EPA-600/1-78-005
January 1973
Environmental Health Effects Research Series
            PREPARATION AND  CHARACTERIZATION
                 OF PARTICULATE  ENVIRONMENTAL
   CONTAMINANTS  FOR  BIOLOGICAL EXPERIMENTS
                                    Health Effects Research Laboratory
                                    Office of Research and Development
                                   U.S. Environmental Protection Agency
                              Research Triangle Park, North Carolina  27711

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                RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology.  Elimination of traditional grouping  was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.  Environmental Health Effects Research
      2.  Environmental Protection Technology
      3.  Ecological Research
      4.  Environmental Monitoring
      5.  Socioeconomic Environmental Studies
      6.  Scientific and Technical Assessment Reports (STAR)
      7.  Interagency Energy-Environment Research and Development
      8.  "Special" Reports
     9.  Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances  or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals  but always with intended application to human health measures.
This document is available to the public tl rough the National Technical Informa-
tion Service, Springfield, Virginia  22161.

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                                          EPA-600/1-78-005
                                          January 1978
PREPARATION AND CHARACTERIZATION OF FINELY DIVIDED
      PARTICIPATE ENVIRONMENTAL CONTAMINANTS FOR
                BIOLOGICAL EXPERIMENTS
                         by

         George Yamate and Hubert Ashley
               IIT Research Institute
              Chicago, Illinois  60616
               Contract No. 68-02-1687
                  Project Officer

                    David Coffin
         Health Effects Research Laboratory
        U.S. Environmental Protection Agency
         Research Triangle Park, N.C. 27711
        U.S. ENVIRONMENTAL PROTECTION AGENCY
         OFFICE OF RESEARCH AND DEVELOPMENT
         HEALTH EFFECTS RESEARCH LABORATORY
         RESEARCH TRIANGLE PARK, N.C. 27711

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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.  Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
                                ii

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                               FOREWORD

     The many benefits of our modern, developing, industrial society are
accompanied by certain hazards.   Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy.  These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.

     The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.  These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants.  The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards.  Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.

     The purpose of this project was to provide a centralized source
for the preparation and characterization of selected particulate
materials for biological experiments.  The particulate materials of
interest were a range of environmental contaminants known or suspected
to detrimentally affect human health.
                                               Director,
                                   Health Effects Research Laboratory
                                 iii

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                                  CONTENTS
Foreword	   iii




Tables  	     v




     1.     Introduction  	     1




     2.     Description of Work Effort	     3




     3.     Results and Conclusions	    21




Appendix A	    22




Appendix B	    23




References	    25
                                      IV

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                                   TABLES
Number                                                                  Page
   1   Sources of Raw Powders .  .  .	   4

   2   Preparations for SUNY-Stony Brook, Series I  	   7

   3   Preparations for IITRI Life Sciencess  Series I 	   8

   4   Trace Chemical Analysis of Parent and  Ground Catalyst Powder .  .  11

   5   Size Distribution of Parent Fly Ash	13

   6   Weight Percent Concentration of Selected Elements in Various
       Fly Ash Samples    <>    	13

   7   Atomic Absorption Analysis of Fly Ash  Fractions  	  16

   8   Number of Fly Ash Particles per Unit Mass of Fly Ash	17

   9   Preparations for SUNY-Stony Brook, Series III  	  19

  10   Trace Chemical Analysis of Metal Oxides	'.....  20
                                      v

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                                 SECTION 1
                                INTRODUCTION

     The objective of this project was to obtain a variety of high purity
particulate substances whose size distribution was reliably and carefully
characterized, and when necessary reduced and/or selectively fractionated
into a size range of interest for various biological experiments.   The exper-
imental protocols employ particulate suspensions for intratracheal instilla-
tions in small animals, for inhalation exposures of animals to aerosols of
these substances5 and for administration of particulates to in vitro cell
culture systems.  In addition, a limited number of different preparations of
selected, finely divided particulate metal or metal oxides in combination with
the polycyclic aromatic hydrocarbon carcinogen, benzo(a)pyrene, (BP) or other
designated carrier material were required for experimental use.  These various
preparations were to be used by several different investigators supported by
EPA contracts, as well as in-house investigators at the National Experimental
Research Center, Research Triangle Park, North Carolina 27711.
     The project was designed to provide a centralized, consistent source for
particle characterization and preparation.  This would eliminate one potential
cause of factitious variability between different investigators seeking to
contrast biological consequences of environmental contaminants.  Assuming a
uniform, highly reproducible particle preparation for all related investiga-
tions will help to provide meaningful data on the basis of which observed dif-
ferences in test effects can be better understood.  In the absence of such
assurance, apparent differences may be due to or masked by variations in the
physical parameters as well as accession or retention in biologically signi-
ficant sites.
     Compounds from the following list were to be characterized, prepared,
and supplied as required by the Project Officer.  These were:

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     Platinum Sulfate - Pt(SO,)2
     Platinum Metal
    *Platinum Oxide - Pt02
     Palladium Metal
     Palladium Oxide - PdO
    *Lead Oxide - PbO
    *Iron Oxide - Fe20~
     Cadmium Oxide
     Nickel Oxide - NiO
     Vanadium - V20c, V203
     Manganese - MnO, Mn02
     Chromium Oxide - Cr203
    *Crude Air Particulate (particle samples collected for EPA by electro-
     static precipitation from air of a large industrial city)
     Each powdered compound was to be prepared approximately in the 0.5-5.0 ym
range.
     Sterile, stable preparations of the four compounds designated above with
an asterisk were to be prepared in combination with benzo(a)pyrene (BP) for
intratracheal instillation.  Precipitation as well as grinding methodology
were used to prepare these combination preparations.  The powdered compounds
both with and without BP were prepared according to the needs and requirements
of the Project Officer, and on a time schedule satisfactory to the contractors
experimental program.

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                                 SECTION 2
                         DESCRIPTION OF WORK EFFORT

PARTICULATE SUBSTANCES OF INTEREST
     The chemicals of interest to the program were obtained from chemical
supply houses and the EPA.  The approximate data on the powders were obtained
by physical and chemical analysis as well as from information supplied by the
manufacturer.  An initial list of sources and data obtained is given in
Table 1.
Classification of Source Powders
     Since the powders in Table 1 contained the desired range of particle
size (0.5-5.0 ym) a cascade sedimentation method was selected to obtain the
size distribution (fraction) of interest.  In an earlier study of a similar
nature, the cascade sedimentation method was found to be highly successful in
obtaining a selected size fraction.  In addition, the method is a classifica-
tion technique based on Stoke's diameter (aerodynamic size) which more closely
resembles the diameter of interest in inhalation studies.  Particles were thus
separated and sized according to their Stoke's diameter.  In this context,
Stoke's diameter is defined as "the diameter of_ the sphere with the same ter-
minal settling velocity as the particle".  Thus, under conditions of any
degree of shape variation, microscope "size" and Stoke1s diameter are not
related.
Cascade'. Sedimentation Method
     The Cascade Sedimentation Method is based on Stoke1s equation

                              ,2   18n h x 108
                              d  =3 	

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                     TABLE 1.  SOURCES OF RAW POWDERS
Powder Supplier
i
Platinum Oxide
Palladium Oxide
Platinum Metal
Palladium Metal
Platinum Sulphate
Lead Chloride
Cadmium Oxide
Manganese Dioxide
Vanadium Pentoxide
Manganous Oxide
Vanadium Trioxide
Ferric Oxide
Chromium Oxide
Nickel
Lead Oxide
Crude Air
Particulate
1
Platinum Chemical
New Jersey
J.T. Baker Chemicals
Chicago
M.C.B. Chemists
Puratek Division
Chicago
Charles Pfizer
Reading, Pa.
Fisher Scientific
Chicago
E.P.A.
Cost
$12/g
$12/g
$12/g
$12/g
$12/g
$6/lb
$17/lb
$6/lb
$12/lb
$l/g
$0.3/g
_!
!

	
Approximate Characteristics
PSR 1/2-20 um; D = 14.9; P = 99.9
PSR 1/2-20 um; D = 8.3; P = 99.9
PSR 1/2-40 p.m; D = 21.4; P = 99.9
PSR 1/2-30 urn; D = 12.0; P = 99.9
D = 5.85
D = 7.0
D = 5.0
D = 3.4
P = 99.9; D = 5.4
P = 99.9; D = 4.9
PSR = 0.5-5 um; D = 5.24; P = 99.8
S.A. = 5 m /g; S = cylindrical
PSR 0.5-5.0 urn; D = 7.5
S.A. = 4 m2/g; S = amorphous
P = 99.8
P = 99.8; D = 9.5
	
Particle  Size  Range = PSR
Surface Area         = S.A.
Shape                = S
Purity               - P
Density              = D

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where
      d = Stoke's diameter in microns
      h = height of fall =10 cms
      r\ = viscosity of IPA @ 25C in poise
     p  = density of particles gm/cc
      S
     pf = density of IPA @ 25C in gms/cc
      g = acceleration due to gravity cms/sec/sec
      t = time in seconds
     The powder was ultrasonically dispersed in a suspending fluid (usually
isopropyl alcohol [IPA]), and introduced into the primary sedimentation flask,
and made up to a volume at which the distance from the suspension surface to
the discharge tap was known (usually equal to 10 cm).  The suspension was well
mixed and allowed to stand for time, tj, calculated from Stoke's equation.
     After time tj, the primary vessel tap was opened and suspension containing
particles of size less than di was slowly run into vessel 2.  Vessel 1 then
contained particles of size greater than di and the fines contained in the
volume between the tap and the vessel base.
     This was then refilled with clean fluid, mixed, and settled again.  Mean-
while, vessel 2 was mixed and allowed to settle 10 cms for time t2 which
corresponded to a da of 5 ym.  After time t2, the contents were run into
vessel 3 where fractionation was performed to time ts which corresponded to a
da of 2 ym.  As ts > t2 > ti careful scheduling permitted the continuous
fractionation and washing of all size fractions to be conducted on a daily
basis, fines needing a longer time to precisely fractionate then coarse par-
ticles.  Frequent examination of the fractions revealed that about 10 washings
were required to produce powders to within each specification.
Toxicity and Reactivity of Powders
     The potentially hazardous and toxic nature of the chemicals listed in
Table 1 require special precautions during handling and use during fractiona-
tion, preparation, and characterization.  For example, platinum or platinum
oxide (PtO_) in powdered form and isopropyl alsohol together under certain

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conditions were found to be highly reactive.  To minimize this hazard, double
distilled water was used as the settling fluid for this powder/fluid combina-
tion.
Powder Characterization
     All prepared powders were characterized as to particle size, surface
area, particle shape, and purity.  Particle size distribution was determined
by sedimentation (Andreasen Pipet Method) or by optical microscopy.  Surface
area measurements were determined by use of the multi-point B.E.T. method.
Particle shape was determined with the scanning electron microscope (SEM).
Purity was determined by semi-quantitative arc emission spectroscopy to detect
contaminants.
PREPARATIONS FOR STATE UNIVERSITY OF NEW YORK AT STONY BROOK
(SUNY-STONY BROOK), SERIES I
     Particulate preparations and suspensions were made for delivery to
Dr. Philip Kane at SUNY-Stony Brook.  Characterized platinum oxide (Pt02),
iron oxide (Fe90~) and lead oxide (PbO) powder, both uncoated and coated with
benzo(a)pyrene (1:1 weight ratio) were prepared and shipped once each three
weeks.  These were shipped dry.  Suspensions of finely divided benzo(a)pyrene
(1.25% by weight) in gel-saline along with gel-saline control samples were
also prepared and shipped once each three weeks.  The suspensions were placed
in separate vials of approximately 20 ml per vial to approximate usage rate
and thus minimize contamination from repeated opening of stock supply.
     Benzo(a)pyrene applied as a coating to metal oxide particles has a ten-
dency to crystallize into needles.  Since this was undesirable in biological
experiments, close coordination was required between the production of the
coated powder and its time of use.  As a precaution against delays in transit,
the carrier's shipping number for each shipment was relayed to the recipient
by telephone.  With this information, a telephone search could be initiated
immediately if the shipment did not arrive when expected.
     Table 2 lists the various preparations delivered during this series to
SUNY-Stony Brook.

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TABLE 2.  PREPARATIONS FOR SUNY-STONY BROOK, SERIES I
Schedule
Date Shipped
1) 01-06-75
2) 01-13-75


3) 01-28-75

4) 02-04-75

5) 02-17-75
6) 02-25-75
7) -03-10-75
8) 03-11-75
9) 03-18-75
10) 03-25-75
11) 03-31-75
12) 04-19-75
13) 05-19-75


14) 06-09-75

15) 07-01-75

16) 07-21-75
17) 07-30-75
18) 07-31-75
19) 08-20-75

20) 09-03-75

21) 09-18-75

B. P. -Coated (1:
Metal Oxide
PbO
Fe203
PtO
PbO
Fe23
Pto2
PbO
~
-
Fe23
PbO
~
Fe23
PbO
-
-
~
Pto2
PbO
PbO
Fe203
Pt02
PbO
Fe23
PbO
Fe23
PbO
-
Fe23
Pto2
PbO
 
Fe23
Pto2
Fe203
Pto2
Pl,0
I'bO
; 1 wt. ratio)
Quantity,
prnms
0.9
0.8
1.0
0.8
0.4
0.9
1.9
1.3
1.6

-
1.9
1.3
2.0
~
1.8
1.3
1.7
-
--
-
1.8
1.3
1.9
1.8
1.3
1.7
1.8
1.5
2.0
1.8
1.3
1.4
1.3
1.8
1.5
-
1.6
1.3
1.5
-
1.9
1.4
1.8
1.5
2.3
2.2
Uncoated
Metnl OxiJe
Fc23
Pto2
PbO


-


Fe23
PbO
Pto2
PbO
-
Fe23
Pto2
PbO
-
~
-
Fe23
PbO
Fe23
pto2
PbO
Fe203
Pto2
PbO
pto 3
PbO
Fe23
PbO
-'-
PbO
--
Fe23
Pto2
Fe23
Pto2
PbO
PbO
Quantity,
grams
0.4
0.4
0.4


-


1.2
1.2
1.2
15.0
1.2
1.2
~
1.2
1.2
1.2
~
~
-
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
-
1.2
1.2
1.2
-
1.2
1.2
1.2
1.2
1.2
1.2
One Week Ball Milled Suspension
0.5% Gel in 100 ml Saline Solution
1.25Z B.P. Control-No. B.P.

60 ml 60 ml




60 ml 60 ml


80 ml 80 ml

80 ml
100 ml
80 ml
80 ml
80 ml 80 ml
80 ml 80 ml


80 ml 80 ml



80 ml 80 ml

80 ml
100 ml 100 ml

80 ml 80 ml

-


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PREPARATIONS FOR IITRI LIFE SCIENCES DIVISION, SERIES I
     Biological experiments in IITRI's Life Sciences Division were supported
by preparations of Platinum Oxide (PtO-) and Palladium Oxide (PdO).   These
powders required size fractionation by the cascade sedimentation process.
     Table 3 is a description of the delivered preparations:
          TABLE 3.  PREPARATIONS FOR IITRI LIFE SCIENCES, SERIES I
         Date Shipped   Metal Oxide   Size Range   Quantity Shipped
           01-04-75         Pt02       1-10 urn         55.4 gm
           03-10-75         Pt02       1-10 ym         72.2 gm
           03-23-75         PdO        1-10 ym         55.5 gm
POWDER PREPARATIONS FOR EPA/CINCINNATI
     Preparation and characterization of submicron Pt/Pd-coated alumina auto-
motive catalytic converter dust were made for Dr. Wellington Moore at EPA/
Cincinnati.  The objective of the work was to produce 1 kg of powder not
exceeding 1 ym in particle size.  A combination of grinding and size fraction-
ation was to be used to achieve this size.  Physical and chemical characteris-
tics pertinent to the powder were to be obtained.
     The Pt/Pd-coated Al-0., catalyst powder was ground by wet ball milling for
two weeks in a 14 cm diameter porcelain milling vessel using 1 cm diameter
porcelain balls.  The ground material was check-d for size by microscopic
examination.
     In addition to microscopic examination, the size of the parent and ground
Al.,0, powder was determined by means of a Micromeritics Model 5000 Sedigraph
particle size analyzer.  In a preliminary step, the density of the powder was
determined to be 3.04 g/cc by means of a liquid pycnometer.  The powder was
dispersed in water (2 g/30 ml) using a Waring blender and 0.1% tetrasodium
pyrophosphate (TSPP) surfactant.

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     The parent powder was too coarse for direct analysis by the Sedigraph.
Therefore, a preliminary wet sieving step was done using a 200 mesh (74 ym)
screen.  It was found that 17,3% by weight was retained on the screen.   The
fraction passing the screen was then run on the Sedigraph.  The combined size
distribution is shown in Figure 1
     Figure 1 shows that the mass medium diameter of the parent material is
2.0 ym (equivalent spherical diameter).  By comparison, the ground material
has 90% by mass below 1.0 yms and 80% by mass is less than 0.3 ym.  The mass
median diameter of the ground material is probably below 0.1 ym.
     Surface area measurements for the ground Al_0_ were run by two methods  
the Mieromeritics Model 2200 and Quantachrome "Monosorb" device.  The results
were:
                                               2
          by Micromeritics Model 2200  92.1 m /g
                                               2
          by Quantichrome "Monosorb"   77.3 m /g
The two values agree reasonably well.  The former value is probably more
nearly correct, since the latter value was obtained during an apparatus
demonstration.
     A pore volume analysis was obtained by mercury porosimeter.  The total
pore volume was found to be 1.12 cc/g.  One half of this volume was attributed
to pores smaller than 0,13 ym.
     The trace chemical analysis on the parent and ground Al 0  was done by
arc-induced emission spectroscopy.   The results are shown in Table A.  It is
seen that the grinding process did not alter the concentrations of the
catalyst materials, palladium, and platinum.  However, there was an enhancement
of the silicon, magnesium, and titanium content, probably due to erosion of
the mill parts.
     As a further chemical test of the parent and ground powders, x-ray
emission spectra were obtained with a scanning electron microscope.  In this
test, the analyst could not detect platinum or palladium on either the parent
or ground Al_0.  Apparently, the catalyst materials are embedded in pores
not accessible to the electron beam used to exite the x-rays.  The analyst did

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o>
N
TJ
OJ
    99


    98



    95



    90
fl  80
g   70
(U
fj
H
W
(U

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TABLE 4.  TRACE CHEMICAL ANALYSIS OF PARENT AND
            GROUND CATALYST POWDER*

Weight Percent of Total
Element
Si
Mg
Ca
Cu
Ti
Sr
Fe
Pt
Pd
Parent
0.1
0.1
0.02
0.002
0.002
0.001
0.1
0.01
0.02
Ground
0.4
0.2
0.02
0.002
0.01
0.001
0.1
0.01
0.02

* Arc-induced emission spectroscopy.
                      11

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find traces of calcium in the parent material and traces of silicon in the
ground material.
     The arc-induced emission spectrograph yields a chemical analysis by
elements which is only semi-quantitative.  More accurate analysis for specific
metals can be obtained by atomic absorption spectrophotometry, as was done for
the parent powder by R.G. Miller of EPA's Environmental Toxicology Laboratory
in Cincinnati.  His results were:  Pt, 0.042% by weight; Pd, 0.016% by weight.
PREPARATIONS FOR IITRI LIFE SCIENCES DIVISION, SERIES II
     Another series of biological experiments conducted by IITRI's Life
Sciences Division under EPA contract required preparations of fractionated fly
ash material both uncoated and coated with a selected metal oxide.  The effect
of particle size as well as the effect of the particle's metal oxide coating
on phagocytosis of fly ash particles by rabbit lung cells were being investi-
gated.
     The size ranges of fly ash required were:  0-2 ym, 2-5 urn, and 5-10 ym.
The Required coating materials were the oxides of nickel, manganese, lead,
cadmium, and vanadium.  A stock of fly ash material was supplied by Dr. Donald
Gardner at EPA/RTP.
     Size classification of fly ash is not straightforward due to the hetero-
geneous nature of the material.  The particles range from glassy silicate
spheres, some of which are hollow, to hematite particles.  Accordingly, the
density varies considerably from particle to particle.  Some particles have
a specific gravity less than unity.  Air classification using a Bahco
Microparticle Classifier (Harry Dietert Co., 9220 Roselawn Ave., Detroit,
Michigan) was adopted.  This technique is sanctioned by the American Society
of Mechanical Engineers in the ASME Powder Test Code No. 28 as the recommended
method for determining the size distribution of fly ash (1).  The results are
shown in Table 5.
     The Bahco Microparticle Classifier has size cut points which closely
match those desired for the fly ash fractions.  Furthermore, the method is an
accepted standard for fly ash fractionation.  For these reasons, the fly ash
fractions delivered to IITRI Life Sciences were obtained using the Bahco.
The size ranges were:

                                      12

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        TABLE 5.  SIZE DISTRIBUTION OF PARENT FLY ASH
        Size Range*, yim

             0-2.2
             2.2-5.0
             5.0-8.0
             over 8.0
Percent of Total Mass
	in Size Range	

          7.0
         35.0
         14.5
         43.3
TABLE 6.   WEIGHT PERCENT CONCENTRATION OF SELECTED ELEMENTS
                IN VARIOUS FLY ASH SAMPLES

Element
Fe
Si
Mg
Al
V
Ca
Cu
Ti
Ni
Mn
Sr
Cr
E.a
Present
Fly Ash,
Emission
Spectroscopy
10.
10.
1.
2.
0.5
1.
1.
1.
0.01
0.05
0.1
0.05
0.05
NBS Fly Ash Standard,
SRM #1633
ORNL NBS
6.37

1.98
12.5
0.024 0.0214
4.34
0.0133 0.0120
0.642
0.0109 0.0098
0.046 0.0493
0.1201 0.1380
0.0138 0.0130
0.278
Ondov

21
1.8
12.7
0.0235
4.7

0.740
0.0098
0.0486
0.1700
0.0127
0.270
Lee, et al
(3)
15.4



0.017



0.015


0.008


 *  Result of Oak Ridge National Laboratory, National of
    Standards and Ondov, et al., Analyses, reported in
    Reference 4.
                               13

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          Fine fraction    0-2.2 ym
          Intermediate     2.2-5.0 ym
          Coarse fraction  5.0-8.0 ym
     It is well to point out that the particle classification obtained by the
Bahco are according to aerodynamic equivalent diameter.  These differ somewhat
from the geometric diameters as determined by microscope.  A large, hollow
particle, for example, behaves aerodynamically like a smaller, solid particle,
and is so classified by the Bahco.  Hence, photomicrographs may show a number
of large, hollow particles in the fine size fraction.
     A sample of the parent fly ash was screened by arc-induced emission
spectroscopy to determine the elemental composition.  The results are shown
in Table 6.  These results are semi-quantitative only, and were done mainly
to provide a base line against which to compare the coated materials.
     Also shown in Table 6 are results of elemental analysis of various fly
ash samples reported in the recent literature (2,3,4,5,6).  The National
Bureau of Standards fly ash standard (Standard Reference Material No. 1633) is
particularly well studied.  These results are shown for comparison only, and
they indicate that substantial differences in composition can exist between
different lots of fly ash.
     References 2 and 3 show that the elemental composition of fly ash varies
with particle size and with point of collection (before or after the emission
control device).   Therefore, different size fractions of fly ash will have
different elemental composition.  This should be kept in mind when inter-
preting the results of biological experiments involving fractionated fly ash.
     The technique devised for coating fly ash with oxides of nickel, lead,
or manganese was patterned after that described by Pupynin, et al. (7).  In
brief, the technique was as follows:  dissolve 0.3 g of the chloride salt of
the desired metal in 200 ml of water; chill the solution to 3C; add 3 g of
the desired fly ash fraction while stirring continuously; add the equivalent
weight of sodium hydroxide, one drop at a time, to precipitate the metal as
fine metal hydroxide; filter the suspension using a Buchner funnel; wash to
remove soluble sodium salts, dry the filter cake in a muffle furnace at 600C
                                     14

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to cause conversion to metal oxide.  The procedure is outlined more completely
in Appendix A.
     Microscope examination of the dried material after crushing the filter
cake repealed a degree of coating which varied with the metal.  Lead oxide
produced the best coating of the four tried.  The lead oxide coatings were of
a quality similar to our benzo(a)pyrene-coated particles.   Vanadium oxide
coating of fly ash was not possible.  The solubility of the oxide prevented
the preparation of a suitable product for biological use.   All attempts to
prepare a precipitate free of soluble vanadium compounds failed.
     An assay of the metal content of the final coated samples delivered to
IITRI Life Sciences was obtained by atomic absorption spectrophotometry.
Approximately 10 mg of each coated fly ash was Weighed accurately and digested
in concentrated nitric plus hydrochloric acid.  These samples were then
brought to 10 ml total volume by adding deionized water.  Similarly, approxi-
mately 25 mg of each uncoated fly ash fraction was weighed, digested, and
brought to 25 ml total volume.  Standard solutions were also prepared for
each metal, and all standards and samples were analyzed by direct aspiration
atomic absorption spectrophotometry.  The results are shown in Table 7.
     During the IITRI Life Sciences experiments with the fly ash fractions, a
question came up concerning the number of particles per unit mass for each of
the fly ash samples.  Therefore, a technique based on microscopy was devised
to obtain this data.  This technique is described in detail in Appendix B.
     The results of these counts are shown in Table 8.  It is noted that in
the small size fraction, the application of the coatings substantially de-
creased the number of particles per unit mass.  With one exception, this de-
crease did not occur for the two larger size fractions.  The microscopic
observations indicated that the decreased number in the coated samples for the
small size fraction was due to aggregates of up to about six fly ash particles.
The uncoated material was well dispersed as single particles.  The particle
size range for the small size fraction, as determined by microscope, was
0.5 to 4.0 ym.
                                     15

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TABLE 7.   ATOMIC ABSORPTION ANALYSIS OF FLY ASH FRACTIONS

Size
Fraction
0.0 - 2.2 ym



2.2 - 5.0 ym



5.0 - 8.0 ym



Element
Pb
Ni
Mn
Cd
Pb
Ni
Mn
Cd
Pb
Ni
Mn
Cd
Concentration of Metal in Weight Percent
Lead Nickel- Manganese-
Uncoated Coated Coated Coated
Fly Ash Fly Ash Fly Ash Fly Ash
<.03 3.85
<.03  2.63
<.01 --  3.22
<.02
<.03 3.84
<.03  2.91
<.01   3.67
<.02
<.03 3.15
<.03  2.77
<.01   2.91
<.02

Cadmium
Coated
Fly Ash


: . 
12.05



11.91



12.30

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        TA-DT r* O
	NUMBER OF FLY ASH PARTICLES PER UNIT MASS  OF FLY ASH	


   Nominal     	Number of Particles per mg  of  Fly  Ash	
    Size       Uncoated    PbO-Coated   NiO-Coated    MnOa-Coated   CdO-Coated
  Fraction     Fly Ash      Fly Ash       Fly Ash      Fly Ash       Fly ASh

0.0 - 2.2 ym   2.3 x 108   1.05 x 108   3.8 x 10?     5.8 x 10?     3.2 x 107

2.2 - 5.0 ym   2.3 x 10?   4.6 x 10?    2.2 x 10?     2.6 x 10?     2.8 x 106

5.0V- 8.0 ym   1.6 x 107   1.2 x 107    4.4 x 106     1.7 x 107     1.3 x 104

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PREPARATIONS FOR SUNY-STONY BROOK, SERIES III
     A second series of preparations for biological experiments was initiated
for Dr. Philip Kane at SUNY-Stony Brook.  A modified milling procedure was
initiated to overcome some of the earlier uncertainties in preparatory tech-
nique.  The essential change in technique for the BP/metal oxide suspensions
was the addition of fractionated metal oxide particles with all other ingredi-
ents except the BP for sterilization in the autoclave prior to the "ball-
milling" step.  The intended benefits in the change of procedure were:
     1.   To minimize contamination.
     2.   To obtain a more uniform BP/metal oxide homogeneity and particle
          distribution.
     To maintain continuity and a basis for comparison, all ingredients were
from the original stock in the Series I preparations.  The BP and platinum
oxide (Pt02) were provided by Dr. Kane.  All glassware, equipment to be in
contact with the suspension, the required quantity of metal oxide, gelatin,
and saline solution were sterilized in the autoclave.  The BP was then added
to the autoclaved suspension prior to milling.  Approximately 100 ml of
milled suspension were required per preparation.  Milling time was seven days.
The milled suspension was then divided into four vials of approximately
20-23 ml of suspension per vial, the balance being retained for particle size
analysis, BP assay, and sterility.
     Data on these preparations are presented in Table 9.  Table 10 presents
results of the spectrochemical analysis for the contaminants present in the
metal oxides used.
     Initially the four vials (80-90 ml) were sealed, taped, and packed with
canned ice in an insulated shipping container for shipment.  Air freight and
air mail special delivery as shipping methods were found to be deficient in
attaining a precise delivery time table.  Express mail service (guaranteed
next day delivery) was found to be the fastest and most reliable shipping
method.  Each vial was individually bagged, then the group bagged into
another plastic container, and the groups bagged into a larger leak-proof
container.  These bags within a bag were then packed snugly in an insulated
shipping container.

                                     18

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TABLE 9.  PREPARATIONS FOR SUNY-STONY BROOK, SERIES III

Date
05-10-76


06-10-71)


07-06-76



07-20-76

07-29-76

08-02-76




08-26-76



09-01-76
09-21-76



Preparation
IA 22 Suspension,
1:1 BP/Fe203
IB 22 Suspension,
1:1 BP/Pt02
1C 22 Suspension,
1:1 BP/PbO
IIA 22 Suspension,
1:1 BP/Fe?03
IIB 22 Suspension,
1:1 BP/Pt02
IIC 22 Suspension,
1:1 BP/PbO
IIIA 22 Suspension,

IIIB 22 Suspension,
1:1 BP/Pt02
IIIC 22 Suspension,
1:1 BP/PbO
IIIA-1 22 Suspension,

IIIA-2 22 Suspension,

IV A 22 Suspension,
1:1 BP/Fe203
IV B 22 Suspension,
1:1 BP/Pt02
IV C 22 Suspension,
1:1 BP/PbO
1:1 BP/Fe203
IHA-4 22 Suspension,
1:1 BP/Fe203
V A 1.52 Suspension,

VB 1.52 Suspension,
1:1 BP/Pt02
V C 1.52 Suspension,
1:1 BP/PbO
V A-l 1.52 Suspension,
1:1 BP/Fe203
VI A 1.52 Suspension,
1:1 BP/I'e203
VI B 1.52 Suspension,
1:1 BP/Pt02
VI C 1.52 Suspension,
1:1 BP/PbO
Particle (Number 2 Less than Stated Size)
0.75P 1.5M 3. 0|i 6. Op 7.3p 12. Op
13.4 24.8 50.2 77.1 91.5 100.0
8.1 23.1 54.3 88.2 95.2 100.0
56.8 74.0 89.9 97.0 99.6 100.0
14.2 25.6 50.3 77.2 91.4 100.0
10.7 24.3 54.9 85.5 93.7 100.0
54.9 72.8 88.1 94.9 97.5 100.0


12.8 36.1 68.4 88.9 96.9 100.0
54.1 73.0 89.2 96.9 98.8 100.0




12.7 26.2 50.1 72.1 86.8 100.0
10.2 26.7 58.0 88.0 95.5 100.0
54.1 72.9 90.7 96.9 99.1 100.0

13.8 25.2 50.2 77.1 91.4 100.0


8.3 30.1 59.4 80.7 90.5 100.0
48.4 70.3 87.0 95.8 98.9 100.0
5.2 21.2 47.9 76.3 91.8 100.0
21.2 28.1 52.7 78.3 91.6 100.0
7.7 16.5 50.0 95.4 100.0
63.1 76.9 92.3 98.1 100.0
Assay ,
B.P.
( Spec tropho tome ter)
2.00
2.10
2.10
2.12
2.12
2.12


2.12
2.12




2.00
2.00
2.00

2.10


1.51
1.50
1.50
1.50
1.50
1.50
2
Metal Oxide
(Gravimetric)
2.03
2.00
2.16
2.23
2.31
2.02


2.00
2.00




2.63
2.00
2.00

2.30


1.54
1.51
1.50
1.50
1.50
1.50

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                                TABLE 10.   TRACE  CHEMICAL ANALYSIS  OF METAL  OXIDES
NJ
O

Fe203
Constituent
Fe
Mn,Zn
Ni,Mg,Ca
Si.Al
Cr,Mo,Co
Cu,Pb
Concentrat ion ,
% (Estimate)
Major
0.05-0.5
0.005-0.5
0.003-0.03
0.001-0.01
0.0005-0.005
PbO
Constituent
Pb
Bi
Si
Ca
Fe
Al,Mg,Cu,Ag
Concentration ,
% (Estimate)
Major
0.01-0.1
0.005-0.05
0.001-0.01
0.0003-0.003
0.0001-0.001
Pt02
Constituent
Pt
Zn
Ni
Cu,Pd,Ca,Fe
Si,Pb,Au,Ag
Ir,Mg,Ru,CL
Concentration ,
% (Estimate)
Major
0.05-0.5
0.03-0.3
0.005-0.05
0.002-0.02
0.001-0.01
                                                                          Al             0.0005-0.005

                                                                          Mn             0.0001-0.001

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                                 SECTION 3
                          RESULTS AND CONCLUSIONS

     Series II in the preparations for SUNY-Stony Brook is still continuing.
Negotiations are in progress for continued funding for this as well as other
preparation and characterization work on particulate contaminants for biologi-
cal experiment.
                                      21

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                                 APPENDIX A

                 PROCEDURE FOR COATING FLY ASH WITH NICKEL,
                         MANGANESE, OR LEAD OXIDES
1.   Use the Bahco air classifier to separate the fly ash into three size
     ranges, each weighing 3 grams:  (0-2.2) ym, (2.2-5.0)  ym, and (5.0-8.0) yra.
2.   Dissolve 0.300 g of a chloride salt in 200 ml HzO from one of the above
     metals for each size fraction to be coated.

3.   Pour in one 3 g size fraction of fly ash and cool mixture to 3C.  Stir
     mixture continuously.

4.   Add equivalent weight of NaOH (drop at a time) to cold stirred mixture
     and continue stirring for one hour.

5.   Filter the metal hydroxide coated fly ash using a Buchner funnel.

6.   Dry the coated material in a muffle furnace at 657 C.  This temperature
     will convert most of the hydroxide to the oxide.

7.   Assay the dried sample to determine amount of metal (Ni, Mn, Cd, or Pb)
     present .
                                     22

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                                 APPENDIX B
              NUMBER OF PARTICLES PER UNIT MASS FOR THE COATED
                       AND UNCOATED FLY ASH FRACTIONS
     The technique for determining the number of particles per unit mass of
fly ash was as follows:  a 3 to 5 mg amount of the fly ash sample was weighed
to the nearest 0.1 mg and suspended in 20 ml of growth medium 199.  This was
shaken vigorously and treated for 15 seconds in an ultrasonic bath to disperse
the fly ash.  The suspending medium, the concentration, and the dispersing
technique was selected to resemble those used in the IITRI Life Sciences
Division experiments.
     Next, a second suspension was made up consisting of paper mulberry pollen
suspended in distilled water at a concentration of about 1.0 rag/ml.  After
vigorous shaking, a drop of the pollen suspension was mounted on a hemacyto-
meter, and the number of particles per ml of suspension was determined.  The
large (16 ym diameter) uniformly sized pollen particles served as an internal
standard in the later counting of fly ash particles.
     A third suspension was then prepared by mixing equal volumes of the fly
ash suspension and the paper mulberry pollen suspension.  A drop of this mixed
suspension was placed on a microscope slide and covered with a cover slip.
After an initial inspection at low magnification to insure that the particles
were dispersed and that the sample was uniformly spread on the slide, counts
were made of both the fly ash and the pollen particles.  The fly ash particles
were counted in several view-fields at 500X, and the pollen particles were
counted in several view-fields at 125X.  The 500X magnification used for fly
ash made it possible to count particles as small as 0.5 ym.
     From the above data, the number, N, of fly ash particles per mg of fly
ash was determined from the equation
                                     23

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                                   nr     s
                                   fa
                               N = 	 <

                                   n     m,.
                                    p     fa
where
     nf  = the number of fly ash particles per unit view-area (from the count

           on the mixed suspension).



      n  = same as above for the pollen particles.



      s  = the number of pollen particles per ml of suspension 2 (from the

       "   hemacytometer count).



     m,.  = mg of fly ash per ml of suspension 1.



(A factor was applied to the number of fly ash and pollen particles counted to


place these numbers on an equal view-area basis.)
                                     24

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                                 REFERENCES
1.    ASME Power Test Code No.  28,  "Determining the Properties  of Fine
     Participate Matter", American Society of Mechanical Engineers,  345  East
     47t:h Street, New York, NY 10017,  1965.

2.    Davison, R.L., Natusch, D.F.S., and Wallace,  J.R.,  "Trace Elements  in Fly
     Ash - Dependence of Concentration on Particle Size", Environ.  Sci.  Tech.,
     81(13) :1107-1113, 1974.

3.    Lee, R.E., Jr., Crist, H.L.,  Riley, A.E., and MacLeod,  K.E., "Concentration
     and Size of Trace Metal Emissions from a Power Plant, a Steel Plant,  and  a
     Cotton Gin", Environ. Sci. Tech., 9^(7)-.643-647, 1974.

4.    Klein, D.H., Andren, A.W., Carter, J.A., Emery, J.F., Feldman,  C.,
     Fulkerson, W., Lyon, W.S., Ogle,  J.C.,  Talmi, Y., Van Hook, R.I.,  and
     Bolton, N., "Pathways of Thirty-Seven Trace Elements Through Coal-Fired
     Power Plant", Environ. Sci. Tech., (10)-.973-979, 1975.

5.    National Bureau of Standards Analysis of NBS fly ash standard,  SRM No. 1633,
     cited in Ref. 4.

6.    Ondov, J.M., Zoller, W.H., Olmez, I., Aeas, N.K., Gordon, G.E., Rancitelli,
     L.A., Abel, K.H., Filby, R.H., Shah, K., and Ragaini, R.C., "Elemental
     Concentrations in the National Bureau of Standards Environmental Coal and
     Fly Ash Standard Reference Materials",  Anal.  Chem., 47^1102, 1975 (results
     cited in Ref. 4).

7.    Pupynin, V.P., Putintsev, V.N., Dragunkina, N.I., Chechanovskii, A.M., and
     Panin, V.V., "Coatings on Ultrasonically Dispersed Particles", See Chemical
     Abstracts, Vol. 74, No. 24, Abstract No. 1295832, June 14, 1971.
                                      25

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
 1. REPORT NO.
  EPA-600/1-78-005
                              2.
                                                           3. RECIPIENT'S ACCESSION-NO.
 4. TITLE AND SUBTITLE
  PREPARATION AND CHARACTERIZATION OF PARTICULATE
  ENVIRONMENTAL  CONTAMINANTS FOR BIOLOGICAL EXPERIMENTS
                                                           5. REPORT DATE
              6. PERFORMING ORGANIZATION CODE
 7. AUTHOR(S)

  George Yamate and  Hubert Ashley
                                                           8. PERFORMING ORGANIZATION REPORT NO.
                  January 1978
9. PERFORMING ORGANIZATION NAME AND ADDRESS

  IIT Research  Institute
  Chicago, IL 60616
              10. PROGRAM ELEMENT NO.

                  EHE625	
              11. CONTRACT/GRANT NO.

                  68-02-1687
 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
RTP.NC
              14. SPONSORING AGENCY CODE

                 EPA 600/11
 15. SUPPLEMENTARY NOTES
 16. ABSTRACT
       The purpose  of  this project was to provide  a  centralized source for the
  preparation and characterization of selected particulate materials for biological
  experiments.  The particulate materials of  interest  were a range of environmental
  contaminants known or suspected to detrimentally affect human health.  The  prepared
  materials were delivered to personnel at the Environmental Protection Agency  and  to
  designated EPA contractors.
       The centralized source approach helped to assure that the results of biological
  experiments conducted at different laboratories  were comparable.  In addition,
  the centralized source approach placed specialists in particle technology at  the
  disposal of biological researchers.  Thus,  more  intricate particle preparation
  procedures could  be  undertaken than would otherwise  be the case.
       Source powders  were collected, size classified, and characterized for  use.
  Preparations were shipped to (1). State University  of New York at Stony Brook  for  two
  long-term series  of  experiments, (2) life sciences Division of IIT Research Institute
  for two series of experiments, and (3) EPA/Cincinnati.
 7.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS  C.  COSATI Field/Group
  chemical analysis
  powder  (particles)
  particles
  air pollution
  fly ash
                             07 C
                             06 L
 8. DISTRIBUTION STATEMENT

  RELEASE TO PUBLIC
 19. SECURITY CLASS (This Report)
  UNCLASSIFIED
21. NO. OF PAGES

       31
                                              20. SECURITY CLASS (This page)
                                               UNCLASSIFIED
                                                                         22. PRICE
EPA Form 2220-1.(19-73). ,
                                            26

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