EPA-600/2-77-178
Revised June 1978
Environmental Protection Technology Series
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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 five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY series. This series describes research performed to develop and
demonstrate instrumentation, equipment, and methodology to repair or prevent
environmental degradation from point and non-point sources of pollution. This
work provides the new or improved technology required for the control and
treatment of pollution sources to meet environmental quality standards.
EPA RE VIEW NOTICE
This report has been reviewed by the U.S. Environmental
Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policy of the Agency, nor does mention of trade
names or commercial products constitute endorsement or
recommendation for use.
This document is available to the public through the National Technical Informa-
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL SCIENCES RESEARCH LABORATORY
RESEARCH TRIANGLE PARK
NORTH CAROLINA 27711
NOTICE OF REVISED PROVISIONAL METHODOLOGY MANUAL
The enclosed copy of the EPA publication "Electron Microscope Measurement
of Airborne Asbestos Concentrations - A Provisional Methodology Manual"
represents a revised edition of document EPA-600/2-77-178 which was first
published in August 1977.
The changes are mostly minor and have been made primarily for the sake of
clarity. The most important changes are in the section (2.5.2.4) on
Fiber Classification Rules, wherein the procedure is described in more
explicit terms and should be easier to follow.
Please discard any copies you may have of the earlier edition.
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EPA-600/2-77-178
Revised June 1978
ELECTRON MICROSCOPE MEASUREMENT
OF AIRBORNE ASBESTOS CONCENTRATIONS
A Provisional Methodology Manual
by
Anant V. Samudra
Colin F. Harwood
John D. Stockham
IIT Research Institute
Chicago, Illinois 60616
Contract No. 68-02-2251
Project Officer
Jack Wagtnan
Director, Emissions Measurement and Characterization Division
Environmental Sciences Research Laboratory
Research Traingle Park, North Carolina 27711
ENVIRONMENTAL SCIENCES RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This manual has been reviewed by the Environmental Sciences Research
Laboratory, U. S. Environmental Protection Agency, and approved for publi-
cation. 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.
11
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FOREWARD
Asbestos or asbestiform minerals include several types or groups of
fibrous crystalline substances with special thermal and electrical
properties that have long encouraged their use in the manufacture of
such products as roofing, insulation, brake linings, fireproof curtains,
etc. Their occurrence as pollutants in the ambient air and in supplies
of food and drinking water has caused considerable concern because
occupational exposures to asbestos have been found to induce mesothelioma
of the pleura and peritoneum, as well as cancer of the lung, esophagus,
and stomach, after latent periods of about 20 to 40 years.
Electron microscopy is currently the principal technique used to
identify and characterize asbestos fibers in ambient air and water
samples. Because of the poor sensitivity and specificity of conven-
tional bulk analytical methods, electron microscopy is also being used
for routine measurement of airborne or waterborne asbestos concentrations.
The several laboratories that perform such analyses generally have
reasonable internal self consistency. However, interlaboratory compari-
sons have shown that the results obtained by the separate laboratories
are often widely different.
This manual describes a provisional optimum electron microscope
procedure for measuring the concentration of asbestos in air samples.
It results from a study, carried out under EPA Contract No. 68-02-2251,
to evaluate the various methods currently in use in the various labora-
tories. Statistical analysis was used to evaluate the effects of the
many interacting sub-procedures and arrive at an optimum composite
procedure.
This manual does not provide the vast amount of data that supports
the provisional methodology. These data are included in the final
report on EPA Contract No. 68-02-2251.
Jack Wagman
Project Officer
111
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ABSTRACT
This manual describes a provisional optimum electron
microscope (EM) procedure for measuring the concentration of
asbestos* fibers** in air samples. The main features of the
method include depositing an air sample on a polycarbonate
membrane filter, examining an EM grid specimen in a transmis-
sion electron microscope (TEM), and verifying fiber identity by
selected area electron diffraction (SAED).
This provisional manual results from a study to develop an
optimum EM procedure for airborne asbestos determination. The
analytical data supporting the provisional methodology are
included in a separate final report.
* Asbestos is used as a collective term for the six minerals:
chrysotile, amosite, crocidolite, and the asbestiform
varieties of anthophyllite, actinolite and tremolite.
** The term fiber is used for a particle with an aspect ratio
3:1 or greater and with substantially parallel sides.
IV
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TABLE OF CONTENTS
1. PROVISIONAL METHODOLOGY - SUMMARY 1
2. METHODOLOGY 2
2.1 Air Sampling 3
2.1.1 Air Sampling Parameters 3
2.1.2 Sample Time Periods 5
2.2 Sample Storage and Transport 6
2.3 Carbon Coating the Filter 6
2.4 Transfer of the Sample to the EM Grid 9
2.5 Examination of the Grid by Transmission
Electron Microscopy 11
2.5.1 Low Magnification 11
2.5.2 High Magnification 12
2.5.2.1 Calibration Magnification at
Fluorescent Screens 12
2.5.2.2 Loading Levels 12
2.5.2.3 Fiber Counting Rules 12
2.5.2.4 Fiber Classification Rule .... 14
2.5.2.5 Counting at Low Loading Level . . 16
2.5.2.6 Counting at Medium Loading Level . 16
2.5.2.7 Counting at High Loading Level . . 17
2.6 Recording of Data 17
2.6.1 Recording Formal 17
2.6.2 Computer Coding Forms 19
2.7 EM Data Analysis 19
2.7.1 Checking Data on Key Punch Cards 19
2.7.2 Separating Very Large Sized Bundles .... 20
2.7.3 Fortran Program for Obtaining
Characterizing Parameters 20
2.7.4 Printout of Results on each TEM Grid ... 20
2.7.5 Summary of Results for a Typical Air Sample 20
2.7.6 Precision of TEM Estimates 21
2.7.7 Analyzing Data on Very Large Bundles
of Fibers 21
v
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TABLE OF CONTENTS (continued)
2.8 Ashing, Bonification, and Reconstitution 22
2.9 Limits of Detection 23
3. PREPARATION OF BLANKS 24
References 27
Appendix A - Instrumentation and Supplies 28
Appendix B - Magnification Calibration 34
Appendix C - Listing for the Fortran Program CONLAB 37
Appendix D - Illustrative Tables 41
vi
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LIST OF TABLES AND FIGURES
Table
1 Suggested Sampling Times for Determining
Airborne Asbestos Concentrations 5
2 Data Recording Sheet 17
3 Minimum Detection Limit Using High-Volume Air
Sampler 23
Figures
1 Modified Jaffe Washer Method 9
2 Two Methods of Examining a Grid 12
Vll
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ELECTRON MICROSCOPE MEASUREMENT
OF AIRBORNE ASBESTOS CONCENTRATIONS
A Provisional Methodology Manual
1. PROVISIONAL METHODOLOGY - SUMMARY
(1) Take an air sample on a polycarbonate membrane
filter, 0.4 ym, using a high-volume or personal sampler.
(2) Coat the filter with a 40 nm thick film of carbon
using a vacuum evaporator.
(3) Transfer the deposit from the polycarbonate filter
to an electron microscope grid using a modified Jaffe washer.
The Jaffe washer is prepared as follows. A 60 or 100 mesh
stainless steel mesh is placed on top of a paper filter
stack or foam sponge contained in a petri dish. Chloroform
is carefully poured into the petri dish until the level is
just touching the stainless steel mesh. A 1 mm x 2 mm portion
of carbon coated polycarbonate filter is placed particle side
down on a 200 mesh carbon coated copper electron microscope
(EM) grid and this pair is placed on the steel mesh. The
1 mm x 2 mm portion is wetted with a 5 y j, drop of chloroform.
The polycarbonate filter will dissolve in about 24 to 48 hours.
(4) Examine the EM grid under low magnification in the
TEM to determine its suitability for high-magnification exam-
ination. Ascertain that the loading is suitable and is uni-
form, that a high number of grid openings have their carbon
film intact, and that the sample is not contaminated.
(5) Systematically scan the EM grid at a magnification
of about 20,OOOX (screen magnification 16.000X) for chrysotile
and possibly a lower magnification for cases where predominant
amphibole fibers are present. Record the length and breadth
of all fibers that have an aspect ratio of greater than 3:1
and have substantially parallel sides. Observe the morphology
of each fiber through the 10X binocular and note whether a
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tubular structure characteristic of chrysotile asbestos is
present. Switch into SAED mode and observe the diffraction
pattern. Note whether the pattern is typical of chrysotile
or amphibole, or whether it is ambiguous or neither chryso-
tile nor amphibole.
(6) Count 100 fibers in several grid openings, or al-
ternatively, count all fibers in at least 10 grid openings.
If more than 300 fibers are observed in one grid opening,
then a more lightly loaded filter sample should be used. If
no other filter sample can be obtained, the available sample
should be transferred onto a 400 mesh grid. Processing of
the sample using ashing and sonification techniques should
be avoided wherever possible.
(7) Fiber number concentration is calculated from the
following equations
Fibers/m3 = Total No. of Fibers
No. of EM Fields
2
Total Effective Filter Area, cm
Area of an EM Field, cm
Volume of Air Sampled, m
Fiber mass for each type of asbestos in the sample is calcul-
ated by assuming that the breadth measurement is a diameter;
thus, the mass can be calculated from
r\
Mass (yg) = - • (length, ym) • (diameter, ym)
4
o _ f-
• (density, g/cm ) • 10
3
The density of chrysotile is assumed to be 2.6 g/cm , and of
o
amphibole 3.0 g/cm . The mass concentration for each type of
asbestos is then calculated from
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Total Mass of all
. Fibers of that Type W
Particular Type Volume of Air Sampled (m^)
(8) Other characterizing parameters of the asbestos
fibers are:
(a) Length and width distributions of chrysotile fibers
(b) Volume distribution of chrysotile fibers
(c) Fiber concentration of other asbestos minerals
(d) Relative proportion of chrysotile fibers with
respect to total number of fibers.
2. METHODOLOGY
2.1 Air Sampling
Collect the sample of airborne asbestos on 0.4 urn pore
size polycarbonate filters using the shiny smooth side as the
particle capture surface. In cases where polycarbonate filters
cannot be used, the air sample may be collected on a high
efficiency membrane filter, e.g. cellulose acetate, which can
then be prepared by using an ashing procedure described in
Section 2.8. Use the high-volume air sampler [1]"', or in
certain instances, the personal dust sampler.[2] When 110V,
60 cycle power supply is available, a variety of other combina-
tions of pumps and filters and other techniques can be used
for air sample collection.[3-5]
2.1.1 Air Sampling Parameters
Sampling rates vary with the type and model of sampler and
with the type and pore size of filter used to collect an air
sample. Typically, a high-volume air sampler fitted with a 20
cm x 25 cm, 0.4 pm pore size, polycarbonate filter will
have a flow rate of about 700 f/min (25 cfm) at a pressure
drop of 145 cm of water across the filter. By comparison, a
* Numbers in brackets denote the literature references.
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personal dust sampler, operated with a 37 mm diameter, 0.4 ym
pore size, polycarbonate filter, is set, by a flow controller,
to sample at a flow rate of 2 £/min. The pressure drop across
the filter is 20.9 cm of water.
The two types of samplers can be compared by dividing
the volumetric flow rate by the effective filtration area of
the filters. The high-volume sampler, with an effective fil-
2 32
tration area of 406.5 cm , operates at a rate of 28.7 cm /cm /sec
while the personal dust sampler, with an effective filtration
+ 2 32
area of 6.7 cm , operates at a rate of 5.0 cm /cm /sec. Thus,
the filtering rate of the high-volume sampler is about five
times higher than that of the personal sampler. Some research
investigators contend that the higher face velocity of the
high-volume sampler results in a lower fiber retention efficiency,
These investigators expect the fibers to align perpendicular
to the collection filter, and hence, better able to penetrate
through the pores in the filter. They recommend collecting
air samples at as low a face velocity as feasible and propor-
tionately extending the sampling time. The optimization study,
upon which this provisional methodology is based, tends to
support the contention but the reason remains obscure.
Personal dust samplers are used frequently to assess re-
spirable dust levels. When used in this mode, they are pre-
ceeded with a nylon cyclone that collects fibers and other
particles with aerodynamic diameters in excess of 10 pm. To
be comparable with the results of the high-volume sampler, it
is recommended that the personal sampler be operated without
the cyclone.
It is recommended that a cellulose acetate membrane fil-
ter with a pore size of 5 pm be used to support the polycar-
bonate filter in the samplers. It should be placed between
f The effective filtration area varies with the style or
manufacturer and hence should be measured.
4
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the polycarbonate filter and the wire mesh filter support of
the high-volume sampler, or the glass frit filter support of
the personal sampler. The cellulose acetate membrane acts
as a diffusion plate and aids in obtaining a uniform deposit
on the polycarbonate filter. It also decreases the possibility
of contaminating the filter with particles from the sampler
frame.
It is recommended that flow rate meters be checked per-
iodically during sampling to ascertain constancy of flow rate.
2.1.2 Sample Time Periods
As a guide, the following time periods are suggested for
the sampling of airborne asbestos. It is recommended that
samples be collected at all three of the suggested time periods
until experience dictates otherwise. Sampling at the three
time periods increases the probability that one of the samples
will be suitably loaded with asbestos to permit quantification
of the asbestos by the direct transfer technique.
Table 1
SUGGESTED SAMPLING TIMES FOR DETERMINING
AIRBORNE ASBESTOS CONCENTRATIONS
Proximity
to Source
Point Source
90 m
Near Source
90-180 m
Distant Source
0.8-1.6 km
Sampler
Type
High-volume
Personal
High-volume
Personal
High-volume
Personal
Suggested Sampling Times, min
15, 30, 60
75, 150, 300
30, 120, 480
150, 600
240, 480, 1440
not recommended
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2.2 Sample Storage and Transport
After acquiring the sample, every precaution must be
taken to assure its integrity and prevent contamination and
loss of fibers until the sample is examined under the elec-
tron microscope. The polycarbonate filter should be removed
immediately from the filter holder with great care and
tacked, with cellophane tape, to the bottom of a clean plas-
tic petri dish. The dish cover should then be secured and
all necessary sample identifying marks and symbols applied
to the cover. With the 20 cm x 25 cm high-volume filters,
it may be necessary to cut the filter into 5 cm x 5 cm seg-
ments and store each segment in separate petri dishes. A
consistent notation must be used so that the location and
orientation of each segment with respect to the original
filter is not lost. It is recommended that the petri dishes
containing the filters be maintained in a horizontal position
at all times during storage and transportation to the analyzing
laboratory. At the present time, there are no reliable
estimates on the loss of fibers from polycarbonate filters
prior to carbon coating the filters in the laboratory.
If other membrane filters, such as cellulose acetate,
are used for collecting airborne particles, it is generally
believed that the storage and transport do not affect these
filters because of their high retention efficiency. However,
there are no reliable data for comparing retention efficiency
of polycarbonate filters and other membrane filters.
Suitable blank and standard filters should be introduced
at this stage in the analytical process and carried through
the remaining procedures along with the samples.
2.3 Carbon Coating the Filter
The polycarbonate filter with the sample deposit and
suitable blanks and standards should be coated with carbon
as soon after sampling is completed as possible. The carbon
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coating forms an almost continuous film over the filter and
bonds the collected particles to the filter surface. Losses
are thus reduced during subsequent handling of the filter,
and during the transfer process to the electron microscope
grid. A carbon film of about 40 nm thickness is most suit-
able. All experimental equipment and supplies are listed in
Appendix A.
It is highly recommended that the handling and processing
of the filters after their receipt by the analyzing laboratory
be conducted in a clean room or clean bench to reduce the
possibility of contamination. Tweezers should be used for
handling the filters; static charge eliminators will facili-
tate handling of the polycarbonate filters by neutralizing
the surface electrostatic charge.
Because a thin, uniform, carbon film is desired, the
coating of the filter deposit with carbon should be carried
out in a vacuum evaporator. Carbon sputtering devices should
be avoided because they produce a film of uneven thickness.
Too thick a film can lead to problems during the subsequent
steps in the procedure, particularly filter dissolution,
fiber sizing, and fiber identification. Electron diffraction
patterns tend to be faint when operating the TEM at less
than 100 KV.
Typically, vacuum evaporators accept samples as large
as 10 cm in diameter. Thus, if the personal sampler was used
for sample collection, the entire filter may be carbon coated
at one time. It is convenient to use the petri dish in which
the polycarbonate filter is being stored. After inspecting
the filter to be sure it is securely tacked to the bottom of
the petri dish, remove the cover and place the bottom of the
dish containing the filter in the vacuum evaporator for coating.
If the airborne asbestos was collected on the 20 cm x 25 cm
polycarbonate filter using the high-volume sampler, the entire
filter cannot be coated at once. Portions, about 2.5 cm x 2.5 cm,
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should be cut from the central region of the filter using
scissors or scalpel. The portions should be tacked with
cellophane tape to a clean glass microscope slide and placed
in the vacuum evaporator for coating.
Any high-vacuum, carbon evaporator may be used to carbon
coat the filters (caution again: carbon sputtering devices
should not be used). Typically, the electrodes are adjusted
to a height of 8-10 cm from the level of the turn-table upon
which the filters are placed. A spectrographically pure car-
bon electrode sharpened to a 0.1 cm neck is used as the
evaporating electrode. The sharpened electrode is placed in
its spring-loaded holder so that the neck rests against the
flat surface of a second graphite electrode. The samples, in
either a petri dish bottom or on a glass slide, are attached
to the turn-table with double-sided cellophane tape.
The manufacturer's instructions should be followed to
obtain a vacuum of about 1 x 10" torr in the bell jar of
the evaporator. With the turn-table in motion, the carbon
neck is evaporated by increasing the electrode current to
about 15 amperes in 10 seconds, followed by 25-30 seconds at
20-25 amperes. If the turn-table is not used during carbon
evaporation, the particulate matter is not coated from all
sides and there is a shadowing effect which is not desirable.
The evaporation should proceed in a series of short bursts
until the neck of the electrode is consumed. Continuous
prolonged evaporation is not recommended since overheating
and consequent polymerization of the polycarbonate filter
may easily occur and impede the subsequent step of dissolving
the filter. The evaporation process may be observed by
viewing the arc through welders goggles. (CAUTION: never
look at the arc without appropriate eye protection.) A rough
3
calculation shows that a graphite neck of 5 mm volume,
when evaporated over a spherical surface of 10 cm radius,
will yield a carbon layer 40 nm thick.
8
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After carbon coating, the vacuum chamber is slowly returned
to atmospheric pressure, the filters are removed and placed in
clean, marked petri dishes, and stored in a clean bench.
2.4 Transfer of the Sample to the EM Grid
The transfer of the collected airborne asbestos from the
coated polycarbonate filter to an electron microscope grid is
accomplished in a clean room or bench using a Jaffe washer
technique [6] with some modification.
Transfer is made in a clean glass petri dish about 10 cm
diameter and 1.5 cm high. A stack of 40 clean, 5% cm diameter
paper filter circles is placed in the dish; alternatively, a 3
cm x 3 cm x 0.6 cm piece of polyurethane foam (like those used as
packing in Polaroid film boxes) may be used. Spectroscopic grade
chloroform is poured into the petri dish until it is level with
the top surface of the paper filter stack or the foam. On top of
the stack or foam a piece of (about 0.6 cm x 0.6 cm) 60-mesh
stainless steel screen is placed. Several transfers may be
completed at one time and a separate piece of mesh is used for
each grid. Details of the modified Jaffe washer and the washing
process are illustrated in Figure 1.
Sections of the carbon-coated polycarbonate filter on
which the sample is deposited are obtained either by using a
punch to punch out 2.3 mm discs or sharp scissors to cut
out approximately 1 mm x 2 mm rectangles. A section is laid
carbon side down on a 200-mesh carbon-coated electron micro-
scope (TEM) grid. (Alternatively, one may use formvar-coated
grids or uncoated TEM grids. Here, the carbon coat on the
polycarbonate filter forms the grid substrate.) Minor overlap
or underlap of the grid by the filter section can be tolerated
since only the central 2-mm portion of the grid is scanned in
the microscope. This pair (TEM grid and filter section) is
picked up with tweezers and carefully placed on the moist
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(a) Plan of Jaffe Washer
Tweezers
(c)
Stainless Mesh
EM Grid
01
Chloroform
Level
40-high stack of
5 cm S&S filters
(b) Elevation of
Jaffe Washer
Nuclepore
Asbestos Fibers
Nuclepore C-coated
Particle Side Down
1 .
Grid
Carbon Coat .
Chlorof orm,_ ^*
\ 1 wash | n n ir
\ X
Carbon
Substrate
Asbestos Fibers
Wet Stack of
Filters in a
Pool, of
Chloroform
'Grid
(d)
Figure 1
MODIFIED JAFFE WASHER METHOD
Plan view
Elevation view of Jaffe washer
(c) Details of placing a specimen
for washing
(d) Principle of the Jaffe method
(a)
(b)
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stainless steel mesh of the Jaffe washer. The 1 mm x 2 mm
section is wetted immediately by a 5 yi drop of chloroform.
When all the samples are in place in the washer, more
chloroform is carefully added to increase the level back to
where it just touches the top of the paper filter stack.
Raising the chloroform level any higher may float the TEM
grid off the mesh or displace the polycarbonate filter section;
neither is desirable. The cover is placed on the washer and
weighted to improve and seal and reduce the evaporation of
the chloroform.
More chloroform should be added periodically to maintain
the level within the washer. After a minimum of 24 hours,
the polycarbonate filter should be completely dissolved. The
TEM grid is removed by picking up the stainless steel mesh
with tweezers and placing it on a clean filter. When all
traces of chloroform have evaporated, the grid may be lifted
from the mesh and examined in the electron microscope or
stored for future examination.
2.5 Examination of the Grid by Transmission
Electron Microscopy
2.5.1 Low Magnification
The grid is observed in the transmission electron micro-
scope at a magnification of 500X to determine its suitability
for detailed study at high magnification. The grid is rejected
if:
(a) The carbon film over a majority of the grid opening
is damaged and not intact. If so, the transfer
step 2.4 must be repeated to obtain a new grid.
(b) The fibers give poor images and poor diffraction
patterns due to organic contamination or inter-
ference due to non-fibrous particles. If so,
the filter may be ashed, redispersed, and re-
filtered (see Section 2.8). Ashing step allows
elimination of organic matter and facilitates
diluting to minimize the interference from other
particles.
11
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2.5.2 High Magnification
2.5.2.1 Calibrating Magnification at Fluorescent Screen
It is important to know the exact value of magnification
at the fluorescent screen for the most common settings of the
electron microscope. The method for calibrating magnification
is illustrated in Appendix B.
2.5.2.2 Loading Levels
The method for examining the grid for fiber counting is
a function of the fiber loading on the filter. Three general-
ized loading levels may be encountered.
(a) Low Loading -- less than 50 fibers in a full grid
opening(80 ym x 80 ym).
(b) Medium Loading -- 50 to 300 fibers in a full grid
opening.
(c) High Loading -- more than 300 fibers per full grid
opening.
2.5.2.3 Fiber Counting Rules
In making a fiber count, the following rules are to be
observed:
(a) A field of view is defined. In some microscopes,
it is convenient to use the central rectangular
portion of the fluorescent screen which is lifted
for photographic purposes [see Figure 2(a)]. On
other microscopes, a scribed circle or the entire
circular screen may be used as the field of view.
The area of the field of view must be accurately
measurable.
(b) All fibers within the field of view are counted and
their length and width estimated and noted.
(c) Fibers which extend beyond the perimeter of the
field of view are counted. The width of these
fibers is measured but their length is measured
as only that portion which lies within the field
of view. Such fibers are noted by the letter "L"
as the length information is recorded, indicating
that it is a limit case [see Figure 2(a)]. In the
final analysis, such fibers are treated as half-
fibers (half-counts).
12
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Field of View:
Fibers 2 & 3 are crossing
the perimeter; the segment
of length inside the field
of view is estimated.
Perimeter
(c) Full Crid Opening:
Method of scanning.
(b) Random Choice of
Field of View
First Pass
Second Pass
Third Pass
Fourth Pass
Figure 2
TWO METHODS OF EXAMINING A GRID
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(d) Tightly bound bundles of fibers are counted as a
single fiber and an estimate made of their average
length and width. Fibers which touch or cross are
counted separately. Some subjective judgement is
required but fortunately, borderline cases are rare.
Notation is also made in recording the data that
the fiber was a bundle.
(e) Selection of the grid opening and the selection of
a field of view within a grid opening should be
done on a random basis [see Figure 2(b)]. This is
important for avoiding biases and to ensure the
statistical validity of the results.
(f) Morphological comparison with standard specimens is
used as a basis for rejecting non-asbestos particles
such as plant parts and diatoms. Where doubt exists,
the electron diffraction pattern of the particles
should be examined.
2.5.2.4 Fiber Classification Rules
Fibers are classified by observation of their morphology
and electron diffraction patterns. It is recommended that
both morphological and diffraction pattern study be done at
zero degree tilt angle.
The following rules should be followed when classifying
a fiber:
(a) Observe a fiber at a TEM screen magnification of
about 16.000X through a 10X binocular. At such
high magnifications, the tubular structure of
chrysotile is usually apparent (compare with
standard specimens). Fibers showing the tubular
structure may be classified tentatively as chrysotile
A few other minerals, e.g. halloysite, show tubular
structure, but can be recognized from chrysotile by
electron diffraction. Amorphous matter can get
inside the tube or cover the chrysotile fiber, and
thus obscure the tubular structure. Therefore,
non-tubular structure does not rule out chrysotile.
Amphibole asbestos fibers usually have a lath or
plate-like shape, are more electron dense, and show
thickness contours and other diffraction contrasts
which change due to beam heating. Morphological
features alone may not be a sufficient basis for
distinguishing chrysotile from amphiboles and other
fibrous minerals.
14
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(b) Electron diffraction patterns from single fibers of
asbestos minerals fall into distinct groups. The
chrysotile asbestos pattern has characteristic streaks
on layer lines other than the central line and some
streaking also on the central line. There are spots of
normal sharpness on the central layer line and on
alternate lines (2nd, 4th, etc.). The repeat distance
between layer lines is about 0.53 nm.
Amphibole asbestos fiber patterns show layer lines
formed by very closely spaced dots, and the repeat
distance between layer lines is also about 0.53 nm.
Streaking in layer lines is occasionally present due
to crystal structure defects.
(c) Transmission electron micrographs and selected area
electron diffraction patterns obtained with standard
samples should be used as guides to fiber identifica-
tion. [7-9]
From the examination of the electron diffraction patterns,
fibers are classified as belonging to one of the following
categories:
Chrysotile
Amphibole group (includes amosite, crocidolite,
anthophyllite, tremolite and actinolite)
Ambiguous (incomplete spot patterns)
Non-asbestos (minerals other than the six
asbestos minerals)
Unknown (no spot pattern)
It should be noted that other particles with fibrous mor-
phology also give layer patterns; for example, pyroxenes. The
complete quantitative indexing and deriving interplanar d-
spacings from diffraction patterns is a time consuming and
complex undertaking and is not feasible for routine analysis.
It is not possible to inspect electron diffraction patterns
for some fibers. There are several reasons for the absence of
a recognizable diffraction pattern. These include contamination
of the fiber, interference from nearby particles, too small a
fiber, too thick a fiber, and non-suitable orientation of the
fiber. Some chrysotile fibers are destroyed in the electron
beam resulting in patterns that fade away within seconds of
15
-------
being formed. Some patterns are very faint and can be seen
only under the binocular microscope. In general, the shortest
available camera length must be used and the objective lens
current may need to be adjusted to give optimum pattern visi-
bility for correct identification. Use of a 20 cm camera
length and a 10X binocular to inspect the SAED pattern on the
tilted screen is recommended.
2.5.2.5. Counting at Low Loading Level
When fewer than 50 fibers per grid opening are encountered,
the preferred counting method is to scan the entire grid opening
and defining the full grid opening as one field. With the
microscope magnification at 20,OOOX, a series of parallel scans
across the grid square are made starting with the top corner of
the square and ending at the bottom [see Figure 2(c)]. (With
the tilting section of the fluorescent screen used as a single
field of view, approximately 300-400 fields will be observed
if the entire grid opening is scanned.) Fibers noted in each
full grid opening (or single field) are classified in accord-
ance with the procedure described above.
Additional grid openings are selected, scanned, and
counted until the total number of fibers counted exceeds 100,
or a minimum of 10 grid openings have been scanned, whichever
occurs first.
2.5.2.6 Counting at Medium Loading Level
When the loading on the filter is in the range of 50 to
300 fibers per grid opening, counting is done on randomly
selected fields of view. At a nominal magnification of 20.000X,
fields are randomly selected within a grid opening until a
total of 20 fibers have been counted, sized, and classified.
(Generally 20-40 fields of view are observed per grid opening.)
After about 20 fibers have been counted, another grid opening
is selected and an additional 20 fibers (approx.) are counted.
This procedure is repeated for 5 grid openings until a minimum
16
-------
of 100 fibers are counted. (When estimating fibers of a par-
ticular type of asbestos, counting is continued until 50-100
fibers of that type are counted.)
2.5.2.7 Counting at High Loading Level
When the fiber loading exceeds 300 fibers per grid
opening, the filter should ideally be rejected in favor of
a filter sample taken for a shorter time period.
If no other filter sample is possible and the number of
fibers above 300 is not too great (up to 400), then a filter
section should be transferred to a 400 mesh grid and the pro-
cedure repeated as for medium filter loading levels. The
400 mesh grid opening is much smaller in area than a 200 mesh
grid opening, and hence can be scanned much faster with less
fatigue for the operator.
When the loading level is so high that fibers touch and
overlap and no other sample is available, then the filter
should be ashed, dispersed, and refiltered to yield a lower
concentration level. Details for this procedure are given
in Section 2.8.
2.6 Recording of Data
It is advantageous to record the TEM data in a systematic
form so that it can be transferred to computer data cards for
statistical analysis.
2.6.1 Recording Format
A suggested data sheet format is shown in Table 2. The
entries at the top describe the sample (identification, the
storage box, and storage location), the sampling parameters
(volume of air sampled, total effective area of the filter),
and the TEM parameters (screen magnification, area of one
2
field of view in cm , etc.).
Column 1 -- EM grid opening identification number
Column 2 -- Identification number for the field of view
17
-------
Table 2
DATA RECORDING SHEET
00
Sample :
Storage
Box No . :
Vol.
of Air Sampled: 9
.2m3
2
Effective Area of Membrane: 406.5 cm
Location in Box:
Magnification: 17,
Area of
Grid
Opening
I.D.
1
and so
One Field:
Field
of View
Number
1
2
3
4
5
on.
000
0.182 x
Fiber
Number
I
2
1
2
3
4
5
6
1
2
3
4
5
1
2
3
1
2
3
10'6 cm2
Cumulative
Fiber Count
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Fiber
Width,
mm
1.0
0.25
0.5
1.0
1.0
0.75
1.0
0.5
0.25
0.75
1.0
1.0
0.25
0.25
0.25
0.5
2.0
1.0
0.5
Fiber
Length,
mm
20
7
17
10
18
22
12
10
24
18
10
8
10
6
20
5
65
10
4
Fiber Identification
by Morphology and
Electron Diffraction
Chrysotile
Ambiguous
Ambiguous
Chrysotile
Chrysotile
Chrysotile
Chrysotile
No Pattern
Chrysotile
Ambiguous
Chrysotile
Chrysotile
No Pattern
Chrysotile
Ambiguous
No Pattern
Chrysotile
Ambiguous
No Pattern
-------
Column 3 -- Fiber sequence number within a given field
of view
Column 4 -- Cumulative number of fibers counted
Column 5 -- Fiber width in mm
Column 6 -- Fiber length in mm
Column 7 -- Fibers extending beyond the perimeter of
the field, marked with L (limiting case)
Column 8 -- Fiber identification, chrysotile, amphibole,
ambiguous, or no pattern or non-asbestos
2.6.2 Computer Coding Forms
A Fortran program has been developed (see Appendix C) to
analyze the data obtained from the electron microscope study.
In order to use this Fortran program, it is recommended that
data from the notes be transferred to IBM computer coding
sheets to facilitate key punching. The coding scheme is given
in Table D-l and an illustration is presented in Table D-2 of
Appendix D. The scheme is sufficiently broad to keep all
relevant information, such as sample code number, laboratory
code number, operator code number, TEM grid number, etc.
Ashing factor refers to the dilution or concentration resulting
from the ashing and reconstitution step. It is defined as the
ratio of the redeposition filter area to the area of the filter
2
segment ashed. For example, if a segment of 5 cm area was
ashed and the ash suspension deposited on 25 mm diameter final
2
filter (effective area 2 cm ), the ashing factor is 0.4. The
area of the field of view when multiplied by the ashing factor
gives the corrected area of the field.
2.7 EM Data Analysis
2.7.1 Checking Data on Key Punch Cards
Key punch cards are checked by obtaining a printout of all
cards as illustrated in Table D-3 of Appendix D. This printout
helps in detecting key-punching errors by comparison with the
coding forms.
19
-------
2.7.2 Separating Very Large Sized Bundles
At present, separating bundles of fibers from the data is
done by inspection of printout of the input data. The computer
program can be modified to exclude the very large sized fibers
from the analysis.
2.7.3 Fortran Program for Obtaining
Characterizing Parameters
Each analyzing laboratory can develop its own computer
program to facilitate statistical analysis and to obtain the
necessary characterizing parameters. One Fortran program
called CONLAB was specially developed at IITRI for obtaining
several important characterizing parameters. The listing for
this program is given in Appendix C. The program gives char-
acterizing parameters for each TEM grid used.
2.7.4 Printout of Results on each TEM Grid
A typical printout of results on each TEM grid (for the
data in Table D-3) is given in Tables D-4 and D-5. The para-
meters shown are:
Fiber counts for each category
Fiber concentration per cm^ of filter
Fiber concentration per m3 of air
Mass concentration per cm^ of filter
Mass concentration per m3 of air
Length (ym) Mean
Std. Deviation
Diameter (vim) Mean
Std. Deviation
Volume (ym)3 Mean
Std. Deviation
2.7.5 Summary of Results for a Typical Air Sample
Summaries of the results are obtained using relevant
quantities from the printouts in Tables D-4 and D-5. Shown
20
-------
in Table D-6 are the characterizing parameters for all fibers
and for chrysotile fibers.
2.7.6 Precision of TEN Estimates
When more than one TEM grid is used, it is possible to
obtain the mean values and 957o confidence levels on the means.
This is done for each important parameter. The method consists
of obtaining the mean, x, the standard error of the mean, SEm,
and t-value [10] (0.025, n - 1) for n - 1 degrees of freedom,
where n = number of TEM grids examined and hence n replicates
available. The 95% confidence limits are given by x + t • (SEm),
In the illustrative case, Table D-6, the following four
parameters are given:
/• o
(1) Fiber number concentration of all fibers, 10 /m of air
-9 33
(2) Volume concentration of all fibers, 10 cm /m of air
fi o
(3) Fiber number concentration of chrysotile, 10 /m of air
3
(4) Mass concentration of chrysotile fibers, yg/m of air
The t-value decreases sharply with greater replication.
For example, t = 12.7 for n = 2 and decreases to 4.3 for n = 3
and to 2.77 for n = 5 and so on. The standard error of the mean
also decreases with greater replication. Hence, to increase the
precision of the TEM estimates, 3 or 4 replicates per sample
should be analyzed.
2.7.7 Analyzing Data on Very Large Bundles of Fibers
Fiber bundles should be reported separately as the number
concentration of large bundles or fiber aggregates (greater than
3 3
1 ym each) per m of air. In general, these are few and these
computations can easily be done using a desk calculator.
No attempt is made to compute either the volume or the
mass of bundles because of the large uncertainty in assigning
dimensions to aggregates.
21
-------
2.8 Ashing, Sonification, and Reconstitution
Some air samples (especially samples collected over several
hours) may contain high levels of organic contaminant. This
organic matter obscures the fibrous particles, and interferes
with the proper counting, sizing, and identification. Such
samples should be ashed and reconstituted. In cases where a
flat, polycarbonate filter cannot be used for initial sample
collection, and a depth filter (cellulose acetate) with high
retention efficiency must be used, the ashing step may be used to
reconstitute the particles on a flat filter for TEM analysis.
The procedure for ashing is as follows:
A section of known area (e.g. 1 cm x 1 cm) is cut from the
polycarbonate or cellulose acetate filter used to collect the air
sample and is placed in a clean glass vial (30 mm diameter x 80
mm high). The membrane is positioned such that the particle
collection side (shiny side) faces the glass wall. The vial is
placed in an upright position in a low-temperature asher. Using
manufacturer's instructions, vacuum is obtained and the filter is
ashed at 40 watts power in oxygen plasma. Oxygen is admitted at
2 psi pressure. Though the membrane vanishes in about a half-
hour, the ashing is continued for about 3-4 hours to ensure
complete ashing. The ashing chamber is allowed to slowly reach
atmospheric pressure. The vial is removed and 10 ml of filtered
distilled water containing 0.1 percent filtered Aerosol OT is
added. The vial is placed in a 100 ml beaker containing 50 ml of
water, and this beaker is placed in a low-energy, ultrasonic
bath. Ultrasonic energy is applied for 15 minutes to disperse
all of the ash.
A 25 mm diameter filtering apparatus is assembled with a 25
mm diameter, 0.1 pm pore size polycarbonate filter with 5 ym pore
size cellulose ester filter backing on the glass frit. Suction
is applied and the filters are recentered if necessary. The
filter funnel is mounted, the vacuum is turned off and suction is
allowed to cease. Two ml of distilled water is added to the
funnel followed by the careful addition of the water containing
22
-------
the dispersed ash. Suction is applied to filter the sample.
The vial should be rinsed with 10 ml of 0.1 percent Aerosol OT
at least twice and the contents carefully transferred to the
filtration funnel before the funnel goes dry. At the end of
filtration, the suction is stopped. The filter is then dried
in still air and stored in a disposable petri dish. After
drying, the filter is ready for carbon-coating (see Section 2.3)
and transfer of the sample to an EM grid (see Section 2.4).
The effective area of the redispersion filter and the
area of the section cut for ashing from the original membrane
must be taken into account when computing the fiber concentra-
tion, etc., in the TEM data analysis.
2.9 Limits of Detection
The minimum detection limit of the electron microscope
method for the counting of airborne asbestos fibers is variable
and depends upon the amount of total extraneous particulate
matter in the sample and the contamination level in the
laboratory environment. This limit also depends on the air
sampling parameters, loading level, and the electron microscope
parameters used.
In this provisional method, 100 fields, each with an area
-6 2
0.18 x 10 cm are scanned. Assuming that a fiber count
has an accuracy of ± 1 fiber, then the detection limit is
Detection Limit - -±- . Area of Filter (cm2)
100 0.18 x 10° (cnT)
Vol. of Air
In some cases (for very lightly loaded samples) when four,
full grid openings are scanned, each grid opening with an
area of 0.72 x 10"^ cm2, the detection limit is
23
-------
Detection Limit = I . Area of Filter (cm2)
4 0.72 x 10 ^ (cnT)
Vol. of Air (m3)
Table 3 gives an indication of the magnitude of the detection
limit, calculated for the high-volume sampler method. It is seen
that the minimum detection limit is lower for very dilute samples.
Examining full grid openings leads to a lower value of minimum
detection limit because of the large area scanned, as compared
with the field of view method. With a given sample, the detection
limit can be lowered considerably but the experimental effort
required also increases. The guidelines of using 100 fields of
view or four full grid openings represent a judicious compromise,
between a reasonable experimental effort and a fairly low value
of the detection limit. Also, using two or more TEM grids will
reduce the detection limit further and also improve the precision
of the estimates.
3. PREPARATION OF BLANKS
Even after taking utmost precautions to avoid asbestos
contamination, one cannot rule out the possiblity of some con-
tamination. It is a good practice to check contamination periodi-
cally by running blank samples.
A blank sample may consist of a clean filter, subjected to
all the processing conducted with an actual air sample. These
may include ashing, resuspension, redeposition, carbon coating,
transferring to TEM grid, and TEM examination.
When analyses of blank samples show significant background
levels of asbestos, these should be subtracted from the values
obtained for field samples. Also, the minimum detection limit
may be calculated as twice or three times the standard deviation
of the blank or background value.
24
-------
Table 3
MINIMUM DETECTION LIMIT USING HIGH-VOLUME AIR SAMPLER
Full Grid Opening**
Vol. of Air Field of View Method* (1 fiber in
Sampling Sampled (1 fiber in 100 fields) 4 grid openings)
Duration m^ million fibers/m^ million fibers /m^
Point
Source
Near
Source
Distant
Source
*
Detectio
*>'-
% hr 21 1.07 0.07
2 hr 84 0.27 0.02
8 hr 336 0.067 0.005
,. . 1 Fiber 406 cm2 1
100 fields 0.18 x 10~6 cm2/field Vol. of Air Sampled, m3
T . . 1 Fiber 406 cm2 1
4 Grids 0.72 x
cm /grid Vol. of Air Sampled, m3
-------
REFERENCES
1. Tentative Method of Analysis for Suspended Particulate
Matter in the Atmosphere (High-Volume Method) 11101-01-70T,
Methods of Air Sampling and Analysis; Intersociety
Committee, American Public Health Association, 1015 18th
Street, N.W., Washington, DC, 1972.
2. Recommended Procedures for Sampling and Counting Asbestos
Fibers; Joint AIHA-ACGIH Aerosol Hazards Evaluation
Committee, American Industrial Hygiene Assoc. Journal,
36(2):83-90, 1975.
3. Standards of Performance for New Stationary Sources;
Federal Register, 36^(247) :24876 ff, December 23, 1971.
4. Standards of Performance for New Stationary Sources;
Federal Register, 42_(160) :41754 ff, August 18, 1977.
5. Ranade, M.B., Sampling Interface for Quantitative Transport
of Aerosols; EPA-650/2-74-016, 1973.
6. Jaffe, M.A., Handling and Washing Fragile Replicas; Proc.
Electron Microscope Society of America, Toronto, Sept.
1948, J. Appl. Phys . , 19_(12) :1189 , 1948.
7. Clark, R.L. and C.O. Ruud, Transmission Electron Microscopy
Standards for Asbestos; Micron, 5_:83-88, Pergamon Press,
1974.
8. Ruud, C.O., C.S. Barrett, P.A. Russell and R.L. Clark,
Selected Area Electron Diffraction and Energy Dispersive
X-ray Analysis for the Identification of Asbestos Fibers,
A Comparison; Micron, 7^(2) : 115-132, 1976.
9. Mueller, P.K., A.E. Alcocer, R.Y. Stanley and G.R. Smith,
Asbestos Fiber Atlas; U.S. Environmental Protection Agency
Publication No. EPA-650/2-75-036, April 1975. National
Technical Information Service, Springfield, Va.
10. Fisher, R.A. and F. Yates, Statistical Tables for
Biological, Agricultural and Medical Research Workers;
Table IV, 6th Ed., Stechert-Hafner, Inc., New York, 1964.
26
-------
Appendix A
INSTRUMENTATION AND SUPPLIES
27
-------
Appendix A
INSTRUMENTATION AND SUPPLIES
A. INSTRUMENTATION
1. Transmission Electron Microscope
A transmission electron microscope should be capable of
100 kv of accelerating voltage, 1 nm resolution, and a magni-
fication range of 300 to 100.000X. The instrument should be
capable of selected area electron diffraction analysis on
areas 300 nm diameter. The fluorescent screen should have
either a millimeter scale, concentric circles of 1, 2, 3,
and 4 cm radii, or other devices to estimate the length and
width of fibrous particles. All modern transmission electron
microscopes meet these requirements.
2. Vacuum Evaporation
A vacuum evaporator is required for depositing a layer
of carbon on the polycarbonate filters and for preparing
carbon-coated EM grids. The evaporator should have a turn-
table for rotating the specimen during coating.
3. Low-Temperature Plasma Asher
A low-temperature plasma asher is required when the
quantities of organic matter in the air sample are very high
and interfere with the detection and identification of
asbestos. Oxygen should be used for plasma ashing. The
sample chamber should be at least 10 cm diameter, so that
glass vials can be positioned vertically (e.g., Plasmod,
Tegal Corporation, Richmond, CA or equivalent).
B. SUPPLIES
1. Jaffe Washer: For dissolving polycarbonate filters
This item is not available commercially. The assembly is
described in Section 2.4 and illustrated in Figure 1.
28
-------
2. Filtering Apparatus: 47 mm filtering funnel
(e.g., Cat. No. XX1504700, Millipore Corp. Order Service Dept.,
Bedford, MA 01730). 25 mm filtering funnel (Cat. No. XX1002500,
Millipore Corp. Order Service Dept., Bedford, MA 01730).
These are used to filter dispersed ash samples.
3. Vacuum Pump: A vacuum pump is needed to filter ash
suspensions. It should provide up to 20 in. of mercury. Such
vacuum pumps are available from any general laboratory supply
house.
4. EM Grids: 200-mesh copper or nickel grids with car-
bon substrate are needed. These grids may be purchased from
manufacturers of electron microscopic supplies (e.g., Cat.
No. 1125, E.F. Fullam, Schenectady, NY) or prepared by standard
electron microscopic grid preparation procedures. Finder grids
may be substituted and are useful if the re-examination of a
specific grid opening is desired (e.g., Cat. No. 1458, H-2
London 200 Finder 'grids, E.F. Fullam, Schenectady, NY or Cat.
No. 17420 200 mesh carbon-coated nickel grids, Ladd Research
Industries, P.O. Box 901, Burlington, VT 05401).
5. Membrane Filters: Polycarbonate
(R)
(a) 47 mm diameter, 0.4 vim pore size Nucleporew
membranes or equivalent.
dS
(b) 37 mm diameter Nuclepore^ membranes for use
with the personal dust samplers.
(c) 25 mm diameter, 0.4 ym pore size Nuclepore^
membranes or equivalent to filter dispersed
ash suspension.
(Rj
(d) 20 cm x 25 cm, 0.4 urn pore size Nuclepore^
membranes or equivalent for collecting air
samples using the high-volume sampler.
6. Membrane Filters: Cellulose acetate (to be used
as backing filters)
(a) 47 mm diameter, 5.0 ym pore size
or equivalent.
29
-------
(b) 37 mm diameter, 5 ym pore size Millipore®
filters or equivalent for use with personal
dust samplers.
(c) 25 mm diameter, 5 ym pore size Millipore®
filters or equivalent.
(d) 20 cm x 25 cm, 5 ym pore size Millipore®
filters or equivalent for use with the
high-volume sampler.
7 . Air Samplers:
(a) High-volume sampler, see reference 1
(e.g., Sierra Instruments, Model 305,
3756 N. Dunlap St., St. Paul, Mn. 55112 or
equivalent).
(b) Personal dust sampler, see reference 2
(e.g., MSA Gravimetric Dust Sampling Kit,
MSA Co., Pittsburgh, Pa. 15208 or equivalent).
8. Glass Vials: 30 mm diameter x 80 mm long; for
holding filter during ashing. 50 ml beakers can ba used
instead of vials.
9. Glass Slides: 5.1 cm x 7.5 cm; for support of
filters during carbon evaporation.
10. Scalpels: With disposable blades and scissors.
11. Tweezers: Several pairs for the many handling
operations.
12. Doublestick Cellophane Tape: To hold filter section
flat on glass slide while carbon coating.
13. Disposable Petri Dishes: 50 mm diameter and 100 mm
diameter for storing membrane filters.
14. Static Eliminator: 500 microcuries PO-210.
(Nuclepore Cat. No. V090POL00101) or equivalent. To eliminate
static charges from membrane filters.
15. Carbon Rods: Spectrochemically pure, 3.0 mm
diameter, 4.6 mm long with 1.0 mm neck. For carbon coating
30
-------
(Cat. No. 42350, Ladd Research Industries, P.O. Box 901,
Burlington, VT 05401 or equivalent).
16. Ultrasonic Bath: (50 watts, 55 KHz). For dis-
persing ashed sample and for general cleaning.
17. Graduated Cylinder: 500 ml
18. 10 y& Microsyringe: For administering drop of
solvent to filter section during sample preparation.
19. Carbon Grating Replica: 2160 lines/mm. For cal-
ibration of EM magnification (e.g., Cat. No. 1002, E.F. Fullam,
Schenectady, NY or equivalent).
20. Specimen Grid Punch: For punching 3 mm diameter
sections from membranes (e.g., Cat. No. 1178, E.F. Fullam,
P.O. Box 444, Schenectady, NY 12301 or Cat. No. 16250, Ladd
Research Industries, P.O. Box 901, Burlington, VT 05401).
21. Screen Supports: Copper or stainless steel;
6 mm x 6 mm, 60-100 mesh. To support specimen grid in Jaffe
washer.
22. Filter Paper: S&S #589 Black Ribbon or equivalent
(5% cm circles). For preparing Jaffe washer.
23. Chloroform: Spectro grade, doubly distilled. For
dissolving polycarbonate filters.
24. Acetone: Reagent grade or better. For cleaning
the various tools.
25. Asbestos: Chrysotile (Canadian), crocidolite,
amosite. UICC (Union International Contre le Cancer) standards
Reference asbestos samples available commercially (e.g., Duke
Standards Company, 455 Sherman Avenue, Palo Alto, CA 94306 or
Particle Information Service, 600 South Springer Road,
Los Altos, CA 94022 or equivalent).
26. Petri Dish: Glass (100 mm diameter x 15 mm high).
For modified Jaffe washer.
31
-------
27. Cleanser: Alconox, Inc., New York, NY 10003 or
equivalent. For cleaning glassware. Add 7.5 g Alconox to
a liter of distilled water.
28. Aerosol OT: 0.1% solution (Cat. No. So-A-292,
Fisher Scientific Co., 711 Forbes Ave., Pittsburgh, PA 15219).
Used as a dispersion medium for ashed filters. Prepare a
0.1% solution by diluting 1 ml of the 10% solution to 100 ml
with distilled water. Filter through 0.1 ym pore size poly-
carbonate filter before using.
29. Parafilm: American Can Company, Neenah, WI Used
as protective covering for clean glassware.
30. Pipettes: Disposable, 5 ml and 50 ml.
31. Distilled or Deionized Water: Filter through 0.1 ym
pore size polycarbonate filter. Used for making all reagents
and for final rinsing of glassware, and for preparing blanks.
31. Storage Box for TEM Grids: Cat. No. E-0174 Grid
Holders, JEOL U.S.A., Inc., 477 Riverside Avenue, Medford,
Mass. 02155 or equivalent.
33. Squeeze Bottles: For keeping double-filtered dis-
tilled water and 0.1 percent Aerosol OT solution.
34. Welders Protective Goggles
32
-------
Appendix B
MAGNIFICATION CALIBRATION
33
-------
Appendix B
MAGNIFICATION CALIBRATION
(1) Align the electron microscope using the instruction
manual provided by the manufacturer.
(2) Insert mag-calibration grating replica (with 54864
lines per inch, or 2160 lines per mm, e.g., Cat. No. 1002,
E.F. Fullam, Schenectady, NY) in the specimen holder.
(3) Switch on the beam, obtain the image of the replica
grating at 20.000X magnification (or the magnification at which
the asbestos samples will be analyzed) and focus.
(4) If the fluorescent screen has scribed circles of
known diameters, proceed as follows. Using stage control,
align one line tangentially to circumference of one circle.
Count the number of lines in a diameter perpendicular to the
lines. In most cases, the other end of the diameter will be
t~ V\ +• V»
in-between the N and N + 1 line. You can estimate the
fractional spacing by eye. Alternatively, one can estimate
the separation between lines using the scribed circles.
(5) If X line spacings span Y mm on the fluorescent
screen using this grating replica, the true magnification is
given by
Y x 2160
M =
X
The readings should be repeated at different locations of the
replica and the average of about 6 readings should be taken
as the representative or true magnification for that setting
of the electron microscope.
34
-------
Line Spacings mm on Screen Magnification
X Y M
9.5 83 18871
9.3 80 18580
7.0 60 18514
8.8 80 19636
9.0 80 19200
9.0 80 19200
Average 19000
On most electron microscopes with large (18 cm dia.)
fluorescent screens, the magnification is substantially con-
stant only within the central 8-10 cm diameter region. Hence,
calibration measurements should be made within this small
region and not over the entire 18 cm diameter.
35
-------
Appendix C
LISTING FOR THE FORTRAN PROGRAM CONLAB FOR
OBTAINING CHARACTERIZING PARAMETERS
36
-------
Appendix C
LISTING FOR THE FORTRAN PROGRAM CONLAB FOR
OBTAINING CHARACTERIZING PARAMETERS
C PROGRAM CONLAB
C ANALYZE FIBER AND MASS CONCENTRATIONS
C
REAL SUMX(7i6)fSUMX2(7i6),CONCT(6)iCONMAS(6J»VOLCT(6)tVOLMASC6)
REAL FIBCTC6)iQTY(7)fDEN(2)»SOEV(7.6)»SLDEV(7f6)iGMN(7»6)
RCAL CVAR(7t6)»MEAN(7i6)»MEAN2(7»6)
DATA I.ICTtPI/l»Ot3.Hl59/
DATA DEN/2.6»3.0/
C QTYC1) LENGTH
C OTY(2) DIAM
C QTY(3) MASS OR VOL
C OTY(«) LOG LENGTH
C OTY(5) LOG 01AM
C OTY(6) LOG MASS
C
C RCAD LENGTHiDIAMiCOMPUTE OTHER DATA» STORE
REAO(5»110»END=190) IGRIO.IFUDfIFSEQ»ICSEQ»DIAMiALENtlOUTi
*INFIB»ILAB»IFIL>IPUNtXMAG(AREAiXASHtXVOLfTAREAtICASE
050 TOTAR'O
TOTCTeO
LCTabO
LCASE»IC.A8C
LLABBILAB
LPUN»IPUN
00 060 I=lf6
FIBCT(I}=0.0
VOLCT(I)aO.O
VOLMAS (I)BO.O
CONCT(I)=0.0
CONMAS(I)=l),0
00 080 J=l«7
SDEVCJ»I)=0,0
SLOCV(JfI)BO.O
QMN(JiI)=0.0
CV*R(JtI)»0.0
QTY(J)aO.O
MCAN2(JiI)sO.O
SUMXCJf I)=0.0
SUMX2(J»I)sO.O
030 COWTINUE
GOTO 1101
100 R|.'AD(5illOtEND=190) I GRID. Tf-'i n , IK5KG i ICbEGIt DI AM, ALFN» I OUT f
*INFIBfILA8tIFIU»IPUN»XMAn,AR!:.\,XA.>'l»XVOI. tTAREA.ICASE
110 FORMAT (I2t2I3tI«i2Fft.Ot lX.T!-2Xf!l»lX».iIl»2Xf2P7.1»3F5.1i5Xtia)
IF ((TCASE.NE,LCASE).OR.(!L*p,NE.LLAB)
* .OR.(IFR.NE.LFIL).OS.(IPU^»1''E.LfJUN))
* GOTO 200
1101 AVOL=XVOL
FILARaTAREA
JLAQ=ILA8
JPUN=IPUN
JPri=IFIL
IF ((IGRID.EO.LGRID).AND.(IFLD.EO,LFLO)> OOTO 1110
TOTAR=TOTAR+APEA*XASH*1.0E-6
WRITE (6tll02)
1102 FORMATC132X)
LCT=LCT*2
LFLO=IFLD
37
-------
Appendix C (continued)
LGRID=IGRID
U10 IFCLCT.LE.SO) GOTO 1112
WRITE (6.1111) ILAB.IFIL.IPUN.LCASE
1111 FORMATC'li.'IOX.IFIBER iND MASS CONCENTRATION I » 15Xf I LAB 'il
*' SAMP Itll.i GRID 1.12,1 CASE i,12/
*SOX. 'DATA LIST'//
*1X.IF1B»SEQ FIELD FLD-SEQ OUMif6Xi
*'LENGTH OUT-COD FIB-COD FLD-MAG «»
*IFLD"AREA ASH-HAS VOL FILT»AREA I /)
LCTaO
1112 IF (INFIB.EQ.O) GOTO 1120
IFIBsINFIB
IFC(INFIB.GE.2).AND.(INFIB.LE.5)) IFIB"INFIB+1
IF(INFIB.EO,5) IFIB=2
IFCINFIB.GE.6) IFIB=5
1120 wRITE(6f 1113) ICSEQ.IFLO.IFSEQ.DIAM.ALENf IOUT i INFIBf XMAGi
*ARL'A'XASH»XVOL»FILAR
1113 FORMAT(3XiI'l»3X.n.SX.l3i2(5XiF6.2)
*3(5X«F7,3).2(4X»F5.1))
LCT=LCT+1
112 IF(IFSEQ.EO.O) GOTO 100
IF UOUT.E0.2) GOTO 115
F1BCT(IFIB)=FIBCT(IFIB5+1.0
GOTO 117
115 FIPCT(IFie)=FIBCTCIFI8)+0,5
ALt'N=ALEN*2.0
117 ALI:N=ALEN/XMAG
DIAMcDIAM/XMAG
OTY(1)=ALEN
QTY(2)=DIAH
QTY(3)=PI*Ai.EN*DIAM*DIAM*l,oi:.-12/4.0
IFC1FIB.GT.2) GOTO 122
QTY(7)=OTY(3)*OtN(IKIB)
C FIMD LOG OF EACh QTY» SUM QTY AMD LOG
122 HO t«9 I=li3
IF (OTYCI)) HO»1'IO,U5
1«0 QTYCI+3)sO
GOTO 1«9 '
QTY(H-3)=ALOG(QTY(I))
CONTINUE
150 DO 159 1=1.7
159 CONTINUE
GOTO 100
190 Il!NO = l
200 DO 205 IFIB=1?5
TOTCTsTOTCT+FIBCTCIFIB)
DO 205 1=1.7
205 CONTINUE
FIBCT(6)=TOTCT
DO 599 IFIB=1.6
IF (FIBCT(IFIB).EO.O.O) GOTO 599
CONCT(IFIB)=FIBCT(IFIB)/TOTAR
VOLCT(IFIB)=CONCT(IFIB)*FILAR/AVOl
IF (IFIB-?) 220.220,210
210 CONMAS(IFIB)=F1BCTCIFIB)*100.0/TOTCT
GOTO 230
220 COMMAS CIFIB)=SUMXC7» IF IB) /TOTAR
VOLMAS(IFIB)=CONMAS(IFI6)*FILAR/AVOL
230 DO 25U I=l«7
206 MEANU«IFI8) = SUMXCI.!FIB)/FIBCTCIFIB)
MEAN2(If IFIB)=SUHX2(I,IFIB)/FIBCTCIFI6)
250 CONTINUE
38
-------
Appendix C (continued)
DO 270 1st, 3
SOGVU»IFIB)sSQRTCABSCMEAN2(I»IFIB)»
* (MEANCIiIFIB))*(MEAN(I,XFI8))))
3LDEV(I,IPIB)sSQRTUes
*15X,' LAB ifljtl SAMPLE '.Ilfi GRID l»I2fl CASE i»I2/
*SOXi 'SUMMARY!//
*jxiiTOTAL AREA SCALED « i,Ei2.««i so CMI/
*1X«ITOTAL »REA FILTE1? = i ,F7. 1 1 BXi ' 80 CMI/
flXilTOTAL FIBER COUNT = I . F?. 1 • 6X » I FIBERS " /
*tx» ITOTAL' voLUMt AIR = I.F;. 1,8X1 ICUBIC METERSI///
*30Xi ICHRYSOTILE 4HPHIBPLE AMBIGUOUS NO PATTERN! •
*! NON-ASBESTOS ALL FIBERSI/)
WRITE (61 607) (FI3CT(IFIB)tIFIBeli6)
60T FORMATClXt 'FIBER COU'JTi/
*IXt i (FIBERS) l.21Xf6(Fa.l.7X)/)
WRITE(6>610) (CONCTCIFTB)iI?IB=li6).(VOLCT(IFlB)fIFlB"li6)
610 FORMATClXt IFIBER COKCuNf RATION i /
*lXtl(FIBER9 PER SO CM OF ULTER) " » 6(E12.«f 3X)/
*lXtKFIBERS PEK CUB t'LTtR Or AI ft ) I i 6 CE1 2.4 1 3X) /)
WRITE (6, 6 13) (CONhASfIFlft)tIFIB=l,5)»(VOLMASMFIB)»IFIBsif2)
6J5 FORMATClXt IMASS COHCINTRi TIOM • 53X •< PERCENT TOTAL FIBERS!/
flXt" (GRAMS PER SO O OF FILTER)!,
*2Xt2(El?.'l.3X) ..l(F6.,"!i !Xi < 7. I t7X)/
*lXtU6RAMS PER CU8 ^-:Tf:R Of AIR) ' » t Xf 2(E12.«i3X)/)
WRITE(6»620)
*(MEAN(«iIFIB)ttFIB-l.i.)i
*(GMN( JtlFIB)
620 rORMATClX, (LENGTH HffANi , 1U»6(F12.«»3X)7
»1X. I (MICRONS) STD D[-:vi ,8Xt&CF12.4»3X)/
*) IX, 'MEAN LOG I ,7X.^(F J2,a,iX)/
*HXf'6EOM MN I i 7X, 6 (,'-'l?, a, -}X)/
WRITE (6, 629) (MEANl^.XFJ.a) ,iFIPsl, 6) t
*(SDEV(2fIFIB),IFIB«lt6)i
*(MEAN(5t IF1B)»IFIB=1»6)|
*(GMN(2tIFIB),IFIBsJf6),
*(CVAR(2,IFIB),IFIB=1»6)
625 FORMAT(lX,iDIAMETfcR MEAN 1 , 11X,6(F12.«» JX)/
*IX,I(MICRONS) STD DEV « ,8X,6(F12.fl»3X)/
*ltXi"MfAN LOPI,7X,6(F12.«»3X)/
»HX»IGEOM MN 'f7Xt6(Fi2.«,3x)/
*11X»"COEF VARi,7Xi6(F12.
-------
Appendix D
ILLUSTRATIVE TABLES
40
-------
Table D-l
ELECTRON MICROSCOPE METHODS FOR ASBESTOS DATA ENTRY FORMAT, PER FIBER
Cols.
1-2
3-5
6-8
9-12
13-18
19-24
26
28
30
32-36
37-43
44-50
51-55
56-60
61-65
71-72
Description of Coding-Sheet Field
EM grid opening ID
EM field ID
Sequence no. of fiber within field'1"'"
Cumulative sequence no. within sample'-'"'"
Diameter in mm (do code decimal point)
Length in mm (do code decimal point)
2 if fiber extends beyond perimeter
1 if a fiber bundle
Fiber type
Case identification:
Col. 32 - lab
33 - filter - sample
34 - punch - grid
35 - instrument type
36 - operator within lab
Magnification in multiples of K = 1000
(do code decimal point)
Area of EM field identified in cols. 3-5
— ft 9
in 10 cm (do code decimal point)
Ashing factor
3
Volume of air sampled in m
Effective area of the original membrane
9
in cm"
Data set code
Permissible Values'
01 to 99
001 to 999
0 to 999
0 to 9999
0.0 or greater
0.0 or greater
0, 2
0, 1
1, 2, 3, etc.
0 to 6
1, 2, etc.
1, 2
1,2,3
1, 2
1.0 to 99.9
0.001 to 999.99
0.1 to 10
1.0 to 100.0
1.0 to 999.9
1 to 99
Right-justify numbers in all fields unless a decimal point is
entered. A blank is equivalent to a zero.
If no fibers are observed in a field, write a one-line record with:
(1) 0 entered for sequence no. of fiber in field and for cumulative
sequence no.
(2) diameter and length fields and also columns 26, 28, and 30 blank
(3) grid opening ID, field ID, case ID, magnification, and area,
entered as usual.
41
-------
1*2 3
Table D-3
COMPUTER PRINTOUT OF DATA CARDS
6
789 10
11
12
15
16
C OJ
J_l
Cl —I
CD -H
o
IA-I
T! 0
•H
» a
O M
1 1
1 1
1 2
1 2
1 ?
1 02
1 02
1 02
1 03
1 03
1 03
1 03
1 03
1 04
1 00
1 04
1 05
1 05
1 05
1 0 6
1 06
1 07
1 OP
1 09
1 "
1 IP
1 IP
1 K'
1 IP
1 1"
2 11
2 11
2 11
2 11
2 12
2 12
12
13
13
1"
15
15
15
15
16
16
17
17
17
17
17
IP
2 IP
2 IP
2 IP
•S. c
• H
• £.
U- U
CD -rj
w 3
1
2
1
2
3
u
5
6
1
c!
3
4
5
1
2
3
1
?
3
1
2
i
1
1
2
1
2
3
4
5
1
2
3
4
1
2
3
1
c1
1
1
2
3
4
1
2
1
2
3
4
5
1
2
3
4
^ -0
-H
. li-,
E
d iw
U 0
1
2
3
4
5
06
07
08
09
10
11
12
13
14
15
16
17
IB
19
20
?1
22
23
2U
2>3
2t>
27
26
39
30
31
32
33
34
35
36
37
38
39
"0
41
42
43
44
45
4b
47
46
49
50
51
52
53
54
55
Fiber 1
1 .
,?5
.5
1.
1.
.75
1.
.5
.25
.75
1.
1.
.?5
.25
.25
.=;
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t- .
1.
.5
1.
,'.
1 .
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1.
1.
1.
.25
.?5
1.
1.
.5
i.
1.
.?
.^5
1.
.2
1.
1.
.5
.25
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.S
.?
1.
1.
.5
.75
. 1
.5
1.5
.2
1.
l-i
CJ
-O
20.
7.
17.
10.
1P.
22.
12.
10.
24.
16,
10.
8.
10.
6.
20.
5.
65.
10,
4,
30.
32.
25.
5.
6.
30.
16.
6.
12.
10.
4.
P.
fc.
5.
6.
4,
4.
5.
18.
12.
20.
1".
12.
P.
3.
5.
3.
25.
20.
6,
8.
12.
30.
P.
P.
1 o.
iJ § >-,
•H 33 a
E .0
•H ^j -H
J I-J (i
1
2
2
1
1
1
1
3
1
2
1
1
3
1
2
3
2 1
2
2
1
1
1
2
2
2 1
2 1
2 1
2 1
2
3
1
1
£
2 2
1
2
3
1
3
1
1
i
2
3
2
3
2 1
2 1
2 1
1
3
1
1
d
2 1
M
•J
M
<4
U
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31111
31 11
31 11
31 11
31 11
31 11
31111
31111
3111
3111
3111
31 11
3111
3111
3111
31 11
3111
3111
Mil
31 11
Jill
3111
31111
31111
31111
31111
31111
31111
Screen
x 1000
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17,
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
O 0)
-H
ra >
o
^ ^
< o
.tee
.161
.162
.162
.162
.162
.18?
.112
.162
.162
.182
.162
.182
.182
.162
.162
.162
.182
. 1 62
.182
.162
.162
.182
.182
.162
.162
.182
.162
.182
.182
.1(2
.182
.162
.182
.162
.182
.162
.162
.182
.182
.162
.162
. 162
.182
.182
.162
.182
.182
.182
.182
.162
.162
.182
.162
.1 62
CO
c
•H
~w
<
.8
.8
.0
.0
.0
.0
.8
.0
.0
.0
.0
.0
.0
.8
.0
.0
.0
.0
.0
.0
.8
.0
.0
.0
.0
.0
.0
.8
.0
.0
.8
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1 .0
1.0
1.8
l.o
0 i u -J
-H a a>
• ex aj ^H
rH E OJ r-4
O C3 1/3 W U
9.2 406.5
9.2 406,5
9.2 406.5
9.2 406,5
9.2 406,5
9.2 406.5
9.2 406,5
9.2 406,5
9.2 406.5
9.2 406.5
9,2 406.5
9,2 406.5
9.2 406.5
9.2 U06.5
9.2 406.5
9.2 406,5
9.2 406,5
9.2 406.5
9,2 406.5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406,5
9.2 406,5
9.2 a06.5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406,5
9.2 406.5
9.2 «06,5
9.2 406.5
9,2 406,5
9.2 406.5
9.2 406.5
9.2 406.5
9.2 406,5
9.2 406,5
9.2 406.5
9,2 406.5
9.2 406.5
9.2 406,5
9,2 406.5
9.2 406.5
9.2 406.5
4.2 406.5
9.2 406.*;
9.2 uOfc.5
9.2 U06.5
9.2 406,5
u
X
OJ
13
C
3
3
3
3
3
3
3
3
i
3
3
3
3
3
3
i
3
3
3
3
3
i
3
3
i
3
3
i
3
3
3
3
3
3
3
3
3
i
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
* See page 44 tor detailed explanation of the column headings.
42
-------
OJ
Table D-2
ILLUSTRATION OF FORTRAN CODING FORM SCHEME
. 182
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3'
3
4
4
4
5
5
5
6
6
7
8
9
9
1
I/-
z
1
2.
~
4
C
6
1
2
3
4
5
1
2
3
1
2
3
1
2
1
1
1
2
1
2
j
4
c
6
7
8
g
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1.
. 25
. 5
1.
1.
. 75
1.
. 5
. 25
. 75
1.
1.
. 25
. 25
. 25
. 5
2.
1.
. 5
1.
2.
1.
. 5
. 25
1.
20 .
7 .
17 .
10 .
18 .
22 .
12 .
10 .
24 .
18 .
10 .
8 .
10 .
6.
1
2
2
1
1
1
1
I 3
( 1
! 2
i
i i
i -•-
I i
20. 1
5. !
65 .
10 .
4 .
30 .
32.
25.
5.
6 .
30.
2
2t
3
1
2
3
1
2
3
1
1
1
2
2
1
1
!
311111 17 .
i
i
. i
j i
i
!
i
!
! I
1
|
i
j i
1 i
|
i
3
j
ii
1. 0
9 . 2
406.5
!
!
-------
Table D-3 (continued)
1* 2
2 19
2 19
2 19
2 20
2 20
2 20
2 20
3 21
3 21
3 21
3 21
3 22
3 22
3 22
3 23
3 23
3 23
3 24
3 24
3 25
3 25
3 26
3 26
3 26
3 27
3 2P
3 ,19
3 29
3 30
3 30
3 30
4 31
4 31
4 31
4 32
4 32
4 32
4 33
4 33
4 34
4 34
4 34
4 35
4 35
u 35
4 35
4 35
4 36
4 36
4 36
4 37
4 37
4 37
4 3P
4 39
4 39
440
440
4 40
3
1
2
3
1
2
3
4
1
i?
3
4
1
2
3
1
?
3
1
2
1
2
1
2
3
1
1
1
2
1
2
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
a
5
1
£
3
1
2
3
1
1
2
1
2
3
4
56
57
56
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
TP
79
PO
PI
P2
63
P4
Pb
P6
P7
Pb
P9
°0
91
92
93
94
95
96
97
98
99
1UO
10 1
102
103
U' 4
105
1<'6
H'7
108
t')9
110
111
112
113
114
5
.5
.25
.25
.1
1.5
.5
.25
.75
.25
.5
.25
.5
.5
.25
.5
.25
.2
1.
1.
1.5
.25
.75
.5
.25
,r,
.5
2.
.?5
1.
.5
.25
3.
.5
.5
1.5
1.
1.
1.
.25
2.
1 .
1.
2.
2.
1.
1 .
.5
.5
.5
.5
.5
.5
.25
1.
1.
.25
,S
2.
.75
6
13.
6.
12.
1,
25.
27.
10.
15.
6.
15.
3.
6.
10,
5.
7,
12.
3.
2*.
30.
6.
a.
13.
1".
5.
26.
15.
17.
3.
3.
4.
4.
IP.
7.
15.
25.
15.
10.
35.
5.
10.
15.
12.
28.
12.
1 1.
6.
1".
13.
4.
to.
9,
5.
6.
45.
25.
5.
17.
12.
5.
1 8 9
1
4
3
2
1
2 i.
2
1
2
2
3
2 1
b
3
1
2
3
1
1
1
3
I
2
3
1
2
1
3
2 1
2
3
1
4
4
1
2 1
4
2 1
i
2 1
2 1
4
2 1
1
4
U
3
2 1
4
3
1
4
3
2 1
1
3
1
1
2 1
10
31111
31111
31111
il 1 11
31111
31111
31111
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
3121 1
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
11211
31211
31211
31211
11211
31211
31211
31211
312H
31211
31211
31211
31211
31211
31211
31211
31211
31211
31211
31 21 1
31211
31211
31211
31211
11
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17,
17.
17.
17.
17.
17.
17,
17.
17.
17.
17.
17.
17.
17,
17.
17.
17.
17.
17.
17.
17.
17.
17.
17.
17,
17.
17,
17.
17,
17.
17.
17,
17.
17.
17.
17.
17.
17,
12
.182
.182
.182
.112
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.18?
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.182
.162
.182
.182
. 162
.182
.182
.182
.182
.182
.182
.182
J,3
1.0
1.0
l.o
1.0
1.0
1.0
1.0
1.0
1.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
l.o
1.0
1.0
1.0
1.0
l.o
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
l.o
1.0
1.0
1,0
1.0
l.o
1.0
1.0
l.o
l.o
l.o
1 .0
1.0
1.0
1.0
14 15
9,2 406,5
9,2 406,5
9.2 4C6.5
9.2 406.9
9.2 406,5
9.2 406.5
9,2 406.5
9.2 406.5
9,2 406,9
Q.2 406, 5
9,2 406,5
0.2 406,5
9.2 406.5
9,2 406,5
9.2 406.5
9.2 406,9
9,2 406,5
9,2 406.5
9,2 406.5
9.2 406.5
9.2 406.5
9,2 406,9
9.2 406.5
9.2 406.5
9.2 406,5
9,2 406.5
9.2 406,5
9.2 406,9
9.2 406.5
9.2 406,5
9.2 406,9
9.2 406.5
9.2 406.5
9.? 406.5
9,2 406,5
9,2 406.9
9.2 406.5
9.2 406.5
9.2 406,5
Q.2 406. •?
9.2 U06.9
9,2 406.5
9.2 406.9
9.2 406,9
9.2 406,5
9.2 406.5
9.2 406,9
9.2 406,5
9.2 406,5
9.2 406.5
9.2 406,5
9.2 406.5
9.2 406.9
9,2 406,5
9.2 406.5
9,2 406,5
9.2 406.5
9.2 406.5
9.2 406,5
16
3
3
3
3
3
3
3
3
3
J
3
3
3
3
3
3
3
3
3
3
±
3
3
3
i
i
3
3
i
3
3
3
3
3
3
i
3
3
3
3
3
3
3
3
i
3
i
i
5
3
i
i
3
3
3
3
3
3
3
* See page 44 for detailed explanation of the column headings.
44
-------
EXPLANATION OF THE COLUMNS IN TABLE D-3
Column 1 -- EM grid opening identification number
Column 2 -- Identification number for the field of view
Column 3 -- Sequence number of a fiber within a given field
of view
Column 4 -- Cumulative number of fibers counted
Column 5 -- Fiber width in mm
Column 6 -- Fiber length in mm
Column 7 -- A '2' indicates the fiber crossing the perimeter
and, hence, one that is counted as a half-fiber
Colunn 8 -- A '!' indicates a bundle
Column 9 -- Fiber type identification code
1 -»• chrysotile
2 -> ambiguous
3 ^ no SAED pattern, etc.
Column 10 -- Case identification. Laboratory code, sample
code, TEM grid index, type of TEM instrument
code, the operator code, etc.
Column 11 -- Magnification at the TEM fluorescent screen in
multiples of 1000
f\ 9
Column 12 -- Area of one field of view in multiples of 10 cm
Column 13 -- Ashing factor, to account for the dilution or con-
centration resulting in the ashing step. In the
procedure without the ashing step, the ashing
factor is taken as 1.0.
Column 14 -- Volume of air sampled in m
2
Column 15 -- Effective area of the original air filter in cm
Colunn 16 -- Index for the data set
45
-------
Table D-4
PRINTOUT FROM PROGRAM COHLAB CHARACTERIZING PARAMETERS PER TEM GRID
FIBER AND MASS CONCENTRATION
SUMMARY
LAB 3 SAMPLE 1 CHID 1 CASE 3
THAL ARE* SCANNED « ,aoo4-.o5 SB CM
T^TAL ARC4 F^LfEk s 406.5 Su CM
T"T»L FIHFR COUNT = S6.5 ' FIBERS
rrTAi.voL'iMf'AiR = <).£ cu "ic METERS
fHBYSOTILt
rise* COUNT
(FT?l:RS) 26.5
CFJPERS OFH SO CM OF FIMFR) .66)8+07
(FI«r»5 F'F" CU6 HETl;R OF AJR) .2924 + 09
Cf.TAi^S PCP S3 CM OF FILTL'H) .1241-06
CSCAMS PCP CUP METER ^F nikj .5482-05
C'i) C^ONS) STO DCV l.i?02
MfAN LOG ,()69fc
GfOM MN 1.0720
COCF VA» 1.2066
"; '. AMI" Tt-P rtFAM .(1 &8 3
"KAN LOG -3,'l2e14
(>FO^ MN .H32b
C'IC-F VAR ?.?4513
VPu'-'^l7 Mf'AA' .7210-14
CCilR CM5 STO JQV ,1554-13
>*f it* LOG -3O.3868
seoM MN .7844-17
CflCF VAR ,3076+07
AMPHIPOLE
.0
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
AMBIGUOUS
19.0
.4745+07
.2097+09
33.63 X
.7152
.6267
-.6538
.5201
.8473
.0279
.0140
-3.9964
.0184
.9282
.5595-15
,«>eB2-15
-37.9861
.3163-16
.4293+04
NQ PATTERN NON-ASRtSTOS
10.0
.2496+07
.1104+09
PERCENT TOTAL FIBERS
17.70 X 1
.4471
.2168
-.9470 -1
.3679
.7397
.0162
.0071
-4.2171 -4
.0147
.5463
.1054-15
.1116-15
-37.2536 -37
.6620-16
.1586+01
1.0
.2498+06
.1104+06
.77 X
.3529
.0000
.0415
.3529
.0000
.0147
.0000
.2195
.0147
.0000
.5995-16
.0000
.3531
.5995-16
.0000
ALL FIBERS
56.5
.1411+06
.6235+09
1.1036
1.1182
-.3733
.6665
1.2524
.0445
.0233
-3.7716
.0230
1.2685
.3b90-14
.1118-13
-36.5023
.1900-16
.7941+05
-------
Table D-5
PRINTOUT FROM PROGRAM CONLAB CHARACTERIZING PARAMETERS PER TEM GRID
FIBER AND MASS CONCENTRATION
SUMMARY
LAd 3 SAMPLE 1 CHID 2 CASE 3
TPT»L ARCA SCANNED = .36«0-(.)5 SC CM
TOTAL AREA H'lLTCH = <*0o.b Sfc C*
TSTi,. Kl'jpiy C^UNT = U7.0 FIRfcHS
TOT*!. VOLI'MK AIR = 9.2 Cu«IC MtTtRS
thfiYSGTILfc
(FIcU'BS) 20.0
(FJrv.RS PpK S" CM OF FIl.TtFR) .5495+07
(r.ijtMS PLP S'.0 CM OF FTLTC") . 1?0«-06
(r.,TiMs PEP CUB METCR CF AISI .5320-05
H'f-Tw MFAN 1.7941
C^KrtPMS) ST.;) OTv l."228
MfAN LOG .1371
C ^EF VAR 1 , 157y
" I A '* E T f. R M p A N . 0 8 6 U
C-JCKCflS) STO I5fv .0182
MfA^ LOG -3.523S
ufcQM MM .0295
rr-tF VAP ^.suuo
Cruf- CM) STO UEv .6552-14
AMPHJPOLF
1.0
!l2l4+08
!lub5-07
.0000
-.5306
.5«82
.0000
.onoo
.OPOO
.3997-15
.POCO
.3997-15
.0000
AMBIGUOUS NO
6.0
.1648+07
.7283+08
PERCENT
12.77 X 25.
.6078
-.6029 -l!
.5472
.6297
.0069 !
-3.7574 «4.
.0233
.3865 .
.3231-15
.2220-15
-35.9903 -37.
.2342-15
.1378+01
PATTERN NON
12.0
.3297+07
.1457+09
TOTAL FIBERS
53 X 17
3284
1410
2002
3011
5105
0169
0056
1226 -3
0162
3146
.1046-15
.1327-15
3179 -34
.6209-16
.1471+01
-ASBESTOS
e.o
.2198+07
.9711+08
.02 X
.5147
.2116
.7549
.4701
.5447
.0441
.0147
.1798
.0416
.4142
.934?-15
.6864-15
.9670
.6387-15
.1562+01
ALL FIBERS
47.0
.1291+08
.5705+09
1.0250
,9549
-.4646
.6262
1.2627
.0522
,0334
•3.6480
,0260
1,3776
.3622-14
.6655-14
-38.5966
.1725-16
.1319+06
-------
Table D-6
SUMMARY OF TEST RESULTS ON ONE AIR SAMPLE (SEE TABLES D-4 AND D-5)
1
Data
Set
Code
3-1
3-2
Mean
Std. Dev.
Std. Error (SEm)
t
t • SEm
95% Conf. Interval
Upper
Lower
2
Number
Cone, of
All Fibers,
106/m3
623.5
570.5
597.0
37.48
26.50
12.706
336.71
933.71
260.29
3
4
5
Size Distribution of
All Fibers
Mean
Length,
ym
1.104
1.025
Mean
Dia. ,
ym
0.044
0.052
Mean
Volume,
10-15cm3
3.590
3.822
6
Volume Cone .
of all
Fibers,
10-15cm3/m3
2238.4
2180.5
2209.4
40.94
28.95
367.84
2577.24
1841.56
7
Number
Cone, of
Chrysotile,
106/m3
292.4
242.8
267.6
35.07
24.80
315.11
582.71
(negative)*
8
9
10
Size Distribution of
Chrysotile
Mean
Length,
ym
1.658
1.794
Mean
Dia. ,
ym
0.068
0.086
Mean
Volume,
10-15cm3
7.210
8.428
11
Chrysotile
Mass Cone.
in Air ,
yg/m3
5.482
5.320
5.401
0.115
0.081
1.029
6.430
4.372
-p-
00
* Negative values are truncated to zero. Such situations are due to limited replication. It is recommended that
at least 3 or 4 TEM grids be examined to substantially improve the precision. t-value decreases sharply to
4.30 for n = 3 and to 2.77 for n = 5. Also the standard error decreases with greater replication.
-------
TECHNICAL REPORT DATA
(Please read Instruction? >.vi the ruirsc before c
1. REPORT NO.
EPA 600/2-77-178
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
ELECTRON MICROSCOPE MEASUREMENT OF AIRBORNE ASBESTOS
CONCENTRATIONS
A Provisional Methodology Manual
7. AUTHOR(S)
Anant V. Samudra and Colin F. Harwood
9. PERFORMING ORGANIZATION NAME AND ADDRESS
5. REPORT DATE
August 1977.Revised June 1978
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO
IIT Research Institute
10 West 35th Street
Chicago, Illinois 60616
10. PROGRAM ELEMENT NO.
1AD712 BA-14 (FY-77)
11. CONTRACT GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Sciences Research Laboratory
Office of Research and Development
U. S. Environmental Protection Agency
Research Triangle Park, N. C. 27711
68-_02-2251_
13 TYPE OF REPORT AND PERIOD COVERED
- RTP, N.C. ; Final 6/75 - 6/77
M4. SPONSORING AGENCY CODE
EPA/600/09
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This manual describes a provisional optimum electron microscope (EM) procedure
for measuring the concentration of asbestos in air samples. The main features of the
method include depositing an air sample on a polycarbonate membrane filter, examining
an EM grid specimen in a transmission electron microscope (TEM), and verifying fiber
identity by selected area electron diffraction (SAED).
This provisional manual results from a study to develop an optimum EM procedure
for airborne asbestos determination. The analytical data supporting the provisional
methodology are included in a separate final report.
17.
a.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
'\Air pollution
Asbestos
^Serpentine
*Anphiboles
Measurement
'''Electron microscopy
"Electron diffraction
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b.IDENTIFIERS/OPEN ENDEDTERMS
Chrysotile
19. SECURITY CLASS (Thn Keuort)
UNCLASSIFIED
c. COS AT I Held/Group
13B
08G
HE
14B
21. NO. OF PAGES
57
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
49
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