PROCEEDINGS
CATALYST RESEARCH PROGRAM
 PLATINUM RESEARCH REVIEW
          CONFERENCE
               Quail Roost Conference Center
                 Rougemont, North Carolina
                      December 3-5,1975
   U.S. Environmental Protection Agency
    Office of Research and Development
    Health Effects Research Laboratory
       Catalyst Research Program
Research Triangle Park, North Carolina  27711

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                 PROCEEDINGS

          CATALYST RESEARCH PROGRAM

     PLATINUM RESEARCH REVIEW CONFERENCE

        Quail Roost Conference Center
         Rougemont, North Carolina
            December 3-5, 1975
                Sponsored by:

    U.S.  Environmental  Protection Agency
     Office of Research and Development
     Health Effects Research Laboratory
         Catalyst Research Program
Research  Triangle Park, North  Carolina  27711
                 April  1976

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                           DISCLAIMER
     This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication.  Mention of trade names or commercial  products does
not constitute endorsement or recommendation for use.
                               n

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                               CONTENTS
I.    Introduction

     A.    Background	       1
     B.    Objectives	       4
II.   Conference Presentations

     A.    Listing of Presentations 	       6
     B.    Introduction to Conference 	      10
     C.    Highlights of Presentations

          1.    Baseline Studies	      10
          2.    Measurement Technology and
                 Emission Characterization 	      13
          3.    Methylation Chemistry 	      20
          4.    Toxicology and Health Effects 	      20
III. Workshop Summaries and Recommendations

     A.    State-of-the-Art and Recommendations
            of the Technology Workshop	      26
     B.    State-of-the-Art and Recommendations
            of the Health Sciences Workshop	      28
IV.  List of Participants	      33
V.   Appendices

     I.    Abstracts of Presentations
     II.  General Information for Investigators
                                  111

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                          I.   INTRODUCTION
A.   Background
     The Catalyst Research Program (CRP) was initiated in FY 1974 by the
Environmental Protection Agency (EPA) Administrator Russell  Train concurrent
with the Agency's decision (Nov. 1973) to permit the use of the noble metal
oxidation catalyst as an emission control device on 1975 model year passenger
vehicles.  Some research had been initiated prior to this specific program
when EPA first learned of the automotive industry's intent to control vehicle
emissions via the oxidation catalyst.  Results of this early research
suggested that, though emissions of hydrocarbons, carbon monoxide, and
certain organics would be dramatically lowered by the catalytic converter,
sulfate emissions would increase, and slight emissions of platinum, palladium,.
and alumina might also be expected.
     The possibility of emissions of platinum and/or palladium was of
concern because this would likely represent the introduction of a "new"
environmental contaminant.  Though both are found in minute quantities in
the earth's crust, known exposure to these elements had been solely
occupational.  It was expected that the active components of the catalyst,
platinum (Pt) and palladium (Pd), would be emitted during vehicle aging as
attrition products.  Laboratory studies had measured small amounts of
platinum from automotive catalysts.  Early studies with prototype catalyst
vehicles indicated measurable platinum quantities were emitted.  Since
platinum is the predominant component of noble metal catalysts, EPA's
research program focused on that element, though palladium was also
studied to a lesser degree in some projects.

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     The Catalyst Research Program's approach to assessment of health
effects of both sulfate and noble metals emissions from the catalytic
converter can be outlined as follows:
     1.   identify emissions
     2.   determine emission factors
     3.   estimate human exposure
     4.   assess public health impact
     5.   evaluate control options
     6.   provide timely reports to provide basis for Agency
          regulatory decisions.
     The study of sulfate emissions from catalytic converter-equipped
vehicles was basically one of assessing the impact of an incremental
contribution from these vehicles to an already existing atmospheric
sulfate burden.  Research on characterization, formation, transport,
ambient measurement, exposure and health effects of sulfate had been
ongoing within EPA's Office of Research and Development for some time.
The challenge was to identify effects of sulfuric acid as opposed to
sulfate as a class.  Study of the health effects of the noble metals,
however, presented an entirely different challenge.  Very little data  on
the toxicology of noble metals existed in the literature, and limits  of
detection through state-of-the-art measurement technology was not suffi-
ciently low to quantify the expected (but undefined) levels in air,
water, soil, and biological tissue.
     The Catalyst Research Program's objective as it pertains to the
noble metals then, was to develop an information base on noble metals,
particularly platinum, in many areas.  Time was at a premium, as the

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                                    3
introduction of catalytic converters was scheduled for the 1975 model
year, to be placed on sale in the fall of 1974.  In California, all
American made 1975 model year automobiles would be equipped with noble
metal catalytic converters; in the other 49 states, some 60% would be so
equipped.  The number of catalyst-equipped vehicles was expected to
increase rapidly over the next ten years, replacing the pre-1975 catalyst
vehicle population at a rate of about ten percent per year.
     The areas of study for the Catalyst Research Program's (CRP). platinum
research effort was set up as shown in Table I.
                                Table I
              Areas of Study, Noble Metal Health Effects
                     EPA Catalyst Research Program
Emission Characterization (Exhaust)             Health Effects Assessment
Measurement Methodology Development             Carcinogenicity
                                                Mutagenicity
     exhaust                                    Cytogenicity
     ambient air, water, soil                   Inhalation Toxicology
     animal & human tissue,                     Sensitization (allergic
       feces, urine                               response)
     standard reference material                Irritation
       development                              Immunology
Bioenvironmental Impact                         Body Burden Tissue Analysis
Methylation Chemistry                           Epidemiology
Population Exposure Studies

     These areas were identified as requiring further research after
receipt of a literature survey conducted by an EPA contractor in FY
1974.  Most of the 20-odd research tasks in the CRP associated with
noble metals, particularly platinum, were initiated in FY 1975.

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     Work on most of the tasks in the CRP's platinum research effort had
been under way for almost a year at the end of fiscal year 1975.  At that
time it became apparent that the data being reported by researchers
should be discussed at an appropriate forum for informative exchange and
discussion.  The decision was made early that only active researchers
performing work under EPA contracts and grants, their project officers,
CRP staff, and a very few non-EPA active researchers would be invited to
participate.  It was decided that the meeting would not be public, as
data presented would eventually be published as EPA reports upon  completion
of the work.  In this way, informal exchange of information regarding
work in progress would be encouraged and discussion would not be  inhibited
by the possibility of "outsiders" drawing premature conclusions from
preliminary research results reported.
B.   Objectives
     The conference presentations and the workshops were divided  into
two broad categories:  (1) analytical and measurement technology, and
(2) health sciences.  The main objectives of the conference were  to bring
together active researchers in the platinum area to:
     1.   exchange knowledge
     2.   identify areas for further study and possible changes in
          emphasis or direction of current study
     3.   recommend priorities
     The two-day conference program combined plenary sessions with intro-
ductory and keynote presentations, presentations of results of data for
platinum research tasks included in the CRP, workshop discussions, presenta-
tion of workshop reports, and recommendations by all participants.

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     The questions to which workshop discussions were addressed were:
     1.   Based upon the current state-of-the-art, what conclusions
          can now be drawn?
     2.   What research gaps exist which must be addressed to assess
          more completely the environmental consequences of platinum?
     3.   What is the priority in filling these gap areas?
     4.   If we (EPA) fill these research gap areas, what additional
          conclusions can be drawn?

                    II.  Conference Presentations
     During the first day of the conference and continuing through the
early part of the second day's session, a total of 25 presentations on
work completed or in progress were made.  A following section, "Highlights
of Presentations," provides a brief description of each project upon
which reports were made.
     The twenty-five conference presentations were organized as follows:
     Baseline Studies - 3 presentations
     Measurement Technology & Emission Characterization - 10 presentations
     Methylation Chemistry  - 1 presentation
     Toxicology & Health Effects  - 11 presentations

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A.   Listing of Presentations

     1.   Welcome and Introductory Remarks

          Mr. John B. Moran
          Environmental Protection Agency

     2.   "Noble Metals:  A lexicological Appraisal of Potential  New
          Environmental Contaminants"

          Dr. Paul E. Brubaker
          Environmental Protection Agency

     3.   *"Review of Literature, Health Effects of Noble Metals, and
          Pre-Catalyst Metals and Pre-Catalyst Baseline Population Studies
          for Determination of Human Body Burden of Platinum and  Palladium"

          Dr. Donald Johnson
          Southwest Research Institute
          Contract # 68-02-1274

          Final Report:  Baseline Levels of Platinum and Palladium in
          Human Tissue
          EPA-600/1-76-019

     4.   "Overview of the Los Angeles Catalyst Study"

          Mr. Charles Rodes
          Environmental Protection Agency

     5.   "Determination of Human Body Burden Baseline Data of Platinum
          Through Autopsy Tissue Analysis"

          Ms. Vandy Duffield
          Environmental Protection Agency

     6.   "Measurement of Platinum from Catalyst-Equipped Vehicles,
          Combustion and Attrition Products"

          Mr. John Sigsby
          Environmental Protection Agency

     7.   "Toxicological Studies of Auto Exhaust Catalyst Components"

          Dr. Jerry Stara
          Environmental Protection Agency

     8.   "The Use of Chemical lonization Mass Spectrometry for the
          Analysis of Platinum and Palladium"

          Dr. Terence Risby
          Pennsylvania State University
          Grant # R-803651

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9.    * "Chemical Transport of Platinum from Automotive
     Catalysts -- A Laboratory Study"

     Dr.  John Hawley
     New York State Department of Environmental  Conservation
     Former Grant # R-801486

     Final Report:  The Chemical Transport of
                    Platinum from Automotive Catalysts

10.  "EPA's Efforts to Determine the Platinum Content of Ambient Air"

     Dr.  Richard Thompson
     Environmental Protection Agency

11.  "Platinum Metals in Air Particulates Near a Catalytic Converter
     Test Site as Measured by Isotope Dilution SSMS"

     Dr.  Joel Carter
     Oak Ridge National Laboratories
     Interagency Agreement # EPA-IA6-D5-0466

12.  "Measurement of Platinum and Palladium in Ambient Air, Soil, and Water'
     "Measurement of Platinum and Palladium in Biological Tissues"

     Dr.  Donald Johnson/Mr. John Prevost
     Southwest Research Institute

13.  "Platinum Analyses in Animal Tissues and Fluids"

     Dr.  Andre LeRoy
     National Institutes of Health

14.  "Spontaneous Deposition Radiochemical Separation for the
     Determination of Platinum, Palladium, and Gold in Biological
     and Environmental Materials"

     Dr.  Donald A. Becker
     National Bureau of Standards

15.  * "Measurement of Platinum in Biological Tissues, Urine,
     and Feces:

     Dr.  Anna Yoakum
     Stewart Laboratories
     Contract # 68-02-0663

     Final Report:  Trace Metal Analysis of Maternal/Fetal Tissue Sets
                    House Dust Specimen, and Human Scalp Hair

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                               8

16.  * "Methylcobalamin:  Methylation of Platinum and Demethylation
     With Lead"

     Dr. Robert Taylor
     Lawrence Livermore Laboratories
     Interagency Agreement # EPA-IAG-D4-0439

     Final Report:  Comparative Methylation Chemistry
                    of Platinum, Palladium, Lead, and
                    Manganese" EPA-600/1-76-016

17.  "Toxicity of Platinum (IV) Salts for Cells of Pulmonary Origin"

     Dr. Mike Waters
     Environmental Protection Agency

18.  "Compare Pulmonary Carcinogenesis of Platinum Group
     Metal Compounds and Lead Compounds in Association
     With Polynuclear Aromatics Using In Vitro Hamster System"

     Dr. Philip Kane
     State University of New York - Stoneybrook
     Contract # 68-02-1299

19.  "Studies of No-Effect Level of Platinum and Palladium Using
     Mouse Pulmonary Infectivity Model"

     Dr. Richard Ehrlich
     IIT Research Institute
     Contract # 68-02-1273

20.  "Compare Relative Toxicities of Platinum Compounds
     Against Lead Compounds Using In Vitro Macrophage System"

     Dr. Donald Gardner, EPA for
     Dr. Eula Bingham
     University of Cincinnati
     Contract # 68-02-1286

21.  "Allergic Induction in Experimental Animals Using
     PtS04 and PdS04"

     Dr. James Taubler
     St. Vincent College
     Grant # R-803036

22.  "The Effects of Platinum Sulfate on Leucocyte
     and Platelet Metabolism and on Immunologic Responses
     in the Rabbit"

     Dr. Kenneth Lunan
     Stanford Research  Institute
     Contract # 68-02-1300

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     23.   "Cytogenetic Analyses of the Effects of Platinum"

          Dr.  Ann Mitchell
          Stanford Research Institute
          Contract # 68-02-1300

     24.   *"Toxicities of Compounds of Platinum and Palladium as Measured
          by Biochemical  Parameters"

          Dr.  David Holbrook
          University of North Carolina

          Final  Report:  Assessment of Toxicity of Automotive
                         Metallic Emissions

                         EPA-600/1-76-010A  Volume I:   Assessment of Fuel
                         Additives Emissions Toxicity  via Selected Assays
                         of Nucleic Acid and Protein Synthesis
                         (Contract # 68-02-1205)

                         EPA-600/1-76-010B  Volume II:   Relative Toxicities
                         of Automotive Metallic Emissions Against Lead
                         Compounds Using Biochemical Parameters
                         (Contract # 68-02-1701)

     25.   "Evaluation of the Mutagenic Potentials of Platinum Compounds"

          Dr.  Shabeg Sandhu
          North  Carolina Central  University
          Grant  # R-803299

     26.   "Development of Sensitive Biochemical and Behavioral Indicators
        o of Trace Substance Exposure"

          Dr.  Edward Massaro
          State  University  of New York - Buffalo
          Contract # 68-02-1768
*These final  reports are available and can be obtained from the project

officers and  grantees listed.   Those reports that have been assigned an

EPA publication number can also be obtained from the National  Technical

Information Service, Springfield, Virginia  22161.

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                                 10

B.   Introduction to Conference
     Welcoming remarks and an overview of the conference objectives were
presented by Mr. John Moran, Director of the Catalyst Research Program.
Dr. Paul Brubaker, of EPA and former project officer for some CRP platinum
contracts, discussed the potential  toxicological  hazards of platinum and
palladium in relation to their use in catalytic converters.
 C.  Highlights of Presentations
     1.    Baseline Studies
          (a)  The Southwest Research Institute (SwRI) study of platinum
and palladium was reported by Dr.  Donald Johnson.   The first phase of
the study was a literature survey performed in early 1974 covering
sources, uses, production, consumption, reported medical cases, and
toxicology of platinum and palladium.  The literature survey concluded
that "no data existed by which an estimate can be made of transfer of
platinum and palladium to the environment.  Investigations show that
only salts of platinum present human health hazards.  Industrial  exposure
to these is limited to the mining and refining of platinum ores and the
preparation of catalysts for the chemical and petroleum refining  industries."
     The second phase of SwRI's study covered several areas:
     (1)  collection and analysis of biological samples (blood, urine,
          hair, and feces) and environmental samples (air, soil,  and
          water) from populations in southern California -- one in an
          urban area near a major freeway, the other in a non-urban
          location.  The urban population studied was near the Los
          Angeles Catalyst Study site which allowed for corroborative
          pollutant and meteorology data comparison.  The site was discussed
          in Mr. Rodes1 presentation.

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                                  11
     (2)   Collection  and  analysis  of  biological  samples  and  environmental
          samples  from three  locations  --  one  at a mining  and  processing
          facility, and two at  platinum/palladium refineries.
     (3)   Limited  autospy samples  were  collected from the  area near  the
          area of  study discussed  in  (1)  and near the mining area  described
          in (2).
     Preliminary conclusions  drawn from the results  of the Pt/Pd  baseline
studies were reported as  follows:
          •    Baseline levels  of  Pt  and  Pd in the environment
               (air,  soil, water)  and in  urban populations are extremely
               low.   Levels  in  nearly all  samples were below the  detection
               limits of  the  analytical methods (which,  however,  were
               not defined).
          t    Examination of ambient air, and blood, urine, and  feces
               from miners for  levels of  Pt and Pd did not provide
               definitive data  on  the relationship of ambient  air to
               body burden,  as  air levels  were nearly always below
               detectable levels,  which,  however, were not defined.
          •    Data from  Pt  and Pd refineries  indicate that measurable
               levels of  Pt  (0.23.- 2.6 yg/liter) and Pd (0.21 -  6.3
               ug/liter)  are  found in the urine of workers exposed to
                                              o                        o
               air containing 0.02 -  0.26 yg/m  Pt and .003 -  0.36 yg/m
               of Pd.  Measurable  levels  were  not found  in the blood of
               these  workers.

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                                  12
          t    Data from the refineries indicate that it is likely that
               measurable levels of Pt and Pd could be seen in the
               general population when ambient levels of these two
               metals approach 0.1 ug/m .  The metals would appear
               initially in urine rather than blood.
          t    Additional data are needed on the distribution of Pt and
               Pd in human tissue other than blood and urine following
               exposure to air containing the 2 metals at the 0.01 - 0.1
               ug/rn .levels.  Additional data to define the,relationships
               between air levels and Pt and Pd and tissue  levels in the
               general population are needed, but development of data
               should be;deferred until there is. more-information available
               on quantities and types of the two me;taIs. emitted from
               the catalytic mufflers.
          (b)  The second baseline presentation, made by Ms. Duffield,
reported data resulting from^analysis,by.;Stewart Laboratories of autopsy
samples collec,tedvby EPA in southern California* the same general area
where two of the populations .studied^in the Southwest Research Institute
work were located.. Results of analysis, for Pt ,in 97 autopsy sets were
presented.  Analysis .was performed by a specially developed ,emission
spectrochemical method. .Almost half.,(46%) of the .individuals studied
were1 found to have detectable'-amounts of platinum in ,one ,or.more tissue
samples. -Levels of platinum in .13 of-21 tissue types were  detected.
The range of Pt concentrations was 0.003 to 1.46. vg/gm,(wet..tissue); the
mean value of detected Pt was 0.16 ug/gm, and the median value of detected
Pt was 0.067 ug/gm.  Surprisingly high values were found in subcutaneous
fat, not previously considered to be a target site.

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                                    13

          (c)   A brief report of set-up and operations at the Los Angeles
Catalyst Study (LACS)  site was presented by Charles Rodes.   This monitoring
site,  which consists of four instrumented stations located in a perpendicular
line across a  major freeway in the Los Angeles area, is an important
support operation to the CRP platinum research effort.  It was at this
location that  a large particulate sample was collected to determine
baseline ambient Pt.   Also, one of the southern California populations
studied in the SwRI human body burden baseline study was located near
downwind of the LACS site, thus presenting corroborative ambient pollutant
and meteorological  data for analysis of the SwRI study results.  If at
some future time it is determined that automotive emissions of Pt are at
a high enough  level to warrant concern for inhalation exposure of this
general population, this site would be an important part of any monitoring
effort initiated by EPA.
     2.   Measurement Technology and Emission Characterization
          (a)   A brief summary of EPA/ORD's attempts to identify platinum
in exhaust particulate emitted by catalyst-equipped vehicles was presented
by Mr. John Sigsby.  Some 20 cars were included in both inhouse and
extramural emissions characterization studies.  No platinum was ever
detected in particulate collected on filters, so that if there is any
platinum in the size range of airborne exhaust particulate, it is below
current detection limits.  These detection limits equate to < 10~
gin/mile in the extramural activities.  On the basis of these measurements,

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                                    14
it is EPA's conclusion that there is at present no risk to public health
presented from exposure to respirable range platinum in catalyst-equipped
vehicle exhaust particulate.  However, no measurements were made with
vehicles other than those operating properly and with unleaded fuel.
     Platinum has been identified in the larger size range of exhaust
particulate (that portion of the exhaust which would normally deposit
upon the roadway or near the roadway).  Analysis was performed on tube
sweepings collected from the bottom of dilution tunnels in which the
exhaust is diluted prior to collection of samples for both particulate
and gaseous analysis.  The collections were made after many runs, often
with a variety of properly operating catalyst vehicles.  One sample was
split and analyzed by a variety of techniques, others were only analyzed
by atomic absorption.  The material collected was extremely heterogeneous
consisting of a wide range of sizes including a few whole catalyst
pellets.  The range of Pt content found in various samples and fractions
ranged from 0.034 to 635 yg/gm of collected material.  The higher values
were related to whole pellets and pellet fragments and corresponds with
the initial catalyst loadings.  Such pellets and fragments are usually
only found on initial runs on fresh unused catalysts.  Emission spectroscopy
for chemical analysis (ESCA) results showed that some of the platinum
appeared to be in the +4 oxidation state but no quantisation was possible.
A definitive characterization has not yet been performed.  There is
question as to whether precise identification is necessary or possible
due to the small amounts emitted and to the catalyst matrix with which
it is associated.

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          (b)  Dr. Stara described work performed by EPA's Cincinnati
group wherein animals were exposed to  both catalytic and non-catalytic
automobile exhaust.  In an early exposure experiment with a prototype
catalyst, platinum was found in chamber air near the detection limits of
atomic absorption (AA) only once; in later experiments with production
catalysts, platinum was found once in  a filter placed near the vehicle's
tailpipe, again near the limits of detection for AA.  Four samples of
large particulate collected in the dilution tunnel were analyzed for
platinum by flame atomic absorption at EPA's North Carolina Environmental
Research Center.  Though there were matrix difficulties, valid results
were obtained using standard additives.  Platinum was found in the
samples in concentrations ranging from 60 to 145 yg of Pt per gram of
the samples.  No attempt was made to identify the platinum compound due
to small amount of samples available for analysis.
          (c)  Dr. Risby of Pennsylvania State University presented a
method for analysis of volatile mixtures.  He described his preparation
of volatile chelates of platinum and palladium.  Using a chemical ionization
mass spectrometric method, spectra were established for the noble metal
chelates synthesized.  Limits of detection for the compounds are in the
picogram range.
          (d)  Dr. John Hawley of the  State of New York Department of
Environmental Conservation described results of parallel bench laboratory
and engine stand tests of chemical transport of platinum from noble
metals oxidation automotive catalysts.  Scaled to a 60 mph cruising
automobile, particulate Pt was collected during some laboratory experiments.
Engine stand tests also showed microgram per mile platinum transport

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                                   16    £,.
under certain conditions.  Tests conducted with nitrogen and oxygen as
the carrier gas showed that platinum, in these experiments, was transported
chemically rather than mechanically.  Analysis was performed by x-ray
fluorescence.
          (e)  Dr. Thompson discussed EPA's efforts to determine the
platinum content of ambient air.  In 1969 massive samples of suspended
particulate matter collected from ambient air were analyzed for all
elements having stable nuclides, except the noble gases.  Results of
analysis for platinum by X-Ray Fluorescence (XRF), Optical Emissions
Spectroscopy (OES), Atomic Absorption Spectrometry (AAS), and Neutron
Activation Analysis (NAA) were negative.  In 1975, the recently developed
isotope dilution mass spectrometry technique showed platinum existing at
                   —fi     ^
a level of 2.5 x 10"  ug/m  (based on an assurned air volume obtained by
considering the particulate to have come from air at, an average TSP
loading).
          .(f)  Dr. Opel.Carter described the method by which isotope
dilution spark-source mass specjtrpmetry was .employed at Oak Ridge National
Laboratory for analysis for platinum of.the above-mentioned massive
suspended particulate samples..  To obtain sufficient,sensitivity for
obtaining positive values for platinum, it was necessary to dissolve a
large portion, of the,ashed sample for the purpose of equilibrating
                   1Qfi    10?        Qft
stable.isotopes of    Pt,  u Pt, and_  Ru. .Platinum as a metal prec.ipi.tate,
and gold as a carrier were then added.  Gold and the platinum..group were.
concentrated into .the gold precipitate used ,in the.isotope .dilution;
spark-source mass spectrometry .measurements.  As mentioned earlier,
platinum levels were reported at 2.5 picograms per cubic meter of.air.

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                                  17
          (g)  Dr.  Don Johnson of SwRI presented results of analysis of
air, soil, and water samples collected at the various sites included in
the baseline study mentioned above (see "Baseline Studies").  Using a
method involving digestion extraction and preconcentration followed by
atomic absorption,  "less-than" values were reported for all samples
except air samples  collected inside mining and refining facilities.  Air
concentration levels of platinum ranged from 0.021 - 0.259 ug/m  for
platinum refinery locations (mean of 0.159 ug/m ); only one positive
value was reported  in the mining facility (0.377 ug/m  Pt).  For a more
detailed description of analytical methods employed and limits of detection
applicable for the  various types of samples, see final report.*
     Measurement of platinum in biological samples was reported on by
four conference participants.   Dr. Johnson reported that the method for
biological samples  is similar to that previously discussed for platinum
analysis of environmental samples.  No detectable levels of platinum
were found in individual samples of blood, urine, hair, and feces
collected from the  California residents (both urban and non-urban)
participating in the study.  Analysis of a composite of all blood from
all age groups and  both sexes gave a value of 0.049 ug/100 ml for the
group near the Los  Angeles freeway and 0.180 ug/100 ml for the high
desert non-urban group.
     No detectable  levels of Pt were found in blood, urine, hair, and
feces collected from miners at the mining site.  At the Pt refinery

*Directions for obtaining copy of final report on page 9.

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                                   18
location, positive values were detected for platinum in urine in a range
of .23 - 2.5 yg/liter, where minimum detectable Pt concentration was
0.10 yg/1.
     Autopsy samples were collected from cases, again, near both the Los
Angeles site and near the mining area.  Only "less than" values were
reported from the two groups numbering 10 and 9 cases, respectively,
with the exception of 3 tissue samples from 3 of the nine mining area
cases.  Analysis for platinum in these 3 cases gave Pt values for fat,
lung, and muscle samples at 4.5, 3.7, and 25.0 ppb, respectively.
          (h)  Work in the area of platinum analysis of animal tissue
and fluids performed at the National Institutes of Health was described
           *
by Dr. Andre LeRoy.  An adaptation of the flameles atomic absorption
spectrophotometry method has given quantitative determination of platinum
in biological samples down to about 30 parts per billion.  The NIH
analytical method is similar to the SwRI method for Pt analysis by
atomic absorption, but differs in the digestive and preconcentration
procedures.  In the NIH method, tissues are digested first by nitric
acid, then by a mixture of perchloric and nitric acid.  The digest is
taken up in HC1, aliquots of which are analyzed by comparison with a
working standard.  Aliquots of blood plasma or urine are analyzed directly
for samples containing a minimum Pt content of 2 ng/30ul.  Detection
limits are reported to be approximately 30 ppb.
     An important feature of the method used is the introduction of a
temperature ramp generator developed and built at NIH which replaces the
step-wise temperature change in the graphite furnace with a ramp. Its
use results in a time interval between absorption peaks due to "smoke"
and molecular absorption from the sample combustion and the atomic
absorption by the ground state platinum atoms.

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                                 19
          (i)  Mr.  Donald Becker of National  Bureau of Standards  described
the application of neutron activation analysis coupled with spontaneous
deposition radiochemical  separation to determination of platinum  and
other noble metals in biological and environmental  materials.   Sample
preparation involves lypholization  and wet-ashing with a mixture  of
nitric and perchloric acids on a hot plate, dissolution of the perchlorate
residue in hydrochloric and nitric  acids, and addition of "holdback"
carriers prior to the addition of silver powder to effect deposition.
The silver powder is then counted on a germanium gamma-ray detection
system.  Results of an analysis of bovine liver and orchard leaves gave
platinum values of <1.6 ng/g and <9 ng/g, respectively.  Analysis of
sample splits with Dr. LeRoy of NIH for biological  tissues from animals
which had ingested platinum compounds indicated reasonably good agreement
between the two methods.
          (j)  Stewart Laboratories' literature search for available
methods for analysis of platinum conducted in 1974 and subsequent
development of a method specifically for determination of trace quantities
of platinum, lead, and manganese in biological tissues was presented by
Dr. Anna Yoakum.  The sample preparation for the method involves  lypholization
and wet-ashing prior to spectrochemical analysis.  Samples and standards
are packed into carbon electrodes and analyzed by a total energy  burn in
a controlled argon-oxygen atmosphere with dc arc excitation.

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                                  20
     3.   Methylation Chemistry
          Dr. Robert Taylor of Lawrence Livermore Laboratories described
his work on comparative methylation chemistry of platinum, palladium,
and lead.  He reported that at least one platinum compound, potassium
hexachloraplatinate, can be methylated via the same Vitamin B^2 reaction
by which mercury is methylated.  By labeling both the carbon and the
hydrogen in the methyl group component of methylcobalamin (Vitamin B^
metabolite), Dr. Taylor established that the platinum compound demethylates
the MeB12 to form a methylated,platinum compound.• This methylated
platinum compound, though slightly light-sensitive, appears to be moderately
stable, but has yet to be fully characterized and evaluated for biological
activity. Characterization will be difficult, as the compound cannot be
lypholized without loss of integrity.  Dr. Taylor also reported that
platinic sulfate also demethylates MeB,2, but further work is necessary
to establish whether the methyl, group ;is transferred, to the platinic
sulfate.
     Ip. addition, various Pd, Pb, and Mn compounds showed ^significantly r
lower levels of demethylatio.n activity, in,the.^presence of MeB,'? when
compared to plat.inic sulfate.
     4.   Toxico-logy and Health Effects
        , (a)  Dr. M-ike,Waters of EPA reported results of ire vitro
studies of acute toxicity of platinum (+4) valence compounds uslnig"
rabbit alveolar macrophages and human lung fibroblasts.  Though platinum
dioxide exposure elicited no response, platinum tetrachloride exposure
resulted in significant loss of viability for both macropl^age and fibroblast
cells.  Thymidine uptake and incorporation (DNA synthesis) was shown to
be 50% inhibited at a concentration of 10   M PtCl.; a concentration of

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                                  21
                    _5
approximately 6 x 10  M was  required  to produce  50% inhibition  of uptake
and incorporation of   C uridine (RNA synthesis)  and leucine (protein
synthesis).
          (b)  EPA's Health  Effects  Research  Laboratory in  Cincinnati
has performed a number of studies  on  platinum,  involving both biological
fate and toxic response to various compounds  of  platinum following
various routes of exposure.   Dr. Jerry Stara  reported on the results of
these studies, some highlights  of  which were  as  follows:
          •    Retention, tissue distribution,  and excretion of
               191
                  Pt in rats were  determined  after intravenous, oral,
               and inhalation exposure.  A substantial  portion  of platinum
               is excreted rather  rapidly in  either feces or urine.
               Deposition sites included liver,  adrenal, spleen, kidney,
               lung, or bone.  Lung  clearance after inhalation  exposure
               has a half-time  of  about 8 days.  Platinum was found to
               cross the "placenta!  barrier"  in  small amounts after
               dosing of pregnant  rats.
          •    Platinum salts tested  did not  appear to affect the
               mitochondrial function of the  cell  in various organs.
          •    Platinum salts tested  were 100 to 1000 times less
               active than lead in inhibiting ALA-D enzyme  activity.
          (c)  Dr.  Kane described  the study being conducted by  the State
University of New York at Stoneybrook in which  the potential role of platinum
and lead compounds as co-carcinogens  in respiratory carcinogenesis

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                                  22
is being evaluated.  The metals are to be evaluated in conjunction with
the known carcinogen benzo(a)pyrene (BaP).  Intratracheal  and intragastric
instillation in two groups of hamsters are in progress.  Since this is
of necessity a long-term study, no conclusions can yet be drawn as to
the tumor producing properties of platinum compounds.
          (d)  The studies of no-effect level of Pt and Pd for response
of treated mice to a challenge of influenza virus or Streptococcus sp.
were reported by Dr. Ehrlich of IITRI.  Results indicate that inhalation
of platinum and palladium oxide aerosols diminished survival  time when
challenged with the infectious agent compared with control mice challenged.
Limited experimentation with response to Staphylococcus aureus administered
via intranasal instillation indicated that inhalation of PtO^ aerosol
dimished lung clearance of the infectious agents, presumably by reduction
of function of the alveolar macrophages.
          (e)  Dr. Gardner presented results of work done by Dr. Eula
Bingham at the University of Cincinnati.  The study involved a comparison
and evaluation of cellular response in the lung (especially alveolar
macrophage) to inhalation of platinum and other metal compounds.  Inhalation
exposure to platinum salt did not statistically alter the number of lung
cells, while some other metals administred via inhalation did significantly
alter the number of cells, both positively and negatively.

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                                   23
          (f)  Dr. Taubler reported that studies of immediate and delayed
allergic response to platinum compounds in rabbits, guinea pigs and mice
produced essentially no positive response with metals alone.  However,
when complexed to a protein (albumin) the toxicity of both metals was
potentiated to a degree that the majority of animals died when a subsection
was exposed to platinum and a positive allergic response was elicited
from palladium.
          (g)  Drs. Lunan and Mitchell discussed the several studies
using platinic sulfate being performed at Stanford Research Institute
including effects on leucocyte and platelet metabolism, immunologic
response, and cytogenetic effects in the rabbit.  Preliminary data
showed the following:
            t  Guinea pig skin sensitization studies and rabbit skin
               irritation studies gave no positive response.
            •  Eye irritation experiments with rabbits showed the
               PtCSQ^ should be regarded as a moderate to severe eye
               irritant.
            •  In vivo Pt(SQ4)2 treatment only moderately affected
               leucocyte metabolism.  In the leucocytes from the 1- and
               3-week exposure studies DNA and RNA synthesis, respec-
               tively, were depressed 50%.  In the 3-week exposure plus
               1-month recovery study phospholipid turnover was markedly
               retarded.
            •  In vitro experiments indicated that 2-hour exposures to
                 -4
               10  M platinic sulfate caused 50% depression of DNA
               synthesis in leucocytes.

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                                  24
             e Numerous alterations of platelet metabolism were observed
               in all experiments involving animals exposed to 15 and 20
               mg/kg of Pt(SQ4)2.
            •  Several whole blood values were affected in recovery
               experiments.
          Cytogenetic analyses are in progress, but no data are yet
available.
          (h)  Evaluation of the mutagenic potential of several platinum
compounds in progress at North Carolina Central University was discussed
by Dr. Sandhu.  Both an in vitro system involving mouse lymphoma cells
and an In vivo study of multi-generation growth of Phaseolus aurealis
are being employed in this study.  Work is now in progress, but data are
not yet available.
          (i)  Dr. David Holbro.ok of the University of North Carolina
summarized the results of his studies on the -toxicity of platinum com-
pounds as measured by biochemical parameters.  Studies of both dietary
intake and drinking water administration of platinum salts showed that
high levels of exposure restricted weight gain.,  Dietary administration
did not show any significant effect on organ weights.
          Intraperitoneal injection of platinum salts at high levels
significantly reduced DNA synthesis in several organs.  Also, high
levels of platinum salts administered intraperitoneaMy decreased liver
        . -Ji *           '    •        '
detoxification mechanism activity.  Dietary administration showed no
consistent effect on this function.

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                                  25
          (j)  Mr.  John Prevost and Dr.  Donald Johnson of SwRI discussed
the results of that portion of the baseline studies which examined levels
of platinum in blood, urine, hair, and feces of both groups occupationally
exposed to platinum and those exposed only to general  ambient levels.
In the studies of occupationally exposed subjects, it was established
that exposure to levels of .02 - 0.2 ug/m  or higher can result in
measurable levels of platinum in urine.   No platinum was detected in
blood, hair, or feces of either occupationally on non-occupationally
exposed study participants.  A composite blood sample from two non-
occupationally exposed groups showed Pt levels of .049 and .180 yg/100
milliliters respectively, as discussed above.
          (k)  The last presentation was a report on an experimental
area quite different from its predecessors.  Dr. Massaro of the State
University of New York at Buffalo discussed his work in the tissue/organ
distribution and effect of platinum on behavior, as well as levels of
neurotransmitters, glycolysis, and rates of DNA, RNA, and protein synthesis.
Though the results of the experiments are so far preliminary, it appears
that Na2PtClg has an effect on open-field behavior which would be charac-
terized as depressant.

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                                  26
             III.  Workshop Summaries and Recommendations

A.  State-of-the-Art and Recommendations of the Technology Workshop
     Question 1:    Based upon the current state-of-the-art, what conclu-
                    sions can now be drawn?
          1.   Pt is emitted from motor vehicles equipped with catalysts.
               Emission factors are unknown.  Predominant size is non-
               respirable.  Form is particulate.  Composition is unknown.
          2.   Background exposure to Pt in air, water, and soil is very
               low, but data are incomplete because levels are at or
               below current measurement capabilities.
          3.   Occupational exposures at fairly low levels may result in
               detectable human burdens.                             :
          4.   Autopsy samples from the general population evidence Tow
               level burden of Pt, but exposure route is unknown.
               Living individual human populations do not evidence Pt
               burdens at current limits of detection.
          5.   Current measurement methods are not adequate to establish
               exposure or body burden background.
          6.   Our current knowledge of environmental burdens of Pt and
               distributions thereof are not now adequate to assess
               exposures.

     Question 2:    What research gaps exist which must be addressed to
                    more completely assess the environmental consequences
                    of platinum?

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                             27
     1.    Determine  emitted  Pt  compound(s).
     2.    Develop adequate analytical  techniques  focusing  upon
          concentration  and  sample  preparation  step.   Once com-
          pleted, a  number of analytical  tools  are  adequate to do
          the job.
     3.    Ascertain  Pt concentrations, reactions, and  transport  in
          the ecosystem  (air, soil, sediment, water, localized
          exposure,  tissue,  food,  plants).
     4.    Determine  Pt emission factors,  size distribution and
          form.
     5.    Develop Pt monitoring analytical  capabilities.
     6.    Develop (EPA)  mechanism  to assure operational  quality
          control in multi-task programs  such as  in the  Catalyst
          Research Program.
     7.    Must assure analytical methods  intercomparability.

Question 3:    What  is the  priority in filling  these gap areas?
     1.    Determine  emitted  Pt  compound(s).
     2.    Develop adequate  analytical  techniques  focusing upon
          concentration  and  sample preparation  step.   Once com-
          pleted, a  number  of analytical  tools  are  adequate to do
          the job.
     3.    Develop Pt monitoring analytical  capabilities.
     4.    Develop (EPA)  mechanism  to assure operational  quality
          control in multi-task programs  such as  in this program.

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                                 28
          5.   Assure analytical method intercomparability.
          6.   Ascertain Pt concentrations, reactions, and transport, in
               the ecosystem (air, soil, sediment, water, localized
               exposure, tissue, food, plants).

     Question 4:    If we fill these research gap areas, what additional
                    conclusions can be drawn?
          1.   Determine quantitative environmental Pt distribution.
          2.   Movement of Pt in the ecosystem.
          3.   Composition of Pt,exposure.
          4.   Determine exposures to Pt compounds in the-environment
               (basis'for assessment.of pubI.ICLIhealth consequences).
          5.   Can, in the future, assess quantitative changes in
               exposures.

      f
B.  State-of-the-Art and Recommendations, of the; Health Sciences Workshop
     Question 1:    Based upon the current state-of-the-art, what conclu-
                   • sions can now be drawn?
               From the literature and papers presented,;it is evident
          th'at platinum compounds, a-t some concentrations, can have a
          biological effect, i.e., biochemical, immunolpgical, host
          defense, and physiological.  If such effects;are to be seen in
      ;    the population/they wiImprobably be the result of long-term
          low levels of exposure.  This necessitates the need for further
          testing in the areas of carcinogenesis, mutagenesis, immunological,
          behavioral, and biological screening (in vitro test).

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                             29
          At the present time,  the levels of Pt in air, water, and
     soil appear to be very low.   It will be important to continue
     monitoring atmospheric, soil  and water levels in the future.
     Increases in levels in these media may likely result in some
     biological reaction since  the present concentration is so low.
          There are many areas  that need further investigation. We
     know nothing about turn-over-rate nor what form the substance
     is in the tissue.  Nor do  we know what form the Pt is when
     emitted from the exhaust.   This latter information is vitally
     important to the environmental  toxicologist in performing
     experiments.

Question 2:    What research gaps exist which must be addressed to
               assess more completely the environmental consequences
               of platinum?
          The health research needs regarding these potential
     environmental toxicants is great and to fulfill all goals
     would require a great deal of time and money.  There are many
     "gaps," but a few essential  areas which should be investigated
     first are listed below:
     1.   Most relevant to EPA  mission are studies which will
          investigate the long-term, low concentration effects
          resulting from exposure to platinum compounds.
     2.   Studies should be conducted to determine deposition and
          target site of action of these agents (through different
          exposure routes).

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                             30
     3.   Interaction studies of Pt with other agents that are
          being emitted from the exhaust (HpSO.).
     4.   Characterization of emission/attrition products, and of
          ambient samples.
     5.   Correlation of in vitro test with whole animal studies.
     6.   Greater involvement of EPA ecological team in following
          the biotransformation of Pt in food chain and water.
          Coupled with this would be controlled feeding studies.
     7.   Investigation into the effects on sensitive populations.
     8.   Continued studies on comparative toxicological effects  of
          selective Pt compounds.
     9.   Long-term epidemiological studies of the population at
          risk, once defined.

Question 3:    What is the priority in filling these gap areas?
          In order to obtain the maximum amount of useful information
     concerning the potential toxicity of these agents, it is
     necessary to study simultaneously the areas of epidemiology,
     environmental assessment, and the biological effects.  The
     group's recommendation for toxicological testing is listed as
     follows:
     1.   Develop information on what concentrations exist in man
          (non-occupational).  Evidence suggests that Pt is in
          tissue, but how did it get there (food chain, H,,0?)  In
          what form is the Pt?

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                             31
     2.    Because  of the  time  it takes  to  obtain  the  information,
          long-term, low-level  research studies  should  begin  soon
          (carcinogenesis,  mutagenesis, host-mediated immunity,
          etc.)
     3.    Development of  model  systems  for study  of the sensitive
          population (neonates, hypersensitive,  developing embryo).
          This can be the basis for future epidemiological  studies.
     4.    Research on the interaction of Pt with  other exhaust
          emissions such  as H2S04,  NO^.
     5.    Measurement of  Pt in food chain  (milk  samples, etc.).
     6.    Study of deposition  and target site of Pt once it has
          entered  the body by  various routes of  exposure.

          It was also indicated that there must  be a close working
     relationship between the  various scientific disciplines  as
     well as between the  contractor and the funding agency.  Meetings
     such as this  one bring together individuals  with similar
     interest and  permit  very  meaningful scientific dialogue.

Question 4:     If we fill these research gap areas, what additional
               conclusions can be drawn?
          If the above-suggested research  is performed, we will  be
     able to obtain useful  information upon which to base decisions
     as  to the need for any regulatory action.  We will be able  to
     evaluate the  potential health  hazard(s) resulting from any
     predicted increase in levels of exposure to platinum or  platinum
     compounds.   Finally, the  data  generated in  these experiments

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                        32
would provide us with scientific models which could be used in
future toxicological screening of new environmental pollutants.
Evaluation could be made more quickly and accurately of possible
adverse effects on public health.

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                                    33

                             Participants

                  Platinum Research Review Conference
Mr. Donald A. Becker
Room A 309
Bldg. 222
National Bureau of Standards
Washington, D. C.  20234

Dr. Arthur Bierman
Lawrence Livermore Laboratories
Livermore, California  94550

Dr. Paul E. Brubaker
Environmental Protection Agency      (formerly Criteria and Special Studies
RD-683                                  Office, HERL, EPA, RTP, NC)
Washington, D. C.  20460

Dr. Robert Bruce
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

Mr. Joseph Bumgarner
Environmental Monitoring and Support Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

Dr. Joel Carter
Oak Ridge National Laboratories
Box Y, Building 9735
Oak Ridge, Tennessee  37830

Ms. Vandy Duffield
Health Effects Research Laboratory
Environmental Protection Agency
Research Trianlge Park, North Carolina  27711

Dr. Richard Ehrlich
I IT Research Institute
10 W. 35th Street
Chicago, Illinois  60616

Dr. Edward Faeder
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

Dr. Donald Gardner
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

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                                    34

Mr. Herman Gibb
Office of Health and Ecological Effects
Environmental Protection Agency
Washington, D. C.  20460

Dr. George Goldstein
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

Dr. John Hawley
New York State Department of
Environmental Conservation
50 Wolf Road
Albany, New York  12233

Dr. David Hoi brook
Department of Biochemistry
School of Medicine
University of North Carolina
Chapel Hill, North Carolina  27514

Dr. Donald Johnson
Southwest Research Institute
8500 Culebra Road
San Antonio, Texas  78284

Dr. Philip Kane
State University of New York
Department of Pathology
Health Sciences Center
Stoneybrook, New York  11790
        *
Dr. Andre LeRoy
Bldg.  13
Room 3W 13
National Institutes of Health
Bethesda, Maryland 20014

Dr. Kenneth Lunan
Stanford Research Institute
333 Ravenswood Avenue
Menlo Park, California  94025

Dr. Edward J. Massaro
State University of New York - Buffalo
Dept.  of Biochemistry
School of Medicine
Buffalo, New York  14212

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                                   35


Dr. Robert Miller
Health Effects Research Laboratory
Environmental Protection Agency
Cincinnati, Ohio  45268

Dr. Ann Mitchell
Stanford Research Institute
333 Ravenswood Avenue
Menlo Park, California  94025

Dr. Wellington Moore
Health Effects Research Laboratory
Environmental Protection Agency
Cincinnati, Ohio  45268

Mr. John B. Moran
Director, Monitoring Technology Division
Environmental Protection Agency            (formerly Director, Catalyst
RD-680                                      Research Program, HERL, EPA,
Washington, DC  20460                       RTP, NC)

Mr. John Prevost
Southwest Research Institute
8500 Culebra Road
San Antonio, Texas  78284

Dr. Terence Risby
Pennsylvania State University
424 Davey Laboratory
University Park, Pennsylvania  16802

Mr. Charles Rodes
Environmental Monitoring and Support Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

Dr. Shabeg Sandhu
North Carolina Central University
P.O. Box 19553
Durham, North Carolina  27707

Mr. John Sigsby
Environmental Sciences Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

Dr. Jerry Stara
Health Effects Research Laboratory
Environmental Protection Agency
Cincinnati, Ohio  45268

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                                     36

Ms. Peggy Stewart
Stewart Laboratories, Inc.
5815 Middlebrook Pike
Knoxville, Tennessee  27921

Dr. James Taubler
St. Vincent1s College
Biology Department
Latrobe, Pennsylvania  15651

Dr. Robert Taylor
Lawrence Livermore Laboratories
Livermore, California  94550

Dr. Richard Thompson
Environmental Monitoring and Support Laboratory
Environmental Proteciton Agency
Research Triangle Park, North Carolina  27711

Dr. Michael Waters
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina  27711

Dr. Anna Yoakum
Stewart Laboratories, INc.
5815 Middlebrook Pike
Knoxville, Tennessee  27921

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                              APPENDICES
 I.   Abstracts of Presentations
II.   General  Information for Investigators
          A.    Platinum Chemistry Pertinent to the Catalyst Program
          B.    Spectrophtometric Verification of PtCU"

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          APPENDIX I
Abstracts of Presentations

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                                                                         Environmental Health. Perspectives
                                                                         Vol. 10. pp. 39-56. 1975
Noble  Metals:  A  Toxicological  Appraisal  of
Potential  New  Environmental  Contaminants
by Paul E. Brubaker,* John B. Moran,* Kenneth Bridbord,* and F. Gordon
Hueter*
           The public health benefits expected by reducing known hazardous emissions from mobile
         sources should not be compromised by increasing levels of other potentially hazardous
         unregulated emissions. Catalytic converters are going to be used to meet  the statutory
         requirements  on carbon  monoxide and  hydrocarbon emissions  from light duty motor
         vehicles. Platinum and palladium metals are the catalytic materials to be used in these
         emission control devices. Preliminary experimental evidence and analysis of the impact
         of these control devices on the future use and demand for platinum indicates that this
         mutal may appear at  detectable levels in the environment by the end of this decade. At
         the present time, platinum and palladium are not present in the  public environment and
         represent potentially  new environmental contaminants as a consequence of use of this
         new abatement control technology. There  is  relatively little information  available to
         adequately assess the potential health hazards that may be associated with exposure to
         these metals and their compounds. Analysis of the environmental problems and concerns
         associated with possible  new  environmental contaminants arc discussed.  Limited esti-
         mates are made on community exposure by use of a  meteorological dispersion model.
         Biodegradatiotr potential and  attention is  also given to the limited toxicological infor-
         mation available.
  * Special  Studies Staff,  Office of the Director,
National Environmental  Research Center, Research
Triangle Park, North  Carolina  27711.
                                                  1-A

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   Review of Literature,  Health Effects of Noble Metals
   and Pre-Catalyst Baseline Population Studies  for
   Determination of Human Body Burden of Pt and Pd.
                     Dr. Donald E. Johnson
       A summary of the literature review of the supply and demand
for platinum and palladium will be presented.  This review includes
the prediction of the  impact of the catalytic muffler on these markets.
Data on the health effects from the two metals will also be summarized.
The justification for  a baseline study and for surveys to help understand
the relationship of exposure to the two metals and body burdens will
be presented.
                                 2-A

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  OVERVIEW OF THE LOS ANGELES CATALYST STUDY
                      by
                Charles E. Rodes
Environmental Monitoring and Support Laboratory
        Environmental Monitoring Branch
Research Triangle Park, North Carolina 27711
          Summary of Presentation at
      Platinum Research Review Conference
         Quail Roost Conference Center
               December 4,
                         3-A

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     The Los Angeles Catalyst Study (LACS) is a field monitoring
program which was designed to determine the impact of catalyst equipped
cars on the ambient air in the vicinity of a Los Angeles freeway.
The principal pollutants of interest in this study are H^SO.  and
sulfate aerosols.  Since an acceptable monitoring technique is presently
not available for HpSO* aerosol, associated pollutants and parameters
such as Pb, SCL, CO, and meterology are monitored to allow "surrogate"
predictions of the expected ambient HpSO^ concentrations.
     The site selected for the LACS is adjacent to the San Diego
Freeway approximately 0.5 miles north of the Wilshire Blvd interchange.
See Figure 1 for the location in Los Angeles and Figures 2 and 3
for the layout of the sites and the location of the instrumentation.
Site selection criteria included: (1) a traffic flow (180-200,000
cars per day), (2) prevailing cross-freeway winds, (3) lew S02 background
levels, and (4) site accessibility.  Sampling sites were established
on a perpindiculer line to the freeway to allow the contributions
from the freeway to be determined by simultaneous up-wind and down-
wind measurements.  A system has been recently installed to provide
information on the traffic speed and count by lane.
     The LACS has been on-line continuously since June, 1974, to
obtain data before and after the introduction of the catalyst cars.
An annual report of the data collected during the first year  was
                                     4-A

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prepared in July, 1975.   Some typical  data for the May,  1975,  are
shown in Tables 1 through 9.   Tables 1 through 5 show data from the
1500-1900 hour interval  (evening rush-hour) when there is not  only
peak traffic flow, but also consistent perpindicular cross-freeway
winds.  Tables 6 through 9 are hourly data reduced to 24-hour  averages,
Figure 4 is a summary of the prevailing wind directions  and wind
speeds for May, 1975.  Table 10 is a sample of the hourly traffic
count data for November 10, 11, and 12, 1975.
     The Pb data in Table 2 shows a monthly average contribution
                           o
by the freeway of 7.40 vg/m  (Site C minus Site A).  This can  be
compared to a yearly average contribution of 5.83 pg/m .
     There is no on-going program to measure Pt in the LACS program,
because of analytical sensitivity limitations.  A single large dust
sample was collected from the air conditioning system of a building
near the LACS site during the pre-catalyst period.  This sample was
analyzed for Pt by the Analytical Chemistry Branch (ACB)  of EMSL/RTP
and a subsequent report is available.   There are only two projects
on-site from which the Pt data could be obtained.  One is through
analyses of soil and vegatation samples at various distances from
the freeway.  The other would be to composite into one large sample
all of the aerosol material collected for at least 6 months by the
Batelle Massive Aerosol  Sampler.  These samplers shown in Figure
5 are capable of collecting over one gram of size fractioned aerosol
per day.  The mass breakdown by stage of a typical collection  is
shown in Table 11.  The aerosol samples presently collected by the
Battelle Samplers are being characterized by ACB for epidemiological
studies.  Since only two of these samplers are available at present,
this approach is impossible at this time.
                                          5-A

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cr>
                                            -.   '.  V.-—,   SAMPLING SITES
                                            Figure  1   LACS   Site Location  in Los Angeles

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  CO Analyzer
  Total  Sulfur Analyzer;
  •NO/NO- Analyzer

  24-hr  S02 Bubbler

  24-hr  Hi-Vol
  4-hr Hi-Vol
  4-hr Membrane
  24-hr  Cascade
  Aiibient Temp, and Dewpolnt
  Wind Speed
  Wind Direction
  Massive Aerosol  Sample:
  24-hr Dichotomous Sampler
  0  Analyzer
                             B" site
                              ""
                         24 hr,
                          4 hr,
                         24 hr,
Hi-Vol
Hi-Vol
Membrane
   A
-<&
                                                                       •f
Sepulveda Blvd
                                                                                   "110'
                                        ''C" site
CO Analyzer
Total Sulfur  Analyzer
;iO/M02 Analyzer
24-hr S02 Bubbler-

24-hr Hi-Vol
4-hr Hi-Vol
4-hr Membrane
24-hr Cascade
'Massive aerosol  sample
24-hr Dichotomous Sampler

83 Analyzer
                             "D" site


                          24  hr. Hi-Vol
                          4  hr. Hi-Vol
                          24  hr. Membrane
                          Wind  Speed
                          Wind  Direction
                           Figure  3   LACS Study Site Composition .and Elevation

-------
                         I HO"»l'(T AI. PNOTLCTJON A&lNCT
                         LOS ANOIES C*T*LT5T STUOT
                         Sl'LFATl
                   ~~~I2S039IOI3
                    UG/CU MCTEH  (25 C)
                                                   Table-!-
15/01/75
15/02/75
15/03/75
IS/OS/7S 	
15/05/75
15/06/75
15/07/75
I5/OB/75 _. 	
15/09/75
IS/ U/ 75 _ 	 	 .
15/1/75
IS/ 2/75
IS/ 3/75
)5 / s / 7 5 ,
15 / 5/75
IS/ 6/75
IS/ 7/75
IS/ 8/75
IS/1 9/75
15/20/75 '
1S/2J/75
JS/22/75
iS/23/75
JS/2S/75
15/25/75
JS/26/75
JS/27/7S
iS/26/75
15/29/75
3S/3U/7S
15/31/75
ivCSAGE
'.AX. '
1 I N .
ilC, OlV.
,'OTAL DBS.
22.8* ''^
7.2
4.7
I I • 6
IS.S
I3«0
13-1
17.1 ~"
37.5*
|6>S
IM.6 .__
27.6
20.2
11.8 ' '
5.8
7 .S
100
1 1 • S
1 I .6
12.9
IS .8
22O
30.9
30.9
36.5*
2l«6
IV. 8
13. S
17O
37.5
S.B
8.6| |
29
u.a 	 sm.c-.-.-sm.o..;
2U.2 . 25.3 26.0
26.9 25. S
52 O» S9.2* ' SB.O*
9.t ||.S 12,9
9.N II «S |2.2
II. 0 • |2>6 IS. 9
20. S
IS.S
21 >l
12.0
I'O
SO. 3 	
M.8
13.8
26. &
71 .0
13.6
6.5
9.2
10.6
12.2
IS.S
15.9
25.6
33.6
30.7
S| .9 .
22*8
22.6
13.7
20. I
52.3
6.5
0.95
30
|7.S
J8O
220 "
|S .6
|7.8
3'« 6
2I*S
21>2
30.0
2I>6
21 >2
20.9
23.1
| S.S
|5.6
21*2
2^.9
37.9
35.3
3V. 7
29.5
25.6
22.2
23.5
S9.2
II. s
a, 96
31
7,6
6,9
.
|S, 9
20.7
20,3
27,2
20.5
ISO
21.2
|2,0
11,6 j
17,7
|9.8
21,9
23.|
36.0
35.6
37.9
28*0
2S.S
21.5
8, A
S.s"
2,2
2.7
• 0.5
5.1
2,5
3.0
. 2«
. 2,
3.
-1 •
• -o.
• 1.0
O.B
-I.I
0.7
1.8
0.3
0,6
1.5
2.5
1*1
	 3.3
2.7
. -O.I
S.S
1.2
2.B
" 0.3
21. S 1.7
SB.O * S.S
6.9 . | . 6
9»S5 " '1,91
28 29
$•*
'2.5*
• • . • *— •
SO
S,7
1*0
2,0
, 5.3
S.2
S.S
0.7
2.1
6,0
6,6
6.S
IS.l*
IS. 7*
. • "S.S
S.2
7.6
6.3
6, S
0,6
7,0
S.S
3,2
7.V
5,6
	 	 8,8
5.2
15,7
"0.6
"".' 3,56
'29
0,A C,»
	 DCkXA — .'v-BUXt...
3.2 -2»B
-3.0
5.7 2-1
S.S 2*0
30 |.6
-7.S '•
_ -4,0
.22.5*
S.2
5,7
-OO
00
. 30
9,5
|3. B
1,7
	 o.s
4.1
•"" 4.9 "
7-1
' " ' O.fl " ' '
5. 1
' "' S.9
l.s
4.S
s.7
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2.7
13. B
.22.5
4,79
26
•3'0
2>H
2«*
4.7
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3>S
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7.6
|S«S*
1 3 « 9 o
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3>S
6*1
3*8
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• 2«6
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S,6
6*7
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30
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s.sv
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6.
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2.
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-0.7
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0.5
-1 .7
M.s
•i .0
-0.6 — — • • -
2O " "
-21.6
Si/6-
       vo
IATIO
                                                            1,34
     cotrr
                                                0.97
0,90
|,2S


Oi 46
                       1.17
1.07
0.7U

0.75
 0.9|
•i.bb

 O.b1
s:
ss:
 IS  3lUN|.rICANT  AT  P»0.9S
:  IS  SI&Nlr ICANT  AT P-0,99
                     " ATYPICAL T'TMCY  COUlO'BC  QUESTIONABLE'

-------
      LOS ANCLES  CATALTST  SluOT

      LEAP                 (I5-I7J
"""   '      |2|289201J"~~
 UC/CU ritTEH  (25  O
                                                            Table 2



... 	 ..',:-• — SlIE-A ._ _S1IE B
05/01/75
OS/02/75
05/OJ/75
OS/OM/75
05/05/75
05/06/75
05/07/75
OS/Oo/75
05/09/75
05/10/75
05/1 I /75
05/12/75
05/1 J/75
OS/Js/75
05/ IS/75
05/16/75
C5/I 7/75
CS/18/75
05/19/75
05/20/75
05/2J/75
05/22/75
05/23/75
05/24/75
05/25/75
05/26/75
05/27/75
OS/28/75
05/29/75
05/JO/75
OS/Jl/75
AytHAtt
MAX,
H i N .
510, DtV.
TOTAL OBS
U.IO


0,30
	 o.io
O.JO
o.io 	
U.IO
0.60
0 • JO
' '•"* 0.10
O.bO
0.50
U.JO
"u.io "
0.20
0.20
0.2U
0.20
1.70*
"0.20
0.10
• "•" ' o.io 	
0. 70
O.JO
O.JU
0.10 "
o.io
- - - • • o.20
U.JO
	 0.20
O.J7
' ""' 1 .70 ""
O.IO
O.JO
. 2V
0.10

1 .00
0.70
0.60
0.50
U.JO
O.IO
I. HO
0.60
,0 . S 0 "
1 >00
o.oo
o.oo
o.oo '
O.IO
O.IO
O.JO
O.IO
1 .50*
0.50
O.JO
o.oo
0 .10
0.20
o.io
O.JO
0-20
0.50
O.JO
0.20'
0-50
1.50 "
0.00
O.B2
30

	 Sllt.C.
5.60
6.90
". 11,50
V . 10
•*—-•• l 6, JQ
7. JO
",'" 6.10"
. .6.30
V 5.90
JJ.JO*
n » 20
* v.iu
6.20
6.20
6 .00
6.80
J~ V.BO "
8-60
.8.50
6 «5U'
6. JO
6.00
5.6U
IJ.70
" io.«o
8>5U
" 	 7. JO
6*20
"" 6«VO
6'00
||.OU
7.«6
•--; u.jo -
5.60
2 « 1 V
31
• "'• " B(A "'
*t"I"
... _siit. o.. ..—DELIA . _OE£I«_.
	 3.70
1.90
6.30
1.10
3.10
1.60
1.50
: 1.30
-— — -- ,( ..

_ .
6.20
~- " 1.00
3.60
. " 3.VO
1.70
. 5,00
1.30
1.90
I.JO
" 3.80.
1.00
" 	 i.vo
65.80*
" 5.VO
I.JO
'"" 1.70
1.10
1.10 ""
S.JO
6.UO ""
O.UO

• v . ' ~" • '
o.id
c 0.50 ~"
0.20
-u.ov "~
0.00"
~: 0.80 '" ""
U.JO
0,10
0.20
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O.IO
-O.QV
2.bO*
O.JO
--O.OV
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-U.|V
"-o.uv "'
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~ ' o. jo —
o.oo
"" ~u.oo
"6.7V'"" 0. I 1
45.80 " 2.80
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28 29 •
5.20


V.|0
A. 20
7,00
5.70
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5,30
|3.ou*
JU,80
6. 30
5.70
5.90
5 ,60
6.60
V.60
b,10
b, 30
1.80
6,10
5,60
5.20
I 1 .00
tO.so
«.20
6,90
6,|0
4.70
5.70
|U.80
7.10
|3.00
i.80
.IB
2V
OiA
C,H O.B O.C
..,.0[tI4......0CLlA 	 _ DELIA _ __ UtLIi 	
	 3.30


1.10
3.00
1.30
1.10
1.20 .
... . t—

~ ~L ~t~-m ' •••
5.10
3.50
3.30
• — j.so
1.50
" "" 1.80 "'•'
1.10
~ 1,70 ~~
.2.60
"3.60
3.60
""'""""1.50
65.10*
	 . 5.60 '"
1.00
1.30
I.JO
	 " 1.20
S.OO
	 5.80 " "
• * 6.52
	 65.10
2.60
" JI.V7
26
5.20 3.30

I0.5Q
8.70
5.70
6.60
5.«0 ' ~
6. Ju
.50
1 .70.
1 .7u
.10
.iu - -
.20
,UU
6.70
V.7o
8. JO
8 . 10
2.UQ.
S.b'u
5.7u
5,6J
11. 30
10. 60 "•"•
U.IO
7.UO ~
6, DO
6.10
5.>U
10. bQ
7.J9
12.70
2,00
2.J8
JU

5.30
3.70
2. 50
1. |0
1.20
1.20
.


5«20
1.00
3.40
3.90
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« .90
• 00
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• • 1 V
• JO
.70
.90
65> 10*
5 . / 0
1.20
l.tl)
1.2U
3.70 ' •
5*00
S.bO ,
4.J9 .
45. SO
-O.lV
U. B1
27
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-5. IV
.1 . V9
• J. IV
.2.69
-1 .bV
.1 «V9


*
.2.bV
-2.1V
• 2.bV
-2. 09
-2.0V
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• •«,29
-3.5V
-2. |9
-2 . s9
. I , V9
-0.6V
bl.10*
.l.bV
.1. |V
-2.5V
• 1.79
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.l.VV
.U.bJ
51.10
• 5,1V
JO.bl * "
2o
RATJO  

CORR COEfF St DirFERCNCE IS SIQNirJCANT AT P.O.95 ss: oirrcRENce is SIGNIHCANT *T P"0,vv I. 35 1.08 0.64 2U.V2 ' l«.2lbS •0,02 J8.07 2.765 0.21 IS. 52 I6.V5SS •O.UJ 13.10 •0.01 U.b6 • U.1U U.16 STARRCDI*) VALUES ARE ATYPICAL! THEY COULD BE QUESTlONABLI


-------
         O
LOS ANGLES
                  PRO T t C T I ON «6lNCY
                  CATA.LTST
                         SUSPENOEO PART.
                   .....         I | 10171 013
                    UG/Cu HEUR  I2b C)
                            115-191
              a  -
            T.eb]e»_l_.
                                                         O


Oi/OI /7S
OS/02/75
Oi/03/75
OS/CN/7S
OS/OS/ 75
CS/Ci/75
C5/C7/75
OS/OE/75
OS/09/75
OS/ O/.'S
CS/ 1/75
CS/ 2/75
OS/ 3/75
CO/ N./75
0 5 / ->/75
CS/ 6/?5
OS/ 7/75
OS/ 8/75
OS/ It 75
CS/2'j/75
CS/2 1 /75
0 '.• / 1 2 I 7 S
OS/23/75
OS/ZM//5
05/25/75
05/26/75
OS/27/75
OS/28//5
0 S / 2 V / 7 S
OS/ 30/75
05/31/75

.SilC.
97


116
" ' 91
67
112
68
92
••."' * S3
89
129
62
66
83
73
71
36
31
99
V3
88
75
62
80
93
78
106
83
1 13


1.. -SHE 6 ... SJ
" ' 107

135
130
107
52
1 18
77
1J1 •
17
90
• 113
73
83
93
79
79
1 1
• ~ 17
127
99
9b
89 ~
62
83
100
31
136
88
101
SI

— ' 6, A

•• O,A-
"c"»"
U.B
LIE.C - -SilElO— __OCLIA 	 __OtClA 	 	 OtLIa 	 ULLlA 	 ^OClIi.
151
173
182
192
1 37
112
137
123
169
127
1S9

133
137
113
137
135
96
111
<6B
1 18
|5U
13B
121
136
1 1fl
139
167
161
129
1 1»
|16
1 13 '
1 17
170
— 131
72
._. J22
106 .,


f
• 161 •
	 132
139
137
136
	 123
70
9b
1 16
150 '
117
	 Ib2
101
"ill
131
' ~" ' | 3 I
169
|13
121
- - ,j
10


11
13
-n
6
7
322
-5
1
11
II
17
10
6
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5
13
28
6
7
"11
0
3
7
3
30
5
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2
' 57


76
1J
15
	 25
55
• , 77
71
70
85
7|
71
• ' ' • 60
61
"6|
60
80
67
..... ss
62
" '' ' 63
62
56
55
....... 4j
6|
— 76
16
67
	 H9 	


51
37
25
• — — jo. •'"
36
" *'
i
• * ~ ....
55
' 	 70
73
	 56 	
65
11 "• 17
51
61
17
" ' "* 57
57
.... . . j y
12 .
31
38
	 53
63
' — ' 60
• 8 •
11
i>

17
62
3U
6U
l'
16
-211
80
6V
71
60
5**
50
58
56
54
6/
1 1
l7
5*
1*
6*
53
is
5*
31
7i
2&
6>.
• ~ 37

11
10
21
10
' ~ " 1
27
• ~

.••
11
-- . s,
• 56
"" 16
59
11
% 1»
5 1
17
5 |
52
~ 63
12
i 1
31
' ' 50
33
"~ 55
20
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o,c
	 	 uuu». .......
• 7
-27
•32
•21
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•- , ' o - i
2
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A v £ -< « 01.
81
    100
132
18
61
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MA* .
KIN.
STO.
TOTAL
*,'»
RATIO
T
COR«
S: D|
ss: o


otv.
06S.

— J
1

cocrr
Tf CRC
irrcR
129 ' •"'
31
23.08 65
27

• 	 .__...._... .


N« IS SIGNIFICANT
ENCC IS SIGNIFICANT
11 - ~"211""
11 96
.32 21. VJ
30 31

-- 	 •• • 	 	


AT P.O. 95
AT P-0.77
181 :""322""
90 -11
21.07 S7.18"
2b 27

	 	 U2H-
1.70
0.16


65 —
16
~|5.05 ""
27

	 I.7B "
22.ISSS
0,77


	 77 	
a
|7« 16 "
26

	 1.62 - ~
J1.77SS
0.71


• 60 "
• 2ii
'56. i 8
3-0

-: i.ii •
1.27SS
0.5J


                                                                                                           '  63
                                                                                                              1
                                                                                                           |1.tt4
                                                                                                             27
                                                                                                           'I.3I
                                                                                                           11.JVS3

                                                                                                            0.81
                                                                                                     •I I
                                                                                                    -32
                                                                                                   M.57
                                                                                                    0.9 I
                                                                                                   -b. J6SS

                                                                                                    0.87

-------
                          NITRATE
                               1230692313
                                 «25 C'l
                                                           	Table 4.

05/01/75%
05/02/75
05/03/75
05/01/75
' 05/05/75
05/06/75 	
"Ob/07/75"
Ob/08/75
05/09/75
OS/IU/7S
05/1 1/75
05/12/7b
CS/13/75
OS/li/75
05/15/75
05/16/75.
Ob/ t 7/7b
05/1 8/75,
OS/1 9/75
Ob/20/75
05/21/75
05/22/75
05/23/75 • " .
OS/2H/75
OS/2i>/ >S. " ~"
Ob/26/75 '
05/27/75
Ob/2«/75. .
Ob/29/75
05/30/75
OS/Jl/75

AvERAQt
fl » Jt ,
HIN, .
sru. OEV» ""
T°T*^OBS. ....
RATIO 7* "
T **
CORR C&EFF
..SIZE. A
21.02

3.13
" 2.H8
B.iQ
21.67
10.25
1 1 .73
a, J7
"IB.UO
2 >. v 5
• 8.11
9,88
19.18
IU.6.1
1 o . d |
3. SI
2.10
6.17
""II .06
7.35
" 'l i . "<0
9.B7
1 i,b6
lb.27
' IU.52
16.38
B, V j
9.31
6.!>9

11.21
23.V5
2,10
~ "5,72
29


.....51U.B 	
23.12
21.70
3,7]
2. VI "•'
	 7.13 	
"22.06 "
10.07
ll.bb """"'
8,09
"-" 17.11
•__' 	 28 . 6 3 • 	
7 . 7 1

... _
10.1H'
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3>65
1.87
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7.6|
11-06
V 02SS
0,9«
O.A
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0.90
O.bl
0.35
• 1 ,6B
.0,99


1 ""^
.3,19*
" 0.11
0,88
0,07 ~
.20
.10 "
,6b
•' ,16 •"
,bu
,24 ' '"
,15
" 0,65 "
• 3"* b 1 •
"' 0,62
i.i.i
.0.28 •
2,81
' 0,58
~ i *2»
1,19 —

0.15
.3.51
" ' | ,-11 —
26
t,:07 •••"
i .' !''%'& 0 '' ' '
0^9*
C,U O,B
.01118. ... ..DELIA .
"ru»l!5
3.9l»
•0.37
•U.62
0.^6
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1.36
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u . 2- 1
•O.I |
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I .08
2,25
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2.26
1*32
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1 , *0
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2.2)
2.b7
1 «'9
- 2.27
•
1.24
3.VH
•0.62
I.U6
2?
' 1*13
4,275$
-1*18
2.27
0.30
0. | 1
l>32
•2* 07
•0.81
"


•8. | 7«
"1.11

.
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0.64
0.91
.U
.B3
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• (9
•19
• .23
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> 1 3
• >27
• b6
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.72

0.31
2.27
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2.21
kb
0.79
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0.36
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0.02 —
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• U. 74
-1 .07 '
• 1 . J6
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.0.91 '
O.oS •
.B.Ub
i.B3 -;•- ~
'" — ..
u , v ) .— -
0.97
s: DIFFERENCE is SIGNIFICANT  AT  r
55!  D1>FEKE*CE IS 3J&NIF1CANT  AT.P*0.99        -"          "     .,'."

        • I'" v"ALOCS"*'HE "ATYPICAL I'lHtT" COULO Bf QUESTIONABLC""7"" T-T—

-------


..->._...;.
CNVjMO.-'nCNTAL
LOS ANGLES
•
AMMONIUM
• • 123019
: UG/CU MLTE1 (25
'
1
05/01/75
_ 09/07/75
05/03/75
05/01/75
05/05/75
	 05/06/75
05/07/T5
C5/08/75
05/07/75
_ 05/1 0/75
" C5/ 1 1 //S
CS/ 1 2/75
C5/ 1 3/75
_ C5/M/75
C5/I 5/75
,_ 05/16/75
"05/17/75'
_ 05/18/75
C5/ 1 V/75
0 5 / 2 o / f 5
" 05/2J/75
C5/22/ ?5*
Ob'2j//S
05/2^/75
05/25/75
05/26/7S
05/27/75
05/2S/7S
" 03/29/75
05/ 30/75
~05/3|/75
AVt KAGL
" MAX .
• n IN.
570. DEY,
TOTAi. DBS.
XV w
RATIO i
T >
CORR COEFF
:• . . •
..^".^.sixc.t
0.10
0.21
0.21
0-21
0.03
• 	 U.OO
O.UO
	 O.UO
- • u.oi
l.9|
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	 0.29
0,18
	 0. 30
"~ 0.10
	 0.06
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0.29
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O.UO
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0.00
u. u
0>21
0.23
0.39
0.05
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0.35
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. 0.00
0.00
0.12
0.30
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0.23
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1,16
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0.30
30


PROTECTION
CATALYST ST
1
2013
Cl
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0.71
0.73
2>03*
0*53
0. t« .
0*10
. U'31
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0.16
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0.26
0>32
0>73
0,33
0*51
0.15
" ' 0 • 3 I
• 0> 10
0.39
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0,*6
0.30
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1,13
0.19
0«27
0«27
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3.12
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0*60
31

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U.31
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0,27
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0,29
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0.31
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. !•*!•_
.U.OI
0.03
0.25
0.03
0.1| .
0.09
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.0. 1 8
0.39
0.31
0,26 .
0.30
' 0,20
0,21
0.6tt
0,71
O.ti
O.oi
0.20 -' '
0.28
1.21
-U.|8
U.26
"29
2,55
&.73SS 	
0,92

OiA
..OCLXi...
0.31
0.25
0.22
0.13
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_ 0,16
_ O.b8«
»U,27> *
.0.28*
0.02
0.02
0,|5
0,|9
0.33 ":
O.OI
0,17
0,29
0,13
0.25
0.18 ~~
0,20
0.31
0,12
0,21
0.03
	 o.oi '
0.16
0,58
.0.28
0.19
26
' 2.12
• , 7 i e«
0.90
•
C,H
' UELI&
U,l|
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0.32
0.^5
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0*26
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0. 79
0.«3
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U.UI
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0.35
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-0.6 t •
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U.V3
5: ojrrcRCNCc is SIGNIFICANT AT P»o.9s
SSJ OJfFEHENCE IS SIGNIFICANT AT P'0.9?
"ST A^ £0 ( .r
                  A«E"" ATYPICAL! "THEY COULD' BE OUCST (ONAbLC ------

-------

	 1.
•V?
05/OJ/75
05/02/75
05/03/75
05/OH/75
05/05/75
05/06/75
05/07/75
05/08/75
05/09/75
os/io/;s
05/1 1/75
05/1 2/75
C5/I3/7S .
C5/ M/75
C5/I5/75
05 / I 6/7S
C5/I7/75
Ci'/ J 8/75"
05/19/75 -T
C5/20/75
05/2 1 /7&1
C5/22/75
05/23/7S
: 5 / ~ H / 7 1,
3-3/2 7," 7 5 ~
; 5 / 2 9 / •«
35/JO/75
,5/31/75. .
IV"*G£
tAX.
• TO. OtV.
•\.^.:'
*T>£JJ!"
o«« coirr
*"% ""' SVlf
^ (/ft/CV flCTI
31U 4 51!
e
|B
	 0
17
It)
30 •
8
0
8
21
0
t
	 6 	
2
3
3
18
... 25
It
21
_'_ 	 IB
2
2
1 1
30
_ P
3> 28


• V
^ *

1210JVJ.OJ7 - •
CH I2S' CJ
[i a silt c '•• sue 0'.'
2H
26
6.
21
39
.. 15
16
30 '
26
17
13
• _; . • ._ 	
8
1?
.... 	 «7 ... 	 	 _.„
31
3?
"•'" * ' ' 38 /' "" " "." "
30
..... . 28,'. ' ""' "" ,
' .. 2 J
;, ,15 .
23
1
4.
J • IU .
27
^ "

. . •
:._.. T.-._ •. . -.. .• ^
Table 6 ••;..'•'
DiA C,A DiA C,t» Ui6 0,C
"»6 ' ' '"•"
10
16 • ......
S
f ^ 	 	 ' 	 	
... / >« •_.''.•
.-- . " a -..-_. .._...._.
10
.... 17 . 	
»o '•.-...
"" 	 It " ' 	 '"• "' ' • •"• •' '
6 • i
16
13 •
M '••''.
^ .. .._........ 	 ..,. .. 	 	 •-..'- "
9
..•••' . '
: 	 '•.- 	 •"' 11 ' 	 — .. — ' 	 -. 	 .--•.-.-.
;••;.'." . 23 • -. 	 • . ••• • - • • '- 	 ----- ^
1 • . •
. J3 ... . ... . . ....
*
•" " • 	 :: 23 	 *""• ' -.- • — • 	 •-
•••;•& ' , • .
_A_ 	 <•> •_. 	 "2*1 . . • 	 . . • . _ 	 	 	 	 . _. . .

'. i^'.loss
a,as .
             is  sicmricANi  AT p«o.9s
              IS  SlONirjCANT  »T >»0t»?
•*ftREO(»)  V*LUES"~/iRL~ATTPICAU"THET COULO 8C OUESTIONA8LI

-------
                           KONf INTAI. PROMOTION AGlNCY
                          LOJ  ANGLES CATALYST 5TUOT
                                  ~ MONOXIDE
                     -.___.	^ CAR a ON
                         ~" "'   S2 101 I 1071
                     PARTS PER MILLION
MOURLT
                                                                 Tabe 7
                                                               B,A
                                                                            C,A
                                                                                      O.A
                                                              Oib
35/01/75
35/02/75
)5/03/75 '
)5/Ot/75 	
55/05/75
15/06/75
)5/0;/?5
35/07/75
15/10/75 	 ;_.-
35/ 1/75
jb/ 2/>5
:=/ 1/75
3S/ 5/75
05/ 7/75 .
05/ 8/75
0^/19/75
05/20/75
CS/21/75
Orj/:2/7S
05/TJ/75
05/2S/75
05/25/75
C5/2A/7S
05/27/75"
OS/23/75
05/27/75 '
05/30/75
05/J1/75
AVL^UE
HA X .
n 1 u .
STO. Otv,
TOT AC OB5.
1.5
2.6
0.7
J.7
3.7*
0.9
.2.2
1 .9
0.7
0.9
U-6
0.7
0*6
0*5
• 1
.8
.1
.5
.0
.0
0.5
0.7
0>6
0.7
0.3
1 >2
'3.7
0.3
0.78
30
3.1
H.S
H.6
2.5*
" 	 2.9
1.3
6.3*
'• M.9
	 1.8 	 	 -,_
6.1

6«H*
5>6
3.9 '
» 3.8
,S . 8
M.S
• "~ 	 3.8
•(••J 	 ._
1.7
3.2
• 3.0
S.7
S.O

3.6 '
.-••H
6.S
2.5
0«95
29
j'.e . "
3.4 . V<.
' '•' . " "' '• ' 1.2 "" " 	 .
•..:.,.«,
2'» . . . ' ' ' • .-;.
ill T. . ' •
.
5.0 ' .
3.2 . ,
3.2 .•',,.'
' ' 	 "" '" " 1.H ' • '^ 	
3.5 ;.)' . ' " '
: ' .'" " " '-2.o;-"' 	 "V " " 	 ~ 	
2.8
.'. 	 	 3.6 " " 	 ' ..'''.'•'' " . • •- -
3t7 .
' ' " 2.2 - ' ' ' ' - '
2.5
; .; " ;;;; . ' • •
3.6 •
T 3.3 . . " ^
3.0 • •• .
	 " ' 5.0 ' 	
.0.5.
1.08 ' ' -
".28 . ....
       tn
        i
3; ClFFERtNCC'lS  SIONIFJCANT AT P»0.95
ss: oifTEHCNce  is SIONIFICANT AT p»o.99
~5TA^RtOV»T"V»LUtS "AHt." ATYPICAL" I" THEY  cbuLb"BC
                                                                                                                         (I

-------
     _  V-
L05 ASULCS CATALTSr  STUPT


                     HOU«UT

. • '.
. .
05/31/75
05/02/75
05/03/-7S .
05/01/75
05/05/75
05/06/75
05/07/75
05/08/75
05/09/75
OS/.|'0/75
05/1 1/75
OS/J2/7b
OS/"l3/75
OS/1 N/75
05/15/75
U5/ 16/75
OV/'J ;/7.5 "
05/10/75 .
DS/Jf/75
35/20/75
15/71 /7S
S/i J/?5 "
J5/2N/7S
15/15/75
,5/26/75
15/27/75
!'s'/2'8/75
.'J/2V/7S
•5/30/75 '
5/3J/75 .
vLRjkGt
IN. '
TU« OEV.
3T*L 06S, 	
(^^
I T I 0
PARTS

,.S1.C-,..



0.12
O.J1
0.13
O.U7
0.05
0.02
0.02
	 0.00
0 .00
0.00
O.UO
O.OH
0.10
0.10
0.09
O.OH
0.00
u.oo
O.UO
0.01 .
0.03
u.oo
0,01
0413
u.oo
O.OH
22

1260 II 107 J . . . '"" " " " " ' —
PC* MILLION ; . . . - . . .
• *
..sin. D 	 sm.c-....5iic.o.,
0,22
0.25
0.27 .
0*22
0,17 ' "
0.29
0.28
1 0»27
0.3)
0.27
0,17
0.29
	 	 	 o.32 	 	 •....;..
- 	 0.33 ""' *
0«3?
0.30 '
0.29 -1" """
0.36
0«27
0»38
0>32
' " "" '•" ". o • 2 a ' ' ' "~" ;
0.30
0.35 |

. , 0.30
0.17
6|A t»A 0»A C»k 0,8 P,C
.
. . ' '•

• • •
.." • • U.OB- ' . '^ • . _ v .. r. . _ .
0.20 ' •
Ll . 1 II
t ^ » 1 '~
fl.30 ' .
0.30 --...-.--
""-"•"• OJ33 -." * ~~ ~ " 	 	 •
0,39
U, «»8
0.35
U.20 ' .
r ' 0.20
•--•--• ' 0.20 •'•' '" ' " 	 " 	
0.. 3.t •
0.23
O..3b ""• 	 ~ •" 	 "
0.33
0,28 " •."-"- ' . . 	
0.27
u.35 ; '" •• • ~ j • • '• - ••- - •'•
0..2.B ' ......
0>0«
o.o; O.io • .
:;"- ' ' ': •' ••-••-•-;-- -,••_•-:-,,- 	 -•-•-. 4. 7s •-/•.- -7-v ' • ' -• 	 ••-
cocrr
                                               >u.ss
         .15 SIGNIFICANT AT pio.95
  FERE Ncc'is SUN iriCANT AT'-.-P»O.?V
  i •~>~TAlVC5~'AHE~ATTPiCAL'r THCV "COuLD"BC~QUCST IONABLT

-------
•^ 	

Nftno&tN oioxloC HOURLY I . 	 	 .
^
	 " S' 12602110M ....
• 	 - 	 PARTS PCR MILLION • . Table 9 • .....

>/C|/7S
i/02/75 	
1/03/75
1/01/75
. /O i/ 7 5
./07/75
./on/7s
, .' U ? / 7 5
./ 1/75
/ 7 / 7 c
,/ J/75
,/ 5/75
./ 6/75 	
i / 3/75
./ v/75
. / 2 0 / 7 5
,/2|/75
./22/75
./2J/75
1/21/75
•/26/75
i/27/75
./2B/75
i / 2 9 / 7 5 	 " "
>/30/75
. / 3 I / 7 S
(EKAGE
IX . "
rc. DCv.
)TAL OBSt 	
~^i . 	
JKR cocrr
: DIFFERENCE is
S ! OlFfEKE''CE I
'"'•-

0.01
0.03
0.01
0 >t)6*
0.01
0.01
0.02
0.02
0.01
0.00
0.02
0.02
O.U2
0.03
0.02
0.03
0.02
U.OI
0.01
U.03
U.03
O.U2
0.02
'0.06
0.00
O.UI
22

SIGN)
S SUN
	 .. B|A C,» 0»4 C,» U.d U,C
0.05 •
' • 0.05 .. .. 	 	 	 -* 	 . — . - 	
0.05 . • ' •
0«03« . ...'. .__. ... ; 	 _..-,.- . - .......
0«03» '
0*05 , , ,. , -, - - — - . 	
0.05 ' .
0.05 ' 	 	 r. — .. ..-. ,' .... — • • 	
""" " • 0-05 "»0l . .
o.'ai ' 0»oi 	 ^ ... 	 	 . _ .
0.05 . . • ' 0,01
0.05 0.00*
0.05
0«05 °«01 . 	 . ... ._ . 	 -
0.05 "'•' " ' J}«01 '
o»os- 9'03 -^ 	 	 ...._' .. . 	
' " 0.06 """ "" W'01 • , • •
0.01 u«03
.. ^ o .05 U«OS
' -. 0.03 0.02 ... .. ... . _ '
• • •• - 	 " • O.os 	 0.02
0.01 0.03 . 	
• — * - ' o.OI " ' • • 0.02
0.05 • _. _ .. y.-u2 ... . 	 	 ....- ' . - —
1 '" ' ' o»U6 " ^'03
O.US 0.01 	 	 . .... .. . 	 .
" 0«06 O.OS . .
0.05 . ' . V'01 _ . . . ._ 	 	 . . 	 	 ____
' 0.06 ' V 0.03
0»07« 0.05 ' •
— - - • 0.06 U.oi . ...
' O.U5 0.03 .
..._._ . o>07 .0.05
0.03 0.00 . . .
o.oo - 9«oi
31 22 	 . . _ . 	

	 -.'• 	 — •' 	 	 '• 	 '1.96
|0.i|SS
	 	 u.o? 	 	 ._. ... .. ... _ _.._ 	 _.
MCANT AT p.o.ts .. 	 '__ 	 	 	 - - . - 	 	 — .'....-.-- --• .- 	 	
IF1CAHT AT P»0t9? 	 ~~ 	 " •

— —

—

—
_ —
._ . '- j-
-
... 	

	
VALVES- ARE ATfPJCAL rTMET"cOyL'D BT'ouEST 10NABLE

-------
                            	F-igure 4  .
              HIND  D-IRECTION(XFREQUENCY)  RND  flVG  SPEED
                   BY  SflflPLING  INTERVRLtSITE  R)   >
              05/75
                                                             07/11/75
6-10
8-11
3-7
C-S3
       100.

       35

       60
       o.

       30.

       SO.
       sc.
       52.
                15.31!
          3.0'
                       9.3 IE.
          3.0 r
                4.7 2    5.3 *  .  4.7 X
                                     3.3
                                                  44.
                                                                      flVG KS
                                                                   3.S PPH
                                                         9.0 r
                                                                4.7 S
                                                                   5.0 HfH
                                                         2-0 X    0.7 X
                                                  72.3X
                                           14'.; v.
            •>.   o-o x   0.7 •/.    O.T x    0.
                                                                   5-6 fl?H
                                                        . 1
                                                       ' 0-7
40.

CO.
-*. I;. 4 >.
". „ I PUT
                  . 1 >.
d_^i_^
                                     4-t-'
                 N ,      lit
                                                         9.7
                                                                .-) X
                                     SF   . . S ,   "   SH
                                                               •-NI, ' '

-------
                                   Table 10
                   HOURLY  VALUE  LOGOUT.ROUTINE
YEAR
1975
1975
1975
1975
1975
19:7^1-
1 97-5^ c
1975
19<75-e
1975
1975
1975
1975
1975
i95H:tc~
1975
1975
1975
1975
1975

1975
1975
DAY
314
314
3 1 4
3.1.4
3 :l 4
314
314
314
314
315
315
315
315
315
- "315
-nctf'15
315
315
315
315
315

31'5
315
315
TIME
1 5 : 00
16: 00
1 7 : oo
IB: oo
1.9:00
20:00
21 :oo
. 22:00.
23:00,,
00:00
01 :oo
,* .02:00 f
. V>3:ob"
04:00
^ti5-{^)~0v-
l67YooJ
< 108*00 (
09:00
10:00
1 1 tOO

12:00
13:00
14:00
NORTH BOUND
05815
07308
07461
07064
06110
04244
03094
03218
02846
02314
01338
	 00797
004J.O
00309 •'•-
-r^-.c 00352" "
''""' U'"03:tV6
-;..--•: 7 :0509 7 ri^.ss)
05163. '
O53T49.-.1
05204

04992
05153
SOUTH BOUND
04625
05060
. 04850
04925
04184
03294
02081
02265
02194
01370
00797
00399
00281 v.
00281
- * •-' v -00433* ,. :«.
, ...... .01781 ^ . . -
~ H '''\ '06891 •-•-•«'--•-•
~ •'« i ;,.-:, 1078' IS -'• --^ '.:.
07631 	
06635 5.655 f.
05675

04868
04149 	 ""
04795
                            TEN MINUTE VALUES PER LANE
.*.)
                    HOURLY VALUE LOGOUT ROUTINE
r
r
YEAR
1975
1975
1975
'1973'
1975
1975
1375
1975
1975
1975
1775
1975
1975
1775
1975
1975
1975
1975
1975
1975
1975
1975
1975
1975
 DAY
 315
 315
 315
'315
 315
 315
 315
 315
 315
 315
 315
 315
 315
 315
 316
 316
 316
 316
 316
 316
 31.6
" 316
 316
 31'6
.TIME
 10:00
 ll'.OO
 12:00

 1 1:00
 15:00
 16: 00
 17:00
 IB: oo
 19:00
 20.: oo
 21:00
 22:00
 23: oo
 00:00
 01 :oo
 02:00
 03: oo
 04:00
 6 s: oo
.06: oo
 
-------
  ; \  -
Figure 5
                                     Scolping stage,
                                     cut off *  20/i
JlJUXlU
••:7I;
                                  3.5/1 stager
                                       stage
                                  Electrostatic
                                  precipitotor
            20-A

-------
      Table 11





PARTICULATE COLLECTION AT L.  A.  SITE
Site Location Si
Site A


3.
i.
< 1.
zc Fraction
5-20
7-3.
7 urn
p.m (1st
5 |itn ( 2 nd
stage)
stage)
(electrostatic


plates)
Tine I'criod
~ A
~ A
- A
days (100
days (100
days (100
hr)
hr)
hr)
Total
Site C
(downwind)

3.
I.
< 1.
5-20
7-3.
7 urn
p,m (1st
5 \im (2nd
stage)
stage)
(electrostatic


plates)
~ A
~ A
~ A
days (100
days (100
days (100
hr)
hr)
hr)
Mass
1
0
3
6
l
0
A
.AAO
.8A5
.730
.015
.2A5
.9A5
.A95
g
g
g

g
g
g
           Total                           6.685 g
             21-A

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               DETERMINATION OF HUMAN BODY BURDEN  BASELINE
            DATA OF  PLATINUM THROUGH AUTOPSY TISSUE ANALYSIS
 by F.  Vandiver  P.  Duffield,* Anna  Yoakum,** Joseph  Bumgarner,*  and
                            John B.  Moran*
      Results  of  analysis  for  platinum  in  97  autopsy  sets are  presented.
 Analysis  was  performed  by specially  developed  emission  spectrochemical
 method.   Almost  half  of individuals  studied  were  found  to  have  detected
 platinum  in one  or more tissue  samples.   Deposition  of  platinum in  13
 of 21  tissue  types is reported.   Surprisingly  high values  are observed
 in subcutaneous  fat,  previously not  considered to be a  target site  for
 platinum  deposition.  These data will  serve  as an estimated human tissue
 burden baseline  for platinum  in EPA's  Catalyst Research Program.
 *Environmental  Research  Center,  Environmental  Protection  Agency,  Research
  Triangle Park,  N.C.

**Stewart Laboratories,  Inc.,  5815 Middlebrook  Pike,  Knoxville,  Tennessee

-------
Measurement of Platinum from Catalyst-Equipped Vehicles, Combustion
                       and Attrition Products

                           Mr. John Sigsby
             Environmental Sciences Research Laboratory
                   Environmental Protection Agency
            Research Triangle Park, North Carolina  27711
                Notes used in presentation (attached).
                                  24-A

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            PRODUCTION  CATALYSTS  1975-1976  CARS
ro
en
i
MONOLITHS



   BASE  MATERIAL



     CORDIERITE  ( 4 C M G F E 1 0 ' 4 AL2 0 3'10 Si 02 ' H20)



     R I L A R I T E (K20 '4CAO'2AL203'24Si02'H20)



   WASH  COAT  Y - A L U M I N A



   CHLOROPLATINIC  ACID  (H 2 P T C L 6)  ADDED



PACKED  BED



   BEAD  AND   EXTRUDED  PELLETS



     Y ALUM I NA-.H I GH  PURITY

-------
                            CATALYSTS
      PACKED  BED - GM - AMC
PO
CTi
TWO  SI.ZES  160 C LI, IN,  -  1 8 " L 0 N G - V E G A  AND  ANC (6CYL.)


            2 6 0 C U , IN,*  24 "LONG-ALL G M  BUT  VEGA
                                       %

B.E.A D, S, . AND  PELLETS  INT E0R C H A NrG E A B L E


* 3 KG  OF  PACK I NG


   A M C.  D I F F E R E f! T  F 0 R H U L A T I OJ! .THAN 6 M


  .,0.5 TROY  OZ ,  ( 1 , 56 GRAMS )

-------
                          CATALYSTS
ro
ONOLITHS

  FORD-ENGELHARDT  ( 6 0 % )  - P T / P D  AND  PT
    f-1 A T H E Y  BISHOP  ( '4 0 I ) - P T / R H - 1 9 7 5

                          PT/PD - 1976

    -0,05  TROY  0 Z ,  ( 1 , 5 6 G M s )  ME T A L
    CYLINDER  5 "  D I A , - 1 3 "  LONG

  ALSO  USED  BY  MANY  FOREIGN  M A N U F A C T U R E R S  ( V W E T c, )

  CHRYSLER - UOP

    V A R I A B L E - C 0 MP 0 S I T I 0 N  AND  LOADINGS

    OVAL  8 "  WIDE  -15-18"  LONG  (MUCH  LARGER
                             THAN  ABOVE  FORD)

-------
                                               ANALYSIS OF TUBE SWEEPINGS
CO
I
Sample
A
B-E
F
F
F
Laboratory
EPA-ESRL
EPA-EMSL
SWRI
Stewart
Livermore
Method
Wet AA
Wet AA
Flame! ess AA
Activation Analysis
Emission etc.
AA
ESCA
Pt vg/gm
«
245 - 635
60 - 145
0.034
2.44
0.165
100
West Pt+4

-------
F i Iter
Air
  in
         Engine
      C y n G ~prr,e t e r
f\j
vo
cng i
                                             Figure 2
                                     Flow Dioqrom for Engine Exhoust
                                         Porticulote Collection
                                                                         Air
                                                                         out
                               I ns trimen t
                                  and
                               Control Room
•„ .  . ^
?/ i x i n g
  i fe
                                                 Part iculote
                                               rav i r,2 tr i c  Fallout
                                                                 Flow —>-
                                                                Control
i ! i >




4












i











O
^i V i. /

\
$
\ /
\ 1 /
Scmpl ing Slits
- — ' oil Pipe




N
\,
X
~s
\
\
^>>
"X

Anderson _^
Seporctor
"•»•
•'•—S tender d Muffler


/^

i
19 . S°0tt R=seorch ins, Killinor^
•v
^
•^
™ ond ,'02 F.lter
Anclys i s



















Ai
| Pu










i
y






l_
                                      F isher  Gas  Port it i oner
                                         CO,  C02,  N2,  02

                                      Beckmcn 109A
                                      Total Hydrocarbon
                                      Analyzer
             Exhaust P ipe
                                                 Flow Meter —>
                                                                         Vacuum
                                                                         • Pump
                                                                                                  n
                                                                                                     •o
                                                                                                     O
                                                                                                     I
                                                                                                Kenoneter

-------
                SNAIL.  P ARTICULATE  EMISSIONS
      SOURCE
     EXXON
     EPA-RTP
                       DETECTION  LIMIT

             < 1 0 " 7 G M / M I L E -  < 1 0 ' 5 M G R / c M -
                                 X-RAY AND CHEMICAL
                                       •
             < 1 0 ~ 5 G M / M I L E -  < 1 C " LI u G R / c M - X-RAY
oo
o
1 , 5 6 •  G R A M S  TOTAL  LOADING   -  50,000 MILES OPERATION
            1,56 G M
         50,000   MILES
                    = 3,1 x  1 0"5  G M/M I L E

-------
       lexicological Studies of Auto Exhaust Catalyst Components
                          Dr.  Jerry Stara
                   Environmental  Protection Agency
                          Cincinnati,  Ohio
     The attached reports were used as the basis for Dr. Stara's
presentation.
                               31-

-------
                                                                              B.4
                Comparative Effects of  Intratracheally Administered
              Sulfuric Acid ar.d Palladium Sulfate on  Pulmonary Free Cells
                                K.  I. Campbell

                                E.  L. George

                                T.  Grumbles,  and

                                Y.  Y. Yang




                                   SUMMARY
    The comparative  acute  toxicity  of  sulfuric  acid  (H^SCM)  and  palladium
sulfate (PdS04)  to pulmonary  free cells  was  investigated  in  rats  using  the
intratracheal  route  of administration.   These materials were of  interest
because of their potential  emission as  constituents  of exhaust from
automobiles using high-sulfur fuel  and  noble-metal catalytic devices.   The
pulmonary free cells (PFC),  normally primarily  alveolar rr.acrophages,  are  an
important component  of pulmonary defense and clearance functions.

    H2S04 solution was administered in  a dose of  0.45  mg  [S04~2j/kg body
weight to one  test group (5  rats each  group) and  an  aqueous  solution  of
hydrous PdSG/i  was given to the other test group at a [SO/j-Zjdose  rate
equivalent to  that of the  H2S04 group  and [Pd^jdose of 0.5  mg/kg.   The control
group received deionized water (H^O) only.   Dose  volumes  ranged  from  0.1  to
0.14 ml per ret, and body  v/eights averaged 188  gm.   One day  later the PFC
werg collected by saline endobronchial  lavage.  Total  and differential  cell
counts, cell viability, phagocytic  activity, and  lung  and body weights  were
determined.

    With respect to  several  criteria,  there  were  marked and  significant treat-
ment effects in the  FdS04-exposed animals but not in those exposed to H2S04.
Noted effects  included the following:   increased  total numbers of free  cells;
increased sub-populations  of  both viable and nonviable nucleated  cells,
phagocytic and nonphagocytic  macrophages, polymorphonuclear  leukocytes, and
red blood cells; increased numbers  of  phagocytized  test-spheres;  increased
dried lung mass.  There were  no apparent effects  on  proportions  of cells
nonviable, on  proportional  phagocytic  activity  (spheres per  cell), or on
cell size (diameter).
                                        33-A
                                        -acfr;   -

-------
    The results indicate definite effects by PdSO/; on the pulmonary free
cell system suggestive of a recruitment of alveolar macrophagr:S and/or
retarded cellular clearance activity, of leukocytic infiltration, and of
hemorrhage and perhaps cdc-r^a.  These effects are regarded as .'having toxic
implication for palladium compounds  (especially soluble and  ionizable)
gaining access to the lungs' terminal air spaces, and 'being  associated
with the cation moiety rather than the anion [SO/j'^Jor acidity of the material.
                                             34 -A

-------
                                                             B.5

                                                       P75-31.6
EFFECTS OF VARIOUS  METAL SULFATES on SUCCINATE-DEPENDENT RESPIRATION
                 S.  D.  LEE, M. RICHARDS, L. MCMILLAN
                U.S.  ENVIRONMENTAL PROTECTION AGENCY
                         CINCINNATI/ OHIO
                      R,  SHELLEY, V. N, FINELLI
                      UNIVERSITY OF CINCINNATI
                          CINCINNATI, OHIO
        For Presentation at the 68th Annual Meeting of the
                  Air Pollution Control Association
        Boston, Massachusetts            June 15-20,1975
                       35-A

-------
                                                                     75-31.6

   UTECrs OF VARIOUS MJ7TAI, SULFATES ON SlA-acn Monitor wis used to measure .the
O2~uptake.   V.'c tested the effects of various sulfatcs,  such  as cadriur1.,
palladiin;., rar.grmeeo, r.?.crnesiur.,- calciii:', sodium, and iuncniurr. on  the system.
The results  indicated that the sulfate icn in tissue slices  or in  hcnocenatc
did not affect the respiratory chain.  However, ancr.g the cations, Cd4^
appeared to  be the most potent inhibitor.  Cacri'-m inlubited the 02-uptaXe by
approyjurately 501 at 2 x 10~7  H and 100; at 3.3 x 10~5  M.  Othsr catioj^.s did
not shov: iriiibitorj'  effects at sini laj- concentrations.  To achieve a JC*
inhibition by Pdr/D^  noire than  10"^ K w?.s reruircd.  Cadmirn  ion, a kno-.n
potent inhibitor  of  the nitcchonJrial rcs:;iratcry chain, was utilized in this
opprijncnt as a reference to>:icr.nt.  CdSO^j V.TIS found to ho manyfold rore
toxic to the respiratory chain than KSO4.  Other cations, such as KnJ~1', ivq"t~f,
Ca4^, were not inhibitory at oor.centraticns up '_o 10-3  M, but appeared to have
a slight stinulatcry effect.  Ce4"*"1", r:a+, ?r.d 13'.^ did  not cause any effect.
Intragastric acninistration cf a single and nultiple dose cf CdSC^, PttS^Jj.
,£ind PdSO4 at 3 ?jnd 60 tr-ole per kg body v.vi^ht, did not affect succir.ate-
depcr.dont respiration in any of the organ tissues tested  (kicr.ey,  liver, heart,
and lung).   It is probable that, under these experimental conditions, these
rrotals were  not absorbed in significant arotmts to cause any effect en the
mitochonirial respiration of soft tissue orcans.  The in vitro o^rirL-nent
suggests tJxat at  least Cd^"1", the nost potent inhibitor  tested, should have
impaired this cnzyne systen if absorbed in significant  quantity and accumulated
in the tested organs.
                          36-A

-------
              INTERACTION OF METAL BINDING AGENTS FROM
                COMBUSTION PRODUCTS V.'ITH TRACE METALS
      L. V7. Michael,  V.  N. Finelli, V. J. Elia, H. G. Petering
                 Department of Environmental Health
           Kettering  Laboratory, University of Cincinnati
                       Cincinnati, Ohio 45267
                                and
                   S. D.  Lee and K.  I.  Campbell
               National  Environmental Research Center
                  Environmental Protection Agency
                      Cincinnati, Ohio 45268
                              ABSTRACT
   Metal binding agents  present in the environment may react with trace
metals thus altering  essential biological functions.  Combustion pro-
ducts from tobacco, gasoline and coal as well as from other pyrolytic
processes are ubiquitous sources of such material.  We have demon-
strated the potential for such interactions in biological systems by
comparing the copper  binding activity of condensates from selected
sources.
   The reactivity with other metals was also examined.  The isolated
metal binding fractions  have been tested for effects on metalloenzyraes
and mitochondrial respiratory activity.  In addition, the efficacy of
                                                              *
this concept was tested  in vivo using a chelating agent, 3-ethoxy-2~
oxobutyraldehyde • bis(thiosemicarbazone), (H-KTS).  H-KTS has been
found to alter cholesterol metabolism In a manner similar to that ob-
served in copper deficient rats.
                                    37-A

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    CHEMICAL CHARACTERIZATION AND  BIOCHEMICAL TOXICITY






   OF ORGANIC AND  INORGANIC  COMPONENTS  OF AUTO EXHAUST
     Contract No. 68-03-2011, Project No.  Cl-7^-C217
Annual Progress Report - June 20,  197*4  to June  20,  1975
                              38-A

-------
A.  INTRODUCTION
     This is the first annual report on research progress
achieved betv;een June 20, 197^ to June 20, 1975 carried out
under contract No.  68-03-2011, Project No. CI-7^-0217 of NERC/EPA,
Cincinnati.   The research data reported herein'^are the result of a
coordinated  and collaborative effort betv;een this laboratory and
ETRL project Officers, Si Due Lee, Ph.D., and Kirby I. Campbell,
D.V.M.
     During  this year we have achieved substantial progress in
the following studies:
     1.  We, have characterized the auto exhaust gas emissions
(AEGC)  from  engines with and without catalytic converter as to
titratable acidity, copper binding activity and total organic
carbon.
     2.  We  have isolated from the whole exhaust condensates the
protonated and non-protonated ligand fractions and tested these
metal-binding fractions as inhibitors of tyrosinase, a copper
containing metalloenzyme.
     3«  We  have assessed certain .biological effects in rats
exposed to irradiated and non-irradiated auto exhaust emissions
and to single components of auto exhaust, such as carbon monoxide
and sulfuric acid mist.  Lipid metabolism, serum and liver lipids,
hematologic  effects and organ damage were the parameters studied
in the exposed animals.
     4.  We  have investigated the effects of noble metals, plati-
num and palladium,  and of other heavy metals, cadmium and mercury,
on 6-aminolevulinate dehydratase  (ALA-D) of rat erythrocytes, a
zinc and -SH containing enzyme, and on succinate-dependent respir-
ation of rat lung,  kidney, heart and liver homogenates.  The effects
of the above toxicants have been evaluated and compared in these
metal sensitive systems.
     5.  We  have initiated nutritional studies to evaluate the
effects of components of exhaust emissions on essential trace
metal metabolism and to determine the protective roles that cer-
tain micro-nutrients may play against the toxicity of these agents.
                              on A

-------
H.  BIOCHEMICAL STUDIES
Exposure studies were conducted at the ETRL/NERC facilities in
Cincinnati in collaboration with Dr» S.D. Lee.
     1.  Biological Effects of Auto Emissions
     V7e have participated in a series of studies designed by ETRL,
"Toxicity Assessment of Mobile Emissions" (TAME).  The purpose of  ,
these experiments was to determine the efficiency of oxidizing
catalysts in reducing certain pollutants.  The qualitative and
quantitative alterations of emissions due to the introduction of
an oxidizing catalytic converter to the automobile exhaust system,
along with the possibility of introducing nev; pollutants such as
platinum and palladium in the environment, called for toxicity
studies in animals exposed to engine exhaust emissions.  A series
of exposure experiments, whose characteristics and conditions are
reported in Table I, were conducted to evaluate the toxicity of-
irradiated (I) and non-irradiated (N-I) exhaust emissions from
engines with and without converter.  Other variables in these
experiments were the length of exposure, the sulfur content of.
the fuel and the type of engine.  The animals used in the first
experiments(TAME1s I,J, and K) were Sprague-Dawley lactating fe-
male rats.  In the remaining experiments, adult male Sprague-
Dawley rats were used.
     To assess the effects of a catalytic converter on the toxicity
of engine exhaust emissions, two pairs of exposure experiments can
be utilized:  TAME I vs.. TAME J and TAME 0 vs. TAME N.  The first
pair of experiments was conducted to determine the effects in
lactating female rats of short term exposure  (7 days, 2^ hours/day)
to emissions from an engine with and without converter, TAME J
and TAME I, respectively.  The other, pair of experiments, TAME N
and TAME 0 were designed to compare the effects in adult male rats
exposed to the emissions of an engine (1975 Ford, UOO CID)
equipped with a converter and tuned as specified by the manufacturer.
The conditions in both experiments were kept the same with the exception
that in TAME 0 the converter was removed from the exhaust train.
In these two latter experiments the animals were fed a semipurified
                               40-A

-------
diet with controlled  levels of essential trace minerals,
especially copper and zinc (Table II), to study the impact of engine
emissions on certain  aspects of the metabolism of these trace
metals.   The other experimental animals v;ere fed commercial chow.
     The data collected from the various experiments are reported
in Tables III through XVII.  An overall analysis of the results
suggests that various biological parameters have been significantly
altered  in the animals exposed to non-catalytic emissions (TAME I
and TAME 0 experiments).   However, some slight alterations and
a trend  of higher serum cholesterol were also noticed in some
of the rats exposed to catalytic emissions.  In summary, we have
observed the following changes:
     a.   As shown in  Table III, TAME I animals (lactating female
rats), both I and N-I groups, showed a dramatic loss of body
weight.   This effect  was more severe in the I group.  In TAME J.
the exposed animals also showed a loss in body weight, although
the effect was less pronounced in this study than in the preced-
ing one.  In TAME K the animals exposed to the engine emissions
gained more weight than the control animals.  No significant
effects  on growth were observed in the other TAME studies, except
for the  rats exposed  to the non-catalytic emissions in TAME 0
where a significantly slower growth was evident in N-I and I groups.
The growth patterns of the animals in the first 3 TAME studies
cannot be compared with those of the remaining experiments since
two different animal  models were used; lactating rats were utilized
in the earlier studies while male rats were chosen thereafter.
Rats used in TAME N and TAME 0 seemed to show better growth
patterns than the others.  This observation may be explained by the
fact that the animals in these studies were fed a semipurified diet
which met all the NRG requirements in both macro and micro dietary
constituents (Table II).
     b.   Elevation of serum lactate dehydrogenase (LDH), which
may indicate possible tissue damage, was observed in I and N-I
groups of TAME I.  No effects were present in the experiments
where a catalytic converter was used (Table IV).
                              41-A
                               /y /

-------
     c.  In all the experiments serum glutamate-oxaloacetate
 transaminase (GOT) was normal in the exposed animals.   This obser-
 vation, along with the elevation in serum  LDH  in TAME  I animals,
 may suggest that the target organs of toxic components of.non-
 catalytic emissions may be the lung and/or trie, kidney, probably
 excluding the heart and the liver.
     d.  Serum lysozyme was measured only  in TAME  J  and TAME "K.
 An elevation of serum lysozyme activity would  have indicated  a
 possible effect on the lungs.  However, the exposed  animals in
 these experiments did not show any alteration  of this  parameter.
 Analysis of urine lysozyme in the same groups  of rats  were not
 conclusive, since the values obtained for  controls and exposed
 animals we.re scattered and yielded a large' standard  deviation.
 A significant increase of lysozyme .activity in the urine of -the
 exposed animals would have suggested kidney damage .•
     e.  Blood analyses showed that hematocrit and hemoglobin-
 fables VIII and IX) were significantly-elevated in  the animals
 exposed to non-catalytic emissions (.TAME I and TAME  0).  A "poly-
 cytemic response (Erythrocytosis^) >may have occurred  in these
 animals since the levels of CO in the exposure chambers were
 elevated.  The increase in hematocrit was  not  due  to'dehydration
 since serum albumin was found to'be^normal-in  the  exposed animals.
     f.  The effects of engine emissions on lipid  metabolism, as
 shown in Tables X through XV, can be summarized as follows:   N-I
 and I groups of TAME .L sho.wed 'an- elevation of  serum  triglycerides
which may be statistically significant.
     Animals in TAME N and TAME 0 had higher serum triglycerides
 than those in TAME M.  This effect may be  due  to the dietary  change
 (semipurified diet vs. laboratory chow).
     There seems to be a trend of higher serum cholesterol in animals
 exposed to either catalytic or to non-catalytic emissions.  How'ever,
 this effect is not. statistically significant except  in TAME N where
 N-I and I groups are statistically different from  control, p<0.01.
     There is no difference  in total  liver lipids  and  liver  phospho-
 lipids between exposed and control animals. -' However,  'liver 'chol-
 esterol  seemed to be  affected in TAME  0 where  the  value for  the
 I  group  is significantly  lower than  that  for control.
                               42-A

-------
     The in vivo incorporation of acetate-1-  C into liver lipids
 van not affected in any of the exposure studies.
     g.  Serum zinc and copper v;ere analyzed in the experimental-
 animals of TAME N and  TAME 0.   In the animals exposed to non-
 cntalytic emissions serum zinc was elevated ^'-concurrently serum
                                             f
 copper decreased in the sarne animals.  No alterations of these
 parameters were noted  in the animals exposed to catalytic emissions.
     h.  Erythrocyte 6-aminolevulinate dehydratase (ALA-D) was
 substantially elevated in the  I and N-I groups of TAME 0.  ALA-D
 is a zinc dependent enzyme involved in the synthesis of heme.  This
 elevation may be due to an alteration in the metabolism of zinc
 or to the 'presence in  the exposed rats of more immature red blood
 cells which may ccntain a higher concentration of ALA-D.
 2.  Effects of Carbon  Monoxide Exposure
     Four groups of 20 adult male Sprague-Dawley rats were
 exposed to atmospheres'containing various concentrations of carbon
monoxide (CO) for periods of 2 .and 4 weeks.  The carbon monoxide
 levels in the four exposure chambers were 0, 57-5» 172.5* 517-5
mG/m  (0,50,150, and U50 ppm).  The following parameters were
measured:  body weight, lung,  liver, kidney and heart weight,
serum enzymes (GOT, LDH, and HBD), hematocrit, hemoglobin,
serum albumin, serum cholesterol and triglycerides.
     a.  Gross biological effects — In the animals exposed to
the various levels of  CO for 2 and 4 weeks there was no significant
effect in body weight  gain when compared to the control rats.
Serum GOT, LDH and HBD were not affected in any of the groups, thus
confirming that CO exposure, at these levels and conditions, did
not cause cellular damages in rat organs such as liver, lung,
kidney and heart (see  Appendices 1 and 2).  From the organ weight
measurements, we found that the heart was the only organ affected
by CO exposure.  There was a significant increase in the heart
weight of animals exposed to CO (Figure 1).  Our results agree
with those reported by Theodor et al. (J. Occup. Med. _13:2U2, 1971)
These authors attributed the cardiac hypertrophy to an increase
 in cardiac work due to an elevation in blood volume and viscosity
 (High hematocrit).
                             43-A

-------
     b.   Effects  on hematocrit.  hemoglobin and serum albumin-- An
elevation in hematocrit and hemoglobin levels was observed after
?. weeks  of CO exposure (Figures  2 and 3).  A follow-up study on-
hcnatocrit was performed after termination of CO exposure.  Figure
*rshows  a drastic increase in hematocrit during CO exposure and a
                                               •f
p.radual  return to normality during the following recovery period
of 'I weeks.   Serum albumin was not altered in the exposed rats.
   (  c.   Effects  on serum cholesterol and triqlycerides — There
nppcars  to be a trend  to lower serum cholesterol values in the
CO exposed animals which may be  significant only in the group
exposed  to the highest level of  carbon monoxide (Table XVIII).
Ho significant effects were evident on serum triglycerides in
the animals exposed to any concentration of CO (Table XIX).
     Furthermore, during TAME 0  experiments we exposed a group of
six rats fed semipurified diet to CO alone.  In this study the '.CO
levels were kept  similar to those observed in the whole emission
                                i        o
exposure chambers, that is, 230  - 46 mg/m  (see TAME 0 aerometry
data).  The results reported in  Table XX show that hematocrit and
hemoglobin in the CO exposed animals are substantially higher than
control.  Other parameters affected in the exposed animals were
ALA-D activity, serum zinc and liver cholesterol.  Again, as in
the previous experiment, we noticed that the CO exposed rats
showed a slight reduction in serum and liver cholesterol.
Elevations in hematocrit and hemoglobin  (erythrocytosis) were
reconfirmed in this study.  An increase of ALA-D activity in the CO
exposed  animals may be explained either by the alteration of zinc
metabolism, since  this enzyme is  Zn-dependent (Finelli et al.,
Biochem. Biophys. Res. Comm. 60:1^18, 197^5 see Appendix 3),
or by a  possible  increase in number of immature red blood cells
which may contain a higher concentration of ALA-D.
3.  Effects of Sulfuric Acid Exposure
     In  TAME M and TAME N studies, run concurrently with exhaust
emissions exposures, two groups  of adult male Sprague-Dawley rats
                                     o
were exposed to H0SO..  mist, 6.59 mg/m° for 90 days and of 6.35
    o            d.  "i
mg/m  for 28 days, respectively.  Growth rates, hematocrit and
hemoglobin levels were not altered in the animals exposed to
HgSOjj (Table XXX).  Tables XXII, XXIII, and XXIV show the effects
of fUSO^ exposure on the lipid metabolism.  Of the various para-
meters measured only the level of phospholipids seemed to be
significantly altered.  The liver phospholipids in the H

-------
 exposed croup of TAME  N  v/ere 6 6$ higher than control.  However,
 no significant alterations  v/ere noticed in the in vivo incorpor-
 ation of acetate into  liver lipids.                                 •
 A.  Effects of Intraqastric Administration of Pt, Pd and Cd salts
 on SuccJnate-Stiniulated  0 -uptake
     Adult Sprague-Dawley' rats (300 to 400 g) were exposed to
 ulnglc and multiple  intragastric injections of platinum, palla-
 dium and cadmium chlorides  and sulfates.  In the single dose exper-''•
 Incnt v;e used two concentration of the chlorides (40 and 80 jamols/kg
 body weight), and the animals were killed 24, 48 and 72 hours after
 exposure.  Succinate-stimulated Op-uptake was tested in the homo-
 gcnntes from liver,  heart,  lung, and kidney (Table XXIV).  No pro-
 nounced effects were observed in the rats exposed to 40 and 80
/imols PtKpClg after  24 and  48 hours post administration.  Cadmium
 chloride seems to enhance Op-uptake in liver, heart and kidney-
 nt both concentrations,  72  hours post-injection.  This effect
 could be explained by  a  possible uncoupling of the mitochondrial
 oxidative phosphorylation.   A depression of Op-uptake in lung homogena
wns observed with CdClp  24  hours post-exposure; however, this effect
decreases at 48 and  72 hours, incubation time.  Further studies
using isolated mitochondria may be required to confirm these
findings arid elucidate the  mechanism by which Cd   affects the
mitochondria electron  transport.  In vitro studies conducted by
other investigators  and  by us have shown Cd   to be one of the most
toxic metals affecting the  succinate dependent respiration.
     In the multiple-dose experiments groups of rats received
daily 8 and 80 juunols of  the compounds per Kg body weight for
4 consecutive days.  The animals were sacrificed 50 hours after
        •
the last exposure.  Succinate-dependent respiration in the homo-
genates of the various organs was not significantly altered by
any of the compounds used (Table XXVI).  These findings suggest
that intragastric administration of Pt and Pd salts do not affect
the mitochondrial function of the cell in various organs.  The
reason for the lack  of detectable effects may lie in the low
 intestinal absorbtion  of these compounds  (see W. Moore, D.
 Hysell, W. Crocker and J. Stara  in Toxological Studies of
                             45-A

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 I'al Indium and Platinum Document,
 5.  Effect of PdClp Injected  Intravenously into Rats on
    *              t_
    Succinate-stimulated  0  -uptake in Tissues
    *~~ "~                     t_
     We measured the succinate  stimulated respiration in homo-
 eenales of'liver and heart  tissues of rats which had been  in-
 jected bi-weekly with various doses of PdCl?.  The total dose, of  •
 PdCl  in saline solution  received  by five experimental rats
 consisted of 6.5 mg/kg body weight injected in 7 portions  (3
 Injections of 0.5 mg, 3 of  1.0  mg  and 1 of 2 mg/kg of body weight).
 The control group (5 rats)  received at the same time injecticns
 of saline solution.  Four weeks after the last injection the
 animals were..sacrificed',  and  the  liver and heart were homogenized.
 The O-up'.take-values for  the  experimental and control groups
 are, reported in .Table'XXVIl1.  l There "was no significant -effect
 ofiRdClp exposure on either heart  or Driver 0 -uptake.
 6. 'In-.Vitro Effects of Various^ Metal's on  (S-Aminolevulinate
    Dehydra ta'se">-'.Act-i'Vi-ty  '-
    :The. following^compounds  were  tested in vitro on the erythro-
                        '  ..  i  ....-..•..'.         '       • '     '
 cyto - 6-amlnolevuMrr.a-te de'hytrata!se '(ALA-D) activity:  HgCl2,
 ClUHgGl', PtgCLjij, "HgPtcTg-1, CuCl'2,"znCl2", and PbClg.  Lead ion  is
 a known inhibitor of this enzyme;  thereforev we compared the
 effects of all the"1 other  metals to the inhibitory capacity of Pb
At concentration'" 6-f ' 10" M PbClp there was-approximately 50$
                            ....*-   •     '.-
 inhibition of ALA-D activity.   Le'ss inhibitory were CuClp, HgCl  ,
                                    •- -. •> •    '  >      •'•.«:   •   2
and  CdC'l0 which produced  the  same  extent" of inhibition at  concen-
        ^               -c;       -h              .       "i" ••  '
 trations:>ra-1ging from 10 '^  to 10   M.  These metals v;ere 10, to 100
 times  .less inhibitory than  lead.   The least ALA-D inhibition was
                                   r                 -    ..•••'
 produced by :.the( following'compounds :  'PtK Cl^, H PtCl^-, PdCl_,
and  CHQHgCl.   This group  of compounds 'produced 50^ ALA-D inhibition
      J                      .    -l\      _^j
at concentrations ranging from  10    to 10  M; that is, they.are
 100  to 1,000. times less active  than lead. " Zinc ion has been found
 to activate ALA-D.' The in vitro  effects Of various metals on
 ALA-D  are reported in Figures 5 and' 6.
    We had previously shown  that ALA-D is a zinc dependent enzyme
 (Table  X%VII) and now have  observed both in vivo and in vitro
                  I i        »^              .       .,•.'•           •
 interaction of Zn _,  and .rPb .;  on the activity of this enzyme
 (see Appendix ^).
                                  •\-»'
                              46-A

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     We  have plotted  erythrocytc  ALA-D activity versus time
        7)  for  two groups  of rats fed a semipurified diet low
In zinc  aMd supplemented with Zn  in deionized drinking water      • '
(?.5 and ^40 ppm, respectively);  from the seventh to the ninth
week one half of the  animals from each group deceived 200 ppm
  j 1                                          *
Pb   in  drinking water.
     In  all animals there  was a  dramatic loss of erythrocyte 'ALA-D
activity with aging.   The  animals in group II, which received
a lov/ zinc  diet, showed considerable lower enzymatic activity
than those  in group I,  (statistically significant, p^0.05)»
The addition of lead  to the  drinking water caused a reduction
In enzymatic activity to zero in  the low zinc group and almost
to zero  in  the  zinc-fortified group.  Following the removal of
the lead exposure, ALA-D activity in rats fed the higher zinc
level returned  to normal values  more quickly than in those en  '.
                         • X
lower zinc  diet.  Five weeks after withdrawal of lead the enzyme
Activity in the lead-treated low- zinc group was still significantly
lower than  in the control  group,  whereas there appeared to be no
significant difference between lead-treated and control groups in
the, animals fed a higher zinc diet.
     The effects on erythrocyte  ALA-D activity by in vitro addi-
tion of  ZnClp to blood obtained  from rats fed low and high zinc
diet with and without lead exposure are reported in Figure 8.
In all cases zinc reactivates the lead inhibited enzyme; at
10~ M ZnClp ALA-D values are similar in lead-exposed and control
animals.
     In  another experiment,  rats  were fed for one month laboratory
chow containing 500 ppm lead (as  lead acetate).  Two months after
withdrawal  of dietary lead the average level of lead in the blood
of these animals was  about 50 jug/lOO ml and ALA-D activity ranged
between  10$'and 20^ of normal.  Figure 9 shows the activation of
ALA-D in the blood of one  of these animals by the in vitro addition
                                                           -U
of ZnClpj again we noticed that  ALA-D activity peaked at 10  M ZnClp,
Flcure 10 show  the ALA-D activation by ZnCl0 in adult rat blood
                                            -S
with and without the  in vitro addition of 10 -'M PbCl0.  Between
  _h       _c         	                 -     2
10   and 10 -'M.  ZnClp, the  enzyme  activity reached about 150$
                             47-A

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i.
[  of control in both incubation mixtures.
i      The data lend further support to the reports  that ALA-D
  IP A zinc-dependent enzyme.  The return of ALA-D activity  to
:  f.orr.nl after termination of lead exposure o'ccured  more slowly
                                              i
  In tlio low zinc group.  The inhibition of ALA-D by lead  may
  therefore be due to an antagonistic effect of  lead on zinc
  rotnbolism resulting in a functional impairment of this  enzyme.     .-,
      These findings may be of great importance as  a means  of
  Improving plumbism therapy and in determining  the  exposure level
  of the toxicant in relation to the nutritional state of  the
  exposed population.
  C.  Chemical Studies
      1.  Collection
           a.) Sources
               Chemical studies of auto exhaust were conducted  on
  materials condensed from a 1972 Chevrolet V-8  350  CID engine
  operated  at 30 MPH steady state (collection  sequences I  through
  IV), and  a 1975 Ford V-8 400 CID production  "California" engine
  (all subsequent collections).  The latter engine was equipped  with
  an Engelhard monolith catalytic converter on American Lava substrate.
 Runs V and VII through X were conducted at 20  mph  and run  VI  at
 25 niph all at steady state.  Engine performance data for the
 collection series are shown in Table XXIX.   Collections  were  con-
 ducted  at ETRL/NERC facilities in Cincinnati under the direction
 of Dr.  Campbell.
           b) Traps
             A system of two consecutive traps  (A and B)  were
 employed  for trapping auto exhaust gas condensate  (AEGC).   The
  first trap (A) was cooled with water and ice and the second trap
  (B) was cooled with a slurry of acetone and  dry ice.  A  third
  acetone scrubber (C) was used in some collections. During run I
  and part  of run VI traps A and B contained acetone.
      Samples were collected adjacent to the  converter on the  pre-
  treatment side  (engine side of the catalytic converter)  for non-
  catalytic exhaust  (NC) and the post treatment  side for catalytic
  exhaust (C).
                               48-A

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       2.   Chemical  Parameters
       a)   Total  Organic  Carbon
        Total  organic  carbon was  determined with a Dohrmann
Rnvirotech  Organic Analyzer, model DC-50,  on AEGC as shown in
                                             t
Tnble XXX,  as a measure of material collected.'.  In general, the
cnrbon content  of AEGC  was greater in NC-AEGC than C-AEGC as
anticipated.  Large  variations were observed between collections.
Results in  this report  are reported relative to the volume of
exhaust passed  through  the collection system as reported by
ETRL/EPA Cincinnati  in  Table XXIX.
       b) Apidity
        The acidity of AEGC was measured  by titration with sodium
hydroxide and by  pH  measurements of the condensate as received.
These data  are  shown in Table XXXI.  The titration showed two
readily identifiable inflections.   These were indicated by differ-
ential plots of the  titration curve,   pH/  vol. NaOH versus
volume of NaOH.   Catalytic AEGC  was approximately three times as
acidic as NC-AEGC.   The slope of the titration curve is indicative
of 'a buffering  capacity  in addition to a high concentration of
acidic capacity functional groups  vrhich is consistent with the
low  pH of the condensate.
       c)   Copper Binding Activity
       1.   Extractable  Copper
        The ability of AEGC to  effect the distribution of copper
between aqueous and  organic phases (Michael, et al. Environ.
Sci.  Tech.  5:2^9, 1971) was used as an assay for determining the
efficiency  of exhaust collection.   The range of values reported
in Table XXXII  suggests that comparisons between collection periods
or other collection  variables must be made with caution.
     Wet and dry  trapping systems  shown in Table XXXII, refer to
the  presence or absence of an acetone scrubbing solutions in the
sample collection system.   The presence of a scrubber increased the
trapping efficiency  as  determined  by extraction.
     The results  of  extraction studies show in general that more
copper was  extracted by NC-AEGC  than by C-AEGC.   These results are
based on liters of exhaust passed  through the collection system.
                              49-A

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     The extraction procedure is a  simple  or  one  stage equil-
 ibration between the immiscible solvents in the presence of
 excess copper.  Thus, this technique does not  reflect the amount
 of material v:ith potential total dynamic interaction in a system
 w»»ore the subject material is in continuous coatact v;ith fresh
 reitcrial, which is. the usual case in vivo.  What  has been
 demonstrated is the formation of complexes which  are capable "of crossing
 A  phase barrier.  Even if these are ion pairs, the transport
 across the phase boundary shovis the potential for forming lipid
 soluble metal binding complexes or  neutral complexes which might
 be able to cross a cell membrane.
           «
     ii-.  Total Copper Binding Activity
       Total copper binding activity was determined,by .measuring
 the copper stripped from a carboxymethylcellulo,se .ehromatographic
 columns in the  Cu (II) form,... (Finelli  et,al., Environ. Sci. Tech.,
 6:7^0,  1972).  The.total activity represents  the  copper eluted by
 both ethanol and ammoniac a,! eth.an.pl.   In general  the-catalytic
 exhaust bound, more copper.,. The variations between, runs was
 greater than the difference between the total Cu.  binding-..capacity
 of O and, NC-AEGC as shown in Table XXXIII.
     iii.  Fractionation of AEGC
       Condensates vrere transferred to A-meithyl-2.r-p.entanone (MIBK)
 solutions by treating with   2,2-dimethoxypropane and expess>fIBK
 and reducing the.rvolume ,of,aquepus  solution.   The MIBK.solutions
 of AEGC were fractionated ,on the. CMC-Cu columns,  which were   . -.
described, in the above section. • Ethanol had  previously .been .shown
 to elute copper binding compounds with:displaceable protons. -•
Non-protonated ligands which fornucharged  complexes with  Cu (II)
were  displaced by anunoriiacal-'e.thanol eluent.   Fractionation data
are., summarized in. Table , XXXIV,.
     Complexing agents with, displaceable protons  predominated in
both  NC- and C-AEGC.. Catalytic AEGC's greater total copper rbinding
activity is attributable to complex-ing1-agents with displ-aceable.
protons:  The level-of neutral complexing^agents-was about the
 same  in .paired collections of C- and NC-AEGC.
d.   Enzyme Inhibition Studies
     The effect of fractionated and non-fractionated C- and NC-
AEGC, of run vn,on the activity of tyrosinase (Finelli, et al.,
                               50-A

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 7ox. Applied  Pharm.,  27:*415,  197*0, a copper enzyme, has been
 Investigated.   The  fractions  tested include the non-metal
 binding fraction, F, ,  the complexes eluted from CMC-Cu columns
 with ethanol,  F , and  those eluted by ammonial ethanol, F,..
                                          •    -   »      .  O
 Ihccc fractions v;ere  stripped of copper on CMd-,HT column .to
 yield metal  free fractions, F^, F^, and Fg respectively.  The
 Kinetic studies of  tyrosinase yielded typical noncompetitive
 inhibition graphs when reciprocal velocity was plotted versus
 reciprocal substrate  concentration.  The activity of fractions
 and nonfractionated AEGC' is  shown in Table XXXV.
     The differences  in activity between the activities of copper
 complexes of  run VII  C-AEGC follows that anticipated for fraction
 F_ followed by the  metal free fraction F(-J namely, the inhibition
 of the free  complex,  FJ-, is greater than the metal bound complex^
                      5                       •
 F?.  The unanticipated inhibitory activity of the F^ fractions -of
 both C- and  NC-AEGC is attributed in part to the same trend observed
 in blanks; namely,  an inhibitor is being eluted from the CMC-Cu
 column.
     The differences  in activity of nonfractionated AEGC and the
 fractionated  material suggests additional interactions which are
unknown. A material  balance  is required to elucidate this problem.
c.  AEGC Stability
     Attempts to correlate collection, fractionation or enzyme
activity have been  frustrated by the lack of an adequate monitor
or assay for  a material balance.  Copper binding activity varies
with time.  Although  samples  could be stored under refrigeration,
when samples  were maintained  at room temperature the copper
binding activity increased.  Both total and extractable copper
binding activity increased.  Therefore, the interpretations and
 interrelationships  between collection sequences must be made
           «
with caution.
                               51-A

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!  D.   Slr.nl Ticr-nce of Results
       1. Biological Studies
        The in vivo studies on the biological effects  of  engine
  enlr.cions have shown that an oxidizing catalytic converter  was
  very effective in preventing in exposed rats loss of  body weight,
  or^nn damage and hematologic changes all of which were observed
  In  rats exposed to non-catalytic emissions.  Some of  these
  adverse effects, such as elevation in hematocrit and  hemoglobin
  levels and cardiac hypertrophy, were due solely to  the high
  concentrations of carbon monoxide in the exposure chambers,
  while other, effects, such as impaired growth and organ damage
  no  shown by elevation of LDH in the serum, must be  attributed
  to  other components of automobile engine exhaust' emissions.  In
  conclusion these results indicate that the introduction  of  a
 xatalytic converter in the engine exhaust train not only elimin-
  ated the deleterious effects of carbon-monoxide, but  also the
  effects of other toxic constituents of engine emissions  not
  presently identified.
      The in vitro studies have shown that platinum  and palladium
                                                           i
  compounds, important components of catalytic converters  and
  possibly new pollutants, seem to be less toxic than other heavy
  metals such as cadmium and lead when tested on succinate-dependent
  respiration and ALA-D activity.  However, these studies  alone
                            t       ,             ; * '      *
  are insufficient for concluding that platinum and palladium are
  relatively non-toxic and therefore not a threat to  the
  environment.                                          .
      Dietary zinc has been found to'be protective against the
  in vivo inhibition of ALA-D by lead.  This Pb-Zn interaction has
  been shown also in vitro.  These findings may"be of great import-
  ance in assessing the exposure-level of the toxicant  in'felation
  to the nutritional state of the exposed population.  Furthermore,
  zinc, administered with the chelating agents now used in plumbism
  therapy, may be more beneficial in  lead detoxification treatment.
      Serum zinc and copper have been found altered  in the animals
  exposed to nori-c'atalytic emissionsi therefore an in vivo inter-
  action between these essential metals and engine exhaust
                                52-A

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pollutants has been established.   Carefully planned nutritional
•tudics may be an aid  in  the  studies of subtle effects of
pollutants.
     2.   Chemical Studies
      An interaction  betv;een AEGC and copper has been shown by
tho formation of copper complexes.  A portion of these complexes
could be  distributed between  iraniscible solvents indicating
thnt neutral complexes were formed.   The in vitro demonstration
of synthesis and mobility suggests the potential for biological
Interaction.
     Fractipnation of  C-  and  NC-AEGC showed that compounds with
dir.placeable protons comprised the major portion of the ligands
which formed copper complexes.  This is consistent with the
presence  of an appreciable level of titratable protons in
AEGC samples.
     Studies of tyrosinase activity show greater inhibition by
C-AEGC than NC-AEGC.   The copper binding fractions of C-AEGC
nlco showed greater inhibition of tyrosinase activity than
NC-AEGC.
                              . 53-A

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           Biological Fate of  a Single Administration

                of  1!"Pt in  Rats Following Different   '

                          Routes of  Exposure

                       W. MOORE, JR.. D. HYSELL.
                       W. CROCKER. AND J. STARA

       U. S. l-Mvirniiiiienltil Protection Agency. Office nf Research IJeveliipiiieni.
National l-'.nvironnicnial Research Center. Environmental To.ricnlti.vy Research Laboratory-.
                                Cincinnati. Ohio
                              Received May 6. 1974

  The rclcnliim. tissue clislribulion. and excrelion of l!"Pt in ;idull rats was determined fol-
lowing oral, intravenous (IV). and intratracheal administration. The highest retention was
obtained following IV dosing, and lowest retention (less than O.S^f-) occurred after oral
dosing. Tissues containing the highest concentrations of ""Pi following IV administration
wore I he kidney, adrenal, spleen, and liver. Following a  single oral dose: almost all of the
""I1! was excreted in the fcccs due to nonahsorplion. whereas after IV dosing, similar quan-
tities were excreted in both the urine and feces. Following IV dosing of pregnant rats. '"Pi
was found in all the fetuses: however, (he amount was small.
                                 55-A

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Preliminary  Studies  on  the  Toxicity  and
Metabolism  of  Palladium  and  Platinum
               *.
by  W.  Moore,   D. Hysell,   L Hall,  K.  Campbell,   and J. Stara
          Preliminary data a IT given on the Ml-- of PdCI- following different routes of exposure
         and on (lie 1,1) of PlCI, following inlra*cuoiis exposure.
          The retention, (issue distribution, and excretion of '"'Td and "'PI in rats was deter-
         mined following oral, in! ravenous, int ratraclu'al, and inhalation exposure.  The highest
         retention  for holh '"'I'd nnd "''Pt was ohtainrd following intravenous dosing, and  the
         lowest retention occurred after oral dosing. Following n single oral dose, almost all of the
         '''I'd and '"'Pt was excreted in the feces due to nonabsorplion, whereas after intravenous
         dosing, similar <|iianlilies were excreted in holh the urine and feces. Tissues containing
         the highest concentrations of these metals were Hie kidney, spleen and liver. Following
         inlrateiioiis dosing of pregnant rats, a small amount  of '"Td and ""Pi was found in  the
         fetuses.
      * U.S. Environmental Protection Ajrency, National
    Environmental   Research  Center,  Environmental
    Toxicology  Research Laboratory,  Cincinnati, Ohio
    46268.
                                           57-A

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                                                                    B.I
   WHOLE  BODY  RETENTION  IN  RATS  OF  DIFFERENT  191Pt  COMPOUNDS
                 FOLLOWING  INHALATION  EXPOSURE
          W.  Moore,  Jr.,*  M.  Malanchuk,*  W.  Crocker,*
            D.  Hysell,*  A.  Cohen,*  and  J.  F.  Stara*

 (This  manuscript  has  been submitted  for  publication  in
  Environmental  Health Prospectives)
* U.  S.  Environmental  Protection Agency,  National  Environmental
  Research Center,  Environmental Toxicology Research Laboratory,
                       Cincinnati,  Ohio 45268
                             59-A

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     The whole body retention, excretion,  lung clearance,  distribution
                                                                           <
and concentration of 191Pt in other tissues was determined in rats

following a single inhalation exposure to different chemical forms of

191Pt.  The chemical forms of 191Pt used in study were 191PtCl4, 191Pt(S04)2i

191PtO and 191Pt metal.  Immediately after exposure most of the 191Pt

was found in the gastrointestinal and respiratory tract.  Movement of

the 191Pt through the gastrointestinal tract was rapid with most of the

l9^Pt being eliminated within 24 hours after exposure.  Lung clearance

was much slower.with a clearance .half-time of about 8 days.  In addition

to the lungs, kidney and bone contained the highest concentrations of
                                60-A

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                                                                  B.2
 INHIBITION  OF  HYDROXYPROL1NE   SYNTHESIS   BY   PALLADIUM   IONS
   Rao S.  Rapalca,*   Keith R.  Sorcnsen,   Si Duk Lee**   and

                   Rajcnclra  S.  Bhatnagar***
            Laboratory of Connective Tissue Biochemistry
               Scliool  of Dentistry,  630 Sciences
                University of California
               San Francisco,  California  94143
  ^Present address:   Albert Einstein Medical Center
                     Northern Division
                     York and Tabor Roads
                     Philadelphia,  Pa.  19141

 **Dr.  Lee's Address is:   Health  Effects Research Laboratory
                          Environmental Research Center
     •—•""             '    Environmental Protection Agency
                          Cincinnati, Ohio  45268

***Author to whom reprint requests should be sent.
                                61-

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                                SENARY





     Palladium ions, administered as PclSO^ markedly affect, tlic



incorporation of L- (3,^-^IL) -proline into non-dialyzablc fractions



in 10-day chick embryo cartilage explants with a 55-65% reduction



in the concentration range 0.06-0.6 jnM.  Under these conditions



the synthesis of 3H-hydroxyproline was nearly completely inhibited.



Experiments with prolyl hydroxylase indicated a strong irreversible



inhibition of the enzyme with a.competition between Fe2+ and, Pd"+.



The Ki for the inhibit ion-was 0.02 mM.. , Tliese studies suggest tliat



Pd?+ may inhibit, collagen synthesis by replacing Fe2+ in the



active site of prolyl hydraxylase.  .Tliese studies., also point to a



potential mechanism of Pd2t  toxicity.
                             i 62-A

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                                                          13.3
PALI ADI IJM LNTF.RFERL7S WITH CONNECTIVE




  TISSUE mCRQMOLECULAR SYNTHESIS
                     63-A

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ABSTRACT






     Palladium is a component of catalytic converters installed in auto-



mobiles to reduce hydrocarbon coicentrations.  There is a liklihood that



Pd may enter the environment as a result of attrition and disposal of



such devices, however, little information is available on its toxicity.



Studies reported here suggest tliat Pd^* ions may be highly toxic to con-



nective tissues and cause inhibition of macromolecular synthesis, if



these ions reach the target systems.
                                     64-A




                                        /

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                                                                              B.4
                 Comparative  Effects of  Intratracheally Administered
              Sulfuric Acid and  Palladium Sulfate on  Pulmonary Free Cells
                                 K.  I, Campbell

                                 E.  L. George

                                 T.  Grumbles,  and

                                 Y.  Y. Yang




                                   SUMMARY
    The comparative acute toxicity of sulfuric  acid  (H2S04)  and  palladium
sulfate (PdS04)  to pulmonary free cells  was  investigated  in  rats using  the
intratracheal route of administration.   These materials v;ere of  interest
because of their potential  emission as  constituents  of exhaust from
automobiles using high-sulfur fuel and  noble-metal catalytic devices.   The
pulmonary free cells (PFC), normally primarily  alveolar macrophages,  are an
important component of pulmonary defense and clearance functions.

    H2S04 solution was administered in  a dose of 0.45 mg  [S04~2J/kg body
weight to one test group (5 rats each group) and an  aqueous  solution  of
hydrous PdSC4 was given to the other test group at a [$04-2]dose rate
equivalent to that of the H2S04 group and [Pd+2] dose of 0.5  mg/kg.   The control
group received deionized water (^O) only.   Dose volumes  ranged  from  0.1  to
0.14 ml per ret, and body weights, averaged 188  gm.   One day  later the PFC
were1 collected by saline endobronchial  lavage.   Total and differential  cell
counts, cell viability, phagocytic activity, and lung and body weights  were
determined.

    With respect to several criteria, there  were marked and  significant treat-
ment effects in the PdS04-exposed animals but not in those exposed to H2S04.
Noted effects included the following:  increased total numbers of free  cells;
increased sub-populations of both viable and nonviable nucleated cells,
phagocytic and nonphagocytic macrophages, polymorphonuclear  leukocytes, and
red blood cells; increased numbers of phagocytized  test-spheres; increased
dried lung mass.  There were no apparent effects on  proportions  of cells
nonviable, on proportional  phagocytic activity  (spheres per  cell), or on
cell size (diameter).
                                        65-A

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    The results indicate definite effects by PdS04 on the pulmonary free
cell system suggestive of a recruitment of alveolar macrophages and/or
retarded cellular clearance activity, of leukocytic infiltration, and of
hemorrhage and perhaps ederra.  These effects are regarded as .'having toxic
implication for palladium compounds (especially soluble and iohizable)
gaining access to the lungs' terminal air spaces, end being associated
with the cation moiety rather than the anion [SO^Jor acidity of the material
                                        66-A-

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                                                             B.5
EFFECTS OF VARIOUS  METAL SULFATES ON SUCCINATE-BEPENDENT RESPIRATION
                 S.  D.  LEE, M. RICHARDS, L. MCMILLAN
                U.S.  ENVIRONMENTAL PROTECTION AGENCY
                          CINCINNATI, OHIO
                      R.  SHELLEY, V, N. FINELLI
                      UNIVERSITY OF CINCINNATI
                          CINCINNATI, OHIO
        For Presentation at the 68th Annual Meeting of the

                  Air Pollution Control Association

        Boston, Massachusetts            June 15-20,1975
             67-A

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                                                                   75-31.6

   ITTECTS OF VARIOUS MCTAL SULFATES ON SIKXBJATE-DEPE.'DRTT RESPIRATION


      S. D. Lee, H. Richards, I,. 1-fcMillan; FPA, Cincinnati, Ohio
 K. Karaffa and V. Finelli; University of Cincinnati, Cincinnati, Ohio


     The use of catalytic converters to control hydrocarbons and carbon
monoxide in the autcmobile exhaust emissions resulted in an increased output
of sulfatc(s).  This, along with possible oiussions of noble metals  from the
converter prcrpted us to test conparative toxicity of the various sulfates in
an enzyne system.  Succinate-depcndent respiration w.is tested in rat liver
slices incubated in Krebs-Rinqer phosphate buffer and in liver horogenate
incubated in 0.25M pliopphatc buffer, pi! 1.4,  A Clark-type oxygen electrode
attached to a YSI Model 53 Biological Oxygon Monitor was used to measure .the
O2-uptake.  Ke tested the effects of various sulfatcs, such as cadmium,
palladium, rar.gnnese, ragnesium, calcium, sodium, and arnonium on the system.
The results indicated, that the sulfate ion in tissue slices or in hcrogenate
did not affect the respiratory chain.  However, anor.g the cations, Cd++
appeared to bt the most potent inhibitor.  Cadr-ium inliibited the O2~uptake by
approximately 50% at 2 x 10~7 M and 100% at 3.3 x 10~5 M.  Othsr cations did
not show inhibitor^' effects at sinilar ccnccntrationE.  To achieve a 505
inhibition by PdSO^ more than 10"^ M was rerruircd.  Cadnium ion, a kna-.n
potent inhibitor ct the nitcchondrial respiratory chain, was utilized in this
operiment as a reference toxic.-'.nt.  CdSOfl was found to he manyfold  more
toxic to the respiratory chain than PdSO*).  Other cations, such as Mn4"1", t-'q**,
Ca"1"*;, were not inhibitory at concentrations up to 10-3 M, but appeared to have
a slight stinulatory effect.  Ce+^f, Na+, and 13'.^ did not cause any effect.
Intragastric administration of a single and mltiple dose of CdSO^,  Pt(SOjj)2,
.and PdSO^ at 0 and 60 unole per kg body weight, did not affect succinate-
dependent respiration in any of the organ tissues tested  (kidney, liver, heart,
and lung).  It is probable that, under these experimental conditions, these
metals were not absorbed in significant amounts to cause any effect  on the
mitochondria! respiration of soft tissue organs.  The in vitro experiment
suggests that at  least Cd4"1", the most potent inhibitor tested, should have
impaired this enzyme system if absorbed in significant quantity and  accumulated
in the tested organs.
               68-A

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              INTERACTION OF METAL BINDING AGENTS FROM

                COMBUSTION PRODUCTS WITH TRACE METALS
      L. W. Michael, V. N. Finelli, V. J. Elia, H..G. Petering
                 Department of Environmental Health
           Kettering Laboratory, University of Cincinnati
                       Cincinnati, Ohio 45267                      "  .'

                                and
                    S.  D.  Lee and K.  I.  Campbell

               National Environmental Research Center
                  Environmental Protection Agency
                      Cincinnati, Ohio 45268
                              ABSTRACT

   Metal binding agents present in the environment may react with trace

metals thus altering essential biological functions.  Combustion pro-
                                                  i

ducts from tobacco, gasoline and coal as well as from other pyrolytic

processes are ubiquitous sources of such material.  We have demon-

strated the potential for such interactions in biological systems by

comparing the copper binding activity of condensates from selected

sources.

   The reactivity with other metals was also examined.  The isolated

metal binding fractions have been tested for effects on metalloenzymes

and mitochondrial respiratory activity.  In addition, the efficacy of
                                                              •
this concept was tested in vivo using a chelating agent, 3~ethoxy-2-

oxobutyraldehyde • bis(thiosemicarbazone),  (H-KTS).  H-KTS has been

found to alter cholesterol metabolism In a manner similar to that ob-
                                                                    •
served in copper deficient rats.
                                  69-A

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                                                                            B.7
    PUU-DNAKY BIOCHEMICAL 7\LTERATIONS RESULTING FROM OZONE EXPOSURE



            Mohaivniad G. Mustafa
            Center for the Health Sciences
            University of California School of Medicajie
            Los Angeles, California  90024

            Si Duk Lee
            National Environmental Research Center
            United States Environmental Protection Agency
            Cincinnati, Ohio  45268


     ABSTRACT.  1-iatabolic response of lung tissue to ozone was studied in rats

and monkeys after exposure of animals to various levels of ozone (0.1 to 0.8

ppm) for 1 to 30 days.  In rats, 0.8 ppm ozone exposure resulted in a 40-50%

augmentation of oxygen utilization in lung homogenate in the presence of an

added substrate (e.g. succinate or 2-oxoglutarate).  Activities of marker

enzymes, viz., mitochondria! succinate-cytoclirome c reductase, microsomal

NADPH-cytochrome £ reductase and cytosolic glucose-6-phosphate dehydrogenase,

increased maximally  (40-70% over control) after 3 to 4 days of exposure, and

remained elevated throughout the continuous exposure for 30 days.  In monkeys,

the observations were the same except that the magnitude of biochemical

changes was relatively smaller.  Exposure of animals to lower levels of ozone

resulted in proportionately smaller biochemical changes in the lung, and

ozone effects were detectable for up to 0.2 ppm level.  While 0.1 ppm ozone

exposure was ineffective, dietary deficiency of vitamin E, a natural anti-

oxidant, increased the sensitivity of rat lungs to this concentration of ozone.

The results suggest that low-level ozone exposures may cause metabolic

alterations in the lung, and that dietary supplementation of vitamin E may

offer protection against oxidant stress.
                                     71-A

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The Use of Chemical lonization Mass Spectrometry for the Analysis
                    of Platinum and Palladium.

Terence H. Risby, Department of Chemistry, Pennsylvania State
University, University Park, Pennsylvania, 16802.
     The preparation of the l,l,l-trifluoro-2,4-pentanedionates of
platinum and palladium will be presented.  This procedure can be
used for the direct analysis of Pt or Pd in unknown samples.

     The chemical ionization mass spectra for these complexes will
also be reported together with the predicted mass spectra based on
the natural isotropic abundancies of the metals, 0  , C13 and H2.

     The C.I. mass spectrometer used in this study has been modified
so that solutions of the chelates can be injected directly into the
mass spectrometer.

     Preliminary sensitivity studies suggest that the limit of
detection for these complexes is of the order of 1 x 10~12 g.
                              73-A

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                           Chemical Transport of Platinum
                             from Automotive Catalysts
                                A Laboratory Study*

                                     John Hawley
                                   New York State
                      Department of Environmental Conservation
        Laboratory  tests  with  a  noble metal  beaded  oxidative  automotive  catalyst
        indicate  chemical  transport  of  platinum.  Samples  from  the  exhaust  gases
        of  a  furnace  employed  to house  and heat  the catalytic reactor  were
        filtered  through  glass and quartz fiber  mats which were then  subjected
        to  x-ray  fluorescence  analysis.  Scaled  to  a 60  mph cruising  automobile,
        particulate platinum in  microgram per  mile  quantities was collected
        during  some experiments. Ethylene dibromide (5  ppm)  and carbon  monoxide
        (3%)  as additives and  air and nitrogen as carrier  gases were  studied  in
        all combinations  in the  final experiments.   No platinum transport was
        induced,  even at  elevated temperatures,  with pure  nitrogen  carrier  gas,
        whatever  the  additive.  Otherwise, oxygen was the  most  important
        component in  determining platinum transport from aged catalyst samples;
        bromine enhanced  transport in the presence  of CO,  but depressed  trans-
        port  when alone at higher temperatures.

        Parallel  engine test stand experiments with exhaust converters containing
        the same  catalyst material showed microgram per  mile  platinum transport
        with  ethylene dibromide  added to the fuel and reactor temperatures
        raised  via  modified timing and  carburetion.  Earlier  engine experiments
        at  lower  temperatures  showed no transport either with or without ethylene
        dibromide.

        This  report was submitted in fulfillment of EPA  Grant No. R-801486  by
        the Division  of Air Resources of the New York State Department of
        Environmental Conservation under the partial sponsorship of the
        Environmental Protection Agency.  Work was  completed  as of  July  1,  1975.
Based upon the Final Report on EPA Grant No. R-801486 by R. D. Harris, R. E. Gibbs
and N. Kolak.
                                            75-/1

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                                                                         NOV  2 4  i9/b
      Platinum Metals in Air Particulates Near A Catalytic Converter Test
                    Site As  Measured by Isotope Dilution SSMS*

                        J. A.  Carter and W.  R. Musick

      Composite dust samples weighing about one kilogram were supplied by
Dr. R. Thompson, EPA-RTP,  for Pt,  Pd, and Ru analyses.   The origin of the mate-
rial was airborne dust particulates  collected by filter banks which were located
about 1.4 km from a catalytic converter test facility.  The sample was a blackish-
gray mish-mash of particulate matter.   The major constituents of the ashed com-
posite (50% wt loss) were  Si,  Al,  Pb, Ca, Fe, Na, Zn, K, Mg, and Ti.  Many minor
constituents, including Pt,  Pd,  and  Ru may be determined by excitation with radio-
frequency spark followed by  spectrometric measurement of the mass resolved ion
beams.  This technique of  spark-source mass spectrometry when applied to the
composite was not sufficiently sensitive; only Au was detected at the 1 ppm level.
Therefore, to obtain adequate sensitive and quantitative Pt, Pd, and Ru results,
we dissolved a large sample  so we  could equilibrate enriched stable isotopes of
   Pt,    Pd and   Ru, with  each of  these isotopically unaltered elements present
in the samples prior to platinum metal enrichment as a metal precipitate.  Gold
was added as a carrier.  Gold and  the platinum group elements were concentrated
into the Au precipitate which was  subsequently used in the isotope dilution spark-
source mass spectrometry measurements.
      Recovery of the Au by  weight was near theoretical for blank acid samples,
but was always less than 50  percent  for the sample solutions.  This fact did not
alter the validity of the  results, however, since equilibrium was established prior
to the precipitation process.
      The results by isotope dilution spark-source mass spectrometry in picograms
per cubic meter of air are as follow:  Pt-2.5, Pd-0.5, and Ru-<0.1.  These results
assume an average air burden of  50 yg M  .
*Work supported by  the  Environmental Protection Agency under EPA Interagency  Agree-
 ment D5-0466 with  ERDA.   ORNL is operated by the l.friion Carbide Corporation for ERDA.
                          By occeptDnco ol thio nrtlclo, tho publl .FIT or
                          recipient Pcknowl.-oVos iho U.S. Government's
                          rlcht to retain a non - exclusive, royalty - (rao
                          license In and to any copyrleht covering the
                          article.

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    Measurement of Pt and Pd in Ambient Air, Soil and Water


                     Dr. Donald E.  Johnson
       Methods for collection and storage of these samples will be
presented along with the analytical methods. The assay methods
emphasize atomic absorption with a graphite furnace.
                            79-A

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        Measurement of Pt and Pd in biological Tissues


                       Dr. Donald E. Johnson
       Methods for collection, storage, sample preparation and
analysis will be presented.  The limits of sensitivity for each type of
sample will be given.
                                80-A

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             Platinum Analyses in Animal Tissues and Fluids
                      *                  *             .        **
        Andre F. LeRoy , Walter S. Friauf , Charles L. Litterst  ,
                     **                    **                       *
     Theodore E. Gram  , Anthony M. Guarino  , and Robert L. Dedrick
Introduction
     Interaction of platinum and its complexes with biological systems
has become a matter of increasing interest and importance.  Individuals
may be exposed for therapeutic purposes in the treatment of cancer and
through environmental contamination from vehicular emission control
devices or some industrial processes.
     We have adapted flameless atomic absorption spectrophotometry
(AAS) to the quantitative determination of Pt in biological tissues
and fluids down to  ~30 ppb.  The methodology has been .applied to
observation of the distribution and disposition of the anti-cancer
drug cis-^PtC^CNH^^J > DDP, in several animal species following intra-
venous administration.
Analytical Considerations
     Therapeutic doses of DDP are of the order of 1 mg/kg; so, analytical
methods must be capable of quantitating platinum at concentrations less
than 1 ppm.  Samples from animals subjected only to environmental
exposure would be expected to contain much less, e.g., <2 ppb .
     *Biomedical Engineering and Instrumentation Branch, Division of
Research Services, National Institutes of Health, Bethesda, Maryland,
      **
U.S.    Laboratory of Toxicology, National Cancer Institute, Bethesda,
Maryland, U.S.  Decker, D.A., LaFleur, P.O.,  LeRoy, A.F.  "Spontan-
eous Deposition Radiochemical Separation for Platinum Determination in
Biological Materials."  Presented at the 19th Annual Meeting on Analytical
Chemistry and Nuclear Technology, October 14-16, 1975 Gatlinburg,
Tennessee, U.S.
                                  81-A

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                                                             2.
     Radioisotopes of platinum can be incorporated in the drug to
trace its distribution after administration, but the practical
considerations of isotopic abundance, decay energy, half-life,
purification of labeled preparation and cost limit such applications.
     Reported analytical methods were generally developed for ore
and alloy assays; such materials are usually more concentrated and
less complicated matrices than biological materials.  Of the
available analytical methods, AAS offers a relative simplicity of
equipment and lends itself to routine application to a variety of
biological specimens.  Ease of application of the technique and
accuracy of the measurements depend on the tissue and concentrations
obtaining.
Equipment and Methods
     Samples are atomized flamelessly at approximately 2700° in a
heated graphite atomizer (Perkin-Elmer, HGA-2000).  Absorbance is
measured at 265.9 nm against a deuterium arc reference beam with a
Perkin-Elmer 303 Atomic Absorption Spectrophotometer and displayed on
an Omniscribe strip chart recorder (Model 5213-12).  Aliquots of
samples requiring no pretreatment (urine, blood plasma) can be analyzed
by direct combustion in the furnace so long as the volume (typically
30 ul) introduced contains a minimum of 2 ng platinum.  Tissues requiring
pretreatment are weighed, freeze-dried, and triturated to yield
homogeneous samples.  Each sample is liquified and partially digested by
heating with 60% HNO, in a Teflon beaker.  Digestion is completed with
addition of a mixture of 3 parts HC104 (70%) to 2 parts HN03 (60%)
(approximately 20 ml per gram of tissue).  Appropriate safety measures
should be taken when using HCIO^ to oxidize organic material.  When
the digest is almost dry, the residue is taken up in 0.1 M HC1,
evaporated and resolubilized in a known volume of the HC1.  Aliquots
of this digest are then analyzed by comparison with working standards
prepared by dilution of a 1000 ppm hexachloroplatinic acid standard.
The method of additions has been applied successfully for analysis of
platinum in some biological materials.
                                  82-A

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                                                             3.
     The deuterium arc reference serves as a basis of correction for
broad band absorption but may be insufficient for tissues or digests
that produce profuse smoke near the atomization temperature.  A modi-
fication of the HGA-2000 Temperature Controller, designed and built
at NIH, permits control of the rate of temperature rise between the
plateaus for drying, charring, and atomizing the sample.  Use of the
temperature ramp generator produces a time interval between the
•appearance of the smoke peak and the platinum peak.
Results
     Precision and accuracy of flameless AAS are satisfactory for most
tissues at the levels observed in the distribution studies (8 - 0.03 ppm,
wet weight basis).  Immediately after drug administration in the dog,
the platinum concentration rises sharply in the kidney, liver, skin,
muscle and urine.  Platinum levels in the brain remain low during the-
entire interval studied.  Rapid urinary clearance of about two-hour
duration is followed by a low clearance rate measured for twelve more
days.  A pharmacokinetic model is being developed based on the data
obtained.
     Flameless atomic absorption spectrophotometry results obtained
using the temperature ramp generator compare favorably with preliminary
data by the more sensitive neutron activation analysis.  Biological
Standard Reference Materials certified for platinum are not yet
available from the National Bureau of Standards.  Their availability
may permit modification of sample preparation and the AAS procedure to
increase its sensitivity further and render it suitable for determination
of the low levels in the environment.
                                83-A

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      Spontaneous Deposition Radiochemical Separation
   for the Determination of Platinum, Palladium and Gold
         in Biological and Environmental Materials
         D. A. Becker, P. D. LaFleur, and A. LeRoy*
                Analytical Chemistry Division
                National Bureau of Standards
                    Washington, DC  20234

The spontaneous deposition technique has be-en used a number
of times for radiochemical separations in activation analysis
(1, 2).  It has also been used as a preirradiation chemical
separation method (3).  Although this technique has been used
for the analysis of one or more noble metals from inorganic
materials  (2) and metals (3) , it apparently has not been
applied to the determination of noble metals in biological
and environmental materials.

The use of platinum, palladium and possibly other noble metals
in motor vehicle emission control systems has recently been made
public.  It thus becomes imperative that accurate and sensitive
methods for the determination of these elements be available
for the evaluation of environmental baseline levels and any real
or imagined increases in these levels.

In addition, there is currently medical research on the use
of platinum compounds as chemotherapeutic agents in the treatment
for certain types of malignant growth.  In order to adequately
evaluate the metabolic pathways and rate of discharge of these
compounds  from laboratory test animals, it is necessary to have
an analytical method which is both accurate and precise.

*Guest worker at NBS; from the National Institutes of Health,
 Biomedical Engineering and Instrumentation Branch, Division
 of Research Services.
                              85-A

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The neutron activation analysis method developed and described
here is capable of quantitatively determining gold, platinum
and palladium in a wide variety of biological and environmental
matrices.  The nuclear characteristics of the radioisotopes
used in this procedure are found in Table 1.  As shown, the
isotope used for the determination of platinum is the gold-199
daughter from platinum-199.

In addition to the matrices described here, this method is
currently being applied also to the analysis of several new
biological standard materials currently undergoing certification,
a fresh water sediment being evaluated as a potential reference
material, and an urban air particulate material.

Experimental
All samples were prepared for analysis by lyophilization.  Dry
samples weighing from twenty to two-hundred milligrams each were
placed into precleaned, polyethylene snap cap vials for neutron
irradiation.  The facility used was the NBS nuclear reactor,
with irradiati.on periods of up to four hours at a thermal
neutron flux of 5.6 X 1013 n-cnr2 sec-1.

The chemical separation technique employed involved wet-ashing
the irradiated biological or environmental sample with approxi-
mately ten milliliters of a three to one mixture of concentrated
nitric and perchloric acids.  This sample dissolution took
place in the presence of about one milligram each of platinum,
gold and palladium carriers, and about five milligrams of copper
holdback carrier.  The samples were slowly wet-ashed on a hot
plate to fumes of perchloric acid, then taken almost to dryness.
After.cooling, they were taken.up in about three milliliters
of a three to one mixture of concentrated hydrochloric and nitric
acids until complete dissolution occurs.  An additional five
                                  86-A

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milligrams of copper holdback carrier was added at this time,
along with several milligrams of scandium and phosphate holdback
carriers.  Samples were then diluted to approximately twenty
milliliters, and quantitatively transferred into appropriately
sized disposable plastic containers (e.g. centrifuge tubes,
bottles, etc.).  At this point, 200 milligrams of finely
divided silver metal powder was added, and the solution-powder
mixture stirred for approximately five minutes using a magnetic
stirring bar with high agitation.  The silver powder should
swirl throughout the entire solution during this period.
        i

After spontaneous deposition onto the silver powder is concluded,
the solution with silver powder is quantitatively transferred
to a standard radiochemical filter chimney with a filter capable
of retaining fine particle size material.  In this work, glass
fiber filter pads were used.  The silver powder is washed
several times with distilled water, and great effort is taken
to ensure that all of the silver particles have oeen transferred
to the filter.  At this point, the silver powder on the filter
paper is removed from the filter chimney and carefully transferred
to a plastic disposable petri dish and mounted for subsequent
counting.  Separation of the silver by centrifugation was also
investigated, but filtration was found to be more effective
in removing the silver powder fines from the solution.

Aliquots of the dissolved standards are processed in exactly
the same way in order to check radiochemical recovery.  In
addition, duplicate aliquots are added directly to filter paper
in the disposable petri dishes producing a geometry similar
to that of the processed sample.

Results and Discussion
Tracer studies made using this radiochemical separation
procedure showed quantitative recovery (greater than 99.5%) for
                                  /
the gold-198,  gold-199, and palladium-109.

                                  87-A

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This analytical system has been evaluated through application
to the determination of gold, platinum, and palladium in
several available biological and environmental materials.
Results on the analysis of a bovine animal tissue (liver) gave
values of 1.8 ng/g for gold, less than 1.6 ng/g for platinum,
and less than 1 ng/g for palladium.  Results for the analysis
of a powdered botanical material (leaves) gave results of 0.2
ng/g for gold, less than 9 ng/g for platinum, and less than
1 ng/g'for palladium.  These analyses were made using a neutron
flux of 5.6 X 10l3 n-cm-2sec-1 for four hours, and counting
both on a 6% efficient Ge(Li) detector (gold-198, gold-199)
or LEPS System (109-palladium).  For the botanical material,
the less sensitive 208 keV gamma ray peak was used due to an
interference with the 158 keV gamma ray.  This interference
was apparently due to the scandium-47 daughter from neutron
irradiation of natura1ly stable calcium-47.  Later determinations
using  scandium holdback carrier eliminated this interference
problem.

In conclusion, the spontaneous deposition technique has been
demonstrated for the first time to be an effective radidchemical
separation method for biological and environmental samples,
and neutron activation combined with this separation technique
appears to be a very useful method for the determination of
platinum, palladium, gold, and possibly other noble metals
in biological and environmental materials.
                                88-A

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                          References
1.  W. D. Kinard,  D.  A. Becker,  and P.  D. LaFleur,  "The
       Determination  of Indium  and Copper in  SRM  High
       Purity  Gold Wire by Neutron Activation Analysis,"
       in Activation  Analysis Section:   Summary of  Activi-
       ties, edited by P. D. LaFleur.and D. A. Becker, NBS
       TecTT. Note  548  (1970) pp.  66-69.

2.  D. A. Becker,  "Trace Analysis for Platinum in Glasses
       by Neutron  Activation,"  Anal. Chem. Acta 61, 1 (1972)

3.  K. S. Park,  R. Gijbels,  and J. Hoste, "Neutron  Activa-
       tion Analysis  of Palladium, Platinum,  and  Rhodium in
       Lead Foam," J. Radioanal.  Chem.  5, 31  (1970).
          Table 1.    Radioisotopes Used for the Analyses
     Element
    Determined

    Gold

    Platinum

    Palladium
Radioisotope
   Used
198

199
Au
               Photon Energy
412 keV
  Au (daughter)  158 keV (208 keV)
109
   Pd
             88 koV
Half-Life

  2.69 d

  3.14 d

 13.S  h
                                 89-A

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                     PRESENTATION SUMMARY:  ANNA M. YOAKUM


    Topic:  Measurement Technology and Emmission Characterization.

Sub-topic:  Measurement of Pt in Biological Tissues, Urine, and Feces.



     A historical presentation of the involvement of Stewart Laboratories, Inc,

in the topic under consideration will be presented.  An evaluation of

methodology currently employed in autopsy tissue analysis (1) will be

discussed as it relates to future research goals.
     (1)  Yoakum, A. M., Stewart, P. L., and Sterrett, J. E.,  "Method
Development and Subsequent Survey Analysis of Biological Tissues for
Pt, Pb, and Mn Content."  Env. Health Prospectives, Vol. 10, pp 85-93,
1975.
                                  91 -A

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                Methylcobalamin:  Methylation of Platinum


                       and Demethylation with Lead


                               R.T. Taylor

             Biomedical and Environmental Research Division
                     Lawrence Livermore Laboratory
                        University of California
                     Livermore, California  94550
     A study was carried out to evaluate the potential for platinum,
palladium, lead, and maganese salts to be biochemically methylated.
Methylation is an important well -recognized determinant of metal  toxicity,
examples being the increased hazard of methyl  mercury over mercuric  salts
and the apparently reduced toxicity of methyl  arsenic acid relative  to
arsenite and arsenate.  The above metals are associated with emissions
arising from the use of automotive fuels, fuel additives, and catalytic
control devices and are therefore of special concern to the Environmental
Protection Agency.  In this study methyl cobal ami n (MeB-12), a functional
derivative of vitamin B-12, was used as a biological alkylating agent.

   + Salts of Pt, Pd, and Pb and  oxides of Pb all containing the metal in
a 4  valence state were observed  to demethylate MeB-12 under weakly  acidic
conditions.  Pt(SO^)2 was unique  in that it rapidly demethylated MeB-12 at
pH 7.0.  Inorganic salts and oxides of manganese were unreactive at  all
pHs tested (2.0-7.0); soluble Pb2+ salts, suspensions of PtOp, and KpPtC
alone were likewise inactive.  Demethylation of [Me-'4C]MeB-T2 with  Pb4"1"
oxides was accompanied by a proportionate volatilization of the label  and
only unreacted radioactive MeB-12 was detected upon paper electrophoresis.
In contrast, acidic demethylation  with KpPtClg and Pt(SO/i)2 occurs with a
significant recovery of the Me-'4C label, 75-85% and 37% respectively.
The reaction between MeB-12 and H^PtCls has been studied in more detail as
described below.
     Incubation of yM levels of I^PtCls and methyl cobal ami n (MeB-12)  results
in the complete conversion of MeB-12 to aquoB-12.   Demethylation is optimal
at approximately pH 2.0 and is accelerated by the  addition of K^PtCld.   The
reaction is stoichiometric between MeB-12 and the  K2?tCl6 added (1:1).
Isosbestic points at 492 nm, 367 nm, 335 nm during  the course of the
reaction indicate that MeB-12 is demethylated to aquoB-12 with no
accumulation of corrinoid intermediates.  Higher alkyl cobal ami ns and
                                    93-A

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                                    -2-
Mecobinamide react at much slower rates compared to MeB-12.   Incubation of
40 yM K2PtCl6 with either 40 yM [Me-14c]MeB-12 or [Me-3H]MeB-12 followed by
lyophilization converts 70% of the label to a stable form which is associated
with Pt upon subsequent paper chromatography and electrophoresis.   There is
no preferential loss of -fy relative to ^C in the reaction product.  Difference
spectra indicated that the Pt reaction product had a 260 nm absorption
                              of [Me-'4C]MeB-12 and I
maximum.  When 50 ymoles each of [Me-C]MeB-12 and I^PtCls were reacted
and subjected to Sephadex G-15 chromatography the l^c-label eluted with 260 nm
absorbing material.  Further chromatography on Sephadex G-15 and CM-cellulose
yields a labeled UV-absorbing product with a 14C/Pt ratio of 0,9-1.2.   Our
overall recovery was 36-42% based on the '^C.  The l^C-Pt product has
absorption maxima at 260 nm and 208 nm with a minimum at 240 nm
(A24Q nm/A260 nm = 0-5)-  Proton NMR spectroscopy confirmed the presence
of an H-C-Pt covalent bonding pattern (J for "H, 195pt = 78.2 Hz; T for
194pt-Me + 196pt-Me = 6.956).  This Me-Pt compound is somewhat light-
sensitive; however, its moderate stability in aqueous solutions to NaCl ,
temperature, and pH suggest that it could have biological activity.
                                  94-A

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        TOXICITY OF PLATINUM (IV) SALTS FOR CELLS OF PULMONARY ORIGIN


M. D. Waters, T. 0. Vaughan, D. R. Abernethy, H. R.  Garland, C. C. Cox, and
D. L. Coffin, Environmental Protection Agency, Environmental Research Center
Research Triangle Park NC 27711


    The acute toxicity of tetravalent platinum was studied j_n vitro using rabbit

alveolar macrophages and human lung fibroblasts (Strain WI-38).  Alveolar

macrophages obtained by saline lung lavage were exposed in tissue culture for

a period of 20 hours to platinum dioxide or platinum tetrachloride.  There was

no evidence of dissolution of platinum dioxide in the test system and, although

the particles were actively phagocytized, no cellular toxicity was observed at

concentrations as high as 500 yg per ml of culture medium.  Platinum tetra-

chloride, on the other hand, was soluble in the test system and after a 20-hour

exposure, resulted in loss of viability (failure to exclude trypan blue) in 50%
                                                       -4
of the cells at a concentration of approximately 3 x 10  M.  Additional tests

indicated that death of macrophages was preceded by a decrease in total cellular

ATP and a reduction in phagocytic activity as compared to controls.  After a 20-

hour exposure, human lung fibroblasts were rendered nonviable by platinum tetra-

chloride at concentrations comparable to those which affected rabbit alveolar

macrophages.  Additional studies in the fibroblast system demonstrated that

platinum tetrachloride produced a rapid concentration-dependent decrease in

  'C-thymidine uptake and incorporation into acid precipitable material.  After

20 hours, these activities were 50% inhibited as compared to controls at a

platinum tetrachloride concentration of approximately 10~ M.  A higher concentration

(approximately 6 x 10~ M) was required to inhibit the uptake and incorporation

of   C-uridine and leucine by 50%.  Studies on the ability of human lung

fibroblasts to recover following acute exposure to and removal of platinum

tetrachloride indicated the reversibility of the inhibitory effects of this

compound on DNA, RNA and protein biosynthesis.


                                        9S-A

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   Compare Pulmonary Carcinogenesis of Platinum Group Metal  Compounds
   and Lead Compounds in Association with Polynuclear Aromatics  Using
                       In vitro Hamster System

                            Or. Philip Kane
              State University of New York -  Stoneybrook
               The purpose of this contract is to evaluate the potential role

of platinum and lead compounds as cofactors in respiratory carcinogenesis.

The data derived from this investigation will be useful to EPA in evaluating

the hitman health hazard associated with platinum compounds emitted into the

environment in automotive exhaust.  Such emissions have been suggested to be

a potential consequence of catalyst attrition in automobiles equipped with

platinum based catalytic exhaust converters.  Results will aid in assessing

the potential hazards of airborne particulate lead derived from combustion

of gasoline containing lead compounds as anti-knock additives.  These metals

are to be evaluated for their potential cofactor role in the induction of

neoplasia, particularly of the respiratory tract, in conjunction with the

ubiquitous environmental contaminant, and known carcinogen, benzo(a)pyrene.

The contract effort is designed to permit an assessment of the relative

hazard of platinum compounds in comparison to lead compounds as environmental

contaminants derived from automotive exhaust.

        The scope of work for this contract effort entails repetitive admini-

stration by intratracheal instillation of the metal compounds under study

to Syrian Golden Hamsters.  Each metal compound is to be administered alone

and in combination with benzo(a)pyrene as a saline suspension.  Instillations

of benzoCa)pyrene alone, and of the combination of benzo(a)pyrene with iron

oxide (Fe203) are to be administered.  The latter combination has been

extensively characterized in the past, and is expected to be a potent inducer

of pulmonary neoplasia when administered via the endotracheal route.  Test

compounds are to be administered to animals once weekly for at least 16

weeks, after which animals will be maintained for their natural lifetime.

                                 97-A

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>ross and hlstopathologic evaluation of lesions produced will provide the




indpoint of the study.




        Additional studies to be conducted during the next 24 months are




mtlined in the continuation of the contract dated January, 1976.  These




Include:  1) evaluation of transpulmonary absorption of lead or platinum





-------
           STUDIES OF NO-EFFECT LEVEL OF Pt and Pd USING
                 MOUSE PULMONARY INFECTIVITY MODEL
                          Richard Ehrlich
                      IIT Research Institute
                        Chicago, IL 60616
                               SUMMARY
     Mice were exposed to aerosols of platinum dioxide (Pt02) and
palladium oxide (PdO) in respirable particle size, and challenged
with airborne influenza A/2/Taiwan virus or Streptococcus sp.,
Lancefield Group C.  Parameters investigated were mortality and
survival rates, pulmonary consolidation, lung edema, histopathological
changes in lungs by light and scanning electron microscopy, and
phagocytic activity of alveolar macrophages.

     Mice challenged with Streptococcus at 1 or 5 hr after exposure
to Pt02 or PdO aerosol showed significantly increased mortality rates
and decreased survival time, compared to control mice challenged only
with the infectious agent.  These effects were not seen when
influenza virus was used as the infectious agent.  Aerosol concen-
trations to which the mice were exposed ranged from 181 to 316 mg/m3
of Pt02 and from 156 to 287 mg/m3 of PdO.  This resulted in estimated
total inhaled doses ranging from 650 to 2500 yg.  Atomic absorption
analyses indicated that between 75 to 90% of the metals was retained
in lungs for at least 14 days after inhalation of Pt02 or PdO aerosols

     Results of limited experiments indicated that inhalation of
Pt02 aerosol, markedly diminished the ability of lungs to clear intra-
nasally instilled Staphylococcus aureus.  This suggests that pulmonary
inactivation of bacteria in alveolar macrophages was significantly
altered by exposure to Pt02.
Studies supported by EPA Contract No. 68-02-1273.  Presented at
Catalyst Research Program; Platinum Research Review Conference.
Rougemont, NC, December 3-5, 1975.
                                    99-A

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":^""-y~^^i^-v«.J^ri^-\r«*.--'*
•j^t'S^^TtSS ;'~~ *• ^- f -•- LI
                   iU.v,  DIOXIDE
                 100-A

-------
3
    60

    50

"   AO
u
«   30
4J
I   20
        w
        u
            10
                         1 hr
                     PdO	»- Strep
                                                  5 hr
                                              PdO'
•Strep
                     690  1100  1800
                                             690  870 1160 1800
                                    Inhaled Dose, yg
                                                                                  * p <0.05
                                                                                 ** p <0.01
                  EXCESS MORTALITY  AMONG MICE  EXPOSED TO 'PdO AND CHALLENGE WITH STREPTOCOCCUS
                                                                                                                       1
                                                                                                                        n>
                                                                                                                        -4:

-------
o
ro
        100




         90



         80




         70
         60
c
o
«H


g  50
     j
   40




   30




   20




   10


    0
                        Intratracheal

                        in Hamsters
                   PtO,
                           24   A8


                           Hours
                                72
                                             Aerosol

                                             in Mice
PtO,
PdO
                                                 14  days
             RETENTION OF PLATINUM AND PALLADIUM IN LUNGS OF. ANIMALS

             EXPOSED TO PtO, AND PdO
u.
£
^

3\
»*r
I
r»
(J
r '
i

-------
o
co
       n
       05
       3


      I
60





50
          30
20
10
                           1 hr
                     PtO,
                     •Strep
                              ***  '***
                    650 950  1450 1950
                                                5 hr
PtO
                                           650  950 1450 1950
                                                                                  * p^ 0.1


                                                                                 ** p ^ 0.05


                                                                                *** p .£ 0.01
                                          Inhaled Dose,
                 EXCESS MORTALITY AMONG MICE EXPOSED TO Pt02 AND CHALLENGED WITH STREPTOCOCCUS

-------
                                TABLE 1
             BACTERICIDAL ACTIVITY IN  LUNGS  OF  MICE  AFTER
                 INHALATION  OF  METAL COMPOUND AEROSOLS

                                      BACTERIA  RETAINED  (%)
f COMPOUND
  CONTROL
  CONTROL
MID
HS_
2090
0
1935
0
GEOMETRIC MEAN
39,6
13,4
31,8
46,2
SE
8,0
2,3
4,8
5,6
.NO, LUNGS
5
4
12
6

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        COMPARE RELATIVE TOXICITIES OF Pt COMPOUNDS AGAINST Pb
              COMPOUNDS USING IN VITRO MACROPHAGE SYSTEM

                          Dr. Donald Gardner
                    Environmental  Protection Agency
                  Health Effects Research Laboratory

(Summary of work performed by Dr.  Eula Bingham, University of Cincinnati)

     This study compared and evaluated the cellular response of the lung,
especially the pulmonary alveolar macrophage, to the effect of inhalation
of Pt and other metal  compounds using the alveolar macrophage (AM)  as a
biological indicator of toxic effects.  In this study young rats were
exposed to an aerosol  (150 ug/m )  of either PtCl, NiCl,  NiO, Pb-O.,,
PbCl or CdO for 6 hours/day, 5 days/week.  At various time intervals, the
animals were removed from the chamber, sacrificed and the lungs lavaged
so as to isolate the pulmonary cells (AM).  The amount of metal deposited
in the lung was determined by atomic absorption technique.  Some of these
substances that were tested (CdO,  NiCl2> PdCl, and PtCl) did not statis-
tically alter the number of lung cells while NiO caused  an increase and
Pb203 caused a depression in number of lavable pulmonary cells.  This
indicates that one cannot assume that the inhalation of  particles per se
induces a non-specific increase in pulmonary cells.  In  these experiments
the number of isolated cells were dependent on the specific metallic parti-
cle inhaled.  The reaction of the lung to these particulates may depend
upon a number of factors such as solubility, physical properties, on
specific metals.  Also determined in these studies were  the enzymatic activity
i.e. acid phosphatase, alkaline phosphatase, and lysozyme in alveolar macro-
phages exposed to platinum.  Preliminary analysis indicates an increase in
cellular lysozyme and acid phosphatase and no apparent effect on alkaline
phosphatase.
                               /

-------
                         Dr. James H. Taubler
                         St. Vincent's College
             Allergic Induction in Experimental
                Animals Using PtSO^ and PdSO^
     Various routes and metal concentrations were used to induce
an allergic state in rabbits, guinea pigs and mice.  Subcutaneous
and intravenous injections of 0.3,  0.1 and 0.05 mg/ml of platinum
or palladium sulfate three times per week for four weeks failed
to induce either immediate or delayed allergy as determined by
skin test in rabbits and guinea pigs or by footpad test in mice.
Skin exposure to 0.25g and O.lg paste applied once a week for five
weeks also failed to induce the allergic state.  Intravenous
injection of a palladium-albumin complex failed to induce allergy
also.  However, subcutaneous injection of the palladium-albumin
complex induced a delayed type allergy in guinea pigs.  Passive
transfer via spleen cells was effected.
     Additionally, the blood, urine and splenic tissues were
assayed by Atomic Absorption Spectrophotometry for platinum and
palladium.  When the metals were injected intravenously no trace
of the metals was found.  In rabbits and guinea pigs subcutaneously
injected with platinum, no metal was found in serum or urine.
However, in a similar experiment using palladium, guinea pigs
showed significant levels in the urine.  Regarding splenic uptake
of the metal, only platinum was found in the spleens of rabbits,
guinea pigs and mice subcutaneously injected with PtSO^.
     To date the following conclusions can be made:
1)  There is no allergic induction in rabbits, guinea pigs or
mice injected subcutaneously or intravenously with platinum or
palladium sulfate.
                              107-A

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2)  In animals injected intravenously with PtSOjL or PdSO^, no
levels of metals can be found in the serum, urine or spleen.
3)  In animals injected subcutaneously with PtSO^ or PdSO/^ only
palladium enters urine of rabbits and guinea pigs.
*O  In guinea pigs subcutaneously injected with a palladium-
albumin complex, a delayed type allergy develops which can be
passively transferred via lymphoid cells.
                              108-A

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    THE EFFECTS OF PLATINUM SULFATE ON LEUCOCYTE AND PLATELET METABOLISM
                 AND ON IMMUNOLOGIC RESPONSES IN THE RABBIT

                          Kenneth D. Lunan,  Ph.D.
                    Senior Biochemist and Program Manager
                     Environmental Biochemistry Program
                       Biomedical Research Department
                                    and
                          Ted A.  Jorgenson,  M.B.A.
                               Toxicologist
                           Toxicology Department
                          Life Sciences Division
                        Stanford  Research Institute
     This research is a comprehensive study of biochemical,  physiologi-

cal, immunological,  and mutagenic effects induced in blood leucocytes,

blood platelets, and major organs of the mammalian body by exposure to

platinum sulfate.  This material may be a major attrition product of

catalytic converters, and large segments of the population may be

exposed to it in the future.  In addition,  this research may serve to

establish the feasibility of using formed elements of the blood,  an

easily accessible body tissue,  as monitors of the body burden of  heavy

metals.

     Rabbits were injected intraperitoneally daily with 2, 15, or 20

mg/kg of Pt(804)2 f°r 1 or 3 weeks and then sacrificed.  Others were

allowed to recover for 1 week,  1 month, or 3 months before sacrifice.

Control animals treated with saline were included.  Leucocytes and

platelets were isolated and examined by numerous criteria for effects

due to Pt(SO4)2 treatment.  The ip LD50 for Pt(SO4)2 in rabbits was

found to be 210 mg/kg, with a range of 134 to 322 at the top 95% con-

fidence limits.
                                   109-A

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     Skin and eye irritation studies in rabbits and skin sensitization




studies in guinea -pigs were also performed.  Pt(SO4)2 was applied at a




concentration of 0.5 g/sq in. to abraded and unabraded skin patches




for up to 72 hours.  No effect of Pt(S04)2 was noted.  Guinea pigs




were injected subdermally with a 0.1% solution of Pt(SO4)2 on alter-




nate days for 20 days.  Two weeks after the last injection, an addi-




tional challenge injection was given.  Examination of the injection




site 24 hours after each injection indicated that Pt(S04)2 did not




cause skin sensitization in the guinea pig-  Eye irritation studies




were performed by instilling 0.1 g of Pt(SO4)2 into the conjunctival




sac of the eye of the rabbit.  The material was then washed out after




30 seconds or 5 minutes.  In other rabbits, the material was not




washed out.  The cornea, iris, and conjunctiva were examined after




1 and 4 hours and then daily for 14 days.  The rabbits whose eyes were




washed 30 seconds after instillation of the test material showed a




transitory effect and were generally normal after 9 days.  However,




the eyes of rabbits in the 5-minute wash and no-wash groups were more




severely damaged and had not returned to normal by 14 days.  In many




animals, the amount of conjunctival swelling was so great that either




the eye surface could not be observed at all or only a small portion




of the cornea and iris could be viewed for scoring.  Thus, Pt(S04)2




should be regarded as a moderate to severe eye irritant, depending on




wash conditions.






     In the leucocyte experiments, the majority of the effects were




observed in the exposed animals from two treatment groups—the one
                                    110-A

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subjected to 3 weeks of treatment with Pt(SO4)2 at 20 mg/kg and the




one allowed 1 month to recover after 3 weeks of treatment with Pt(SO4)2




at 15 mg/kg.  In the former,  blood values were not affected.  RNA syn-




thesis was depressed (DNA synthesis was depressed after 1 week of




treatment).  Also,  protein turnover after 24 hours was retarded.  In




the latter group,  only one labeling parameter was affected—the phos-




pholipid turnover,  which was markedly retarded after 24 hours.  The




other values affected in this experiment were blood parameters.  The




mean corpuscular hemoglobin concentration and the lymphocyte count




were low, whereas the eosinophile count was markedly high.  In general,




in vivo Pt(SO4)2 treatment had only modest effects on leucocyte




metabolism.





     Several preliminary iji vitro exposure experiments indicate that a




2-hour exposure to 10~4 M Pt(SO4)2 caused a 50% depression of DNA




synthesis.





     Numerous alterations of platelet metabolism were observed in all




experiments involving animals exposed to 15 and 20-mg/kg of Pt(SO )2.




5-Hydroxytryptamine uptake was stimulated by 1 and 3 weeks of Pt(S04)2




treatment.  The stimulation remained after 1 week of recovery but was




gone after  1 month of recovery.  0-Glucuronidase activity was elevated




in the platelets from these two treatment groups and had returned to




normal after 1 week of recovery.  Aryl sulfatase was elevated in the




animals  treated 1 week and after 1 week and 1 month of recovery.




Finally,  phospholipid synthesis was depressed after 1 week of treat-




ment, stimulated after 3 weeks of  treatment, and again depressed after




1 month  and 3 months  of recovery.






                                    ni-A

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     Several whole blood values were affected in the 1-week, 1-month,




and 3-month recovery experiments.  Generally red blood cell and plate-




let counts were elevated, and hemoglobin and hematocrits were low.




Also, the volume, hemoglobin content, and hemoglobin concentration of




the average red blood cells were low.  Thus, Pt(S04)2 produced a func-




tional anemia that did not appear until 1 week after treatment had




ceased and that persisted for at least 3 months-.

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             Cytogenetic Analyses of the Effects of Platinum

                           Ann D. Mitchell
                  Biochemical Cytogenetics Program
                   Biomedical Research Department
                     Stanford Research Institute
                    Menlo Park,  California  94025
     Cytogenetic analysis is one of the most widely used methods for
assessing the cellular damage caused by environmental pollutants.
The examination of chromosomal aberrations in cells after in vivo
exposure to environmental pollutants can reflect both the direct
action of mutagenic agents and the indirect effects of an altered
metabolism.  The observation of Cytogenetic damage occurring in a
controlled in vitro environment can be of value for determining the
mechanisms of possible mutagenic action.

     This study was proposed because:  A search of available litera-
ture had failed to reveal any previous testing of platinum salts for
mutagenic activity, although certain other metals had been shown to
induce chromosome breaks and aberrations and/or mutations; some
researchers indicated that certain platinum complexes having a
marked antitumor activity may induce a primary lesion in DMA through
the formation of an intrastrand purine dimer; and some investigators
suggested that metal ions may enhance the aberration frequency of
clastogenic agents by complexing with the broken chromosomes,
thereby preventing the repair of the breaks.

     We are conducting the following seven experiments to determine
whether platinum induces chromosomal aberrations or prevents the
repair of Cytogenetic damage induced by a known mutagenic agent,
ethylmethanesulfonate (EMS):  (l) Examination of chromosomes from
rabbit leucocytes exposed in vitro to Pt(SO4)2, with one-half of the
samples also exposed to EMS; (2) a similar ±t\ vitro experiment using
human leucocytes; (3) examination of chromosomes from leucocytes of
rabbits exposed ii\ vivo to Pt(SO4)2 for 1 and 3 weeks, with EMS
added ±r\ vitro to one-half of the samples; (4) a similar experiment
using leucocytes from rabbits recovering for various lengths of time
from a high dosage of Pt(SO4)2; (5) examination of chromosomes from
bone marrow cells of rats exposed to acute and chronic dosages of
Pt(SO4)2; (6) chromosome banding analysis of the more significant
samples from the previous studies; and (?) use of an assay system
developed at SRI to evaluate the effects of Pt(SO4)2 in inducing
and/or inhibiting repair of DNA damage.
                                  113-A

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       Toxicities of Compounds of Platinum and Palladium
              as Measured by Biochemical Parameters
                     David J.  Holbrook, Jr.
                   University of North Carolina
     Studies have been conducted on the effect of the dietary administration
of salts of lead, manganese, platinum and palladium on the following:
the growth rate of male rats, the organ weight of five tissues (liver,
kidney, spleen, heart and testis), and the tissue content of DNA, RNA and
protein.  In general, dietary levels of PbCl2, PdCl2, PdO and PdSO^ greater
than 10 mmoles/kg feed were necessary to restrict the weight gain of the
rats.  Soluble salts of Pt*+, namely PtCl4 and Pt(804)2-4H20, at levels of
approximately 2 mmoles/liter drinking fluid, were sufficient to restrict
the weight gain of treated rats.  Dietary PbCl2 markedly increased the
size of kidneys in treated rats.  In almost all other studies, however,
the dietary administration of salts of lead, manganese, platinum or
palladium did not markedly or consisently alter the organ weights of the
metal-treated rats.  The dietary administration of PbCl2, PtCl4 or Pt(S04>2
  4H20 for 4 weeks did not alter the content of DNA, RNA or protein in
liver, kidney or spleen (when the content is expressed per gram of wet
tissue).

     The intraperitoneal administration of PtCl^ or Pd(N03>2 at levels
of 28 or 56 ymoles/kg body weight decreased the thymidine incorporation
into DNA of spleen, liver, kidney and testis.  Spleen was most sensitive
to both the platinum and the palladium salt.  In liver, DNA syntheses in
parenchymal cells and stromal cells were about equally sensitive to PtCl4.
The administration of PtCl4 or Pd(N03)2 did not decrease the entrance of
the labeled precursor into the tissue, and PtCl4 did not alter the relative
amounts of thymidine and its phosphates in liver or spleen.

     The effects of various salts of platinum or palladium were determined
on the parameters of the microsomal mixed-function oxidase system from
rat liver.  The intraperitoneal injection of PtCl^ or Pd(N03)2 at 56
umoles/kg, Increased the hexobarbital-induced sleeping time in vivo and
generally decreased the aminopyrine demethylase in vitro and the microsomal
content of cytochrome P-450.  The dietary administration of various salts
of Ft or Pd for one week generally decreased or had no effect on the para-
meters of drug metabolism by isolated microsomes and after 4 or more weeks
generally had no effect on, or increased, the parameters.  The addition of
0.15-0.2 mM PtCl4 or 0.2-0.3 mM Pd(N03)2 to the incubation medium (containing
5 mM MgCl2> inhibited the aminopyrine demethylase of isolated hepatic
microsomes by approximately 50%.
                                   115-A

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      Studies of Platinum and Palladium in Human Tissue
      Using Selected Populations Including Occupationally
      Exposed Individuals
          Mr. John Prevost and Dr. Donald Johnson
       Mr. Prevost will present information on the selection of sites
for the baseline and occupationally exposed populations, on the
recruitment of study participants and on the description of the study
participants.  Dr. Johnson will present a summary of the results for
Pt, Pd and Pb in these populations.
                               H7-A

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      Evaluation of the Mutagenio Potentials of Platinum Compounds
Shahbeg Sandhu
Department of Biology
N. C. Central University
Durham, North Carolina
     This study was initiated to screen a number of platinum com-

pounds for their possible mutagenic effects.  An established cell

line, L5178Y mouse lymphoma cells, is employed as a test system.

These cells are known to be diploid at thymidine fcinase (TK)

locus.  The selective media for isolating TK competent cells

(TK •••/- and TK -:/') and TK deficient cells  (TK -/-) has also

been developed.  The compound under scrutiny is considered to

be mutagenic if it has the ability to change the TK competent

cells to TK deficient cells and virj? vprsa^ with frequency com-

parable to that of the known mutagens.

     In addition to the jai vitro testing, presoaked and dry seeds

of PhaspoluR atirnalja are exposed to different concentrations of

platinum compounds.  The data is recorded on germination, charact

teristics, growth and various morphological characters in M^, Mg

and Mg generations.  Currently the following compounds are under

study in our laboratory:

     Platinum diammino dichloride, Ammonium chloroplatinite,
     Platinum dinitrodichloride  (cis and trans isomers), Ammonium
     chloroplatinite, and Platinum chloride.
                              119-A

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    DEVELOPMENT OF SENSITIVE BIOCHEMICAL AND

BEHAVIORAL INDICATORS OF TRACE SUBSTANCE EXPOSURE
              Dr.  Edward J.  Massaro
           Department of Biochemistry
     State University of New York at Buffalo
            Buffalo,  New York  14214
                      121-A

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     In adult male mice (Swiss-Webster CFW strain:  Carsworth Farms, New




York, New York), the tissue/organ distribution and effects of platinum on:




(i) levels of selected neurotransmitters; (ii) energy production through




glycolysis and; (iii) rates of DNA, RNA and protein synthesis are being




investigated.  Simultaneously, and coordinated with these studies, behav-




ioral analysis, including open-field, passive avoidance, maternal and social




behavior are being undertaken.




     Three routes of administration are being investigated:  intragastric -




because of its possible environmental relevance; and intravenous and sub-




cutaneous - for monitoring the effects of rapid and slow sytemic distri-




bution, respectively.  The latter two routes also will avoid confounding due




to possible binding of the compound to food materials; microfloral inter-




actions; etc. inherent in the IG route of administration.  Seven day LD,-0




data are being established to serve as toxicity parameters.




     Originally, Ft as Na_PtCl, was being investigated.  LDc0 levels for




this compound have only been estimated (90% confidence intervals by probit




analysis).  However, ultra-accurately defined toxicity levels are not nec-




essary for the rough definition of experimental parameters and pilot studies




of open-field behavioral effects and tissue/organ distribution at the ap-




proximate LD1f. and LD   levels have been initiated.  Reasonable estimations




of the LD,_ and LD-j. levels for each compound for each route of administra-




tion were made by simple linear regression of % responding on dose.




     The open-field behavior  (ambulations, rearings) of the animals was ob-




served at four time periods postadministration  (4 hours, 1 day, 3 days and




seven days).  Tissue/organ distribution of Pt is undergoing analysis.
                                     123-A

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                                     -2-
     Ten animals were assigned to each treatment group.  Injection solu-



tions were prepared from a 50 ppm Pt stock solution, which was adjusted



to pH 7.4 with NaOH, and diluted with phosphate buffered saline (PBS:



0.14 M NaCl, 0.01 M NaPO,, pH 7.4) to the appropriate concentrations.



For purposes of simplification, each time group was run in a block starting



with the four hour groups and preceding in order to the seven day groups.



When the three day and seven day groups were observed; inordinately high



death rates were encountered compared with the originally estimated LD



levels.  Concievably, the LDin and LD_,. dose levels could have been mis-



calculated; but no miscalculations were discovered.  Nevertheless, the LD



level was reestimated taking into account the new lethality data and another



open-field study was undertaken employing the reestimated LD9t-'  Again,



there were excessive deaths at 3 and 7 days.  Rechecking each step of the



methodology revealed that the pH of the injection solutions had drifted



with time from 7.4 to approximately 4.0.  This down-shift was confirmed in



a separate experiment.  The drift probably results from a slow hydrolysis


            2-           2-
of the PtCl,   to PtC1..0H   liberating HC1.  Ex post facto, it was learned
           0          J	


that such an hydrolysis effect occurs with other inorganic chlorides.  Al-



though we have uncovered no information in the literature on this point,



it seems reasonable to assume that pH drift was responsible for the time-



dependent alterations in toxicity of our injecting solution.



     In view of this technical difficulty, interpretations of the results



of the pilot open-field study are confounded; but the data were subjected



to analyses of variance.



     Four behavioral response variables were measured during the 5 minute



trial in the open field:  ambulations  (automatically recorded); rearings;
                                    124-A

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                                     -3-
initial latency (in the six inch center circle); and the number of recros-

sings of the center circle during the trial.  The analysis was, therefore,

of a 3 (route) by 4 (dose) by 4 (time) multivariate design and The Multi-

variance (J.D. Finn, 1972)* computer program was used to perform the analysis.

Since  certain cells had less than 10 animals due to deaths, the design was

nonorthogonal which precludes exact significance tests of main effects in

the presence of significant interactions.  Similar two way Anovas also were

conducted for each time since the experimental conditions were more homo-

geneous within each time.

     From the overall analyses, most of the possible significant** effects

were seen in the ambulations and rearings variables.  For the route effects,

IG had the highest scores across variables; IV had the lowest; and SC was

intermediate.  For the dose effect, ambulations and rearings were depressed

at all dose levels compared to saline controls.  The time effect was not

"significant".  The route by dose interaction was the only "significant"

interaction.  In the two way analyses at each time, the dose effect was

"significant" throughout while the route effect was "significant" only at

the first three times and diminished with time as would be expected.  The

route by time interaction also was "significant" only at the first three

times.

     In spite of the experimental and statistical ambiguity of this pilot

study, we believe the results and our own observations of the animals

strongly suggest that Na.PtCl, has an effect on behavior which would be

characterized as depressant.  The effect appears most strongly shortly
     *Finn, J.D. (1972) "Multivariance", National Educational Resources,
      Inc., Ann Arbor, Michigan,
    **Not certainly significant because of the experimental and analytic
      difficulties mentioned above.


                                      125-A

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                                     -4-
after administration and diminishes with time.   The effect also depends




on route of administration:  IV, the route of most rapid uptake, exhibits




the greatest effects;  IG,  the route of slowest uptake, exhibits the smallest




effects.
                                      126-A

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                             APPENDIX II

         Platinum Chemistry Pertinent to the Catalyst Program

     During general  discussion at the conference, the need was expressed
for a short summary of useful information on platinum chemistry.   Rather
than provide the requested information separately to the investigators
who expressed interest, it is hereby presented for general use.  Questions
should be directed to the author, Dr. Richard Thompson, Environmental
Monitoring and Support Laboratory, Environmental  Protection Agency, MD-78,
Research Triangle Park, North Carolina  27711.
                                 127-A

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                               Appendix  IIA


         Platinum Chemistry Pertinent to the Catalyst Program

     The catalyst program must necessarily be concerned in part with the
transition metals, since one of their characteristic properties (not
shared with representative metals) 1s catalytic activity.   The use of
platinum palladium and other transition metals 1s based primarily on
catalytic activity, an empirical property.  Because of the known chemical
nature of these elements and their compounds, we may make estimations of
expected chemical behavior in the environment with some confidence in
most cases.  It 1s of Interest that prior to the use of platinum as a
catalyst in automobiles the environmental levels 1n matrices such as
atmospheric particulate matter were so low as to defy detection by all
methods attempted other than Isotope dilution mass spectrometry, even
when a kilogram sample was taken.

     Elemental platinum is Inert to simple acids, but will react with
aqua regia (3:1 concentrated HC1:HNO,), chlorine, bromine, sulfur and
fused alkali and cyanide.  None of these substances exist naturally.
The oxides are not thermally stable, decomposing below 300°C.  (A temper-
ature of about 650°C  is attained in automotive catalytic converters).
Biological activity is not expected to result in formation of compounds
from elemental platinum.

     One of the notable properties of the transition metals 1s that they
form many and stable coordination complexes.  These can be visualized as
being formed by the coordination of Ions or molecules with metal Ions.
The mechanism of this coordination is the donation of an electron pair
by tne ligand (1on or molecule) to the bare metal ion.  As an illustration,
consider "Pt(SO,)V which 1s said to be deliquescent.  This can be
                         +                               B
pictured as a [Pt(H00)g]4  species accompanied by two SO^ .  The hexa-
coordinated platinum ion can be shown 1n crystalline species to be
octahedral, and all six of the Ugand positions are equivalent.  If one
                                  128-A

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dissolves Pt(SO.)2 in water and allows the solution to stand, In time,
the [Pt(H20)6]4  Ion will  yield Pt(OH)4 '  nH20 + 4H* which can be expressed
as Pt02 * nH20; heating the solution will  accelerate the process.

     In the catalyst program, we are Interested In the environmental
changes that will  ensue from the introduction of platinum and its compounds
(and other catalyst metals) into the environment.  Unfortunately, the
exact nature of the species emitted from a converter 1s not knowny but
Is most likely to be the element, or oxides and halides.  These can be
used in inhalation studies, but for other in vivo experiments a soluble
form is desirable.

     At the Quail  Roost Conference, it became painfully obvious that
commercial samples of "platinum sulfate" and "platinum chloride" were
most likely hydrolytic end products resulting from the reaction of these
deliquescent substances with water from the air.

     A possible approach for in vivo studies could utilize fresh solutions
of M2PtCl6 (where H is K*, Na+, NH4* etc.)  The (PtClg)* 1on 1s stable
1n cold water for some time and the addition of M Cl (if not objectionable)
will further stabilize the complex In solution by forming a pool of
chloride Ions to compete with water (and Its constituent ions) as a
Ugand.  Furthermore, the ion Is colored, and absorbs light very effec-
tively 1n the near ultraviolet.  From the literature :

                             [PtCl6]"

          Wave length (nm)          e            6"           6
               453
               353
               262               24,540        2,250        2,300

where e is the molar absorptivity and 6~ and 6  are the half-band widths
towards Increasing and decreasing values.

                                    129-A
50
790
24,540
1,900
1,700
2,250

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     Therefore absorbance 9 (cone) (path length) (absorptivity) and 1n a
1 cm cell, a 4 x 10   M solution of hPtCl  would have an absorbance of
0.1 at 262 run.  One could thus measure the absorption at 262 ran prior to
administration to test animals to ascertain whether or not the solution
1s        *
     1.  C. K. Jorgensen, Acta Ch1m. Scand. JjD, 518 (1956).
                                  130-A

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                             Appendix IIB


               Spectrophotometric Verification of PtC1g~
1.   Obtain a supply of sodium hexachloroplatinate(IV) (Na^PtClg) or
     chloroplatlnic acid (H^PtClg).  Standard solutions typically used
     for calibration of instruments are available.  Be certain from the
     supplier (got a guarantee) that the contents are the chemical form
     cited above.  [A SPEX Industries standard available in our labora-
     tory was suitable.]  Such solutions typically contain 5000 yg Pt/ml .

2.   Obtain clean glassware of the proper si^e.  Make up only as much
     solution as will be used within 4 hours or solution preparation.
     These solutions will  deteriorate.  Therefore do not store them.

     If for exerr.ple one liter of final solution were desired, the standard
     were 5000 ug/ml and the final solution concentration desired v/as
     5 yg/ml  a one nil pipette and a one liter volumetric flask are needed.

3.   Prepare the solvent ty be used for diluting the standard.  (It is
     assumed here that -isotonic saline will be used.)  Do not use plain
     water.

4.   Prepare solution.  Pipette the correct number of ml of standard
     solution into the volumetric flask and dilute to volume with isotonic
     sarine.   Mix by inverting the flask about 10 times.

5.   I.ocate0a spectrophotometer capable of measuring absorbance at 262 nn
     (2620 A) such as a Gary Model 14 or a Beckman DLL  Th? 262 nm wave-
     length is below the range obtainable on inexpensive colorimeters
     such as the Spectronics , Co lemons, and Turners.
     Instructions which follow are for a Gary 14.

6.   While "power" switch  is off, install a 0-1 absorbance slide-wire
     cartridge being careful  not to scratch the wire spindle.

7.   Turn on coolant watr.-r supply to UV lamp before tiTnin-j on instrument
     "power" switch and UV lamp switch.  Allow lainp and irisvrunieni tc
     warm-up for 1/2 hour.

8.   Set "Source" ar.d '"Detector", selectors to "UV," "IR det" to "out,"
     and pull-out phototube shutter.

9.   Set wavelength to 262 nm (2620 ft)  with dial  approaching 262 from
     higher numbers.

10.  Fill two 1-cm (10 run) optical  cuvette cells  about 3/4 full  with
     same solvent to be used for diluting Pt standard.  T?p cells to
     dislodge any air hubbies, and  v.-'ipe optical  surfaces clean with
     soft tissue.
                                  131-A

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                                  -2-
11.  With "master" switch off (but "power" switch on), position the two
     optical cells (both containing solvent) in the two optical beams
     such that light will pass through the unfrosted sides of the
     cuvettes.

12.  With lids on the optic chambers, turn on "master" control and with
     "slit" program and "pen" systems in "on" position set pen at 0.000
     absorbance 'level on chart recorder using "balance" dial.

13.  If pen movement is more than 0.005 absorb?r,ce, pen dampening can
     be set higher (on Gary 14 the dampening control knob is located
     on back panel behind the swing-out recorder).

14.  With "master" switch off ("power" switch left  on), remove cell from
     "sample" optical chamber, rinse out cell repeatedly with deionizcd
     water and finally with acetone to dry it.

15.  Pour 5 ug/nil Pt solution into clean, dry cuvette cell (used earlier
     with solvent to set zero absorbance).  Tap cuvette to dislodge any
     visible air bubbles, and wipe outer optical surfaces clean with
     soft tissue.

16.  Place cuvette with Pt solution in "sample" optical chamber and
     close chamber tightly with lid.

17.  Turn "master" switch on and record position of recorder pen to
     determine absorbance value.
18,
Observe pen position as wavelength dial  is turned slowly in both
directions (50 rim) to locate maximum absorbance value.
19.  If maximum absorbance is located at different wavelength setting
     than used in step £14 above, rinse "sample" cuvette repeatedly
     in dc.-ionized >..'nter and refill with solvent (after drying cell
     with acetone) to verify that ebsorbance v.ith solvent in both
     cells reads 0.000 at this other wavelength setting.  If not 0.000
     absorbance add or subtract reading (as appropriate) to maximum
     found in #15.

20.  UV scan for 5 yg/ml Pt in water (from Spex Indust.  Std.) is
     attached.  NaCl (0.9%) or HC1 (2N) did not alter this  scan when
     measurements were conducted within 1/2 hour after preparation.
     The absorbance at 2400 fl and at 2850 fl should both  be  very nearly
     equal and be equal to 1/2 of the maximum absorbance which should
     occur at 2620  .
                                   132-A

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                                  -3-
21.  The expected absorbance at 2620 $ for an expected concentration should
     scale linearly as follows for a 1 cm path length cell.

               Cone.                         Absorbance
               1.0 yg/ml                         0.125
               2.5 yg/ml                         0.313
               5.0 pg/'ral                         0.625
               7.5 i:g/inl                          .938

     Values shoi'ld be within about 15%'of the above.  1 cm path length cells
     are standard.  However, if path lengths differ values for absorbance
     scale linearly,  [e.g., for a 1/2 cm path length cell 8 ug/ml solutions
     have an absorbsnce of 0.50.  A 2.5 y.g/ml solution in a 2 cm cell should
     have an absorbance of .625.]

22.  If solutions more concentrated than 7.5 yg/ml  are to be utilized
     prepare then at the  desired concentration.  Take a small sample, of
     known vclurr.e e.rd dilute it in a volumetric flask with normal saline
     so that the f'iral concentration is less than 7.5 yg/ml.  Calculate
     the conceivLration expected in this diluted sample as

                                          sample) = conc> Qf di]uted ^
                      or
                         V W I U i: I *i- V I
     Examine this diluted sample in the spectrophotometer.  Absorbance
     should cicree with that expected for the calculated concentration.
                                    133-A

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                                   — '1
                       0-7
          o.c
                                                         .- 1
t -

          0.0
         •o./.
          0.
                                   \.."-"•;
                                         .._ .1	j

                                                     !_.

                                                          Cj
                                                          r»
                                                                     134-A

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                                   TECHNICAL REPORT DATA
                            (/'lease read /nUnictioris on the reverse before completing)
 I. REPORT NO.
4. TITLE AND SUBTITLE
                                                           3. RECIPIENT'S ACCESSION>NO.
  Proceedings  -  Platinum Research Review  Conference
                                                           5. REPORT DATE
                                                               April  1976
                                                           6. PERFORMING ORGANIZATION CODE
7. AUTHORIS)
                                                           8. PERFORMING ORGANIZATION REPORT NO.
p PERFORMING ORGANIZATION NAME AND ADDRESS
  Catalyst Research Program
  Criteria and  Special  Studies Office
  Health  Effects  Research Laboratory
  Research Triangle Park, N.C. 27711
             10. PROGRAM ELEMENT NO.

                 1AA601
             1 1. CONTRACT/GRANT NO.
 12. SPONSORING AGENCY NAME AND ADDRESS
   Health  Effects Research Laboratory
   Office  of  Research and Development
   U.S.  Environmental Protection Agency
   Research Triangle Park. N.C. 27711
             13. TYPE OF REPORT AND PERIOD COVERED
             14. SPONSORING AGENCY CODE

                 EPA-ORD
15. SUPPLEMENTARY NOTES
  This  is  an unpublished report.
16. ABSTRACT
         The  Catalyst Research Program was  initiated in FY 1974 by  the  EPA
   Administrator,  Russell E. Train, concurrent with the Agency's Nov. 1973 decision
   to  permit  the use of noble metal oxidation catalyst as an emission control  device
   on  1975 model year passenger vehicles.   Early research suggested  that  slight
   emissions  of platinum and palladium might  be emitted.
         The  CRP's objective as it pertains to the noble metals, then,  was to  develop
   an  information base on noble metals,  particularly platinum, in many  areas.
         The  areas of study for the CRP's platinum research effort  include:  emissions
   characterization, measurement methodology  development, bioenvironmental  impact,
   health effects assessment, body burden tissue analysis, and epidemiology.
         After the platinum research effort had been under way
   apparent that the data being reported by researchers should
   appropriate forum for informative exchange and discussion.
         The  conference presentations, areas  for further study
   emphasis or direction of current study,  and priorities for
   are briefly discussed in this report.
                   for a year, it became
                   be discussed at an

                   and possible changes in
                  these recommendations
17.
                               KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
   catalytic converters
   precious  metals
   platinum
   palladium
                                             h.IDENTIFIERS/OPEN ENDED TERMS
                           c.  COSATI Hclil/Cioup

                             06 B
13. DISTRIBUTION STATEMENT

   UNPUBLISHED
19. SECURITY CLASS fflm Heporl)

      UNCLASSIFIED
21. NO. OF PAGES
     154
                                             20. SECURITY CLASS (This page)

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                                                                        22. PRICE
EPA Form 2220-1 (9-73)
                                              135-A

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