, a pulver-
 ized dry ice bath at -7SCC and a liquid  nitrogen bath
 regulated to -175"C to fractionate the contaminants in
 the air stream.  Materials which are not concentrated
 are oxygen, nitrogen and compounds which  have  suffi-
 cient vapor  pressure at -175CC to pass through the
 system.
   The air stream,  consisting  of atmospheric gas,  first
 enters a flow meter (Figure 1) and  then passes to the
 first  trapping cylinder,  which is maintained at a  tem-
 perature of 0~C with ice water. The gas, having passed
 through the ice bath trapping cylinder, flows through a
 heated  inlet  into the trapping cylinder,  maintained at
 -7SCC with pulverized dry ice. The pulverized dry ice
 reouires occasional tamnine to ensure  contact with the
 wall of the trapping cylinder.  The gas then passes to
 a trapping cylinder maintained at -175"C where many
 of the materials not previously removed  from the gas
 stream are condensed.  The remaining gas is  conducted
 to the  vacuum  inlet of a circulating pump  and ex-
 hausted  by the pump  into the atmosphere or into  a
 closed ecological  system.
  The trapping cylinders (Figure 2) are stainless  steel
 with an  internal  volume of 150 cc.  The  cylinders are
 S'ted with Swagelock connections, modified pipe fittings
 and  needle valves.  Teflon and stainless  steel arc  used
 throughout the  system to  minimize  catalytic conver-
 sions and contamination of samples.  A thermocouple is
 mounted  through  a tapped port in th*5 bottom of the
 cylinder.  Temperatures are  monitored  with  copper-
 constantan thermocouples on a pyrometer calibrated in
 degrees centigrade.
  In the -7SCC trapping cylinder there  is a rapid ice
 formation in  the inlet tube. This formation, due to the
  Fig. 1.  diagrammatic flow  representation, multi-stage  cryo-
genic trapping system.
                           Reprinti-d from Acrosjiacc Medicine, Vol. 36, No. 9, September 1965

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                     . A MULTI-STAGE CRYOGENIC  TRAPPING SYSTEM—CON KLE, ET AL
                                        -64-
  Fig. 2.  Trapping  cylinders, multi-stage cryogenic trapping
 system.

 temperature gradient along  (or  down) the  entrance
 tube, is prevented  by heating the entrance  tube.  The
 heater consists  of  a 1/16-inch stainless steel  rod in-
 serted through a Teflon insulated  Swagelock fitting at
 the top of the cylinder. It is positioned in  the center
 of the entrance  tube and projects to the bottom where
 it is  bent at a  90°  angle.  The  rod is threaded  and
 secured to the wall  of the entrance tube with a  nut.
 This connection serves as the electrical contact between
 the cylinder and the rod.  Electrical  energy  from a
 variable  transformer is applied across  the primary  of
 a filament transformer, the secondary of which is  con-
 nected to the rod and cylinder wall. Sufficient energy
 is applied to the healei to prevent the formation of ice
 in the tube without affecting the  operational tempera-
 ture of the trap.
  The final trapping cylinder is  controlled  at a tem-
 perature of -175CC in order  to prevent the formation
 and entrapment of liquid oxygen  (-1S3?C at standard
 pressure).  The  presence of  liquid oxygen in  the  trap
 would present an explosive hazard for personnel han-
dling the cylinders,  and  it  would make available a
supply of oxygen for degradation  of the original con-
taminants and the formation of new compounds.
  This cylinder  is  positioned with two glass-phenolic
rings  in a well which is surrounded by liquid  nitrogen
 (Figure 3).  A  flow of  dry  warm nitrogen  from  the
bottom  of the well  controls the  temperature of  the
 trapping cylinder. The flow of gaseous nitrogen is regu-
lated with a micrometer needle valve. One of the glass-
phenolic rings covers the top of the well to maintain a
positive pressure  of gaseous nitrogen and prevent back
870   Aerospace Medicine • September 1965
 diffusion of atmospheric air and  liquid oxygen  forma-
 tion.
   The well is positioned by a fitted lid for the  Dewar
 flask. .The lid also contains a vent, a well for the liquid
 nitrogen level  sensor  and the liquid nitrogen filling
 device.
   A liquid nitrogen level control device was developed
 to simplify the operation of the system (Figure 4).  The
 level controller uses a  thermister  as a sensor so that  a
 change in resistance of  the thermister, in or out of liquid
 nitrogen, results in a change in  current.  The change
 in current operates a  control meter which actuates  a
 solenoid operated liquid nitrogen transfer valve.
   The trapping system was desicned for use in studies
 of trace contaminants in simulated  space cabin  atmos-
 pheres but  may be used in any situation where there
 is a desire to concentrate atmospheric contaminants for
 identification and  quantisation.  The unit  is  portable
 and easily  operated, making air contaminant studies
 feasible  in  locations which  do not  have  facilities  for
 analysis of complex chemical mixtures.  The multi-stage
 cryogenic trapping system (Figure 5) is put into oper-
 ation by connecting liquid nitrogen and gaseous nitro-
 gen  to their respective inputs and  providing 110  volt
 60 cycle power.  The liquid  nitrogen controller is acti-
 vated  and the -175:C  trap  Dewar filled,  ice water is
 placed in the 0°C trap Dewar and pulverized  dry ice
 in the -IS'C  trap  Dewar.  Caseous nitrogen  flow is
 adjusted  to  achieve the desired temperature in   the
 liquid  nitroeen tran well. Thf  «\-<;tpm i«
                   VCMT-
                                    _T" LIOUlO NITROGEN
                                     SUPPLY
                              TO THERMOCOUPLE READOUT
   IIOUIO NITROGEN
   LEVEL SENSOR
                                OEWAR FLASK
           LIQUID NITROGEN TRAP
              — CROSS SECTION	

  Fig. 3.  Liquid nitrogen trap, multi-stage cryogenic trapping
system.

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                      A MULTI-STAGE CRYOGENIC  TRAPPING SYSTEM—CONKLE, ET  AL
                                        -65-
   Fig. 4. Sche-
 matic of liquid
 nitrogen level
 controller.
a box 86 X 66 x 61  centimeters.  The  unit requires  25
liters of liquid nitrogen,  11.3  kilograms  dry  ice, 11.3
kilograms ice and 1.41 standard cubic meters of gaseous
nitrogen for six hours' operation.  The  need for a gase-
ous nitrogen  source  may be eliminated by heating the
boil-off of liquid  nitrogen to provide the required dry
warm nitrogen for the control of the trapping cylinder
temperature.
  Preliminary evaluation  of the  multi-stace  cryogenic
trapping system was made  using a  255-liter chamber.
The chamber was evacuated and 5 microliters ol acetone
 JJ  1  rpl   1    1    r      t    '• l 1 tr\ T^  'II*   of 1
of Hg with nitrogen.  The  amount  of acetone  in the
chamber was determined by extraction of  a sample into
a 10-meter multi-path infra red  cell,  and quantitated
by infra red absorbance (Figure  6).
  The trapping system  was attached  to  the  chamber
and operated  for six hours.  The  flow  through the sys-
tem was indicated by a variable-area  flow meter.  At
the end of the trapping period the amount of acetone
in the chamber and  the trapping cylinders was deter-
mined by infra red absorbance.


           RESULTS AND DISCUSSION
  Information gained during  the  preliminary tests  of
the system (Table I) indicate  that a useful tool for the
study  of  trace  contaminants  was developed.  Initial
chamber, final chamber and the -175:C trapping cylin-
der contents and  concentrations  were  determined by
infra red absorbancc and are reported in milligrams  or
parts per million.
  The  amount of acetone in the chamber at any time
t can be expressed by the 1st  order reaction equation:
   Fig. 5.  Portable multi-stage cryogenic trapping syiteni.

                 -kt
        C, = C0e                      (equation 1)
where
  Ct = amount of acetone in the chamber at time t  in
      minutes.
  C0 = initial  amount of acetone in the chamber.
  k = a constant.
  e=2.71S2S.
  t = trapping time  in minutes.
  Assuming complete removal of acetone from  the gas
passing through the trapping system, a theoretical value
for k in  equation 1 was determined.  The  amount of
acetone removed in successive one minute  increments
was summatcd for a period of ten minutes.  The result-
ant was subtracted  from the initial amount  of acetone
                     TABLE i. RESULTS OF PRELIMINARY EVALUATION: EFFICIENCY AND RECOVERY

                                    Amount of Acttone     Amount of Acetone in     Percent    Percent
                         Flow  Time  in Chamber (mg.)  175°C Trapiiini; Cylinder (m?.)  Recovery   Efficiency
                     Run cc/min  min    Initial  Final   .Measured  Predicted Calculated   	F_      _F
                      A   B    C    .  D      E       F      C      D-E     D-E      C
1
2
3
380
300
300
360
SCO
360
4.072
4.210
4.210
2.610
3.117
2.900
1.50
1.07
1.19
1.702
1.457
1.457
1. 49 1
1.093
1.310
102.6
97.9
90.8
88.1
73.4
81.7
                                                                     Aerospoce Medicine  • Scprembcr  1965   871

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                    A MULTI-STAGE CRYOGENIC TRAPPING  SYSTEM—CONKLE.  ET AL
                                                                                         -66-
                                                   IT •-,• '-i,'-i -nrpV,
       -i-i
              -J -
             .  i
                    I   I
                      T~n~t:T~t~!"F~T  -Tt:1-rr\
                 -;• rJ7{-:l-j4-!-T-t™H-  -|~[~-f"Hf\
                 "1" ^^tTVtT7"'"  ~ llillli
4000   MOO
             - -l-i-
            .1 i  J.
            •'  I   I
            1800 ^ZOOO
                                                             -r
BOO   rroo ~^~ BOO '  ooo   uoo
' woo   eoo   eoo
                                                                        Fig. 6. Typical initial and  final infra
                                                                      red scans.
                    •  •    i                   i     '       i      II
                    . _._._._ -J. FINAL  - -i	1" ,- <— T	t—-II-
                    I  '    !   i     •   I   •  '   I  I   I    !   I  ,   '  I
                        i     I   ,     I   [  •   I  I   - I    •  I      II
           •o -t -,~.-  -:T - f -r-

              I      '  I -      I       «• !   :  •'  ' I
             _'....i_.*i  I_L._. _'_ J	.  __i	^J—li
 4000
       J2CO
In nVifnin HIP amount of arr>tonr> in tlip rJininhcr n
ten minutes. The initial amount of acetone in the cham-
ber (C0), the final amount oi acetone in  the chamber
('Ct)  and the  time  (t = 10 minutes) were  substituted
in equation 1 to obtain k.
  To  predict the  amount of acetone  in the chamber
after a trapping period of 360 minutes the initial amount
(C0), the  trapping time (t = 360 minutes) and the
theoretical value for k were substituted in equation J.
  The predicted amount of acetone in the -175:C trap-
ping cylinder was determined by subtraction  of the
predicted final amount of acetone  (C,) from the initial
amount of acetone (C0) in the chamber.
  The multi-stage  cryogenic trapping system efficiency
was determined using equation 2.
                  amount of acetone in -175°C
Per cent efficiency = trapping cylinder
                  	X 100
                  predicted amount of acetone
                  in —175°C trapping cylinder
                                     (equation 2)
  The values obtained for the efficiency of  the  system
were 73.4, 81.7, and 88.1 per  cent (Table  I). A varia-
                                                   tion of plus or minus 10 per cent in flow gives values
                                                   of 76.2 'or S9.S per  cent for the 81.7 per cent value.
                                                   Creator accuracy in  the control of  flow and the incor-
                                                   poration of a  mass flow meter will  decrease the varia-
                                                   tion  in  values  obtained.  The change in amount  of
                                                   acetone from  the  initial to  final chamber values and
                                                   the amount of acetone in the -175:C trapping cylinder
                                                   were used to determine the recovery of the system. The
                                                   indicated recovery of the system was 102.6, 97.9 and
                                                   SO.S per cent.
                                                     The  results indicate that  the average concentration
                                                   in the chamber can be directly estimated.  The amount
                                                   of acetone present in the -175'C trapping cylinder, the
                                                   flow rate of the  sample gas through the system and the
                                                  . total trapping time were used to calc'.'hte the average
                                                   acetone, concentration in the chamber during the trap-
                                                   ping period.  This is compared in  Table II with  the
                                                   result determined by averaging the initial and final con-
                                                   centrations of  the chamber.
                                                     Water vapor  is concentrated in  the 0:C and  the
                                                   -78CC  trapping cylinders (Figure  7).  Tin's aids  in
                                                   analysis by minimizing the  interference of water absoqv
                                                   tion bands in the -175:C trapping cylinder sample.
                        TABLE II. RESULTS OF PRELIMINARY EVALUATION: CONCENTRATION
872  Aerosnarp
Run
A
1
2
3
Flow
cc/min
B
3EO
300
300
Time
min
C
SCO
360
360
Chamber Concentration (ppm)
Initial Final Average
D E D + E

6.65
6.S8
6.88

4.25
5.08
4.73
2
5.45
5.98
5.80
-175'C
Trapping
Cylinder
Concentration
(ppm)
F
4164
2970
3304
Estimated
Chamber
Concentration
(ppm)
G
4.56
4.12
4.58
Sample
Concentration
2F
D + E
7C4.04
496.66
SOT. 16

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   A MULTI-STAGE  CRYOGENIC TRAPPING SYSTEM—CONKLE, ET AL
                                                                             -67-
      —:—J    —,-  .  ,  	
      :-,. i   rp1  i  cr:
      	1	£0 r-i	rl— l«	
                                        ;•  r  j   : ;.;_  ;M	[  ;       • -•
 4OOO   3200    2800   200O    BOO   TOO   BOO    BOO    ICO    BOO   800   800
                   ;  1  !•!--(-'  ' ' I', i  • :.  -;-f   }
. , .,y ,.
M i ;
i... - . i
i
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1
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f '-!•-•
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: . . i ,
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i; .- ;•;•-;••
:.,/.,• . •'
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                                                                    s
 4000   3200    2800   2OOO    DOO   (700   BOO    BOO    ICO    COO   ftOO   600
   -
. 4000
       3200
              iSOO   2OOO
                           1400
                                 r/oo
                                      BOO
                                            DOO
                                                  100
                                                         COO
                                                               800
                                                                    600
                                        TABLE HI.  DISTRIBUTION OF COMPOUNDS BY TRAPPING
                                                     CYLINDER TEMPERATURE
                       Fig. 7.  Typical 0:C,  -7S:C and -J75:C trapping cylinder  infra red scans.

Acetone is present in all  three traps but concentration
occurs in the -175rC trapping cylinder.
  The acetone concentration was several hundred times
greater in  the -1T5:C trapping cylinder than  in  the
chamber after trapping.  This  degree of concentration
aids identification of materials present in trace amounts.
  The multi-stage cryogenic trapping system will con-
centrate a compound if the vapor pressure at the trap
temperature  is  less than its  partial pressure  in  the
sample stream. Partial separation of compounds occurs
due to the different operational temperatures  of  the
three traps.  This separation simplifies identification and
quantitation.
  After liquification and/or solidification  there  is  no
significant  loss from a  trap  by transfer  of participate
material in the form of fog or snow.  This is minimized
by  the  construction of the trapping cylinders.
Multi-Stage Cryogenic Trapping Cylinder

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                                                      -68-
  Table III depicts  the distribution  of several com-
pounds as a function of the temperature at which they
are expected to be concentrated in significant quanti-
ties. Substances are identified in each column accord-
ing to the state in which they exist at that temperature,
either as a h'quid(L) or as a solid(S).  Any material
existing  as  a solid at a given  temperature  will not  be
found concentrated in a succeeding trapping  cylinder.

                 ACKNOWLEDGMENTS
  The  authors wish to thank Richard  Dawson, Binclectronics,
for assembly  of the liquid nitrogen level control unit, James D.
Wise, Machine Shop, for the construction of special equipment,
F. Duepner and Dr. B. E. Welch for their  encouragement and
suggestions.
                    REFERENCES

1. Fnosr, A. A. and PEARSON, R. C.: Kinetics and Mechanism.
     John Wiley & Sons, Inc., New York, New York, p. 13.
     1953.
2. JOHNSON, J. E.:  Nuclear Submarine Atmospheres, Analysis
     and  Removal  of Organic Contaminants.  NRL  Report
     5800. 1962.
3. JOHNSON, J.  E.:  Atmosphere  Monitoring in the  Nuclear
     Submarine.  Presented at the USN-LMSC Toxicity Sym-
     posium in Palo Alto, Calif., July 1963.
4. SAUNDEHS, R.  A.:  Analysis of the Spacecraft Atmosphere.
     NRL Report 5816, 1962.
5. WEUER, T. B., DICKEY, J.  E., JACKSON, N. N.,  REGISTER,
     J. W. and CONKLE, J. P.: Monitoring of Trace Contami-
     nants in Simulated Manned Spacecraft. Aerospace Mcd.,
     35:148-152.

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